University of Florida Thesis Or Dissertation Formatting

University of Florida Thesis Or Dissertation Formatting

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 REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1 © 2010 Cortney Michael Bouldin 2 To my wife and family 3 ACKNOWLEDGMENTS Without the moral and material support of my wife and family, this work would have been impossible. 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. Harfe’s 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 Maatouk, 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 Andrew McMahon for conversations and sharing unpublished data, which helped to shape the progress of this work.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. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 7 LIST OF FIGURES .............................................................................................................. 8 ABSTRACT........................................................................................................................ 10 CHAPTER 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 Molecules Expressed within the ZPA ................................................................................................................... 17 Mechanisms of Action for Shh ............................................................................. 17 2 NETWORKS OF SIGNALING WITHIN THE DEVELOPING LIMB .......................... 21 Establishment 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 Mouse Alleles and Breeding ................................................................................ 36 Fate Mapping, β-Galactosidase Detection and Immunohistochemistry ............. 36 Whole Mount RNA in situ Hybridization and AER Measurement ....................... 37 5 Bead Implantation and LysoTracker Analysis ..................................................... 38 4 ABERRANT FGF 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 APPENDIX 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 6 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 Gene Expression, Vital Dye Staining .......................... 77 B-1 Oligonucleotides used as genotyping primers. ..................................................... 89 C-1 Probes used for RNA

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