The Role of Hoxa2 and Characterization of its New Downstream Targets in Murine Palatogenesis A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Pharmacy and Nutrition University of Saskatchewan Saskatoon By Tara Marie Smith © Copyright Tara Marie Smith, September 2009, All right reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Doctor of Philosophy degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying this thesis in any manner, in whole or in part, for scholarly purposes may be granted by Dr. Adil J. Nazarali, the professor who supervised my thesis work, or in their absence, by the Dean of the College of Pharmacy and Nutrition in which my thesis work was done. It is understood that any copying or publication, or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make use of material in this thesis in whole or in part should be addressed to: Dean of the College of Pharmacy and Nutrition University of Saskatchewan 110 Science Place Saskatoon, Saskatchewan S7N 5C9 i Abstract Hoxa2 null embryos display a high incidence of cleft secondary palate which has previously been described as secondary to altered tongue development. The experiments described in this thesis demonstrate that expression of Hoxa2 does occur within the developing palate, with the highest levels appearing in the early stages of palatogenesis (E12.5 and E13.5). Increased cell proliferation was observed throughout the palate in the absence of Hoxa2, without a detectable difference in apoptosis or the ability of the shelves to fuse. In addition, the palate shelves of the null embryos failed to elevate above the tongue, suggesting a mechanism by which the increased cell proliferation results in cleft palate. Numerous downstream targets of Hoxa2 were also identified in the palate (Msx1, Bmp4, Barx1, Ptx1, Six2, Lef1 and Tbx1). In all cases, Hoxa2 appears to act as a transcriptional repressor. Increases in palatal Msx1, Bmp4 and Barx1 expression have all been previously described to lead to increases in cell proliferation. Hoxa2, Ptx1, Lef1 and Tbx1 may be involved in a novel pathway that regulates proliferation in the palate. In addition, three novel gene targets were identified in the palate, Six2, Fgf8 and Htra3. Together these data show that there is a direct role for Hoxa2 in regulating palate development, apparently through regulating the expression of downstream genes involved in maintaining normal cell proliferation rates. ii Acknowledgments This thesis would not have been possible without the support of many individuals. I would like to thank Dr. Adil Nazarali for his guidance and support over the past five years, in both my professional and personal life. I would also like to thank my committee members, Dr. William Kulyk, Dr. Baljit Singh and Dr. Phyllis Paterson for their support and suggestions throughout this process. Also thanks to my external examiner Dr. Scott Lozanoff for taking time out of his busy schedule to review my research and act on my committee. I would like to thank the members of my lab especially LaRhonda Sobchishin. She provided constant encouragement and support and acted as a wealth of technical information. Thank you to my fellow graduate students especially Erin Prosser-Loose and Barbara McEwen with whom I have developed a special friendship due to the incredible ups and downs that grad school as put us through together. iii Dedication This is dedicated to my ever supportive family. To my mother, Elaine Smith, who taught me how to be a strong, confident woman that when she puts her mind to it can do anything. To my father, Curt Smith, who has been behind me everyday with love and encouragement. To my sister, Shari Smith, who constantly reminds me what is important in life. Finally to my husband, Scott Caswell, who has been there for me through all the ups and downs of the past couple of years, and whose confidence in me has never waivered. iv Table of Contents Permission to Use ...................................................................................................... i Abstract ..................................................................................................................... ii Acknowledgements ...................................................................................................iii Dedication ................................................................................................................ iv Table of Contents ...................................................................................................... v List of Figures ............................................................................................................ x List of Abbreviations .................................................................................................xii 1.0 Introduction ......................................................................................................... 1 2.0 Hypothesis and Objectives .................................................................................. 3 3.0 Literature Review ................................................................................................ 4 3.1 Overview of Craniofacial Development ...................................................... 4 3.2 Cranial Neural Crest Cell Migration ............................................................ 4 3.3 Facial Development .................................................................................. 10 3.3.1 Secondary Palate Development .................................................. 12 3.3.1.1 Cleft Palate .................................................................. 17 3.4 Molecular Signalling within the Palate ...................................................... 19 3.4.1 Gene Signalling that Regulates Proliferation in the Palate ......... 22 3.4.1.1 Msx1 Signalling Pathway .............................................. 22 3.4.1.2 Bone Morphogenic Protein Signalling Pathway ............ 26 3.4.1.3 Sonic Hedgehog Signalling Pathway ............................ 28 3.4.1.4 Sprouty2 Signalling Pathway ........................................ 30 v 3.4.1.5 Barx1 Signalling Pathway ............................................. 31 3.4.1.6 Tbx22 Signalling Pathway ............................................ 32 3.4.1.7 Fgf10 Signalling Pathway ............................................. 34 3.4.1.8 Fgf Receptors ............................................................... 36 3.4.1.9 Shox2 Signalling Pathway ............................................ 36 3.4.2 Regulation of Palatal Shelf Fusion............................................. 37 3.4.2.1 Transforming Growth Factors Signalling Pathway ......... 38 3.4.2.2 Epidermal Growth Factor Signalling Pathway ................ 40 3.4.2.3 Meox2 Signalling Pathway ............................................. 42 3.4.2.4 Fgf10 Signalling Pathway .............................................. 43 1.4.3 Cell Migration............................................................................. 43 3.5 Homeobox genes .......................................................................................... 46 3.5.1 Hoxa2 ......................................................................................... 53 3.5.1.1 Regulation of Hoxa2 Expression .................................. 54 3.5.1.2 Hoxa2 Mutant Phenotypes ........................................... 56 3.5.1.3 Hoxa2‟s Involvement in Palate Development ............... 58 3.5.1.4 Putative Downstream Targets of Hoxa2 in the Palate .. 59 3.5.1.4.1 Msx1 .............................................................. 59 3.5.1.4.2 Htra3 .............................................................. 60 3.5.1.4.3 Six2 ................................................................ 61 3.5.1.4.4 Ptx1 ............................................................... 62 3.6 Summary ....................................................................................... 63 vi 4.0 Materials and Methods ...................................................................................... 65 4.1 Hoxa2 Transgenic Mice ................................................................. 65 4.1.1 PCR Analysis of Genotype .......................................... 65 4.2 Immunohistochemical Analysis.................................................... 66 4.2.1 Fgf8, Six2 and Activated Caspase 3 ............................. 66 4.2.2 Htra3 and Hoxa2 .......................................................... 69 4.3 Cell Proliferation Assay ................................................................. 70 4.4 TUNEL Assay ................................................................................ 71 4.5 Whole Palate Organ Cultures ........................................................ 72 4.6 Western Blot Analysis .................................................................... 76 4.7 RNA Isolation and Reverse Transcription ...................................... 78 4.8 Quantitative Real Time RT-PCR...................................................