MAPPING GAPING LIDS: A MUTATION CAUSING OPEN EYELIDS AT BIRTH IN MOUSE by KATHLEEN GRACE BANKS B.Sc, The University of British Columbia in association with the University College of the Cariboo, 1996. A THESIS SUMBITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty of Medicine; Department of Medical Genetics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1999 © Kathleen Grace Banks, 1999 UBC Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 11/19/99 ABSTRACT Gaping lids (gp) is an autosomal recessive mutation that arose spontaneously in the C57BL/6-ax strain of mice and is now maintained in the inbred strain GP/Bc. The purpose of this study included the mapping of the gp mutation, analysis of its segregation after two outcrosses to normal (non-open eyelid) strains and characterization of the mutant phenotype. The main objective, to map the gp locus, was undertaken based on the hypothesis that the loci with mutations that cause open eyelids with simple Mendelian transmission patterns may also be loci involved in the open eyelids traits with more complex inheritance. Since these mutations are viable, they are good models for studying the interaction of multiple loci in a genetically complex birth defect. The gaping lids mutation was mapped close to the centromere at the proximal end of chromosome 11, employing PCR amplification of informative SSLP marker loci, initially using 41 gaping lids F2 progeny from a cross of GP/Bc to the normal strain CBA/J, followed by a refinement of the region using 23 gaping lids F2 progeny from a second outcross of GP/Bc to ICR/Be, another normal inbred strain. Based on the recombination breakpoints, gp is within 10 cM of Dl lMit80 and 2 cM of Dl lMit71. The epidermal growth factor receptor gene (Egfr) was also mapped to mid-chromosome 11 between Dl lMit226 and Dl lMitl51, employing PCR amplification of SSLP markers, using mice carrying a null allele at this locus. This map location supports the finding that gp and Egfr are not allelic as determined by a complementation test between Egfr+I~ and GP/Bc. gp showed reduced penetrance, 82% and 33% in the outcrosses to CBA/J and ICR/Be, respectively, but it was determined that this was likely due to suppressors-of ii open eyelids loci introduced by the normal strains and not prenatal death of gp/gp progeny. The only phenotypic anomaly associated with this mutation appears to be eyelids at birth, due to failure of normal eyelid closure during late gestation. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vi List of Figures viii List of Appendices x List of Abbreviations xii Acknowledgments xiii CHAPTER I: INTRODUCTION I: History of gaping lids 1 II: Review of eyelid development and open eyelids at birth mutations 2 A. Eyelid development 2 B. Genes expressed in the developing eyelids 5 C. Open eyelids at birth mutants 7 III: Mouse mapping 14 A. Overview and history 14 B. Polymerase Chain Reaction and Simple Sequence Length 31 Polymorphisms C. Review of mouse maps 33 IV: Rationale and approach to this study 36 CHAPTER II: GENERAL METHODS AND MATERIALS I: gaping lids: Scientific progress before and during this study 38 A. The cross to CBA/J 38 B. The cross to ICR/Be 39 II: Mouse stocks and maintenance 40 III: Technical methods 42 A. GP/Bc study 42 B. Egfr study 46 CHAPTER III: PHENOTYPIC INVESTIGATIONS I: Introduction 49 II: Rationale, Materials and Approach 49 III: Results 50 CHAPTER IV: MAPPING GAPING LIDS I: Introduction 55 iv II: Rationale, Materials and Approach 55 A. Experimental design 55 B. Analysis of genetic transmission/penetrance 60 C. Molecular investigations 61 III: Results 65 A. Segregation studies 65 B. Mapping studies 67 I. GP/Bc x CBA/J crosses 67 II. GP/Bc x ICR/Be cross 77 C. Analysis of genetic transmission/penetrance after crosses to 84 CBA/J and ICR/Be D. Molecular investigations 85 CHAPTER V: MAPPING EGFR I: Introduction 90 II: Rationale, Materials and Approach 90 A. Experimental design 90 I. Egfr7BXA-2 x SWV/Bc cross 91 III: Results 93 A. Egfr7BXA-2 x SWV/Bc cross 93 CHAPTER VI: CORRECTING THE MGI MAP 101 CHAPTER VII: DISCUSSION I: Segregation studies 106 A. CBA/J cross 107 B. ICR/Be cross 107 C. Applications of the threshold model 110 II: Mapping studies 114 A. GP/Bc study 114 B. Egfr study 123 III: Phenotypic investigations 124 IV: Conclusions 126 Literature Cited 127 Appendices 141 v LIST OF TABLES Table 1 Genes expressed in the developing eyelids. 6-7 Table 2 Open eyelids at birth mutations in mouse. 15-28 (a) nonsyndromic (b) syndromic (c) ectopic gene expression (d) strains with susceptibility to open eyelids (e) chromosomal Table 3 Frequency of open eyelids in newborns from GP/Bc x CBA/J cross. 39 Table 4 Frequency of open eyelids in newborns from GP/Bc x ICR/Be cross. 40 Table 5 (a) Measurements of palpebral opening and eye in GP/Bc, CBA/J and 52 (GP/Bc x CBA/J) Fl autopsied animals, (b) Measurements of palpebral opening and eye in GP/Bc and AXB- 23/Pgn animals. Table 6 Comparison ofSSLP marker loci map position between the Mouse 64 Genome Informatics Database (MGI), Massachusetts Institute of Technology/Research Genetics (MIT) and the European Collaborative Interspecific Backcross panel (EUCIB-BSB). Table 7 (a) Data for GP/Bc x CBA/J F2 affected and normal progeny - 11 77 complete litters (b) Segregation at markers closest to gp, Dl lMit62 and Dl lMit226, 77 in F2 from GP/Bc x CBA/J in 11 complete litters. Table 8 Segregation of alleles at D11 Mit74 in normal F2s in GP/Bc x ICR/Be 83 cross. Table 9 (a) Data for GP/Bc x ICR/Be F2 affected and normal progeny - 6 83 complete litters (b) Segregation at marker closest to gp, Dl lMit74, in F2 from GP/Bc 84 x ICR/Be in 6 complete litters. Table 10 Haplotype analysis of GP/Bc versus C57BL/6J DNA. 89 Table 11 Segregation of alleles at Dl 1 Mit 149 in open eyelie/pinhole F2 mice 101 from GP/Bc x CBA/J cross. Table 12 Outline of modifier scenarios in GP/Bc x ICR/Be F2 109 vi Table 13 Loci in the region of gaping lids - between the centromere and 116-117 DllMit80 on Chr 11. vii LIST OF FIGURES Figure 1 (a) Scanning electron microscope picture of dl6 GP/Bc fetal eye. 54 (b) Close up view of inner canthus of dl6 GP/Bc fetal eye. Figure 2 Comparison of informative SSLP marker loci locations used in 62 GP/Bc x CBA/J and ICR/Be crosses, where marker identification numbers follow the format Dl lMit##. Figure 3 Comparison of SSLP marker loci map locations used in GP/Bc x 63 CBA/J and GP/Bc x ICR/Be crosses on MGI, MIT and EUCIB (BSB) maps. Figure 4 Mapping matrix of open eyelid F2 mice in GP/BC x CBA/J cross. 69 Data does not include pinhole F2 mice. Figure 5 Location of markers used in GP/Bc x CBA/J cross. 70 Figure 6 Pictures of representative gels of D11 Mit62; GP/Bc, CBA/J, F1 and 71 panel of open eyelid and pinhole F2 animals. Figure 7 Pictures of representative gels of Dl lMit226; GP/Bc, CBA/J, Fl and 72 panel of open eyelid and pinhole F2 animals. Figure 8 Map location of gaping lids and distances between markers as 74 determined in GP/Bc x CBA/J cross. Figure 9 Mapping matrix of "pinhole" F2 mice from GP/Bc x CBA/J cross. 75 Figure 10 Mapping matrix of normal F2s in GP/Bc x CBA/J cross. 76 Figure 11 Mapping matrix of open eyelid F2 mice in GP/Bc x ICR/Be cross. 79 Figure 12 Locations of markers used in GP/Bc x ICR/Be cross. 80 Figure 13 Pictures of representative gels of D11 Mit74; GP/Bc, ICR/Be, F1 and 81 panel of open eyelid F2 animals. Figure 14 Map location of gaping lids and distances between markers as 82 determined in GP/Bc x ICR/Be cross. Figure 15 Comparison of SSLP marker map locations between CBA/J cross, 86 ICR/Be cross and MGI, MIT, and EUCIB. viii Figure 16 Picture of representative agarose gel of primers which amplify the 94 Egfr null and wildtype alleles. Figure 17 Locations of markers used in (Egfr7BXA-2)Fl x SWV/Bc special 95 testcross. Figure 18 Outline of (Egfr7BXA-2)F 1 x SWV/Bc special testcross. 96 Figure 19 Haplotypes of Egfr +/+ and Egfr +/" mice in (Egfr7BXA-2)F 1 x 97 SWV/Bc special testcross. Figure 20 Location of Egfr locus based on (Egfr7BXA-2)F 1 x SWV/Bc special 99 testcross.