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Molecular Interactions Between the ZPA and the AER in the Developing Vertebrate Limb

Permanent Link: http://ufdc.ufl.edu/UFE0041555/00001

Material Information

Title: Molecular Interactions Between the ZPA and the AER in the Developing Vertebrate Limb
Physical Description: 1 online resource (104 p.)
Language: english
Creator: Bouldin, Cortney
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: apoptosis, development, fgf, limb, polydactyly, shh
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The development of a vertebrate embryo is a complex process marked by several morphogenetic events, which create a highly reproducible pattern. The vertebrate limb has emerged as a model for studying pattern formation in the embryo mainly because limb manipulations do not affect embryo survival. Within the developing limb, experimental manipulation of the embryo resulted in the identification of the classical signaling centers known as the Zone of Polarizing Activity (ZPA) and the Apical Ectodermal Ridge (AER). The molecular signals required for function of the ZPA and AER have been identified. They are Sonic hedgehog (Shh) from the ZPA and Fibroblast Growth Factors (Fgfs) from the AER. The functions of each of these molecules are now beginning to be understood. Analysis of Shh and hedgehog (Hh) signaling target genes has shown that Hh activation in the limb bud mesoderm is required for normal limb development. It has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. Contrary to previous reports, we have identified SHH protein and targets of Hh signaling present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning we removed Smoothened (Smo), an essential component of the Hh signaling pathway, from the AER. Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development. The formation of supernumerary digits, or polydactyly, is a common congenital malformation. In addition to our studies investigating the role of hedghog signaling in the AER, we characterized a spontaneous chicken mutant, known as Dorking. The hindlimbs of Dorkings form a preaxial supernumerary digit. During the early stages of limb development ectopic expression of several genes, including Sonic Hedgehog (Shh) and Fibroblast Growth Factor 4 (Fgf4), was found in Dorking hindlimbs. In addition to ectopic gene expression, a decrease in cell death in the anterior of the developing Dorking hindlimb was observed. Further molecular investigation revealed that ectopic Fgf4 expression was initiated and maintained independent of ectopic Shh. Inhibition of Fgf signaling but not hedgehog signaling was capable of restoring the normal anterior domain of cell death in Dorking hindlimbs. Our data indicate that in Dorking chickens, preaxial polydactyly is initiated independent of Shh. The sum of all findings is discussed within the context of establishing appropriate gene expression patterns, cell number and digit number in the developing limb.
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.
Statement of Responsibility: by Cortney Bouldin.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Harfe, Brian D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041555:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041555/00001

Material Information

Title: Molecular Interactions Between the ZPA and the AER in the Developing Vertebrate Limb
Physical Description: 1 online resource (104 p.)
Language: english
Creator: Bouldin, Cortney
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: apoptosis, development, fgf, limb, polydactyly, shh
Genetics (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The development of a vertebrate embryo is a complex process marked by several morphogenetic events, which create a highly reproducible pattern. The vertebrate limb has emerged as a model for studying pattern formation in the embryo mainly because limb manipulations do not affect embryo survival. Within the developing limb, experimental manipulation of the embryo resulted in the identification of the classical signaling centers known as the Zone of Polarizing Activity (ZPA) and the Apical Ectodermal Ridge (AER). The molecular signals required for function of the ZPA and AER have been identified. They are Sonic hedgehog (Shh) from the ZPA and Fibroblast Growth Factors (Fgfs) from the AER. The functions of each of these molecules are now beginning to be understood. Analysis of Shh and hedgehog (Hh) signaling target genes has shown that Hh activation in the limb bud mesoderm is required for normal limb development. It has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. Contrary to previous reports, we have identified SHH protein and targets of Hh signaling present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning we removed Smoothened (Smo), an essential component of the Hh signaling pathway, from the AER. Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development. The formation of supernumerary digits, or polydactyly, is a common congenital malformation. In addition to our studies investigating the role of hedghog signaling in the AER, we characterized a spontaneous chicken mutant, known as Dorking. The hindlimbs of Dorkings form a preaxial supernumerary digit. During the early stages of limb development ectopic expression of several genes, including Sonic Hedgehog (Shh) and Fibroblast Growth Factor 4 (Fgf4), was found in Dorking hindlimbs. In addition to ectopic gene expression, a decrease in cell death in the anterior of the developing Dorking hindlimb was observed. Further molecular investigation revealed that ectopic Fgf4 expression was initiated and maintained independent of ectopic Shh. Inhibition of Fgf signaling but not hedgehog signaling was capable of restoring the normal anterior domain of cell death in Dorking hindlimbs. Our data indicate that in Dorking chickens, preaxial polydactyly is initiated independent of Shh. The sum of all findings is discussed within the context of establishing appropriate gene expression patterns, cell number and digit number in the developing limb.
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.
Statement of Responsibility: by Cortney Bouldin.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Harfe, Brian D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041555:00001


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1 MOLECULAR INTERACTIONS BETWEEN THE ZPA AND THE AER IN THE DEVELOPING VERTEBRATE LIMB By CORTNEY MICHAEL BOULDIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE RE QUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Cortney Michael Bouldin

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3 To my wife and family

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4 ACKNOWLEDGMENTS Without the moral and material support of my wife and family, this work would have been impo ssible. For all of their love, I am eternally grateful. I offer a special thank you to my wife, Erin DeFries Bouldin, both for allowing me the space to pursue dissertation work and for tutoring me in the subtleties of statistical analysis. I thank my mentor Dr. Brian Harfe for introducing me to the fascinating field of developmental genetics. Dr. Harfes enthusiasm and unique ability to provide both support and freedom have been invaluable throughout my graduate career. I am also grateful to the members of my advisory committee of Dr. Martin Cohn, Dr. Jorg Bungert, Dr. S. Paul Oh for their guidance and support. Innumerable teachers throughout my education have inspired me to follow a career in science. In particular, I thank Mr. Terry Nusbaum for instilling an early interest in the biological sciences, and Drs. Brian Cain and Thomas Yang for introducing me to the wonders of laboratory science outside of a classroom. I thank all the members of the Harfe Lab, including but not limited to Jason Rock, Danielle M aatouk, Kyunsuk Choi, Jennifer Maier, Kendra McKee, Ben Cole, John Palsis and Bhavana Vangara, for friendship, technical assistance and countless helpful discussions. I thank the members of the Cohn lab for friendship and helpful discussion. Outside of the University of Florida, I thank Dr. Amel Gritli -Linde, Dr. William Scott, Jr., Matthew McFarlane, Dr. Steve Vokes and Dr. Andrew McMahon for conversations and sharing unpublished data, which helped to shape the progress of this work. Finally, I am indebted to Joyce Connors, Michelle Ramsey and Jenneene Spencer for their outstanding ability to manage the practical aspects of graduate work and the staff at Morningside Nature Center for access to their Dorking chickens.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 7 LIST OF FIGURES .............................................................................................................. 8 ABSTRACT ........................................................................................................................ 10 C H APT ER 1 THE MOLECULAR BASIS OF LIMB PATTERNING ................................................. 12 Sustained Outgrowth of the Vertebrate Limb ............................................................ 13 The Apical Ectodermal Ridge and the Progress Zone Model ............................ 13 A Molecular Model for Limb Outgrowth ............................................................... 15 Patterning within a Developing Vertebrate Limb ....................................................... 17 The Zone of Polarizing Activity (ZPA) and Mol ecules Expressed within the ZPA ................................................................................................................... 17 Mechanisms of Action for Shh ............................................................................. 17 2 NETWORKS OF SIGNALING WITHIN THE DEVELOPING LIMB .......................... 21 Estab lishment of the AER .......................................................................................... 21 Establishment of the ZPA ........................................................................................... 21 Interactions between the ZPA and the AER .............................................................. 22 Roles for Gene Expression Networks within the Developing Limb ........................... 23 3 THE SHH SIGNALING PATHWAY IS PRESENT AND REQUIRED WITHIN THE VERTEBRATE LIMB BUD APICAL ECTODERMAL RIDGE FOR NORMAL AUTOPOD PATTERNING ......................................................................................... 25 Results ........................................................................................................................ 27 SHH Protein and Components of the Hedgehog Signaling Pathway are Present in the AER ........................................................................................... 27 Hedgehog Signaling in the AER is Required for Formation of a Normal Autopod ............................................................................................................. 30 AER Length is Controlled by Hh Signaling within the AER ................................ 31 Hedgehog Signaling in the AER Regulates the Shh/Grem1/Fgf Feedback Loop .................................................................................................................. 32 Discussion ................................................................................................................... 33 Experimental Procedures ........................................................................................... 36 M ouse Alleles and Breeding ................................................................................ 36 Fate Mapping, -Galactosidase Detection and Immunohistochemistry ............. 36 Whole Mount RNA in situ Hybridization and AER Measurement ....................... 37

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6 Bead Implantation and LysoTracker Analysis ..................................................... 38 4 ABERRANT FG F SIGNALING, INDEPENDENT OF ECTOPIC HEDGEHOG SIGNALING, INITIATES PREAXIAL POLYDACTYLY IN DORKING CHICKENS .. 54 Introduction ................................................................................................................. 54 Results ........................................................................................................................ 56 Dorking Chickens have Partially Penetrant Preaxial Polydactyly in the Hindlimb but not in the Forelimb. ..................................................................... 56 Ectopic Hedgehog Signaling Occurs in the Anterior of Late Stage Dorking Hindlimbs .......................................................................................................... 57 Gene Expression in the AER is Expanded in Dorking Hindlimbs ...................... 58 Ectopic Fgf4 Precedes Ectopic Shh in Dorking Hindlimbs ................................. 58 Extended Expression of Fgf4 in the Dorking Hindlimb is Maintained Independent of Hedgehog Signaling ................................................................ 59 Cell Death is Decreased in the Anterior Necrotic Zone of Dorking Hindlimbs ... 60 Inhibition of Ectopic Fgf but not Hedgehog Signaling in the Dorking Hindlimb can Rescue the Reduction of Cell Death Present in the Anterior Necrotic Zone .................................................................................................... 61 Discussion ................................................................................................................... 62 Multiple Roles for FGFs in the Formation of Supernumerary Digits in Dorkings ............................................................................................................ 62 The Role of Shh in Dorking Polydactyly .............................................................. 64 Potential Genetic Causes for Dorking Polydactyly ............................................. 66 Experimental Procedures ........................................................................................... 66 Embryo Collection ................................................................................................ 66 Skeletal Preparation ............................................................................................. 67 Whole Mount RNA in situ Hybridization .............................................................. 67 RNA Isolation, cDNA Synthesis and RT -PCR .................................................... 68 LysoTracker and Drug Treatments ...................................................................... 68 5 CONCLUDING REMARKS ........................................................................................ 78 APPEN D I X A CRE RECOMBINASE EXPRESSION DRIVEN BY THE PEAK7 ELEMENT OF THE PTCH1 PROMOTER .......................................................................................... 83 B OLIGONUCLEOTIDES USED AS GENOTYPING PRIMERS .................................. 88 C PROBES USED FOR RNA IN SITU HYBRIDIZATION ............................................ 90 LIST OF REFERENCES ................................................................................................... 91 BIOGRAPHICAL SKETCH .............................................................................................. 104

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7 LIST OF TABLES Table page 3 -1 Frequency of skeletal element number in forelimbs and hindlimbs of control and mutant animals. ............................................................................................... 53 4 -1 Summary of skeletal phenotypes among 35 Dorking incross progeny. ............... 76 4 -2 Summary of Variability of Gen e Expression, Vital Dye Staining .......................... 77 B-1 Oligonucleotides used as genotyping primers. ..................................................... 89 C -1 Probes used for RNA in situ hybridiz ation. ............................................................ 90

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8 LIST OF FIGURES Figure page 3 -1 SHH and components of the hedgehog signaling pathway are present in the limb bud ectoderm .................................................................................................. 39 3 -2 SHH immuno histochemistry on E10.5 embryos ................................................... 40 3 -3 Ptch1:LacZ expression in hindlimb buds ............................................................... 41 3 -4 Smo expression in the embryo including the AER of the developing limb ........... 42 3 -5 Msx2 -Cre is expressed robustly in the AER and not in the limb bud mesoderm ............................................................................................................... 43 3 -6 Hedgehog signal transduction in the AER has a functional role in limb patterning ................................................................................................................ 44 3 -7 Ptch1 i s lost from the AER of Msx2 -Cre;Smoflox/flox limb buds at E10.5 ............... 45 3 -8 Extra condensations appear on Msx2 -Cre;Smoflox/flox limbs between E13.5 and E16.5 ............................................................................................................... 46 3 -9 Fgf8 is expressed within the morphologically distinct AER in the developing limb ......................................................................................................................... 47 3 -10 Removal of hedgehog signaling in the AER results in an increase in AER length ...................................................................................................................... 48 3 -11 Fgf4 is excluded from the posterior AER after treatment with SHH soaked beads ...................................................................................................................... 49 3 -12 Analysis of cell death in limbs that have been treated with SHH soaked beads ...................................................................................................................... 50 3 -13 Gene expression in Msx2 -Cre;Smoflox/flox limbs .................................................... 51 3 -14 Model of stochastic control of AER length by hedgehog signal transduction within the AER ........................................................................................................ 52 4 -1 Dorking chicken hindlimbs have an ectopic preaxial digit II ................................. 70 4 -2 Ectopic hedgehog signaling and Fgf expression occurs in the Dorking hindlimb .................................................................................................................. 71 4 -3 Additional ectopic gene expression in Dorkings ................................................... 72

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9 4 -4 Ectopic Fgf4 expression in the Dorking hindlimb precedes and is independent of ectopic Shh ................................................................................... 73 4 -5 Cell death is decreased in the anterior necrotic zone (ANZ) of Dorking hindlimbs ................................................................................................................. 74 4 -6 Inhibition of FGF but not Hedgehog signaling rescues cell death in the anterior necrotic zone ............................................................................................. 75 A-1 -Galactosidase activity driven by the Ptch1:LacZ allele and the Ptch1:Peak7_LacZ allele at E10.5 ........................................................................ 86 A-2 Activation of cre recombinase in tissues with both the Ptch1:Peak7CreERt2 and the R26R allele at E11.5 ................................................................................. 87

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10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MOLECULAR INTERACTIONS BETWEEN THE ZPA AND THE AER IN THE DEVELOPING VERTEBRATE LIMB By Cortney Michael Bouldin May 2010 Chair: Brian D. Harfe Major: Medical Sciences Genetics The development of a vertebrate embryo is a complex process marked by several morphogenetic events, which create a highly reproducible pattern. The vertebrate limb has emerged as a model for studying pattern formation in the embryo mainly because limb manipulations do not affect embryo survival. Within the developing limb, experimental manipulation of the embryo resulted in the identification of the classical signaling centers known as the Zone of Polarizing Activity (ZPA) and the Apical Ectoderma l Ridge (AER). The molecular signals required for function of the ZPA and AER have been identified. They are Sonic hedgehog ( Shh) from the ZPA and Fibroblast Growth Factors (Fgfs ) from the AER. The functions of each of these molecules are now beginning to be understood. Analysis of Shh and hedgehog (Hh) signaling target genes has shown that Hh activation in the limb bud mesoderm is required for normal limb development. It has been stated that Hh signaling in the limb bud ectoderm cannot occur because compo nents of the Hh signaling pathway and Hh target genes have not been found in this tissue. Contrary to previous reports, we have identified SHH protein and targets of Hh signaling present in the limb bud ectoderm including the apex of the bud. To directly

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11 test whether ectodermal Hh signaling was required for normal limb patterning we removed Smoothened ( Smo ), an essential component of the Hh signaling pathway, from the AER. Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development. The formation of sup ernumerary digits, or polydactyly, is a common congenital malformation. In addition to our studies investigating the role of hedghog signaling in the AER, we characterized a spontaneous chicken mutant, known as Dorking. The hindlimbs of Dorkings f orm a pr eaxial supernumerary digit. During the early stages of limb development ectopic expression of several genes, including Sonic Hedgehog (Shh ) and Fibroblast Growth Factor 4 (Fgf4 ), was found in Dorking hindlimbs. In addition to ectopic gene expression, a d ecrease in cell death in the anterior of the developing Dorking hindlimb was observed. Further molecular investigation revealed that ectopic Fgf4 expression was initiated and maintained independent of ectopic Shh Inhibition of Fgf signaling but not hedge hog signaling was capable of restoring the normal anterior domain of cell death in Dorking hindlimbs. Our data indicate that in Dorking chickens, preaxial polydactyly is initiated independent of Shh The sum of all findings is discussed within the context of establishing appropriate gene expression patterns, cell number and digit number in the developing limb.

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12 CHAPTER 1 THE MOLECULAR BASIS OF LIMB PATTERNING The formation of the vertebrate limb is a reproducible process that creates a complex organ. As th e limb is patterned, it must be shaped along three axes. Using ones own arm as an example, the limb forms from shoulder to digit tip along the proximal to distal axis, from the thumb to small finger along the anterior to posterior axis and from the back o f the hand to the palm along the dorsal to ventral axis. When the limb begins to grow out from the body wall, the developing limb is composed of two cell types: loosely compacted mesenchymal cells derived from the mesoderm and tightly associated epithelial cells derived from the ectoderm. By removing large pieces of the developing limb, Ross Harrison observed that the remaining mesodermal cells were capable of substituting for the removed cells (Harrison, 1918) indicating that the cells of the limb are multipotent. Separation of mesoderm from ectoderm follo wed by mixing and reshaping of the randomized mesoderm into a bud which was then covered by ectoderm resulted in the formation of limbs with multiple long bone and digit elements (MacCabe et al., 1973; Zwilling, 1964) By establishing that mesodermal cells are interchangeable, these results indicate that pattern within the limb is established by cell -to -cell signaling in a process known as regulative development. In the past two decades, a complex program of signalin g molecules has been identified which regulate the positional identity of mesodermal cells within the developing limb. The combination of a large theoretical base formed by experimental embryology and recent advances in genetics and molecular biology has r evealed much about cell signaling within the context of limb outgrowth and digit formation. This chapter

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13 will outline a framework for an understanding of limb development. As limb patterning is a vast subject, special focus will be placed on two specific r egions of the developing limb bud, the Apical Ectodermal Ridge (AER) and the Zone of Polarizing Activity (ZPA). Sustained Outgrowth of the Vertebrate Limb The Apical Ectodermal Ridge and the Progress Zone Model After the limb mesoderm is specified, outgrowth occurs. A morphologically distinct layer of epithelium that forms at the distal most extent of the limb, known as the Apical Ectodermal Ridge (AER), is required for limb outgrowth. First insight into the function of the AER during limb development came from experiments in which the AER was removed resulting in a truncated limb (Saunders, 1948) Early removal of the AER resulted in a more severe truncation than late removal, indicating that the time of exposure to the AER was critical in determining how much of the limb formed (Summerbell et al., 1973) Cells in close proximity to the AER divide rapidly (Summerbell et al., 1973) As limb growth continues, cells move further from the AER and their rate of division slows. Because of its ability to facilitate cell division, the zone of cells in close proximity to the AER was termed the progress zone. The progress zone model of proximal -distal axis formation states that cells in the progress zone sense the amount of time that cells spend in proximity of the AER. After growth removes cells from the influence of the AER, cells become specified (Summe rbell et al., 1973) Specification of cells by removal from the progress zone would suggest that targeted disruption of the cells in the progress zone at intermediate stages of development should result in loss of limb structures. In support of the progre ss zone model of limb patterning, irradiation of cells in the progress zone results in loss of

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14 intermediate structures (Wolpert et al., 1979) One interpretation of this result is that after irradiation the progress zone requires time to repopulate and during this time there are no cells excluded from the influence of the AER. After the progress zone has returned to its normal size, cells are once again excluded from the influence of the AER and cell specification continues. Recent reanal ysis of these experiments provided a unique interpretation of these results. Sox9, a homeobox containing transcription factor, is required for the condensation of cells into cartilage and the formation of endochondral bone (Akiyama et al., 2002; Barna and Niswander, 2007) Using Sox9 as a marker of chondrocyte specification irradiated limbs were shown to lose cells that already expressed Sox9 and had been specified as chondrocytes (Galloway et al., 2009) According to this interpretation of irradiating limbs, the cells that are mitotically arrested by irradiat ion are not undifferentiated occupants of the progress zone, but cells that had already been specified to form cartilage. After reanalysis of limb irradiation, the progress zone model still offers explanation for limb truncations, which are observed after AER removal (Summerbell et al., 1973) After AER removal, large amounts of cell death are observed in a region as far as 200 microns from the distal tip of the limb (Dudley et al., 2002; Rowe et al., 1982) Because the 200 microns of cells undergoing apoptosis are observed regardless of the limb stage, an alternative explanation for the limb truncation phenotypes observed after AER removal could be that cell death eliminates progenitors required to form the various segments of the limb.

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15 A Molecular Model for Limb Outgrowth On the molecular level, the function of the AER is to provide the limb with Fibroblast Growth Factors (FGFs). Placement of beads soaked in FGF protein on a developing limb bud lacking an AER rescued outgrowth (Niswander et al., 1993) Four Fgf family members are expressed in the mouse AER Fgf4, Fgf8, Fgf9 and Fgf17 (Mar tin, 1998) Removal of just two of these, Fgf4 and Fgf8, limited limb bud outgrowth (Boulet et al., 2004; Sun et al., 2002) Consistent with cell death after AER removal, genetic removal of Fgf4 and Fgf8 resulted i n cell death, but not in the distal portion of the limb (Sun e t al., 2002) Recent studies have further clarified the role of Fgfs secreted from the AER in proximal distal outgrowth. In the mouse AER, four Fgfs are expressed, but they are not expressed at the same level (Mariani et al., 2008) Because Fgfs in the AER can functionally substitute for each anot her (Lu et al., 2006) removal of multiple Fgf family members from the AER created an allelic series where developing limb buds were expos ed to a variety of FGF concentrations from the AER (Mariani et al., 2008) The extreme example, removal of both Fgf4 and Fgf8, which are expressed at the highest level, results in a failure of the limbs to grow out. When Fgf8, the most robustly expressed Fgf was reduced in combination with removal of any of the other Fgfs found in the AER, outgrowth continued with a reduction in digit number. These results indicate that in addition to being permissive for outgrowth, Fgfs expressed in the AER are instructive in limb patterning (Mariani et al., 2008) Additionally, changes in the level of FGF a ffect the expression of one member of a family of proteins known as Meis genes (Mariani et al., 2008) These experiments and others involving retinoic acid have placed the Meis genes at the center of a molecular

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16 model for limb outgrowth. Treatment of regenerating limbs with retinoic acid induces proximal identity in regenerating tissue (Maden, 1982) Synthesis of retinoic acid occurs in the proximal portion o f the limb and in the tissue adjacent to the limb, known as the flank (Mercader et al., 2000) Retinoic acid specifies limb tissue through the expression of Meis genes. When the limb is first formed, the Meis genes are expressed throughout the limb bud mesoderm. Proliferation regulated by Fg fs at the distal extent of the limbs separates cells from the influence of retinoic acid, silencing the expression of the Meis genes (Mercader et al., 2000) The developing limb can be divided into three sections. From proximal to distal, they are the stylopod, the zeugopod and the autopod. Hoxa11 and Hoxa13 have been used to mark the zeugopod and autopod (Yokouchi et al., 1991) Using Meis Hoxa11 and Hoxa13 as markers of the stylopod, zeugopod and autopod respectively, a model of proximal distal outgrowth has been proposed that involves progressive initiation and expansion of the domains of expression. This model relies on the spatial relationship of cells to Fgfs from the AER and retinoic acid from the flank for establishing each of these domains (Tabin and Wolpert, 2007) While a molecular model of proximal distal patterning represents a potential to reconcile the known data relating to proximal -distal patterning and Meis genes have the potential to be instructive in patterning (Mercader et al., 1999) Hoxa11 and Hoxa13 are not instructive. Removal of the Hox 11 paralog group results in the production of a normal limb (Wellik et al., 2002) and removal of Hoxa13 results in loss of only the first digit of the limb rather than loss of the entire autopod (Fromental -Ramain et al., 1996) Confirmation of this mechanism of proximal distal patterning will require ident ification of

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17 molecules such as the Meis genes, which are instructive in the formation of the zeugopod and autopod. Patterning within a Developing Vertebrate Limb The Zone of Polarizing Activity (ZPA) and Molecules Expressed within the ZPA As the limb grow s, anterior to posterior patterning must be established. By looking at a fully formed forelimb, a thumb distinguishes the anterior from the posterior. The anatomical differences between the anterior and posterior of an adult limb are controlled by a small portion of mesodermal tissue in the posterior of the limb bud known as the zone of polarizing activity (ZPA). When grafted to the anterior of the limb, the ZPA disrupts the asymmetric limb pattern and creates a limb with a mirror image duplication of the distal skeletal elements (Saunders and Gasseling, 1968) A vertebrate homolog of the Drosophila hedgehog gene, Sonic Hedgehog ( Shh ), was identified and found to be expressed in the ZPA (Riddle et al., 1993) Placement of cells expressing Shh into the anterior of the developing limb reproduced the mirror image digit dupli cation phenotype seen upon grafting of cells located in ZPA to the anterior of the limb bud (Riddle et al., 1993) Genetic removal of Shh from the developing limb resulted in a limb with post erior defects (Chiang et al., 1996) indicating that Shh is required for the function of the ZPA and proper anterior to posterior patterning of the developing limb. Mechanisms of Action for Shh After the discovery of the ZPA, anterio r to posterior patterning of the vertebrate digits was proposed to be established by a gradient of morphogen diffused from the ZPA (Wolpert, 1969) Cells exposed to high concentrations of morphogen would be established as posterior and cells exposed to low concentrations would be patterned as

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18 anterior. Consistent with the formation of a morphogen gradient, placement of increasing numbers of cells from the ZPA into the anterior of the limb created digit duplications of increasing severit y (Tickle, 1981) Additionally, increasing concentrations of SHH in the anterior of a developing limb were capable of inducing mirror image duplic ations with increasing numbers of digits with increasingly posterior identity (Yang et al., 1997) A morphogen model of Shh function requires that SHH be secreted to establish a gradient of SHH protein that can be interpreted by the cells of the limb field to specify different cell fates (Wolpert, 1969) Through a series of immunohistochemical analyses, a gradient of SHH protein has been identi fied in the posterior of the developing limb (Gritli -Linde et al., 2001) Further, analysis of Shh expression and targets of hedgehog signaling revealed that Shh activated targets of the hedgehog signaling pathway s everal cell layers away from the domain of expression (Lewis et al., 2001) To identify the fate of cells from the ZPA, cells that have expressed Shh were traced throughout limb development (Harfe et al., 2004) Descendants of Shh expressing cells were found to give rise to the posterior half of the limb. Labeling Shh expressing cells later in development revealed that lateexpressing Shh descendants were restricted more post eriorly and gave rise to a smaller portion of the limb. Comparison of cells labeled at early and late stages revealed that cells in the posterior of the limb were exposed to SHH for longer amounts of time than more anterior cells. Since cells that give ris e to the posterior most digits are exposed to SHH for the longest amount of time, a model was proposed whereby increasingly posterior digits are specified by increasing time of exposure to SHH (Harfe et al., 2004)

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19 If the duration of SHH exposure dictates posterior digit patterning, deletion of Shh at progressively earlier time points should result in progressive loss of digits from the posterior of the limb. In mice limbs, deletion of Shh at progressively earlier t ime points resulted in concurrently more severe digit loss (Zhu et al., 2008) However, when the skeletons of the limbs were analyzed, the digits that were lost were found to be medial digits rather than posterior digits. Shh deletion at progressively earlier time points resulted in loss of digits in an order reverse of ossification (Zhu et al., 2008) The interpretation of this data is that only when the limb field is large enough can cells ossify to form digits. Because Shh is capable of controlli ng growth through modulating the expression of genes, which promote the G1-S transition (Roy and Ingham, 2002) limbs that lose Shh may no longer contain enough cells to form the appropriate number of digits. These data i ndicate that one of the primary roles of Shh is to regulate growth and limb field expansion. Similar to the data uncovered in mice, the data from Towers, et. al. using the chick model system revealed an involvement for Shh in the regulation of growth in th e chick limb. Increased Shh signaling increased expression of cyclin genes, which resulted in increased cell division (Towers et al., 2008) However, in contrast to the results obtained from mouse, the data from Towers et. al. reveal a continued late involvement of Shh in patterning. By blocking cell cycle progression within the limb, Towers et. al. demonstrated that only a single digit formed. However, the identity of the single digit mo re closely resembled a digit with a posterior identity. In these experiments, Shh was still capable of affecting pattern and promoting posterior digit identity in the absence of cell proliferation in the limb (Towers et al., 2008)

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20 Currently, many questions remain about the function of Shh in anterior to posterior pattering of the limb. For example, it is unclear why the studies in the chick reveal a role for Shh in both patterning and growth, while in the mouse limb Shh appears to be mainly required for growth. Because digit identity in the mouse is more difficult to assign than in the chick, one possibility is that the digit identity in the mouse experiments are variable, or misassigned. This seems unlikely because several methods were used to check digit identity (Zhu et al., 2008) A second possibility is that there are differences in the abilities of mouse limbs and chick limbs to respond to Shh In Chapter 3, we identify an essential role for Shh signaling in the ectoderm of the developing limb. A key in reconciling the function of Shh into a model will be separating the roles of Shh in the mesoderm and the ectoderm, and characterizing the role this signaling pathway plays in each individually.

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21 CHAPTER 2 NETWORKS OF SIGNALIN G WITHIN THE DEVELOPING LIMB Throughout development, the limb bud integrates inf ormation from multiple signaling pathways for proper patterning. This chapter will focus on the interactions between these signaling molecules and the effect of their temporal and spatial regulation on limb development. Establishment of the AER The initiat ion of limb outgrowth results from a series of epithelial mesenchymal interactions mediated by FGF and WNT proteins. Expression of Fgf10 in the lateral plate mesoderm elicits a response in the overlying ectoderm (Ohu chi et al., 1997) The ectoderm responds by activating wnt3a (Kawakami et al., 2001; Kengaku et al., 1998; Ohuchi et al., 1997) At the boundary between the dorsal and ventral limb bud ectoderm, the AER is formed (Altabef et al., 1997) Formation of the AER then initiates Fgf8 expression (Kawakami et al., 2001; Kengaku et al., 1998; Ohuchi et al., 1997) Without Fgf8 expression, Fgf10 in the mesenchyme cannot be maintained (Boulet et al., 2004; Sun et al., 2002) Through interactions between FGF and WNT proteins, the AER is properly positioned and maintained by Fgf10 from the underlying mesoderm. Establishment of the ZPA The expression of Shh in the ZPA is similarly induced through interaction between multiple signaling pathways. Retinoic acid is a known inducer of Shh e xpression (Riddle et al., 1993) The involvement of retinoic acid in regulating Shh expression was further confirmed by inhibition of either retinoic acid synthesis (Stratford et al., 1996) or retinoic acid reception (Helms et al., 1996) which results in loss of Shh expression. Because retinoic acid induces expression of Hox genes including Hoxb8 (Charite et al., 1994; Lu

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22 et al., 1997) retinoic acid was proposed to induce expression of Shh with Hoxb8 acting as an intermediate. However, genetic removal of Hox 8 paralogs does not block expression of Shh (van den Akker et al., 2001) suggesting that some factor other than Hoxb8 is required for Shh expression. Another molecule induced by retinoic acid is Hand2 (Fernandez -Teran et al., 2000) Genetic removal of Hand2 results in the loss of Shh (Charite et al., 2000) Because Hand2 is dependent on Fgf expression (Fernandez Teran et al., 2000) the intersection of retinoic acid, Hand2 expression and Fgf expression initiates expression of Shh (Cohn, 2000) Interactions between the ZPA and the AER After the AER is induced and the ZPA initiates Shh expression, interactions between SHH from the mesenchyme and FGFs from the AER are required to maintain gene expression from these two signaling centers. Implantation of a source of SHH in the anterior of the developing limb increased expression of Fgf4 in the AER (Laufer et al., 1994) and placement of a source of FGF in the posterior of the limb (Yang and Niswander, 199 5) but not the anterior of the limb (Niswander et al., 1994) expanded Shh expression. Additionally, genetic removal of Fgf8 reduces the expression of Shh (Lewandoski et al., 2000) and genetic removal of Shh reduces the expression of Fgf4 (Chiang et al., 2001) Interactions between Shh and Fgfs are mediated by Grem1 Grem1 expression is eliminated in ld mutants (Zuniga et al., 2004) Analyis of ld mutant limbs revealed reduced expression of Shh and Fgf4 (Haramis et al., 1995) Incorporation of Grem1 into the interactions between SHH and Fgfs lead to the initial description of the Shh/Grem1/Fgf feedback loop, in which SHH signals to Grem1 which then signals to Fgfs in the AER in a positive manner.

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23 Because Grem1 is an antagonist of Bone Morphogenetic Proteins ( Bmps ), Bmps are also involved in the Shh/Grem1/Fgf feedback loop. Three Bmps are expressed in the limb bud: Bmp2, Bmp4 and Bmp7 (Robert, 2007) Genetic removal of a Bmp receptor from the AER results in loss of outgrowth (Pajni -Underwood et al., 2007) Removal of two Bmp ligands specifically from the AER of the developing limb resulted in outgrowth of the limb but defects in limb patterning (Maatouk et al., 2009) In contrast to limb buds that lack Bmp receptor in the AER, which fail to grow out, limbs missing Bmp ligands in the AER could be analyzed for changes in Grem1 expression. Consistent with a role for Bmps within the AER mediating interactions with Grem1, Grem1 expression was reduced concomitant with a reduction of Bmps in the AER (Maatouk et al., 2009) Roles for Gene Expression Networks within the Developing Limb Formation of new outgrowths from the embryo require the establishment of tissue within three dimensions. Interaction between multiple signaling pathways allow unique mechanisms to control pattern within the limb bud. For example, expansion of different domains of gene expression in the mouse and chick create physical boundaries for the various signaling molecules of the limb. As expression domains are separated, the Shh/Grem1/Fgf regulatory loop breaks down in the chick limb beginning with Grem1 (Scherz et al., 2004) and in the mouse limb beginning with Fgf4 (Verheyden and Sun, 2008) From these initially reduced domai ns of expression the whole regulatory loop is down-regulated. Overlapping regulation through networks of signaling pathways ensures the appropriate termination of gene expression in the developing limb. Another potential reason for regulatory networks is t hat these interactions exist to correct any stochastic changes in gene expression that might occur throughout development of the limb. For example, Grem1 interacts with Bmp4 (Benaz et et al.,

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24 2009) By analyzing the time required to induce changes in gene expression, Grem1 and Bmp4 interactions were identified as a separate regulatory loop within the Shh/Grem1/Fgf feedback loop. Using multiple regulatory loops within the limb bud, v ariations in gene expression between limbs, individuals and environmental conditions are overcome and a limb with a normal pattern is formed (Mackem and Lewandoski, 2009) Within this work, we present evidence for both the coordination of gene expression as a mechanism for regulating the proper patterni ng of the limb within three dimensions (Chapter 3 and 4) and evidence for a regulatory loop to fine-tune levels of gene expression within the limb (Chapter 3).

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25 CHAPTER 3 THE SHH SIGNALING PA THWAY IS PRESENT AND REQUIRED WITHIN THE VERTEBRATE LIMB BUD APIC AL ECTODERMAL RIDGE FOR NORMAL AUTOPOD PATTERNING Within the developing limb bud, exposure to SHH controls number and identity of digits produced. Proper digit pattering requires both the establishment of a concentration dependent gradient of SHH (Yang et al., 1997) and a time dependent gradient of exposure to SHH (Ahn and Joyner, 2004; Harfe et al., 2004) The ability of SH H to pattern the forming digits is coupled to the ability of SHH to control cell proliferation (Towers et al., 2008; Zhu et al., 2008) and a full complement of digits requires tight control over the concentration and timing of Shh expression. Although SHH is capable of limiting its own expression (Sanz -Ezquerro and Tickle, 2000) the molecular mechanism responsible for sensing the amount of SH H from the ZPA remains unknown. Through both short -range and long-range signaling (Goetz et al., 2002) secretion of SHH pro tein from Shh producing cells activates a cellular signal transduction pathway. Binding of SHH protein to the transmembrane protein PTCH1 activates the Hh signaling pathway (Fuse et al., 1999) Once SHH binds to PTCH1, inhibition of another transmembrane bound protein, SMO, is relieved (Murone et al., 1999) Through a complex of proteins, SMO modulates proteolytic cleavage of members of the Ci/Gli family of transcription factors (Hooper and Scott 2005) GLI transcription factors then directly activate transcription of hedgehog target genes, such as Gli1 and Ptch1 (Vokes et al., 2007; Vokes et al., 2008) Within the limb, SHH is involved in a network of p ositive gene interactions known as the Shh/Grem1/Fgf feedback loop. SHH is capable of upregulating Fgf4 expression

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26 in the AER (Lauf er et al., 1994) Conversely, changes in FGF4 can upregulate Shh expression (Yang and Niswander, 1995) The Bmp antagonist, Gremlin1, is an intermediate for the interactions between Shh and Fgf4 (Khokha et al., 2003; Panman et al., 2006) Spatial disruption of the positive interactions of the Shh/Grem1/Fgf feedback loop has been shown to terminate feedback loop gene expression (Scherz et al., 2004; Verheyden and Sun, 2008) Regulation of the Shh/Grem1/Fgf feedback loop is essential for proper patterning of the limb. Robust regulation of genes involved in the loop is mediated by Gremlin1 and Bmp4 (Benazet et al., 2009) In addition to mutual positive interactions within the Shh/Grem1/Fgf feedback loop, the Etv4/5 genes, which are targets of Fgf signaling, are required for prop er spatial restriction of Shh in the limb bud mesenchyme (Mao et al., 2009; Zhang et al., 2009) Although coordinated regulation of limb development by interactions between the ZPA and AER have been documented (Niswander, 2002) the role Hh signaling may play within the limb bud ectoderm is unknown. This is due in part to reports that components required for Hh signaling are not found in the ectoderm of the vertebrate limb bud (Pearse et al., 2001; Quirk et al., 1997) Recently, the treatment of limb buds with the teratogen acetazolamide has uncovered a potential role for Hh signaling in the limb ectoderm. In one study, in utero exposure to acetazolamide r esulted in limbs with ectrodactyly, phenotypically similar to limbs mutant for Shh However, Shh expression in treated limbs was not substantially different from control limbs suggesting that loss of digits in these animals may instead have resulted from d efects in the ability of cells to respond to SHH (Bell et al., 1999) A series of grafting experiments suggested that the limb bud ectoderm and not the mesoderm was defective in Hh signaling in these

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27 experiments (Bell et al., 1999) Microarray experiments in which components of the Hh signaling pathway, but not Shh were identified in the limb bud ectoderm provided further evidence that the Hh signaling pathway may be present in the limb bud ectoderm (Bell et al., 2005) In this report, we demonstrate that SHH protein is present in the limb bud ectoderm and the Hh signaling pathway is required for normal autopod patterning to occur. Removal of Smo an essential component of Shh signal transduction, resulted in the disruption of normal digit patterning and formation of additional cartilaginous condensations. Through analysis of mice that lacked Hh signal transduction in the AER and over expression experiments using the chick model system, we show that the length of the AER is regulated by the Hh signaling pathway. Additionally, Hh signaling within the AER is necessary to refine the expression of genes involved in the Shh/Grem1/Fgf fe edback loop. These results uncover a novel role for Hh signal transduction within the vertebrate limb ectoderm and provide further insight into the molecular mechanism responsible for coordinating interactions between the ZPA and AER. Results SHH Protein a nd Components of the Hedgehog Signaling Pathway are Present in the AER The secreted protein SHH is produced in the mesenchyme of the limb bud ZPA. Once produced, SHH can spread many cell diameters from its source (Goetz et al., 2002) For the Hh signaling pathway to be active in the ectoderm of the developing limb, SHH protein should be present in this tissue. To investigate S HH protein localization in the limb bud ectoderm, immunohistochemistry using an antibody raised against SHH

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28 was performed. At E10.5, SHH immunostaining was observed in both the posterior mesoderm and the posterior ectoderm of the limb bud including the AE R (Fig. 3 1A -B) (Gritli -Linde et al., 2001) SHH immunostaining was not present in the anterior mesoderm or ectoderm (Fig.31C). The two additional Hh homologs present in vertebrates, Dhh and Ihh are not expressed concomitant with Shh in the limb bud (Bitgood and McMahon, 1995) To confirm that the staining observed was specific for SHH protein, a section from a limb bud that lacked SHH ( Shh / ) was analyzed in a manner identical to wild type limb buds. Shh / were found to lack immunostaining in both the mesoderm and the ectoderm (Fig. 3-1D). Sections through different regions of the embryo and limb bud confirmed immunoreactivity of SHH in the AER and specificity of the antibody to regions of the embryo known to express SHH (Fig. 3-2). If Hh signaling is active within a given tissue, transcriptional targets of this pathway should be present. Active Hh signaling transcriptionally activates expression of the transcription factor Gli1 (Marigo et al., 1996a) Using a tamoxifeninducibl e Gli1CreERT2 allele (Ahn and Joyner, 2004) in combination with the cre-inducible R26R reporter allele (Soriano, 1999) we fate mapped cells that activate Gli1 in the mouse limb. Administration of tamoxifen at E9.5 resulted in detection of -galactosidase activity in the autopod and zeugopod including ectoderm in embryos at E12.5 (Fig. 31E -G) (Ahn and Joyner, 2004) To determine if the ectodermal staining observed in Gli1CreERT2; R26R embryos required SHH, the cells activating Gli1 were analyzed in Shh null ( Shh / ) embryos. In Shh null limb buds the ectoderm was negative for galactosidase activity (Fig. 3 -1H & I) indicating that ectodermal Gli1 expression required

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29 activation of the Hh signaling pathway by SHH. Cells surrounding the early stage cartilaginous skeletal elements in the limb mesoderm activated the Hh signaling pathway in the absence of SHH indicating Gli1 expressing cells were present in this region of the limb (Fig. 3 1H). This staining is likely the result of IHH activation of the Hh signaling pathway since Ihh is expressed in chondrocytes (Bitgood and McMahon, 1995; St -Jacques et al., 1999) These data indicate that ectodermal cells have activated the Hh signaling pathway in response to SHH produced from the ZPA. To confirm that Hh signaling is present in the limb bud ectoderm we analyzed a second Hh pathway target gene, Ptch1 (Marigo et al., 1996b) Mice with a LacZ cassette inserted downstream of the endogenous Ptch1 promoter produce galactosidase activity in cells that have activated transcription from the Ptch1 locus (Goodrich et al., 1997) In t he limbs of embryos containing the Ptch1:LacZ allele, galactosidase activity was detected at E11.5 in the posterior limb bud ectoderm including the posterior AER (Fig. 3-1J & K). Further analysis revealed that galactosidase activity initially appeared punctate in the posterior of the limb including the ectoderm at E10.0 with robust expression in the ectoderm observed from E10.5 through E12.5 (Fig. 33). Sectioned limbs contained galactosidase activity in both the posterior mesoderm and ectoderm (Fig. 3 1L). Examination of sections revealed detectable galactosidase activity in the posterior AER, the posterior dorsal and ventral ectoderm, and the ectoderm of the posterior margin (the space from the posterior AER to the flank; Fig. 3 1L and 3-3). These data indicate that SHH is present and capable of activating the Hh signaling cascade within the limb bud ectoderm.

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30 Hedgehog Signaling in the AER is Required for Formation of a Normal Autopod Although Hh signal transduction was found in several regions of t he ectoderm, we chose to focus specifically on activation of the Hh signaling pathway in the AER because of the importance of the AER in limb development. SMO, a transmembrane protein required for active Hh signaling (Zhang et al., 2001) was detected in the limb bud ectoderm at E11.5 (Fig. 34). The Msx2 -Cre allele expresses cre recombinase in the AER but not in the limb mesoderm (Fig. 3 -5) (Barrow et al ., 2003) o determine if Hh signaling in the AER played a functional role in limb patterning, we removed Smo from the AER using Msx2 -Cre and a conditional allele for Smo (see Experimental Procedures for details). Embryos that were homozygous for the Smof lox allele and contained the Msx2 -Cre transgene have lost SMO from the AER. As a result of SMO loss, Hh signaling was absent. Loss of Hh signaling in the ectoderm of embryos in which Smo was removed from the AER was confirmed by performing Ptch1 in situ h ybridizations using BM purple as a substrate (see Experimental Procedures). Whole mount RNA in situ hybridizations to detect Ptch1 at E11.5 revealed expression in the posterior AER of control limbs but not in the AER of Msx2 -Cre;Smoflox/flox limbs at the s ame somite stage (ss) (Fig. 3 -6A D). Sectioned tissue revealed that in Msx2 -Cre;Smoflox/flox embryos at E10.5 (Fig. 37) and E11.5 (Fig 3-6E & F) Ptch1 mRNA was absent from the AER and ventral ectoderm. Ptch1 was still expressed in the dorsal ectoderm in addition to the ectoderm proximal to the posterior AER. In situ hybridization with Ptch1 riboprobe indicated that Msx2 Cre;Smoflox/flox limbs lacked the ability to activate the Hh signaling pathway within the AER.

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31 To determine whether Hh signaling within the AER played a functional role in the establishment of limb pattern, Msx2 -Cre;Smoflox/flox and wild type skeletons were examined. Skeletal analysis revealed that the stylopodal and zeugopodal elements of both Msx2 -Cre;Smoflox/flox and control limbs were normal. The autopods of the hindlimbs and forelimbs of Msx2 -Cre;Smoflox/flox animals exhibited extra cartilaginous condensations (Fig. 3-6G -N), which were highly penetrant (Table 3-1). Extra cartilaginous condensations were investigated using both Sox9 expre ssion and bright field microscopy, and were found to be formed between E13.5 and E16.5 (Fig. 3-8). These data indicate that Hh signaling within the AER is necessary for the formation of a properly patterned limb autopod. Msx2 -Cre;Smoflox/flox mice die at birth, which limited our ability to perform a detailed analysis of extra digit condensations. AER Length is Controlled by Hh Signaling within the AER Signals from the ZPA have been postulated to control the length and shape of the AER (Lee and Tickle, 1985; Todt and Fallon, 1984) To investigate if Hh signaling within the limb bud ectoderm regulated the length of the AER, Fgf8 expression was examined and compared in Msx2 -Cre;Smoflox/flox and control limbs. Fgf8 is ex pressed throughout the entire AER and can be used to measure the length of this structure (Fig. 3 -9) (Crossley and Martin, 1995; Mahmood et al., 1995) The Straighten plugin for NIH ImageJ was used to convert the c urved AER into a straight -line, which was then measured (see Methods). Somite matched Msx2 -Cre;Smoflox/flox forelimbs (50ss, n=9, Fig. 3 10B) and hindlimbs (50ss, n=10, Fig. 310D), and control forelimbs (50ss, n=4, Fig. 3 10A) and hindlimbs (50ss, n=7, Fi g. 3 -10C) were collected and analyzed for Fgf8 expression. Stained AERs were then straightened and measured (Fig. 3-10E). AERs in which Hh signaling was removed were found to be on average 700 microns (forelimbs)

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32 and 260 microns (hindlimbs) longer than nor mal limbs at the same somite stage. The means were compared using a t test (Welchs, unpaired), which revealed the increase in AER length in the Msx2 -Cre;Smoflox/flox limbs to be statistically significant (forelimbs, p = 0.03 and hindlimbs, p < 0.0001). Ou r experiments using the mouse model system indicated that loss of Hh signaling from the AER resulted in an increase in AER length. Using the chick model system, we investigated the effects of elevated amounts of Hh protein on AER length. A SHH -soaked bead placed just anterior to the normal ZPA was used to expand the domain of SHH protein (Fig. 3-10F). After 24 hours of treatment with a SHH -soaked bead, the length of the AER was decreased compared to the untreated contralateral limb buds or PBS treated limb buds as measured by Fgf8 expression (3 of 4 treated limbs; Fig. 3 -10G -J) and Fgf4 expression (Fig. 3-11). Consistent with published reports (Sanz -Ezquerro and Tickle, 2000) placemen t of a SHH -soaked bead in the posterior limb bud mesenchyme resulted in an increase in cell death in both the AER and the mesoderm of the limb bud (Fig. 3 12) but did not cause defects in the patterning of limb skeletal elements indicating that the limb bu d recovers from transient exposure to elevated levels of SHH (data not shown). Hedgehog Signaling in the AER Regulates the Shh/Grem1/Fgf Feedback Loop The expression of the genes involved in the Shh/Grem1/Fgf feedback loop is dynamic and dependent on the timing and levels of other components in the regulatory network (Benazet et al., 2009; Panman et al., 2006; Scherz et al., 2004; Verheyden and Sun, 2008) To investigate the effect of Hh signaling within the AER o n the components of the Shh/Grem1/Fgf feedback loop, double RNA in situ hybridizations for Shh and Fgf4 or Fgf8 were performed on normal and Msx2 -Cre;Smoflox/flox embryos. Fgf4

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33 expression was elevated in Msx2 -Cre;Smoflox/flox limbs compared to wild type li mbs (Fig. 3 -13A & B). Expression of Fgf8, which interacts with Fgf4 (Lewandoski et al., 2000) was prolonged in Msx2 -Cre;Smoflox/flox limbs (Fig. 3 -13G & H). Consistent with a link between Fgfs and the expression of Shh (Lewandoski et al., 2000; Sun et al., 2000) loss of Hh signaling in the AER resulted in increased expression of Shh in the underlying mesenchyme for longer amounts of time than in somite matched control limbs (Fig. 313A -D). Analysis of Msx2 -Cre;Smoflox/flox limbs for Gremlin1 expression revealed that the domain of expression was more broad when compared to somite matched control limbs (Fig. 3-13E & F). These data indicate that proper regulation of the Shh/Grem1/Fgf feedback loop requires activation of the Hh signaling pathway in the limb bud AER. Discussion Within the vertebrate limb mesoderm, several different mechanisms of patterning have been reported for SHH. SHH acts through a concentration gradient (Yang et al., 1997) and a temporal gradient (Harfe et al., 2004) to establish both digit identity and number. Additionally, through regulation of genes that promote the G1-S transition, SHH affects limb patterning by controlling growth (Towers et al., 2008; Zhu et al., 2008) In this study, we present evidence that Hh signaling is present in the AER and is required within the AER for correct patterning of the vertebrate autopod. Activation of the Hh signaling pathway was detected in several domains within the posterior ectoderm of the developing limb. Because Shh expression has not been detected by RNA i n situ hybridization (Riddle et al., 1993) nor microarrays (Bell et al., 2005) in the limb bud ectoderm, Hh signals to this tissue through a paracrine mechanism. Targets of the Hh signaling pathway were found in the AER, the dorsal and

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34 ventral ectoderm, and the ectoderm of the posterior margin (ectoderm between the AER and the flank). After removal of Hh signaling from the AER, Hh signaling remained in ectodermal cells juxtaposed to the posterior AER. The lack of a stronger phenotype upon removal of Hh signaling from the AER may be due to the presence of Hh responding cells in the posterior margin. Active hedgehog signaling in this region of the limb bud may be able to compensate for a loss of Hh signaling in the AER. Removal of Hh signaling from both the AER and the posterior margin has the potential to result in a more significant expansion of the AER and an increase in the severity of polydactyly. Up on removal of Hh signaling from the AER, we observed changes in gene expression of components of the Shh/Grem1/Fgf feedback loop. Previously, Shh expression in the ZPA was found to be regulated by Fgfs expressed in the AER (Lewandoski et al., 2000; Lu et al., 2006) In addition, Gremlin1 expression in the mesoderm is controlled by SHH (Nissim et al., 2006; Vokes et al., 2008) In our experiments, removal of Hh signaling from the AER increased the length of the AER, resulting in an increase in the number of cells capable of expressing Fgfs Because recombinase expression was specific to the ectoderm, the observed alterations in components of the Shh/Grem1/Fgf feedback loop are predicted to result from an increase in the length of the AER caused by a loss of Hh signal transduction within this tissue. The amount of SHH produced in the limb bud has been shown to control the number of Shh expressing cells in the ZPA through cell death (Sanz Ezquerro and Tickle, 2000) Consistent with the observations of Sanz Ezquerro et. al., our experiments increasing SHH in the posterior of the chick limb induced cell death. In

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35 addition to an increase in cell death, we identified a reduction in AER length, detected by Fgf8 and Fgf4, expression after elevating SHH protein levels in the limb bud mesenchyme. Our experiments, in which we either genetically removed Hh signaling fr om the AER or ectopically elevated SHH protein in the limb mesenchyme, indicate that Hh signaling in the AER refines the length of the AER. We propose that changes to the AER in response to the Hh signaling pathway during normal limb development refine the initial levels of SHH produced by the ZPA through changes induced by Fgf expression. During normal limb development, the initial levels of SHH produced by the ZPA is likely to vary between embryos and possibly even within the limbs buds of a given embryo. To ensure normal patterning, the pool of Shh expressing cells has to be refined to produce an exact and reproducible amount of SHH in every limb bud. We propose that during normal development, if the original pool of cells in the ZPA produces too much SHH there is a corresponding increase in Hh signaling in the AER resulting in a decrease in AER length. SHH producing cells are then removed from the ZPA (Sanz Ezquerro and Tickle, 2000 ). If too little SHH is produced, Hh signaling in the AER decreases, resulting in an increase in AER length and a corresponding increase in SHH production in the ZPA (see model in Fig. 3 -14). In our model, the ability of the AER to directly respond to SH H facilitates the production of a reproducible concentration of SHH in every limb bud within a given species. In normal limbs, the proposed changes in the length of the AER and SHH produced from the ZPA are most likely very subtle since large alterations i n either SHH protein or AER length would result in defects in autopod patterning.

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36 Regulation of Shh expression in the mesoderm of fins and appendages has been identified as a potential source of morphological change over the course of evolution (Dahn et al., 2007) We propose that the response to Hh signaling within the ectod erm could also function as a source of evolutionary change. Since Hh signaling in the ectoderm is required for patterning the autopod, it is possible that by changing the ability of limb bud ectodermal cells to respond to SHH, digit number could be altered during evolution. Experimental Procedures Mouse Alleles and Breeding The Shh, Gli1 CreERT2, R26R Ptch1:LacZ, Msx2 -Cre and Smoflox alleles were maintained and genotyped as previously described (Ahn and Joyner, 20 04; Chiang et al., 1996; Goodrich et al., 1997; Soriano, 1999; Sun et al., 2000; Zhang et al., 2001) Genotyping was performed on DNA isolated from tail or yolk sack tissue. Embryos harvested at E12.5 or earlier were staged by counting somites. For experi ments involving embryos lacking Smo in the AER, control embryos lacked the Msx2 -Cre allele and were either heterozygous or homozygous for the Smoflox allele. Both genetic combinations were phenotypically indistinguishable from normal mice. Alleles were mai ntained on mixed backgrounds, and animals were handled in accordance with the University of Florida IACUC. Fate Mapping, -Galactosidase Detection and Immunohistochemistry Tamoxifen was administered in corn oil (Sigma C 8267) at a final concentration of 2 0 mg/ml by oral gavage as previously described (Ahn and Joyner, 2004) For whole mount detection of -galactosidase activity, embryos were harvested and fixed

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37 overnight in 0.2% formaldehyde. After fixation, -galac tosidase activity was detected as previously described (Harfe et al., 2004) For galactosidase detection on sections, embryos were fixed and cryoprotected with 30% Sucrose in PBS. After embedding in O.C.T. compou nd (Sakura 4583), cryoprotected embryos were sectioned at 12 microns. Slide mounted tissue was washed two times with PBS before being incubated overnight at 37 C in staining solution [1 mg/ml X -gal, 5 mM K3Fe(CN)6 and 5 mM K4Fe(CN)6 in concentrated rinse buffer (0.1 M NaH2PO4, pH 7.4, 2 mM MgCl2, 0.2% Igepal, 0.1% sodium deoxycholate)]. Stained tissue was rinsed three times with PBS, dehydrated and mounted with Permount (Fisher SP15-500). SHH immunohistochemistry was performed as previously described (GritliLinde et al., 2001) Whole Mount RNA in situ Hybridization and AER Measurement Embryos harvested for in situ hybridization were incubated overnight in 4% formaldehyde at 4 C. Section and whole mount RNA in sit u hybridizations were performed according to a standard protocol (Wilkinson and Nieto, 1993) with the exception that BM Purple (Roche, 1442074) was used in place of NBT and BCIP to develop Ptch1 in situs Smo riboprobe (used in Fi g. 3-4) synthesis was performed on a plasmid constructed by RT -PCR using the following primers: forward, 5 ttcccagggttgaagacag, reverse, 5 cacgttgtagcgcaaag. AER length was calculated by importing pictures into NIH ImageJ (Rasband, 1997 -2007) A calibration slide was measured at the same magnification to set the scale. The Straighten plugin was used to turn the c urved AERs into measurable straight -lines (Kocsis et al., 1991) The mean AER length of Msx2 -Cre;Smoflox/ flox forelimbs (50ss, n=9) and hindlimbs (50ss, n=10),

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38 and control forelimbs (50ss, n=4) and hindlimbs (50ss, n=7) was collected, and a t -test (Welchs, unpaired) was used to test the significance ( = 0.05). Bead Implantation and LysoTracker Analysis Af fi -gel beads (Bio Rad, 153-7302) were soaked in hrSHH (1mg/mL in PBS; R & D Systems, 1845-SH) or PBS for at least one hour. Chicks were staged according to the staging guide by Hamburger and Hamilton (Hamburger and Hamilton, 1951) Eggs w ere fenestrated with laminectomy forceps. At HH stage 20, the amnionic raphe was separated and a bead was transferred to the egg using watchmakers forceps. Next, a microdissection needle was used to create a small hole in the mesenchyme proximal to the AE R in a region just anterior to the ZPA. The bead was then inserted into the space created by the microdissecting needle, and the embryos were placed at 37 C overnight. Embryos were harvested after 24 hours of incubation and fixed in 4% formaldehyde at 4 C overnight before further analysis. LysoTracker (Molecular Probes, L7528), an acidophilic dye which accumulates in the lysosmes of apoptotic cells and phagocytic cells engulfing apoptotic cells (Zucker et al., 1999) was used as previously described (Bouldin and Harfe, 2009)

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39 Figure 31. SHH and components of the hedgehog signaling pathway are present in the limb bud ectoderm. Shh immun ohistochemistry. A) A t low magnification, SHH is detected in a graded fashion specificall y in the posterior of the limb. B) A t high magnification, SHH was visible in the posterior limb bud ectoderm including the AER. C) SHH was not visible in the anterior limb bud ectoderm In B & C, the arrows highlight the AER, and the white dashed lines separate t he mesoderm from the ectoderm. D) Limb bud tissue from a Shh / limb, which was mounted on the same slide as the tissue shown in images A -C, ha d no detectable SHH. Fate mapping of cells responding to Hh signal transduction in limbs exposed to tamoxifen at E 9.5 and harvested at E12.5. S ectioned limbs of Gli1 -CreERT2; R26R embryos showed descendants of cells responding to Hh signal transduction in the posterior mes oderm and pos terior ectoderm of the E) autopod and zeugopod F) at low magnification and G) high magnification. Sectioned Shh / limbs containing the Gli1 -CreERT2 and R26R alleles contained no cells that had responded to Hh signaling in the ectoderm of th e zeugopod H) at low magnification and I) high magnification. -galactosidase activity in limbs containing the Ptch1:LacZ allele Whole mount hindlimbs at E11.5 showed active Hh signaling in the posterior limb mesoderm and ectoderm including the AER J) at low magnification and K) high magnification. L) Sectioned hindlimbs confirmed the presence of galactosidase activ ity in the posterior ectoderm ( arrowhead denotes staining in the ectoderm). The plane of section of L is illustrated by a black line in K. Im ages in A -D provided by Amel -Gritli Linde and images in E -I provided by Sohyun Ahn.

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40 Figure 32. SHH immunohistochemistry on E10.5 embryos. To confirm the ectodermal immunoreactivity of SHH and the specificity of the antibody, limb buds were analyzed fro m different planes of section. Embryos at E10.5 cut at the transverse plane of section showed immunoreactivity of SHH in several tissues known to be positive for SHH including the gut (G), the notochord (N), the neural tube (NT) and the posterior region of the limb including the ectoderm A) at low magnification and B) high magnification. Similarly, embryos at E10.5 cut on an oblique plane of section showed the immunoreactivity of SHH in tissues known to express Shh and in the posterior region of the limb inc luding the ectoderm C) at low magnification and D) high magnification The box in C is enlarged in D. The arrow in D highlights SHH immunostaining in the AER. Images provided courtesy of Amel Gritli Linde.

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41 Figure 33. Ptch1:LacZ expression in hindlimb b uds. To expand our analysis of Ptch1 expression in the limb bud ectoderm, we analyzed Ptch1:LacZ embryos at various stages. A) At E10.0, -Galactosidase activity was punctate in the post erior of the developing limb at low magnification. B) At a higher magn ification -Galactosidase activity was present in the posterior of the developing limb including the ectoderm (highlighted by an arrowhead). At low magnification C) in E10.5 and E) E11.5 llimbs, -Galactosidase activity was present in the posterior of the limb. At h igh magnification in D) E10.5 and F) E11.5 robust staining was visible in the ectoderm ( highlighted by an arrowhead). G) At E12.5, -Galactosidase activity was no longer posteriorly restricted in the mesoderm and was found surrounding proliferating chondrocytes, a known source of Ihh H) At high magnification in the E12.5 limb -Galactosidase activity persisted in the posterior ectoderm (highlighted by arrowheads). Additional sagittal sections from embryos containing the Ptch1:LacZ allele at E11.5 were analyzed in detail on sections. I) A schematic of the posterior of the developing limb bud showing approximate loc ations of sectioned tissue J) In a section from the posterior margin (the posterior region from the flank to the AER), galactosida se activity was detectable in the posterior ectoderm including portions of the dorsal and ventral ectoderm K) In a section from the proximal portion of the posterior AER, galactosidase activity was detectable in t he ectoderm including the AER L) In a s ection from a more distal portion of the posterior AER revealed galactosidase act ivity exclusively in the AER Arrows mark the dorsal and ventral extremes of detectable galactosidase activity in the ectoderm.

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42 Figure 34. Smo expression in the embryo including the AER of the developing limb. To investigate the presence of Smo in the embryo, a series of RNA in situ hybridizations were performed using a riboprobe designed to detect Smo A) At 37ss, embryos hybridized to a sens e probe revealed no stainin g however, B) embryos stained with an antisense probe expressed Smo throughout the embryo including C) in the limb Tissue harvested at 50ss and sectioned revealed staining throughout the mesoderm and ectoderm of the limb including the AER D) at low magn ification and E) high magnification. The arrow in E highlights Smo expression in the AER.

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43 Figure 35. Msx2 -Cre is expressed robustly in the AER and not in the limb bud mesoderm. To investigate the expression of Msx2 -Cre in the developing limb, mating s chemes were designed to incorporate the Rosa26 reporter into animals with the Msx2 -Cre allele. In these limbs, cells that have galactosidase activity have at some time during development expressed cre recombinase from the Msx2 -Cre allele At E10.5, Msx2 -Cre was expressed in the AER of both A) forelimbs and B) hindlimbs C & D) Sagittal sections of E10.5 limbs revealed -galactosidase activity in the AER and ventral ectoder m of two different limbs In all of the limbs analyzed, galactosidase activity was restricted to the ectoderm.

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44 Figure 36. Hedgehog signal transduction in the AER has a functional role in limb patterning. RNA in situ hybridizations with control and Msx2 -Cre;Smoflox/flox limbs using a riboprobe for Ptc h1. Ptch1 was expressed throug hout the posterior of the control limb at 47ss including the ectoderm A) at low magnification and C) high magnification In Msx2 -Cre;Smoflox/flox limbs at 47ss, Ptch1 is expressed in the posterior limb bud mesoderm and in the most proximal region of the po sterior ectoderm, b ut is not expressed in the AER B) at low magnification and C) high magnification (arrowhead denotes loss of expression in the AER while the arrow marks expression in ectoderm adjacent to the AER). F) Sectioned limbs revealed that Ptch1 w as absent from the AER of Msx2 -Cre;Smoflox/flox limbs but E) present in all posterior ectoderm of control limbs ( arrowhead denotes loss of expression in the AER while the arrow marks expression in ectoderm adjacent to the AER ). In C F, the white dashed li nes highlight the visible boundaries of the mesoderm and ectoderm. In C & D, the solid white lines highlight the posterior edge of the limb. Skeletal analysis of P0 limbs of control forelimbs G) at low magnification and I) high magnification and mutant for elimbs H) at low magnifcation and J) high magnification. Skeletal analysis of P0 limbs of control hindlimbs K) at low magnification and M) high magnification and mutant forelimbs L) at low magnifcation and N) high magnification. and hindlimbs Insets in J and N show the supernumerary cartilaginous condensations found in Msx2 -Cre;Smoflox/flox animals. s = scapula, ss = spine of the scapula, h = humerus, dt = deltoid tuberosity, r = radius, u = ulna, a = autopod, cb = carpal bones, mc = metacarpals, il = ilium is = ischium, f = femur, p = patella, t = tibia, fi = fibula, tb = tarsal bones, mt = metatarsals.

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45 Figure 37. Ptch1 is lost from the AER of Msx2 -Cre;Smoflox / flox limb buds at E10.5. To expand our analysis of the loss of Shh signaling in the developing limb ectoderm, we analyzed expression of Ptch1 by whole mount RNA in situ hybridization. Ptch1 was expressed throughout the posterior of the control limb at 35ss including the ectoderm A) by whole mount and C) section. B) In Msx2 -Cre;Smoflox/flox limbs at 35ss, Ptch1 was expressed in the posterior limb bud mesoderm and in the most proximal region of the posterior ectoderm, but was not expressed in the AER. Arrowhead denotes loss of expression in the AER while the arrow marks expression in ectoderm adjacent to the AER. C) Sectioned limbs revealed that Ptch1 was present in posterior ectoderm of control limbs, D) but absent from the AER of Msx2 -Cre;Smoflox/flox limbs (arrowhead denotes loss of expression in the AER while the arrow marks expression in ectoderm adjacent to the AER).

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46 Figure 38. Extra condensations appear on Msx2 -Cre;Smoflox/flox limbs between E13.5 and E16.5. To further investigate the extra cartilaginous condensations formed in Msx2 -Cre;Smoflox/flox limbs, limbs were analyzed at E13.5 for ex pression of Sox9 and at E16.5 by bright field microcopy. Sox9 expression A) in control forelimbs and B) Msx2 -Cre;Smoflox/flox forelimbs and C) control hindlimbs and D) Msx2 -Cre;Smoflox/flox hindlimbs by whole mount RNA in situ hybridization were similar. H owever, analysis of A) control forelimbs and B) Msx2 -Cre;Smoflox/flox forelimbs and C) control hindlimbs and D) Msx2 Cre;Smoflox/flox hindlimbs forelimbs (E & F) and hindlimbs (G & H) by bright field microscopy revealed an extra cartilaginous condensation at E16.5 in the posterior of Msx2 -Cre;Smoflox/flox limbs.

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47 Figure 39. Fgf8 is expressed within the morphologically distinct AER in the developing limb. To confirm previous reports that Fgf8 was expressed throughout the entire AER, limbs were analyzed by RNA in situ hybridization for expression of Fgf8. A) Analysis of Fgf8 expression revealed robust expression throughout the AER in 43ss hindlimbs. B) Analysis of Fgf8 expression on serial sections from embryos at 39ss revealed that when the ectoderm was a simple epithelial layer, no Fgf8 was expressed. C & D) Fgf8 was expressed when the epithelium was stratified. White dashed line denotes the boundary between mesenchyme and epithelium. B -D were taken at the same

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48 Figure 310. Removal of hedgehog signaling in the AER results in an increase in AER length. Double RNA in situ hybridizations on A) control forelimbs, B ) Msx2 Cre;Smoflox/flox forelimbs,C) control hindlimbs and D) Msx2 -Cre;Smoflox/flox hindlimbs using a riboprobe against Fgf8 and Shh at 50ss. Both forelimbs and hindlimbs. AERs were larger in Msx2 -Cre;Smoflox/flox embryos than in somite matched controls. E ) Average AER length after measurement of Fgf8 expression in Msx2 -Cre;Smoflox/flox limbs and control limbs using the Straighten plugin for NIH ImageJ. Data are represented as means and the error bars represent the SEM. A t -test (Welchs, unpaired) indicat ed that these differences were statistically significant (* = p = 0.03 and = p < 0.0001). F) A diagram of chick limbs with a bead soaked in SHH implanted just anterior to the ZPA in the distal limb bud mesenchyme. G) Fgf8 expression was decreased after 2 4 hours of treatment with a SHH soaked bead beginning at HH 20, H) when compared to an untreated contralateral control limb. The arrow in C highlights the loss of posterior AER in the treated limb bud. I) Fgf8 expression after 24 hours of treatment with a PBS soaked bead was unchanged, J) when compared to a contralateral control limb. The proximal location of the beads 24 hours after implantation is a result of outgrowth of the limb bud.

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49 Figure 311. Fgf4 is excluded from the posterior AER after treatment with SHH soaked beads. To determine if increased SHH protein in the posterior of the limb altered the domain of Fgf4 expression, we treated limbs with beads soaked in SHH (see Experimental Procedures for more detail). A) After 24 hours of treatment with a SHH soaked bead, Fgf4 expression was reduced in the posterior AER, B) when compared to the contralateral control. C) PBS soaked beads had no effect on the expression of Fgf4, D) when compared to the contralateral control.

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50 Figure 312. Analysis of cell death in limbs that have been treated with SHH soaked beads. To determine if increased SHH protein in the posterior of the limb induced changes in cell death, we analyzed cell death using LysoTracker red in limbs treated with a SHH soaked bead (see Experi mental Procedures for more detail). A) Analysis of cell death in limbs after treatment with PBS or C) in an untreated contralateral control revealed cell death in the anterior necrotic zone and the AER. B) After 24 hours of treatment with a SHH soaked bead, cell death was noticeably increased in the posterior of the limb including the mesoderm and AER (9/13 limbs). In A & B, white circles depict bead placement. The black arrow in B highlights cell death in the posterior of the limb bud and the white arrowhead highlights cell death in the posterior AER. To determine the effect of the SHH bead on the Fgf8 expression, we analyzed Fgf8 expression in the same limb buds used in A -C. D) Fgf8 expression was unaffected by treatment with a PBS soaked bead or F) in a c ontralateral control limb. E) Treatment with a SHH soaked bead reduced the domain of Fgf8 expression in the developing limb bud. Merged images reveal that G) in a PBS treated and I) a contralateral control limb, cell death can be detected at the anterior and posterior extremes of the domain of Fgf8 expression. H) Treatment with a SHH soaked bead resulted in a noticeable increase in cell death with much of the observed cell death occurring at the anterior and posterior extremes of the domain of Fgf8 expressi on.

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51 Figure 313. Gene expression in Msx2 -Cre;Smoflox/flox limbs. Double labeled RNA in situ hybridizations of A) control and B) Msx2 -Cre;Smoflox/flox hindlimbs for Shh (red) and Fgf4 (purple), revealed an increase in Fgf4 and Shh expression at 39ss. Analysis of Fgf8 expression in C) 61ss control hindlimbs and D) 63ss Msx2 -Cre;Smoflox/flox hindlimbs revealed that Fgf8 expression was prolonged in Msx2 -Cre;Smoflox/flox compared to control limbs. Arrows in D denote prolonged expression of Fgf8. Expression of Gremlin was expanded at 50ss in F) Msx2 -Cre;Smoflox/flox compared to E) control hindlimbs. White lines show the width of the domain of Gremlin expression.

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52 Figure 314. Model of stochastic control of AER length by hedgehog signal transduction within the AER. A) During normal development a population of cells within the ZPA expresses Shh Response to Hh signaling within the ectoderm limits or expands the length of the AER. B) If levels of SHH protein in the AER are elevated, the AER is shortened due t o elevated Hh signaling in this tissue. Shortening of the AER results in a corresponding decrease in mesenchymal Shh expression. C) If levels of SHH protein is decreased or absent from the AER, decreased Hh signaling occurs in the AER and the AER expands in length. As a result of a longer AER, there is a corresponding increase in Shh expression in the underlying mesenchyme. This buffering model ensures that the amount of SHH produced from the ZPA is appropriate and results in a reproducible digit pattern. In A -C only the posterior of the limb bud is diagramed. Green in the mesenchyme shows the level of SHH produced in the ZPA, while red in the AER denotes Hh signaling. Dark blue denotes the posterior AER, where we identified targets of Hh signaling. The mo re anterior AER is show as light blue.

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53 Table 3 1. Frequency of skeletal element number in forelimbs and hindlimbs of control and mutant animals. Genotype # of Limbs Observed Digits in the Autopod n (percent) Six Five Less than five Forelimb Control 18 0 18 (100%) 0 Msx2 Cre;Smoflox/flox 10 8 (80%) 2 (20%) 0 Hindlimb Control 18 0 18 (100%) 0 Msx2 Cre;Smoflox/flox 10 9 (90%) 1 (10%) 0 Samples were analyzed immediately after fixation and before skeletal preparation due to potential loss of extra tissue during the clearing process.

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54 CHAPTER 4 ABERRANT FGF SIGNALI NG, INDEPENDENT OF E CTOPIC HEDGEHOG SIGNALING, INITIATES PREAXIAL POLYDACTYLY IN DORKING CHICKENS Introduction Polydactyly, the most frequent congenital hand malformation observed in humans, has a prevalence between 5 and 19 per 10,000 live births, (Sesgin and Stark, 1961; Zguricas et al., 1999) Over one-hundred clinically distinct disorders have polydactyly as a symptom and, approximately two-thirds have an unidentified molecular cause (Biesecker, 2002) A more complete un derstanding of polydactyly relies on a better understanding of limb development and the etiology of this disorder. Outgrowth and the initial steps of limb patterning are controlled by at least two classically described signaling centers: the zone of polar izing activity (ZPA) and the apical ectodermal ridge (AER) (Saunders, 1948; Saunders and Gasseling, 1968) The ZPA is a posterior portion of mesenchyme, which when transplanted to the anterior of a host limb produces mirror image duplications (Saunders and Gasseling, 1968) The molecule responsible for mediating ZPA activity has been identified as SHH. Further, implantat ion of a source of SHH protein in the anterior of a developing limb bud induces the formation of preaxial extra digits (Riddle et al., 1993) The AER is a distal portion of the ectoderm, which is required for limb outgrowth (Saunders, 1948) One of the primary functions of the AER is to express Fibroblast growth factors (FGFs) [reviewed in (Martin, 1998) ]. The requirement for AER -FGFs has been demonstrated by placing FGF soaked beads on the distal end of limbs in which the AER had been removed. In these experiments, FGF protein sustained limb outgrowth (Fallon et al ., 1994; Niswander et al., 1993) Genetic removal of two of the four Fgfs expressed in the mouse AER results in limb truncation (Boulet et al., 2004;

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55 Sun et al., 2002) Recent studies indicate that the AER -FGFs ar e functionally equivalent, and differences in expression level, timing of expression and receptor affinity make the AER -FGFs unique during limb patterning (Lu et al., 2006; Mariani et al., 2008) Identification and characterization of the molecules required for ZPA and AER function have indicated that these genes are involved in a feedback loop. For example, expression of three of the four ectodermally expressed Fgfs in mouse require Shh for their maintenance (Chiang et al., 1996; Sun et al., 2000) while removal of multiple Fgfs from the AER results in a loss of Shh (Mariani et al., 2008; Sun et al., 2002) Interestingly, SHH is capable of inducing expression of Fgf4. For example, placement of a source of SHH in the anterior of the limb bud causes Fgf4 to expand anteriorly in the AER (Laufer et al., 1994; Niswander et al., 1994) The coordinated regulation of gene expression between the AER and the underlying mesenchyme provides a mechanism for integrating patterning along multiple axes of development (Niswander, 2002) Also, coordinate regulation provides a mechanism for the termination of gene expression (Scherz et al., 2004; Verheyden and Sun, 2008) Changes in expression of Shh and Fgfs can be used to describe the etiology of many of the characterized cases of polydactyly. Several mutants with preaxial polydactyly have been identified in which anterior expression of Shh occurred. Additionally, these mutants w ith preaxial polydactyly have been shown to have Fgf4 ectopically expanded anteriorly in the AER (Chan et al., 1995; Masuya et al., 1997; Masuya et al., 1995; Sharpe et al., 1999) Genetic analysis has revealed that two of these mutants, Hemimelic extra toes and Sasquatch, had mutations in an enhancer

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56 element upstream of the Shh locus resulting in ectopic anterior expression of Shh (Lettice et al., 2003; Lettice et al., 2002) The goal of the present study was to use the Dorking breed of chickens to identify molecular pathways responsible for polydactyly in vertebrates. Differences in Shh and Fgf expression in addition to cell death were found in Dorking hindlimbs when compared to wild type. By taking advantage of the in ovo development of chickens, we uncovered a molecular mechanism in which polydactyly was initiated independently of ectopic Shh Results Dorking Chickens have Partially Penetrant Preaxial Polydactyly in the Hindlimb but not in the Forelimb. Polydactyly in Dorkings has been previously reported to exhibit a dominant mode of inheritance with variable penetrance (Punnett and Pease, 1929) An outcross of our population confirmed the dominance of the Dorking phenotype with 92% (n=12) of offspring showing polydactyly. To characterize the hindlimbs of Dorkings (Fig. 4-1A, B), HH36 embryos wer e processed to visualize cartilage. Alcian blue staining of hindlimbs revealed that Dorking autopods had a preaxial ectopic digit (Fig. 4-1C, D). The extra digit was typically fused to the base of the hallux, or first digit ray. The digits of chicken hi ndlimbs each have a distinct number of phalanges. By counting the phalanges of a digit ray, identity can be inferred. In the chicken hindlimb, the most anterior digit, digit I, has two phalanges while digit II has three phalanges; this pattern continues through digit IV, which has five phalanges. To further characterize the extra digits in Dorking hindlimbs, the phalanges of the extra digit were counted and compared to wild type (Fig. 41C,D and Table 41).

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57 To address the issue of variable penetrance, 35 Dorking embryos were analyzed. In Dorkings, 81% (n=70) of hindlimbs had some degree of polydactyly with no defect observed in any of the forelimbs analyzed (n=70). Of the hindlimbs containing an extra digit, 70% (n=70) resembled a digit II (three phal anges were visible). In instances when a digit II did not occur, ectopic digits resembled a digit I (10%; 7/70 limbs), or a blip (1.4%; 1/70 limbs). Finally, of the 70 hindlimbs analyzed, 19% (n=70) showed no polydactyly and resembled wild type limbs (s ummarized in Table 4 1). In addition to providing data specific to our population, these data confirm previous observations made 80 years ago regarding the penetrance of Dorking polydactyly (Punnett and Pease, 1929) Ectopic Hedgehog Signaling Occurs in the Anterior of Late Stage Dorking Hindlimbs To determine if ectopic hedgehog signaling was present in Dorking embryos, Shh expres sion was examined using whole mount RNA in situ hybridizations. From HH21 to HH25, normal posterior expression of Shh was observed in all Dorkings (Fig. 42A,B and data not shown). However at HH26, a small region of ectopic Shh was detected in the anter ior of Dorking hindlimbs (Fig. 4-2C,D). In HH26 hindlimbs, 70% (n=10) contained ectopic Shh expression. No abnormal Shh expression was observed in the forelimb at any stage examined (data not shown). To investigate if the Shh present in the anterior of D orking hindlimbs was activating the hedgehog signaling pathway, we examined expression of Ptc1 and Bmp2 targets of hedgehog signaling (Laufer et al., 1994; Marigo et al., 1996b) At HH21, normal Ptc1 expression was observed (Fig. 4-2E,F). In the Dorking hindlimb at HH25, one stage prior to visible ectopic Shh expression, ectopic Ptc1 was observed (Fig. 42

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58 G, H). Similar to that observed for Ptc1 normal expression of Bmp2 was detected at HH21 (Fig. 4 2I,J). At HH 26, when ectopic Shh was first observed, Bmp2 was ectopically expressed in the anterior of Dorking hindlimbs (Fig. 4-2K,L). Additional targets of the hedgehog pathway, Gli1 and HoxD13, were also ectopically expressed at HH26 (Fig. 43). These data demonst rated that Shh and hedgehog target genes were ectopically expressed in Dorking hindlimb mesenchyme. Gene Expression in the AER is Expanded in Dorking Hindlimbs Fgf4 is normally restricted to the posterior region of the AER, however, anterior expansion o f Fgf4 has been observed previously in several mutants with preaxial polydactyly (Masuya et al., 1995; Sharpe et al., 1999) Analysis of Dorking hindlimbs by whole mount RNA in situ hybridization at HH21 revealed t hat Fgf4 was anteriorly expanded in Dorking hindlimbs (Fig. 4-2M,N). By stage HH25, Fgf4 expression ceased in wild type embryos, however, Fgf4 expression persisted in Dorking hindlimbs (Fig. 4 2O,P). A second FGF, Fgf8, has also been shown to be expanded in mouse mutants with polydactyly (Yada et al., 2002) An analysis of wild type and Dorking limb buds demonstrated that Fgf8 and Sp8 an activator of Fgf8 (Bell et al., 2003; Kawakami et al., 2004) were slightly expanded in the anterior of Dorking hindlimbs (Fig. 4 -3). These data suggest that, in addition t o anterior expansion of Fgf4, the overall length of the AER was expanded in Dorkings. Ectopic Fgf4 Precedes Ectopic Shh in Dorking Hindlimbs In situ hybridization data suggested that in Dorking hindlimbs ectopic Fgf expression preceded ectopic expression of Shh and hedgehog target genes by 5 HH stages (approximately 24 hours). One limitation of RNA in situ hybridization is that this

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59 technique cannot detect low levels of gene expression. To confirm that ectopic Fgf4 expression in Dorking hindlimbs preceded ectopic Shh, we used RT PCR, a more sensitive technique, to detect Fgf4 and Shh expression in Dorking limbs. Dorking and wild type hindlimb or forelimb buds at HH21 were removed and trisected into anterior, middle and posterior segments (Fig. 4 -4A). The m iddle portion of the limb was discarded and gene expression in the anterior and posterior segments was analyzed. In all posterior pools analyzed at HH21, both Shh and Fgf4 were present (Fig. 44B). In anterior cDNA pools made from either wild type or Dork ing forelimbs, neither Shh nor Fgf4 was detected. In the hindlimb, cDNA from the anterior of wild type limb buds expressed neither Fgf4 nor Shh transcripts However, in the Dorking hindlimb Fgf4 but not Shh was detected in the anterior limb bud (Fig. 4-4B ). These data confirmed our in situ hybridization experiments and indicated that Fgf4 was ectopically expressed in the anterior of Dorking hindlimbs prior to Shh expression. Extended Expression of Fgf4 in the Dorking Hindlimb is Maintained Independent of H edgehog Signaling In Dorking hindlimbs, we have demonstrated that Fgf4 exhibits a temporal shift in expression. Cyclopamine is a steroidal alkaloid, which inhibits hedgehog signaling by blocking Smoothened (Smo ) (C hen et al., 2002; Incardona et al., 1998) To determine if extended Fgf4 expression in Dorking hindlimbs required Shh hedgehog signaling was inhibited in Dorking hindlimbs using cyclopamine. Treatment of Dorking limbs with cyclopamine resulted in a red uction in Ptc1 expression in the hindlimb (5/6 limbs) whereas none of the mock treated limbs (0/8 limbs) showed reduced Ptc1 expression (Fig. 4 -4C F). These results indicated that our treatment method was capable of blocking hedgehog signaling in Dorking hindlimbs (p=0.002).

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60 Analysis of different limbs treated in an identical manner revealed that Dorking limbs retained ectopic Fgf4 expression both after treatment with cyclopamine (12/15 limbs) and HBC (8/11 limbs; Fig. 4-4G -J). Comparison of these propor tions revealed that the differences were not statistically significant (p=0.69). The treatment of wild type limbs with cyclopamine at a stage when Fgf4 was expected to be expressed resulted in a loss of Fgf4 (p=0.011; data not shown). These data indicated that prolonged maintenance of Fgf4 expression in the AER of Dorking hindlimbs did not require hedgehog signaling. Cell Death is Decreased in the Anterior Necrotic Zone of Dorking Hindlimbs Loss of programmed cell death has been observed in chickens with p olydactyly (Dvorak and Fallon, 1991; Hinchliffe and Thorogood, 1974) To investigate apoptosis in Dorking hindlimbs, we used LysoTracker Red, an acidotropic dye that accumulates in the lysosomes of apoptotic cells and phagocytic cells engulfing apoptotic cells (Zucker et al., 1999) Analysis of the anterior necrotic zone (ANZ) of Dorking hindlimbs after staining by LysoTracker Red revealed that Dorking hindlimbs had a reduced domain of cell death (Fig. 45A -D). To confirm the observed decrease in cell death, we performed whole mount RNA in situ hybridizations using a riboprobe against Dkk1 Dkk1 is a Wnt antagonist, which is spatially and functionally associated with programmed cell death in the developing limb (Grotewold and Ruther, 2002) An examination of Dkk1 expression in Dorking hindlimbs revealed that transcripts were down-regulated in the anterior necrotic zone compared to age matched wild type limb buds (Fig. 4-5E -H). The decrease in Dkk1 expression was consistent with a loss of cell death in the ANZ in Dorking hindlimbs.

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61 Inhibition of Ectopic Fgf but not Hedgehog Signaling in the Dorking Hindlimb can Rescue the Reduction of Cell Death Present in the Anterior Necrotic Zone Increases to the amount of both FGF and SHH in the ante rior of a developing limb have been shown to inhibit cell death in the anterior necrotic zone (Montero et al., 2001; Sanz -Ezquerro and Tickle, 2000) To separate whether ectopic activation of the Fgf or Shh signali ng pathway played a role in changes to cell death in Dorking hindlimbs, each pathway was independently inhibited followed by an analysis of cell death. After inhibition of hedgehog signaling with cyclopamine, 10 of 12 limbs analyzed (83%) had a domain of cell death that was comparable to Dorkings (Fig. 4-6A -D). The number of limbs with a Dorkinglike domain of cell death after cyclopamine treatment was similar to untreated limbs (see Table 42). These data indicate that inhibition of Shh signaling in Dor king hindlimbs is not capable of restoring a normal domain of cell death. To determine if FGF signaling was required to block cell death in the anterior necrotic zone of Dorking hindlimbs, SU5402, a small molecule inhibitor that inhibits all FGF signalin g by blocking tyrosine phosphorylation of FGF receptors was used (Mohammadi et al., 1997) Inhibition of anterior FGF signaling in Dorking hindlimbs resulted in an increase in the domain of cell death in 50% (n=10) of limbs analyzed (Fig. 4 -6E H). In limbs that had rescued cell death, an increase in LysoTracker staining was observed directly over the SU5402 coated bead. Treatment with a DMSO soaked bead resulted in a Dorking expression pattern in 100% (n=7) of lim bs analyzed. These data indicated that treatment with SU5402 had a significant effect on cell death in the developing Dorking hindlimb (p=0.019) and suggests that increases in FGF signaling but not hedgehog signaling are capable of inhibiting cell death i n the anterior necrotic zone of Dorkings hindlimbs.

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62 Discussion Polydactyly is a common congenital malformation with multiple potential causes. Because human embryos are difficult to obtain for studies of the molecular origins of polydactyly, our knowledge of the molecular etiology of polydactyly relies in part on experiments in model systems. The first recorded description of polydactyly in the chicken model system was in Columellas Latin text De Rei Rustica [translated to English in (Columella, 1941) ]. Although they did not bear the name in Roman times, the polydactylous chickens described in Columellas text are thought to be Dorkings. Since the Roman era, Reginald Punnett, among others, have been interested in the genetics of Dorking polydactyly (Darwin, 1890; Punnett and Pease, 1929) However, prior to the present study the developmental basis of the extra digit in Dorkings has remained uninvestigated. To better understand the potential origins of polydactyly in vertebrates, we used modern genetic and molecular approaches to investigate the signaling pathways responsible for causing polydactyly in Dorking chickens. Our data indicate that the ectopic preaxial digit found in the Dorking hindlimb initiates from ectopic expression of Fgfs in the AER, which occurs independent of anterior Shh expression. Multiple Roles for FGFs in the Formation of Supernumerary Digits in Dorkings Thr ough gene expression analysis, we found several differences in Dorking hindlimbs when compared to wild type hindlimbs. Most notable were the differences in Shh and Fgf expression during hindlimb development. Ectopic Shh is known to be capable of inducing ectopic Fgf4 (Laufer et al., 1994; Niswander et al., 1994) Our data revealed that Fgf4 expression preceded Shh expression in the Dorking hindlimb. In addition, Shh is required for the maintenance of Fgf4 (Chiang et al., 2001) In Dorkings,

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63 treatment with cyclopamine revealed that hedgehog signaling was not required for the maintenance of ectopic Fgf4 expression. These data suggest that Fgf4 and possibly additional factors are ectopically expressed independent of Shh in the anterior of the Dorking AER. AER -Fgfs have been hypothesized to establish progenitor numbers in the developing limb (Boulet et al., 2004; Sun et al., 2002) One interesting problem with this hypothesis is that after total AER -Fgf removal, autopodal skeletal elements are lost, however, cel l death is in a location too proximal to affect autopod progenitor number (Mariani et al., 2008; Sato et al., 2007) Although the mechanism that causes cell death in a proximal domain is unclear, it is known that c hanges in Fgf signaling are capable of affecting autopod progenitors. For example, partial removal of receptors required for Fgf signaling results in distal cell death, which causes a loss of autopodal skeletal elements (Verheyden et al., 2005) Clearly, the location of cell death after changes in Fgf signaling is important for interpretation of the Dorking phenotype. Based on fate mapping studies, the decrease in cell death in the anterior necrotic zone observed in Dorkings occur in a region of the limb where autopod progenitor cells are located (Vargesson et al., 1997) We propose that changes in cell death present in Dorkings are capable of altering autopod progenitor cell numbers, resulting in red uced cell death and polydactyly. In addition to a role for Fgfs in the inhibition of cell death, Fgfs have been shown to act as a chemoattractant (Li and Muneoka, 1999). It has been suggested that through migration of new cells towards the AER, ectopic Fgf expression may result in preaxial polydactyly (Tanaka et al., 2000) It is possible that in Dorkings, ectopic Fgf expression

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64 attracts cells to the anterior of the developing limb. In combination with a reduction in cell death, attraction of additional cells could greatly increase the number of autopod progenitors in the anterior of the Dorking hindlimb resulting in polydactyly. In this work, we have demonstrated that inhibiting Fgf signaling in Dorkings results in an increase in cell death. We hypothesize that changes in Fgf signaling in Dorking hindlimbs causes an increase in the number of autopod progenitors. Contrary to this hypothesis, over expression of AER -Fgfs in mice leads to postaxial polydactyly (Lu et al., 2006) not the preaxial polydactyly found in Dorkings. We propose that these differences may arise because mouse limb buds have a necrotic zone, which appears later and more distall y than the anterior necrotic zone in chick limb buds (Fernandez Teran et al., 2006) Such differences in cell death between the mouse and chick limb buds may be responsible for the differences in polydactyly observed upon over expression of Fgfs T he Role of Shh in Dorking Polydactyly Our data revealed a role for ectopic Fgfs in initiating Dorking polydactyly. In addition to ectopic Fgf expression, ectopic Shh was also observed. Concentration and time of exposure to a source of SHH protein determin es identity of extra digits (Yang et al., 1997) Additionally, the cells that compose a normal digit II in mice have been shown to require response to SHH (Ahn and Joyner, 2004; Harfe et al., 2004) In Dorkings, variable expression of Shh (see Table 2) offers a potential explanation for the differences observed in digit identity. Because the formation of a normal digit I is independent of SHH (Chiang et al., 1996; Ros et al., 2003) and viral over expression of Fgf2 has been shown to res ult in the formation of extra digit Is (Riley et al., 1993) we propose that in embryos, which ectopically express Fgfs but not ectopic Shh, the limb

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65 field is expanded. The increased number of progenitors result in an extra digit I in the absence of Shh When anterior Shh is expressed in addition to ectopic Fgfs an extra digit II forms in Dorking hindlimbs. Ideally, analysis of gene expression in an early limb bud and the resulting skeletal pattern should be analyzed in the same limb. Since it is not feasible to perform gene expression studies on living chick limb buds, we have compared the percentage of embryos containing an ectopic digit II and ectopic Shh expression to examine the role of Shh in patterning ectopic digits in Dorkings. In both cases, 70% of the analyzed embryos had either an ectopic digit II or ectopic Shh expression in the anterior hindlimb (see Tables 1 and 2). In Dorkings, 10% of the analyzed skeletal preparations exhibited an ectopic digit I instead of a di git II. In our analysis of Fgf4 expression, we observed 16% more embryos with ectopic Fgf4 expression than embryos with ectopic Shh expression. While consistent with our hypothesis that formation of an ectopic digit I forms in the absence of ectopic Shh in Dorkings, these data do not rule out the possibility that low levels of Shh may be responsible for polydactyly in Dorking hindlimbs. Additionally, high doses of transient Shh may be present in embryos where an ectopic digit I was observed. However, i nspection of 56 limbs revealed no Shh expression between HH stages 20 -25. Because we observed changes in cell death prior to detection of Shh or Ptc1 we favor a model where extra digits in Dorkings are initiated independent of anterior Shh expression. In this model, ectopic Shh only plays a role in specifying digit II and not in initiating polydactyly.

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66 Potential Genetic Causes for Dorking Polydactyly The genetic lesion(s) responsible for formation of the ectopic postaxial digit in Dorkings is unknown. Our data suggests that ectopic digit formation in Dorkings is due to misexpression of Fgfs in the AER, in particular anterior expansion of Fgf4 within the AER. The DNA elements responsible for Fgf4 expression have been identified in mouse (Fraidenraich et al., 1998) By searching the recently completed G. gallus genome, we identified a DNA region homologous to the mouse Fgf4 enhancer. Within this DNA element, ten single nucleotide polymorphisms were identified through comparison of the Dorking sequence with wild type sequence derived from Red Jungle Fowl (C.B. and B.D.H., unpublished results). We are curr ently investigating the potential role of these SNPs in altering transcription factor binding to the Fgf4 enhancer. While it is possible that a mutation in the Dorking version of this DNA element is responsible for ectopic Fgf4 expression our data favor a model in which a mutation in a factor upstream of Fgf expression produces the ectopic postaxial digit found in Dorkings. In part, our hypothesis is based on our findings that both Fgf8 and Sp8 in addition to Fgf4 are ectopically expressed in the AER of D orking hindlimb buds. Since ectopic Fgf4 expression does not induce ectopic Fgf8 expression (Lu et al., 2006) we propose that a mutation upstream of Fgf expression is most likely responsible for the presence of an ectopic postaxial digit in Dorkings. Experimental Procedures Embryo Collection Dorkings and Dominique chickens used for this study were maintained according to common standards and practices at Morningside Nature Center, Gainesville F lorida. Dominique chickens have a normal number of digits and were used as wild type

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67 controls for gene expression and cell death analysis. Photographs of live roosters were taken on-site with a Nikon Coolpix 7900 digital camera. Eggs were collected every morning by the staff at Morningside Nature Center and stored at room temperature for no longer than a week. In the lab, eggs were incubated at 37 C with supplemental water for humidity. Prior to harvest, eggs were fenestrated with blunted forceps and sta ged according to the staging guide published by Hamburger and Hamilton (HH) (Hamburger and Hamilton, 1951) Skeletal Preparation For skeletal preparation, HH stage 36 embryos were collected and fixed overnight in 4% formaldehyde in PBS. After evisceration, embryos were incubated in an alcian blue staining solution (70% Ethanol, 30% Acetic Acid and 0.02% w/v Alcian Blue) for 12 days. Embryos were t hen cleared overnight in 0.5% KOH before being stored in 80% glycerol. After embryos were cleared, phalanges were counted to determine digit identity. Whole Mount RNA in situ Hybridization Harvested embryos were incubated overnight in 4% formaldehyde at 4 C. In situ hybridization was performed as described previously (Wilkinson and Neito, 1993) Plasmids for generating the following riboprobes have been previously described: Shh (Roelink et al., 1994) Ptc1 (Marigo et al., 1996b) Bmp2 (Francis et al., 1994) Fgf4 (Niswander et al., 1994) Gli1 (Marigo et al., 1996a) HoxD13 (Izpisua-Belmonte et al., 1991) and Fgf8 (Crossley et al., 1996) Plasmid for generating Sp8 riboprobes was constructed by RT -PCR using the following primers: F 5 -ACCTGCAACAAGATCGGC and R 5 -TCCATGAGGTGCTGCCC.

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68 RNA Isolation, cDNA Synthesis and RT PCR Two independent pools of Dorking tissue and one pool of wild type tissue each consisting of limbs from 10 embryos were collected. Briefly, trisected limbs were harvested into ice -cold TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was isolated according to the manufacturers protocol. cDNA was synthesized from 2 RNA using AMV rev erse transcriptase (Roche Applied Science, Indianapolis, IN) and oligo dT oligonucleotides. Equal quantities of cDNA were used in each RT -PCR reaction. The primers used for RT -PCR were as follows: Shh (F 5 CGGGAGCTGACAGACTGATG, R 5 -CTGATTTCGCTGCCACTGAG ), Fgf4 (F 5 GCGACTACCTCCTGGGCATC, R 5 -CCGTTTTTGCTCAGGGCTATG) and GAPDH (F 5 -GACGGCAGGTATTAGTTCAAGG, R 5 -ACCATCAAGTCCACAACACGGTTGCTG). LysoTracker and Drug Treatments For LysoTracker (Molecular Probes L 7528) analysis, embryos were harvested into PBS and incubated with 5 l/ml LysoTracker solution in PBS at 37C for 30 min. After staining, embryos were rinsed in PBS, fixed in 4% formaldehyhde in PBS overnight at 4C and placed into 100% methanol at 20C for long-term storage. Cyclopamine (Sigma Alde rich) was dissolved in 45% HBC as described previously (Incardona et al., 1998) Cyclopamine was administered by separating the amnion adjacent to the hindlimbs using forceps. Sterile pipettes were used to direct 5 l of HBC or HBC plus cyclopamine (1mg/mL) onto the hindlimbs of HH23 Dorking embryos. Embryos were then allowed to develop for 20 hours at 37C and harvested. SU5402 (Merck Chemicals Ltd., Nottingham) was dissolved in DMSO. Ag 1 X2 (Bio -Rad Laboratories, Hercules, CA) formate -derived beads were incubat ed in SU5402 (10mM) or DMSO (control) alone for 1hr. Beads were then implanted in the anterior of HH24

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69 Dorking hindlimbs for 10 hours at 37C. The proportion of drug treated limbs similar to control treated limbs was compared using a z -score. A priori st atistical significance was set at a level of 0.05, and all tests were two -sided.

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70 Figure 41. Dorking chicken hindlimbs have an ectopic preaxial digit II. Digital images were taken of A) wild type and B) Dorking hindlimbs. In A and B, Digits are number ed according to location. An asterisk denotes the presence of an ectopic digit; Sp denotes the location of a spur (keratinous outgrowth), which is present in the hindlimbs of the Dominique roosters (wild type; see Experimental Procedures). Insets in A and B are of the adult wild type Dominique and Dorking chicken, respectively. Skeletal preparations performed on C) wild type and D) Dorking HH36 hindlimbs. Limbs are oriented with the most anterior digit at the top of the panel. In C and D, digit numbe rs have been assigned according to phalanx number. The ectopic Dorking digit present in panel D resembles a digit II since it has 3 phalanges and is fused to the hallux.

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71 Figure 42. Ectopic hedgehog signaling and Fgf expression occurs in the Dorking hi ndlimb. Whole mount RNA in situ hybridizations using riboprobes against Shh in A) HH21 wild type limbs B) HH21 Dorking hindlimbs, C) HH26 wild type hindlimbs and D) HH26 Dorking hindlimbs. Whole mount RNA in situ hybridizations using riboprobes against Ptc 1 in E) HH21 wild type limbs F) HH21 Dorking hindlimbs, G) HH25 wild type hindlimbs and H) HH25 Dorking hindlimbs Whole mount RNA in situ hybridizations using riboprobes against Bmp2 in I) HH21 wild type limbs J) HH21 Dorking hindlimbs, K) HH25 wild type hindlimbs and L) HH25 Dorking hindlimbs Whole mount RNA in situ hybridizations using riboprobes against Fgf4 in M) HH21 wild type limbs N) HH21 Dorking hindlimbs, O) HH25 wild type hindlimbs and P) Dorking At HH25. Arrows indicate ectopic expression. Insets in O & P are end on views of hindlimbs.

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72 Figure 43. Additional ectopic gene expression in Dorkings. Whole mount RNA in situ hybridizations using riboprobes against Gli1 in A) HH21 wild type limbs B) HH21 Dorking hindlimbs, C) HH26 wild type hindlimb s and D) HH26 Dorking hindlimbs. Whole mount RNA in situ hybridizations using riboprobes against Ptc1 in E) HH21 wild type limbs F) HH21 Dorking hindlimbs, G) HH25 wild HoxD13 hindlimbs and H) HH25 Dorking hindlimbs Whole mount RNA in situ hybridizations using riboprobes against Fgf8 at HH21 ventral views of I) wild type hindlimbs J) Dorking hindlimbs, and oblique views of K) HH21 wild type hindlimbs and L) HH21 Dorking hindlimbs Whole mount RNA in situ hybridizations using riboprobes against Sp8 at HH21 ventral views of I) wild type hindlimbs J) Dorking hindlimbs, and oblique views of K) HH21 wild type hindlimbs and L) HH21 Dorking hindlimbs Arrows indicate ectopic expression. Insets in O & P are end on views of hindlimbs. Boxes in K and L and O and P are the same size and were used to demonstrate expansion of Fgf8 and Sp8 in Dorking hindlimbs. The top of the boxes was placed where the anterior limb bud was attached to the body wall, or flank. For all genes examined, no ectopic expression was observed in D orking forelimbs.

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73 Figure 44. Ectopic Fgf4 expression in the Dorking hindlimb precedes and is independent of ectopic Shh A) Schematic diagram of the strategy used to isolate RNA from forelimbs and hindlimbs. Limbs were trisected and RNA pooled from th e anterior (A) and posterior (P) regions of the limb bud (see Experimental Procedures). The middle portion was discarded. B) PCR analysis of Fgf4 and Shh expression in the anterior and posterior of Dorking and wild type fore and hindlimbs demonstrated th at Fgf4 was expressed in the anterior region of the Dorking hindlimb prior to Shh expression (noted by an asterisk). GAPDH was used as a loading control. In situ hybridization analyzing Ptc1 expression in C) wild type hindlimbs, D) untreated Dorking hindl imbs, E) mock treated with 45% HBC Dorking hindlimbs and F) Cyclopamine treated Dorking hindlimbs. In situ hybridization analyzing Fgf4 expression in C) wild type hindlimbs, D) untreated Dorking hindlimbs, E) mock treated with 45% HBC Dorking hindlimbs and F) Cyclopamine treated Dorking hindlimbs. The arrows in D & E marks ectopic expression. Arrows in H -J highlight expression of Fgf4.

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74 Figure 45. Cell death is decreased in the anterior necrotic zone (ANZ) of Dorking hindlimbs. Ventral views of LysoTrack er red staining in A) wild type and B) Dorking hindlimbs. Anterior views of LysoTracker red staining in C) wild type and D) Dorking hindlimbs. Ventral views of in situ hybridizations against Dkk1 in E) wild type and F) Dorking hindlimbs. Anterior views of in situ hybridizations against Dkk1 expression in G) wild type and H) Dorking hindlimbs.

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75 Figure 46. Inhibition of FGF but not Hedgehog signaling rescues cell death in the anterior necrotic zone. Cyclopamine treatment of Dorking hindlimbs followed by Ly soTracker Red to visualize cell death. A) Wild type limbs had increased cell death in the anterior necrotic zone compared to either B) untreated or C) mock HBC treated Dorking hindlimbs. D) Treatment of Dorkings hindlimbs with Cyclopamine dissolved in HBC resulted in an inability to rescue cell death in 83% (n=12) of limbs analyzed. To illustrate the domain of cell death in the anterior necrotic zone, white boxes with identical dimensions were placed on the images. SU5402 inhibition of Tyrosine Kinase recep tors followed by staining with LysoTracker Red to assay the amount of cell death present in the ANZ. E) Wild type limbs had increased cell death in the anterior necrotic zone compared to either F) untreated or G) mock PBS treated Dorking hindlimbs. H) Afte r implantation of a SU5402 soaked bead, cell death was greatly increased in 56% (n=9) of limbs analyzed. In G and H the location of the beads used in the experiments are represented by white circles. All panels are views of the anterior edge of the hindli mb with the ventral surface facing left.

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76 Table 4 1: Summary of skeletal phenotypes among 35 Dorking incross progeny. Individuals observed Number of Digit IIs Number of Digit Is Number of Blebs No extra digit Both Legs with Extra Digit 2s 23 46 0 0 0 Both Legs with Extra Digit 1s 2 0 4 0 0 One Leg with Extra Digit 2 *, One Leg Extra Digit 1 2 2 2 0 0 One Leg with Extra Digit 2 *, One Leg with no Extra Digit 1 1 0 0 1 One Leg with Extra Digit 1 One Leg with no Extra Digit 1 0 1 0 1 One Leg with Extra Bleb One Leg with no Extra Digit 1 0 0 1 1 No Extra Digits on Either Leg 5 0 0 0 10 Total Number of Limbs 49 7 1 13 Percent of the Total 70% 10% 1.4% 18.6% A digit II was defined as a supernumerary digit with three cartilagi nous condensations. A digit I was defined as a supernumerary digit with two cartilaginous condensations. A bleb was defined as a singular supernumerary condensation of cartilage.

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77 Table 4 2: Summary of Variability of Gene Expression, Vital Dye Staini ng Similar to the percent of limbs with a digit II (70% [49 of 70 limbs]; Table 1). Similar to the percent of limbs with polydactyly (81.4% [57 of 70 limbs]; Table 1). Marker Analyzed Stage Analyzed Total Number of Limbs Analyzed Percent of Limbs Different from Normal Shh HH20 25 56 0.00% HH26 10 70.00%* Ptc1 HH21+ 10 0.00% HH25 8 50.00% Bmp2 HH26 8 25.00% Fgf4 HH21 20 35.00% HH25+ 12 83.33% LysoTracker H H25 12 83.33% Dkk1 HH24+ 10 80.00%

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78 CHAPTER 5 CONCLUDING REMARKS The developm ent of the vertebrate limb requires precise control of cellular and molecular events. Signaling centers required within the developing limb for proper pattern formation have been identified by tissue grafting and removal. More recently, molecules such as Shh and Fgf have been shown to be required for the function of the ZPA and AER respectively The field of limb development currently focuses on an integration of the theoretical framework with discoveries elucidating the role of signaling pathways in patter n formation (Chapter 1). Since its identification by John Saunders and Mary Gasseling, the ZPA has been a focus of research investigating pattern formation in the limb. In this work, we identified a unique area of the limb bud ectoderm that responds to a s ignal produced from the ZPA (Chapter 3). We present evidence that the area of hedgehog signaling pathway activation in the ectoderm is required for proper autopod patterning. In addition, we presented evidence that activation of the hedgehog signaling path way in the AER of the developing vertebrate limb is involved in refining levels of Shh expression from the underlying mesoderm. In addition to a functional role for hedgehog signaling within the AER, we identified a response to the hedgehog signaling path way outside of the AER in the ectoderm between the AER and the flank (known as the posterior margin), and in the dorsal and ventral posterior ectoderm. For a more complete understanding of the role hedgehog signaling plays in the ectoderm of the developing limb, we are interested in removing active hedgehog signaling from the remaining portions of the ectoderm. However, no cre recombinase alleles were available at the time of our study to identify a role for

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79 hedgehog signaling in the ectoderm outside the AE R. Two available cre alleles, Msx2 Cre and Brn4-Cre are expressed in the AER and the ventral ectoderm (Ahn et al., 2001; Sun et al., 2000) We selected Msx2 -Cre for analysis in Chapter 3, but these cre alleles maint ain gene expression in the dorsal ectoderm and the posterior margin, and are not suitable for a complete ectoderm removal of hedgehog signaling. We also considered the epithelial K14 -Cre (Dassule et al., 2000) fo r use to remove Smo from the entire ectoderm. However, removal of hedgehog signaling using K14 -Cre has been reported to have no effect on the limb (Gritli-Linde et al., 2007) This could be a result of the limited number of limb cells that express K14-Cre at E10.5 when Shh is expressed (MGI Database). The RAR Cre is expressed throughout the limb bud ectoderm. Unfortunately, this allele is also expressed in the mesoderm of the limb (Barrow et al., 2003) confounding interpretation of removal of hedgehog signaling from the ectoderm. A large -scale screening of GLI binding sites revealed several GLI binding sites upstream of the Shh target Ptch1 (Vokes et al. 2007) One of these GLI -binding sites, known as Ptch1:Peak7, was capable of driving expression of a reporter throughout the ectoderm of the developing limb without inducing expression in the mesoderm (Appendix A). Using this element to drive expression of cre recombinase, we can express cre recombinase in any portion of the ectoderm that activates the hedgehog signaling pathway. Removal of Smo using this cre recombinase from the entire region of the ectoderm that contains active hedgehog signaling will p rovide information about the function of hedgehog signaling in the entire posterior ectoderm. Implantation of a source of retinoic acid, a known inducer of Shh in the posterior limb bud decreases the length of the AER and anterior placement increases the length

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80 of the AER (Lee and Tickle, 1985) suggesting differences in the way that the anterior and posterior portions of the limb respond to Shh In Chapter 3, we identify active hedgehog signaling in the posterior AER which limits the length of the AER. Although outside of the scope of this work, differences in the ability of the AER to respond to hedgehog signaling could explain differences in response i n the anterior and posterior limb to active hedgehog signaling. Because Shh can control growth through the modulation of genes, which promote G1-S transition (Roy and Ingham, 2002) a lack of response in the AER could res ult in an increase in growth without increased hedgehog signaling restricting the AER, as is the case in the posterior (Chapter 3). A lack of response to hedgehog signaling in the anterior AER could occur through either restricted diffusion of hedgehog p rotein to the anterior AER or loss of an essential component of hedgehog signal transduction in the anterior AER. An activated form of Smoothened (Smo) has been isolated from basal cell carcinomas (Xie et al., 1998) In mice, Smo with an activating mutation has been inserted into the ubiquitously expressed Rosa26 locus. A floxed stop cassette blocks expression in the absence of cre recombinase. However, cre recombinase expression allows constitutive activation of hedgehog signaling (Mao et al., 2006 ). The ability of the anterior AER to activate hedgehog signaling can be tested by forcing active hedgehog signaling throughout the AER, and then determining the effect on limb pattern and gene expression. Expression of the activated form of Smo throughout the anterior AER would allow us to determine if the anterior AER was capable of responding to hedgehog signaling. The ability of SHH to diffuse to the anterior AER could then be tested by implanting beads into the anterior

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81 of the limb, and analyzing limbs for SHH protein and targets of hedgehog signaling within the ectoderm. From early stages of limb condensation to outgrowth from the body wall, many molecules and pathways have been described within the context of limb development. An important question i s how the molecular information within the limb is converted into digit condensations. In culture, limb ectoderm inhibits chondrogenesis (Solursh et al., 1981) Thi s effect is mediated by WNT and FGF diffusion from the ectoderm, which inhibit the expression of Sox9 (ten Berge et al., 2008) Because Sox9 is required for the condensation of chondrogenic cells (Barna and Niswander, 2007) cells proximity to the ectoderm, which acts as a source of Wnts and Fgfs, inhibits their ability to form cartilage. These findings have interesting implications for the studies reported in this work. In Chapters 3 and 4, extra condensations form as a result of an increase in AER -Fgfs and changes in cell death. One effect of a limb field expanded by an increase in proli feration or a decrease in death is an increase in the volume of cells in the limb. If there is a threshold distance from the ectoderm for cells to be outside the influence of WNTs and FGFs to activate Sox9 expression, an increase in limb volume could increase the number of cells that are an appropriate distance from the developing ectoderm to form cartilage. After exhaustion of WNTs and FGFs, the increased number of progenitors could result in the ectopic cartilage condensations observed in Chapters 3 and 4. In this work, we have identified unique molecular interactions involved in patterning the vertebrate limb. Activation of hedgehog signaling in the ectoderm (Chapter 3) and inhibition of cell death in the anterior necrotic zone of Dorking

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82 polydactylous mutants (Chapter 4) represent novel mechanisms for the autopod to refine gene expression, cell number within the limb field, and establish an appropriate array of digits.

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83 APPENDIX A CRE RECOMBINASE EXPR ESSION DRIVEN BY THE PEAK7 ELEMENT OF THE PTCH1 PROMO TER For years, developmental biologists were limited in their study of genes with embryonic lethal phenotypes. To overcome lethality associated with important developmental genes, conditional knockout technology was developed to remove genes from specific tissues (Lewandoski, 2001) Using the bacteriophage protein cre recombinase, sequences of DNA flanked by LoxP sites can be removed in any tissue that expresses the cre recombinase. In general, promoter fragments ex pressed in tissues of interest are used to drive the expression of cre recombinase (Lewandoski, 2001) A limiting factor for conditional knockout technology is the availability of alleles driving expression of cre r ecombinase. As we report in Chapter 3, Shh activates the hedgehog signaling pathway within the posterior ectoderm of the developing vertebrate limb. A functional role for the hedgehog signaling pathway was demonstrated in the AER by removing an essential component of the hedgehog signaling pathway, Smo Loss of hedgehog signaling from the AER resulted in skeletal defects. Our analysis showed hedgehog signaling pathway activation throughout the posterior ectoderm. Using an AER specific cre recombinase to re move hedgehog signaling from cells in the limb allowed the limb ectoderm, aside from the AER, to respond to hedgehog signaling. A complete analysis of the function of the hedgehog signaling pathway in the developing limb ectoderm requires more complete rem oval of Smo from the entire ectoderm. Currently, there are no alleles capable of driving expression of cre recombinase in the early ectoderm that are not also expressed in the mesoderm of the developing limb.

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84 Analysis of binding sites of the GLI proteins identified six GLI -binding regions upstream of the transcriptional start of Ptch1 (Vokes et al., 2007) Expanded analysis of the data set revealed an additional GLI -binding domain. Because it was the seventh peak of GLI binding activity this 788bp fragm ent was named Ptch1:Peak7 Ptch1:Peak7 is located 154.2 Kb upstream of the promoter of Ptch1 Expression of a reporter driven by the Ptch1:Peak7 regulatory element revealed activation of Ptch1:Peak7 specifically in the ectoderm at E10.5 (Figure A -1). The use of an eGFP -cre recombinase fused to an estrogen receptor ligand binding domain (eEGFPCreErt2) allows temporal control of cre activity by requiring tamoxifen induced nuclear translocation of the cre recombinase for gene removal (Lewandoski, 2001) To drive expression of cre recombinase specifically in the ectoderm of the developing limb, we used the Ptch1:Peak7 element. The eGFPCreErt2 fusion protein has been previously shown to have activity both in vitro and in vivo (Kobayashi et al., 2008) Linear constructs were injected by pronuclear injection. After inject ion into the male pronuclei of embryos, potential founders were identified by PCR genotyping. In total, four founders were identified as positive for the Ptch1:Peak7CreERt2construct. These founders were crossed to the R26 reporter ( R26R ) and expression of cre recombinase was confirmed by expression of -galactosidease. (Soriano, 1999) Currently, one founder has been analyzed in detail. Embryos produced from this foun der with a female positive for the R26R allele treated with tamoxifen (2mg/40g body weight) at E9.5, exhibited -galactosidase activity in several portions of the embryo at E11.5 including the lung, the floor plate of the neural tube, a subset of cranial n erves

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85 and the posterior ectoderm of the limb bud (Figure A -2). In combination with the conditional Smo allele, the Ptch1:Peak7CreERt2 allele will allow us to analyze limbs that are unable to respond to hedgehog signaling throughout the posterior ectoderm d uring most of the developmental stages of the limb. In addition, these transgenics represents the first use of a portion of Ptch1 to activate cre recombinase expression. The Ptch1:Peak7CreERt2 line can be used in tandem with the Shhgfpcre line (Harfe, 2004) to analyze gene removal in cells that express Shh and a subset of cells that respond to hedgehog signaling.

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86 Figure A 1. -Galactosidase activity driven by the Ptch1:LacZ allele and the Ptch1:Peak7_LacZ allele at E10.5. Comparison of -Galactosidase in A) whole embryos with Ptch1:LacZ to B) whole embryos with the Ptch1:Peak7_LacZ allele reveal that the Peak7 enhancer element is in a subset of cells which express Ptch1 (detected by -Galactosidase activity). Galactosidase in limb buds is restricted to the posterior of both C) Ptch1:LacZ and D) Ptch1:Peak7_LacZ limbs. Stained sections reveal -Galactosidase throughout the posterior of E) Ptch1:LacZ limbs but only in the posterior ectoderm of F) Ptch1:Peak7_LacZ limbs. Images provided courtesy of Matthew McFarlane and Andy McMahon.

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87 Figure A 2. Activation of cre recombinase in tissues with both the Ptch1:Peak7CreERt2 and the R26R allele at E11.5. Comparison of whole embryos A) without cre recombinase and B) with Ptch1:Peak7CreERt2reveal cre recombinase expression within the same tissues that express Ptch1:Peak7_LacZ (compare Fig. A 3B to Fig. A 1B). C) Dissection of the embryo revealed cre recombinase activity in the lung and surrounding the neural tube (marked by arrows). D) Dissection of a more caudal portion of the embryo revealed cre recombinase activity in the floor plate of the neural tube and posterior to the neural tube. Cre recombinase activity could also be detected in E) the posterior of the forelimb bud and in a few cells in F) the posterior o f the hindlimb bud.

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88 APPENDIX B OLIGONUCLEOTIDES USE D AS GENOTYPING PRIM ERS

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89 Table B -1. Oligonucleotides used as genotyping primers. Allele Primer Names Tm ( C) Mutant Band Size (bp) Normal Band Size (bp) Primer Sequence (5 >3) Rosa26R SorGF1 SorGR1 SorG R2 62 250 500 AAAGTCGCTCTGAGTTGTTAT GGAGCGGGAGAAATGGATATG GCGAAGAGTTTGTCCTCAACC Ptch1:LacZ PtchlacZASF1 PtchlacZASR1 67 310 GCAAAGACCTCGGGACTCAC AAGGCGATTAAGTTGGGTAACG Msx2Cre MSX2F2 CreB1 MSX 56 ~500 GACTTTTCAGTTTGGGCG AACATCTTCAGGTTCTGCGG Smo flox SmoMutF2 SmoMutR2 65 ~500 GGAGCTCGAGGAGGGACGTG GCGCTACCGGTGGATGTGG Smo IMR1834 IMR1835 62 200 CCACTGCGAGCCTTTGCGCTAC CCCATCACCTCCGCGTCGCA Shh flox Shh1015 Shh1016 65 520 450 ATGCTGGCTCGCCTGGCTGTGGAA GAAGAGATCAAGGCAAGCTCTGGC Shh ShhGenoF ShhGenoR2 470 GACCCAACTCCGATGTGTTCCGT TCTTCCCTTCATATCTGCCGC Cre BHCreF2 BHCreR1 65 260 TGACGGTGGGAGAATGTTAAT GCCGTAAATCAATCGATGAGT GFP GFP2s GFP2aS 62 570 ATGGTGAGCAAGGGCGAGGAG CGATGGGGGTGTTCTGCTGGTAGT LacZ GeoF GeoR 62 270 GTCGTTTTACAACGTCGTGACT GATGGGCGCATCGTAACCGTGC Ert2Cre ERT2Cre F1 ERT2Cre R1 65 400 ATGGATTTCCGTCTCTGGTG AAGGTTGGCAGCTCTCATGT Ptch1:Peak7_Ert2 Peak7F1 Peak7R1 62 500 GCCTCTAATCACCCACCCTTC GGCTGCTCAGTTTGGATGTTC Ptch1:Peak7_eGFPCre Peak7eGFPF1 Peak7eGFPR1 62 480 CTGAAGGGCTGGGACTAT GA AAGTCGTGCTGCTTCATGTG

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90 APPENDIX C PROBES USED FOR RNA IN SITU HYBRIDIZATION Table C -1. Probes used for RNA in situ hybridization. Gene Name Organism Plasmid Number Antisense Enzyme Antisense Polymerase Smo Mouse BH185 SalI T7 Ptch1 Mouse BH141 BamHI T 3 Sox9 Mouse BH165 HindIII T7 Fgf8 Mouse BH99 XhoI Sp6 Fgf8 Chicken BH12 EcoRI T7 Fgf4 Chicken BH11 EcoRI T7 Shh Mouse BH39 HindIII T3 Fgf4 Mouse BH144 BamHI T3 Gremlin1 Mouse BH75 PstI T3 Shh Chicken BH16 SalI Sp6 Ptc1 Chicken BH17 SalI T3 Bmp2 Chicken BH18 HindIII T3 Gli1 Chicken BH100 SacI T7 HoxD13 Chicken BH167 EcoRV T3 Sp8 Chicken BH30 NotI T3 Dkk1 Chicken BH151 NcoI Sp6

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103 K14-Cre recombinase expression in E10.5 limbs was retrieved from the Mouse Genome Database (MGD), (K14 -Cre MGI ID:2445832), Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Maine World Wide Web (URL: http://www.informatics.jax.org). [January, 2010]

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104 BIOGRAPHICAL SKETCH Cortney Bouldin earned his Bachelor of Science in microbiology and cell sciences from the University of Florida in 200 3. Prior to graduate school, he worked on the function of F1F0 ATP synthase in E. coli and the effects of protein restricted diets on the methylation status of DNA within developing embryos. During his graduate career, Cortney studied pattern formation in the developing vertebrate limb. Specifically, he identified a role for Shh signaling in the ectoderm of the developing limb and studied the molecular mechanisms involved in the formation of polydactyly. In his research, Cortney applied a methodology developed by Eva Kocsis to measure the lengths of protein molecules in electron micrographs to measure the lengths of Apical Ectodermal Ridges in the limb bud. Additionally, he participated in the creation of a cre recombinase allele, which is expressed in a subset of cells that express the hedgehog target Ptch1 Cortney has been an active member in the Society for Developmental Biology where he has received travel awards to attend annual meetings and awards in best student poster competitions. He also attended t he 10th International Congress on Limb Development and Regeneration in Madrid, Spain where he gave an oral presentation. Cortney recieved his Ph.D. from the University of Florida in the Spring of 2010, and intends to continue his work investigating mechani sms of pattern formation with a special focus on the early steps of embryo development including gastrulation and axis elongation.