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Endothelin and Endothelin Receptors in the Fish Gill

Permanent Link: http://ufdc.ufl.edu/UFE0022082/00001

Material Information

Title: Endothelin and Endothelin Receptors in the Fish Gill
Physical Description: 1 online resource (159 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: endothelin, evolution, fish, gill, ion, physiology
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Researchers have been studying osmoregulation in fishes over the past 150 years. The one area that had received little attention is the role of paracrines/autocrines in the local regulation of ion transport, specifically in the teleost gill. The focus of my dissertation work was on the peptide, endothelin-1, and the components necessary for EDN1 signaling, including the enzyme that makes active EDN1, ECE1, and the sites of action, the EDN receptors (EDNRS). When I started this work, there were about a dozen papers on the effects of endogenous mammalian EDN1 on fish blood vessel tone or the effects of bolus injections of mammalian EDN1 on the cardiovascular system of fishes. The purposes of my dissertation work were to 1) localize the aforementioned components of EDN1 in the teleost gill; 2) determine if these components are regulated by environmental salinity, thus giving us some insight into whether or not EDN1 is involved in regulation of ion balance in fishes; 3) determine the evolutionary relationship among the protein sequences for the EDNs, ECEs, and EDNRs. Through the use of molecular biology and immunohistochemistry, I determined that EDN1, ECE1 and the EDNRs are expressed in the euryhaline killifish (Fundulus heteroclitus) and longhorn sculpin (Myoxocephalus octodecemspinosus) gills. From my localization studies, I modeled the putative functions of EDN1, and proposed it acts as a paracrine and autocrine in the fish gill. The localization of the EDNRs in the gill suggests that EDN1 signaling is involved in regulation of mitochondrion-rich cell functions, regulation of lamellar pillar cell tone (and ultimately perfusion of lamellae), and clearance of excess EDN1. With the sequences obtained from the first part of my dissertation, I was able to determine the effects of environmental salinity on mRNA expression of each of these EDN1 signaling components using real-time quantitative PCR in both of these fishes. I was also able to measure protein level differences in these experiments. These mRNAs/proteins are regulated by hyperosmotic and hypo-osmotic stress, further suggesting that they are involved in not only ion balance, but also volume regulation. In addition, EDN1 signaling is postulated to be involved in cell survival during osmotic stress. Finally, through the use of phylogenetics and bioinformatics, I determined that EDN1 and the EDNRs are vertebrate specific proteins, supporting the working hypothesis that EDN1 signaling was an important innovation leading to the development of the jaws and radiation of the vertebrates. Although ECE is found in Archaea, Bacteria and Eukarya, it is hypothesized to have shifted from being a monomer and general protease in all non-vertebrate organisms, to a dimer and a specific EDN1 protease in the vertebrates. Finally, the EDNRB2 was originally classified as an avian-specific EDNR; however, it is found in all non-therian gnathostomes, and I believe deleted from the therian genome 150 mya. From my Rate Shift Analysis of the EDNRA and EDNRB1, I hypothesize that therian EDNRA have different functions than the EDNRA in non-therian gnathostomes, but that the EDNRB1 is well conserved over gnathostome evolution.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Evans, David H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022082:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022082/00001

Material Information

Title: Endothelin and Endothelin Receptors in the Fish Gill
Physical Description: 1 online resource (159 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: endothelin, evolution, fish, gill, ion, physiology
Zoology -- Dissertations, Academic -- UF
Genre: Zoology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Researchers have been studying osmoregulation in fishes over the past 150 years. The one area that had received little attention is the role of paracrines/autocrines in the local regulation of ion transport, specifically in the teleost gill. The focus of my dissertation work was on the peptide, endothelin-1, and the components necessary for EDN1 signaling, including the enzyme that makes active EDN1, ECE1, and the sites of action, the EDN receptors (EDNRS). When I started this work, there were about a dozen papers on the effects of endogenous mammalian EDN1 on fish blood vessel tone or the effects of bolus injections of mammalian EDN1 on the cardiovascular system of fishes. The purposes of my dissertation work were to 1) localize the aforementioned components of EDN1 in the teleost gill; 2) determine if these components are regulated by environmental salinity, thus giving us some insight into whether or not EDN1 is involved in regulation of ion balance in fishes; 3) determine the evolutionary relationship among the protein sequences for the EDNs, ECEs, and EDNRs. Through the use of molecular biology and immunohistochemistry, I determined that EDN1, ECE1 and the EDNRs are expressed in the euryhaline killifish (Fundulus heteroclitus) and longhorn sculpin (Myoxocephalus octodecemspinosus) gills. From my localization studies, I modeled the putative functions of EDN1, and proposed it acts as a paracrine and autocrine in the fish gill. The localization of the EDNRs in the gill suggests that EDN1 signaling is involved in regulation of mitochondrion-rich cell functions, regulation of lamellar pillar cell tone (and ultimately perfusion of lamellae), and clearance of excess EDN1. With the sequences obtained from the first part of my dissertation, I was able to determine the effects of environmental salinity on mRNA expression of each of these EDN1 signaling components using real-time quantitative PCR in both of these fishes. I was also able to measure protein level differences in these experiments. These mRNAs/proteins are regulated by hyperosmotic and hypo-osmotic stress, further suggesting that they are involved in not only ion balance, but also volume regulation. In addition, EDN1 signaling is postulated to be involved in cell survival during osmotic stress. Finally, through the use of phylogenetics and bioinformatics, I determined that EDN1 and the EDNRs are vertebrate specific proteins, supporting the working hypothesis that EDN1 signaling was an important innovation leading to the development of the jaws and radiation of the vertebrates. Although ECE is found in Archaea, Bacteria and Eukarya, it is hypothesized to have shifted from being a monomer and general protease in all non-vertebrate organisms, to a dimer and a specific EDN1 protease in the vertebrates. Finally, the EDNRB2 was originally classified as an avian-specific EDNR; however, it is found in all non-therian gnathostomes, and I believe deleted from the therian genome 150 mya. From my Rate Shift Analysis of the EDNRA and EDNRB1, I hypothesize that therian EDNRA have different functions than the EDNRA in non-therian gnathostomes, but that the EDNRB1 is well conserved over gnathostome evolution.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Evans, David H.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022082:00001


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ENDOTHELIN AND ENDOTHELIN RECEPTORS IN THE FISH GILL By KELLY ANNE HYNDMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Kelly Anne Hyndman 2

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To my family and friends who have continued to encouraged me through this crazy learning phase of my career. 3

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ACKNOWLEDGMENTS I thank my advisor, David Evans, for fully supporting my project and taking me to the Mount Desert Biological Laborat ory in Salisbury Cove, ME. My experiences at MDIBL have greatly affected the way I think a bout science and I am so grateful for that. I would like to thank Drs. Peter Piermarini and Keith Choe, for teachin g me most of the techniques presented in my dissertation. With their guidan ce and patience, I (hopefully) have mastered these techniques as they did. Next, I would like to thank my committee members, Drs. Cohn, Guillette, Julian, Miyamoto, and Wood. Each one of them opened th eir labs to me and helped me throughout my dissertation work. Thank yous go to the Cohn lab for teaching me in situ hybridization; the Guillette lab for allowing me to use their embe dding set up, histological supplies and quantitative real-time PCR machine; the Julian lab for teachin g me dot blots; the Miyamoto lab for throwing me into the world of bioinformatics and phylogeny (Chapter 3 is so diffe rent from my usual work); and the Wood lab, although I did not include my prostaglandin work in this dissertation, Dr. Wood taught me how to extrac t prostaglandins and prepare them for EIAs. All of these colleagues have challenged me and helped me develop and complete a dissertation that I am really proud of. I have been fortunate that my project was fully funded by Dr. Evans NSF grants, so thank you to him for that. In addition, I must thank the grants I received from Sigma Xi and the Department of Zoologys Riewald Award. I am gr ateful to the Department of Zoology, College of Liberal Arts and Sciences, a nd the Graduate Student Council fo r the numerous travel grants I have received over my tenure here at the University of Florida. I would like to thank my parents, Jim and Susa n, and my sister Jennifer. Their love and support has been important during my time in gradua te school. Finally, I would like to thank my 4

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5 grandpa Sinclair, who always encouraged me to pursue my dreams, no matter how crazy or farfetched they may have been.

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 LIST OF ABBREVIATIONS........................................................................................................12 ABSTRACT...................................................................................................................................13 CHAPTER 1 INTRODUCTION................................................................................................................. .15 Endothelins.............................................................................................................................18 Endothelin Receptors........................................................................................................... ...19 Medical Importance of Endothelin-1......................................................................................21 Cardiovascular Effects of Endothelin in Fishes......................................................................21 Endothelin Effects of Ion Transport.......................................................................................24 Overview of Dissertation Research........................................................................................25 2 ENDOTHELIN AND ENDOTHELIN CONVERTING ENZYME-1 IN THE FISH GILL: EVOLUTIONARY AND PH YSIOLOGICAL PERSPECTIVES..............................28 Introduction................................................................................................................... ..........28 Methods..................................................................................................................................29 Fish Maintenance.............................................................................................................29 EDN1 cDNA....................................................................................................................29 ECE cDNA......................................................................................................................30 Sequence and Phylogenetic Analysis..............................................................................31 Multiple Tissue Semi-Quantitative PCR.........................................................................31 Salinity Challenges..........................................................................................................32 Quantitative Real-Time PCR...........................................................................................32 Statistics...........................................................................................................................33 Tissue Preparation for In Situ Hybridization and Immunohistochemistry......................34 In Situ Hybridization.......................................................................................................34 Immunohistochemistry....................................................................................................36 Results.....................................................................................................................................36 Sequence Analyses..........................................................................................................36 Tissue Distributions.........................................................................................................37 In Situ Hybridization.......................................................................................................37 Immunohistochemistry....................................................................................................38 Salinity Acclimations......................................................................................................38 Discussion...............................................................................................................................39 6

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Endothelin Sequences......................................................................................................39 Gill Expression of EDN1 mRNA and ProEDN1 Protein................................................40 Acute and Chronic Salinity Acclimations.......................................................................41 Evolution of EDNs and ECE...........................................................................................43 Tentative Model for EDN1 Signaling in the Killifish Gill..............................................45 Note.................................................................................................................................46 3 FUNCTIONAL AND GENOMIC STUDY OF THE ENDOTHELIN RECEPTORS..........59 Introduction................................................................................................................... ..........59 Methods..................................................................................................................................61 Molecular Cloning, Sequencing and Tissue Distribution................................................61 Data mining and multiple sequence Alignment..............................................................62 Phylogenetic Analyses.....................................................................................................63 Synteny............................................................................................................................64 Rate Shift Analysis..........................................................................................................64 Results.....................................................................................................................................65 Killifish Endothelin Receptors........................................................................................65 Phylogeny........................................................................................................................66 Synteny............................................................................................................................68 Rate Shift Analysis..........................................................................................................69 Discussion...............................................................................................................................70 4 EFFECTS OF ENVIRONMENTAL SA LINITY ON GILL ENDOTHELIN RECEPTOR EXPRESSION IN THE KILLIFISH Fundulus heteroclitus .............................86 Introduction................................................................................................................... ..........86 Methods..................................................................................................................................88 Fish Maintenance.............................................................................................................88 Salinity Challenges..........................................................................................................88 Quantitative Real-Time PCR...........................................................................................88 Immunohistochemistry and Immunoblotting..................................................................89 Statistics...........................................................................................................................89 Results.....................................................................................................................................90 Endothelin Receptor mRNA Levels................................................................................90 Endothelin Receptor Protein Concentrations..................................................................91 Immunohistochemistry....................................................................................................91 Discussion...............................................................................................................................91 5 SHORT TERM LOW-SALINITY TOLER ANCE BY THE LONGHORN SCULPIN, Myoxocephalus octodecemspinosus .....................................................................................104 Introduction................................................................................................................... ........104 Methods................................................................................................................................106 Low Salinity Acclimation..............................................................................................106 Plasma Chemistry..........................................................................................................107 Molecular Techniques...................................................................................................107 7

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8 Quantitative Real-Time PCR.........................................................................................108 Immunohistochemistry..................................................................................................109 Western and Dot Blotting..............................................................................................110 Antibodies..................................................................................................................... .111 Gill Na+, K+-ATPase Activity.......................................................................................112 Statistics.........................................................................................................................112 Results...................................................................................................................................113 Plasma Chemistry and Gill Na+, K+-ATPase Activity..................................................113 Immunolocalization of CFTR, NKA, and NKCC1.......................................................113 Westerns and Dot Blots.................................................................................................114 Quantitative Real-Time PCR.........................................................................................115 Discussion.............................................................................................................................115 6 EFFECTS OF LOW ENVIRONMENTA L SALINITY ON ENDOTHELIN RECEPTOR AND ENDOTHELIN CONVERTING ENZYME-1 MRNA IN THE GILL OF THE LONGHORN SCULPIN, Myoxocephalus octodecemspinosus ..................124 Introduction................................................................................................................... ........124 Methods................................................................................................................................126 Molecular Sequencing and Tissue Distribution............................................................126 Salinity Challenge.........................................................................................................127 Quantitative Real-Time PCR.........................................................................................128 Statistics.........................................................................................................................129 Results...................................................................................................................................129 Phylogenetic Relationships...........................................................................................129 Longhorn Sculpin Tissue Distribution..........................................................................130 Quantitative Real-Time PCR.........................................................................................130 Discussion.............................................................................................................................130 7 CONCLUSIONS.................................................................................................................. 138 APPENDIX: TISSUE DISTRIBUTION OF THE LAMPREY ENDOTHELIN A RECEPTOR..........................................................................................................................144 LIST OF REFERENCES.............................................................................................................145 BIOGRAPHICAL SKETCH.......................................................................................................159

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LIST OF TABLES Table page 2-1 Primers used for cloning, tissue distri butions and quantitat ive real-time PCR.................47 3-1 A summary of the binding profiles, and embryonic and adult tissue distribution of the gnathostome EDNRs....................................................................................................75 3-2 Degenerate and non-degenera te primers used to sequen ce the killifish endothelin receptors...................................................................................................................... .......77 3-3 Specific killifish primers used in the tissue distribution experiments...............................78 3-4 Contingency tables summarizing the results of the rate shift analysis..............................79 4-1 Non-degenerate primers used in the quantitative real-time PCR experiments..................97 5-1 Primers used in sequencing and quantit ative real-time PCR of longhorn sculpin Na+, K+-ATPase, Na+, K+, 2Clcotransporter, and the cystic fibrosis transmembrane conductance regulator......................................................................................................120 5-2 Plasma parameters and gill Na+, K+-ATPase for longhorn sculpin acclimated to 100, 20 or 10% SW for 24 or 72 hours....................................................................................121 6-1 Primers used the tissue distribution a nd quantitative real-time PCR analyses..............134 9

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LIST OF FIGURES Figure page 2-1 Maximum likelihood analyses of the vertebrate preproendothelin amino acid sequences...................................................................................................................... .....48 2-2 An alignment of vertebrate pr eproendothelin-1 protein sequences...................................50 2-3 Maximum likelihood analyses of the ECE family of proteins...........................................51 2-4 Tissue distribution of killifish EDN1A, EDN1B, and ECE1 ..............................................53 2-5 Representative pictures of in situ hybridization of EDN1A and EDN1B mRNA in lamellar cross-sections of the seawater killifish gill..........................................................54 2-6 Representative pictures of killifish lamellar cross-sections, labeled with antiproendothelin-1 (brown) and anti-NKA (blue)..................................................................55 2-7 Acute changes in killifish gill E DN1A, EDN1B and ECE1 mRNA levels as determined by quantitative Real Time-PCR......................................................................56 2-8 Chronic changes in gill EDN1A, EDN1B and ECE1 mRNA levels as measured by quantitative Real Time-PCR..............................................................................................57 2-9 A model of paracrine and autocrin e EDN1 signaling in the fish gill.................................58 3-1 An illustration of A) type I and B) type II sites determined by a rate shift analysis........80 3-2 mRNA tissue distribu tion of the killifish EDNRA EDNRB1, and EDNRB2 using duplexing, multi-tissue PCR..............................................................................................81 3-3 A maximum likelihood tree of the gnathostome EDNRs..................................................82 3-4 Genetic maps of the genes (surrounding the EDNRB2 in non-therian gnathostomes, and hypothesized location in the therians..........................................................................84 3-5 Illustration of the human A) EDNRA and B) EDNRB1....................................................85 4-1 Relative gill mRNA levels for endothelin receptors from killifish acclimating to seawater (SW) or fresh water (FW) over a 24 h period.....................................................98 4-2 Relative gill mRNA levels for the endothelin receptors from the killifish (n=5-6) acclimated for 30 days to either seawater..........................................................................99 4-3 Western blots from s eawater killifish gills......................................................................100 4-4 Killifish gill protein level differences of the endothelin receptors during acclimation to SW or FW.................................................................................................................... 101 10

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11 4-5 Representative light micrographs of the immunolocalization of the endothelin A receptor (EDNRA) in the killifish gill.............................................................................102 4-6 Representative light micrographs of the immunolocalization of the endothelin B receptors (EDNRB) in the killifish gill............................................................................103 5-1 Representative light micrographs of th e immunolocalization of the CFTR, NKA, and NKCC1 in the sculpin gill ..............................................................................................122 5-2 Longhorn sculpin gill CFTR, NKA a nd NKCC1 expression levels following acclimation to 100, 20 or 10% seawater..........................................................................123 6-1 Maximum likelihood analysis of the endot helin receptors including our new sculpin EDNRA, EDNRB1 and EDNR B2 protein sequences.....................................................135 6-2 mRNA tissue distribution of the endothelin receptors from the longhorn sculpin..........136 6-3 Longhorn sculpin gill A) EDNRA, B) EDNRB1, C) EDNRB2 and D) ECE1 expression levels following acclimation to 100, 20 or 10% seawater (SW) for 24 and 72 hours (h)......................................................................................................................137

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LIST OF ABBREVIATIONS aa amino acid bp base pair CT cycle threshold DEPC diethylpyrocarbonate water DIG digoxigenin EDN endothelin EDNR endothelin receptor FW fresh water h hour/hours indel insertion or deletion min minutes ORF open reading frame PBS phosphate buffered saline RPA16 RNA polymeras e I 16kDa polypeptide s.e.m. standard error of the mean sec seconds SPRY3 sprouty-3 SRX6c sarafotoxin 6c SSC sodium chloride, sodium citrate solution SW seawater SYBL1 synaptobrevin-like 1 TBS tris-buffered saline TMLHE trimethyllysine hydroxylase, epsilon U units 12

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENDOTHELIN AND ENDOTHELIN RECEPTORS IN THE FISH GILL By Kelly Anne Hyndman May 2008 Chair: David Evans Major: Zoology Researchers have been studying os moregulation in fishes over th e past 150 years. The one area that had received little attention is the role of paracrines/autocrines in the local regulation of ion transport, specifically in the teleost gill. The focus of my dissertation work was on the peptide, endothelin-1, and the components nece ssary for EDN1 signaling, including the enzyme that makes active EDN1, ECE1, and the sites of action, the EDN receptors (EDNRS). When I started this work, there were about a dozen papers on the effects of endogenous mammalian EDN1 on fish blood vessel tone or the effects of bolus injec tions of mammalian EDN1 on the cardiovascular system of fishes. The purposes of my dissertation work were to 1) localize the aforementioned components of EDN1 in the teleost gill; 2) determine if these components are regulated by environmental salinit y, thus giving us some insight into whether or not EDN1 is involved in regulation of ion balance in fishes; 3) determine the evolutionary relationship among the protein sequences for the EDNs, ECEs, and EDNRs. Through the use of molecular biology and immunohistochemistry, I determined that EDN1, ECE1 and the EDNRs are expre ssed in the euryha line killifish ( Fundulus heteroclitus ) and longhorn sculpin ( Myoxocephalus octodecemspinosus) gills. From my localization studies, I modeled the putative functions of EDN1, and propos ed it acts as a paracrine and autocrine in the 13

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14 fish gill. The localization of the EDNRs in the gill suggests that EDN1 signaling is involved in regulation of mitochondrion-rich cell functions, regulation of lamellar pillar cell tone (and ultimately perfusion of lamellae), and clearance of excess EDN1. With the sequences obtained from the first part of my dissertation, I was able to determine the effects of environmental salinity on mRNA expression of each of these EDN1 signaling components using real-time quantitative PCR in both of these fishes. I was also able to measure protein level differences in these experiments. These mRNAs/proteins are regulated by hyperosmotic and hypo-osmotic stress, further sugge sting that they are i nvolved in not only ion balance, but also volume regulati on. In addition, EDN1 signaling is postulated to be involved in cell survival during osmotic stress. Finally, through the use of phylogenetics and bioi nformatics, I determined that EDN1 and the EDNRs are vertebrate specific proteins, supporting the working hypothesis that EDN1 signaling was an important innovatio n leading to the development of the jaws and radiation of the vertebrates. Although ECE is found in Archaea, Bacteria and Eukarya, it is hypothesized to have shifted from being a monomer and general protease in all non-vertebrate organisms, to a dimer and a specific EDN1 protease in the verteb rates. Finally, the EDNRB2 was originally classified as an avian-specific EDNR; however, it is found in all non-ther ian gnathostomes, and I believe deleted from the therian genome 150 mya. From my Rate Shift Analysis of the EDNRA and EDNRB1, I hypothesize that therian EDNRA have different functions than the EDNRA in non-therian gnathostomes, but that the EDNRB1 is well conserved over gnathostome evolution.

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CHAPTER 1 INTRODUCTION Teleost fishes are the most spec ious group of vertebrates, and they have a diverse range of habitat use. Some are stenohaline freshwater or stenohaline marine, a nd these fishes can only maintain ion balance over a small range of envi ronmental salinities. A third broad category is the euryhaline fishes, which can tolerate change s in environmental salinity. The osmoregulation of teleost fishes has been studied for over the past 150 years, and due to space limitations, I will briefly review teleost osmoregulation here (see these reviews Karnaky, 1998; Marshall and Farrell, 2006). Depending on the salinity of their environm ent, fishes must overcome physiological challenges. For example, freshwater fishes are hyperosmotic to their environment, and consequently they tend to gain water and lose ions. To compensate for this water load, they tend to produce a dilute urine and uptak e ions at the gill. In cont rast, seawater fishes are hypoosmotic to their environment, and they tend to lo se water and gain excess salts. To combat this problem, marine fishes drink the seawater and transport salts acr oss the gut epithelium, creating an osmotic gradient favorable for water to follow. These excess salts are transported in the blood to the gills were they are actively excret ed. Euryhaline fishes like the killifish ( Fundulus heteroclitus ), experience large changes in environm ental salinity over th e course of a day (Marshall, 2003), and they must regulate ion uptake or excretion to maintain proper ion balance. The killifish does this by rapidly upor down-regu lating gill ion transporte r density (Choe et al., 2006; Marshall et al., 1999; Scott et al., 2004; Scott and Schulte, 2005). The fish gill is a multifunctional organ that is the main site of ion and acid/base regulation, gas exchange, and nitrogenous waste excretion. The gill epithelium is in direct contact with the environment, and it receives 100% of the cardi ac output (Olson, 2002). In the gill, there are 15

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specialized cells that are involve d in ion transport termed the m itochondrion-rich cell (MRC, also called the chloride cell). These cells are characterized by many mitochondria and ion transporting proteins, and an extensive basola teral membrane. The MRCs have distinct freshwaterand seawater-type morphologi es (Katoh and Kaneko, 2003; Perry, 1997). Freshwater MRCs have a flattened apical memb rane with many microvilli. In contrast, the seawater MRC has an apical crypt, with no micr ovilli, and they are generally larger in size (Katoh and Kaneko, 2003; Perry, 1997) These cells also have di fferent distribut ions of ion transporters. Freshwater MRCs f unction in ion uptake and this i nvolves ion transporters such as, the apically expressed V-ATPase, HCO3 -/Cl-, Na+/H+ exchangers, and a hypothesized Na+ channel, and the basolaterally expressed Na+, K+-ATPase (NKA) (Evans et al., 2005). The seawater MRC functions in ion excretion a nd apically expresses the cystic fibrosis transmembrane conductance regulator (a chloride channel), and basolaterally expresses the Na+/K+/2Clcotransporter, inward rectifier K+ channel (eKir), and NKA (Evans et al., 2005). The hormonal regulation of osmoregulati on has been extensively studied (see these reviews McCormick, 1995; McCormick and Bradsh aw, 2006). Generally, prolactin is the hormone necessary for freshwater acclimation, and cortisol is the hormone necessary for seawater acclimation (McCormick and Bradshaw, 2006) In fishes acclimating to fresh water, prolactin stimulates an increase in the fres hwater-type MRC morphology, and stimulates an increase in Na+, and Cluptake (Evans, 2002; Lee et al., 2006; McCormick and Bradshaw, 2006). Although cortisol is classified as the seawater-adapting hormone (McCormick and Bradshaw, 2006), it is also is important in fres hwater acclimation. Co rtisol injections in freshwater-acclimated fishes leads to NA+ and Cluptake (Perry, 1997), freshwater-type MRC morphology and an increase in Na+, K+-ATPase activity (Dang et al., 2000). During seawater 16

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acclimation, the cortisol and growth hormone/insulin-like growth factor axis is stimulated. In the killifish, one hour post a fresh wate r to seawater transfer, there is a sevenfold increase in plasma cortisol concentration (Marshall et al., 1999), and in the tilapia (Oreochromis mossambicus) it is released within minutes of a salinity transfer (Hegab and Hanke, 1984) (it maybe released within minutes in the killifish but this has not been test ed). In addition, growth hormone is released and works synergistically with cortisol to increase gill Na+, K+, 2Cldensity and increase gill Na+, K+-ATPase activity (Pelis and Mc Cormick, 2001). Also, these hormones stimulate development of the seawater-type MRC (Sakamoto and McCormick, 2006). Recently, an new osmosensing transcription factor, OSTF1, was cloned and char acterized from the tilapia (Fiol and Kultz, 2005). Although there is little evidence that the OSTF1 actually senses changes in osmolality, it is rapidly upregulated within minutes of transf erring the tilapia to seawater. Fiol and Kultz (2005) predict that the effects of cortisol are mediated via the OSTF1 and they are currently testing this hypothesis. In addition to the few hormones that were described above, there are many other hormones that are believed to be involved in the osmore gulation of fishes. These include natriurectic peptides, urotensin II, thyroid hormones, arginine vasotocin, a ngiotensin II, insulin, vasoactive intestinal peptide (and likely others that are yet to be tested) (Evans, 2002; McCormick and Bradshaw, 2006). The one area of teleost osmoregulation that has not been extensively studied is the role of locally produced signaling molecu les in the gill. In addition to the aforementioned endocrine factors, there may also be paracr ines and/or autocrines that are involved in the regulation of ion balance. A study from our lab suggests that th e peptide endothelin-1 (E DN1), the gas nitric oxide (NO), and the arachidonic acid-d erived prostaglandins (likely PGE2) can regulate gill ion 17

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transport (Evans et al., 2004). Evans et al. (2004) determined that EDN1 inhibits net chloride transport (as measured by the inhibition of short circuit current) by the killifish opercular epithelium. This single layer sheet of epith elium is a commonly used model tissue for the seawater teleost gill, because it contains ma ny MRCs like the gill (Karnaky and Kinter, 1977; Karnaky et al., 1977). The inhibition of chlori de transport was mediated through the EDN-1 stimulation of NO and PGE2 (Evans et al., 2004). The mechanism of how the EDN1-NO-PGE2 axis inhibits ion transport is unc lear. For my dissertation, I focu sed on EDN1 in the fish gill. For more information on prostaglandins and NO in the killifish, see Choe et al. (2006) and Hyndman et al. (2006). Endothelins Endothelins (EDNs) are a family cardiovascular peptides with three isoforms, EDN1, EDN2 and EDN3 (Inoue et al., 1989; Yanagisawa et al., 1988c) and the genes encoding these proteins are located on different chromosomes (Masaki, 1993). They are regarded as the most potent vasoconstrictors yet iden tified. Endothelins are translated as ~200 amino acid (aa) preproendothelins (preproEDN) that are initially cleaved by a furin-like enzyme (Yanagisawa et al., 1988c) to form the relatively inactive 38 aa proendothelin (proEDN, also known as BigEDN) (Kimura et al., 1989). Pr oendothelin is further cleaved to form the active 21 aa EDN by the endothelin converting enzyme (ECE-1 and/or EC E-2) (Shimada et al., 1994; Xu et al., 1994). Endothelin-1 release is stimulated by various factors including hypoxia, cortisol, growth factors, sheer stress, calcium ionophores, cytoki nes and endotoxins (Levin, 1995). When I began this work, only a few endothe lin-like peptide sequences were available from non-mammalian vertebrates. These includ ed the rainbow trout (Wang et al., 1999), frog ( Rana ridibula ) (Wang et al., 2000) and the American alligator ( Alligator mississipiensis ) (Platzack et al., 2002). These were peptide sequ ences, as the coding sequences for these EDNs 18

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had not been determined. In addi tion, there is a family of cardi otoxic peptides, the sarafotoxins, that shares 70% primary sequence identity a nd structural identity with mammalian EDN1 (Lee and Chiappinelli, 1989). The sarafoxtoins are found in the venom of the Israeli burrowing asp ( Actractapis engaddensis ), and there is little evidence to suggest they are homologous to the EDNs. The most parsimonious hypothesis is th at they occurred thr ough convergent evolution (Froy and Gurevitz, 1998). Endothelin Receptors Endothelins bind to a specific group of seven transmembran e domain, G-protein-coupled receptors (GPRCs) that are part of the rhodopsin/ -adrenergic GPRC famil y. The classification of the EDN GPCRs has been quite confusing. Fo r example, most researchers acknowledge that there are two EDN-specific receptors (EDNRs) termed, EDNRA (Arai et al., 1990), EDNRB (I will refer to this receptor as its official name EDNRB1) (Sakurai et al., 1990); however, a third EDNR, an amphibian specific EDNRC, was clone d and characterized from melanophores (Karne et al., 1993). In 1998, a fourth GPCR was cl oned and characterized from the chicken ( Gallus gallus) and quail ( Coturnix japonica ), and termed the avian-specific EDNRB2. Traditionally, the EDNRs were classified ba sed upon their pharmacological profiles (see Table 3-1). For example, mammalian EDNRA preferentially bind s EDN1 and EDN2 over EDN3 (Arai et al., 1990). In contrast, mammalian EDNRB1 binds all three EDNs with equal affinity and binds the sarafotoxins, which are EDNRB1 sp ecific agonists (Lecoin et al., 1998; Sakurai et al., 1990). In addition to these EDNRs, pharmacological and physiological studies in mammals suggest that there are multiple EDNRB-type receptors. Fo r example, EDN1 binding to vascular smooth muscle EDNRB1 results in muscle contractions (Yanagisawa et al., 1988a; Yanagisawa et al., 1988b). On the contrary, EDN1 binding of endothe lial EDNRB1 stimulates NO and prostacyclin 19

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production (De Nucci et al., 1988a ; De Nucci et al., 1988b), and subsequent vasodilation of smooth muscle cells. Yet, there is no molecu lar evidence for two EDNRB-type genes in the mammals (Pollock and Highsmith, 1998)(Chapter 3) Most likely these differing responses are due to splice variants of the mammalian E DNRB1(Elshourbagy et al., 1996). Unlike the mammals, the birds have a third receptor, the EDNR B2. This receptor binds all three EDNs with equal affinity (like the EDNRB1), but it has a ve ry low affinity for the sarafotoxins (like the EDNRA) (Lecoin et al., 1998). In addition, the prim ary sequence of this receptor is more similar to mammalian EDNRB1 than EDNRA thus, Lecoin et al. (1998) termed it the EDNRB2. Finally, the amphibian-specific E DNRC binds preferentially binds EDN3 over EDN1 (Karne et al., 1993). As with the EDNs, when I started my disse rtation there was only pharmacological and physiological evidence for the EDNRs in the fish es. For example, physiological studies have suggested that the aortic vascular smooth muscle of the dogfish shark ( Squalus acanthias) has EDNRB-like receptors (Evans et al., 1996), but that hagfish ( Myxine glutinosa) sea lamprey ( Petromyzon marinus ), and eel ( Anguilla rostrata ) aortic vascular smooth muscles contain EDNRA-like receptors (Evans and Harrie, 2001). In addition, pharmacological studies using receptor binding assays demonstrated EDNRB-like receptors in the dogfish gill(Evans and Gunderson, 1999), but autoradiographic studies s howed EDNRA-like receptors in the trout ( Oncorhynchus mykiss) gill (Lodhi et al., 1995). The tr out EDNRA-like receptors were specifically localized in the gi ll lamellae to a region termed th e lamellar sinusoid by the authors (Lodhi et al., 1995). The characterization of th e EDNR subtypes may be species and protocol specific. During my dissertation work, the E DNRB and EDNRA were immunolocalized in the gills of the cod ( Gadus morhua) (using heterologous antibodies) (Stenslokken et al., 2006), and 20

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EDNRA in the gill of the tiger pufferfish ( Takifugu rubripes ) (using a homologous antibody) (Sultana et al., 2007). With the advancemen t of high throughput sequencing and genome sequencing, EDNR sequences can be found in all vertebrates and in Chapter 3 I explore the phylogenetic and evolutionary history of the EDNRs. Medical Importance of Endothelin-1 A large portion of EDN research has gone into understanding the pa thologies associated with errors in EDN signaling. These condi tions include hypertension, atherosclerosis (Shreenivas and Oparil, 2007), congestive heart failure (Ange rio, 2005), and glomerulonephritis (Richter, 2006). About 73 million Americans currently suffer from hypertension, and about 60% of them are under treatment for this disease (American Heart Associat ion high blood pressure statistics), including EDNR antagonists (e.g. bo setan and ambrisentan). There are also developmental and genetic diseases associated with components of the EDN signaling cascades. This includes Hirschsprung disease, a developmen tal disorder that causes improper innervation of the gut resulting in a condition called anga nglionic megacolon (Baynash et al., 1994; Puffenberger et al., 1994). Initially, the EDN system was described as a regu lator of vascular tone ; however, it is quite evident that this system has many functions A recent boom of research has been in understanding EDN signaling during development. Endothelin-1 and the EDNRs are necessary for craniofacial development, and gut innervatio n. This has been demonstrated in mammals and fishes (see Clouthier and Schilling, 2004) and Cl outhier and Schilling (2 004) have hypothesized that EDN1 signaling in jaw development was a gnathostome innovation. Cardiovascular Effects of Endothelin in Fishes Over the past 20 years, the mammalian EDN system has been extensively studied (see reviews Gandhi et al., 1994; La and Reid, 1995; Masaki, 1998; Duru et al., 2001) but 21

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comparatively little is known about EDNs in non-mammalian vertebrates such as the fishes. Olson et al. (1991) evaluated th e cardiovascular effects of ET in trout using cannulated dorsal aortas, in situ perfused hearts, isolated perfused gills, perfused trunks, and isolated systemic vascular rings. They determined that intraarterial bolus injections of mammalian EDN1 (667 pmolkg-1) produced a triphasic (increas e-decrease-increase) response in dorsal aorta pressure (PDA), and continuous infusions produced an incr eased mean perfusion pressure in a dosedependent manner. Hoagland et al. (2000) replicated this study using EDN1 isolated from the trout. They showed that intra-arte rial bolus injections of trout EDN1 (667 pmolkg-1) produced a triphasic response in PDA, and that continuous infusions prod uced a dose-dependent increase in PDA, just as Olson et al. (1991) showed with mammalian EDN1. In a similar study, LeMevel et al. (1999) found that intracerebroventricular or in tra-arterial injections of mammalian EDN1 (86 pmolkg-1) produced a transient increase in PDA, but a triphasic response was not observed further suggesting dose-dependent effects of EDN1 on PDA. Additionally, Olson et al. (1991) found that EDN1 increased mean perfusion pressure in isol ated gills in a dose-depe ndent manner and that the half-maximal effective concentration (EC50) was <10-8 M. Other studies have examined constriction of is olated blood vessels in fishes (Poder et al., 1991; Sverdrup et al., 1994; Evans et al., 1996; Ev ans and Harrie, 2001). In the dogfish shark, mammalian EDN1 caused significant constriction of aortic vascular rings, with an EC50 of 10-9 M (Evans et al., 1996). Evans a nd Harrie (2001) showed that vent ral aortic rings, from hagfish, lamprey, and eels constric ted in response to 0.1 M mammalian EDN1. Wang et al. (1999) compared the constrictive re sponses of isolated fish and rat vascular rings to trout EDN1 and mammalian EDN1. They found that trout anterior cardinal veins and branchial arteries were more sensitive to mammalian EDN1 than trout EDN1. The increased 22

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sensitivity to mammalian EDN1 was hypothesized to be due to slower degradation of the mammalian EDN1compared to the native trout EDN1 (Wang et al., 1999). Degradation of EDN1 has been proposed to be mediated through membrane bound me talloproteinases and internal lysosomes (Jackman et al ., 1993). In rats and guinea pigs the pulmonary circuit rapidly removes 60% of radioiodinated EDN1 and EDN3 in one minute (see La and Reid, 1995). In humans, 53% of EDN1 is removed by pulmonary clearance and this mediated through EDNRB1 (see La and Reid, 1995). In fishes, 55% of an EDN1 bolus was removed during a single pass through the gills (Olson, 1998). Presently, it is unknown which receptors mediate this clearance in fishes. Endothelin induced vasoconstriction may have a large effect on gill haemodynamics. The gills are highly vascularized and are perfused by the entire cardiac output (Olson, 1998); thus, any change in blood flow or pressure would greatly influence thei r functions. The physiological function(s) of EDN1 in fish g ills is unknown. It has been hypothe sized that EDN1 redistributes lamellar blood flow, since a ventra l aortic injection of EDN1 cause d constriction of pillar cells in the gill lamellae of trout and cod (Nilsson a nd Sundin, 1998; Stenslokken et al., 1999). This contraction resulted in a shift of intralamellar bl ood flow to the outer marginal channels. Pillar cells contain contractile elemen ts and although they do not appear to be innervated, hormones like EDN1 may signal pillar cells to contract (or dilate depending on the signal, Bettex-Galland and Hughes, 1973)(Mistry et al., 2004). Sundin and Nilsson (1998) found no evidence that the lamellar arterioles or filamental arteries (affe rent or efferent) were constricted by mammalian EDN1. It appears the pillar cells may regulate microcirculation through the gill lamellae. From these studies, it appears that EDN1 may be one of the paracrine/autocrines controlling this system. 23

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Endothelin Effects of Ion Transport In addition to cardiovascular responses, EDN1 has been shown to have effects on renal transport in mammals (Garvin a nd Sanders, 1991; Plato et al., 2000; Zeidel, 1993). In the rat proximal tubule, EDN1 inhibited Na+, K+-ATPase activity and bicarbon ate transport (Garvin and Sanders, 1991). In the rabbit inner medu llary collecting duct, EDN1 inhibited Na+-K+-ATPase activity and lead to sodium natr iuresis (Zeidel et al., 1989). In the rat thick asce nding limb of the loop of Henle, EDN1 inhibited net chloride flux and this was mediated by an EDNRB1 (Plato et al., 2000). Most recently, a series of papers has emerged from Donald Kohans lab at the University of Utah. They have developed co llecting duct-specific EDN1, EDNRB1, or EDNRA knockout mice (Ahn et al., 2004; Ge et al., 2005a; Ge et al., 2006; Ge et al., 2005b). From their studies, they determined that during salt loading (through diet), EDN1 and EDNRB1 are necessary for sodium excretion. As a consequence, EDN1 and EDNRB1 collecting ductknockout mice were severely hypertensive after salt loading (Ahn et al., 2 004; Ge et al., 2006). The EDNRA collecting duct-knockouts were no diffe rent from control animals, suggesting that EDNRA in the mammalian collecting duct is not involved in blood pressu re regulation (or sodium excretion) (Ge et al., 2005b). Endothelin inhibition of solute transport also has been de monstrated in fishes. As mentioned above, mammalian EDN1 inhibits net ch loride transport in th e killifish opercular epithelium (Evans et al., 2004). In addition, th e EDNRB1 agonist, sarafo toxin S6c (SRX S6c), also inhibited the net chloride transport in this preparation, suggesting that EDN1 is acting through an EDNRB1; however, there are no EDNRA a gonists, thus it has not been determined if EDNRA also affect ion transport in the killifish. Incubation of mammalian EDN1 with proximal tubules from the killifish kidney led to inhibition of transport by the multidrug resistan ce protein 2 (Mrp-2) in the tubules (Notenboom 24

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et al., 2005; Notenboom et al., 2002 ). The Mrp-2 is found in the luminal membrane of the proximal tubules and transports a wide range of chemicals from lipophilic organic anions to polypeptides (Notenboom et al., 2002). Incubation of tubules with a mammalian EDNRB1 antagonist and EDN1 led to EDN1 mediated i nhibition of Mrp-2 tran sport in the proximal tubules, thus suggesting that EDNR B1 are involved in the signali ng cascade. Other than these few studies, the effects of EDN1 on transport in fishes are relatively unexplored. Overview of Dissertation Research When I started my dissertation I had three ma in objectives: 1) de scribe the endothelin system from the teleost gill; 2) determine the effects of changing envi ronmental salinity on gill EDN1, ECE1, and EDNRs in two teleost with diffe ring degrees of euryhalinity, with the aim to elucidate the function of EDN signaling in the gill; 3) determine the evolutionary and phylogenetic relationships among the EDNs ECEs, and EDNRs, respectively. The species used in my experiments were the killifish ( Fundulus heteroclitus Linnaeus) and the longhorn sculpin ( Myoxocephalus octodecemspinosus Mitchill). The killifish is an excellent osmoregulator that to lerates direct transfers betw een fresh and seawater, giving researchers the opportunity to test the extreme effects of salinity acc limation. They are a commonly used model vertebrate to test a wi de variety of physiol ogical, ecological, and epidemiological questions (Burnett et al., 2007). The longhorn sculpin is cl assified as a marine teleost; however Claiborne et al. (1994) determined that these fish can tolerate direct transfer to 20% seawater indefinitely, and they can tolerate days in 8% and 4% seawater. This implies that they have some degree of euryhalinity. Interestingly, fishermen in the Gulf of Maine have described finding longhorn sculpin in estuaries during high tides, further suggesting some level of environmental salinity tole rance (Bigelow and Schroeder, 2002). These two fishes are found in the waters surrounding Mount Desert Island, ME (where we have a su mmer lab), and afford 25

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an interesting comparative system to test questions of the effect of changing environmental salinity on the gill EDN signaling axis. In Chapter 2, I described EDN1 and ECE1 from the killifish. In a ddition, I explored the phylogenetic relationships among the EDNs and EC Es, respectively, and determined the effects of different environmental salinity on these genes/ proteins. This work was recently published in the Journal of Experimental Biology, Endothe lin-1 and the endothelin converting enzyme in the fish gill: physiological and evolutionary perspectives by Hyndman and Evans (2007). In Chapter 3, I determined the functional and genomic relationships of the EDNRs. This chapter is completely different from all the othe r chapters, as it dives d eep into the world of phylogenetics, bioinformatics, and genomics (three words I thought I would never say). In addition, I introduce the complete EDNR sequences from the killifish. This chapter will be refined for submission to PNAS over the next few months. In Chapter 4, I described the EDNRs from the killifish and determined the effects of varying environmental salinity on these recepto rs. Using molecular biology and protein biochemistry, I determined that changing e nvironmental salinity changes EDNR mRNA and protein concentrations in the gill. This work will be submitted to the Journal of Experimental Biology in the near future. In Chapter 5, I determined the effects of low environmental salinity on the longhorn sculpin. Unlike the killifish, the effects of hypo-osmotic environments on plasma osmolality, ions, and gill ion transporter de nsity has not been determined fo r the longhorn sculpin. This chapter was important for setting up the context of our EDN signaling effects in Chapter 5. This study is currently under review with the Journal of Experimental Biology 26

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27 In Chapter 6, I described the effects of low environmental salinity on ECE1 and the EDNRs in the gills of the longhorn sculpin. Usi ng molecular techniques, I sequenced portions of ECE1 and the EDNR s and determined that this modera tely euryhaline fish up-regulates EDNRB2 and EDNRB1 during acclimation to hypo-osmotic environments. Finally, in Chapter 7 I summarize the findings from my dissertation work, and revisit my three main objectives, tying a ll the chapters together.

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CHAPTER 2 ENDOTHELIN AND ENDOTHELIN CONVERTI NG ENZYME-1 IN THE FISH GILL: EVOLUTIONARY AND PHYSIO LOGICAL PERSPECTIVES Introduction Endothelin (EDN) is a family of three auto crine/paracrine peptides (EDN1, EDN2 and EDN3) that function in a variety of physiological processes such as the regulation of vascular tone (Yanagisawa et al., 1988a) a nd natruresis in the kidney (Zei del et al., 1989). Endothelins are translated as ~200 amino acid (aa) preproendo thelins (preproEDN) that are initially cleaved by a furin-like enzyme (Yanagisawa et al., 1988 a) to form the relatively inactive 38 aa proendothelin (proEDN, also known as Big-EDN) (Kimura et al., 1989). Proendothelin is further cleaved to form the active 21 aa EDN by the endothelin converting enzyme (ECE-1 and/or ECE-2) (Shimada et al., 1994; Xu et al., 1994). In mammals, EDNs actions are mediated via two G-protein coupled receptors: endothe lin A receptor (EDNRA), which preferentially binds EDN1 (Arai et al., 1990), and endothelin B1 receptor (EDNRB1), which binds all three EDNs with equal affinity (Sakurai et al., 1990) In non-mammalian vertebrates, EDNs also equally bind to a third G-protein-coupled recep tor, endothelin B2 receptor (EDNRB2) (Lecoin et al., 1998), and in amphibians EDN3 binds to an am phibian-specific receptor termed endothelin C receptor (EDNRC) (Karne et al., 1993). Preproendothelin genes have been found in al l major gnathostome clades (Fig. 2-1), and evidence for EDN1 regulation of vascular tone ha s been shown in fishes (Evans, 2001; Evans et al., 1996; Evans and Harrie, 2001; Olson et al., 1991; Wang et al., 2001). In addition, EDN1 inhibition of transport by the multidrug resistance-association protein was demonstrated in shark ( Squalus acanthias ) rectal tubules (Miller et al., 2002) and killifish ( Fundulus heteroclitus ) renal tubules(Masereeuw et al., 2000). Recently, Evan s et al. (2004)determined that exogenous 28

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(mammalian) EDN1 inhibited net chloride transpor t in the killifish opercular epithelium, a tissue used as a model for the SW teleos t gill (Karnaky et al., 1977). In te leosts, the gill is the main site for ion balance, nitrogen excreti on, acid/base regulation and gas ex change (Evans et al., 2005). Estuarine euryhaline fishes like the killifish ( Fundulus heteroclitus ) encounter varying environmental salinities throughout the day (Marsha ll, 2003), resulting in a net gain or loss of ions depending on the water salinity; thus the re gulation of gill ion transport is an important mechanism to maintain ionic homeostasis. Evans et al. (2004) hypothesized that EDN1 signaling cascades in the gill may be a local regulator of ion balance in fishes. Thus, the purpose of this study was to determine if EDN1 and ECE1are produced in the killifish, and secondarily determine if environmental salinity regulates gill EDN1 and/or ECE1 mRNA expression. We were also interested in determining the phylogene tic/evolutionary relations hips of the EDNs and ECE family of proteins. Methods Fish Maintenance Killifish, Fundulus heteroclitus were trapped in North East Creek, Mount Desert Island, ME, and maintained in free flowing, 31 ppt seaw ater (SW) tanks at th e Mount Desert Island Biological Laboratory, under a natural summer photoperiod, before be ing shipped to the University of Florida. There they were main tained in 32 ppt, 23C SW, under a 12 light:12 dark photoperiod. Tank pH was maintained between 7. 8 and 8.0, and ammonia, nitrate, and nitrite levels were below 1 ppm. The fish were fed comme rcial fish pellets to sa tiation every other day. EDN1 cDNA All protocols and procedures were approved by IACUC at the University of Florida. Molecular protocols of Hyndman et al. (2006) we re used. Killifish were decapitated, and the gills of the right side were removed and snap frozen in liquid nitrogen. Total RNA was then 29

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isolated with TRI-reagent (Sigma, St. Louis, MO ), and 5 and 3 RACE cDNA was synthesized from 4 g of total RNA with a GeneRacer Kit (Invitrogen, Carlsbad, CA) according to the manufacturers protocols. Published degenerate reverse EDN1 primers by Wang et al. (2006) were used in our initial 5 Touchdown RACE PCR following Invitroge ns protocols. The polymerase used was 0.625 U of Ex Taq, hot start, DNA polymerase (Takara Bio, Madison, WI) and the reactions were run in an Express ther mocycler (ThermoHybaid, Franklin, MA). The PCR parameters were: 94C for 2 min, 5 cycles of 94C for 30 sec, 72C for 30 sec, 5 cycles of 94C for 30 sec, 70C for 30 sec, 30 cycles of 94C for 30 seconds, 45C for 30 sec, 72C 30 sec, and a final 72C for 5 minutes. PCR products were visualized by ethidium bromide staining in 1.5% agarose gels ligated into pCR4-TOPO vectors, and transformed into TOP10 chemically competent cells using a TOPO TA Cloning Kit for sequencing (Invitrogen). Plasmid DNA was then sequenced in both directi ons at the Marine DNA Sequencing Facility at the Mount Desert Island Biological Laboratory (Salisbury Cove, ME). Once we had the 5 end, specific killifish EDN1 primers were designed (Table 21) and 3 Touchdown RACE PCR was performed to complete the cDNA sequences. The PCR parameters were: 94C for 2 min, 5 cycles of 94C 30 sec, 72C for 30 sec, 5 cycles of 94C for 30 sec, 70C for 1 min, 30 cycles of 94C for 30 sec, 50C for 30 sec, 72C for 1 mi n, and a final 72C for 10 min. PCR products were cloned and sequenced like above. ECE cDNA To get ECE cDNA, 4 g of total gill RNA (extracted as described above) was reverse transcribed using the First-strand cDNA Superscript III reverse transcriptase kit (Invitrogen) using oligo-dT primers. Initial degenerate primers used to get ECE were designed using CODEHOP (Rose et al., 2003) and are record ed in Table 2-1. PCRs were run on 0.5 l of the 30

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olgio-dT cDNA, with 0.625 U of Ex taq, hot start (Takara) and standard cycling parameters. PCR products were cloned and sequenced as described above. Sequence and Phylogenetic Analysis Sequence results for each transcript were a ssembled with GeneTools software (BioTools Inc., Edmonton, Alberta) and killifish EDN1 and ECE1 nucleotide sequences were searched for open reading frames (ORFs). The resulting amino acid translations were analyzed with the basic local alignment search tool (BLAST) on the National Center for Biotechnology Information (NCBI) website. The predicted amino acid sequ ences were aligned wi th other full-length vertebrate EDN or ECE proteins using Clustal X (Chenna et al., 2003). All sequences were taken from GenBank or the Genome projects in Ensembl (e:!44 April 2007). Preproendothelin-1 sequences from each major vertebra te clade (mammals to teleosts ) were separately aligned, and similarities among the sequences highli ghted with GeneDoc (available at http://www.psc.edu/biomed/genedoc ), including the expected clea vage sites for furin and ECE (Opgenorth et al., 1992; Yanagisa wa et al., 1988a). To determine the relationship among our sequences and those from other organisms, E DN and ECE alignments were exported to PHYML (Guindon et al., 2005) and a Fast Maximum-Like lihood test was performed, following the WAG model of amino acid substitutions and a gamm a calculated of 1.023, and 1.03, respectively. Branches were then tested for statistical si gnificance by bootstrapping with 500 replicates. Multiple Tissue Semi-Quantitative PCR To determine the distribution of EDN1A, EDN1B and ECE1 mRNA among tissues, relative duplexing semi-quantitative PCR was pe rformed on total RNA from gill, opercular membrane, brain, heart, stomach, intestine, and kidney tissue as described previously (Choe et al., 2005; Choe et al., 2004). Briefly, cDNA was pr oduced from the tissues of a SW killifish as described above, but random hexamer primers ( not oligo-dT primers) were used so that 31

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ribosomal and messenger RNA would be reverse tran scribed. Non-degenerate primer pairs were designed to amplify a product with high effici ency (e.g., high melting temperature) and to minimize the chance of amplifying contaminatin g genomic DNA, the primer pair was designed to include at least one intron-exon boundary when possible (Table 2-1). A QuantumRNA 18S internal standard primer kit (A mbion, Woodward Austin, TX) was us ed to control for variability in cDNA quality and quantity between the different tissues tested. Duplexing PCR with primers for 18S and either EDN1A, EDN1B or ECE1 were then optimized to ensure that the reactions were terminated during the exponential phase. Las tly, the products were vi sualized by ethidium bromide staining in 1.5% agarose gels and digi tized using the Biorad Gel Doc XR System. Salinity Challenges Killifish were acclimated to SW (approximate concentrations in mmol l: Na+ 517, Ca2+ 9, K+ 12, Cl 486) (Choe and Evans, 2003) or fresh wate r (FW) (Gainesville dechlorinated tap water approximate concentrations in mmol l: Na+ 4, Ca2 + 1, K+ 0.03, Cl 0.40) (Choe and Evans, 2003) for 2 weeks, at which point the SW killifish were transferred into FW (SW to FW) and the FW killifish were transferred into SW (FW to SW). An additional set of killifish were removed and replaced into SW or FW as sham controls (SW to SW and FW to FW, respectively). Immediately after transfer, 5 or 6 killifish from each treatment were sacrificed, gills excised and snap frozen for RNA extrac tion and cDNA synthesis. Killifish (n=5 or 6/treatment) were further sacrificed at 3, 8 and 24 h post transfer (acute acclimations), as well as, 30 days post transfer (chronic acclimation). R NA was extracted from all of the samples and oligo-dT cDNA synthesized as described above. Quantitative Real-Time PCR To determine the effects of envir onmental salinity on killifish gill EDN1A, EDN1B and ECE1 mRNA levels, quantitative real-time PC R (qRT-PCR) was performed. Nondegenerate 32

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primers were designed to amplify a product between 50-100 bp across a predicted intron-exon boundary (Table 2-1). L8 was used as an internal control gene as previo usly described (Choe et al., 2006; Choe et al., 2005). Each sa mple was run in triplicate using 2 l of 1/10 diluted original cDNA, 7.4 pmol of primers and SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 l. The cycling parameters used were: an initial denaturing step of 95C for 10 min, 40 cycles of 95C fo r 35 sec, 60C for 30 sec and 72C for 30 sec followed by a melting curve analysis to ensure only one product was amplified. Random samples were also sequenced following qRT-PCR confirming amplification of the target of interest. To determine the de gree of possible genomic contam ination, qRT-PCR was run using RNA samples that were not reverse transcribed, and we determin ed that there was no genomic contamination. All qRT-PCRs were run on a MyiQ quantitative thermocycler (Biorad, Hercules, CA). Each primer pairs efficiency was determin ed by performing a 10 fold dilution curve using plasmid cDNA. Efficiency (E) for each primer pair was calculated using the equation: E=-1+10(-1/slope) where slope was the slope of the dilution curve. Each CT value was subtracted from a randomly chosen contro l sample resulting in a CT, and were analyzed using the Pfaffl equation (Pfaffl, 2001): ratio= E CT target/ E CT L8. Each Pfaffl ratio was then standardized to the average chronic seawater Pfaffl ratio. Statistics Data are expressed as mean +/s.e.m. For the qRT-PCR data, a 2-Factor ANOVA was performed to determine whether effect of envi ronmental salinity over time differed between SW to FW transfers and SW to SW shams or FW to SW transfers and FW to FW shams. If statistical significance was found a 1-Factor ANOVA was run to determine the effect of time over a 33

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treatment group. Finally, all time points were compared to sham time points with unpaired Ttests to determine if salinity tr ansfers altered mRNA expression. All values which did not meet homogeneity or equal variance tests were log transformed to meet the assumptions of the ANOVA. =0.05. Tissue Preparation for In Situ Hybridization and Immunohistochemistry Killifish gills were fixed in 4% paraformaldehyde in 10 mM phosphate buffered saline (PBS) pH=7.3, for 24 h, dehydrated in an increas ing concentration of ethanol, cleared in Citrisolv (Fisher Scientific, Pittsburgh, PA), and embedded in paraffin wax. The tissue blocks were cut at 7 microns, placed on Superfrost Plus slides (Fisher Scientific), and heated at 37C overnight. In Situ Hybridization mRNA for EDN1A, EDN1B, and sodium, potassium ATPase ( NKA ) mRNA were visualized using in situ hybridization. An ECE1 mRNA probe was not made because our partial sequence of that transcript was fr om the middle of the sequence, a region that in other fishes is >65% identical to ECE2, and we were afraid of the potential of cross-reactivity of this probe. Specific digoxigenin (DIG)-RNA probe s (sense and antisense) were made against the 3 end of the transcripts (including UTR for the EDN s, these regions were <60% identical). For EDN1A the probe was made from position 520 to the end of the transcript incl uding poly A tail (420 bps long). The EDN1B probe was made from position 515 up to and including the poly A tail (419 bps). Both of these transcripts were cloned as described a bove. A killifish NKA mRNA probe was also made based upon the complete ki llifish NKA sequence (AY057072). The probe was made from bps 915 to 3115. This transcript wa s also cloned and sequenced to ensure it was indeed NKA. All of the transcripts were linearized by T3/T7 PCR amplification from the plasmids containing the sequences of interest Dig-RNA probes were generated by incubating 34

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100-200 ng of the linearized transcripts with the DIG RNA Labeling mix (Roche Applied Science, Indianapolis, IN) following manufactur ers protocols, at 37C for 16 h followed by treatment with DNAse for 1 h at 37C. The DI G-RNA probes were purified using mini Quick Spin RNA columns (Roche) following the manu facturers instruc tions, eluted in 80 l of DEPC water and stored at -80C until use. To determine which gill cells expressed EDN1A or EDN1B mRNA, gill tissue slides were rehydrated in two changes of Citrisolv, followe d by incubation in a series of decreasing concentration of ethanol washes. The slides we re placed in sterile 10 mM PBS and post fixed in 4% PFA for 10 minutes at room te mperature (25C). Following this the slides were rinsed in sterile 10 mM PBS and incuba ted in proteinase K (5 g ml-1) at room temperature for 5 minutes. Again, they were washed in 10 mM PBS and post fixed in 4% PFA for 10 minutes to inactivate the proteinase K. After a final PBS wash, the s lides were incubated in prehybridization solution (50% formamide, 10% dextran sulphate, 2% Blocking reagent, 0.1% CHAPS, 1% Tween 20, 5 mM EDTA pH=8.0, 5X SSC, 50 g ml-1 heparin, 1 mg ml-1 tRNA, in DEPC-water) for 2 h at room temperature. Next 200 to 500 ng of DIG-RNA probes were added to fresh prehybridization solution and the slides were left to incubate at 60C for 18-24 h. Following this, the tissues were washed for 30 minutes in: 2X SSC at room temperature, 2X SSC at 60C, two 0.2X SSC 60C washes, one 0.2X SSC at room temperature and KTB (50 mM Tris pH=7.5, 100 mM NaCl, and 10 mM KCl) at room temperatur e. The tissues were then blocked in 20% normal goat serum diluted in KTB for 1 h at room temperature and incubated in 7.5 U ml-1 of sheep anti-Digoxigenin-AP, Fab fragments (Roche, diluted in NGS) overnight at 4C. The slides were then washed in 3 changes of KTB and incubated in alkaline phosphatase buffer (100 mM Tris pH = 9.5, 100 mM NaCl, 50 mM MgCl2) for 30 minutes at room temperature. Visualization 35

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of the probes was achieved by incubating the ti ssues in BCIP/NBT Substrate Kit, 5-bromo-4chloro-3-indolyl phosphate/ni troblue tetrazolium (Vector Labs, Burlingame, CA) with levamisole following manufacturers instructions, at room temperature un til the signal developed (2-6 h). Images were captured using an Olym pus BX60 light microscope with a Hitachi KP-D50 digital camera. Image contrast and brightness were adjusted with P hotoshop CS (Adobe, San Jose, CA). Immunohistochemistry Slides were analyzed following the methods of Piermarini et al. (2002) and Hyndman et al.(2006). Slides with chronic SW and FW accl imated killifish gill tissue were incubated in primary antibodies: polyclonal, anti-human -proEDN1 (1/1000 diluted in NGS) (Phoenix Pharmaceutical, Burlingame, CA) made against th e complete 38 aa of human proEDN1, which is 74% identical to both killifish EDN1s. Monoclonal, anti-NKA ( 5, 1/1000) was developed by Dr. Douglas Fambrough, and was obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the National Institute of Child Health and Human Development of the University of Iowa, Depart ment of Biological Sciences, Iowa City, IA 52242, USA. Results Sequence Analyses From the killifish gill we have sequenced two EDN1 transcripts and have designated them EDN1A (accession EU009474) and EDN1B (accession EU009475). EDN1A is 917 bp with an ambiguous base at position 762 (C or T), and a pr edicted ORF of 483 bp that translates into a preproendothelin-1A (preproEDN1A) of 189 aa. EDN1B is 912 bp with a predicted ORF of 429 bp that translates into a prepro endothelin-1B (preproEDN1B) of 143 aa. The predicted cleavage sites in the preproEDN1s for furin and ECE ar e depicted in Fig. 2-2A. As found in other 36

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vertebrate preproEDN1 peptides, preproEDN1A and preproEDN1B contain the dibasic cleavage sites for furin and the conserved Trp21-Val22 cleavage site for ECE (Fig. 2-2A). The two active (21 aa) killifish EDN1s are 80% identical to hum an EDN1 and are 100% id entical to each other (Fig. 2-2B). As seen in Fig. 2-1, our kill ifish preproEDN1s group with the other fish preproEDN1 sequences and group outside of the fish preproEDN2 and preproEDN3 sequences. We were unable to find a preproEDN orthologue in the Ciona intestinalis or Branchiostoma genomes, suggesting that the EDNs are found only in vertebrates. We have sequenced 1696 bps from the middle of the killifish ECE1 cDNA (accession EU009476). This translates into 565 aa from the killifish ECE1. Endothelin converting enzymes are found in all organism including Bacteria a nd Archaea (Fig. 2-3). As seen in Fig. 2-3, the non-vertebrate ECEs are not well resolved (i.e. la ncelet ECE groups with locust and sea urchin, while sea quirt groups with hydra) and although there is no clear explanation for this, Fig. 2-3 shows three distinct ECE clades : ECE1, ECE2 and non-vertebrate ECE. Our partial sequence of ECE1 from the killifish groups with the othe r fish ECE1 sequences confirming it is ECE1. Tissue Distributions Using duplexing relative semi -quantitative PCR we found EDN1A mRNA in the gill, opercular epithelium, brain, heart, stomach, inte stine and kidney of the killifish (Fig. 2-4). Relatively high expression was found in the gill, brain and kidney. EDN1B mRNA was not found in the opercular ep ithelium, and had very little expre ssion in the gill, but was highly expressed in the brain, kidney and intestine. Finally, ECE1 mRNA was found in all of the tissues tested, with highest expression in th e stomach, intestine, and gill (Fig. 24). In situ Hybridization EDN1A mRNA was localized to gill epithelial cells in the interlamellar region (Fig. 2-5A). These cells were large, round and were disperse d along the entire length of the filament (not 37

PAGE 38

shown). NKA mRNA was also found in epithelial cells in the interlamellar region (Fig. 2-5C) but were concentrated only near the afferent fila mental artery (trailing edge of the filament, not shown). The morphology of these NKA positive cells are consistent with mitochondrion-rich cells of the killifish gill (Choe et al., 2005; Hyndman et al., 2006; Katoh et al., 2001; Marshall, 2003). In contrast, EDN1B mRNA expression was rare (as was seen in the EDN1B tissue distribution described above); howev er it was found in some lamellar pillar cells (Fig. 2-5D, F) and in epithelial cells adjacent to the environment (Fig. 2-5D). Incubation of slides with sense probes produced a little non-sp ecific staining (Fig. 5B, E). Immunohistochemistry Proendothelin-1, the precursor to active EDN1, was immunolocali zed to epithelial cells in the interlamellar region of the killifish gill, a nd on the lamellar pillar cells (Fig. 2-6A, C). Proendotheln-1 immunoreactivity was seen in ep ithelial cells adjacent to cells immunoreactive for NKA (Fig. 2-6A, C), and these cells share si milar morphology to NECs described in fishes (Goniakowska-Witalinska et al., 1995; Mauceri et al., 1999; Zaccone et al ., 1992; Zaccone et al., 1996). This immunolocaliza tion matches the killifish EDN1 mRNA localization shown in Fig. 2-5. Slides incubated in NGS and double labeled with NKA were done as negative controls, and showed no non-specific staining of the proEDN1 secondary antibody and chromagen (Fig. 2-6B, D). Salinity Acclimations When killifish were transferred from FW to SW there were no statistically significant changes in EDN1A or EDN1B mRNA levels over 24 h (acute acclimation) compared to sham (FW to FW) transfers (Fig. 2-7A, C). ECE1 mRNA increased foura nd sixfold compared with sham ECE1 mRNA levels at 8 and 24 h post a FW to SW transfer (Fig. 2-7E). EDN1A mRNA levels did not change significantly with acute acclimation from SW to FW compared to sham 38

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(SW to SW) treatments (Fig. 2-7B), but 24 h EDN1B mRNA levels were almost threefold higher compared to sham EDN1B mRNA levels at 24 h (Fig. 2-7D). ECE1 mRNA levels were twofold higher after 3 and 24 h of acclima tion to FW but did not differ from sham values at 8 h post transfer (Fig. 2-7F). With ch ronic acclimation (30 days), ther e were no statistical differences between the SW and FW acclimated killifish for EDN1A, EDN1B or ECE1 mRNA levels (Fig. 2-8). In addition, preproEDN1 (protein) immu nolocalization did not differ between fish chronically acclimated to FW (Fig. 2-6A, B) or SW (Fig. 2-6C, D). Discussion Endothelin Sequences We have sequenced two cDNAs for EDN1 from the killifish gill. These two transcripts have identical 5 prime ends until position 510 where th ere is an AC insertion resulting in a frame shift mutation in EDN1A that changes the stop codon T GA to ACT GAG. Conversely, EDN1B may have lost an AC at position 510 resulting in a stop codon and a truncated preproEDN. Past this insertion/deletion (indel), the two transcript s differ along the rest of the sequence, but are only 5 bps different in total length. This suggest s that the two transcri pts are from duplicated EDN1 genes and are not transcript variants. We did not find duplicate EDN1 genes in the Takifugu Tetraodon or Danio genomes, suggesting the EDN1 duplication was a Fundulus specific event (please see note at end of manuscrip t). As a consequence of the indel at position 510, the predicted preproEDN-1a is 46 aa longe r than preproEDN-1b. Even though there are differences between the killifish preproEDN-1s the proEDN-1s and EDN1s are 100% identical. Why would a tissue (cell) produce two different mRNAs if the e nd protein translated is 100% identical? We hypothesize that these two EDN1s ha ve remained in the killifish because they have different regulatory pathwa ys, and/or are stimulated by different signals. This hypothesis might suggest that the EDN1s would have different tissue/cellula r distributions. Supporting this 39

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hypothesis, from our tissue distribu tion analysis (Fig. 2-4), we found EDN1A was ubiquitously expressed in all the tissues tested, with relative ly high expression in the gill, brain, and kidney. Conversely, EDN1B was found in very low levels in th e gill and opercular epithelium, but relatively high in the brain. Nevertheless, until complete killifish EDN1A and EDN1B genomic sequences are determined, it is un clear how and what factors may regulate these genes, and thus suggest why these two have been retained in the killifish. Gill Expression of EDN1 mRNA and ProEDN1 Protein In the killifish gill we found EDN1A mRNA expression in epithelial cells of the interlamellar region, and EDN1B mRNA expression was found in pillar cells, and in cells adjacent to the environment, in the interlame llar region. Not only are these two transcripts expressed in different levels with in a tissue (Fig. 4), they are al so expressed in different cells within the gill. From our immunohistoc hemical experiments, we found proEDN1 immunoreactivity in epithelia l cells adjacent to the NKA immunoreactive cells. NKA is commonly used as a marker for the ion transpor ting, mitochondrion-rich cell (MRC) of the fish gill (Evans et al., 2005; Katoh et al., 2001; Marshall, 2003). Proendothelin immunoreactivity was also found on pillar cells, wh ich is in agreement with our in situ hybridization findings. Zaccone et al. (1996) immunoloca lized proEDN to gill neuroendocri ne cells (NECs) of the eel ( Conger congo) catfish ( Heteropneustes fossilis ) and dogfish ( Scyliorhfnus canicula ), and the morphology of these cells matches that of th e proEDN immunoreactive epithelial cells in the killifish gill. Endothelin production in the pillar cells was suspected by Sundin and Nilsson (1998) and Stenslokken et al. (1999). They showed that in fusion mammalian EDN1 into the lamellae of the rainbow trout resulted in a constric tion of the vascular sheet (of the lamellae) and that this was likely due to constriction of th e pillar cells. The authors hypothe sized that hormonal control of 40

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pillar cell tone may be one mechanism to match respiratory needs of a fish while minimizing ion fluxes. There is no evidence that pillar cells ar e innervated; thus endocri ne/paracrine/autocrine signaling molecules may be the regulators of pill ar cell tone. Pillar cells contain contracting filamentous material (Bettex-Galland and Hughe s, 1973) and recently Mistry et al. (2004) described an actin-binding protein, FHL5, that is highly expressed in thes e cells, suggesting that they are capable of contraction. St enslokken et al. (1999) demonstrated in vivo using video microscopy that pillar cells do contract with EDN1 infusion. Recently, EDN receptors, EDNRA and EDNRB, were immunolocalized in the fish gill. Stenslokken et al. (2006), found EDNRB throughout the gill vascul ature, NECs, and pillar cells of the cod ( Gadus morhua). EDNRA was described in nerve fibers runni ng along the length of the filament and innervating the gill vasculature (Stenslokken et al., 2006). In the tiger pufferfish ( Takifugu rubripes ) EDNRA was found on the pillar cells and in erythrocytes (Sulta na et al., 2007). Studies from our lab in the killifish have found EDNRBs throughout the gill vasculature and pillar cells, and EDNRAs on the mitochondrion-rich cell (Chapter 4). Evid ently, there is species specific EDN receptor distribution in the gill of fishes. Acute and Chronic Salinity Acclimations Killifish usually live in estuaries where th ere are rapid changes in environmental conditions such as salinity and temperature (Mar shall, 2003). We tested the effects of rapid changes of environmental salinity on mRNA expression of EDN1A, EDN1B and ECE1 EDN1 transcript levels did not change with chronic acclimation to FW or SW (Fig. 2-8). In addition, proEDN1 immunoreactivity in the gill did not diffe r between SW and FW acclimated killifish (Fig. 2-6). However, EDN1B and ECE1 mRNA levels increase with acute FW acclimation suggesting that more active EDN1 protein is prod uced. Evans et al.(2004), demonstrated that 108 M mammalian EDN1 can inhibit net chloride trans port in the killifish opercular epithelium, and 41

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this is predominately due to stimulation of cy clooxygenase (COX) and subsequent prostaglandin production. These findings suggest that during transfer to a hypo-osmotic environment, EDN1B and ECE1 protein levels increase resulting in an increase in active EDN1 that could potentially inhibit net chloride transport, helping the fish retain ions. However, we cannot rule out that EDN1 signaling in the gill is different from wh at was described in the killifish operculum by Evans et al. 2004 (see below). In addition, it is un determined how volume stress, like that occurs during a rapid change to a hypo-osmotic enviro nment, effects blood flow through the gill. EDN1B was found on gill pillar cells, and may play a role in regulating blood flow during blood volume increases however this is an unexplored area of fish gill physiology. Although we found an increase in EDN1 during acclimation to FW, we unexpectedly found a sixfold increase in ECE1 mRNA levels with acute SW acc limation suggesting that there is an increase in ECE1 production during this period. This in turn would result in more EDN1 production because the proteolytic cleavage of pr oEDN1 to EDN1 by ECE1 is a rate limiting step (D'Orleans-Juste et al., 2003; Ikeda et al., 2002). Our atte mpts to measure EDN1 production in the fish gill by enzyme immunoassay, Tris-Tricine Western blot ting, and MALDI-FTMS mass spectrophotometry, were unsuccessful, but measuremen ts of EDN1 levels are necessary to fully understand the role of EDN1 cell signaling in the fi sh gill. Recently, Choe et al. (2006), showed a threefold increase in COX-2 mRNA levels in th e killifish gill, 3 h post a FW to SW and a threefold increase 3 h post a SW to FW transf er, and hypothesized the increase in COX-2 is an important mechanism for gill ce ll survival during osmotic stress. A similar result has been demonstrated in mammalian medullary interstitial cells (that experience large changes in osmotic stress), which require functional COX-2 to su rvive (Hao et al., 1999; Hao et al., 2000). Medullary interstitial cells also contain EDNR s but do not produce EDN1 (Dean et al., 1996). 42

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Endothelin has been shown to stimulate COX-2 in a variety of mammalian tissues (Chen et al., 2003; Hughes et al., 1995), and EDN1 signali ng via endothelial E NDRB results in the production of prostacyclin (Hir ata et al., 1993; Warner et al ., 1989). Taken together, our findings suggest that during rapid changes in environmental salinity, gill cell survival during this osmotic stress may be accomplished by increased EDN1 production and subsequent stimulation of COX production of prostaglandins. To the best of our knowledge, it is unclear what aids cell survival during salt or water load in fishes, a nd it is plausible that si nce EDN1 and ECE1 are ubiquitously expressed that this may be a more gl obal change in their sign aling patterns, and that this is not a gill specific phenomenon; however th is is yet to be determined and experiments testing these hypotheses are needed. In addition, studies blocking aspects of EDN1 signaling in the gill and subjecting these fish to salinity challenge s are vital in understanding EDN1 function during osmotic stress. This technique has b een successful in mice models, where kidney collecting duct EDN1 (or EDNRB1) knockout mice w ho are fed a high salt diet are unable to excrete the excess Na+ accumulated and are severely hypertensive (Ahn et al., 2004; Ge et al., 2006) suggesting that EDN1 is necessary for salt excretion in mammals. Applications of these types of techniques to fish models are necessa ry to full understand the in vivo role to EDN signaling in the fish gill. Evolution of EDNs and ECE In searching of the completed genome projec ts, we were unable to find EDN orthologues in any organism basal to the teleost fishes. Fr om the maximum likelihood analyses presented in Fig. 1, there are three distinct groups representing: preproEDN1, preproEDN2 and preproEDN3 in vertebrates. Evans and Harrie (2001) showed that aortic vascular smooth muscle rings from the sea lamprey and Atlantic hagfish constrict in response to mammalian EDN1, suggesting that the receptors and EDN1 are expressed endogenously in these basal, Agnathan vertebrates. 43

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Physiological responses to EDN1 have also been demonstrated in the spiny dogfish shark (Evans et al., 1996; Evans and Gunderson, 1999) ag ain suggesting that EDN1 is produced endogenously. Currently, the exact evol utionary history of this family of peptides is not clear. EDN sequences from hagfish, lamprey, and sharks are necessary to determine when these peptides arose, and to determine if it was due to gene/genome duplications or other evolutionary events over vertebrate evoluti on. Interestingly, mutations in EDN1, endothelin receptors (EDNRs), or ECE result is severe craniof acial developmental abnormalities, and these phenotypes are often lethal (Bra nd et al., 1998; Clouthier and Sc hilling, 2004; Kurihara et al., 1994; Nair et al., 2007), sugge sting the EDN signaling is necessary for development in vertebrates, and it may be a key innovation in the radiation of vertebrates. Unlike EDNs which are only found in verteb rates, ECE is found in all organisms, including Bacteria and Archaea. In Fig. 2-3, our maximum likelihood analyses reveals three distinct groups of ECEs: proka ryote, fungal, and invertebrate ECE, vertebrate ECE1 and vertebrate ECE2. This suggests a gene duplicati on event sometime after the chordate-vertebrate split, but before the teleost radiation. Because there is no molecular evidence for EDNs or EDNRs in animals basal to the vertebrates why w ould they have an ECE? Endothelin converting enzymes are zinc-dependant metalloendoproteases a nd part of the Neprelysin and Kell family (Shimada et al., 1994; Xu et al., 1994). In vertebrates, ECE can function as a monomer or dimer; however for effective proteolytic cleav age of proEDN1 to EDN 1, dimerization at Cys412 is preferential (Shimada et al ., 1996). In contrast, hydra ECE (Z hang et al., 2001) and the other invertebrate, fungal and proka ryote ECEs are missing Cys412 and are believed to function as monomers (Zhang et al., 2001). Vertebrate ECE has been shown to cleave peptides other than proEDN including bradykinin, angiotensin I, a nd substance P (Hoang and Turner, 1997; Johnson 44

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et al., 1999), suggesting that it may a be a gene ralist protease. Alt hough the native substrates cleaved by ECE in non-vertebrate organisms are unde termined, it is plausible that ECE originally cleaved substrates found in all organisms, and dur ing vertebrate evolution started functioning as a dimer and preferentially cleaving proEDN. Tentative Model for EDN1 Sign aling in the Killifish Gill To summarize our findings, we propose the fo llowing model (Fig. 2-9) of paracrine and autocrine EDN1 signaling in the fish gill. Diagrammed is a lamellar cross-section of the gill (same orientation as the gills in Figs 2-5 and 2-6) with pillar cells (PCs) highlighted in grey and adjacent pavement cells (PVCs) in white. In th e intralemallar region there are two MRCs and an NEC above the gill vasculature. Cyclooxygenase-2 (COX-2) and neuronal nitric oxide synthase (nNOS) were previously immunolo calized in the killifish gill, to MRCs (Choe et al., 2006) and NECs, nerve fibers and lamellar arterioles (Hyndman et al., 2006), respectively. NKA was immunolocalized to the basolateral membrane of the MRC (Choe et al., 2006; Hyndman et al., 2006; see Katoh et al., 2001) and the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), to the apical membrane of the M RC (Katoh et al., 2001). From our studies and others, EDNRB were found throughout the gill vasc ulature (Hyndman and Evans, unpublished; Stenslokken et al. 2006), and EDNRB and EDNR A were on the pillar cells depending on the species (Hyndman and Evans, unpublished Stenslokken et al., 2006; Sultana et al., 2007). EDNRA were found on MRCs in the kill ifish gill (Chapter 4). Here we present EDN1 expression in cells adjacent to the MRC (l ikely NECs) and pillar cells. This suggests a paracrine role of EDN1 signaling given that it is produced in the NEC and can bind to receptors on the adjacent MRCs (Fig. 2-9, pathway #1) where it potentially stimulates COX-2 activity resulting in cell survival during osmotic stre ss and/or alter ion transport by the MRC as previously hypothesized (Evans et al., 2004). EDN1 can also potentially act as a paracrine 45

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binding to EDNRB on the gill va sculature and lamellar arteriol es, suggesting it can regulate perfusion of the lamellae (Fig. 2-9, pathway #1). It also can act as an autocrine on the pillar cells, further supporting the role of regulation of local perfusion across a lamella to meet the respiratory needs of the fish (Fig. 2-9, path way #2) (Stenslokken et al., 1999; Sundin and Nilsson, 1998). It may also help maintain lame lla integrity during rapid increases in plasma volume during exposure to a hypoosmotic environment. This is the first m odel to depict EDN1 signaling in the fish gill, and in the future, studi es determining the specif ic function of EDN1 in the gill and whole fish are n ecessary to understand its role in normal fish physiology. Note When this paper was under review a new vers ion of ENSEMBL was released and in it, I found duplicate EDN1 genes in the fish genomes. T hus in contrast to what is stated above, the EDN1A and EDN1B is likely the result of the teleost-specific genome duplica tion and not just a killifish specific gene duplication. 46

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Table 2-1. Primers used for cloning, tissue distri butions (td) and quantit ative real-time PCR (q) Name Orientation Primer (5' to 3' orientation) 3' EDN1F1 sense GAC GCT CT C CTG GAT CCT CTG CAC AGC T 3' EDN1F2 sense CAA CAA GCG CTG CTC CTG CGC AAC TTT C ECEF1* sense CCG GAC TGT CGA CCC ATG YSA NGA YTT ECEF2* sense CCT GCA TGA ATG AGA CCA AGA THG ARG ARY T ECER1* antisense CGC ACT TGT GTG GCG GRT TCA TNG G td EDN1a F1 sense CAG TAA CAG AAC CTC TGG CGG A td EDN1a R1 antisense TCC ACG CAG CTG CTA TCA TT td EDN1b F1 sense GTC GGT GTC TGC GTG AAA ATG A td EDN1b R1 antisense C AG ACC GGA GGA TGA AGT TCA G td ECE F1 sense TCC CCT TTC TTC ACT GTG TTT G td ECE R1 antisense CTT CTG GAG GTA CTC TTT GGC A q EDN1a F1 sense TCC AGA GAG CGA AGA GCA TTC C q EDN1a R1 antisense TGA CTC TAT CCG TTT TTG GTG C q EDN1b F1 sense TGA ACT TCA TCC TCC GGT CTG q EDN1b R1 antisense TTT GTC TTC AGC TGC CAC ATG q ECE1 F1 sense CCT GTG ACA CTC AGC GAA TCA q ECE1R1 antisense CTT CTG GAG GTA CTC TTT GGC A q L8 F1 sense CGT TTC AAG AAA AGG ACG GAG C q L8 R1 antisense GGC GCT GCA GGT GGC GTA G Denotes degenerate primers Denotes primers used in RACE PCR. 47

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Figure 2-1. Maximum likelihood analyses of th e vertebrate preproendothelin amino acid sequences. Following the WAG model of am ino acid substitutions (Whelan and Goldman, 2001) and a gamma=1.023, there are three distinct groups of preproendothelins, preproendothelin-1 (abbreviated as only EDN1 to save space), preproendothelin-2 (EDN2) and prepro endothelin-3 (EDN3). Numbers at nodes represent the percent bootstrap (BS=500 replications). GenBank accession or Ensembl numbers: Chicken EDN1, XP _418943; chicken EDN2, XP_417707; chicken EDN3, XP_001231488; frog EDN1, AAS13535. 1; frog EDN3, AAS13536.1; human EDN1, NP_001946; human EDN2, NP_001947; human EDN3, NP_000105; killifish EDN1A, EU009474; killifish EDN1 B, EU009475; medaka EDN1, ENSORLP00000011633; medaka EDN2, EN SORLP00000010557; medaka EDN3, ENSORLP00000011814; mouse EDN1, NP_034234; mouse EDN2, P22389; mouse EDN3, NP_031929; rat EDN1, NP_036680; rat EDN2, NP_ 036681; rat EDN3, NP_001071118; salmon EDN1, BAF30875.1; Takifugu EDN2, NEWSINFRUP00000182956; Takifugu EDN3, NEWSINFRUP00000181774; Tetraodon EDN3, GSTENT00028275001; Tetraodon EDN2, GSTENT00026224001; Zebrafish EDN1, NP_571594; Zebrafish EDN2, NP_001038650. Scale bar represents the number of replacements per site. 48

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0.2 Zebrafish EDN1 Salmon EDN1 Killifish Killifish 99 99 96 Frog EDN1 Chicken EDN1 Human EDN1 Mouse EDN1 Rat EDN1 72 95 82 96 Medaka EDN3 Takifugu EDN3 Tetraodon EDN3 89 49 Chicken EDN3 Frog EDN3 Human EDN3 Rat EDN3 Mouse EDN3 91 95 55 48 94 Zebrafish EDN2 Medaka EDN2 Tetraodon EDN2 Takifugu EDN2 86 73 68 Chicken EDN2 Human EDN2 Mouse EDN2 Rat EDN2 89 96 92 83 65 49

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Figure 2-2. An alignment of vertebrate preproen dothelin-1 protein sequences. A) Similar amino acid residues (based on the BLOSUM 62 score table) are highlighted in gray, as compared to human preproendothelin-1. The cleavage sites for furin ( ) and ECE1 () are indicated. B) predicted active E DN1 structure from the killifish, modeled after Webb (1997). Amino acids different from human EDN1 are highlighted in gray. Two disulfide bonds are indicated at Cys1 to Cys15 and Cys3 to Cys10. Accession numbers are listed in Fig. 1. 40 80 120 Human : MDYLLMIFSLLFVACQGAPET------AVLG--AELSAVGENGGEKPTPSPPWRLRRSKRCSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSP-RSKRALE-NLLPTKATDRENRCQ : 110 Opossum : MDYFQMIFSLLFVVFQGAPDT-------AFG--AELSTEAQAGGDNQPPSAPWRPRRSKRCSCSSLLDKECVYFCLLDIIWINTPEHTVPYGLGSPSRSKRSLE-DSPLTKPADSRKRCQ : 110 Platypus : MDYFQMIFSLLFVVFQGALETAVL------G--ADLSTGIGTGVEVHPPPAPWRPRRTKRCSCSSLLDKECVYFCHLDIIWINTPEHTVPYGLGGPSRSKRALQ-DSFPAKQSDGNNRCQ : 111 Chicken : MDCSRLFLPLLVALCPA-----------LL----PAAPGAEVNAASPPSPAAASHRRARRCSCSSLLDEECVYFCHLDIIWINTPEKTVPYGLGGPSRSRRSLK-DIMPEMLAGASSRCR : 104 Frog : MD-LQMIVSVLVVLVQGVSATESLSDSARLTNEPPAAAAATQTASHRSSGAPWRPRRVKRCSCSSLMDKECVYFCHLDIIWINTPERTVPYGLGGP-RMKRALQ-DNDQEKLSEPAGRCL : 117 Zebrafish : -MHLRIIFPVLTMLTSGFFD--------------FGAPASL-----GPGTAPARHSRNKRCSCASFLDKECVYFCHLDIIWVNTPERTVSYGLGNAPRKKRSV-TEPVVL---ES--RCK : 94 Killifish EDN1A : -MDLKARISVLSLTLSWILCT------------ALSAPAAEPLSASAAAVAPQRHVRNKRCSCATFLDKECVYFCHLDIIWVNTPERVVSYGLGNAPRAKRAL-SDSMAT--ARA-PRCR : 103 Killifish EDN1B : -MDLKARISVLSLTLSWILCT------------ALSAPAAEPLSASAAAVAPQRHVRNKRCSCATFLDKECVYFCHLDIIWVNTPERVVSYGLGNAPRAKRAL-SDSMAT--ARA-PRCR : 103 160 200 Human : CASQKDKKCWNFCQAGKELRAEDIMEKDWNNHKKGKDC--SKLGKKCIYQQLVRGRKIRRSSEEHLRQTRSETMRNSVKSSFHDPKLKGNPSRERYVTHNRAHW-----------: 212 Opossum : CASRKDKKCWVFCQAGKELWAQNTLKKAWNEPKKIKDCIGH--GLKCVQQQLVNRKKMRR----------LETISNSIKASFHIAKQRAELHKEKKITNNRTHQKQSIWDSLKTTS : 214 Platypus : CANQKDKKCWDFCQAGKELWAQNTLEKGRKQLKKGEQCAD--LGLKCVYWQLVNRRKMRR----------MEAIGNRIKAAFNFAKLKAELHMAKKVTHNRAH------------: 202 Chicken : CASQRDKKCLNFCQTGKDLWAQSTAEKTSRHRNKAGGC----IGPKCMNQQFVDSRKMKR----------LEAVGNSIKASFSIAKLKAELQKGRKLKHNRASKRQSIWKSLKTF: 205 Frog : CAKRKDKKCMDFCHATAEPSVQPSVAKDSRQVQQATKCLGLRLGQQCIQKQHHRNKVMKR----------SESIKQSIKNSFAFASLMNKPNEARETSHLWMHKNWGVWKHRKTTS : 223 Zebrafish : CADSQDKTCSSFCQADSALQFKAASDRAIR-AAQGHDCA----GKQCKHKLAETQTKIRRLRTTKQT----DVLSGKKKIQLLLEKWRMRRNHRSQAWISENS------------: 188 Killifish EDN1A : CLRENDSSCVDFC-----LRSETAAGARIR--SPGHTRS------------EDTGGPDRTEGTSTTNRARPAALRAVLRTRLQLEKWAARRHHRARAWQGESRAS----------: 189 Killifish EDN1B : CLRENDSSCVDFC-----LRSETAAGARIR--SPGHTRS------------EDTGGPDR--------------------------------------------------------: 143 Active EDN1 ProEDN A F L B T A C C C C S L D K E V Y F N H D I I W C 50

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Figure 2-3. Maximum likelihood analyses of th e ECE family of prot eins following the WAG model of amino acid substitutions (W helan and Goldman, 2001), and a gamma distribution of 1.03. There ar e three distinct groups of ECE: ECE1, ECE2 and nonvertebrate ECE. Accession numbers: Ar chaea ECE, NP_616924; Bacteria ECE, NP_812722; chicken ECE1, NP_990048; chicken ECE2, ENSGALP00000010123; frog ECE1, AAH46653; frog ECE2, ENSXETP00000037627; fungus ECE, XP_754379; human ECE1, NP_001388; hum an ECE2, NP_055508; hydra ECE, AAD46624; killifish ECE1, EU009476; la ncelet ECE, 86342 scaffold_150000101; locust ECE, AAN73018; medaka ECE2, ENSORLP00000025753; medaka ECE1, ENSORLP00000021332; mouse ECE1, NP _955011; mouse ECE2, NP_647454; opossum ECE1, ENSMODP00000019967; opossum ECE2, ENSMODP00000002887; platypus ECE1, ENSOANP00000023244; platypus ECE2, ENSOANP00000003016; rat ECE1, NP_446048; rat ECE2, NP_001002815; sea squirt ECE, ENSCSAVP00000016300; sea ur chin ECE, XP_798822; stickleback ECE1, ENSGACP00000006069; stickl eback ECE2, ENSGACP00000005922; Takifugu ECE1, NEWSINFRUP00000136873; Takifugu ECE2, NEWSINFRUP00000151424; Tetraodon ECE1, GSTENP00006535001; Tetraodon ECE2, CAG02177; zebrafish ECE1, XP_694687. S cale bar represents the number of replacements per site. 51

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Human ECE1 0.2 Sea urchin ECE Locust ECE Lancelet ECE 51 69 Sea squirt ECE Hydra ECE Fungus ECE Bacteria ECE Archaea ECE 100 90 45 94 100 Tetraodon ECE1 Takifugu ECE1 100 Zebrafish ECE1 Stickleback ECE1 Medaka ECE1 Killifish ECE1 59 57 66 99 FrogECE1 Chicken ECE1 Opossum ECE1 Platypus ECE1 55 Rat ECE1 Mouse ECE1 100 98 100 77 91 99 Medaka ECE2 Stickleback EC2 65 Tetraodon ECE2 Takifugu ECE2 100 100 Frog ECE2 Chicken ECE2 Opossum ECE2 Platypus ECE2 HumanECE2 Rat ECE2 Mouse ECE2 99 88 82 100 91 100 100 52

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18SOp. epithelium Gill Brain Heart Stomach Intestine Kidney EDN1A EDN1B ECE1 18SOp. epithelium Gill Brain Heart Stomach Intestine Kidney EDN1A EDN1B ECE1 Figure 2-4. Tissue distribution of killifish EDN1A, EDN1B, and ECE1 determined by duplexing semi-quantitative PCR with 18S as a internal control. 53

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B C EDN1Sense NKA A F EDN1 EDN1 D E Sense Figure 2-5. Representa tive pictures of in situ hybridization of EDN1A and EDN1B mRNA in lamellar cross-sections of the seawater killifish gill. A) The EDN1A antisense probe was localized to epithelial cells in the interl amellar region of the gill. Little staining was seen in the gill when the EDN1A sense probe was used (B). C) The sodium, potassium ATPase ( NKA ) antisense probe was localized to mitochondrion-rich cells. D) EDN1B antisense probes bound to pillar cells and epithelial cells adjacent to the environment. Little staining was seen with the sense probe (E). F) A magnification of the pillar cell EDN1B staining. Scale bar = 50 M. 54

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C SW A FW B FW D SW Figure 2-6. Representative pictu r es o f killif ish la m ellar cross sections, la beled with a n tiproendothelin-1 (brown) and anti-NKA (blue) A and B). Gill sections from a chronic (>3 0day) f r esh water ac clim ated killif ish, and C and D) a chron i c SW acclim ated killifish. B and D) were inc ubated in norm al goat serum as a negativ e control for proendothelin immunoreactiv ity, and doubled labeled with anti-NKA (blue) to illustrate the position of th e m itochondrion-rich cells (MRC) in the gill. Proendothelin immunoreactivity was f ound in a cell adjacent to the NKA immunoreactivity (MRC ) and on gill pillar ce lls in both the F W and SW killifish. The immunoreactivity p r esented here m a tc hes the in situ hyb ridization of m R NA probes for EDN1s and NKA in Figures 5 and 6. Scale = 50 M. 55

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Figure 2-7. Acute changes in killifish g ill EDN1A, EDN1B and ECE1 mRNA levels as determined by quantitative Real Time-PCR. FW to SW transfers are indicated by solid lines and circles () and FW to FW sham contro ls are represented by open circles ( ) and dotted lines (A, C, E). SW to FW transfers are represented by solid lines and squares ( ) and SW to SW sham controls by dotted lines and open boxes ( ) (B, D, F). N=5 or 6 killifish per time and treatment and mean +/s.e.m. are represent. Note the y axis is logarithmic. All values are normalized to L8 and standardized to chronic SW mRNA leve ls (Fig. 2-9). A and B) EDN1A mRNA levels, C and D) EDN1B mRNA levels and E, F) ECE1 mRNA levels. The asterisks (*) indicate statistical significance p <0.05 for that time point compared to sham value. 56

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Figure 2-8. Chronic changes in gill EDN1A, EDN1B and ECE1 mRNA levels as measured by quantitative Real Time-PCR. Killifish (n = 6) were transferred from FW to SW (SW treatment, black bars) or SW to FW (FW treatment, gray bars) and sacrificed 30 days later. Mean +/s.e.m. represented. Note the y axis is logarithmic. No significant changes in mRNA level were found be tween the SW and FW killifish for EDN1A, EDN1B or ECE1 57

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58 Lamella EDNR Figure 2-9. A model of paracrine and autocrine E DN1 signaling in the fish gill. Diagrammed is a lamellar cross-section (see Figs 5-7) with pillar cells (PCs) in gray, pavement cells (PVCs) and a lamellar arteriole (LA) adjacen t to the interlamellar region of the gill containing mitochondrion rich-cells (MRCs), a neuroendocrine cell (NEC), and the gill vasculature. Cyclooxygenase-2 (C OX-2) and neuronal nitric oxide were previously immunolocalized in the killifish gill (Choe et al., 2006; Hyndman et al., 2006). Sodium, potassium ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) are used as markers for the MRC (Katoh et al., 2001). See text for details. EDNRB COX-2 nNOS NKA NKA COX-2 EDNRB Gill vasculature EDNRB EDNR nNOS CFT R CFT R 1. 2. 2. P V C PC MRC NEC Interlamell a r region En v i ronment EDN1 EDN1 EDN1 MRC LA EDNRA EDNRA 1.

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CHAPTER 3 FUNCTIONAL AND GENOMIC STUDY OF THE ENDOTHELIN RECEPTORS Introduction Endothelins (EDNs) are small secreted peptid es found in all gnathostomes (Hyndman and Evans, 2007; Yanagisawa et al., 1988a). Th ey have many diverse physiological functions including: regulation of vascul ar tone (La and Reid, 1995; Yana gisawa et al., 1988a; Yanagisawa et al., 1988b); alteration of ion transport (Evans et al., 2004; Ga rvin and Sanders, 1991; Prasanna et al., 2001; Zeidel et al., 1989); and migration of neural crest cells (NCCs) during craniofacial development (Clouthier and Schilling, 2004; Kuri hara et al., 1994). Traditionally researchers have acknowledged that EDN signals via two G protein coupled receptors (GPCRs), EDNRA and EDNRB1. These receptors are coded by separate genes, on different chromosomes (Arai et al., 1993; Hosoda et al., 1992) and have different pharmacological profiles (Table 3-1). In nonmammalian species, the classification of the ED NRs has been somewhat confusing. In the African clawed frog ( Xenopus laevis ) three EDNRs have been described: EDNRAx from the heart (Kumar et al., 1994); EDNRBx from the liver (Nambi et al., 1994); and EDNRC from melanophores (Karne et al., 1993). In addition, from the chicken (Gallus gallus ) and quail ( Coturnix japonica ) an avian-specific receptor EDNRB2 was cloned and characterized (Lecoin et al., 1998). The classificat ion of these GPCRs is generally based upon sequence homology and pharmacological profiles (Table 3-1) compared to the mammalian EDNRA and EDNRBs. For example, EDNRB2 was named such because the primary sequence was more closely related to human EDNRB1 than EDNRA or frog EDNRC; however it was pharmacologically different from human EDNRB1 (Lecoin et al., 1998). This classification system depicts a series of species-specific EDNRs in non-mammalian gna thostomes; however, our knowledge of nonmammalian EDNRs is quite limited. A thorough phyl ogeny of this gene family would help 59

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elucidate the evolutionary relati onships among these receptors, and it will help us better classify the receptors. For example, it will determine if frog EDNRAx is an orthologous or paralgous gene of the mammalian EDNRA. The majority of EDN research has been in the biomedical field, because numerous pathologies have been linked to problems with EDN signaling. These conditions include hypertension, atherosclerosis (Shreenivas and Oparil, 2007), congestive heart failure (Angerio, 2005), and glomerulonephritis (Richter, 2006). The use of mammalian and non-mammalian model organisms has been imperative in unders tanding EDN signaling cascades. For example, experiments using chicks ( Gallus gallus) (e.g. Kanzawa et al., 2002; e.g. Miller et al., 2003; Nagy and Goldstein, 2006) and zebrafish (Danio rerio) (e.g. Clouthier and Schilling, 2004; Kimmel et al., 2003; Miller et al ., 2000; Miller et al., 2003; e.g. Na ir et al., 2007; Walker et al., 2006) have highlighted the importance of EDN in embryonic development. Yet, given our knowledge of EDN signaling during devel opment and mammalian physiology, we know comparatively little about the function of EDN in non-mammalian adult organisms. We recently described the phylogenetic re lationships among the EDNs (Hyndman and Evans, 2007), and explored the effects of environmental salinity on EDN gene expression from the euryhaline teleost, the common killifish ( Fundulus heteroclitus ). The killifish is a model organism used in a diverse range of studies in cluding: ecological, epidemiological, evolutionary, physiological, and toxicological (Burnett et al., 2 007). In addition, they have been valuable in understanding drug transport by multi-drug resistance proteins (Masereeuw et al., 2000; Notenboom et al., 2005; Notenboom et al., 2004; Terlouw et al., 2001). These fish are abundantly distributed in the estu aries along the eastern coast of the US (Bigelow and Schroeder, 60

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2002; Marshall, 2003), and because they are small (~3 inches) and easily maintained in aquaria, they are an ideal species to us e in laboratory experiments. The purposes of this study were to: 1) orga nize the classification of the gnathostome EDNRs; 2) complete a thorough phylogenetic analysis of the EDNRs; and 3) determine if there are functional shifts between the therian mammal and non-therian EDNRS. Using the killifish as our model organism, we sequenced cDNA fo r three EDNRs and putatively called them EDNRA EDNRB1, and EDNRB2 From our complete phylogenetic an alysis, we determined there are three distinct groups of gnathostome EDNRs: EDNRA, EDNRB1 and EDNRB2. Interestingly, we did not find an EDNRB2 orthologue from any therian mammal, suggesting a loss of this receptor in this lineage. We used synteny to de termine if the therian EDNRB2 was lost due to mutation or deletion. To understand better the po tential consequence of not having EDNRB2 in the therians, we determined the replacement rate at each amino acid position, and determined if there were significant changes th ese rates between the therian and non-therian gnathostomes. We then mapped these sites to known regions of the EDNRA and EDNRB1 that are necessary for EDN binding, and tested the hypothesis that therian EDNRS have undergone rapid remodeling of functionally important sites as a consequence of losing EDNRB2. Our results suggest that therian EDNRA have been remode led, but that EDNRB1 has remained conserved throughout gnathostome evolution. Methods Molecular Cloning, Sequencin g, and Tissue Distribution The RT-PCR, cloning, and sequencing protocols of Hyndman and Evans (2007) were used to sequence cDNA for the killifis h EDNRs. Degenerate primer s were designed against highly conserved regions for all EDNRs a nd are recorded in Table 3-2. In itial sequences were identify using homology and BLAST (Wheeler et al., 2007). To determine the tissue expression of the 61

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EDNRs in an adult gnathostome, random hexame r cDNA was made from RNA extracted from the opercular epithelium, gill, brain, heart, stomac h, intestine and kidney of an adult killifish as previously described (Hyndman and Evans, 2007). Gene specific primer pairs were designed to amplify a product with high efficiency (e.g., hi gh melting temperature), and to minimize the chance of amplifying contaminating genomic DNA, the primer pair was designed to include at least one exon-exon boundary when possible (Table 3-3). Multi -tissue duplexing-PCR was run using gene-specific primers and QuantumRNA 18S internal standard primers (Ambion, Woodward Austin, TX) to control for variabi lity in cDNA quality and quantity among the different tissues. The duplexing-PCRs were optimi zed to ensure they were terminated during the exponential phase. The products were visualized by ethidium bromide staining in 1.5% agarose gels and digitized using the Biorad Gel Doc XR System. Data Mining and Multiple Sequence Alignment We used sequence homology to find all of the EDNRs in GenBank (Wheeler et al., 2007) and the completed genomes in Ensembl (e! 44, Ap ril 2007) (Hubbard et al ., 2007; Spudich et al., 2007). A number of BLAST searches were comp leted using the following query sequences: Killifish EDNRA (EU391601); Killifish EDNRB1 (EU391602); killif ish EDNRB2(EU391603); Chicken EDNRB2 (NP_989451); Frog EDNRC (P32940); Human EDNRA (NP_001948; and Human EDNRB (NP_000106). This collection of sequences was refi ned to include only a single protein sequence for EDNR per species. If ther e were splice variants, only the longest one was included in the dataset. We preferentially sele cted RefSeq sequences over others because these sequences are curated. Finally, a ny therian species that did not have complete sequence data for EDNRA and EDNRB1 was not included in the analysis. With these criteria, we were left with a 62

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phylogenetically enriched (species and protein) dataset, while lim iting the size to run a complete and thorough phylogene tic analysis. A recent phylogenetic analysis of a porti on of human GPCR was completed, and the results suggest that the endothelin B receptor-like proteins (GPR37 and GPR37L) are the outgroup to the EDNRs (Fredriksson et al., 2003). Using the aforementioned protocol we compiled a list of sequences to include as the outgroup. All th e sequences were aligned with CLUSTALX (Chenna et al., 2003; Larkin et al., 2007) and visually inspected to ensure no gaps were inserted into known structural regions (i.e. transmembrane domains) (Pollock and Highsmith, 1998). Phylogenetic Analyses The phylogenetic relationships among the EDNRs were determined by fast maximum likelihood analyses (ML) with PHYML (Guindon et al., 2005). The evolutionary model used was the Whelan and Goldman (WAG) model for amino acid replacements (Whelan and Goldman, 2001), with a free gamma distribution parameter optimized using eight rate categories to account for rate heterogeneity across positions. The propor tion of invariable sites was calculated as zero during likelihood analyses, thus this parameter was excluded from the final analysis. The robustness of the ML tree was evaluate by non-parametric bootstrapping = 1000. This analysis was performed with and without the outgroup, and we determined that when the outgroup was included, there was a destabilization of the optimal tree. This was likely because of the great divergence time among the sequences and this issue was recently discussed by (Shavit et al., 2007). Thus, we ran the ingr oup (study group) and outgroup ML separately and tried Lundberg rooting (Lundberg, 1972); a met hod of rooting the st udy group, after you run your analysis. 63

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In addition to our ML analysis, we performe d a Bayesian analysis (BA) with MrBayes (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) with our study group only. Using the same evolutionary model as in out ML analysis, we ran a Monte Carlo Markov Chain using 3 hot chains and 1 cold ch ain (Huelsenbeck et al., 2001), for 20 million generations, with a sample frequency of 500, and a burnin of 10%. The 50% majority consensus tree was compiled. Synteny Using NCBIs Mapviewer (Wheeler et al ., 2007) and Ensembl (Hubbard et al., 2007; Spudich et al., 2007), we determined the location of EDNRB2 in the Gallus gallus (Chicken) genome. This gene is on chromosome 4, at bp 11255609-11264791. The genes surrounding EDNRB2 are listed in figure 4. These include, upstream trimethyllysine hydroxylase, epsilon ( TMLHE), and downstream RNA polymerase I 16kDa polypeptide ( RPA16) and synaptobrevinlike 1 ( SYBL1 ). We then used these genes as markers for the position of EDNRB2, and searched for them in the Western clawed frog, platypus, opossum, mouse, rat, macaque, and human genomes. Only in the Western clawed frog, platypus, opossum, macaque, and human genomes were some of these genes mapped, so we restricted our searches to these organisms. Using this gene order, we calculated gene distances among our aforementioned markers. In the opossum, macaque, and human genomes we did not find EDNRB2. We preformed BLAST searches using any unidentified loci or pseudogenes from these ge nomes to determine if they had any significant homology to the EDNRB2 Rate Shift Analyses To test the significance of losing the EDNRB2 to the therians, we te sted the hypothesis that the remaining therian EDNRs have undergone change s at the sequence level related to functional shifts. This was determined using Knudsen and Miyamotos (2003) rate shift analysis (RSA). The RSA involved likelihood-ratio tests (LRT), on a site-by-site basi s, to test for two types of 64

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rate shifts in a gene family: Type I or Type II (Fig.3-1). As illustrate d in Figure 3-1, a Type I sites is defined as a homologous position that is fixed for one subfamily, but variable in the second subfamily. A type II site is a homologous s ite that is fixed for di fferent amino acids in both subfamilies (Fig. 3-1). This analysis also tests for slow evolving sites (conserved) between both subfamilies. We defined our two subfamilies based upon the split of the last gnathostome lineage where we found EDNRB2 and the lineag e where it was putatively lost; 1) all the gnathostomes basal to the loss of EDNRB2 (thus th e platypus to teleosts), and 2) all the therian mammals (opossum to human). Using the EDNRA sequences from our multi-sequence alignment, and the EDNRA grouping from the ML tree (Fig. 3-3), the RSA program developed by Knudsen et al. (2003), was used to determined the number of Type I and/ or II (Type I/II) and conserved sites. This analysis was repeated using the EDNRB1 portion of the multi-sequence alignment, and EDNRB1 grouping from the ML tree (Fig. 3-3). From the literature, we compiled a list of f unctionally important sites necessary for EDN1 binding (the endogenous ligand) for EDNRA (Adachi et al., 1993; Breu et al., 1995) and EDNRB1 (Wada et al., 1995). From those studies, we determined that EDNRA has a specific set of 26 amino acids (T50-I70, position based on human sequence ,Fig. 3-5A) in the N-terminal necessary for EDN1 binding (Adachi et al., 1993 ) In addition, site mutagenesis studies determined that G97, K140, K159, Q165, and F315 are necessary for EDN1 binding (Breu et al., 1995). EDNRB1s have a region of 60 amino acids, I138 to I197) that are necessary for EDN1 binding (Wada et al., 1995) (Fig. 3-5B). Results Killifish Endothelin Receptors We initially sequenced three EDNR cDNAs from the killifish g ill using degenerate primers made against conserved regions of the vertebrate EDNRs. These sequences were compared, 65

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using sequence homology and BLAST, to the know n gnathostome EDNRs, and determined that we had sequenced killifish: EDNRA (2543 bp), EDNRB1 (1629 bp), and EDNRB2 (1470 bp). These sequences were searched for open r eading frames (ORF) using BioTools (Edmonton, Alberta) ORF tool and compared to other EDNR s. The putative killifish EDNRA has an ORF of 1278 bp, EDNRB1 1251 bp, and EDNRB2 1242 bp. From our phylogenetic analysis (see below), our putative killifish E DNRs (protein sequence) form gr oups with the other gnathostome EDNRA, EDNRB1 and EDNRB2, respectively (Fig. 3-3). Killif ish EDNRA (EU391601) shares 55% amino acid identity to the killifish EDNRBs and is 63% identical to human EDNRA. Killifish EDNRB1 (EU391602) is 63% identical to killifish EDNRB2, and 70% identical to human EDNRB1. Killifish EDNRB2 (EU391603) is 63% identical to human EDNRB1 and 68% identical to quail EDNRB2. The tissue distribution of the EDNR s in the adult killifish was determined, and EDNRA and EDNRB2 were ubiquitously expressed in the opercular epithelium, gill, brain, heart, stomach, intestine, and kidney (Fig. 3-2). EDNRB1 was found in all those tis sues except the heart. EDNRA had relatively high expression in the heart and kidney, EDNRB1 was highest in the gill, brain, and kidney, and EDNRB2 was highest in the gill, brain, heart, and kidney (Fig. 3-2). Phylogeny Initially, we tried to root our phylogenetic tree using Lundbe rg rooting (Lundberg, 1972). We artificially placed the outgroup ML tree to eac h of the branches lead ing to the three EDNR groups (therefore, three artificial trees), and determined the ML sc ore of each of these artificial trees (arrows in Fig. 3-3 depict the artificial roots). All three ML scores were very similar; however, the outgroup branching from the EDNRB2 gr ouping was slightly better, thus to test the statistical significance of this branch we us ed an approximate likelihood ratio test (PHYMLaRLT). The analysis determined that there wa s weak support for this branch (probability = 66

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0.270); thus, we restricted our analyses to the study group, because of th e inability to robustly root the outgroup. By using homology and BLAST searches of NC BIs Protein database, and the completed genome projects (in Ensembl e!44, April 2007), we did not find any EDNR in any animal basal to the teleost fishes. In addition, when EDNR B2 or EDNRC sequences were queried (see above) in BLAST, we found frog EDNRC, all the non-ther ian EDNRs, but the only therian sequences we found were EDNRA and EDNRB1. Thus, we c onclude that if ther ian EDNRB2 or EDNRC were present, we would have found them. The results of our complete phylogenetic analyses of the gnathostome EDNRs are depicted in Figure 3-3. The log-likelihood of this tree was -18202, with a gamma distribution parameter calculated as 0.617. Our bayesian tree was very similar to this tree, and the percent posteri or probabilities of each node are recorded in Figure 3-3. There were three, distinct groups found, representing the EDNRA, EDNRB1 and EDNRB2. The support for these groups was high (bootstrap> 99%, and posterior probab ility=100%). Within each group, the majority of the nodes followed the classic species relationships (i.e. human and chimp grouped together, outside of the macaque, Fig. 3-3). The EDNRA group contains a set of teleost-sp ecific EDNRA gene dupl ications, likely the result of a teleost specific genom e duplication that occurred afte r their split from the tetrapods (Volff, 2005). We named these duplicates E DNRA2 (Fig. 3-3). The African clawed frog EDNRAx (Kumar et al., 1994) grouped with 60 % support to the African clawed frog EDNRA. These also grouped with the Western clawed frog E DNRA, and this is outside of the rest of the tetrapod EDNRA, and teleost EDNRAs. We ha ve termed these proteins EDNRA (AAH44316) and EDNRA2b (AAA19570.1), because they are of the A subtype, but the duplicate is independent of the duplicate EDNR A2s found in the teleost fishes. 67

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The EDNRB1 group, also contains a group of te leost-specific EDNRB1 gene duplications, and we have named them EDNRB1b (Fig. 3-3). Interestingly, we found other duplicate EDNRB1s; the zebrafish has a duplicated EDNRB 1b protein we have termed EDNRB1c, and the opossum has a duplicate EDNRB1 that groups with the opossum EDNRB1, out side of the teleost EDNRB1bs. We have termed this protein E DNRB1d, and it appears to be an opossum specific duplicate; no other therian mammal has a dupl icate EDNRB1. The Western clawed frog EDNRB grouped with the tetrapod EDNRB1, suggesti ng it is an orthologous gene. We did not find an African clawed frog EDNRB1. Unlike EDNRA and EDNRB1, we did not fi nd a teleost-specific duplication of the EDNRB2; however, we did find a stickleback specific EDNRB2 duplicate (named EDNRB2b) (Fig. 3-3). To our surprise, we did not find a zeb rafish EDNRB2, suggesting that they have lost this receptor. Interestingly, the African cl awed frog EDNRC grouped with 100% support with the African clawed frog EDNRB2 (Fig. 3-3), suggesting it is a duplicate receptor. Again, we were unable to find a ther ian EDNRB2 orthologue. Synteny Using the gene order of TMLHE, EDNRB2, RPA16 and SYBL1 as determined by the chicken genome (chromosome 4), we searched the other vertebrate genomes for this same gene order. Similar gene orders were found on th e Western clawed frog (scaffold 258), platypus (Ultra519), opossum (chromosome X), macaque (chromosome X) and human (chromosome X). The total gene distan ces from the end of TMLHE to the beginning of SYBL1 were calculated and the chicken genome has the smallest distance, followed by the opossum, Western clawed frog, macaque, human, and finally the platypus has the la rgest distance (Fig. 3-4) Other genes located between these markers in the therian genomes include some pseudogenes; however, none of 68

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these regions of DNA showed homology to the EDNRB2 In addition, we identified some unnamed loci using homology and BLAS T searches (Fig 3-4. asterisks). Rate Shift Analysis The results from the rate shift analysis (RSA ) are depicted in Figures 5A and B. For EDNRA, between the therian and non-therian gnat hostome subfamilies, there were 35 Type I/II and 119 conserved positions out of a total of 425 informative sites. Based upon published mutagenesis and bind domain experiments complete d in therians, we determined that EDNRAs have 31 functionally important sites necessary for EDN1 binding (Adachi et al., 1993; Breu et al., 1995). As tabulated in Table 3-4A, 6/31 functionally important pos itions were type I/II sites, and 5/31 were conserved, functionally important pos itions (Table 3-4C). All of these 6 type I/II sites were located within one consecutive region of 26 ami no acids that is known to be necessary for EDN1 binding in therians (Adach i et al., 1993). We de termined there was a greater than expected concentra tion of the 6 type I/II sites w ithin this 26 amino acid binding region (the G-test was used because it is less sensitive than the Chi-squared to small column totals, G= 4.54, p=0.022) (Table 3-4B). In additi on, there were significantly less conserved sites within this 26 amino acid region (2/26) th an expected (G=35.34, p<0.0001) (Table 3-4C). With the EDNRB1, between the therian and nontherian gnathostomes, there were 70 type I/II and 221 conserved positions out of a total of 567 informative sites. Wada et al. (1995) determined that a region of 60 amino acids was necessary for EDN1 binding, thus these sites were considered functionally important. Within this region of 60 positions, the RSA determined there was only 1 type I/II site, and 49 conserved sites (Fig. 3-5B, Table 3-4A, C). This was a significantly lower than expected number of type I/II sites in this functionally important region (Table 3-4A, G=6.50, p=0.01) and a significantly hi gher number of conserved sites (Table 3-4C, G=35.34, p<0.0001). 69

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Discussion Here we present three new EDNR sequences from the adult killifish, a commonly used model organism in a variety of physiological, eco logical and epidemiological studies (Burnett et al., 2007). We are the first to determine the tissu e distribution of all three EDNRs from an adult animal; all other studies have looked at the three EDNR s distribution during embryonic development (e.g. Lecoin et al., 1998; Nataf et al., 1996) or distribution of EDNRA and EDNRB1 in mammals (Molenaar et al., 1993; e.g. Ogawa et al., 1991). All three EDNR mRNAs are ubiquitously expressed in the adul t killifish tissues tested, with the exception of EDNRB1 that was not in the killifish heart. These expres sion patterns are similar to those described in mammals, where the EDNRA is the predominant fo rm in the heart (Molenaar et al., 1993), and the EDNRB1 is highly expressed in the brain, lung, and kidney (O gawa et al., 1991). The fish gill is the main site of nitroge n excretion, and gas exchange (Eva ns et al., 2005), functioning like the mammalian kidney and lung, so it is not surprising that EDNRs are highly expressed in the gill. This study is the first to co mplete a thorough phylogenetic an alysis of the gnathostome EDNRs. There are three, distinct groups of EDNRs representing the EDNRA, EDNRB1 and EDNRB2/EDNRC (Fig. 3-3). In ad dition, there appears to be a teleost-specific duplication of the EDNRA and EDNRB1, that is likely a result of the teleost specific genome duplication after their split from the tetrapods (Volff, 2005). The EDNRs are gnathostome specific, with no orthologous genes in the early chordates ( Ciona intestinalis Branchiostoma floridae ). These results are in agreement with our recent analysis of the evolution of the EDNs. We could not find a homologous EDN in any animal basal to th e teleost fishes (Hyndman and Evans, 2007). Physiological studies strongly suggest that thes e EDN and the EDNRs are present in the early vertebrates: hagfish, lamprey, a nd cartilaginous fishes (Evans et al., 1996; Evans and Gunderson, 70

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1999; Evans and Harrie, 2001); however until these genomes are sequenced and fully annotated, it is unclear if they have all three EDNRs. Preliminary searching of the ratfish and lamprey genomes, found small fragments of some highly homologous regions of the EDNRS, but these fragments are too small to determine if they are EDNRA, EDNRB1 or EDNRB2. When these genomes are completed, it will help to elucidate the question of the orig in of the EDNRs. Previously, a novel African clawed frog EDNRAx was ch aracterized from the heart (Kumar et al., 1994). As seen in Figure 3-3, this receptor is a duplicate EDNRA, grouping with a second EDNRA from the African clawed frog. Th is also groups with the Western clawed frog, and the other tetrapod EDNRAs. Searching of the Western clawed frog genome revealed only one EDNRA gene, suggesting the duplicate EDNRAx may be specific to the African clawed frog and not the whole genus (Xenopus). We propose this receptor should be reclassified as EDNRA2b. The novel African clawed frog EDNRBx, de scribed by Nambi et al. (1994) was based upon pharmacology, and not sequence data. From our BLAST searches, we could not find a homologous African clawed frog EDNRB1; however we did find an EDNRB1 from the Western clawed frog. Thus, it is possible that Nambi et al. (1994) were characterizing the African clawed frog EDNRB2 and not a novel EDNRBx. The EDNR B2 has been called the avian-specific EDNR (Lecoin et al., 1998), but th is statement is clearly not true The EDNRB2 is found in all non-therian gnathostomes (for which we have sequen ce data) except the zebrafish (Fig. 3-3). We also found a duplicate stickleback EDNRB2 (E DNRB2b), but unlike the EDNRA and EDNRB1 (Fig. 3-3), we did not find a teleost specific duplication of the EDNRB2. The African clawed frog EDNRC, first described by Karne et al. (1 993), groups with high support to the African clawed frog and Western clawed frog EDNRB2 and non-therian EDNRB2s, suggesting it is a 71

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duplicate of the EDNRB2 gene. The name EDNRB2 is a bit of a misnom er. Lecoine et al. 1998 originally termed it because the novel quail EDNR sequence was more similar on an amino acid level EDNRB1 than EDNRA or EDNRC, bu t it had a distinct pharmacological profile (Table 3-1). The EDNRB2 is a distinct re ceptor, coded by its own gene, on a different chromosome from EDNRA and EDNRB1 (Wheeler et al., 2007) and groups with the frog EDNRC. The term EDNRC would be more appropriate for this gene, emphasizing the independence of this receptor from EDNRA and EDNRB1. Th e majority of these novel EDNR discoveries were before the advent of genome projects and high throughput sequencing, so it is not surprising that we are reclassifyi ng some of these receptors based upon molecular and phylogenetic data. These early studies do highlight the important point th at not all EDNRs have the same pharmacological profile; however, we ar gue that does not necessarily mean they are novel. A common assumption is that all gnathostome EDNRs will be pharmacologically identical to the mammalian EDNRA and EDNRB1. This is not the case (see Table 3-1) and caution should be used when describing the ba sal gnathostome receptors in this fashion. One of our most interesting discoveries wa s the lack of EDNRB2 sequences from the therian mammals. We exhaustively searched GenBank and the therian genome projects, and could only find EDNRA and EDNRB1, suggesting that EDNRB2 was lost about 150 MYA. Interestingly, we did not find a zebrafish or opossum EDNRB2, but both of these animals have a duplicate EDNRB1 (opossum EDNRB1d and zebrafi sh EDNRB1c) (fig. 3), leading us to question whether the initial loss of the EDNRB2 lead to a duplication of the EDNRB1 in these organisms? To determine if EDNRB2 was lost by mutation or deletion, we used the conserved gene order of genes that surround EDNRB2 : TMLHE, RPA16, and SYBL1 This gene order was 72

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similar in the Western clawed frog, chicke n, and platypus genomes. A fourth gene, sprouty 3 ( SPRY3) was found after TMLHE in the chicken, but was not found in the frog or platypus contigs. In the opossum, macaque, and human genomes TMLHE SPRY3 and SYBL1 were conserved. In addition, in th e therians, other genes and pseudogenes were found between TMLHE and SYBL1 In the opossum, the genes at loc100024257 and loc100024284 were not homologous to any other vertebrate gene s. The pseudogenes (loc100024309 and loc100024329) were similar to alpha-tubulin c left lip and palate associated transmembrane protein 1, and coiled coil domain containing 127 In the macaque and human, in the approximate region where EDNRB2 would be expected to be located, there is a gene, AMDP1 None of these proteins from the therians share any common characteristics with the GPRC with seven transmembrane domains. Thus, this region appears to be one of active chromosomal ch ange, resulting in the loss/gain of genes. We therefore hypothesize that EDNRB2 was lost in the therians and has not simply mutated to an unrecognizable state. The loss of the EDNRB2 from the therian mammals is intriguing, and we hypothesized that perhaps the remaining ther ian EDNRs have undergone shifts in replacement rates of amino acids that are necessary for EDN binding. If true this would suggest a functional shift in these receptors, perhaps resulting in a new range of func tions for these receptors to compensate for the loss of EDNRB2. Using an analys is of the site-by-site replacement rate, we determined that therian EDNRAs have more Type I/II sites in a region necessary for EDN1 binding (Fig.3-5A). On contrary, the therian EDNRB1 is highly c onserved across the gnathos tomes (Fig. 3-5B). These results suggest that therian EDNRA have been remodeled, while EDNRB1 have remained conserved. Thus, we hypothesize th at EDNRA have different functions in therians compared to non-therian gnathostomes. Our results set up a framework for future comparative studies among 73

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the therians and non-therians to help elucidate the pot ential functional differences of the EDNRs in these animals. One limitation to EDN research is that there is littl e information about the function of EDNRs in the non-therian gnathosto mes. All EDNR antagonists and agonists are based upon mammalian studies, and as summarized in Table 1, not all EDNRs share a common binding profile, making this a complex system to understand. Studies determining the physiological role of the EDNRS in non-th erian gnathostomes are greatly needed. In conclusion, the majority of gnathostomes have three EDNRS: EDNRA, EDNRB1, and EDNRB2; however, the therian mammals have lost the EDNRB2. Also, teleost fishes have duplicate EDNRA and EDNRB1 genes that are co -orthologous to sing le copy tetrapod EDNRA and EDNRB1, and this may be a remnant of a te leost specific genome duplication after the split from the tetrapods (Volff, 2005). The EDNRB2 has been largely ignored in the literature. In order to understand vertebrate evolution, more EDN/EDNR research is necessary, because this signaling system is thought to be a key innovation in the radiation of vertebrates and the development of jaws (Clouthier and Schilling, 2004). In addition, in the future caution should be used in extrapolating findings in model, nontherian organisms, si nce there is clearly heterogeneity between the presence and expression of the EDNRs. 74

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Table 3-1. A summary of the bi nding profiles, and embryonic and adult tissue distribution of the gnathostome EDNRs. EDN= endothelin, NCC= neural crest cell, SRXc= sarafotoxin 6c, ? = undetermined. Protein Species Binding Profile Embryonic Tissue Distribution Adult Tissue Distribution References EDNRA Human EDN1=EDN2>>>ED N3 Ubiquitous Ubiquitous Adachi et al. 1991 Brand et al. 1998 Rat EDN1=EDN2>>>ED N3 ? Ubiquitous Hori et al. 1992, Lin et al. 1991 Cow EDN1>END2>>ED N3>>>SRXc ? Ubiquitous Arai et al. 1990 Chicken EDN1=EDN2>>>ED N3 NC-derived ectomesenchyme, branchial arches melanocytes, lung* Kempf et al. 1998, Scarparo et al. 2007, Gomez et al. 2007 Quail ? ? ? EDNRAx Western Clawed frog EDN1>>>EDN3>>> >> SRXc ? oocyte follicle, lung, heart* Kumar et al. 1993, Kumar et al. 1994 EDNRA Zebrafish ? migrating NCCs, ectomesenchymal cells of pharyngeal arches ? Clouthier and Schilling 2004 EDNRB1 Human EDN1=EDN2=EDN3 = SRXc Ubiquitous Ubiquitous Brand et al. 1998, Ogawa et al. 1991, Sakamoto et al. 1991 Rat EDN1=EDN2=EDN3 = SRXc ? Ubiquitous Hori et al. 1992, Sakauri et al. 1990 Cow EDN1=EDN3, EDN2? SRXC? ? Lung* Kozuka et al. 1991 Chicken ? NCC emigrating from neural tube, dorsal root ganglion, gut ? Nataf et al. 1996, Lecoin et al. 1998 Quail ? NCC emigrating from neural tube, dorsal root ganglion, gut ? Nataf et al. 1996, Lecoin et al. 1998 Western Clawed frog ? ? ? EDNRB1 Zebrafish ? NCC (melanocytes), xanthophore and iridophore precursor cells, ? Parichy et al. 2000 EDNRB2 Chicken EDN1=EDN2=EDN3 >>>SRXc NCC migrating in dorsolateral pathway, kidney, skin, feather buds, mesenchyme of branchial arches ? Nataf et al. 1996, Kempf et al. 1998, Lecoin et al. 1998 Quail EDN1=EDN2=EDN3 >>>SRXc NCC migrating in dorsolateral pathway, kidney, skin, feather buds ? Nataf et al. 1996, Lecoin et al. 1998 75

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Table 3-1. Continued Protein Species Binding Profile Embryonic Tissue Distribution Adult Tissue Distribution References Western Clawed frog ? ? ? Zebrafish ? ? ? EDNRBx Western Clawed frog EDN1=EDN3>>>> SRXc, EDN2? ? Liver* Nambi et al. 1994 EDNRC Western Clawed frog EDN3>EDN1, EDN2? ? Melanophores* Karne et al. 1993 *these are the only tissues tested thus far > represents one order of magnitude 76

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Table 3-2. Degenerate and non-de generate primers used to seque nce the killifish endothelin receptors. Primer 5' to 3' Orientation EDNRF1 AGC CGA CAG AGA TTA GAT ACT CCT TYA ART AYR EDNRF2 CGC TAC TAC GAA TTA TTT ATC AGA ACA ART GYA TGM G EDNRF3 GCA GTT CTA TCA GGA CGC GAA RGA YTG GTG G EDNRR1 TTT GGA AAC CAA GTA T AG TTG CTA TAG GRT TDA TRA C EDNRAR2 TCT TCC ACT GGA TGG AGG TGM CRT TNA YNG G EDNRB1R1 AGC TTC ATG CAG GAC TGC TTN TCR TCC AT exEDNRAR1 GCA GAA GAC AGC TTT GGC AAC exEDNRAR2 TTT CAG GT G TTC ACT GAG CGC rEDNRAF1 TCC CTT TGC ACC TCA GCA GGA TCC rEDNRAF2 ATT ACT TCG GCA TCA ACC TGG CGA C rEDNRAF4 GGC ATC AAC CTG GCG ACA ATC AAC T rEDNRAR1 TTC CGG TGG TTC AGC ATC TCA CA rEDNRB1F1 TGG TGT TCG CTC TGT GCT GGC TTC C rEDNRB1F2 AAC CGC TGC GAG CTG CTC AGT TTC T rEDNRB1R1 ACG CCG ATG CCT TTG ATT CGA CTC C rEDNRB1R2 CCC CAC GGA CGC TTT CTG GAC AA rEDNRB2F1 GCA TGC GGA TCG CAC TGA ACG ATC rEDNRB2F2 CTC AGC CGC ATC CTG AAG AAA ACC TT rEDNRB2R1 CCGTTCCTCAT GCACTTGTTCCTGTAGA rEDNRB2R2 CCACGAAGATCAGGCAGGAAATGATGGT 77

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Table 3-3. Specific killifish primers used in the tissue distribution (td) experiments. Primer 5' to 3' Orientation tdEDNRAF1 AAA AGC CCG GAA CCC AAC A tdEDNRAR1 GGA CAT CAT TCT CCC TGA CAG C tdEDNRB1F1 TTC TGG ACA AAC GCT CCG T tdEDNRB1R1 TCC CGC CGC TGC TTA ATA T tdEDNRB2F1 TGT GCC CTG AGC ATT GAC C tdEDNRB2R1 AAC ACC GTC TTC GCC ACT TC 78

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Table 3-4. Contingency tabl es summarizing the results of the rate shift analysis (Fig. 5A and B). Observed (expected) numbers recorded. Significance was determined using the Gtest and Chi-squared di stribution (see text). A Type I/ II at functionally important positions Type I/ II at other positions Type I/ II Total EDNRA 6 (2.3) 29 (32.6) 35 EDNRB1 1 (4.7) 69 (65.3) 70 7 98 105 B Type I/II sites in the EDNRA Non type I/II sites in the EDNRA Total EDN1 Binding Domain 6 (2.1) 20 (23.8) 26 Other sites 29 (32.8) 370 (366.1) 399 35 390 425 C Conserved sites at functionally important positions Other sites at functionally important positions Total EDNRA 5 (18.6) 26 (12.7) 31 EDNRB1 49 (36) 11 (24.7) 60 54 37 90 79

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A. Type 1 site B. Type 2 site W Therian S W W T Y Y Y Y Y Y Y Y W W W W W Therian Non-therian Non-therian Figure 3-1. An illustration of A) type I and B) type II sites determined by a rate shift analysis (Knudsen et al., 2003). Depi cted is the phylogenetic re lationship of a homologous site between two subfamili es: therian mammals and nontherian gnathostomes. Arrows indicate reroo ting of the tree along the internode connecting ther ians to other non-therian gnathostomes 80

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EDNRAHeart Gill Kidney Brain Stomach Intestine Op. epithelium 18S EDNRB1 EDNRB2 EDNRAHeart Gill Kidney Brain Stomach Intestine Op. epithelium 18S EDNRB1 EDNRB2 Figure 3-2. mRNA tissue dist ribution of the killifish EDNRA EDNRB1, and EDNRB2 using duplexing, multi-tissue PCR. 18S primers were used as an internal control gene and the 18S amplicon displayed equa l intensity for each tissue. 81

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Figure 3-3. A maximum likelihood tree of the gnathostome EDNRs. Numbers at the arrows represent the maximum likelihood score for the attachment of the outgroup to the maximum likelihood study group tree (see text for explanation). The numbers at the nodes represent percent bootstrap/ posterior probability from our bayesian analysis. Nodes that were not represented in our Ba yesian analysis are represented by (-). Green represents EDNRA, light gree n EDRNA2, blue EDNRB1, light blue EDNRB1b and red EDNRB2. GB= GenBank and EN= Ensembl. 82

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83 EDNRB1b EDNRB2 EDNRB1 EDNRA EDNRA2Medaka (EN-ENSORLP00000022248) Western clawed frog (GB-NP_001072676.1) African clawed frog EDNRC (GB-P32940) African clawed frog (GB-AAH48223) 96/100 100/100 Platypus (EN-ENSOANP00000013874) Chicken (GB-NP_989451) Quail (GB-BAF42697 99/100 94/100 99/100 Stickleback EDNRB2b (EN-ENSGACP00000024567) Stickleback (EN-ENSGACP00000024566) 100/100 Killifish (GB-EU391603) Medaka (GB-BAB20097) 95/100 Tiger pufferfish (EN-NEWSINFRUP00000150746) Green spotted pufferfish (EN-GSTENP00004209001) 99/100 54/55 99/100 99/100 Metatherian EDNRB2??? Eutherian EDNRB2??? -33672.791 Zebrafish (GB-XP_688565) Zebrafish EDNRB1c (EN-ENSDARP00000070019) 100/100 Green spotted pufferfish (EN-GSTENP00026083001) Tiger pufferfish (EN-SINFRUP00000163212) 100/100 100/100 47/65 Zebrafish (GB-NP_571272) Killifish (GB-EU391602) Stickleback (EN-ENSGACP00000017059) Medaka ( EN-ENSORLP00000013867) Spotted green pufferfish (EN-GSTENP00015077001) Tiger pufferfish (EN-SINFRUP00000141006) 98/100 42/54 61/100 98/100 46/56 90/99 Western clawed frog (GB-NP_001072476) Chicken (GB-XP_417001) Platypus (GB-ENSOANP00000007537) Opossum (GB-XP_001367638) Opossum EDNRB1d (EN-ENSMODP00000001834) 100/100 Rat (GB-NP_059029) Mouse (GB-NP_031930) 100/100 Cat (EN-ENSFCAP00000008108) Cow (GB-NP_776734) Pig (GB-NP_001033091) 89/100 66/98 Dog (GB-NP_001010943) Macaque (GB-XP_001089047) Chimpanzee (GB-XP_509693) Human (GB-NP_000106) 98/99 80/99 79/100 49/76 99/94 53/89/94 52/58 66/96 99/100 -33673. 282 Western clawed frog (GB-NP_001072275) African clawed frog (GB-AAH44316) 100/100 45/Platypus (EN-ENSOANP00000008365) Opossum (EN-ENSMODP00000000789) 100/100 Macaque (GB-XP_001098723) Chimpanzee (GB-XP_001149597) Human (GB-NP_001948) 90/100 Mouse (GB-NP_034462) Rat (GB-NP_036682) 100/100 Cat (EN-ENSFCAP00000014287) Dog (GB-XP_853465) 55/100 Cow (GB-NP_776733) Pig (GB-NP_999394) 61/100 43/99 45/100 100/100 77/100 94/100 99/100 -33673.029 Tiger pufferfish (EN-SINFRUP00000157598) Green spotted pufferfish (EN-GSTENP00021215001) 99/100 99/100 Stickleback (EN-ENSGACP00000022740) Green spotted pufferfish (EN-GSTENP00032885001) Tiger pufferfish (EN-SINFRUP00000133804) 89/100 Zebrafish (GB-XP_687609) Medaka (EN-ENSORLP00000009301) Killifish (GB-EU391601) 96/100 48/93 28/83/99 96/100 Chicken (GB-NP_989450) 100/100 (EN-ENSORLP00000003753) Medaka 0.4 Replacements/site African clawed frog EDNRA2b (GB-AAA19570.1) 60/99EDNRB1b EDNRB2 EDNRB1 EDNRA EDNRA2Medaka (EN-ENSORLP00000022248) Western clawed frog (GB-NP_001072676.1) African clawed frog EDNRC (GB-P32940) African clawed frog (GB-AAH48223) 96/100 100/100 Platypus (EN-ENSOANP00000013874) Chicken (GB-NP_989451) Quail (GB-BAF42697 99/100 94/100 99/100 Stickleback EDNRB2b (EN-ENSGACP00000024567) Stickleback (EN-ENSGACP00000024566) 100/100 Killifish (GB-EU391603) Medaka (GB-BAB20097) 95/100 Tiger pufferfish (EN-NEWSINFRUP00000150746) Green spotted pufferfish (EN-GSTENP00004209001) 99/100 54/55 99/100 99/100 Metatherian EDNRB2??? Eutherian EDNRB2??? -33672.791 Zebrafish (GB-XP_688565) Zebrafish EDNRB1c (EN-ENSDARP00000070019) 100/100 Green spotted pufferfish (EN-GSTENP00026083001) Tiger pufferfish (EN-SINFRUP00000163212) 100/100 100/100 47/65 Zebrafish (GB-NP_571272) Killifish (GB-EU391602) Stickleback (EN-ENSGACP00000017059) Medaka ( EN-ENSORLP00000013867) Spotted green pufferfish (EN-GSTENP00015077001) Tiger pufferfish (EN-SINFRUP00000141006) 98/100 42/54 61/100 98/100 46/56 90/99 Western clawed frog (GB-NP_001072476) Chicken (GB-XP_417001) Platypus (GB-ENSOANP00000007537) Opossum (GB-XP_001367638) Opossum EDNRB1d (EN-ENSMODP00000001834) 100/100 Rat (GB-NP_059029) Mouse (GB-NP_031930) 100/100 Cat (EN-ENSFCAP00000008108) Cow (GB-NP_776734) Pig (GB-NP_001033091) 89/100 66/98 Dog (GB-NP_001010943) Macaque (GB-XP_001089047) Chimpanzee (GB-XP_509693) Human (GB-NP_000106) 98/99 80/99 79/100 49/76 99/94 53/89/94 52/58 66/96 99/100 -33673. 282 Western clawed frog (GB-NP_001072275) African clawed frog (GB-AAH44316) 100/100 45/Platypus (EN-ENSOANP00000008365) Opossum (EN-ENSMODP00000000789) 100/100 Macaque (GB-XP_001098723) Chimpanzee (GB-XP_001149597) Human (GB-NP_001948) 90/100 Mouse (GB-NP_034462) Rat (GB-NP_036682) 100/100 Cat (EN-ENSFCAP00000014287) Dog (GB-XP_853465) 55/100 Cow (GB-NP_776733) Pig (GB-NP_999394) 61/100 43/99 45/100 100/100 77/100 94/100 99/100 -33673.029 Tiger pufferfish (EN-SINFRUP00000157598) Green spotted pufferfish (EN-GSTENP00021215001) 99/100 99/100 Stickleback (EN-ENSGACP00000022740) Green spotted pufferfish (EN-GSTENP00032885001) Tiger pufferfish (EN-SINFRUP00000133804) 89/100 Zebrafish (GB-XP_687609) Medaka (EN-ENSORLP00000009301) Killifish (GB-EU391601) 96/100 48/93 28/83/99 96/100 Chicken (GB-NP_989450) 100/100 (EN-ENSORLP00000003753) Medaka 0.4 Replacements/site 0.4 Replacements/site African clawed frog EDNRA2b (GB-AAA19570.1) 60/99

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Platypus : Ultra519TMLHE* EDNRB2SYBL1-prov RNA-polymerase 1, 16 kDa* Brn-3 SYBL1* TMLHE Opossum : Chromosome XLOC1000024257? LOC100024309# SPRY3 LOC100024284? LOC100024329# SYBL1* Macaque : Chromosome XTMLHE SPRY3 SYBL1 AMDP1# NIP7 Human : Chromosome XTMLHE SYBL1 SPRY3 AMDP1# 100K bp Western clawed frog : scaffold 258TMLHE EDNRB2 SYBL1 RNA-polymerase 1, 16 kDaChicken : Chromosome 4 TMLHE SPRY3 EDNRB2 SYBL1 RNA-polymerase 1, 16 kDa Platypus : Ultra519TMLHE* EDNRB2SYBL1-prov RNA-polymerase 1, 16 kDa* Brn-3 SYBL1* Platypus : Ultra519TMLHE* EDNRB2SYBL1-prov RNA-polymerase 1, 16 kDa* Brn-3 SYBL1* TMLHE Opossum : Chromosome XLOC1000024257? LOC100024309# SPRY3 LOC100024284? LOC100024329# SYBL1* TMLHE Opossum : Chromosome XLOC1000024257? LOC100024309# SPRY3 LOC100024284? LOC100024329# SYBL1* Macaque : Chromosome XTMLHE SPRY3 SYBL1 AMDP1# NIP7 Macaque : Chromosome XTMLHE SPRY3 SYBL1 AMDP1# NIP7 Human : Chromosome XTMLHE SYBL1 SPRY3 AMDP1# Human : Chromosome XTMLHE SYBL1 SPRY3 AMDP1# 100K bp 100K bp 100K bp Western clawed frog : scaffold 258TMLHE EDNRB2 SYBL1 RNA-polymerase 1, 16 kDa Western clawed frog : scaffold 258TMLHE EDNRB2 SYBL1 RNA-polymerase 1, 16 kDaChicken : Chromosome 4 TMLHE SPRY3 EDNRB2 SYBL1 RNA-polymerase 1, 16 kDaChicken : Chromosome 4 TMLHE SPRY3 EDNRB2 SYBL1 RNA-polymerase 1, 16 kDa TMLHE SPRY3 EDNRB2 SYBL1 RNA-polymerase 1, 16 kDa Figure 3-4. Genetic maps of the genes (def ined by NCBI) surrounding the EDNRB2 in nontherian gnathostomes, and hypothesized locatio n in the therians. represent gene identities identified by our analyses. # represents pse udogenes as defined by NCBI, and ? represents sequences with no simila rity to any other vertebrate gene (as determined by BLAST searches) 84

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85 A. Human EDNRA E N S M E T L C L R S A G V L A L W F C V I S D N P S L N T S Y R H V D D F T T R F N L F S L E T G V T T H Q P T G N S P L V L M H N Y C P Q T Q K F A S T I K S Y R A C S W S R V A T G Q G I P R D K N R C R V Y I N T V I S C T I F I V G M V G N A T L R L I Y I Q N K R N G P V F K L P I N V I D L I Y V L G D L S L A A I L A A F W D H N D F H K K L F P F L Q K S S V G I T V L N L C A L V F G T L V I P M L N A T S K F F F V M E A I G A I P S F I L W I L I V S I A I E Q G E Y R E M E Y F Q D V K D W W L F G F Y F C M P L V C T A I F Y T L M T C M E L R N N R G F V E S L R I A L K E R R Q L K E H S K K T V S R I L H L L C W F F A L P L V V V F C I M E N L L S F L L Y L M D I G I T N L A M N S P C I N I A L Y N K F K K S C F G S C L C S Q Y C C C K S L M T S V P I S T G N M Q W K N H D Q N R D T N H N S S H K D N A V C M L G L C V D P V T Y S M N Extracellular Intracellular M Q P P P S L G C I P R V L A V L A R L A C G L S R F G R E E G W P D R A T P L Q L P T M I E A T P T K T L K W P A L S Q N S G S L A P A E V K P P I S G A T R D G P P R T I S P E I P G Q C P K E T F K I R D R R A V L I S W S R I K G D Y D P N R C A A Y I N T V V S C L V F V L G I I G N S I L L R I I Y N K K C M R N G P L K Y V N I P I D I V I H L L D G L A L S A I L I L W E P F G A E S Y L V P F I Q K A S V G I T V L S L C A L S C K M W P K G V I T K C L L H P V I I D F G I A E P V A L V V S V V W I L V I E V A G Y K M D T Q K T Y F A M F Q A K D W W L F S F Y F C L P L A I T A F F Y T L M T C M E L K R S K G L V E M Q I A L N T V R E R K Q H L D L T L K L I R S L H L P L W C L A F V L V L C F V N Q Y N L L S F L L V L D Y I G I N M A S L N S C I N P I A L Y N K F R K S C F K C L C C E F S Q C W E K Q S L E E K F K L C S Q K A N D H G Y D N S S R F N K Y S S S N T K AType I&II [Slower subfamily] Type I [Faster subfamily] Type I [Slower subfamily] Type II Type I&II [Slower subfamily] Functionally important sites Slow evolving Extracellular Intracellular B. Human EDNRB1 A. Human EDNRA E N S M E T L C L R S A G V L A L W F C V I S D N P M E T L C L R S A G V L A L W F C V I S D N P S L N T S Y R H V D D F T T R F N L F S L E T G V T T H Q P T G N S P L V L M H N Y C P Q T Q K F A S T I K S Y R A C S W S R V A T G Q G I P R D K N R C R V Y I N T V I S C T I F I V G M V G N A T L R L I Y I Q N K R N G P V F K L P I N V I D L I Y V L G D L S L A A I L A A F W D H N D F H K K L F P F L Q K S S V G I T V L N L C A L V F G T L V I P M L N A T S K F F F V M E A I G A I P S F I L W I L I V S I A I E Q G E Y R E M E Y F Q D V K D W W L F G F Y G F Y F C M P F C M P L V C L V C T A I F T A I F Y T L M T C M E L R N N R G F V E S L R I A L K E R R Q L K E H S K K T V S R I L H L L C W F F A L P L V V V F C I M E N L L S F L L Y L M D I G I T N L A M N S P C I N I A L Y N K F K K S C F G S C L C S Q Y C C C K S L M T S V P I S T G N M Q W K N H D Q N R D T N H N S S H K D N A V C M L G L C V D P V T Y S M N S M N Extracellular Intracellular M Q P P P S L M Q P P P S L G C I P R V L A V L A R L A C G L S R F G R E F G R E E G W P D R A T P L Q L P T M I E A T P T K T L K W P A L S Q N S G K W P A L S Q N S G S L A P A L A P A E V K P P I S G A T R D G P P R T I S P E I P G Q C P K E T F K I R D R R A V L I S W S R I K S W S R I K G D Y D P N R C A A Y I N T V V S C L V F V L G I I G N S I L L R I I Y N K K C M R N G P L K Y V N I P I D I V I H L L D G L A L S A I L I L W E P F G A E S Y L V P F L V P F I Q K A S V G A S V G I T V I T V L S L C L S L C A L S C K M W P K G V I T K C L L H P V I I D F G I A E P V A L V V S V V W I L V I E V A G Y K M D T Q K T Q K T Y F A M F Q Y F A M F Q A K D W W L F S F Y F C L P L A I T A F F Y T L M T C M E L K R S K G L V E M Q I A L N T V R E R K Q H L D L T L K L I R S L H L P L W C L A F V L V L C F V N Q Y N L L S F L L V L D Y I G I N M A S L N S C I N P I A L Y N K F R K S C F K C L C C E F S Q C W E K Q S L E E K F K L C S Q K A N D H G Y D N S S R F N K Y S S S N T K AType I&II [Slower subfamily] Type I [Faster subfamily] Type I [Slower subfamily] Type II Type I&II [Slower subfamily] Functionally important sites Slow evolving Type I&II [Slower subfamily] Type I [Faster subfamily] Type I [Slower subfamily] Type II Type I&II [Slower subfamily] Functionally important sites Slow evolving Extracellular Intracellular B. Human EDNRB1 Figure 3-5. Illustration of th e human A) EDNRA and B) EDNRB1. Residues necessary for endothelin-1 binding are hi ghlighted in red. See text for explanation.

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CHAPTER 4 EFFECTS OF ENVIRONMEN TAL SALINITY ON GILL ENDOTHELIN RECEPTOR EXPRESSION IN THE KILLIFISH FUNDULUS HETEROCLITUS Introduction The endothelin (EDN) family of paracrine pe ptides consists of three isoforms, EDN1, EDN2 and EDN3, (Inoue et al., 1989; Yanagisawa et al., 1988a), and is found only in vertebrates (Hyndman and Evans, 2007). These proteins ar e involved in diverse physiological functions including: regulation of vascular tone (La and Reid, 1995; Yanagisawa et al., 1988a; Yanagisawa et al., 1988b), alteration of ion tr ansport (Ahn et al., 2004; Evans et al., 2004; Garvin and Sanders, 1991; Ge et al., 2006; Prasanna et al., 2 001; Zeidel et al., 1989), and direction of migration of ne ural crest cell during craniofaci al development (Clouthier and Schilling, 2004; Kurihara et al., 1994). Endothe lins bind to three G-pr otein-coupled receptors: endothelin A receptor (EDNRA) (Arai et al., 1990) endothelin B1 receptor (EDNRB1) (Sakurai et al., 1990), and the endothelin B2 receptor (EDNRB2) (Lecoin et al., 1998). We recently determined that there are teleost specific dupl ications of the EDNRA (EDNRA2) and EDNRB1 (EDNRB1B), and multiple species have duplicate e ndothelin receptors (i.e. Western clawed frog has EDNRC that is a duplicate EDNRB2) (Chapter 3). Endothelin receptors (EDNRs) have been charac terized in some fishes, but the results are often species specific. For example, pharmacological studies have suggested that the aortic vascular smooth muscle of the dogfish shark ( Squalus acanthias) has EDNRB-like receptors (Evans et al., 1996), but that eel, lamprey, and hagfish aortic vascular smooth muscle apparently contains EDNRA-like receptors (Evans and Harri e, 2001). In addition, pharmacological studies using receptor binding assays demonstrated EDNRB-like receptors in the dogfish gill (Evans and Gunderson, 1999), but autoradiographic studie s showed EDNRA thr oughout the vasculature of the trout ( Oncoryhynchus mykiss ) gill (Lodhi et al., 1995). Recently, EDNRBs were 86

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immunolocalized in filamental arteries, lamella r arterioles and pillar cells of the cod ( Gadus morhua) gill and EDNRA were found on bran chial nerves thr oughout the filaments (Stenslokken et al., 2006). Fi nally, EDNRA were immunolocali zed to pillar cells in the Takifugu rubripes gill (Sultana et al., 2007). The characterization of the EDNRs may be species and protocol specific. We recently sequenced gill cDNA for EDNRA (accession EU391601), EDNRB1 (accession EU391602), and EDRNB2 (accession EU391603) (Chapter 3) from the euryhaline killifish, Fundulus heteroclitus demonstrating that all three EDNRs are present in the fish gill; however, the specific localization of these receptors has not be en determined in the killifish. Thus, by using molecular techniques, the question of EDNR expression and tissue distribution in these fishes can be resolved, and help one to unders tand the earlier pharmacological, autoradiographical and immunohi stochemical experiments (Evans et al., 1996; Evans and Gunderson, 1999; Evans and Harrie, 2001; Lodhi et al., 1995). In addition to these studies, Evans et al. (2004) determined that EDN1 or the EDNRB1 agonist, sarafotoxin S6c (SRX S6c), inhibited net chloride transport as measured by a reduction of the short circuit current in the killifish opercular epithelium (a model for the SW teleost gill (Karnaky et al., 1977)). Thus in addition to cardiovascular functions, EDN1 and the EDNRs are involved in the regulation of gill ion transport in marine fishes. Killifish are euryhaline fish, distributed in the coastal waters from Florida to Newfoundland (Bigelow and Schroeder, 2002). They live in a harsh environment, where there are daily changes in environmental salinity a nd temperature (Marshall, 2003). They are a commonly used model species test a variety of ecological, epidemiological, and physiological questions (Burnett et al ., 2007). Killifish have been instrume ntal in understanding the effects of changing environmental salinity on fish physiology, because they can tolera te direct transfers 87

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between fresh and seawater (Marshall, 2003) The purposes of this study were to immunolocalize the EDNRs in the killifish gill and to determine the effects of environmental salinity on gill EDNR expression, in an effort to begin to elucidate the putative role of EDN1 signaling in the fish gill. Methods Fish Maintenance The University of Florida and Mount Dese rt Island Biologica l Laboratory (MDIBL) Institutional Animal Care and Use Committee approved all protocols. Killifish were trapped in the brackish waters of North East Creek, Mount Desert Island, ME, and maintained in freeflowing, 15C, 31 ppt seawater (SW) for three mont hs before being transported to the University of Florida. There they were maintain in 3 ft circular tanks, in 20C, 32 ppt SW or dechlorinated Gainesville Fl tap water for 30 days before experi mentation. Fish were fed commercial pellets to satiation every other day, and ammonia, nitrites, and nitrates were below 0.1 ppt, and pH was maintained between 7.8-8.0. Salinity Challenges The killifish salinity challenge experimental design was described in our earlier study (Hyndman and Evans (2007)). In brief, killifish were subjected to one of four treatments: 1) SW to FW transfer; 2) SW sham (SW to SW); 3) FW to SW; or 4) FW sham (FW to FW). At 0, 3, 8, 24 h (acute acclimation) and 30 days (chronic acclim ation) after transfer, 8 or 9 killifish were sacrificed by decapitation, and the gills from the right side snap frozen for RNA analysis, and the left side snap frozen for West ern blotting. In addition, from 3 killifish, gill arch 2 from both sides was fixed in 4% paraformaldehyde (in 10 mM phosphate buffered saline, PBS) for 24 h at 4C for immunohistochemical analysis. 88

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Quantitative Real-Time PCR We previously sequenced killifish cDNA for EDNRA EDNRB1, and EDNRB2 (Chapter 3). From these sequences, we designed non-de generate primers for qua ntitative real-time PCR (qRT-PCR) (Table 4-1). These primers were designed to amplify a product of 50-100 bp, across a predicted exon-exon boundary to prevent amplification of genomic DNA. All reactions were run in duplicate, and all values were normali zed to L8 mRNA values (Choe et al., 2005) and standardize to a cDNA standard curve as previously described by Hyndman and Evans (2007). With each reaction a melting curve analysis wa s completed to ensure only one product was amplified. In addition, we sequenced samples and confirmed that we had am plified the target of interest. Finally, qRT-PCRs were run using R NA instead of cDNA as a negative control, which confirmed that there was no genomic contamination. Immunohistochemistry and Immunoblotting The immunohistochemical methods of Piermarini et al. (2002) and Hyndman and Evans (2007) were used. Five slides per animal ( each slide was about 100 microns deeper into the filament) were analyzed. In addition, Western blots were made from gill samples using the protocols of Piermarini et al. (2002) and Hyndman et al. (2006). Finally, to accurately quantify protein level differences, we made immunoblots following the methods of Joyner-Matos et al. (2006). In all of these protocols, we used the following antibodies: antirat EDNRA (1/500, Alomone Laboratories, Jerusalem); anti-rat E DNRB (1/1000, Alomone Laboratories). Currently a commercial antibody that can discriminate be tween EDNRB1 and EDNRB2 is not available; thus we used a non-specific EDNRB antibody. Negative controls were run using peptideabsorbed antibodies (1 g peptide/1 g antibody, mixed overnight, shaking, 4C, following the manufacturers protocol). A ll of the protocols were run us ing the preabsorbed antibody to determine any non-specific binding. To lo calize the EDNR-expressing cells relative to 89

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mitochondrion-rich (ion transporting) cells (MRC s), slides were double-la beled with the EDNR antibodies and with anti-chicken Na+,K+-ATPase as previously described (Hyndman et al., 2006). This antiNa+,K+-ATPase is a common marker for the MRC in the killifish gill (Choe et al., 2006; Hyndman et al., 2006; Hyndman and Evans, 2007; e.g. Katoh and Kaneko, 2003)Westerns and dot blots were di gitized using a flat bed scanner, and analyzed using Biorads Quantity One software (Hercules, CA). The brightness and contrast of each slide image was adjusted with Photoshop CS3 (Adobe, San Jose, CA). Statistics All values were tested for normality and equal variance and if these were not met, the values were log-transformed for statistical analysis. Quantita tive real-time PCR values were tested using two-factor ANOVA (t reatment and time), and when significance was found specific differences between sham and treatment were determined using unpaired, two-tailed, T-test ( =0.05). Protein level differen ces were analyzed with one-f actor ANOVAs and Dunnetts post hoc test. Chronic qRT-PCR and protein level differences were analyz ed using unpaired, twotailed, T-tests to test for differences between the SW and FW treatments. Results Endothelin Receptor mRNA Levels In killifish transferred from FW to SW, there was a significant, threefold increase in gill EDNRA mRNA levels only at 24 h after transfer (p=0.03) (Fig. 4-1A). There was, however, a significant, twofold increase in gill EDNRB1 mRNA compared to sham at 3 h (p<0.001), 8 h (p=0.003), and 24 h (p=0.016) after tr ansfer (Fig. 4-1C). Gill EDNRB2 mRNA levels increased 3 h after transfer (p<0.001), but the levels were not different from sham by 8 and 24 h (Fig. 41E). In killifish transferred from SW to FW, there were no signifi cant changes in gill EDNRA or EDNRB2 mRNA levels compared to sham over the 24 h acclimation period (Fig. 4-1B, F). 90

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There was, however, a significant increase in gill EDNRB1 mRNA 3 h after transfer (p<0.001), these levels were not different from sham at 8 or 24 h after transfer (Fi g. 4-1D). After chronic acclimation (30 days) to SW or FW, there were no differences in EDNRB1 or EDNRB2 mRNA levels; however, gill EDNRA mRNA were 55% lower in the FW killifish compared to SW killifish (p=0.022) (Fig. 4-2). Endothelin Receptor Protein Concentrations Using Western blots made from SW killifis h gills, we found a si ngle ~37-kDa band with the anti-EDNRA, and a single band of ~40-kDa w ith anti-EDNRB (Fig. 4-3). As we found for EDNRA mRNA, there was a threefold increase in E DNRA compared to control (time zero) only at 24 h after a FW to SW transf er (p=0.014) (Fig. 4-4A). Likewi se, there was a twofold increase in EDNRB in the killifish gill compared to contro l at 24 h after transfer (p=0.002) (Fig. 4-4C). After a SW to FW transfer, there was a significa nt decrease in EDNRA prot ein level (3 and 8 h) (p<0.001); however, by 24 h EDNRA protein levels re turn to control values (Fig. 4-4B). There was no significant change in EDNRB protein level af ter a SW to FW transfer (Fig. 4-4D). After chronic acclimation to SW or FW, there was a significant 60% decrease in gill EDNRA protein levels in the FW killifish compared to the SW killifish (p=0.004) (Fig. 4-4E). There were no statistical differences between SW and FW chr onic acclimated killifish gill EDNRB levels (Fig. 4-4E). Immunohistochemistry In the gill, epithelial cell s in the interlamellar region were immunopositive for EDNRA (Fig. 4-5). In addition, on the afferent side of the filament where there are no lamellae, there were many, large ovoid, cells immunopositive for EDNRA (Fig. 4-5D, E, H, I). All of the EDNRA-immunoreactive cells (-IR ) were also immunopositive for NKA (Fig. 4-5C, E, G, I), suggesting that EDNRA is expressed in the m itochondrion-rich cell (ion transporting) (MRC). 91

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There were no immulocalization differences betw een SW control gills (Fig. 4-5A-C) and gills from killifish acclimated to SW for 24 h (Fig. 45D, E); however, 24 h after SW to FW transfer, the EDNRA immunoreactivity became punctate, and less diffuse throughout the cell compared to the other treatments (Fig. 4-5H, I). In addition, gills from killifish acclimated to FW for 30 days, had a shift in EDNRA distribut ion. Compared to SW gills where the EDNRA-IR took up the whole cell (Fig 4-5A, D), in FW gills there was a shift to only EDNRA along the basal aspect of the cell (Fig. 4-5F, G). Negative controls usi ng a peptide-absorbed an tibody were double labeled with anti-NKA, and showed no nonspecific bi nding of the EDNRA antibody (Fig. 4-5B). Throughout the gill vasculatur e there was EDNRB-IR (Fig. 4-6A, C-H), including the prelamellar arterioles (Fig. 4-6E, F arrows). In addition, EDNRB-IR was found on lamellar pillar cells (Fig. 4-6D). Unlike EDNRA, the ED NRB-IR did not colocalize to the same cell as the NKA-IR (Fig. 4-6C, E, F, H). There we re no obvious immunolocalization differences between the SW (Fig. 4-6A, B) and FW chronically acclimated gills (Fig. 4-6G, H). Also, there were no obvious localization difference 24 h after a SW or FW transfer (Fig. 4-6E, F). A negative control using peptid e absorbed EDNRB antibody disp layed no immunoreactivity (Fig. 4-6B). Discussion The killifish is an excellent osmoregulator, cap able of tolerating direct transfers from FW to SW and SW to FW (Hyndman and Evans, 2007; Marshall, 2003). We have previously hypothesized that endothelin-1 (EDN1) signaling cascades may be a local regulator of gill ion transport, because EDN1 inhibits net chloride transport in the killifish opercular epithelium (Evans et al., 2004), and we coul d localize EDN1 (mRNA and protein) in the gill of this species (Hyndman and Evans, 2007). In the present work, we have found that 24 h after a FW to SW transfer, there was a significan t threefold increase in gill EDNRA mRNA and protein, and a 92

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significant twofold increase in gill EDNRB1 mRNA and protein. Our earlier study (Hyndman and Evans, 2007) determined that after a FW to SW transfer there wa s a significant, sixfold increase in killifish gill endothelin converting enzyme 1 ( ECE1) mRNA (but no change in gill EDN1 mRNA), suggesting that ther e will be an increase in ECE1 protein. Given that the cleavage of proendothelin to EDN1 by ECE1 is the rate limiting step in active EDN1 production (D'Orleans-Juste et al., 2003; Ikeda et al., 2002) one would predict that an increase in ECE1 would result in more active EDN1. Therefore, during short-term hyper-o smotic stress, EDN1 signaling is probably regulated th rough changes in the expression of both the receptors and the ligand. Killifish that were acclimated to FW 24 h after transfer from SW had no significant changes in gill EDNRA, EDNRB1 or EDNRB2 mRNA (or EDNRA and E DNRB protein levels); however, we previously determined that there is a threef old increase in gill EDN1B mRNA and ECE1 mRNA 24 h after a SW to FW transfer (Hyndman and Evans, 2007). This again suggests that more EDN1 protein will be produced, because there is more EDN1 and an increase in ECE1. Therefore, during short-term acclimation to hypo-o smotic environments this system is probably regulated by changes in the ligand only. With chronic acclimation (30 days) to FW, we found a significant 55% decrease in killifish gill EDNRA mRNA and protein, but no significant changes in EDNRB1 or EDNRB2 mRNA or protein, compared to the chronically acclimated SW killifish. We previously determined that these chronic acclimations did not change EDN1A EDN1B or ECE1 mRNA levels (Hyndman and Evans, 2007). Therefore, during long-term acclimation it appears liga nd levels are similar but that there are significantly fewer EDNRAs. 93

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Given our findings of changes to the E DN1 system in the killifish gill during hyperosmotic and hypo-osmotic stress, we can speculate on the function of EDN1 in the fish gill. First, our finding of EDNRA on the MRC, and short term and long term changes in EDNRA expression (mRNA, protein and localization) we hypothesize that EDN1 regulates MRC function via the EDNRA. This may include the regulation of ion transport by this cell. Our lab previously hypothesized that in the gill, EDN1 signaling stimul ates cyclooxygenase-2 (COX-2) production of prostaglandins, and this subsequent ly leads to an inhibition of net chloride transport (Evans et al., 2004). We also previ ously reported that COX2 is expressed in the killifish MRC, and that COX-2 mRNA levels are significantly lower in chronically acclimated, FW killifish (Choe et al., 2006). Collectively wi th our current findings, it appears that FW killifish have less EDNRAs and COX-2, suggesti ng that this EDN1 signaling axis may function in the long-term as a fine-tune control of ion balance, acting lik e a brake, during osmoregulation in hyper-osmotic salinities. The EDNRBs were found throughout the gill vasc ulature, including prelamellar arterioles, and on lamellar pillar cells. Gi ven the epithelial cell localizati on of EDN1 (Hyndman and Evans, 2007), it may function as a paracrine regulator of gill vascular tone, and perfusion of lamellae through control of the tone of the prelamellar arteriole; however, th ere is no evidence that EDN1 contracts filamental arteries or lamellar arteriol es (Stenslokken et al., 1999; Stenslokken et al., 2006). An alternative hypothesis is that the EDNRBs found on th e filamental arteries act as clearance receptors for EDN1. In mammals, EDNRB1 functions as a clearance receptor in the pulmonary circuit (see La and Reid, 1995). In fi shes, 55% of an EDN1 bolus is removed during a single pass through the gills (Olson, 1998). Thus we postulate that gill vascular EDNRB are the clearance receptors in the killifish. 94

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Given that EDN1 (Hyndman and Evans, 2007) and EDNRB were both found on the lamellar pillar cells (Fig. 4-6D), it seems plausibl e that EDN1 is acting as an autocrine on these cells, potentially regulating pill ar cell contractility. It was hypothesized that EDN1 can redistribute and regulate lamellar bl ood flow through the lamellae of the fish gill (Stenslokken et al., 1999; Sundin and Nilss on, 1998). In the trout ( Onycorhynchus mykiss ) and cod, injections of EDN1 into the ventral aorta resulted in constr iction of pillar cells, resulting in a shift of intralamellar blood flow to the outer marginal channels (Stenslokken et al., 1999; Sundin and Nilsson, 1998). Recently EDNRB-like receptors were immunolocalized to the cod pillar cells (Stenslokken et al., 2006). Pillar cells contai n contractile elements and are not innervated (Bettex-Galland and Hughes, 1973; Mistry et al., 2004); thus para crine peptides, like EDN1, may cause pillar cells to contract, and our data support this hypothesis. Recently, EDNRA were found on the pillar cells of the tiger pufferfish ( Takifugu rubripes) using a homologous pufferfish EDNRA antibody, and these receptors were capable of increasing intracellular calcium in vitro, suggesting that they cause contracti on of pillar cells (Sultana et al., 2007). Thus, perhaps the gill EDNR distribution is species-specific. Determining the cellular distribution of these receptors in more fi shes will elucidate any such patterns. An alternative hypothesis is that EDN1 signa ling is involved in maintenance of cellular and tissue integrity durin g volume expansion. Recently, Mistry et al., (2004) sequenced and characterized an actin-binding prot ein, FHL5, from the pillar cells of the tiger pufferfish. They determined that EDN1 and volume expansion (from isotonic dextransaline) both stimulate FHL5 expression in the lamellar pillar cells. In a comparative study between normal and hypertensive rats, volume expansion stimulated EDN1 production in both groups (Abdel-Sayed et al., 2003). In agreement w ith this finding, Wongmekiat and Johns (2003), determined during 95

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volume expansion in normal, lean rats, that EDN1 signaling (likely acting via and EDNRA) leads to diuresis and natriuresis (but this was impaired in obese rats). Thus, collectively we hypothesize that during volume expansion (as occu rs during rapid transfer from SW to FW) EDN1 signaling is involved in maintaining pillar cell integrity through th e stimulation of an increase in the actin binding pr otein FHL5 and regulation of pill ar cell tone. An alternative hypothesis is that initially a volum e load leads to a stretch respons e by the pillar cell, and this increases FHL5 and EDN1 expression in this cell, and subsequent synergis tic contraction of the pillar cell to maintain lamellar integrity. Furthe r experiments used to determine the relationships between EDN1 and volume expansion are needed. In summary, all the components necessary fo r EDN1 production and action (the receptors) are present in the killifish gill. We have recen tly determined that these same proteins are found in the sculpin gill (accessions EU440324-EU44032), and that EDNRs are present in the dogfish shark (accessions EU440328 and EU440329) a nd sea lamprey (accession EU440327) ( Petromyzon marinus ) gills, thus we conclude this signaling molecule is actively expressed in the early vertebrates (Appendix A, Hyndman and Evans, unpublished). We have previously shown that environmental salinity regulates the expr ession of EDN1, ECE1 (Hyndman and Evans, 2007), and present here that salinit y also regulates the EDNRs in th e killifish, but no clear picture has emerged of the role that EDN plays in the re sponse to salinity changes. Studies determining the effects of other factors, like hypoxia, and functional studies (using morpholinos, silencing RNAs or knockout models) will be integral in our understanding of the regulation and potential functions of EDN1 in the gill. 96

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Table 4-1. Non-degenerate primers used in th e quantitative real-time PCR (q) experiments. Primer 5 to 3 Orientation EDNRAqF1 GCA TCA ACC TGG CGA CAA T EDNRAqR1 CAG CAG CAC AAA CAC GAC TTG EDNRB1qF1 CTG ATG ACC TGC GAG ATG CTA A EDNRB1R1 TCC GCG CGC TGC TTA ATA T EDNRB2qF1 CCT GCG AGA TGC TGA GTC G 97

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04812162024 Relative mRNA levelTime post transfer (hr) 048121620 24 *D. EDNRB1 F. EDNRB2 B. EDNRA 04812162024 SW to FW SW to SW 0.1 1.0 5.0 04812162024* *C. EDNRB1 A. EDNRA 0.1 1.0 04812162024 5.0* FW to SW FW to FW 0.1 1.0 04812162024 5.0*E. EDNRB2 04812162024 Relative mRNA levelTime post transfer (hr) 048121620 24 *D. EDNRB1 F. EDNRB2 B. EDNRA 04812162024 SW to FW SW to SW 0.1 1.0 5.0 04812162024* *C. EDNRB1 A. EDNRA 0.1 1.0 04812162024 5.0* FW to SW FW to FW 0.1 1.0 04812162024 5.0*E. EDNRB2 Figure 4-1. Relative gill mRNA levels for endothelin receptors from killifish acclimating to seawater (SW) or fresh water (FW) over a 24 h period (n=5-6 fish/treatment). Dotted lines and closed symbols represent shams, and solid lines and open symbols represent treatments. A, C, E) fish transferred from FW to SW ( ) or maintained in FW (sham, ), and B, D, F) fish transferred from SW to FW ( ), or maintained in SW (sham, ). A, B) EDNRA C, D) EDNRB1, and E, F) EDNRB2. All values are normalized to L8 mRNA levels and are made relative to chr onic SW levels (Fig. 4-2). Asterisks (*) represents statistically significant differences compared to sham determined by 2Factor ANOVA and unpaired, two-tailed, T-tests (c ompared to sham). Mean s.e.m. Note that the scale is logarithmic. 98

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0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 EDNRAEDNRB1EDNRB2Relative to SW mRNA level SW FW p=0.02 p=0.11 p=0.45 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 EDNRAEDNRB1EDNRB2Relative to SW mRNA level SW FW p=0.02 p=0.11 p=0.45 Figure 4-2. Relative gill mRNA levels for the endothelin receptors from the killifish (n=5-6) acclimated for 30 days to either seawater (SW, black bars) or fresh water (FW, grey bars). All values relative to SW. Statistical significance was determined using unpaired, two-tailed, T-tests comparing SW to FW treatments (p values listed on figure). Mean s.e.m. 99

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EDNRA EDNRB Std.195 117 97 50 38 29 20 1 23 EDNRA EDNRB Std.195 117 97 50 38 29 20 1 23 Figure 4-3. Western blots from seawater killifis h gills. Lane 1 is a Coomassie blue stained ladder. Lane 2 is a representative blot stained with anti-EDNRA. Lane 3 is a representative blot stained with anti-EDNRB. 100

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B. EDNRA SW to FW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0481216202Relative intensity (Umgtotal pr-1)4 D. EDNRB SW to FW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0481216202 Time (h)Relative intensity (Umgtotal pr-1)4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 EDNRAEDNRB ProteinRelative to SW protein levels (RU mg total pr-1) SW FW p=0.004 p=0.45E. Chronic levels 0.0 1.0 2.0 3.0 4.0 5.0 04812162024Relative intensity (Umgtotal pr-1)A. EDNRA FW to SW C. EDNRB FW to SW 0.0 0.5 1.0 1.5 2.0 2.5 04812162024 Time (h)Relative intensity (Umgtotal pr-1) p=0.014* p < 0.001 p< 0.00 1* p=0.002 B. EDNRA SW to FW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0481216202Relative intensity (Umgtotal pr-1)4 D. EDNRB SW to FW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0481216202 Time (h)Relative intensity (Umgtotal pr-1)4 D. EDNRB SW to FW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0481216202 Time (h)Relative intensity (Umgtotal pr-1)4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 EDNRAEDNRB ProteinRelative to SW protein levels (RU mg total pr-1) SW FW p=0.004 p=0.45E. Chronic levels 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 EDNRAEDNRB ProteinRelative to SW protein levels (RU mg total pr-1) SW FW p=0.004 p=0.45 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 EDNRAEDNRB ProteinRelative to SW protein levels (RU mg total pr-1) SW FW p=0.004 p=0.45E. Chronic levels 0.0 1.0 2.0 3.0 4.0 5.0 04812162024Relative intensity (Umgtotal pr-1)A. EDNRA FW to SW C. EDNRB FW to SW 0.0 0.5 1.0 1.5 2.0 2.5 04812162024 Time (h)Relative intensity (Umgtotal pr-1) C. EDNRB FW to SW 0.0 0.5 1.0 1.5 2.0 2.5 04812162024 Time (h)Relative intensity (Umgtotal pr-1) p=0.014* p < 0.001 p< 0.00 1* p=0.002 Figure 4-4. Killifish gill protein level differences of the endothe lin receptors during acclimation to SW or FW (n=5-6/treatment). EDNRA levels during acclimation to SW (A, open circles) or FW (B, open squa res). EDNRB levels during acclimation to SW (C, open circles) or FW (D, open squares). These m eans are relative to time zero. Statistical significance was determined using ANOVA and Dunnetts post hoc test (p values listed on figure). E) Chronic (30 days) acc limations to either SW or FW, and all values are relative to SW values and statistical significance was determined using unpaired, two tailed, T-tests. Mean s.e.m. 101

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ABC DEF G HI ABC DEF G HI Figure 4-5. Representative light micrographs of the immunolo calization of the endothelin A receptor (EDNRA) in the killifish gill. AC) seawater (SW) chronically (30 days) acclimated fish. D, E) Gills from fish acclimated to SW for 24 h. F, G) Gills from fish chronically acclimated to FW. H, I) Gills from fish acclimated to FW for 24 h. A, D, F, H) EDNRA immunoreactivity (brown color). B) A section of gill 7 microns deeper into the gill from A and C, inc ubated in peptide-absorbed anti-EDNRA (no staining observed) and double-la beled with antiNa+, K+-ATPase (blue). C, E, G, I) Gill sections double labeled with anti-E DNRA (brown) and anti-Na+, K+-ATPase (blue). The immunoreactivity is in the same cell giving a grey appearance to the cell. Scale bar = 50 m 102

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103 A B C D E F G H A B C D E F G H Figure 4-6. Representative light micrographs of the immunolo calization of the endothelin B receptors (EDNRB) in the killifish gill. A-D) Seawater (SW), chronically (30 days) acclimated fish. E) A gill filament from a fish acclimated to SW for 24 h. F) A gill filament from a fish acclimated to fresh water (FW) for 24 h. G, H) FW chronically acclimated fish. A, G) Immunoreactivity for EDNRB (brown). B) A section 7 microns away from A or C incubated in peptide-absorbed antibody as a negative control (no staining observed). C, E, F, H) Filaments immunoreactive for EDNRB (brown) and Na+, K+-ATPase (blue). D) Magnifica tion of lamellar pillar cells immunopositive for EDNRB. Arrows are pointing to prelamellar arterioles immunopositive for EDNRB. Scale bar = 50 m.

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CHAPTER 5 SHORT TERM LOW-SALINITY TOLERANC E BY THE LONGHORN SCULPIN, MYOXOCEPHALUS OCTODECEMSPINOSUS Introduction Sculpin (Scorpaeniformes: Cottidae) are a sp ecious (~400 species (Nelson, 2006)) group of teleost fishes that displays a great variety in ha bitat use including: rive rs, demersal freshwater lakes, inshore coastal marine ar eas (including brackish waters), and demersal-marine areas. The genus Myoxocephalus consists of 17 species, of which ni ne are demersal-marine fishes, five enter brackish water, and three spend a portion of their life in fresh water (Froese and Pauly, 2000). The longhorn sculpin, Myoxocephalus octodecemspinosus is distributed in coastal waters from Virginia to Newfoundland, Canada (Big elow and Schroeder, 2002). Although they are primarily distributed in marine waters, they ha ve been found entering estuaries during high tides, but never in fresh water (Bigelow and Schroeder, 2002), suggesting they have some level of low salinity tolerance. The only laboratory study to subject l onghorn sculpin to low salinity challenges was conducted by Claiborne and colleague s (1994). They tested the effects of 4, 8, 20% seawater (SW) acclimations, and acid loads on longhorn sculpin, and determined that in 4 and 8% SW they lose Clto a lethal level by 48 and 60 h, resp ectively. In 20% SW, there is an initial decrease in plasma Cl-, but by 72 h this values has returned to control values; however, they did not determine what effect low envi ronmental salinity had on gill ion transporter expression. In teleost fishes, ion balance is regulated by specialized epithelial cells in the gill called mitochondrion-rich cells (MRCs, also termed chloride cells). As the name implies, they contain a high density of mitochondria, as well as ion transporters and channe ls necessary for ion movement. These include the basolateral membrane proteins: Na+, K+-ATPase (NKA) and Na+K+-2Clcotransporter (NKCC1); and the apical membra ne chloride channel, the cystic fibrosis 104

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transmembrane conductance regulator (CFTR) (recently reviewed by Evans et al., 2005). Studies in euryhaline fi shes, like the killifish ( Fundulus heteroclitus ), or anadromous fishes like the rainbow trout ( Oncorhynchus mykiss), brown trout ( Salmo trutta ) or Atlantic salmon ( Salmo salar), have explored the effects of changes in environmental salinity on gill ion transporter density (e.g. Hiroi and McCormick, 2007; e.g. Ma ncera and McCormick, 2000; Marshall et al., 2002; Pelis et al., 2001; Scott et al ., 2004; Seidelin et al., 2000; Si nger et al., 2002). In general, when these fishes move from fresh/brackish waters to marine waters, there is a rapid increase in plasma [Na+], [Cl-] and total osmolality (e.g. Scott et al., 2004; Seidelin et al., 2000), and to help maintain ion homeostasis, euryhaline fishes up-regulate gill NKA, NKCC1, CFTR, and other transporters and channels. The opposite occurs when going from marine waters to brackish/ fresh; there is a loss of ions, and generally, thes e ion transporters are down regulated. In addition to turning off SW osmoregulatory proteins, FW ion transporters are upregulated during FW acclimation. These include Na+-H+ exchangers (NHEs), V+ -H+-ATPase, and Cl-/HCO3 exchangers, and these are also necessary for proper acid-base regulation ( e.g. Evans et al., 2005). Recently, NKA, Na+-H+ exchanger-2, and V+ -H+-ATPase were immunolocalized to MRCs in the longhorn sculpin gill (Catches et al., 2006); however the effects of salinity on the expression on these ion transporters in the l onghorn sculpin is yet to be elucidated. The purpose of this study was to explore the effects of low environmental salinity on SW osmoregulatory ion transporter expr ession and distribution in the l onghorn sculpin gill to further our understanding of why they are not found in FW We hypothesize that longhorn sculpin can only tolerate low salinity environments for shor t periods (days) because they are unable to properly regulate these ion transpor ters necessary for ion homeostasis. 105

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Methods The following experiments were conducted in August of 2006 and 2007, and all methods were approved by Institutional Anim al Care and Use Committee at the University of Florida and the Mount Desert Island Biological Laborat ory (MDIBL, Salisbury Cove, ME). Longhorn sculpin, Myoxocephalus octodecemspinosus (Mitchill), were collect ed by local fishermen in Frenchman Bay, ME. Longhorn scul pin were transported to MDIB L and maintained in 6 ft circular tanks with free-flowing seawater (SW) from Frenchman Bay, under a natural summer photoperiod, and fed squid every other day. All an imals were fasted 48 h (h) prior to and during experimentation. Low Salinity Acclimation In a preliminary experiment, we maintained two longhorn scul pin in 10% SW for 6 days before seeing visual signs of st ress (sluggish behavior, color cha nge, lack of righting) (data not shown). Thus, we terminated the full experime nt after 3 days (72 h), when there were no obvious signs of stress. Longhorn sculpin were randomly assigned to one of four treatments: 24 h sham (100% SW); 24 h 10% SW; 72 h sham (100% SW); or 72 h 10% SW. Each sculpin was placed in five gallons of the appropriate, aera ted solution in a 10-gallon bucket and air. The buckets were maintained in a trough with free fl owing SW (15C) for temperature control. For experiments that lasted longer than 24 h, 50% of the water was replaced in each bucket daily Due to the confined space, only 8 fish were run in each experiment (3 shams, 5 treatments); thus the experiment was run four times (10% SW a nd 20% SW for 24 or 72 h). The buckets were kept in a trough with ~3 inches of flowing SW from Frenchman Bay, and this kept the bucket water temperature at 16 1C. 20% SW was made by mixing 2 parts SW with 8 parts of dechlorinated FW (by bubbling air into a bucket of FW for 24 h). The salinity was confirmed by measuring the osmolality with a Wescor Va por Pressure osmometer 5520 (Logan, UT). A 106

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similar experiment was repeated using 20% SW and 100% SW (sham) as the treatments. After 24 or 72 h, fish were anesthet ized in either100% SW, 20% SW or 10% SW (depending on the assigned treatment) with 0.379 g l-1 benezocaine (initially dissolved in absolute ethanol, final concentration of ethanol was 0.1%) (Sigma, St Louis, MO). Blood (0.5-1.0 ml) was taken from the bulbus arteriosus, and then the fish was p ithed. Half of the filaments of second gill arch were cut off and snap frozen for RNA extr action and the other half was fixed in 4% paraformaldehyde in 10 mM phosphate buffered saline (PBS, pH = 7.3) for immunohistochemical analyses. The rest of the filaments from the 7 gill arches were cut off the arch into a dish of 10 mM PBS, mixed, divided into two tubes, and snap frozen for protein analyses. Plasma Chemistry Blood samples were immediately spun at 1000 rp m for 5 min at 4C, and the plasma was aliquoted and frozen (-20C) until analyzed. Total plasma osmolality was measured using a Wescor Vapor Pressure Osmometer (Logan, UT). Plasma sodium and potassium were measured using an IL943 Automatic Flame Photometer (Instrumentation Laborat ory, Lexington, MA) and chloride by the Labconco Digital Chloridometer (K ansas City, MO). All samples were measured in triplicate. Molecular Techniques RNA was extracted using TRI Reagent (Sigma) as previously descri bed in Hyndman and Evans (2007). RNA pellets were reconstituted in 10 l of diethyl pyrocar bonate (DEPC) treated water, and the concentration of RNA measur ed using a Nanodrop ND-1000 spectrophotometer (Fisher Scientific, Wilmington, DE). Total RNA (5 g) was reverse transcribed using a First 107

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Strand cDNA Synthesis kit with Superscript III (Invitrogen, Carlsbad, CA), following the manufacturers instructions. Degenerate primers were designed using CODEHOP (Rose et al., 2003) to amplify, NKA NKCC1 and CFTR (Table 5-1). The polymerase used was 0.625 Units of Ex Taq, hot start, DNA polymerase (Takara Bio, Madison, WI) and the reactions were run in a Px2 thermocycler (Thermo Fisher Scientific, Waltham MA). The PCR parameters were: 94C for 2 min, 40 cycles of 94C for 30 sec, 45-60C (gradient) for 30 sec, 72C 30 sec, and a final 72C for 5 minutes. These products were then singly nested with a primer listed in Table 5-1, and the PCR was run using the PCR product from the first PCR. With all transcripts, there was a bright single band, and these transcripts were ligated into pCR4-TOPO vectors, and transformed into TOP10 chemically competent cells using a TOPO TA Cloning Kit for sequencing (Invitrogen). Cells were grown on agar plates with Kanamycin (500x) antibiotic and positiv e colonies were grown in LB broth (20 g l-1) over night at 37C, while shaking. Plasmids were extracted from the cells using a miniprep kit (Roche Applied Science, In dianapolis, IN), and sequenced at the Marine DNA Sequencing center at MDIBL. These partial sequences have been deposited into GenBank (accession numbers): NKA #EU391598; CFTR #EU391599; and NKCC1 #EU391600. Quantitative Real-Time PCR To determine the effects of dilute environments on longhorn sculpin gill CFTR NKA and NKCC1 mRNA levels, quantitative real-time PCR (qRT-PCR) was performed. Non-degenerate primers were designed to amplify a product between 50-100 bp (Table 5-1). L8 was used as an internal control gene as previous ly described (Choe et al., 2006; Choe et al., 2005). Each sample was run in duplicate using 2 l of 1/10 diluted original oligo-dt cDNA, 7.4 pmol of primers and SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in a total volume of 25 l. 108

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The cycling parameters used were: an initial denaturing step of 95C for 10 minutes, 40 cycles of: 95C for 35 sec, 60C for 30 sec and 72C for 30 sec, followed by a melting curve analysis to ensure only one product was amplified. Random samples were also sequenced following qRTPCR confirming amplification of the target of interest. To determine the degree of possible genomic contamination, qRT-PCR was run using RNA samples that were not reverse transcribed, and we determined that there was no genomic contamination. All qRT-PCRs were run on a MyiQ quantitative thermocycler (Biorad). Each primer pairs efficiency was determin ed by performing a 10-fold dilution curve using plasmid cDNA. Efficiency (E) for each primer pair was calculated using the equation: E=-1+10(-1/slope) where slope was the slope of the dilution curve. Each cycle threshold (CT) value was subtracted from a randomly c hosen control sample resulting in a CT, and were analyzed using the Pfaffl e quation (Pfaffl, 2001): ratio= E CT target/ E CT L8. Each Pfaffl ratio was then standardized to th e average sham Pfaffl ratio. Immunohistochemistry A portion of the second gill arch from the l onghorn sculpin (see above) was fixed in 4% paraformaldehyde in 10 mM PBS, for 24 h, dehydrat ed in an increasing c oncentration of ethanol series, cleared in Citrisolv (Fis her Scientific, Pittsburgh, PA), and embedded in paraffin wax. The tissue blocks were cut at 7 microns, placed on Superfrost Plus slides (Fisher Scientific), and heated at 37C for 30 min. Slides were analy zed following the methods of Piermarini et al. (2002) and Hyndman et al.(2006). In short, five slides/animal for each treatment were rehydrated, blocked with 3% H2O2 in water for 30 minutes, and washed in 10 mM PBS. Next, there was a 20 min protein block with Biogenexs protein block (BPB; Biogenex, San Ramon, CA), followed by 10 mM PBS washes. Finally, slides were incubated in primary antibody (see below), overnight at 4C. The primary anti body was washed off with 10 mM PBS, and the 109

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immunoreactivity was visualized using Biogenexs Super Sensitive Link-Label IHC Detection System. The chromagens used in this study were 3, 3 -diaminobenzidine tetrahydrochloride (DAB; brown color; Biogenex), Vector SG (blue color, Vector Laborator ies, Burlingame, CA), and Vector VIP (purple color, Vector Laboratories). Following this, some sections were doublelabeled with a second primary antibody, following the same procedures. Western and Dot Blotting Western blots were made following the methods of Hyndman and Evans (2007). Gills were homogenized in 2 ml of ice-cold homogenization buffer (250 mM sucrose, 30 mM Tris, 1 mM EDTA, 0.5% of Sigmas prot ease inhibitor cocktail, and 100 g ml1 phenylmethylsulfonyl fluoride; pH 7.8). The homogenates were centr ifuged at 14 000 rpm for 10 min at 4C and the supernatant decanted. Protein content of the supernatant was measured using Pierces BCA protein assay kit (Rockford, IL). A portion of the supernatant was diluted with an equal portion of Laemmli sample buffer with 0.01 % bromophenol blue and 2% -mercaptoethanol (Laemmli, 1970) and heated at 65C for 10 min. Twenty-fiv e micrograms of protein was separated using SDS-PAGE (10% Tris-HCl gels, Biorad, Hercules, CA) for 2 h at 100 V and then transferred to an Immuno-blot PVDF membrane according to the manufacturer's protocol (Biorad). Next the membrane was placed in blotto, 5% non-fat dry milk in 10 mM Tris buffered saline (TBS: 25 mmol l1 Tris, 150 mmol l1 NaCl; pH 7.4) for 1 h at room temperature (~25C), shaking, and then placed in primary antibody and incubated at room temperature, overnight. Next the membrane was washed in thee changes of 10 mM TBS with 1% Tween 20 (TBST) and incubated in 1/3000 goat anti-mouse alkaline phos photase secondary (Biorad) diluted in blotto for 1 h at room temperature while shaking. Agai n, the membrane was washed in thee changes of 10 mM TBST. The membrane was developed using a chemiluminescent signal (Bio-Rad, 110

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Hercules, CA) following the manufacturers in structions, and developed on ECL Hyperfilm (Amersham, Piscataway, NJ). All films were digitized using a flat bed scanner. Dot blots were used to accurately quantify ion transporter protein level differences among our treatments, and the methods of Joyner-Matos et al. (2006)were used. In short, gills were homogenized and centrifuged as desc ribed above. The supernatant was heated at 65C for 15 min, and then diluted to 2.5 g l-1 in 10 mM TBS, and continued heating at 65C until blotted. Another randomly picked control sample was diluted (in 10 mM TBS) out in a series of 2-fold dilutions to make an 8-point dilution curve. Proteins were blotted in 1 l dots (thus 2.5 g of protein), in triplicate onto dry ni trocellulose membrane (Millipore, Billerica, MA) and left to air dry for 10-20 min. Next, the membrane was plac ed in blotto and followed the aforementioned western incubation protocol. The developed filmed was digitized using a flat bed scanner, and dot density determined using Biorads Quantity One software. All values were standardized to the dilution curve, and made relative to protein content (relativ e units mg protein-1). Antibodies Monoclonal, anti-chicken NKA ( 5, 1/1000) was developed by Dr. D. Fambrough, and monoclonal, anti-human NKCC1 (T4, 1/500) was de veloped by Drs. Lytle and B. Forbush III, and were obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the National Institute of Child Health and Human Development of the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. Monoclonal anti-human CFTR (1/500 ) (R&D systems, Minneapolis, MN) was made against the c-terminal of human CFTR and is ~61% identical to te leost CFTR (Katoh and Kaneko, 2003; Singer et al., 1998). All antibodies were diluted in BPB. 111

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Gill Na+, K+-ATPase Activity Na+,K+ -ATPase (NKA) activity was measured using a NADH-linked, spectrophotometric microassay, similar to the one developed by McCormick (1993). Briefly, gills were homogenized on ice with a polytron in 125 l of 5X SEID buffer ( 250 mM sucrose, 10 mM Na2EDTA, 50 mM imidazole, 0.05% de oxycholic acid, pH=7.3) and 1 ml 1X SEID (diluted in SEI: 250 mM sucrose, 10 mM Na2EDTA, 50 mM imidazole). Samp les were centrifuged at 3000 g for 30 sec at 4 C to remove a ny large particulates. The protei n content of the supernatant was determined using Pierces BCA Protei n Assay. In a 96-well microplate, 10 l of the supernatants were added to 200 l of reaction mixture (80 mM NaCl, 20 mM KCl, 5 mM MgCl2, 50 mM imidazole, 3 mM ATP, 2 mM phosphoenol pyruvate, 0.2 mM N ADH, 3.1 U/ml lactic dehydrogenase, 3.84 U/ml pyruvate kinase) either w ith or without 1 mM ouabain. All samples were run in triplicate. The pl ate was read every ten seconds, fo r a total of 20 minutes, at 25 C on a Biorad Benchmark Plus microplate reader (340 nm). An ADP standard curve was also run for every lot of reaction mixture to determine the extinction coefficient for the ADP-dependent conversion of NADH to NAD+ (used in our final calculation of NKA activity). The difference in the slopes (the rate of [NADH] reduction) between the non-ouabain and ouabain wells was calculated for each sample. These values were standardized to the ADP standard curve and normalized to to tal protein content per sample ( mol ADP mg protein-1h-1) (McCormick, 1993). Statistics Plasma chemistry and gill NKA activity data are displayed as mean s.e.m. All other data were made relative to the mean sham value fo r each time, and are displayed as relative means s.e.m. With the plasma, qRT-PCR and dot blot data, statistical differences among the treatments 112

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were assessed by 2-Factor ANOV As (for salinity and time), fo llowed by unpaired, two-tailed Ttests to determine differences compared to sh am treatments (100% SW). The 10% and 20% SW experiments were not run concurrently (see abov e); however the shams within each time point (24 or 72 h) did not differ significantly, thus they were combined in the 100% sham mean that is reported (n = 6). Statistical si gnificance was set at alpha = 0.05. All statistics were run using SPSS (v.15, Chicago, IL). Results Plasma Chemistry and Gill Na+, K+-ATPase Activity Total plasma osmolality, sodium, potassium, and chloride concentrations did not differ between the 24 h and 72 h 100% SW (sham) treatment s (Table 5-2). These parameters were not different between the sham and 20% SW at 24 or 72 h; however plasma osmolality and sodium were 14% lower after 24 h acclimation to 10% SW (p <0.001), and 22% lower after 72 h compared to sham (p <0.001). Plasma potassi um decreased 24% with 24 h acclimation to 10% SW (p = 0.009) and this decrease was maintained at 72 h. Chloride also decreased 20% and 27% with 24 and 72 h acclimation to 10 % SW, respectively (p <0.001). Longhorn sculpin acclimated to 10% or 20% SW for 24 or 72 h did not ha ve a significant change in gill NKA activity compared to sham values (Table 5-2). Immunolocalization of CFTR, NKA, and NKCC1 Longhorn sculpin gills from all of the treatm ents were immunopositive for CFTR, NKA, and NKCC1 (Fig. 5-1). Epithel ial cells in the interlamella r region were immunopositive for all three transporters. CFTR was f ound on the apical membrane, as indicated by a small, brown dot near to the edge of the epithelial cells (Fig. 5-1A, D, G). NKA and NKC C1 were basolaterally immunolocalized (Fig. 5-1B, C, E, F, H). Longhorn sculpin that were acclimated to 20% (data not shown) or 10% SW for 24 h (Fig. 5-1D-F) ha d similar immunostaining patterns as the shams 113

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(Fig. 5-1A-C). These immunoreactive patterns were also found with longhorn sculpin acclimated to 20% (data not shown) or 10% SW for 72 h. Gills from longhorn sculpin that were acclimated to 10% SW for 72 h, were single-labele d for CFTR (Fig. 5-1G) and then the adjacent section (7 microns further) was double-labeled wi th CFTR and NKA (Fig. 5-1H). As shown in Figures 5-1G and 5-1H, CFTR a nd NKA were immunolocalized to th e same epithelial cells, with CFTR staining the apical membrane, and NKA fo und on the basolateral membrane. Likewise, this double labeling was repeated using CF TR and NKCC1, and again CFTR and NKCC colocalized to the same epithelial cell (data not shown). Westerns and Dot Blots The CFTR, NKA and NKCC1 antibodies used in our Western blot experiments yielded bands of the expected molecular weights (Fig. 5-1I). As seen in Figure 5-1I, a single CFTR band of ~140 kDa, and a single NKA band of ~120 kDa were found in the sham sculpin gill. With the anti-NKCC1 antibody, we found two bands of ~200 and ~130 kDa in the longhorn sculpin gill. Because we found only single (CFTR, NKA) or doubl e bands (NKCC1) with our western blots, and these findings mirror those from other teleos ts (Hiroi and McCormic k, 2007; Tipsmark et al., 2002), we quantified protein differences with dot blots (total protein, not proteins separate by molecular weight). The sham treatments for 24 and 72 h were not statistically different from each other for CFTR, NKA, or NKCC protein levels. We did not find any significant cha nges in gill CFTR or NKA protein level with acclimation to 10% or 20% SW for 24 or 72 h compared to their respective sham treatments (Fig. 5-2A, C). Ther e was a significant 2.5-f old increase in NKCC1 protein level with 24 h acclimation to 20% SW (p <0.001) (Fig. 5-2E); however there were no statistically significant changes in NKCC1 protein level within the 72 h treatment. 114

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Quantitative Real-Time PCR Sham treatments for 24 and 72 h were not significantly different for CFTR, NKA, or NKCC1 Gill NKCC1 and CFTR mRNA levels did not significantly change with acclimation to 10% or 20% SW for 24 or 72 h (Fig 5-2B, F). Longhorn sculpin gill NKA mRNA levels were not different from sham with 24 h acclimati on to 20% SW (Fig. 5-2D); however they did increase 3.6-fold after 24 h acclimation to 10% SW (p = 0.001). After 72 h, gill NKA mRNA levels were 2.3-fold higher in the 10% SW treatme nt compared to sham (p = 0.004) (Fig. 5-2D). Discussion This study is the first to examine the effects of low-salinity seawater (SW) osmoregulatory ion transporters, from a marine teleost, in order to determine why they are incapable of inhabiting freshwater environments (FW). In the wild, longhorn sculpin have been found in estuaries during high tides suggesting they have some low-salinity tolerance (Bigelow and Schroeder, 2002). We determined that acclimat ion to 20% SW for 24 or 72 h did not elicit any significant changes in plasma osmolality or ion concentration (Table 5-2), but acclimation to 10% SW resulted in a significant loss of ions This suggests that down to 20% SW, longhorn sculpin can regulate plasma ion concentrations. Claiborne et al. (1994) determined that longhorn sculpin could not survive past 60 h in 8% SW or 48 h in 4% SW, thus we conclude that longhorn sculpin can tolerate salinities down to 8-10% SW for days, but salinities below this level are lethal within a few days. We propose that longhorn sculpin are missing the mechanism that allows euryhaline (or anadromous/ catadromous) fishes to survive in fresh and marine environments--proper regulation of gill ion transp orter densities. Longhorn sculpin do express CFTR, NKA, and NKCC1 in epithelial cells th at match the morphology of the mitochondrionrich cells (MRCs) (e.g. Hiroi and McCormic k, 2007; e.g. Katoh and Kaneko, 2003). Recently, Catches et al. (2006) immunolocalized NKA in the basolateral membrane of the MRC of the SW 115

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longhorn sculpin gill. We determined that CFTR and NKCC1 are also expressed on the MRC as was determined for other teleosts (Hiroi and McCormick, 2007; Katoh and Kaneko, 2003; e.g. Pelis et al., 2001). Presented here, acclimation to 20% did not affect gi ll CFTR or NKA protein levels or immunolocalization of these proteins in the longhorn sculpin gill (Figs 5-1 and 5-2). There was a significant increase in NKCC1 protei n level after 24 h acclim ation to 20% SW, but the NKCC1protein level was not significantly different from sham at 72 h. The increase in NKCC1 protein level while the longhorn sc ulpin were acclimating to a hypoosmotic environment is puzzling, because it is well doc umented that NKCC1 is stimulated by cell shrinking (as occurs during acclimation to marine environments), and is involved in volume regulation in teleosts (see a recent review, Hoff mann et al., 2007). Interestingly, we did not find a significant increase in NKCC1 mRNA at 24 h compared to sham Unfortunately, the time lag between de novo mRNA production and de novo protein production is not known for these transporters, so it is plausible that there was an increase in NKCC1 mRNA hours before our 24 h sampling resulting in more NKCC1 protein at 24 h. It may also be that there are posttranscriptional modifications occu rring, resulting in a change of protein without a change in mRNA for NKCC1. In any event, these longhorn sculpin were able to maintain a plasma osmolality of ~330 mmol kg-1 (this is within the normal range for euryhaline and stenohaline marine species, see Evans et al.(2005)) during these experiments, wit hout any obvious changes in CFTR and NKA ion transporter density, NKA activit y, or localization of all three proteins in the gill. Unlike acclimation to 20% SW, longhorn sculpin acclimated to 10% SW suffered a significant loss of ions. Euryha line, catadromous, or anadromous teleosts that experience changes in environmental salinity regulate gill io n transporters to maintain proper ion balance. 116

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For example, the euryhaline killifish ( Fundulus heteroclitus), down regulates CFTR and NKCC1 mRNA and protein levels within a day or two of entering fresh water (Choe et al., 2006; Katoh and Kaneko, 2003) resulting in a conservation of i ons. Killifish that were transferred from low (0.1 ppt) to high salinity (35 ppt) increased NKA activity 3 h a nd 72 h post transfer (Mancera and McCormick, 2000) to help excrete excess ions Unlike the killifish, the longhorn sculpin did not down regulate CFTR, NKCC1, or NKA during acclimation to 10% SW, and subsequently suffered a significant loss of ions at 24 h and 72 h (Table 5-2). NKA mRNA levels were higher than shams at 24 and 72 h in the 10% SW treat ments, but there was no obvious change in NKA protein level or immunolocalizat ion. Collectively, this could ag ain suggest differences in time lag between de novo production of mRNA and protein. We did not find an increase in NKA activity level either (T able 5-2), so this observed increas e in NKA mRNA is intriguing. An alternative hypothesis is that there is high NKA protein turnover so to maintain constant NKA protein levels would require an increase in NKA mRNA. This hypothesis has been proposed to explain high increases in carbamoyl phosphate synthetase III ( CPSase III ) mRNA in the Gulf toadfish ( Opsanus beta ) to maintain a constant CPSase III activity level during ureagenesis in this fish (Kong et al., 2000). This may also be occurring with the NKA. Unlike euryhaline fishes like the killifis h, the longhorn sculpin does not down-regul ate gill SW osmoregulatory ion transporter densities in dilute environments. Recently a study determined that landlocked, freshwater populations of Atlantic salmon ( Salmo salar ) were not capable of up-regulating NKA, NKCC1 and CFTR during smoltification, as was obs erved in anadromous salmon (Nilsen et al., 2007). Likewise, landlo cked Arctic char ( Salvelinus alpinus ) were incapable of maintaining ion homeostasis during hyperosmotic stress, and were incapable of up regulating NKA (specifically NKA 1b subunit). This is likely because thes e landlocked fishes have completed many 117

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generations in only fresh water, a nd have adapted to a fresh water ex istence. To the best of our knowledge, we are the first to determine physiologically why a marine fish is incapable of surviving in low-salinity environments. In the future, experiments should determine the effect of low-salinity on FW osmoregulatory proteins, such as the Cl-/HCO3 V+-ATPase or NHEs. Catches et al. (2006) determined that NHE2 is expressed in apical membrane of MRCs and V+-ATPase in the basolateral membrane of the SW longhorn sculpin g ill. This suggests that machinery involved in ion transport uptake may be present in the sculpi n; however, if it is expressed properly in the MRC to drive Na+ absorption and/or upregul ated during low-salinity exposure remains to be determined. The exclusion of longhorn sculpin from fresh water is intriguing. There are records of them entering estuaries during high tide (Bigelow and Schroeder, 2002), and the salinity of this environment during this time is likely higher becaus e of the tide. This is probably less of an osmotic challenge than entering during low tide or entering near the fresh water source, and we have determined that down to 20% SW there is no obvious detriment to the fish. Because longhorn sculpin are incapable of properly regulati ng ion transporter densiti es in the gill below 20% SW, they suffer a net loss of ions eventually to a level that is lethal for the fish. There are freshwater Myoxocephalus: M. polyacanthocephalus, M. sinensis and M. thompsonii (Froese and Pauly, 2000). M. polyacanthocephalus is amphidromous, spending a portion of its lifecycle in fresh water and is distributed in the North Pacific; M. sinensis is a demersal-freshwater species found in China; and M. thompsonii is also a demersal, freshwater species distributed from the St. Lawrence River to the Arctic (Froese and Pauly, 2000). Within the sculpin, there are three other genera that have freshwater species: Cottus, Trachidermus, and Messocottus. The genus Cottus 118

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119 is a group of 57 species of freshwater sculpin (Froese and Pauly, 2000), that diverged from the marine sculpin approximately 2-5 mya (Yokoyama and Goto, 2005). It seems plausible that the marine ancestor(s) to the freshwater sculpin ha d the ability to regulat e gill ion transporters density and or activity, and this lead to their inva sion of freshwater habita ts. To help elucidate this question, a complete sculpin phylogeny, mapp ing habitat use to the different species, would be helpful in understanding the evolution of this group of fishes. A portion of the Myoxocephalus phylogeny has been completed using 7/17 species, and it depicts two distinct groups of the Myoxocephalus: Arctic-Atlantic and Pacific gr oups (Kontula and Vainola, 2003). Both groups contain freshwater representatives, suggesting that there were independent invasions of freshwater by Myoxocephalus. In conclusion, the Myoxocephalus is an interesting group to te st mechanistic questions to help understand habitat invasion and use. The lo nghorn sculpin can tolerate short term exposure to low salinity water (<10% SW) for days but not much longer, because they can not regulate ion transporter density or activity, resulting in a signifi cant loss of ions (eventually to a lethal level). The diverse habitat us e of the species of Myoxocephalus, make it an excellent model to complete comparative studies to explore the relationshi p between environmental salinity and gill ion transporter density, furthering our fundamental knowledge of the mechanisms and evolution of salinity tolerance.

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Table 5-1. Primers used in sequencing and qua ntitative real-time P CR of longhorn sculpin Na+, K+-ATPase (NKA), Na+, K+, 2Clcotransporter (NKCC1), and the cystic fibrosis transmembrane conductance regulator (CFT R). The asterisks denote degenerate primers and q represents quantit ative real-time PCR primers. Primer 5' to 3' Orientation CFTRF1* AAA TGT AAC TGC CTC CTG GGA YGA RGG CFTRF2* TCC CCT CAG ACC TCT TGG ATH ATG CC CFTRR1* CCT CGG CTT CCA GCT GTT TNA RYT GYT G NKAF1* GGA TGA ACT AAA GAA GGA AGT AGA TAT GGA YGA YCA YAA NKAR1* CCA GAC AAT TCT TTT TCG CCA TNC KYT T NKAR2* CAC GCC GGT GAT TAT GTG TAT RAA RTG NTC NKCC1F1* CCC CCT CTC AGT CTC GGT TYC ARG TNG A NKCC1F2* CAT CAT TAT GAT ACG CAC ACG AAY CAN TAY TA NKCC1R1* GGA TGT ACC CTC GT A GAG GCT CRT TRT TYT T qCFTRF1 TTC GAC CTC ATT CAG CTC ACA qCFTRR1 TGG CGG CGA TGA AGA TGT A qNKAF1 ACG AAC CGG CCA ACG ATA A qNKAR1 TTG GTA GT A GGA GAA GCA GCC A qNKCC1F1 GGA TTT GTA CGA GGA GGT GGA G qNKCCR1 GCA AAG GCA AAG ATC AGA CCA A 120

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Table 5-2. Plasma parameters and gill Na+, K+-ATPase for longhorn sculpin acclimated to 100, 20 or 10% SW for 24 or 72 hours. Means (s.e.m.) are recorded. Asterisk s represent significant differences from 100% SW within 24 or 72-hour treatments. 24 hour 72 hour 100% SW 20% SW 10% SW 100% SW 20% SW 10% SW Plasma osmolality mOsm kg-1 340.0 (3.2) 331.6 (7.1) 295.2 (8.5)* 329.5 (7.4) 317.3 (10.1) 259.1 (4.7)* Plasma [Na+] mOsm L-1 178.2 (6.38) 162.7 (5.4)* 152.8 (4.2)* 174.5 (3.5) 171.7 (4.1) 134.1 (2.7)* Plasma [K+] mOSm L-1 4.2 (0.2) 3.6 (0.4) 3.1 (0.4)* 4.1 (0.4) 3.4 (0.1) 3.1 (0.2)* Plasma [Cl-] mEquiv L-1 166.1 (2.9) 158.0 (4.8) 133.7 (3.8)* 160 (5.8) 159.3 (5.0) 118.0 (4.2)* Gill Na+, K+,ATPase activity mmol ADP mg pr-1 hr-1 8.0 (0.66) 8.2 (0.7) 8.3 (0.5) 9.3 (1.51) 8.12 (0.7) 8.5 (1.0) 121

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200 97 53 37 29 21 kDa 1 Std. 3 NKA 4 NKCC 2 CFTR A D G B E H C F I 200 97 53 37 29 21 kDa 1 Std. 3 NKA 4 NKCC 2 CFTR 200 97 53 37 29 21 kDa 1 Std. 3 NKA 4 NKCC 2 CFTR A D G B E H C F I Figure 5-1. Representative light micrographs of the immunolocali zation of the cystic fibrosis transmembrane conductance regulator (CFTR, 1A, D, G), Na+, K+-ATPase (NKA, 1B and E), and Na+, K+, 2Clcotransporter (NKCC1, 1C and F). The gills are from longhorn sculpin acclimated to: 100% SW for 72 h (A-C); 10% SW for 24 h (D-F); and 10% SW for 72 h (G and H). CFTR was immunolocalized to the apical membrane, and NKA and NKCC1 to the basolateral membrane of epithelial cells of the interlamellar region. H) Is the next seri al section of gill (7 microns deeper) from G) and is double labeled with anti-CFTR, indicated by the arrows (brown, apical membrane), and anti-NKA (blue, basolatera l membrane), and shows that CFTR and NKA are expressed in the same epithelial ce lls. I) Western blot s of longhorn sculpin gills acclimated to 100% SW. The first la ne is the molecular mass ladder (std), second lane is a blot incubated with anti-CFT R, third lane is a bl ot incubated in antiNKA and fourth lane is a blot incubated with antiNKCC. Scale bar = 50 m. 122

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123 A CFTR protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 24 72Relative intensity (RU mg pr-1) 100% SW 20% SW 10% SW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72Relative intensity (RU mg pr-1)C. NKA protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 24 72Relative mRNA LevelB. CFTR mRNARelative mRNA Level 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72F. NKCC mRNATime post transfer (h)E. NKCC protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 24 72 Time post transfer (h)Relative intensity (RU mg pr-1)* p=0.01 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 24 72Relative mRNA LevelD. NKA mRNA* P=0.011 p=0.02 p=0.011 A CFTR protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 24 72Relative intensity (RU mg pr-1) A CFTR protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 24 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 24 72Relative intensity (RU mg pr-1) 100% SW 20% SW 10% SW 100% SW 20% SW 10% SW 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72Relative intensity (RU mg pr-1)C. NKA protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72Relative intensity (RU mg pr-1)C. NKA protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 24 72Relative mRNA Level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 24 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 24 72Relative mRNA LevelB. CFTR mRNARelative mRNA Level 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72F. NKCC mRNATime post transfer (h)Relative mRNA Level 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72F. NKCC mRNARelative mRNA Level 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72Relative mRNA Level 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72F. NKCC mRNATime post transfer (h)E. NKCC protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 24 72 Time post transfer (h)Relative intensity (RU mg pr-1)* p=0.01E. NKCC protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 24 72 Time post transfer (h)Relative intensity (RU mg pr-1)E. NKCC protein 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 24 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 24 72 Time post transfer (h)Relative intensity (RU mg pr-1)* p=0.01 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 24 72Relative mRNA LevelD. NKA mRNA* P=0.011 p=0.02 p=0.011 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 24 72Relative mRNA LevelD. NKA mRNA 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 24 72Relative mRNA Level 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 24 72 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 24 72Relative mRNA LevelD. NKA mRNA* P=0.011 p=0.02 p=0.011 Figure 5-2. Longhorn sculpin gill CFTR, NK A and NKCC1 expression levels following acclimation to 100, 20 or 10% seawater (SW) for 24 and 72 hours (h). A, C, E) are protein levels determined from immunoblots (dot blots). B, D, F) mRNA levels determined by quantitative Real-Time PCR. mRNA values are normalized to L8 mRNA levels. All values are relative to the mean sham (100% SW) level at 24 or 72 h, and are mean s.e.m. Asterisks repr esent statistically significant differences compared to the sham value at 24 or 72 h (p values are listed on the graph).

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CHAPTER 6 EFFECTS OF LOW ENVIRONM ENTAL SALINITY ON ENDO THELIN RECEPTOR AND ENDOTHELIN CONVERTING ENZYME-1 MRNA IN THE GILL OF THE LONGHORN SCULPIN, MYOXOCEPHALUS OCTODECEMSPINOSUS Introduction The small secreted protein, endothelin-1 (EDN1), is found in all gnathostomes (Hyndman and Evans, 2007), and it acts to regulate vascular tone (La and Reid, 1995; Yanagisawa et al., 1988a; Yanagisawa et al., 1988b), modulate ion transport (Ahn et al., 2004; Evans et al., 2004; Garvin and Sanders, 1991; Ge et al., 2006; Prasanna et al., 2001; Zeidel et al., 1989), and direct the migration of neural crest cell during cranio facial development (Clout hier and Schilling, 2004; Kurihara et al., 1994). Endothelin -1s actions are mediated via two G-protein-coupled receptors (GPCR) in mammals, EDNRA and EDNRB1. In non-therian tetrapods, a third GPRC, EDNRB2, is involved in EDN1 signaling, and the teleosts are unique because they have an additional EDNRA (termed EDNRA2) and EDNRB1 (termed EDNRB1b) (C hapter 3), likely a result of the teleost specific genome duplicati on (Volff, 2005). Endothelin-1 is produced through two cleavage events involving the enzyme furi n (Yanagisawa et al., 1988c) and the endothelin converting enzyme-1 (ECE1) (Shimada et al., 1994; Xu et al., 1994). The active form of EDN1 is secreted from the cell and acts on neighboring cells as a paracrine or autocrine signaling molecule (Webb, 1997)(Chapters 1 and 4). Evans et al. (2004) were the fi rst to suggest that EDN1 may be a local regulator of ion transport in the fish gill. They determined that in vitro EDN1 inhibits net chloride in the euryhaline killifish (Fundulus heteroclitus ) opercular epithelium (Eva ns et al., 2004), a model tissue for the seawater (SW) teleost gill (Karna ky et al., 1977). Recently, we determined the effects of acclimation to SW or fresh water (FW) in vivo on gill EDN1 ECE1 (Hyndman and Evans, 2007), and endothelin receptor ( EDNR ) (Chapter 4) mRNA levels in the killifish. We 124

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determined that all these components of EDN1 signaling are affected by changes in environmental salinity; however, the physiological ro le of EDN1 in the fish gill is still unclear. One would hypothesize that if EDN1 inhibits ion transport, then during acclimation to SW where there is an increase in plasma osmolality (Marsha ll et al., 1999), that EDN1 levels would be low, allowing the fish to excrete excess ions and return to ion homeostas is. Conversely, during acclimation to FW, when ions tend to be lost to the environment (Scott et al., 2006), EDN1 levels would be high, resulting in an inhibition of ion transport out of the gill, thus retaining plasma ions. Our experiments in the killifis h suggest that EDN1 levels are highest during acclimation to hyperosmotic environments, opposite to our predictions. Thus, it is still unclear what the physiological role of EDN1 is in the gi ll. A recent hypothesis by our lab, is that EDN1 stimulates cyclooxygenase-2 production of prostagla ndins and that these pa racrines aid in cell survival during osmotic stress (Choe et al., 2006). Similar results were found in mammalian medullary interstitial cells that experience rapid changes in extracellular osmolality (Hao et al., 1999; Hao et al., 2000). In an attempt to understand further EDN1s put ative role in the gill we determined the effects of low environmental salinity on the E DN1 system in the moderately euryhaline longhorn sculpin, Myoxocephalus octodecemspinosus. The longhorn sculpin, is a marine fish that is distributed from Virginia to the Arctic, a nd has been found in estu aries during high tides (Bigelow and Schroeder, 2002). We recently repo rted that the longhorn sc ulpin are capable of tolerating low environmental salinity (10-20% SW) for days (Chapter 5). Sculpin in 20% SW can maintain plasma ion homeostasis, while sculpin in 10% SW significantly lose ions over 72 h (Chapter 5). The purposes of this study we re to: 1. sequence scul pin gill cDNA for the EDNR s and ECE1; 2. determine the tissue expression of EDNR mRNA; and 3. determine the effects of 125

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low environmental salinity on EDNR and ECE1 mRNA levels in the gill. By examining the EDN system in a moderately euryhaline fish, we aim to better understand the role EDN may be playing in ion balance in fishes. Methods Longhorn sculpin ( Myoxocephalus octodecemspinosus Mitchill) were purchased from local anglers from Frenchman Ba y, ME. The fish were maintain ed at the Mount Desert Island Biological Laboratory in 3 ft circular tanks supplied with free flowing SW (31 ppt) from Frenchman bay (15C). The fish were fed squid to satiation every other day. Molecular Sequencing a nd Tissue Distribution We previously sequenced killifish (Fundulus heteroclitus Linnaeus) gill cDNA for EDNRA, EDNRB1, and EDNRB2 (Chapter 3) and ECE1 (Hyndman and Evans, 2007). Using the same degenerate primers used to initially se quence these killifish cDNAs (Table 3-2), we sequenced and cloned a portion of the coding sequence for each EDNR and ECE1 from the sculpin gill. These sequences were translated and compared to t hose in NCBIs protein database using BLAST, and using sequence homology, we confirmed we had sequenced the longhorn sculpin orthologues of EDNRA (Accession EU440324), EDNRB1 (Accession EU440325), EDNRB2 (Accession EU44026), and ECE1. In a ddition, to determine which co-orthologous copy of the tetrapod EDNRA and EDNRB1 we had sequenced, and to determine the evolutionary relationship among these sculpin E DNRs, we performed a fast maximum likelihood analysis (Felsenstein, 2004). A portion of the sequences from in our complete phylogenetic analysis of the gnathostome EDNRs (Chapter 3) were used to constructed a multi-sequence alignment using ClustalX (Larkin et al., 2007). Only a portion of the sequences were used because we wanted to run this additional anal ysis in a timely manner, while including all sequences would have extended our analysis an additional 20-30 days. This alignment was 126

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inspected to ensure no gaps were inserted into known structur al regions (i.e. transmembrane domains). Using the WAG model of amino acid substitutions (Whelan and Goldman, 2001), a free gamma distribution parameter, and a fixe d proportion of invarian t sites (Pinv=0), the maximum likelihood (ML) tree was determined. The robustness of the ML tree was evaluate by non-parametric bootstrapping = 1000. Using multi-tissue, duplexing PCR, we determined the relative tissue distribution of the sculpin EDNRs. Following the methods of Choe et al. (2006), the RNA from the sculpin gill, operculum, brain, heart, stomach, intestine, kidney and white muscle was extracted using TRIReagent (Sigma, St. Louis, MS) following ma nufacturers instructions. Total RNA (0.5 g) was reverse transcribed into cDNA using ra ndom hexamer primers and Superscript III (Invitrogen, Carlsbad, CA). These cDNAs were diluted with diethyl pyrocarbonate water. Non-degenerate primer pairs (Table 6-1) were designed to amplify a product with high efficiency (e.g., high melting temperature) and to mini mize the chance of amplifying contaminating genomic DNA; the primer pair was designed to include at l east one exon-exon boundary when possible (Table 6-1). A QuantumRNA 18S internal standard primer kit (Ambion, Woodward Austin, TX) was used to contro l for variability in cDNA quality and the quantity between the different tissues tested. D uplexing PCR with primers for 18S and either EDNRA EDNRB1 or EDNRB2 was optimized to ensure that the reacti ons were terminated during the exponential phase. Lastly, the products were visualized by et hidium bromide staining in 1.5% agarose gels and digitized using the Biorad Gel Do c XR System (Hercules, CA). Salinity Challenge The sculpin salinity challenge was previously described in Chapter 5, and was performed at MDIBL (summer 2006 and 2007). Scul pin (5 or 6) were subjected to one of three treatments: 127

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1) 100% SW (sham) 2) 20% SW; or 3) 10% SW. Each sculpin was placed in five gallons of the appropriate, aerated solution in a 10-gallon bucket and air. The buckets were maintained in a trough with free flowing SW (15C) for temperature control. For experiments that lasted longer than 24 h, 50% of the water was replaced in e ach bucket daily. At 24 or 72 h post transfer, killifish were anesthetized in benezocaine (0.375 g l-1 dissolved initially in ethanol, then dissolved in treatment water, final ethanol concentration 0.1%) and the blood and gills removed (see Chapter 5). These time end points were based upon the time course determined by Claiborne and colleagues (1994). Th ey determined significant decreases in plasma chloride at 24 h and a return to control values at 72 h, thus we used these end poi nts for our experiment. Quantitative Real-Time PCR To determine the effects of dilute environments on longhorn sculpin gill EDNRA EDNRB1, EDNRB2, and ECE1 mRNA levels, quantitative real-time PCR (qRT-PCR) was performed. Non-degenerate primers were de signed to amplify a product between 50-100 bp (Table 6-1). L8 was used as an internal contro l gene as previously described (Choe et al., 2006; Choe et al., 2005). Each sample was run in duplicate using 2 l of 1/10 diluted original oligo-dt cDNA, 7.4 pmol of primers and SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in a total volume of 25 l. The cycling parameters used were: an initial denaturing step of 95C for 10 min, 40 cycles of: 95C for 35 sec, 60C for 30 sec and 72C for 30 sec, followed by a melting curve analysis to ensure only one pr oduct was amplified. Random samples were also sequenced following qRT-PCR, confirming amplificati on of the target of interest. To determine the degree of possible genomic contamination, qR T-PCR was run using RNA samples that were not reverse transcribed; no genomic contaminat ion was found. All qRT-PCRs were run on a MyiQ quantitative thermocycler (Biorad). 128

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Each primer pairs efficiency was determin ed by performing a 10-fold dilution curve using plasmid cDNA. Efficiency (E) for each primer pair was calculated using the equation: E=1+10(-1/slope) where slope was the slope of the dilution curve. Each cycle threshold (CT) value was subtracted from a randomly chosen sham sample resulting in a CT, and were analyzed using the Pfaffl equation (Pfaffl, 2001): ratio= E CT target/ E CT L8. Each Pfaffl ratio was then standardized to the average sham Pfaffl ratio. Statistics Quantitative real-time PCR data was analyzed using 2-Factor ANOVAs (for salinity and time), followed by unpaired, two-ta iled T-tests to determine differences compared to sham treatments (100% SW). Statistical significance was set at alpha = 0.05. All statistics were run using SPSS (v.15, Chicago, IL). Results Phylogenetic Relationships Our predicted sculpin EDNRA, EDNRB1 and EDNRB2, were aligned with other gnathostome EDNRs sequences, and the phylogene tic relationships among these receptors determined. The maximum likelihood score ca lculated was -13083 and a gamma parameter of 0.612. The longhorn sculpin EDNRA grouped with the teleost EDNRAs and the tetrapod EDNRAs, but outside of the teleost specific dup licate of EDNRA2s (Fig. 6-1). Likewise, the EDNRB1 grouped with the teleost EDNRB1s and th e tetrapod EDNRB1s, but outside the teleost specific duplicate of the EDNRB1s. Finally, the longhorn sculpin EDNRB2 grouped with the teleost EDNRB2 and the non-therian gnathostome EDNRB2 (Fig. 6-1); the EDNRB2 has been lost by the therians (Chapter 3). 129

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Longhorn Sculpin Tissue Distribution In the longhorn sculpin, EDNRA, EDNRB1 EDNRB2 mRNA are ubiquitously expressed in the tissues tested (Fig. 6-2). Th ere are relatively high levels of EDNRA in the gill, operculum, heart, and kidney. EDNRB1 was relatively high in the gill, operculum, and heart, and EDNRB2 was highest in the heart, ki dney, and intestine (Fig. 6-2). Quantitative Real-Time PCR Longhorn sculpin gill EDNRA mRNA levels did not differ from 100% SW sham at 24 or 72 h (Fig. 6-3A). Longhorn sculpin acclimated to 20% SW had gill EDNRB1 mRNA levels that were not different from sham at either time; how ever, there was a significan t 2.5-fold increase in gill EDNRB1 after 24 h and 72 h acclimation to 10% SW (Fig. 6-3B). Also, we found a significant increase in gill EDNRB2 after 24 and 72 h acclimation to 20% SW, but 10% SW sculpin were not different from 100% SW shams (Fig. 6-3C). Finally, gill ECE1 mRNA levels did not differ from shams with low-salinity acclimation after 24 or 72 h (Fig. 6-3D). Discussion Endothelin-1 signaling cascades are hypothesized to be local re gulators of ion transport in the fish gill (Evans et al., 2004); however the physiol ogical role of this system in the fish gill is unclear. From our phylogenetic analysis, it was determined that we sequenced a portion of the longhorn sculpin EDNRA, EDNRB1, and EDNRB2, but we did not sequence the teleost specific duplications, EDNRA2 and EDNRB1b (Chapter 3). The sculpin EDNRA, EDNRB1, and EDNRB2 are ubiquitously expressed throughout the fish, with relatively high levels in osmoregulatory tissues such as, the gill and kidney. We recently determined that in the euryhaline killifish EDNRA, EDNRB1 and EDNRB2 are also ubiquitously expressed in these same tissues, suggesting a constitutive role of EDN1 signaling in teleost fishes. One unexplored area of EDN1 research is unders tanding the function of the tele ost specific duplicates, EDNRA2 130

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and EDNRB1b. Teleost fishes have at least five EDNRs, while ma mmals have two. What is the function of these duplicate recepto rs? Are they expressed in the gill? How does environmental salinity affect their expression or immunoloca lization? Does the longhorn sculpin have a duplicate EDNRA2 and EDNRB1b? These are a ll important questions that have not been explored. The longhorn sculpin can tolerate direct transf ers from SW to 20% SW, but cannot survive for more than a few days in salinities less th an 10% SW (Chapter 5, Claiborne et al. 1994), making it a moderate osmoregulator. We determined that ECE1 mRNA levels did not change with acclimation to low salinity water. The c onversion of the EDN1 pr ecursor, proendothelin-1, by ECE1 is a rate limiting step in the production of active EDN1 (D'Orleans-Juste et al., 2003; Ikeda et al., 2002). Here we postulate th at because there was no observed change in ECE1 mRNA there is likely no change in ECE1 pr otein level. Thus, we hypothesize that in the longhorn sculpin, acclimation to 10% or 20% SW does not change active EDN1 levels. In contrast, there were increases in EDNRB1 and EDNRB2 mRNA in the sculpin during acclimation to 10% SW or 20% SW, respectively. It may be that there is an increase in EDN1 signaling during hypo-osmotic stress, because there are more receptors present. If the EDN1 signaling via an EDNRB-type receptor results in the inhibition of ion transport as hypothesized by Evans et al. (2004), then in our 10% SW sculpin one would e xpect that the sculpin would stop losing ions. We previously determined that longhorn sculpin cannot survive in 10% SW more than 6 days, and 24 h after transfer to 10% SW they have a si gnificant decrease in plasma osmolality and ions (Chapter 5). Therefore, we conc lude that the EDNRB1 is not i nvolved in the inhibition of ion transport as hypothesized by Evans et al. (2004). 131

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Twenty percent SW is also hypo-osmotic to the sculpin, but in this sa linity, the fish was capable of maintaining plasma osmolality over the 72 h test period (Chapter 5). There was a significant 1.75-fold increase in EDNRB2 mRNA level during this time. Perhaps the activation of the EDNRB2 can lead to local regulation of ion transport in the l onghorn sculpin, but below 20% SW the sculpin is incapable of up-regulating EDNRB2 and subsequently loses plasma ions. In contrast, during FW acclimation by the killifish, there is no change in EDNRB2 mRNA (Chapter 4), and this fish is capable of returning to contro l plasma ion values during hypoosmotic acclimation, thus the role of EDNRB2 in fish osmoregulation warrants more experimentation. In the killifish, EDNRA are localized on th e MRC, and FW-acclimated killifish have significantly lower EDNRA (mRNA a nd protein) compared to SWacclimated killifish (Chapter 4). We attempted to immunolocalize the EDNRs in the longhorn sculpin gill (as we did in the killifish, Chapter 4); however, our controls (Western blots and peptide-absorbed antibodies) did not work, suggesting these anti bodies were not functioning in the longhorn sculpin. At the mRNA level, though, acclimation to 20% and 10% SW did not elicit any changes in EDNRA mRNA in the sculpin gill. There are two possi ble explanations for this difference: 1. the putative role of EDNRs in regula tion of ion transport is species specific, with EDNRA being the receptor responsible for these effects in the kill ifish, and potentially EDNRB2 fulfilling this role in the sculpin. 2. The sculpin cannot surv ive in <20% SW, and th e regulation of gene expression in this fish is impaired. Perhaps the changes we see are due to a lethal stress and are do not reflect a normal physiological response. In the future, experiments to differentiate between these two hypotheses are needed. 132

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133 In conclusion, the role of EDN1 in the fish gill is still not clear; howev er, it is evident that environmental salinity regulates gill EDNR. It is plausible that the EDN signaling axis is impaired during acclimation to low salinity water, and if this is upstream to the regulation of ion transporter density, it maybe one reason why they cannot maintain ion homeostasis. Future experiments will help el ucidate this hypothesis.

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Table 6-1. Primers used the tissu e distribution (td) and quantita tive real-time PCR (q) analyses. Primer 5' to 3' Oreintation tdEDNRAF1 TAT CTG GTT GCT GTC GCT CTT tdEDNRAR1 CAA CAG GAA ATT GAG CAG CTC tdEDNRB1F1 CAC TCG GAG ACC TGC TAC ACA T tdEDNRB1R1 CCA ACG GCA GGC AGA AAT AT tdEDNRB2F1 TAG ACT GCT GCC GTT CAT CCA tdEDNRB2R1 TTC ATG TGG TCG TTG AGC G qEDNRAF1 CTA TCT GGT TGC TGT CGC TCT T qEDNRAR1 TGC GTA TGG TTT CGT TCC TGT qEDNRB1F1 AAG AGA GGT GGC TAA GAC GGT G qEDNRB1R1 AGG ATA CGG CTG AGA TGG AGA G qEDNRB2F1 TCT GAT CTG GTT GGT CGC TGT qEDNRB2R1 TGT GAC CTC TGT ACG GCA TCT C qECE1F1 GAG ATC GTG GAC TTT GAA ACC A qECE1R1 AGA TCC TTG GCC TCC ATC TTG 134

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Western clawed frog EDNRB1 Zebrafish EDNRB1 Green spotted pufferfish EDNRB1 Sculpin EDNRB1 Medaka EDNRb1 Killifish EDNRB1 Medaka EDNRB1b Green spotted pufferfish EDNRB1b Zebrafish EDNRB1b Zebrafish EDNRB1c EDNRA EDNRB2 EDNRB1 Western clawed frog EDNRA Chicken EDNRA Platypus EDNRA Sculpin EDNRA Green spotted pufferfish EDNRA Zebrafish EDNRA Killifish EDNRA Medaka EDNRA Medaka EDNRA2 Green spotted pufferfish EDNRA2 95 54 36 61 97 99 Human EDNRA Opossum EDNRA 80 97 85 97 100 Western clawed frog EDNRB2 Chicken EDNRB2 Platypus EDNRB2 Green spotted pufferfish EDNRB2 Sculpin EDNRB2 Medaka EDNRB2 Killifish EDNRB2 78 90 100 91 99 99 Chicken EDNRB1 Platypus EDNRB1 Human EDNRB1 Opossum EDNRB1 Opossum EDNRB1d 100 66 92 96 72 86 99 39 100 100 65 98 54 99 0.3 replacements/site Western clawed frog EDNRB1 Zebrafish EDNRB1 Green spotted pufferfish EDNRB1 Sculpin EDNRB1 Medaka EDNRb1 Killifish EDNRB1 Medaka EDNRB1b Green spotted pufferfish EDNRB1b Zebrafish EDNRB1b Zebrafish EDNRB1c EDNRA EDNRB2 EDNRB1 Western clawed frog EDNRA Chicken EDNRA Platypus EDNRA Sculpin EDNRA Green spotted pufferfish EDNRA Zebrafish EDNRA Killifish EDNRA Medaka EDNRA Medaka EDNRA2 Green spotted pufferfish EDNRA2 95 54 36 61 97 99 Human EDNRA Opossum EDNRA 80 97 85 97 100 Western clawed frog EDNRB2 Chicken EDNRB2 Platypus EDNRB2 Green spotted pufferfish EDNRB2 Sculpin EDNRB2 Medaka EDNRB2 Killifish EDNRB2 78 90 100 91 99 99 Chicken EDNRB1 Platypus EDNRB1 Human EDNRB1 Opossum EDNRB1 Opossum EDNRB1d 100 66 92 96 72 86 99 39 100 100 65 98 54 99 0.3 replacements/site 0.3 replacements/site Figure 6-1. Maximum likelihood analysis of the endothelin receptors including our new sculpin EDNRA, EDNRB1 and EDNRB2 protein sequences. Numbers at the nodes represent percent bootstrap. For accession numbers see Figure 3-3. 135

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Op. epithelium Gill Brain Heart Stomach Intestine Kidney EDNRA EDNRB1 EDNRB2 18S Op. epithelium Gill Brain Heart Stomach Intestine Kidney Op. epithelium Gill Brain Heart Stomach Intestine Kidney EDNRA EDNRB1 EDNRB2 18S Figure 6-2. mRNA tissue distri bution of the endothelin recepto rs from the longhorn sculpin determined using duplexing semi-quantitative PCR. 18S was used as an internal control. 136

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137 A. EDNRA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72relative mRNA level 100% 20% 10% B. EDNRB1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72 C. EDNRB2 0.0 0.5 1.0 1.5 2.0 2.5 24 72Time (h)D. ECE1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 24 72Time (h)* p=0.04 p=0.043 *p=0.003* p=0.03 A. EDNRA 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 24 72relative mRNA level 100% 20% 10% 100% 20% 10% B. EDNRB1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72 0.0 0.5 1.0 1.5 2.0 2.5 3.0 24 72 C. EDNRB2 0.0 0.5 1.0 1.5 2.0 2.5 24 72 0.0 0.5 1.0 1.5 2.0 2.5 24 72Time (h)D. ECE1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 24 72 0.0 0.2 0.4 0.6 0.8 1.0 1.2 24 72Time (h)* p=0.04 p=0.043 *p=0.003* p=0.03 Figure 6-3. Longhorn sculpin gill A) EDNRA, B) EDNRB1, C) EDNRB2 and D) ECE1 expression levels following acclimation to 100, 20 or 10% seawater (SW) for 24 and 72 hours (h). mRNA levels determined by quantitative Real-Time PCR. mRNA values are normalized to L8 mRNA levels, and all values are relative to the mean sham (100% SW) level at 24 or 72 h, and are mean s.e.m. Asterisks represent statistically significant differences compared to the sham value at 24 or 72 h (p values are listed on the graph).

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CHAPTER 7 CONCLUSIONS This work is the first to explore fully th e expression of EDN1, ECE1, and the EDNRs in the teleost gill, to test the effects of hyperosmotic and hypo-osmotic environments on gill EDN1 signaling, and completely analyze the e volution of EDNs, ECEs and EDNRs. In the killifish and the longhorn sculpin gills, EDN1, EC E1, and the EDNRs are expressed, demonstrating for the first time that EDN1 signaling occurs in vivo in the fish gill. A summary of the localization of these proteins is found in Figure 2-9. Although, I could not immunolocalize these proteins in the sculpin (because Westerns di d not work with sculpin gills), I was able to sequence ECE1 and EDNR s from the sculpin gill, and a recent papers on EDNRA and EDNRB immunolocalization in the cod and tiger pufferfish gills suggests that this system is active in all teleosts. The fi ndings of EDN1 producing cells and EDNRs in the gill strongly suggest that EDN1 acts as a para crine and autocrine in these fishes. There is mounting evidence that EDN1 acts as an autocrine to regulate pill ar cell tone, and as a re sult regulates blood flow through the lamellae. The finding of EDNRA on th e MRC of the killifish gill suggests a putative role in the regulation of ion transport by the MRC. Interestingly, EDNRA was immunolocalized (using heterologous and homologous antibodies) in two other teleosts, and each had a different EDNRA distribution through the gill. In the cod, EDNRA were found on hypothesized nerve fibers running along the length of the filament (Ste nslokken et al., 2006). In the tiger pufferfish, EDNRA were found on pillar cells. Thus, the dist ribution of EDNRA in the teleost gill may be species-specific given these obs erved differences in localizat ion. The expression of EDNRB throughout the gill suggests that these receptors may act as clearance receptors for the EDNs. More than half of a bolus inject of EDN1 is removed with one pa ss through the gills (Olson, 1998), and likewise in mammals a significant por tion of EDN1 is removed through the lungs by 138

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the EDNRB1 (La and Reid, 1995). Therefore, it seems plausible that either EDNRB1 or EDNRB2 is acting as a clearance receptor in the fish gill. One limitation to my findings was that I was not able to differentiate on the protein level between EDNRB1 and EDNRB2. We did make k illifish-specific EDNR antibodies; however, preliminary experiments were inconclusive. In the future, in situ hybridization will be important in determining the mRNA localization of these receptors. Until more teleosts are tested, and perhaps even more homologous antibodies made (or more in situ studies complete), the generality of my model (Fig. 2-9) is unknown. Howe ver, this is the first model to describe the potential functions of EDN1 in the fish gill usi ng empirical data, and it is a reasonable hypothesis to test in other species. The expression of EDN1, ECE1, and the EDNR s are affected by acclimation to seawater (hyperosmotic environment) and fresh water (hypoosmotic environment). In the killifish, some changes were within 3 h of transfer, while othe rs took at least 24 h to occur, suggesting that EDN1 may be involved in short-term regulation of ion balance as I originally hypothesized. When one compares the extremely euryhaline kil lifish with the moderate ly euryhaline sculpin during acclimation to hypo-osmotic environments, we find that the killifish after 24 h had small changes in EDNR expression (mRNA and protein) while the sculpin had a 2.5-fold increase in EDNRB1 mRNA levels. In 10% SW the sculpin loses ions, likely because it does not downregulate ion transporters necessary for tole rance in hypo-osmotic environments (CFTR and NKCC1) (Chapter 5). In contrast, the kil lifish significantly decreases these same ion transporters, and their plasma ion levels start to return to control values (Choe et al., 2006; Scott et al., 2006). Thus, it is hard to differentia te between normal, physiological responses to changing environmental salinity, and the effects from the sculpin that are not osmoregulating 139

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properly, and ultimately lose ions to a lethal leve l (as the sculpin is doing in 10% SW). If we compared the sculpin in 20% SW and the killifish in fresh water for 24 h, we do see a similar responseno change in EDNRA or EDNRB1 mRNA levels; however, sc ulpin increased in gill EDNRB2 mRNA levels, and unlike the killifish, sculpin were not losing plasma ions during this acclimation period. There are no clear trends betw een these two fishes, but I think an important conclusion is that EDN signaling is present in th e teleost gill, and there are some changes with acclimation to a hypo-osmotic environment. During acclimation to hyperosmotic environmen ts, killifish increas e ECE1, EDNRA, and EDNRB, suggesting that there is more active ED N1 produced (because there is more ECE1) and more receptors to bind EDN1 (Chapters 2 and 4). In addition, chronically acclimated killifish had lower gill EDNRA levels (all other EDN components were unchanged), and the EDNRA was immunolocalized to the MRC (Chapter 4). Thus, I speculate that EDNRA is involved in regulation of ion transport in the killifish gill during hyperosmotic stress. As stressed throughout my dissertation, until functional studies are co mpleted, through the use of tissue-specific knockouts, silencing RNAs, or morpholinos (and likely other techniques), all one can do is speculate on the function of EDN1 in fishes. From my phylogenetic analyses, I determined the evolutionary relationships among the EDNs, ECEs and EDNRs, respectively. The first c onclusion from these studies is that all three EDNs and EDNRs are found in teleos ts, and they are not found in the early chordates (lancelets and sea squirts) or any invertebra tes. The EDN signaling axis is on ly found in the vertebrates. I have sequenced portions of the EDNRs from the sea lamprey (EDNRA EU440327, Appendix A) and shark gill (EDNRA EU440328, and EDNRB1 EU440329), and physiological studies by Evans and Harrie (2001) in the Atla ntic hagfish suggest th at this system was present in the early 140

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vertebrates. This signaling axis is necessary for proper craniofacial development and may have been a crucial step in the development of the ve rtebrate jaws (Clouthier and Schilling, 2004). My results support this hypothesis by determining that EDNs and E DNRs are vertebrate-specific. What was very interesting was to find ECEs in all organisms including prokaryotes. It seems likely that ECE was a general protease and as it evolved, it became specialized for cleaving preferentially the EDNs. ECEs in vertebrates function as dimers, while in all other organisms they function as monomers, so ther e has been a structural and func tional shift of this enzyme in the vertebrates. The second, and in some respects, the most in teresting thing I discovered during the course of my dissertation, was that th ere are three groups of EDNRs. When I started (and even now) most researchers talked about the two EDNRs: EDNRA and EDNRB. Wh at we all missed was in 1998 a third receptor was described (and mislab eled) as the avian-spec ific EDNRB2. Since then about four more avian-specific papers ha ve been published on this receptor. I initially found out about this receptor because it was the first clone I sequenced from the killifish gill. This finding and discussions with Dr. M. Miya moto prompted me to explore the field of bioinformatics and phylogenetics to further und erstand this family of G-protein-coupled receptors. As shown in Figure 33 there are EDNRB2 proteins in the teleosts, amphibians, birds, and platypus, and I determined that EDNRB2 wa s lost by the therian mammals about 150 mya. In addition, an interesting fi nding was that the EDNRA and E DNRB1 are duplicated in the teleost fishes, likely a result of the teleost-specific genome duplication after this split from the tetrapods. In the future, studies should look at these duplicates more closely and determine what function they may play in the teleosts. 141

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With the loss of EDNRB2 in therians, I hypothesized that perhaps the remaining EDNRS had undergone modifications, and had different functions from the non-therian EDNRS to compensate for this missing protein/gene (Chapter 3). Using a Rate Shift Analysis developed by Knudsen et al. (2003) and the known functionally important site s in the mammalian EDNRA and EDNRB1, I determined that the therian EDNRA has 6/21 functionally impor tant sites that are slow evolving Type 1 sites or Type II sites (s ee Fig. 3-1 for a definition) compared to the nontherian gnathostomes. In addition, the EDNRB1 is highly conserved among these gnathostomes, compared to the EDNRA. This suggests that EDNRA may have differe nt functions in the therians than non-therian gnathostomes. My dissertation work is the foundation for the future functional studies needed to determine the function of EDN1 in the fish gi ll. With these sequences from the killifish, morpholinos, knockouts and silencing RNA probes can be made and us ed to test the effects of inhibiting specific steps in EDN1 signaling casc ades. When I started this work, the tiger pufferfish ( Takifugu rubripes) genome was just released, and the zebrafish ( Danio rerio ) genome sequencing was underway. The tiger pufferfish doe s have some level of salinity tolerance; however, the zebrafish is strictly a freshwater sp ecies. Thus, the addition of the sequences from a fish with extreme salinity tolerance, like the killifish, are very useful for future functional studies. In addition, the model presented in Fi gure 2-9 summarizes the localization and signaling patterns of EDN1 in the gill, and is an excellent hypothesis for others to test in their species of interest. Finally, my dissertation work points out some fundamental issues with the way EDN research has been progressing over the past 20 years. First, most people assumed that antagonists and agonists developed for mammals will behave the same in non-mammalian 142

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143 vertebrates. We recently determined this wa s not true for a COX-2 antagonist (Choe et al., 2006), and as tabulate in Table 3-1, the EDNRs have different pharmacology profiles for many animals. Second, there is an assumption that these receptors have the same functions in all organisms, and given that many biomedical studies are conducted in non-mammalian model organisms (chicks, frogs and zebrafish), this is a faulty assumption. My functional analysis of the EDNRA and EDNRB1 suggests that there was a functional shift in th e therian EDNRA, but that the EDNRB1 is well conserved across the gnathostomes. This needs to be tested using welldeveloped, functional studies in non-mammalian vert ebrates. Thus, all one can do is hypothesize as to the function(s) of EDN in the lower vertebrates. Finally, the fact that a third group of EDNRs, the EDNRB2, is still largely ignored need s to be remedied. By exploring the biology of the EDNRB2, it will help researchers understand the evolution of EDNRA and EDNRB1 in the therians. Given the medical importance of thes e receptors, it seems important to understand how their functions have changed or remained cons tant over the past 500 mya. These types of comparative studies can lead to discoveries of new functions, give insight into drugs and treatments of EDN-associated pathologies, a nd further our basic knowledge of this system.

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APPENDIX TISSUE DISTRIBUTION OF THE LA MPREY ENDOTHELIN A RECEPTOR Tissue distribution of the sea lamprey EDNRA mRNA (Accession EU440327). Like the killifish and longhorn sculpin, the lamprey EDNRAs are f ound in all tissues tested. Gill Brain Stomach Intestine Heart Kidney MuscleENDRA 18S Gill Brain Stomach Intestine Heart Kidney Muscle Gill Brain Stomach Intestine Heart Kidney MuscleENDRA 18S 144

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LIST OF REFERENCES Abdel-Sayed, S., Brunner, H. R. and Nussberger, J. (2003). Volume expansion enhances plasma endothelin-1. Am. J. Hypertens. 16, 1057-61. Adachi, M., Hashido, K., Trzeciak, A., Watanabe, T., Furuichi, Y. and Miyamoto, C. (1993). Functional domains of human endothelin receptor. J. Cardiovasc. Pharmacol. 22 Suppl 8, S121-4. Ahn, D., Ge, Y., Stricklett, P. K., Gill, P ., Taylor, D., Hughes, A. K., Yanagisawa, M., Miller, L., Nelson, R. D. and Kohan, D. E. (2004). Collecting ductspecific knockout of endothelin-1 causes hypertensi on and sodium retention. J. Clin. Invest. 114 504-11. Angerio, A. D. (2005). The role of endot helin in heart failure. Crit. Care. Nurs. Q. 28, 355-9. Arai, H., Hori, S., Aramori, I ., Ohkubo, H. and Nakanishi, S. (1990). Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348, 730-2. Arai, H., Nakao, K., Takaya, K., Hosoda, K., Ogawa, Y., Nakanishi, S. and Imura, H. (1993). The human endothelin-B receptor gene. Structural organization and chromosomal assignment. J. Biol. Chem. 268, 3463-70. Baynash, A. G., Hosoda, K., Giaid, A., Rich ardson, J. A., Emoto, N., Hammer, R. E. and Yanagisawa, M. (1994). Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79, 1277-85. Bettex-Galland, M. and Hughes, G. M. (1973). Contractile filament ous material in the pillar cells of fish gills. J. Cell Sci. 13, 359-70. Bigelow, H. B. and Schroeder, W. C. (2002). Fishes of the Gulf of Maine. Caldwell, NJ: Blackburn Press. Brand, M., Le Moullec, J. M., Corvol, P. and Gasc, J. M. (1998). Ontogeny of endothelins-1 and -3, their receptors, and endothelin conve rting enzyme-1 in the early human embryo. J. Clin. Invest. 101, 549-59. Breu, V., Hashido, K., Broger, C., Miyamoto, C., Furuichi, Y., Hayes, A., Kalina, B., Loffler, B. M., Ramuz, H. and Clozel, M. (1995). Separable binding sites for the natural agonist endothelin-1 and the non-peptide antagonist bosentan on human endothelin-A receptors. Eur. J. Biochem. 231, 266-70. Burnett, K. G., Bain, L. J., Baldwin, W. S., Callard, G. V., Cohen, S., Di Giulio, R. T., Evans, D. H., Gomez-Chiarri, M., Hahn, M. E., Hoover, C. A. et al. (2007). Fundulus as the premier teleost model in environm ental biology: Opportunities for new insights using genomics. Com. Biochem. Physiol. D: Genomics and Proteomics 2, 257. 145

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Catches, J. S., Burns, J. M., Edwards, S. L. and Claiborne, J. B. (2006). Na+/H+ antiporter, V-H+-ATPase and Na+/K+-ATPase immunolocalization in a marine teleost ( Myoxocephalus octodecemspinosus ). J. Exp. Biol 209, 3440-7. Chen, D. H., Balyakina, E. V., Lawren ce, M., Christman, B. W. and Meyrick, B. (2003). Cyclooxygenase is regulated by ET-1 and MA PKs in peripheral lung microvascular smooth muscle cells. Am. J. Physiol.Lung Cell. Mol. Physiol. 284, L614-L621. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. and Thompson, J. D. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497-500. Choe, K. P. and Evans, D. H. (2003). Compensation for hypercapnia by a euryhaline elasmobranch: effect of salinity and role s of gills and kidneys in fresh water. J. Exp. Zool. A Comp. Exp. Biol. 297, 52-63. Choe, K. P., Havird, J., Rose, R., Hyndman K., Piermarini, P. and Evans, D. H. (2006). COX2 in a euryhaline teleost, Fundulus heteroclitus : primary sequence, distribution, localization, and potential function in gills during salinity acclimation. J. Exp. Biol. 209 1696-708. Choe, K. P., Kato, A., Hirose, S., Plata, C., Si ndic, A., Romero, M. F., Claiborne, J. B. and Evans, D. H. (2005). NHE3 in an ancestral verteb rate: primary sequence, distribution, localization, and f unction in gills. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289 R1520-34. Choe, K. P., Verlander, J. W., Wingo, C. S. and Evans, D. H. (2004). A putative H+-K+ATPase in the Atlantic stingray, Dasyatis sabina : primary sequence and expression in gills. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R981-91. Claiborne, J. B., Walton, J. and Compton-Mccullough, D. (1994). Acid-base regulation, branchial transfers and renal output in a marine teleost fish (the long-horned sculpin Myoxocephalus Octodecimspinosus ) during exposure to low salinities. J. Exp. Biol. 193 79-95. Clouthier, D. E. and Schilling, T. F. (2004). Understanding endothelin-1 function during craniofacial development in the mouse and zebrafish. Birth Defects Res. C Embryo Today 72, 190-9. D'Orleans-Juste, P., Plante, M., Honore, J. C., Carrier, E. and Labonte, J. (2003). Synthesis and degradation of endothelin-1. Can. J. Physiol. Pharmacol. 81, 503-10. Dang, Z., Balm, P. H., Flik, G., Wende laar Bonga, S. E. and Lock, R. A. (2000). Cortisol increases Na(+)/K(+)-ATPase density in plasma membranes of gill chloride cells in the freshwater tilapia Oreochromis mossambicus J. Exp. Biol. 203 2349-55. 146

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De Nucci, G., Gryglewski, R. J., Warner, T. D. and Vane, J. R. (1988a). Receptor-mediated release of endothelium-derived relaxing factor and prost acyclin from bovine aortic endothelial cells is coupled. Proc. Natl. Acad. Sci. U. S. A. 85, 2334-8. De Nucci, G., Thomas, R., D'orleansjuste, P., Antunes, E., Walder, C., Warner, T. D. and Vane, J. R. (1988b). Pressor Effects of Circul ating Endothelin Are Limited by Its Removal in the Pulmonary Circulation a nd by the Release of Prostacyclin and Endothelium-Derived Relaxing Factor. Proc. Natl. Acad. Sci. U. S. A. 85, 9797-9800. Dean, R., Zhuo, J., Alcorn, D., Casley, D. and Mendelsohn, F. A. (1996). Cellular localization of endothelin receptor subtypes in the rat kidney following in vitro labelling. Clin. Exp. Pharmacol. Physiol. 23, 524-31. Elshourbagy, N. A., Adamou, J. E., Gagnon, A. W., Wu, H. L., Pullen, M. and Nambi, P. (1996). Molecular characterization of a novel human endothelin receptor splice variant. J. Biol. Chem. 271, 25300-7. Evans, D. H. (2001). Vasoactive receptors in abdomi nal blood vessels of the dogfish shark, Squalus acanthias Physiol. Biochem. Zool. 74, 120-6. Evans, D. H. (2002). Cell signaling and ion transpor t across the fish gill epithelium. J. Exp. Zool. 293, 336-47. Evans, D. H. and Gunderson, M. P. (1999). Characterization of an endothelin ET(B) receptor in the gill of the dogfish shark Squalus acanthias J. Exp. Biol. 202, 3605-10. Evans, D. H., Gunderson, M. and Cegelis, C. (1996). ETB-type receptors mediate endothelinstimulated contraction in the aortic vascular smooth muscle of the spiny dogfish shark, Squalus acanthias J. Comp. Physiol. 165, 659-64. Evans, D. H. and Harrie, A. C. (2001). Vasoactivity of the vent ral aorta of the American eel ( Anguilla rostrata) Atlantic hagfish ( Myxine glutinosa ), and sea lamprey ( Petromyzon marinus ). J. Exp. Zool. 289, 273-84. Evans, D. H., Piermarini, P. M. and Choe, K. P. (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregula tion, acid-base regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97-177. Evans, D. H., Rose, R. E., Roeser, J. M. and Stidham, J. D. (2004). NaCl transport across the opercular epithelium of Fundulus heteroclitus is inhibited by an endothelin to NO, superoxide, and prostanoid signaling axis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R560-8. Felsenstein, J. (2004). Inferring phylogenies. Sunde rland, MA.: Sinauer Associates. Fiol, D. F. and Kultz, D. (2005). Rapid hyperosmotic coinduction of two tilapia ( Oreochromis mossambicus ) transcription factors in gill cells. Proc. Natl. Acad. Sci. U. S. A. 102, 927-32. 147

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Fredriksson, R., Lagerstrom, M. C., Lundin, L. G. and Schioth, H. B. (2003). The G-proteincoupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256-72. Froese, R. and Pauly, D. (2000). FishBase 2000: concepts, de sign and data sources, a global information system on fishes. Los Baos, Laguna, Philippines: International Center for Living Aquatic Resources Management. Froy, O. and Gurevitz, M. (1998). Membrane potential modulators: a thread of scarlet from plants to humans. Faseb J. 12, 1793-6. Garvin, J. and Sanders, K. (1991). Endothelin inhibits fluid a nd bicarbonate transport in part by reducing Na+/K+ ATPase activity in the rat proximal straight tubule. J. Am. Soc. Nephrol. 2, 976-82. Ge, Y., Ahn, D., Stricklett, P. K., Hughes, A. K., Yanagisawa, M., Verbalis, J. G. and Kohan, D. E. (2005a). Collecting duct-specifi c knockout of endothelin-1 alters vasopressin regulation of urine osmolality. Am. J. Physiol. Renal. Physiol. 288, F912-20. Ge, Y., Bagnall, A., Stricklett, P. K., Strait, K., Webb, D. J., Kotelevtsev, Y. and Kohan, D. E. (2006). Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention. Am. J. Physiol. Renal. Physiol. 291, F1274-80. Ge, Y., Stricklett, P. K., Hughes, A. K., Yanagisawa, M. and Kohan, D. E. (2005b). Collecting duct-specific knockout of the endothelin A receptor alters renal vasopressin responsiveness, but not sodium excretion or blood pressure. Am. J. Physiol. Renal Physiol. 289, F692-8. Goniakowska-Witalinska, L., Za ccone, G., Fasulo, S., Mauceri, A., Licata, A. and Youson, J. (1995). Neuroendocrine cells in the gills of the bowfin Amia calva. An ultrastructural and immunocytochemical study. Folia Histochem. Cytobiol. 33 171-7. Guindon, S., Lethiec, F., Duroux, P. and Gascuel, O. (2005). PHYML Online--a web server for fast maximum likelihood-ba sed phylogenetic inference. Nucleic Acids Res. 33, W5579. Hao, C. M., Komhoff, M., Guan, Y., Redha, R. and Breyer, M. D. (1999). Selec tive targeting of cyclooxygenase-2 reveals its role in re nal medullary interst itial cell survival. Am. J. Physiol. 277 F352-9. Hao, C. M., Yull, F., Blackwell, T., Komhoff, M., Davis, L. S. and Breyer, M. D. (2000). Dehydration activates an NF-kappaB-driven, COX2-dependent survival mechanism in renal medullary interstitial cells. J. Clin. Invest. 106 973-82. Hegab, S. A. and Hanke, W. (1984). The significance of cortis ol for osmoregulation in carp ( Cyprinus carpio ) and tilapia ( Sarotherodon mossambicus ). Gen. Comp. Endocrinol. 54, 409. 148

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Hirata, Y., Emori, T., Eguchi, S., Kanno, K., Imai, T., Ohta, K. and Marumo, F. (1993). Endothelin Receptor Subtype-B Mediates Synt hesis of Nitric-Oxide by Cultured Bovine Endothelial-Cells. J. Clin. Invest. 91 1367-1373. Hiroi, J. and McCormick, S. D. (2007). Variation in salinity tolerance, gill Na+/K+-ATPase, Na+/K+/2Clcotransporter and mitochondria-r ich cell distribution in three salmonids Salvelinus namaycush Salvelinus fontinalis and Salmo salar J. Exp. Biol. 210, 1015-24. Hoang, M. V. and Turner, A. J. (1997). Novel activity of e ndothelin-converting enzyme: hydrolysis of bradykinin. Biochem. J. 327 (Pt 1) 23-6. Hoffmann, E. K., Schettino, T. and Marshall, W. S. (2007). The role of volume-sensitive ion transport systems in regulation of epithelial transport. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 148, 29-43. Hosoda, K., Nakao, K., Tamura, N., Arai, H ., Ogawa, Y., Suga, S., Nakanishi, S. and Imura, H. (1992). Organization, structure, chromo somal assignment, and expression of the gene encoding the human endothelin-A receptor. J. Biol. Chem. 267, 18797-804. Hubbard, T. J., Aken, B. L., Beal, K., Ballest er, B., Caccamo, M., Chen, Y., Clarke, L., Coates, G., Cunningham, F., Cutts, T. et al. (2007). Ensembl 2007. Nucleic Acids Res. 35, D610-7. Huelsenbeck, J. P. and Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754-5. Huelsenbeck, J. P., Ronquist, F., Nielsen, R. and Bollback, J. P. (2001). Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294, 2310-4. Hughes, A. K., Padilla, E., Kutchera, W. A., Michael, J. R. and Kohan, D. E. (1995). Endothelin-1 induction of cyclooxygenase-2 expression in rat mesangial cells. Kidney Int. 47, 53-61. Hyndman, K. A., Choe, K. P., Havird, J. C., Rose R. E., Piermarini, P. M. and Evans, D. H. (2006). Neuronal nitric oxide syntha se in the gill of the killifish, Fundulus heteroclitus Comp. Biochem. Physiol. B Biochem. Mol. Biol. 144, 510-9. Hyndman, K. A. and Evans, D. H. (2007). Endothelin and endothelin converting enzyme-1 in the fish gill: evolutionary and physiological perspectives. J. Exp. Biol. 210 4286-97. Ikeda, S., Emoto, N., Alimsardjon o, H., Yokoyama, M. and Matsuo, M. (2002). Molecular isolation and characterization of novel four subisoforms of ECE-2. Biochem. Biophys. Res. Commun. 293 421-6. Inoue, A., Yanagisawa, M., Kimura, S., Kasu ya, Y., Miyauchi, T., Goto, K. and Masaki, T. (1989). The human endothelin family: three st ructurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. U. S. A. 86, 2863-7. 149

PAGE 150

Johnson, G. D., Stevenson, T. and Ahn, K. (1999). Hydrolysis of peptide hormones by endothelin-converting enzyme-1. A comparison with neprilysin. J. Biol. Chem. 274, 40538. Joyner-Matos, J., Downs, C. A. and Julian, D. (2006). Increased expression of stress proteins in the surf clam Donax variabilis following hydrogen sulfide exposure. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 145 245-57. Kanzawa, N., Poma, C. P., Takebayashi-Suzuki, K., Diaz, K. G., Layliev, J. and Mikawa, T. (2002). Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression. Development 129, 3185-94. Karnaky, K. G., Jr. (1998). Osmotic and Ionic regulation. In The physiology of fishes (ed. D. H. Evans), pp. 159-178. Boca Raton, FL: CRC Press. Karnaky, K. G., Jr. and Kinter, W. B. (1977). Killifish opercular sk in: a flat epithelium with a high density of chloride cells. J. Exp. Zool. 199, 355-64. Karnaky, K. J., Jr., Degnan, K. J. and Zadunaisky, J. A. (1977). Chloride transport across isolated opercular epithelium of killifish: a membrane rich in chloride cells. Science 195 203-5. Karne, S., Jayawickreme, C. K. and Lerner, M. R. (1993). Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J. Biol. Chem. 268, 19126-33. Katoh, F., Hasegawa, S., Kita, J., Takagi, Y. and Kaneko, T. (2001). Distinct seawater and freshwater types of chlo ride cells in killifish, Fundulus heteroclitus Can. J. Zool. 79, 822829. Katoh, F. and Kaneko, T. (2003). Short-term transformation and long-term replacement of branchial chloride cells in killifish transfe rred from seawater to freshwater, revealed by morphofunctional observations and a newl y established 'time-differential double fluorescent staining' technique. J. Exp. Biol. 206, 4113-23. Kimmel, C. B., Ullmann, B., Walker, M., Miller, C. T. and Crump, J. G. (2003). Endothelin 1-mediated regulation of pharyngeal bone development in zebrafish. Development 130 1339-51. Kimura, S., Kasuya, Y., Sawamura, T., Shinimi, O., Sugita, Y., Yanagisawa, M., Goto, K. and Masaki, T. (1989). Conversion of big endothelin-1 to 21-residue endothelin-1 is essential for expression of full vasoconstrictor activity: structure-activity relationships of big endothelin-1. J. Cardiovasc. Pharmacol. 13 Suppl 5, S5-7; discussion S18. Knudsen, B., Miyamoto, M. M., Laipis, P. J. and Silverman, D. N. (2003). Using evolutionary rates to inves tigate protein functional diverg ence and conservation. A case study of the carbonic anhydrases. Genetics 164, 1261-9. 150

PAGE 151

Kong, H., Kahatapitiya, N., Kingsley, K., Salo W. L., Anderson, P. M., Wang, Y. S. and Walsh, P. J. (2000). Induction of carbamoyl phos phate synthetase III and glutamine synthetase mRNA during confinem ent stress in gulf toadfish ( Opsanus beta ). J. Exp. Biol. 203, 311-20. Kontula, T. and Vainola, R. (2003). Relationships of Palearctic and Nearctic 'glacial relict' Myoxocephalus sculpins from mitochondrial DNA data. Mol. Ecol. 12, 3179-84. Kumar, C., Mwangi, V., Nuthulaganti, P., Wu, H. L., Pullen, M., Brun, K., Aiyar, H., Morris, R. A., Naughton, R. and Nambi, P. (1994). Cloning and characterization of a novel endothelin receptor from Xenopus heart. J. Biol. Chem. 269, 13414-20. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W. H., Kamada, N. et al. (1994). Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368, 703-10. La, M. and Reid, J. J. (1995). Endothelin-1 and the re gulation of vascular tone. Clin. Exp. Pharmacol. Physiol. 22, 315-23. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettiga n, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R. et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-8. Lecoin, L., Sakurai, T., Ngo, M. T., Abe, Y., Yanagisawa, M. and Le Douarin, N. M. (1998). Cloning and characterization of a novel endothe lin receptor subtype in the avian class. Proc. Natl. Acad. Sci. U. S.. A. 95, 3024-9. Lee, C. Y. and Chiappinelli, V. A. (1989). Sequence homology between sarafotoxins S6 and porcine endothelin. Toxicon 27, 277-9. Lee, K. M., Kaneko, T., Katoh, F. and Aida, K. (2006). Prolactin gene expression and gill chloride cell activity in fugu Takifugu rubripes exposed to a hypoosmotic environment. Gen. Comp. Endocrinol. 149, 285-93. Lodhi, K. M., Sakaguchi, H., Hirose, S. and Hagiwara, H. (1995). Localization and characterization of a novel receptor for endothelin in the gills of the rainbow trout. J. Biochem. 118, 376-9. Lundberg, J. G. (1972). Wagner Networks and Ancestors. Systematic Zoology 21, 398-413. Mancera, J. M. and McCormick, S. D. (2000). Rapid activation of gill Na(+),K(+)-ATPase in the euryhaline teleost Fundulus heteroclitus J. Exp. Zool. 287, 263-74. Marshall, W. S. (2003). Rapid regulation of NaCl secr etion by estuarine teleost fish: coping strategies for short-durat ion freshwater exposures. Biochim. Biophys. Acta. 1618, 95-105. 151

PAGE 152

Marshall, W. S., Emberley, T. R., Singer, T. D., Bryson, S. E. and McCormick, S. D. (1999). Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus : a multivariable approach. J. Exp. Biol. 202 (Pt 11), 1535-44. Marshall, W. S. and Farrell, A. P. (2006). Ion transport, Osmoregulation, and Acid-Base Balance. In The physiology of fishes vol. 3 (eds. D. H. Evans and J. B. Claiborne), pp. 177-230. Boca Raton, FL: CRC, Taylor & Francis. Marshall, W. S., Lynch, E. M. and Cozzi, R. R. (2002). Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water. J. Exp. Biol. 205, 1265-73. Masereeuw, R., Terlouw, S. A., van Aubel, R. A., Russel, F. G. and Miller, D. S. (2000). Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol. Pharmacol. 57, 59-67. Mauceri, A., Fasulo, S., Ainis, L., Licata, A., Lauriano, E. R., Martinez, A., Mayer, B. and Zaccone, G. (1999). Neuronal nitric oxide synthase (nNOS) expression in the epithelial neuroendocrine cell system and nerve fi bers in the gill of the catfish, Heteropneustes fossilis Acta. Histochem. 101, 437-48. McCormick, S. D. (1993). Methods for nonlethal gi ll biopsy and measurement of Na+,K+ATPase activity. Can. J. Fish. Aqua. Sci. 50, 656-658. McCormick, S. D. (1995). Hormonal control of gill Na+,K+-ATPase and chloride cell function. In Fish Physiology Ionoregulation: Cellular and Molecular Approaches vol. XIV (eds. C. M. Wood and T. J. Shuttleworth), pp. 285-315. New York: Academic Press. McCormick, S. D. and Bradshaw, D. (2006). Hormonal control of salt and water balance in vertebrates. Gen. Comp. Endocrinol. 147, 3-8. Miller, C. T., Schilling, T. F., Lee, K., Parker, J. and Kimmel, C. B. (2000). sucker encodes a zebrafish Endothelin-1 required for ve ntral pharyngeal arch development. Development 127, 3815-28. Miller, C. T., Yelon, D., Stainier, D. Y. and Kimmel, C. B. (2003). Two endothelin 1 effectors, hand2 and bapx1, pattern ventral pharyngeal cartilage and the jaw joint. Development 130, 1353-65. Miller, D. S., Masereeuw, R. and Karnaky, K. J., Jr. (2002). Regulation of MRP2-mediated transport in shark rectal salt gland tubules. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R774-81. Mistry, A. C., Kato, A., Tran, Y. H., Hond a, S., Tsukada, T., Takei, Y. and Hirose, S. (2004). FHL5, a novel actin-binding protein, is highly expressed in eel gill pillar cells and responds to wall tension. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287 R1141-54. 152

PAGE 153

Molenaar, P., O'Reilly, G., Sharkey, A., Ku c, R. E., Harding, D. P., Plumpton, C., Gresham, G. A. and Davenport, A. P. (1993). Characterizati on and localization of endothelin receptor subtypes in the human atrioventricular conducting system and myocardium. Circ. Res. 72, 526-38. Nagy, N. and Goldstein, A. M. (2006). Endothelin-3 regulates neur al crest cell pr oliferation and differentiation in the hindgut enteric nervous system. Dev. Biol. 293, 203-17. Nair, S., Li, W., Cornell, R. and Schilling, T. F. (2007). Requirements for Endothelin type-A receptors and Endothelin-1 signaling in the facial ectoderm for the patterning of skeletogenic neural cres t cells in zebrafish. Development 134, 335-45. Nambi, P., Pullen, M. and Kumar, C. (1994). Identification of a novel endothelin receptor in Xenopus laevis liver. Neuropeptides 26, 181-5. Nataf, V., Lecoin, L., Eichmann, A. and Le Douarin, N. M. (1996). Endothelin-B receptor is expressed by neural crest cells in the avian embryo. Proc. Natl. Acad. Sci. U. S. A. 93, 9645-50. Nelson, J. S. (2006). Fishes of the world. Hoboken, NJ: John Wiley. Nilsen, T. O., Ebbesson, L. O., Madsen, S. S ., McCormick, S. D., Andersson, E., Bjornsson, B. T., Prunet, P. and Stefansson, S. O. (2007). Differential expression of gill Na+,K+ATPase alphaand beta-subunits, Na+,K+,2Clcotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar J. Exp. Biol. 210, 288596. Nilsson, S. and Sundin, L. (1998). Gill blood flow control. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119, 137-47. Notenboom, S., Miller, D. S., Kuik, L. H., Smits, P., Russel, F. G. and Masereeuw, R. (2005). Short-term exposure of renal proximal tubules to gentamicin increases long-term multidrug resistance protein 2 (Abcc2) tran sport function and reduces nephrotoxicant sensitivity. J. Pharmacol. Exp. Ther. 315, 912-20. Notenboom, S., Miller, D. S., Smits, P., Russel, F. G. and Masereeuw, R. (2002). Role of NO in endothelin-regulated drug transp ort in the renal proximal tubule. Am. J. Physiol. Renal Physiol. 282 F458-64. Notenboom, S., Miller, D. S., Smits, P., Russel, F. G. and Masereeuw, R. (2004). Involvement of guanylyl cyclas e and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule. Am. J. Physiol. Renal Physiol. 287, F33-8. Ogawa, Y., Nakao, K., Arai, H ., Nakagawa, O., Hosoda, K., Suga, S., Nakanishi, S. and Imura, H. (1991). Molecular cloning of a non-is opeptide-selective human endothelin receptor. Biochem. Biophys. Res. Commun. 178, 248-55. 153

PAGE 154

Olson, K. R. (1998). Hormone metabolism by the fish gill. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119, 55-65. Olson, K. R. (2002). Vascular anatomy of the fish gill. J. Exp. Zool. 293, 214-31. Olson, K. R., Duff, D. W., Farrell, A. P., Keen, J., Kellogg, M. D., Kullman, D. and Villa, J. (1991). Cardiovascular effects of endothelin in trout. Am. J. Physiol. 260 H1214-23. Opgenorth, T. J., Wu-Won g, J. R. and Shiosaki, K. (1992). Endothelin-converting enzymes. Faseb J 6, 2653-9. Pelis, R. M. and McCormick, S. D. (2001). Effects of growth hor mone and cortisol on Na(+)K(+)-2Cl(-) cotransporter lo calization and abundance in th e gills of Atlantic salmon. Gen. Comp. Endocrinol. 124, 134-43. Pelis, R. M., Zydlewski, J. and McCormick, S. D. (2001). Gill Na(+)-K(+)-2Cl(-) cotransporter abundance and loca tion in Atlantic salmon: effects of seawater and smolting. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280 R1844-52. Perry, S. F. (1997). The chloride cell: st ructure and function in the gills of freshwater fishes. Annu. Rev. Physiol. 59, 325-47. Pfaffl, M. W. (2001). A new mathematical model for re lative quantification in real-time RTPCR. Nucleic Acids Res. 29, e45. Piermarini, P. M., Verlander, J. W., Royaux, I. E. and Evans, D. H. (2002). Pendrin immunoreactivity in the gill epitheliu m of a euryhaline elasmobranch. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R983-92. Plato, C. F., Pollock, D. M. and Garvin, J. L. (2000). Endothelin inhibits thick ascending limb chloride flux via ETB receptor-mediated NO release. Am. J. Physiol. 279 F326-F333. Platzack, B., Wang, Y., Crossley, D., La nce, V., Hicks, J. W. and Conlon, J. M. (2002). Characterization and cardiovasc ular actions of endothelin1 and endothelin-3 from the American alligator. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, R594-602. Pollock, D. M. and Highsmith, R. F. (1998). Endothelin receptors and signaling mechanisms. Berlin; New York, Georgetown, TX: Springer; Landes Bioscience. Prasanna, G., Dibas, A., Hulet, C. and Yorio, T. (2001). Inhibition of Na(+)/K(+)-atpase by endothelin-1 in human nonpigmen ted ciliary epithelial cells. J. Pharmacol. Exp. Ther. 296, 966-71. Puffenberger, E. G., Hosoda, K., Washington, S. S., Nakao, K., deWit, D., Yanagisawa, M. and Chakravart, A. (1994). A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung's disease. Cell 79, 1257-66. 154

PAGE 155

Richter, C. M. (2006). Role of endothelin in chronic renal failure--developments in renal involvement. Rheumatology 45, iii36-38. Ronquist, F. and Huelsenbeck, J. P. (2003). MrBayes 3: Bayesi an phylogenetic inference under mixed models. Bioinformatics 19, 1572-4. Rose, T. M., Henikoff, J. G. and Henikoff, S. (2003). CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Res. 31, 3763-6. Sakamoto, T. and McCormick, S. D. (2006). Prolactin and growth hormone in fish osmoregulation. Gen. Comp. Endocrinol. 147, 24-30. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miya zaki, H., Kimura, S., Goto, K. and Masaki, T. (1990). Cloning of a cDNA encoding a nonisopeptide-selectiv e subtype of the endothelin receptor. Nature 348, 732-5. Scott, G. R., Richards, J. G., Forbush, B., Isenring, P. and Schulte, P. M. (2004). Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am. J. Physiol. Cell. Physiol. 287 C300-9. Scott, G. R. and Schulte, P. M. (2005). Intraspecific variation in gene expression after seawater transfer in gills of the euryhaline killifish Fundulus heteroclitus. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 141 176-82. Scott, G. R., Schulte, P. M. and Wood, C. M. (2006). Plasticity of osmoregulatory function in the killifish intestine: drinking rates, salt and water transport, and gene expression after freshwater transfer. J. Exp. Biol. 209, 4040-50. Seidelin, M., Madsen, S. S., Blen strup, H. and Tipsmark, C. K. (2000). Time-course changes in the expression of Na+, K+-ATPase in gills and pyloric caeca of brown trout ( Salmo trutta ) during acclimation to seawater. Physiol. Biochem. Zool. 73, 446-53. Shavit, L., Penny, D., Hendy, M. D. and Holland, B. R. (2007). The problem of rooting rapid radiations. Mol. Biol. Evol. 24, 2400-11. Shimada, K., Takahashi, M. and Tanzawa, K. (1994). Cloning and f unctional expression of endothelin-converting enzyme from rat endothelial cells. J. Biol. Chem. 269, 18275-8. Shimada, K., Takahashi, M., Turner, A. J. and Tanzawa, K. (1996). Rat endothelinconverting enzyme-1 forms a dimer through Cy s412 with a similar catalytic mechanism and a distinct substrate binding mechanism compared with neutral endopeptidase-24.11. Biochem. J. 315 (Pt 3) 863-7. Shreenivas, S. and Oparil, S. (2007). The role of endothelin-1 in human hypertension. Clin. Hemorheol. Microcirc. 37 157-78. 155

PAGE 156

Singer, T. D., Clements, K. M., Semple, J. W., Schulte, P. M., Bystri ansky, J. S., Finstad, B., Fleming, I. A. and McKinley, R. S. (2002). Seawater toleran ce and gene expression in two strains of Atlantic salmon smolts. Can. J. Fish. Aqua. Sci. 59, 125-135. Singer, T. D., Tucker, S. J., Marshall, W. S. and Higgins, C. F. (1998). A divergent CFTR homologue: highly regulated salt tran sport in the euryhaline teleost F. heteroclitus Am. J. Physiol. 274 C715-23. Spudich, G., Fernandez-Suarez, X. M. and Birney, E. (2007). Genome browsing with Ensembl: a practical overview. Brief Funct. Gen. Prot. 6, 202-19. Stenslokken, K. O., Sundin, L. and Nilsson, G. E. (1999). Cardiovascular and gill microcirculatory effects of endot helin-1 in atlantic cod: evidence for pillar cell contraction. J. Exp. Biol. 202 (Pt 9) 1151-7. Stenslokken, K. O., Sundin, L. and Nilsson, G. E. (2006). Endothelin receptors in teleost fishes: cardiovascular effects and branchial distribution. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290, R852-60. Sultana, N., Nag, K., Kato, A. and Hirose, S. (2007). Pillar cell and er ythrocyte localization of fugu ET(A) receptor and its implication. Biochem. Biophys. Res. Commun. 355, 149-55. Sundin, L. and Nilsson, G. E. (1998). Endothelin redistributes blood flow through the lamellae of rainbow trout gills. J. Comp. Physiol. 168, 619-623. Terlouw, S. A., Masereeuw, R., Russel, F. G. and Miller, D. S. (2001). Nephrotoxicants induce endothelin release and signaling in renal proximal tubules: effect on drug efflux. Mol. Pharmacol. 59, 1433-40. Tipsmark, C. K., Madsen, S. S., Seidelin, M., Ch ristensen, A. S., Cutler, C. P. and Cramb, G. (2002). Dynamics of Na(+),K(+),2Cl(-) cotransporter and Na(+),K(+)-ATPase expression in the branchial ep ithelium of brown trout (Salmo trutta) and A tlantic salmon ( Salmo salar ). J. Exp. Zool. 293, 106-18. Volff, J. N. (2005). Genome evolution and biodiversity in teleost fish. Heredity 94, 280-94. Wada, K., Hashido, K., Terashima, H., Adachi, M ., Fujii, Y., Hiraoka, O., Furuichi, Y. and Miyamoto, C. (1995). Ligand binding domain of the human endothelin-B subtype receptor. Protein Expr. Purif. 6, 228-36. Walker, M. B., Miller, C. T., Coffin Ta lbot, J., Stock, D. W. and Kimmel, C. B. (2006). Zebrafish furin mutants reveal intricacie s in regulating endo thelin1 signaling in craniofacial patterning. Dev. Biol. 295 194-205. Wang, H., Quan, J., Kotake-Nara, E., Uc hide, T., Andoh, T. and Saida, K. (2006). cDNA cloning and sequence analysis of prep roendothelin-1 (PPET-1) from salmon, Oncorhynchus keta Exp. Biol. Med. (Maywood) 231 709-12. 156

PAGE 157

Wang, Y., Jensen, J., Abel, P. W., Fournier, A., Holmgren, S. and Conlon, J. M. (2001). Effects of trout endothelin on the motility of ga strointestinal smooth muscle from the trout and rat. Gen. Comp. Endocr. 123, 156-62. Wang, Y., Olson, K. R., Smith, M. P., Russell, M. J. and Conlon, J. M. (1999). Purification, structural characterization, and myotropic activity of endothelin from trout, Oncorhynchus mykiss. Am. J. Physiol. 277 R1605-11. Wang, Y., Remy-Jouet, I., Delarue, C., Leto urneau, M., Fournier, A., Vaudry, H. and Conlon, J. M. (2000). Structural characte rization and effects on cor ticosteroid secretion of endothelin-1 and endothelin-3 from the frog Rana ridibunda. J. Mol. Endocrinol. 24, 28593. Warner, T. D., Mitchell, J. A., de Nucci, G. and Vane, J. R. (1989). Endothelin-1 and endothelin-3 release EDRF from isolated perfus ed arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol. 13 Suppl 5, S85-8; discussion S102. Webb, D. J. (1997). Endothelin: from molecule to man. Br. J. Clin. Pharmacol. 44, 9-20. Wheeler, D. L., Barrett, T., Benson, D. A., Bryant, S. H., Canese, K., Chetvernin, V., Church, D. M., Dicuccio, M., Ed gar, R., Federhen, S. et al. (2007). Database resources of the National Center for Biotechnology Information. Nucleic Acids Res Whelan, S. and Goldman, N. (2001). A general empirical mode l of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol. Biol. Evol. 18, 691-9. Wongmekiat, O. and Johns, E. J. (2003). Significance of endotheli n on volume homeostasis in obese Zucker rats. Clin. Exp. Pharmacol. Physiol. 30, 702-8. Xu, D., Emoto, N., Giaid, A., Slaughter, C ., Kaw, S., deWit, D. and Yanagisawa, M. (1994). ECE-1: a membrane-bound metalloprotease that cat alyzes the proteolyti c activation of big endothelin-1. Cell 78, 473-85. Yanagisawa, M., Inoue, A., Ishikawa, T., Ka suya, Y., Kimura, S., Kumagaye, S., Nakajima, K., Watanabe, T. X., Sakakibara, S., Goto, K. et al. (1988a). Primary structure, synthesis, and biological activity of rat endothelin, an e ndothelium-derived vasoconstrictor peptide. Proc. Natl. Acad. Sci. U. S. A. 85, 6964-7. Yanagisawa, M., Kurihara, H., Kimura, S., Goto, K. and Masaki, T. (1988b). A novel peptide vasoconstrictor, endothelin, is produ ced by vascular endothelium and modulates smooth muscle Ca2+ channels. J. Hypertens. Suppl. 6, S188-91. Yanagisawa, M., Kurihara, H., Kimura, S., To mobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988c). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-5. 157

PAGE 158

158 Yokoyama, R. and Goto, A. (2005). Evolutionary history of freshwater sculpins, genus Cottus (Teleostei; Cottidae) and re lated taxa, as inferred from mitochondrial DNA phylogeny. Mol. Phylogenet. Evol. 36 654-68. Zaccone, G., Lauweryns, J. M., Fasulo, S., Tagliafierro, G., Ainis, L. and Licata, A. (1992). Immunocytochemical Localization of Serotoni n and Neuropeptides in the Neuroendocrine Paraneurons of Teleost and Lungfish Gills. Acta. Zool. 73, 177-183. Zaccone, G., Mauceri, A., Fasulo, S., Aini s, L., Lo Cascio, P. and Ricca, M. B. (1996). Localization of immunoreactive endothelin in the neuroendocrine cells of fish gill. Neuropeptides 30, 53-7. Zeidel, M. L. (1993). Hormonal regulation of inner medu llary collecting duct sodium transport. Am. J. Physiol. 265, F159-73. Zeidel, M. L., Brady, H. R., Kone, B. C., Gullans, S. R. and Brenner, B. M. (1989). Endothelin, a peptide in hibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells. Am. J. Physiol. 257 C1101-7. Zhang, J., Leontovich, A. and Sarras, M. P., Jr. (2001). Molecular and f unctional evidence for early divergence of an endothelin-like system during metazoan evolution: analysis of the Cnidarian, hydra. Development 128, 1607-15.

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BIOGRAPHICAL SKETCH Kelly Anne Hyndman was born in Guelph, Ontario, Canada, in 1978. She received her Bachelor of Science Honours, with a specializ ation in marine and fresh water biology in 2001. As an undergraduate, Kelly was awarded an NSE RC Undergraduate Research Experience grant and spent the summer of 1999 working as an aquaculture technician on an Arctic Char ( Salvelinus alpinus ) fish farm (Icy Waters, Limited, Whitehorse, Yukon). Following this, she worked as a research assistant to Dr. James Ballantyne (University of Guelph), examining the role of photoperiod on Na+,K+-ATPase activity in the char and rainbow trout ( Oncorhynchus mykiss). In August, 2001 she entered the graduate program in the Department of Zoology at the University of Florida in Gainesville. In May 2004, she bypassed the M.S. and entered the Ph.D. program in the department. She has spent many summers at the Mount De sert Island Biological Laboratory in Salisbury Cove, ME, where a si gnificant portion of her dissertation was completed. In 2006, she attended the Workshop on Molecular Evolution at the Marine Biology Laboratory, Woods Hole, MA. In October 2008, sh e hopes to start a post doc position; however, the exact location of that has not been determined. 159