Intervertebral Disc Development In Chickens

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Intervertebral Disc Development In Chickens
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english
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Mohiuddin,Yasmin S
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University of Florida
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Degree:
Master's ( M.S.)
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University of Florida
Degree Disciplines:
Medical Sciences, Medicine
Committee Chair:
Harfe, Brian D
Committee Members:
Cohn, Martin J
Renne, Rolf

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Medicine -- Dissertations, Academic -- UF
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Abstract:
Vertebrate vertebral columns are placed under large amounts of stress every day. In order to help relieve this stress between vertebrae, intervertebral discs are present to absorb shock and provide cushioning support; however over time these discs deteriorate which can result in serious medical problems. The intervertebral disc is composed of three parts: a jelly-like center called the nucleus pulposus that is surrounded by the cartilaginous annulus fibrosis. These two structures are bordered by anterior and posterior endplates. In order to obtain a more thorough understanding of disc development, we have investigated the intervertebral region of chickens. With the well-studied mouse model to serve as a comparison, we examined wild-type chicken disc development to determine how disc formation varied throughout vertebrate evolution beginning with chickens. Our initial examination of chickens revealed that mouse and chicken disc development were vastly different. It appears that chicken discs lack a nucleus pulposus. Currently, we are examining factors known to be involved in the differentiation of the mouse nucleus pulposus to determine if these factors play a similar role during chicken intervertebral development. One signaling pathway that we are currently examining in detail is the Sonic Hedgehog (Shh) pathway. Shh is required for differentiation of multiple tissues including the limb, brain, and spinal cord and we have shown that this signaling pathway plays an essential role in formation of the mouse nucleus pulposus. The long-term goal of this project is to understand the molecular mechanisms responsible for forming a normal intervertebral disc so that cell or molecular-based therapies can be developed to relieve back pain.
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In the series University of Florida Digital Collections.
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Statement of Responsibility:
by Yasmin S Mohiuddin.
Thesis:
Thesis (M.S.)--University of Florida, 2011.
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Adviser: Harfe, Brian D.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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1 INTE R VERTEBRAL DISC DEVELOPMENT IN CHICKENS By YASMIN S. MOHIUDDIN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 201 1

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2 2011 Yasmin S. Mohiuddin

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3 To my mom and dad

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4 ACKNOWLEDGEMENTS I thank my mentor Brian Harfe and my other committee members for their guidance. I also thank everyone in the Harfe and Cohn labs for their help along the way I would like to specially thank Kyung Suk Choi and Jen Maier for their assistance in obtaining mouse wil d t ype data. Lastly I thank my f amily and friends for their constant support.

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5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 TABLE OF CONTENTS ................................ ................................ ................................ .. 5 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 8 CHAPTER 1 INTRODUCTION AND AIMS ................................ ................................ .................. 10 Development of the Intervertebral Disc in Vertebrates ................................ ........... 10 Cellular Origin of the Nucleus Pulposus ................................ ................................ .. 11 Avian Vertebral Development ................................ ................................ ................. 11 Composition of the Intervertebral Discs ................................ ................................ .. 12 Sonic Hedgehog ................................ ................................ ................................ ..... 12 Scleraxis ................................ ................................ ................................ ................. 1 3 Aims of Thesis ................................ ................................ ................................ ........ 13 2 METHODS ................................ ................................ ................................ .............. 16 Chicken Incubation and Harvest ................................ ................................ ............. 16 Paraffin Embedding ................................ ................................ ................................ 16 Cryo Embedding ................................ ................................ ................................ .. 17 Histological Staining ................................ ................................ ............................... 17 Probe Preparation ................................ ................................ ................................ ... 17 Whole Mount In situ Hybridization ................................ ................................ .......... 18 Section In situ Hybridization ................................ ................................ .................... 18 3 RESULTS ................................ ................................ ................................ ............... 19 Histological Analysis of intervertebral discs ................................ ............................ 19 Sonic Hedgehog Gene Expression ................................ ................................ ......... 20 Scleraxis Gene Expression ................................ ................................ ..................... 21 4 DISCUSSION ................................ ................................ ................................ ......... 26 Intervertebral Development Analysis Via Collagen Staining ................................ ... 26 Sonic Hedgehog Effect Pre and Postnatal ................................ .............................. 27 Scleraxis Effects and Continuation through Birth ................................ .................... 27 APPENDIX: LIST OF REAGENTS ................................ ................................ ................ 29

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6 LIST OF REFERENCES ................................ ................................ ............................... 31 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 33

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7 LIST OF FIGURES Figure page 1 1 4 Week old mouse intervertebral disc ................................ ................................ 15 1 2 Mouse and proposed chicken intervertebral disc model ................................ ..... 15 3 1 Histological Analysis of Early Chicken Development ................................ .......... 22 3 2 Histological Analysis of Late Chicken De velopment ................................ ........... 23 3 3 Serial Sections of HH35 Chick vertebral column ................................ ................ 23 3 4 Sonic Hedgehog Gene Expression in Early Chicken Development .................... 24 3 5 Sonic H edgehog Gene Expression at HH35 ................................ ..................... 24 3 6 Sonic Hedgehog Gene Expression at HH44. ................................ ..................... 24 3 7 Scleraxis Gene Expression in HH19 and HH27 Chickens ................................ .. 25 3 8 Scleraxis Gene Expression in HH35 and HH44 Chickens. ................................ 25

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8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INT ERVERTEBRAL DISC DEV ELOPMENT IN CHICKENS By Yasmin S. Mohiuddin August 2011 Chair: Brian D. Harfe Major: Medical Science s Vertebrate vertebral columns are placed under large amounts of stress every day. In order to help relieve this stress between vertebrae, intervertebral discs are present to absorb shock and provide cushioning support; however over time the se discs deteriorate which can result in serious medical problems. The intervertebral disc is composed of three parts: a jelly like center called the nucleus pulposus that is surrounded by the cartilaginous annulus fibrosis. Th ese two structures are border ed by anterior and posterior endplates. In order to obtain a more thorough understanding of disc development, we have investigated the intervertebral region of chickens. With the well studied mouse model to serve as a comparison, we examined wild type chic ken disc development to determine how disc formation varied through out vertebrate evolution beginning with chickens Our initial examination of chickens revealed that mouse and chicken disc development were vastly different. It appears that chicken discs l ack a nucleus pulposus. Currently, we are examining factors known to be involved in the differentiation of the mouse nucleus pulposus to determine if these factors play a similar role during chicken intervertebral development. One signaling pathway that we are currently examining in detail is the Sonic Hedgehog (S hh ) pathway.

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9 S hh is required for differentiation of multiple tissues including the limb, brain, and spinal cord and we have shown that this signaling pathway plays an essential role in formation of the mouse nucleus pulposus. The long term goal of this project is to understand the molecular mechanisms responsible for forming a normal intervertebral disc so that cell or molecular based therapies can be developed to relieve back pain.

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10 C HAPTER 1 INTRODUCTION AND AIM S Development of the Intervertebral Disc in Vertebrates Intervertebral disc are structures present in mammalian spinal columns The role of intervertebral discs is to help relieve stress between bony vertebrae. With age these disc s can dete riorate and lose their structural integrity causing severe pain. According to the National Institutes of Health 8 out of 10 people will suffer from back pain at some point during their lives (NIH, 2011) Some of these conditions affecting disc str ucture include bulging, herniated, and thinning disc s In addition to these conditions disc degeneration due to aging still remains one of the primary causes of back pain (Hunter et al. 2003). Development of i ntervertebral discs follows similar pathways in all mammals and results in i ntervertebral discs that are composed of three parts (Alini, 2008) The center of each disc is composed of jelly like material called the nucleus pulposus. Surrounding this structure is the annulus fibrosis which is comprised p rimarily of cartilage. Finally on both sides of the disc are the anterior and posterior endplates (Humzah and Soames, 1988) When examining a completely developed mammalian adult intervertebral disc using collagen staining reagents Picrosirius Red and Alc ian Blue on 4 week old paraffin embedded mice the three components can be easily identified. (Figure 1 1 ) In our laboratory we use the mouse model system to model human disc development and disease. In mice, the embryonic notochord is present from E 7.5 until E12. 5 (Choi, 2008 ) Previous work from ou r laboratory has demonstrated that the embryonic notochord forms the middle part of the disc called the nucleus pulposus (Choi et al, 2008) The nucleus p ul posus persists throughout adult life in both mice and

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11 humans. Figure 1 1 shows the center nucleus pulposu s (NP) surrounded by the collage n rich annulus fibrosis (AF) In blue the anterior and posterior endplates can be seen on their respective sides bordering the vertebral bone. As an organism ages it is t h e cells that compose the nucleus pulposus that have been proposed to be responsible for main t a in in g disc structure These cells which originate from the notochord, may serv e as disc stem cells and may be able to replace damaged or diseased nuclei pulposi throughout life ( Hunter 200 3 ) Cell ular O rigin of the Nucleus Pulposus Using a previously created SHHcre allele (Harfe et al 2004), all cells expressing Sonic Hedgehog were made to express CRE protein, including th ose in the embryonic notochord. These mice were crossed with the CRE reporter lines R26R or R OSA 26R:EYFP Mice containing bot h the Shhcre and a reporter allele activate d reporter expression via CRE recombinase driven by the Shh promoter. All cells expressing Shh and any of their cells descendants are labeled throughout the life of the animal. At E10.5, in embryos in which CRE expression was activated using the Shhcre allele, a long rod like notochord is visible. B y E12.5 the notochord has begun to form bulges and by E15.5 clearly observed nucle i pulposi have formed from the S hh cre expressing cells (Choi et al 2008) Avian Vertebral Development I ntervertebral development in chickens has not been thoroughly documented in the publi shed literature The current model suggests th at the vertebral bodies arise from the somites (Christ, 2000 )

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12 Future vertebral tissue begins as a part of the paraxial mesoderm in a portion known as the segmental plate. Via epithelialization of this tissue, the first 28 pairs of somites are formed by primary gastrulation (Christ, 2000). Avian somites are surrounded by a basement membrane and connected to other structures via the extracellular matrix such as the notochord and neural tube. Between day 2 and 3 of embryonic development the early somites undergo an epithelial to mesenchymal transit ion and begin to form the sclero tome. fissure dividing each one into a cranial and a caudal half ( Christ, 2000 ) When vertebrae are forming, the cranial half of one somite fuses with the caudal half of an adjacent somite and forms one vertebral body and the intervertebral tissue In particular t he intervertebral discs are thought to arise from the somitocoele derived cells which are cells located in the somiti c core that retain their original mesenchymal organization. (Christ et al 200 4 ) Composition of the Intervertebral Disc s Figure 1 2 models the difference between the current mouse disc structure and our proposed model of avian intervertebral structure. T he mouse model is composed of the three structures mentioned previously Currently in avian s it is known this type of disc is not present, but tissue of unknown origin and composition is present between vertebrae One of the goal s of this thesis is to characterize this tissue and determine if there are any similarities to analogous mouse disc structures. Sonic Hedgehog Three homologues of the Hedgehog gene exist in vertebrates Indian Hedgehog, Desert Hedgehog, and the one in particular this study is investigating Sonic Hedgehog

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13 (Shh). Shh plays an essential role as a morphogen in vertebrate development in the limb, brain, spinal cord, and other areas ( McMahon 2003). Excretion of Shh from Sonic Hedgehog producing cells activates the Shh pathway Ext racellular Shh binds with Pat c hed (Fuse, 1999) and activates the trans membrane protein Smoothened (Murone et al 1999 ) In the presence of activated Smoothened, Gli proteins enter the nucleus and act as transcriptional activators ( Hooper and Scott, 2005 ) Scleraxis Scleraxis is a transcription factor that is expressed in developing tendons and the annulus fibrosis (Deries et al 2010) It is expressed in cells that develop into tendon tissue and other muscle attachments. Additionally scleraxis plays a role in mesoderm formation a nd is present in the embryonic mesenchymal tissue during somite and vertebral development (Shukunami et al 2006) Aims of Thesis The goal of this study is to characterize avian vertebral development utilizing the chi cken model to determine if formation of the intervertebral discs has been conserved between mammals and birds. The instances of back pain due to problems with disc maintenance are increasing rapidly in humans Currently, there are very few therapies to aid in disc repair. M ost therapies are geared towards relieving pain associated with a degenerating disc and not the underlying defect In this work we performed experime n ts designed to uncover molecules and genetic pathways that are required for formation of the discs. In Aim 1, I examine d the developing chicken intervertebral disc at various stages using histological stains to determine what tissues were present during these stages.

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14 Intervertebral disc development in mice was examined, and stages were chosen that matched with comparable chicken stages These comparisons were based on limb development. Since the n oto chord forms nucle i pulpo si between E11 .0 and E16 .0 in mice analogous chicken stages preceding, during, and after these time points were chosen for analysis in chickens. The stages chosen were E8.5, E1 2 .5, E14.5 and E18 for mice, which comprise s of stages from pre intervertebral disc formation through the presence of fully formed discs in newborn. For our chicken samples, we chose Hamilton and Ham burg er (HH) stages 19, 27, 35, and 44 (Hamburger and Hamilton 1951) By histologically examining development of the discs at these stages we can determine when structures are forming and there relative shape compared to the previously characterized mouse model. Our second aim is to determine Sonic Hedgehog expression in the notochord and spinal column as the chicken develop s Since Shh is known to be expressed throughout embryonic development in mice, and proposed to help maintain disc structure after they form in adult mammals (Hunter, 2003) observing the duration and loaction of Shh expression in ch i ckens may explain the developmental difference between the mouse and chicken vertebral structure. The last aim is to monitor the expression patter ns of scleraxis in developing chicken embryos. Since scleraxis is expressed in the annulus fibrosis, monitoring the different stages of chicken development will allow us to see if an annulus is present in chickens. If present expression patterns will also show annulus structural changes throughout development allowing for a better understanding of what takes place in the chicken vertebral column during disc formation.

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15 Figure 1 1. 4 Week old mouse int ervertebral disc, stained with picrosirus red (collagens) and alcian b lue (Glycosaminoglycans, GAGs), NP = Nucleus Pulposus, AF = Annulus Fibrosis, V = Vertebrae Figure 1 2 Mouse and p roposed c hicken i ntervertebral d isc m odels. V = Vertebrae NP = Nucleus Pulposus, AF = Annulus Fibrosis. V V AF NP End Plates AF End Plates Uncharacterized Tissue Mouse Chicken

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16 CHAPTER 2 METHODS This chapter contains protocols including a list of solution and reagents used to conduct the experiments fo r this study. Chicken Incubation and Harvest Fertilized chicken eggs were ordered from Charles River and are placed in an incubator at 38 C The eggs were incubated for the time it takes to reach each stage of interest, according to the Hamburg er and Hami lton developmental stages ( Hamburger and Hamilton, 1951). Upon reaching the correct incubation time, the chicken embryos were harvested and fixed overnight in 4% PFA. The chicken samples used for these experiments were harvested at HH 19, 27, 35, and 44. T hese stages were representative of the overall chick growth cycle in an egg, including the time for potential intervertebral disc development. Paraffin Embedding After fixing overnight in 4% PFA, samples were decalcified in CalEx (Fisher) if necessary. Fo r stage HH 35 and HH 44 samples, vertebral columns were dissected out prior to decalcification in order to minimize excess tissue. Samples were washed three times in PBS for 20 minutes each. Following the washes samples were dehydrated in an ethanol series starting with a 30 minute was h in 70% EtO H followed by two 30 minute washes in 95% EtO H and finally completely dehydrated by three 20 minute washes in 100% EtO H The samples then proceed through three 10 minute washes in xylenes to clear the ti ssue Next the tissue was placed in an incubator and set in six 20 minute rinses in blue ribbon embedding material. Finally the samples are embedded in paraffin and stored at room temperature

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17 Cryo Embedding Embryos were removed from overnight 4% PFA fi x and washed in DEPC water twice for five minutes each. Samples were then placed on a rocker in 15% sucrose at room temperature until the embryos sunk followed by a 30% sucrose solution overnight at 4C. The following day the embryos were placed in a 50/5 0 mixture of 30% sucrose and Optimum Cutting Material (OCT) at room temperature approximately 23 C for 3 4 hours. The samples were then embedded in 100% OCT and stored at 80C until needed. Histological Staining Paraffin embedded samples were sectioned using a Leica microtome at 7m sections and floated onto slides. After drying, the slides are placed in a rack and taken through the following series of solutions for the times given: Xylenes 10 minutes 100% Ethanol 10 minutes 95% Ethanol 1 0 minutes 70% Ethanol 10 minutes Distil led Water 5 minutes Alcian Blue (1.0g alcian blue 8GX + 97 ml water) 15 minutes Water 10 minutes Picrosirius Red 45 minutes 0.5% Acidified Water 5 minutes 95% Ethanol 2 minutes 100% Ethanol 2 minutes Xy lenes 5 minutes The slides where then spotted with permount and cover slipped. Slides were examined using Leica DM2500 compound microscope. Probe Preparation Plasmids were taken from Harfe Lab stock and grown on LB Ampicillin plates. Colonies were pi cked and grown in LB media overnight at 37C. Bacterial c olonies were

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18 pelleted and DNA was extracted using Qiagen Miniprep Kit. After purification the DNA was digested and mixed with reagents for probe assembly. Digested DNA was mixed Roche 10x transcripti on buffer, DIG labeled nucleotide mix RNase inhibitor, RNA polymerase and incubated for two hours Pr obe was then purified through Roche mini quick RNA s pin column s and stored at 80C until needed Whole Mount In situ Hybridization Samples selected for whole mount In Situ hybridization were dehydrated after overnight fixation following harvest. Whole mount analysis was only done on HH 19 and HH 27 embryos due to their young age and difficulty of isolating vertebral column. The whole embryos were then dehydrated through a methanol series permeabilized and incubated with probe. After two days of washes the samples were put in a color solution which produce s a dark purple precipitate after reacting with the alkaline phosphatase found in AP DIG labeled antibody to bind to probe and antibody Section In situ Hybridization OCT embedded samples were serially sectioned to 12m and lifted onto slides. Sections were surrounded by a DAKO pen to build a hydrophobic barrier. The sections are acid hydrolyzed with 0.2M HCl, washed with DEPC PBS permeab i lized with Proteinase K and then fixed in 4% PFA. Probe was applied overnight. The following day the slides were passaged through a series of SSC washes, after which the slides were blocked with 10% goat serum and KTBT solution Antibody w as applied overnight at 4C. The last day the slides were placed in a color solution and developed at room temperature.

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19 CHAPTER 3 RESULTS Histological Analysis of intervertebral discs In order to observe formation of chicken intervertebral discs during development histological staining of embryos was conducted with picrosirius red and alcian blue. These reagents stain red for collagen and blue for glyc o s aminoglycans respectively. In the early stages HH19 and HH27 of chick embryo genesis, the notochord running the lateral length of the body was clearly visible (Figure 3 1 ). At our chosen comparable mouse stage E12.5 which is equated to HH27, the notochord is observed in the same form This leads us to conclude that early development in both organisms is similar. As the chicken embryo develop s to stage HH27, the notochord is still distinctly visible, while the vertebral body begins to undergo ossification. The two remaining stages that were examined, HH35 and H H44 are two later stages which correspond to mouse stages in which intervertebral discs have already developed In chickens a t these stages the notochord still persist ed (Fig ure 3 2 ). In contrast no notochord was observed in adult chickens. These data sug gests that the notochord does not exist throughout postnatal life. It is currently unclear the exact developmental stage when this struc ture is no longer present in adult chickens. Unlike the chicken the mouse notochord does not progress in the same manner. In Figure 3 2 mouse vertebral sections at E14.5 and at P0 clearly delineated nuclei pulpos i that arise from the notochord presumabl y through cell migrat ion In chickens, when we examined stage HH35 onward there were noticeabl e collagen rings that formed between ver tebrae. Serial s ections examined from entire

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20 vertebral columns showed the collagenous rings to have a central area, where the notochord passed through to maintain its rod like structure. Sonic Hedgehog Gene Expressi on The second aim of this project was to determine how the signaling protein, Sonic Hedgehog, was expressed during chicken disc formation It is known that SHH is expressed by notochordal cells and the floor plate of the neural tu be (Fan and Tessier, 1994) However it still remains unknown if these structures continue to produce this developmental signal past HH 29 In our experiment we monitored sonic expression through development and expression was found in each stage examined. Sonic Hedgehog expression was found in the notochord at HH19 and in both notochord and floorplate in HH27. As the chick develops the expression persists in the notochord and when sagittal sections are examined in both chick and mouse at comparable g rowth stages of HH35 and E14.5 S h h expression was visible in the chick notochord and mouse nuclei pulposi These data suggest that both groups of cell s had similar cell origins and expressed at least one of the same genes. As the chick develops, the notochord continues to be observed and maintained as a rod like structure, whereas in the mouse at an equivalent developmental stage, notochord cells were observed to form nuclei pulposi within the center of the discs. Examining the notochord structure from HH35 and HH44 embryos, the notochord remained the same. When compared with a P0 mouse, the Shh expression from E14.5 to P0 remains localized to the nuclei pulposi. From these experiments we can conclude that Sonic Hedgehog expression persists in the notochord throughout the entire embryonic development of the chicken. As shown in f igure 3 5 and figure 3 6, expression is present in the mouse at E14.5 and

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21 P0, However the cells that were found to express Shh in mice w ere also found to condense into bulges and eventually form the disc center nucl e i pulposi. Scleraxis Gene Expression In order to understand the function of the rod like notochord that persists scleraxis expression. Scleraxis is a tendon marker that in mice is expressed in the annulus fibrosis, the cartilaginous ring surrounding the nucleus pulpos u s (Deries et al, 2010 ). Using this marker, we examined early stages of disc formation HH19 and HH27 in chicken. At early stage s clear expression was observed in the anterior and posterior borders of the somites. ( Shukunami et al., 200 6 ). In the later stages HH35, of chick development it has previously been shown that scleraxis is expressed in the developing tendon at the anterior and posterior edges of the developing vertebrae (Schweitzer et al, 2001). As seen in figure 6 whole mount RNA in situ hybridization at HH19 there is expression in the tissue immediately surrounding the notochord (arrow ) As the embryo progresses up to the pre disc (E12.5) equi valent at HH27, scleraxis staining was found as published on the anterior and posterior borders of the somites (Figure 3 7 and 3 8 ) In these later stages expression can be seen in bordering the vertebrae. At the final stage examined HH44 scleraxis expression was difficult to interpret due to high background levels. However staining still persisted into this late stage Additional experiments are being conducted to verify its exact expression location at this late stage.

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22 Figure 3 1. Histological a nalysis of early c hicken d evelopment visualized with picrosirius red and alcian blue A) T he vertebral region of chick at HH19. B) The verterbral region of chick at HH27. C) The vertebral region of mouse at e12.5. T he notochord is present in both organisms at HH19 and HH27 in chickens and E12.5 mouse. Mouse E12.5 N C V V V Chick Chick C

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23 Figure 3 2: Histological a nalysis of l ate c hicken d evelopment stained with picrosirius red (collagens) and alcian blue (GAGs) A) Chicken section at HH35. B) Chicken section at HH44. C) Adult chicken section. D) Mouse vertebral section at E14.5. E) Mouse vertebral section at P0 In chickens, the notochord (arrow) pe rsists until birth but is not visible in adults. Rings of collagen (Red labeled with *) were visible from HH35 till birth. NP = nucleus pulposus, V = vertebra Figur e 3 3. Serial sections of HH35 c hick vertebral column stained with picrosirius red and alcian blue to visualize collagenous ring (arrow) present between vertebrae at HH35. A) Dorsal section from HH35 chick. B) Middle section from HH35 chick. C)Ventral section from HH35 chick. ~50 m apar t. NC = Notochord, V = Vertebrae V V V V V V NC NC E14.5 P0 N P V N P N P V N P * A B C D E A B C

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24 Figure 3 4. Sonic h edgehog gene e xpression in early chicken d evelopment. Whole m ount In situ hybridization with Sonic Hedgehog. Expression can clearly be seen in the notochord and floor plate. (NC = notochord) Figure 3 5 Sonic h edgehog Gene Expression at HH35.Sagittal section in situ hybridization of Chick and Mouse at HH35 and E14.5 days of development respectively. NC = Notochord, NP = Nucleus Pulposus. Figure 3 6 Sonic Hedgehog Gene Expression at HH44. Section In situ hybridization. Chick transverse section at HH44 shows notochord (NC) stained positive. P0 Mouse Betagal stained section shows notochord derived nuclei pulposi (NP). Dorsal HH19 HH27 Dorsal Chick HH35 Sagittal Mouse E14.5 Sagittal NP HH44 Chick Transverse Mouse P0 N P N P V NC NC NC Dorsal

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25 Figure 3 7: Scleraxis Gene Expression in HH19 and HH27 Chickens, whole mount in situ hybridization, HH19 Scx expression surrounding notochord. HH27 Scx expression is observed between the somites. NC = notochord Figure 3 8 Scleraxis Gene Expression in HH35 and HH44 Chickens. HH35 Sagittal and HH44 Transverse section in situ Hybridizations. In HH35 scleraxis staining was visible on vertebral borders and in near birth stage HH44, staining was difficult to interpret. HH35 HH44 Transverse V V NC HH19 HH27 NC NC

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26 CHAPTER 4 DISCUSSION Intervertebral D evelopment Analysis V ia Collagen S taining Our experiments investigating intervertebral development in the chicken embryo determined that difference s exist between mammalian intervertebral disc development and avian intervertebral development. In the chicken s we found that the notochord remain ed present at least until birth. Additionally in histological section s it was observed that cartilaginous tissue for ms between the developing vertebrae. In the early stages examined it is difficult to see the developing collagen However upon looking at HH35 and after a ring structure of collagen in the region of the forming disc is evident which may prov ide some typ e of cushioning The notochord was found to persist throughout the length of the vertebral column at these stages As the embryo develops and gains more mobility these collagen rings form to possibly cushion the vertebrae from increased stress due to devel opment and in particular while hatching In contrast in the adult sample no presence of intervertebral rings or notochord was observed It is unclear what happens from the period of birth to an adult chicken but at some developmental stage the notochord deteriorates leaving bony vertebrae. One explanation for the ring s that are present between vertebrae is for stress reduction as the chick develops into an adult As seen in Figure 3 B there is a dark red collagen staining between the bony p rotrusions. While examining the sample it can be seen that the edges have more GAG (alcian blue) staining, however as the section progresses in a ventral to dorsal fashion, there is a clear transition from GAG to a collagen and then back to the GAG, sugges ting that disc structure has a collagen center but on a much smaller scal e than a mammalian disc center

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27 This miniature disc could develop as a stress reducing mechanism, only necessary as the chick ages and deteriorate when the chick matures In contrast as the chick matures into an adult, and the notochord deteriorates, the cells surrounding the notochord may fill the void created by this structure. This causes the ring structures to condense and flatten into the collagen rich tissue found between adult vertebrae. In the future I would like to examine post birth samples and determine the composition of this intervertebral tissue and how it develops post embryonically Sonic Hedgehog Effect Pre and Postnatal As previously shown Sonic Hedgehog was found to be expressed for the first two stages, HH19 and HH27 in the somites (Johnson, 1994) Additionally Shh expression continues until birth throughout the notochord similar to mouse models where expression continues in the nucleus pulposus (DiPaola, 2005). As the chick grows persistent sonic hedgehog signal from the notochord might be a method for the renewal of notochord cells that die from daily stress involving the vertebral column Additionally it seems that chicks have a slower developmental clock, an d the persistence of the notochord into late embryonic stages may correlate with this slower development. It will be an interesting future experiment to see if sonic hedgehog expression continues into adulthood and coincides with notochord disappearance. Scleraxis Effects and Continuation through B irth Scleraxis expression was previously shown in the literature to be expressed in the somites in early s tage chick embryos (Shukunami, 2006). In my experiments, th ose results were confirmed and the early stage s of HH19 and HH27 showed clear evidence of scleraxis expression. In the earliest stage the expression in the vertebral area was limited to the cells surrounding the notochord As the chicks developed scleraxis

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28 expression spread to the areas bordering each vertebra Since scleraxis is a marker of the annulus in the mouse model, and it remains surrounding the notochord the evidence suggests that there is some structure similar to the annulus that runs the outside entire length of the notochord and possibly behaves in a functionally similar manner By retaining a similar structure to an annulus fibrosis, chick vertebral columns may utilize it to lighten the stress present on the rod like notochord. As the chick develops, scleraxis signaling decreases and it may be due to the developing vertebra reducing stress levels enough to the point where an annulus fibrosis structure is no longer necessary. In contrast chicks may also retain the annulus fibrosis but with the vertebrae and collagenous rings helping to red uce stress levels, the annulus tissue plays a minimal role.

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29 A PPENDIX LIST OF REAGENTS This list includes the reagents as they were used in experiments and the companies from which they were bought. Alcian Blue(Acros Organics) o 1.0g alcian blue o 97 ml distilled water o 3 ml glacial acetic acid Ampicillen (Fisher) Acetic Acid (Fisher) Blue Ribbon Embedding Medium (Surgipath) 5 bromo 4 chloro 3 indolyl phosphate (BCIP) (ROCHE) Dako pen (Super HT) DEPC (Applichem) Ethanol (Fisher) Formamide (Fisher) Gelatin (Fisher) Gluteraldehyde (Sigma) Goat Serum (Sigma) Hydrochloric Acid (Fisher) KTBT o Tris ph 7.5 50mM o NaCl o KCl o Tween LB (Fisher) Magnesium Chloride (MgCl 2 ) (Fisher) Methanol Miniprep Kit (Qiagen) Nitroblue Tetrazolium Chloride (NBT) (ROCHE) NaCl (Fisher) NTMT o Tris ph 9.5 50mM o MgCl2 50mM (Fisher) o NaCl 100 mM (Fisher) o Triton 1% (Sigma) Optimal Cutting Temperature Compound (OCT) (Fisher) Paraffin (Fisher) Paraformaldehyde (Fisher) Phosphate Buffered Saline (Fisher) Picrosirius Red (Polyscientific) Permount (F isher)

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30 Proteinase K (Fisher) Tween 20 (Fisher) Xylenes (Fisher)

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31 LIST OF REFERENCES Alini M, Eisenstein S M, Ito K Little C, Kettler AA Masuda K, Melro se J, Ralphs J, Stokes I, Wilke HJ. 2008. Are Animal Models useful for studying human disc disorders/dege ne ration? Eur Spine J 17: 2 19. Choi KS Cohn MJ and Harfe BD. 2008. Identification of Nucleus Pulposus Precursor Cells and Notochordal Remnants in the Mouse: Implications for Disk D egeneration and Chordoma Formation Developmental Dynamics 237: 3953 3958. Choi KS Harfe BD. 2011. Hedgehog signaling is required for formation of the notochord sheath and patterning of nuclei pulposi within the intervertebral disc. Developmental Biology 108(23): 9484 9489. Christ B, Huang R, Scaal M. 2004 Formation and differentiation of the avian sclerotome. Anat Embryol 208:333 350. Christ B, Huang R, Wilting J. 2000. The development of the avian vertebral column. Anat Embryol. 20 2: 179 194. Deries M, Schweitzer R, Duxson MJ. 2010. Developmental fate of the mammalian myot ome. Dev Dyn 239(11): 2898 2910 DiPaola CP Farmer JC, Manova K, Niswander Lee. 2005. Molecular signaling in intervertebral disk development. Journal of Orthopaedi c Research. 1112 1119. Fan CM, Tessier Lavign M. 1994. Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hed gehog homolog. Cell 67:767 774. Fuse N Maiti T, Wang B, Porter JA, Hall TM, Leahy DJ, Beach y PA 1999. Sonic Hedgehog protein signals not as a hydrolyt i c enzyme but as an apparent ligand for patched. Proc Natl Acad Sci USA 96: 10992 10999 Hamburger V, Hamilton H. 1951. A series of normal stages in the development of the chic k embryo. J Morphol. 88:49 92. Harfe BD, Scherz PJ, Nissim S, Tian H, McMahon AP, Tabin CJ. 2004. Evidence for an expansion based temporal shh gradient in specifying vertebrate digi t identities. Cell 118:517 528. Hooper JE, Scott MP. 2005. Communicating with Hedgehogs. Nat Re v Mol Cell Biol 6:306 317 Humzah MD, Soames RW. 1988. Human intervertebral disc: structure and function. Anat Rec. 220: 337 356.

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32 Hunter CJ, Matyas JR, Duncan NA. 2003. The notochordal cell in the nucleus pulposus: a review in the context of tissue engineer ing. Tissue Eng 9:667 677. Johnson RL, Laufer E, Riddle RD, Tabin C. 1994. Ectopic expression of Sonic Hedgehog alters dorsal ventral patterning of somites. Cell 79(7):1165 1173. Lachlan S J Nerukar NL, Choi KS Harfe BD, Elliott DM. Degeneration and re generation of the intervertebral disc: lessons from development. Disease Models & Mechanisms 4 31 41. McMahon AP, Ingham PW, Tabin CJ. 2003. Development roles and clinical significance of hedgehog signaling. Curr. Top. Dev. Biol. 53:1 114 Murone M, Rosenthal A, de Sauvage FJ. 1999. Sonic Hedgehog signaling by the patched smoothened receptor complex. Curr Biol 9: 76 84 National Institutes of Health. Back Pain: Medline Plus. June 3, 2011. http://www.nlm.nih.gov/medlineplus/backpain.html Schweitzer R, Chyung JH, Murtaugh LC, Brent AE, Rosen V, Olson EN, Lassar A, Tabin CJ. 2001. Analysis of the tendon cell fate using Scleraxis, a specific marker for tendons and ligaments. Development 1 28(19): 3855 3866. Setton LA, Chen J. 2006. Mechanobiology of the intervertebral disc and relevance to disc degeneration. J Bone Joint Surg Am 88( Suppl 2):52 57 Shukunami C, Takimoto A, Oro M, Hiraki Y. 2006. Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of teno cytes. Dev Bio 298(1): 234 247. Vokes SA. 2007. Genomic characterization of Gli activator targets in sonic hedgehog mediated neural patterning. Development 134: 1977 1989.

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33 BIOGRAPHICAL SKETCH Yasmin Mohiu ddin received a B achelor of Science, majoring in microbiology and cell s cience from the University of Florida in Gainesville, Florida in 2009. Here she was the recipient of the HHMI Science of Life Award and accepted into the University Scholars program She received her Master of Science majoring in m edical s ciences in 2011.