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Comparative Analysis of Sonic Hedgehog Signalling and the Response to Sonic Hedgehog Signalling in Vertebrate Forelimbs and Hindlimbs

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Comparative Analysis of Sonic Hedgehog Signalling and the Response to Sonic Hedgehog Signalling in Vertebrate Forelimbs and Hindlimbs Comparative analysis of Sonic Hedgehog signalling and the response to Sonic Hedgehog signalling in vertebrate forelimbs and hindlimbs Martin David Carkett Division of Developmental Biology, MRC Francis Crick Institute, Mill Hill Laboratories Mill Hill, London UCL Submitted in 2015 for the degree of doctor of philosophy Declaration I, Martin David Carkett, confirm that the work presented in this thesis is my own and was performed in the laboratory of Dr. Malcolm Logan at the MRC National Institute for Medical Research, the Francis Crick Institute Mill Hill Laboratories and King’s College London. Where information or reagents have been derived from other sources, I confirm that this has been indicated in the thesis. This work has been submitted for the degree of doctor of philosophy. 2 Acknowledgments First and foremost I would like to thank Malcolm for giving me the opportunity to study this project and for giving me both the freedom and support to complete it as best I could. It has been a great experience and I have learnt a lot. I would like to thank fellow Logan lab members Sorrel, Satoko, Ania, Sue, Vero, Natalie and Laurianne for their input, advice and friendship without which this would have been far more difficult and far less fun. Noriaki, James B, James T and Tim have given me valuable advice throughout, for which I thank them too. I would like to mention Alex E, Clem, Jimena, Andy, Manu, Charlie, Alex W and Aina who have been great friends and have made the last four years a far richer experience beyond the lab. Lastly, as ever, my family have offered perspective, support and guidance throughout which has been invaluable. 3 Abstract The Sonic Hedgehog (Shh) morphogen is required to establish anteroposterior (AP) pattern in vertebrate limbs. Limb progenitors exposed to increasing levels or durations of Shh signalling ultimately give rise to progressively more posterior structures. However, how Shh specifies different digit identities at a molecular level is poorly understood and molecular markers of individual digits are yet to be determined. Shh also patterns the dorsoventral axis of the vertebrate neural tube, where desensitisation to Shh signalling via Patched-mediated negative feedback – termed temporal adaptation - is required for correct interpretation of the Shh morphogen gradient. To investigate how limb progenitors respond to, and integrate, different levels and durations of Shh signalling at a molecular level I have developed an ex vivo assay and used RNA-sequencing to examine the immediate transcriptional responses of chick limb progenitors exposed to defined concentrations of Shh over fixed periods of time. I observe that limb progenitors initially respond equivalently to different concentrations of Shh but establish a graded response over time through a variation of a temporal adaptation mechanism in which both signal desensitisation and signal accumulation are required to generate distinct transcriptional outputs. I demonstrate that signal desensitisation is mediated, at least in part, by Patched-mediated negative feedback, but that additional cell-autonomous and noncell-autonomous feedback mechanisms operating through Sufu/Gli and Disp1 also exist. I further use in silico analyses to identify candidate markers of digit identities that are induced by different levels of Shh signalling. I show a subset of candidate markers are expressed in intermediate AP domains, consistent with predictions, and may mark or specify middle digit identities. Finally, I have investigated differences in Shh signalling dynamics and the response to Shh signalling in chick forelimbs and hindlimbs and provide evidence that hindlimbs are patterned by Shh over a shorter period of time. Table of contents DECLARATION 2 ACKNOWLEDGMENTS 3 ABSTRACT 4 TABLE OF CONTENTS 5 LIST OF FIGURES 9 LIST OF TABLES 11 LIST OF ABBREVIATIONS 12 CHAPTER 1: INTRODUCTION 13 1.1 THE EMBRYOLOGICAL ORIGIN AND FORMATION OF VERTEBRATE LIMBS 14 THE SKELETAL STRUCTURE OF THE VERTEBRATE LIMB 14 EMBRYOLOGICAL ORIGIN OF VERTEBRATE LIMBS 16 LIMB BUD INITIATION 17 ORGANISING CENTRES WITHIN THE LIMB 18 1.2 THE SONIC HEDGEHOG (SHH) SIGNALLING PATHWAY 20 THE SHH MORPHOGEN 20 SHH SYNTHESIS AND DISPERSION 21 SHH SIGNALLING IS MEDIATED THROUGH GLI TRANSCRIPTION FACTORS 23 1.3 SONIC HEDGEHOG MORPHOGEN ACTIVITY IN VERTEBRATE LIMBS 27 SHH ACTS TO FORM A COUNTER GRADIENT OF GLI3R IN THE LIMB BUD 30 SHH SIGNALS IN A DOSE AND TIME DEPENDANT MANNER IN VERTEBRATE LIMBS 32 ENCODING DIGIT IDENTITY 39 1.4 INTRA-CELLULAR DYNAMICS OF SHH SIGNALLING: INSIGHTS FROM THE NEURAL TUBE 43 SHH INTERPRETATION BY A TEMPORAL ADAPTATION MECHANISM 43 LIGAND DEPENDENT ANTAGONISM 46 1.5 DIFFERENCES IN SHH SIGNALLING IN VERTEBRATE FORELIMBS AND HINDLIMBS 49 1.6 SUMMARY OF AIMS 52 CHAPTER 2: MATERIALS AND METHODS 54 2.1 CHICK LIMB BUD MEASUREMENTS 55 2.2 QUANTIFYING LIMB BUD CELL NUMBERS 55 2.3 WHOLE MOUNT IN SITU HYBRIDISATIONS 56 2.4 QUANTIFYING ENDOGENOUS EXPRESSION LEVELS OF SHH, PTCH1 AND GLI1 IN CHICK FORELIMBS AND HINDLIMB BUDS 57 5 2.5 QUANTIFYING SHH EXPRESSION AND SHH RESPONSE DOMAINS IN CHICK LIMB BUDS 58 2.6 DISSOCIATED CELL CULTURE 58 2.7 EXPLANT EX VIVO CULTURE 59 2.8 QUANTITATIVE PCR 60 2.9 RNA-SEQUENCING 61 2.10 GENERATION OF GENE LISTS 61 2.11 CHICK IN OVO ELECTROPORATION 64 2.12 CLONING OF CHICK GENES 66 CHAPTER 3: TEMPORAL AND QUANTITATIVE ANALYSIS OF SHH SIGNALLING AND SHH RESPONSE DOMAINS IN CHICKEN FORELIMB AND HINDLIMB BUDS 68 3.1 THE SIZE OF THE HINDLIMB BUD MORPHOGEN FIELD IS LARGER THAN THE FORELIMB BUD THROUGHOUT THE PERIOD OF SHH PATTERNING ACTIVITY 69 3.2 SHH IS EXPRESSED FOR A SHORTER PERIOD OF TIME IN HINDLIMB BUDS THAN IN FORELIMB BUDS 72 3.3 ENDOGENOUS EXPRESSION PROFILES OF SHH, PTCH1 AND GLI1 AT STAGES HH17- HH24 75 3.4 THE RELATIVE SIZE OF THE SHH EXPRESSING DOMAIN OF FORELIMB BUDS IS LARGER THAN THAT OF HINDLIMB BUDS DURING SHH PATTERNING STAGES 78 3.5 THE RELATIVE SIZE OF THE SHH RESPONSE DOMAIN OF FORELIMB BUDS IS LARGER THAN THAT OF HINDLIMB BUDS DURING SHH PATTERNING STAGES 79 CHAPTER 4: TRANSCRIPTOMIC ANALYSIS OF THE RESPONSE OF LIMB PROGENITORS TO SHH SIGNALLING 83 4.1 DISSOCIATED LIMB BUD CELLS EXHIBIT A LIMITED AND INCONSISTENT RESPONSE TO EXOGENOUS SHH 84 4.2 FORELIMB AND HINDLIMB EXPLANTS CULTURED EX VIVO EXHIBIT A ROBUST AND GRADED RESPONSE TO EXOGENOUS SHH 87 4.3 FORELIMB BUD EXPLANTS EXHIBIT A NON-LINEAR GRADED RESPONSE TO THE SHH MORPHOGEN GRADIENT THROUGH SIGNAL ACCUMULATION AND SIGNAL DESENSITISATION 92 4.4 SIGNAL DESENSITISATION IS MEDIATED BY A PTCH1/2 LIGAND DEPENDENT ANTAGONISM 99 4.5 SUFU IS UPREGULATED BUT GLI2-3 AND DISP1 ARE DOWNREGULATED BY SHH SIGNALLING 105 6 4.6 GENES IMPLICATED IN ANTEROPOSTERIOR (AP) IDENTITY EXHIBIT A NON-LINEAR GRADED RESPONSE TO SHH IN FORELIMB AND HINDLIMB EXPLANTS 107 4.7 HINDLIMB EXPLANTS RESPOND MORE RAPIDLY THAN FORELIMB EXPLANTS TO SHH SIGNALLING 111 CHAPTER 5: IN SILICO AND IN SITU ANALYSIS OF THE INTERPRETATION OF GRADED SHH SIGNALLING IN CHICKEN FORELIMBS AND HINDLIMBS 118 5.1 GENES IMPLICATED IN LIMB DEVELOPMENT ARE DIFFERENTIALLY EXPRESSED IN EXPLANTS EXPOSED TO SHH 119 5.2 IN SILICO ANALYSES CAN BE USED TO PREDICT SPECIFIC AP EXPRESSION PATTERNS OF SHH TARGETS IN CHICKEN LIMB BUDS 127 5.3 CANDIDATE TARGETS OF SHH SIGNALLING ARE EXPRESSED IN DISTINCT AP DOMAINS IN CHICKEN LIMB BUDS CONSISTENT WITH PREDICTIONS 136 5.4 ATTEMPT TO MIS-EXPRESS SMOC1 IN THE DEVELOPING FORELIMB BUD 140 5.5 TRANSCRIPTIONAL TARGETS OF SHH SIGNALLING ARE EXPRESSED IN THE PHARYNGEAL ARCHES 143 CHAPTER 6: DISCUSSION 146 6.1 DIFFERENCES IN SHH SIGNALLING BETWEEN CHICKEN FORELIMB AND HINDLIMB BUDS 147 THE CHICKEN HINDLIMB APPEARS TO BE PATTERNED FASTER THAN THE FORELIMB 150 6.2 ESTABLISHING A GRADED RESPONSE TO THE SHH MORPHOGEN GRADIENT IN VERTEBRATE LIMBS 151 6.3 THE ROLE OF FEEDBACK MECHANISMS IN CORRECT MORPHOGEN GRADIENT INTERPRETATION 158 TEMPORAL ADAPTATION BY LIGAND DEPENDENT ANTAGONISM 158 ADDITIONAL FEEDBACK MECHANISMS 161 6.4 TARGETS OF SHH SIGNALLING IN THE LIMB AND ENCODING DIGIT IDENTITY 165 EVALUATION OF IN SILICO ANALYSES 165 ENCODING POSTERIOR DIGIT IDENTITIES 168 ENCODING ANTERIOR DIGIT IDENTITIES 169 ENCODING INTERMEDIATE DIGIT IDENTITIES 173 THE ROLE OF SMOC1 IN SPECIFYING AND MARKING MIDDLE DIGIT IDENTITIES 173 AN OVERVIEW OF SHH TARGETS IN THE LIMB 175 6.5 DIFFERENCES IN THE RESPONSE OF FORELIMB AND HINDLIMB PROGENITORS TO EQUIVALENT SHH SIGNALLING 177 7 6.6 SUMMARY AND FUTURE DIRECTIONS 180 FUTURE LINES OF INVESTIGATION 181 REFERENCES 184 APPENDICES 207 8 List of Figures Figure 1| Overview of limb skeletal structures and the role of Shh in establishing anteroposterior polarity __________________________________________________________________________ 15 Figure 2 | Schematic of the Shh signalling pathway and structure of Gli proteins _____________ 24 Figure 2.1 | The polarising activity of the posterior margin of the limb bud is concentration dependent __________________________________________________________________________________________29 Figure 2.2 | Overview of different models of digit specification in vertebrate limbs by Shh signalling.__________________________________________________________________________________________35 Figure 3 | Schematic of the proposed Gene Regulatory Network (GRN) downstream of Shh signalling in the limb. _____________________________________________________________________________ 41 Figure 4| Schematic of the Temporal adaptation model ________________________________________ 45 Figure 5 | Comparison of the size and cell number of chicken forelimb and hindlimb buds
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