THE USE OF THE SPONTANEOUS BN MOUSE MUTANT, AND TARGETED ALLELES OF SMAD2 AND TGIF TO UNDERSTAND AXIAL SPECIFICATION AND NEURAL DEVELOPMENT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Tessa Lyn Carrel, B.S., M.S. ***** The Ohio State University 2004 Dissertation Committee: Approved by Michael WeinsteinPh.D., Advisor Christine Beattie, Ph.D. _________________________________ Heithem El Hodiri, Ph.D. Advisor Program in Molecular, Cellular Amanda Simcox, Ph.D. and Developmental Biology ABSTRACT To understand the basis of axial specification and neural development, mouse models are commonly utilized. Bent tail (Bn) is a spontaneous mutation on the mouse X chromosome that produces tail deformities and open neural tube defects (NTDs). Analysis of progeny from an intraspecific backcross places the mutation between the microsatellites DXMit166 and DXMit140. Refined genetic and physical mapping of the Bn critical region demonstrate that the mutation is associated with a <170 kb submicroscopic deletion, including the entire Zic3 locus. Human mutations in ZIC3 are associated with left-right axis malformations. The presence of anal and spinal abnormalities in some patients and deletion of Zic3 in Bn mice support a key role for this gene in neural tube development and closure. However, mutations in the ZIC3 gene have yet to be identified in families with X- linked NTDs. Holoprosencephaly (HPE) results from abnormal development of the forebrain. One gene associated with HPE in humans, TG interacting factor (TGIF), was identified by its ability to bind to the retinoid X receptor response element, and has been shown to play a role in regulating TGF-β signaling. HPE is not evident in mice carrying the targeted null allele of Tgif. To elucidate ii whether Tgif in conjunction with reductions in TGF-β signaling can cause HPE, mice that have mutations in both Smad2 and Tgif were generated. Results show that of the Smad2+/-; Tgif+/- and Smad2+/-; Tgif-/- embryos, one third display HPE. Molecular characterization at E9.5 reveals that Shh, Fgf8, Six3 and Zic2 expression are not affected. The forebrain domain of Otx2 expression shows a modest to nearly complete reduction in affected embryos. Published data has shown that introduction of retinoic acid (RA) to cultured embryos or pregnant dams can induce HPE in embryos. Some of these studies have further shown a reduction in rostral Otx2 expression. The known interaction of Tgif and RA signaling led to the evaluation of the possibility that the HPE resulted from altering RA pathways. Analysis of developing embryos provided evidence that genetic deficiencies of Smad2 and Tgif allows for increased sensitivity to RA, suggesting a unique link between genetics and environmental teratogens. iii DEDICATION To my husband, my mother and father, and my brother. Thank you for your patience, love and support. iv ACKNOWLEDGEMENTS I wish to thank my advisor, Dr. Michael Weinstein, for his intellectual support, zeal, and for his patience and guidance. I am grateful to my committee members Dr. Christine Beattie, Dr. Heithem El Hodiri, and Dr. Amanda Simcox for their encouragement. I thank the members of the Weinstein Laboratory, Ye Liu, Mark Hester, Samuel Lasse, Maria Festing, J. Chris Thompson, Andy Chow and Sami Jafaar, for proofreading this dissertation and scientific discussions. I am indebted to those at Columbus Children’s Research Institute for their advice, stimulating conversations and support, especially Dr. David Cunningham, Dr. LaRae Copley, Dr. Heithem El Hodiri, Ms Marsha Lucas and Ms Marlene Parker. I also wish to acknowledge Dr. Gail Herman, whose laboratory the ZIC3 work described in this dissertation was performed. Finally, I wish to thank the editors of The American Journal of Medical Genetics, and Molecular and Human Genetics for their permission to use the previously published text and figures that are included in this dissertation. v VITA October 16, 1974 Born – Columbus, Ohio. 1995 Beta Beta Beta Biology Honorary. 1/96 – 6/97 Senior Thesis, Muskingum College-Advisor Dr. David Quinn. “Role of Vigabatrin in reversing stimulus- evoked afterdischarges in rat hippocampal slices.” 6/96 – 8/96 Internship, South Carolina School of Medicine - Advisors Dr. Leslie S. Jones and Dr. Marsha Welsh. “Spatial/temporal studies of the extracellular matrix of the developing rat brain.” 1997 Sigma Xi. 1997 Sigma Xi Biology Senior Thesis Award. 1997 Muskingum College, New Concord, Ohio. Biology (Chemistry), BS. 1998 Outstanding Graduate Teaching Assistant Award. 2000 21st Annual Children’s Hospital and Children’s Research Institute Research Forum, 1st place award in basic research. 2003 The Ohio State University, Columbus, Ohio. Program in Molecular, Cellular and Developmental Biology, MS. 2002 Children’s Research Institute Cherry Valley Retreat, 1st place, graduate student oral presentations. 9/97 - 6/98 Graduate Teaching Assistant, Biology 113. The Ohio State University - Supervisor Dr. Courtney Smith. vi 4/98 – 3/03 Graduate Research Associate, The Ohio State University - Advisor Dr. Gail E. Herman. “Identification of a deletion of the Zic3 locus associated with the X-linked mouse mutation Bent tail.” 4/03 – present Graduate Student, The Ohio State University - Advisor Dr. Michael Weinstein. “The TGF-β pathway and holoprosencephaly: understanding the role of Tgif and Smad2.” 6/03 - 9/03 Graduate Teaching Assistant, Molecular Genetics 500. The Ohio State University - Supervisor Dr. Andrea Doseff. 1/04 – 3/04 Graduate Teaching Assistant, Introductory Biology Program 101. The Ohio State University – Supervisors, Dr. Cheryl Johnston, Course Coordinator and Dr. Steve Rissing, Director, Introductory Biology Program. PUBLICATIONS Carrel, T., Purandare, S., Harrison, W., Elder, F., Fox, T., Casey, B. and Herman, G. E. “The X-linked mouse mutation Bent tail is associated with a deletion of the Zic3 locus.” Human and Molecular Genetics. 2000. 9(13). 1937-1942. Carrel, T., Moore G. E., Stanier, P. and Herman, G. E. “Lack of mutations in ZIC3 in three families with neural tube defects.” American Journal of Medical Genetics. 2001. 98:283-285. SELECTED MEETING ABSTRACTS Carrel, T., Purandare, S., Harrison, W., Elder, F., Fox, T., Casey, B. and Herman, G. E. “The X-linked mouse mutation Bent tail is associated with a deletion of the Zic3 locus.” Presented at the 50th Annual American Society of Human Genetics. 2000. FIELD OF STUDY Major Field: Molecular, Cellular and Developmental Biology vii TABLE OF CONTENTS Page Abstract ………………………………………………………………………. ii Dedication …………………………………………………………………….. iv Acknowledgements ………………………………………………………….. v Vita ……………………………………………………………………………. vi List of Tables ………………………………………………………………… x List of Figures ……………………………………………………………….. xi List of Abbreviations ………………………………………………………… xiii Chapters: 1 Introduction ……………………………………………………………… 1 1.1 Axial Patterning of the Mouse Embryo ………………………………. 2 1.1.1 Anterior-Posterior Axis Formation …………………………. 2 1.1.2 Dorsal-Ventral Axis Formation ……………………………… 5 1.1.3 Left-Right Axis Formation …………………………………… 6 1.2 Neural Development and Disease …………………………………… 9 1.2.1 Neural Tube Formation and Neural Tube Defects ………. 9 1.2.2 Prosencephalic Development and Holoprosencephaly … 12 1.2.3 Neural Maturation and Related Diseases ………………… 15 2 The X-Linked Mouse Mutation Bent Tail Is Associated With a Deletion of the Zic3 Locus ………………………………………………………….. 21 2.1 Introduction ……………………………………………………… 21 2.2 Results …………………………………………………………… 23 2.2.1 Genetic Mapping of Bn ……………………………… 23 2.2.2 Identification of a Submicroscopic Deletion Associated with the Bn Mutation ……………………………….. 25 2.2.3 Phenotypic Characterization of situs defects in Bn mice ……………………………………………… 26 2.3 Discussion ………………………………………………………. 27 2.4 Materials and Methods ………………………………………… 29 viii 2.4.1 Mouse Strains, Crosses and Genetic Mapping …… 29 2.4.2 Physical Mapping of Bn ……………………………… 29 2.4.3 Phenotypic Characterization ………………………… 30 3. Lack of Mutations in ZIC3 in three X-linked pedigrees with neural tube defects …………………………………………………………………… 39 4. Smad2 and Tgif Work in Concert to Cause Murine Holoprosencephaly ……………………………………………………… 44 4.1 Introduction ……………………………………………………… 44 4.2 Results …………………………………………………………… 47 4.2.1 Frequency and Appearance of Holoprosencephaly in Smad2; Tgif Embryos ……………………………….. 47 4.2.1 Molecular Analysis of Smad2; Tgif Holoprosencephalic Embryos ………………………………………………. 50 4.2.2 Retinoic Acid and Holoprosencephaly in Smad2; Tgif Embryos ………………………………………………. 53 4.3 Conclusions …………………………………………………….. 55 4.4 Materials and Methods ………………………………………… 62 4.4.1 Mice and Matings ……………………………………. 62 4.4.2 Genotyping …………………………………………… 63 4.4.3 Histology ……………………………………………… 63 4.4.4 Hematoxylin and eosin staining ……………………. 64 4.4.5 Bromodeoxyuridine Analysis ……………………….. 65 4.4.6 TUNEL Analysis ……………………………………… 67 4.4.7 Whole Mount In Situ Hybridization ………………… 69 4.4.8 Teratogenic Application of Retinoic Acid …………. 72 5. Discussion ……………………………………………………………….. 94 Works Cited …………………………………………………………………. 98 ix LIST OF TABLES Page 1.1 Order of events of neural development ………………………………… 17 2.1 Nondisjunction in the Bent tail strain …………………………………… 32 2.2 Situs abnormalities in Bn mice …………………………………………. 33 3.1 ZIC3 PCR parameters and primers ……………………………………. 42 4.1 Summary of the evaluation of E10.5 Smad2; Tgif embryos …………. 74 4.2 Malformation frequency in Smad2; Tgif embryos ……………………. 75 4.3 Retinoic