Gene Expression and Regulation in Early Vertebrate Development Xianhui Li
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Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2007 Gene Expression and Regulation in Early Vertebrate Development Xianhui Li Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES Gene Expression and Regulation in Early Vertebrate Development By XIANHUI LI A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science Degree Awarded Fall Semester, 2007 The members of the Committee approve the thesis of Xianhui Li defended on October 18, 2007 Curtis R Altmann Professor Co-Directing Thesis Hank W Bass Professor Co-Directing Thesis Yoichi Kato Committee Member Wu-Min Deng Committee Member Hengli Tang Committee Member Approved: Timothy S. Moerland, Chair, Department of Biological Science The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGEMENTS I am very grateful to Dr. Curtis Altmann and Dr. Hank Bass, my major professors, for their guidance, support, patience and encouragement. This thesis would not have been completed without their wide knowledge and insightful foresight. I also appreciate my committee members, Dr. Yoichi Kato, Dr. Wu-Min Deng, Dr. Hengli Tang, and Dr.Nancy Greenbaum for spending their precious time in discussing my project progress, giving invaluable suggestion, and revising my prospectus and thesis. I thank Dr. George Bates for his support and encouragement. I thank my colleagues: Ailing Zheng, Tyrone Ryba, Malcolm Klein, Barbara Danner, and Tomomi Kiyota for their help. I thank my parents, my siblings, my husband and son for their love, sacrifices, patience, and support. I am appreciative of College of Medicine and Department of Biological Science for their support. iii TABLE OF CONTENTS List of Tables ................................................................................................v List of Figures ................................................................................................vi Abstract ......................................................................................................vii 1. Expression and Functional Analysis of LTBP4 in Early Development ................................................................................................1 Introduction ................................................................................................1 Materials and methods ................................................................................7 Results ................................................................................................13 Discussion ................................................................................................22 2. Identification and Characterization of Downstream Targets of Pax6 in Early Eye Development ................................................................................................30 Introduction ................................................................................................30 Materials and methods ................................................................................32 Results ................................................................................................35 Discussion ................................................................................................45 REFERENCES ................................................................................................55 BIOGRAPHICAL SKETCH ..............................................................................61 iv LIST OF TABLES Table 1: The primers that are used for sequencing xtLTBP4 ..............................8 Table 2: Primers for xLTBP2, xLTBP3 and xLTBP4 .........................................15 Table 3: Percentage of secondary axis induced by microinjection of mRNA for hLTBP4 and xtLTBP4 ..........................................................................24 Table 4: Markers and primer sequences used for the animal cap experiment ....28 Table 5: Primers of transcription factors ............................................................36 Table 6: Primers of Wnt signal genes .................................................................37 Table 7: Primers of receptor genes ......................................................................37 Table 8: Confirmed pax6 upregulated transcription factors by real time PCR....41 Table 9: Confirmed pax6 upregulated Wnt signal genes by real time PCR ........42 Table 10: Confirmed pax6 upregulated receptor genes by real time PCR ..........42 Table 11: Direct targets of Pax6 ..........................................................................47 Table 12: Phenotype of overexpression of LRP11 ..............................................52 v LIST OF FIGURES Figure 1: Secretion, activation and targeting of TGF-β ......................................3 Figure 2: The structure of LTBPs ........................................................................5 Figure 3: Microinjection of Xenopus embryos ...................................................11 Figure 4: Expression of xLTBP2, xLTBP3, and xLTBP4...................................16 Figure 5: Whole amount in situ hybridization of xLTBP2-4...............................16 Figure 6: Sequence alignment of Xenopus tropicalis, human, and mouse LTBP4 ................................................................................................18 Figure 7: xtLTBP4 domain structure. ..................................................................20 Figure 8: Multiple Alignment of TB domains. ....................................................20 Figure 9: In situ hybridization of xLTBP4. .........................................................21 Figure 10: Injection of hLTBP4 induces a second axis. .....................................23 Figure 11: Secondary axis is stained by 12-101 and β-gal. .................................24 Figure 12: hLTBP4 acts synergistically with Xnr-1............................................26 Figure 13: xtLTBP4 enhances the expression of Xnr1........................................27 Figure 14: hLTBP4 enhances Xnr1 in the mesoderm induction in the animal caps ................................................................................................28 Figure 15: Pitx2 is confirmed by one experiment of PCR...................................39 Figure 16: Model of direct target and indirect target ..........................................43 Figure 17: Pax6-GR fusion protein in active condition and inactive condition ..43 Figure 18: Foxd3, a direct target of pax6.............................................................46 Figure 19: Expression of Pax6, Foxd3 and Irx3 in the eye of the mouse............46 Figure 20: Pax6 MO has inhibited exogeneous pax6 in animal caps ..................47 Figure 21: Expression of Xiro3 in Xenopus requires Pax6..................................48 Figure 22: Overexpression of Xiro3 causes misplaced eyes ...............................49 Figure 23: Overexpression of Xiro3 causes ectopic photoreceptor and muller cells ................................................................................................49 Figure 24: Overexpression Xiro3 induced Pitx2, Six3, Otx2 and Xag................50 Figure 25: Overexpression of LRP11 induced ectopic RPE and proximal eye defect ................................................................................................50 Figure 26: Overexpression of LRP11 can induce ectopic photoreceptor cells....51 Figure 27: Spacious expression of LRP11 by in situ hybridization.....................51 Figure 28: Temporal expression of LRP11 by Real time PCR............................52 vi ABSTRACT Gene expression and regulation in early vertebrate development has been examined in Xenopus laevis, a model organism for embryonic developmental biology. Two related projects were carried out and they are described in separate chapters. The first chapter describes the expression and functional analysis of Latent TGF-β Binding Protein 4 (LTBP4) in early development. LTBP4 is expressed in the organizer which is located in the dorsal mesoderm and induces a secondary axis upon overexpressed on the ventral side. The data indicate that LTBP4 enhances the nodal signaling pathway which induces mesoderm in the early development. The second chapter describes the identification and characterization of downstream targets of Paired Box Gene 6 (Pax6) in early eye development. Three groups of Pax6 upregulated genes, identified from a DNA microarray screen, are confirmed by real time PCR. Among the Pax6-activated genes are transcription factors, the genes that are involved in Wnt signaling pathway which is network of proteins implicated in embryogenesis and cancer, and receptor genes. Importantly, seven different genes have been identified as direct targets of Pax6 based on their mRNA accumulation patterns. In addition, functional analysis of some Pax6 regulated genes such as Xiro3 and LRP11 is presented in the second chapter. vii CHAPTER 1 EXPRESSION AND FUNCTIONAL ANALYSIS OF LTBP4 IN EARLY DEVELOPMENT Introduction The vertebrate embryonic development starts as a fertilized egg, followed by a rapid cell division. The dorsoventral polarity is established immediately after fertilization in Xenopus (reviewed by De Robertis et al., 2000). Once fertilized, the cortex rotates. This rotation