THE GENOMIC STRUCTURE OF THE ZEBRAFISH wnt8b GENE by Yvonne Marie Beckham Department of Zoology Submitted in partial fulfilment of the requirements for the degree of Master of Science Faculty of Graduate Studies The University of Western Ontario London, Ontario December 1997 O Yvonne Marie Beckham, 1997 National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliographie Services seMces bibliographiques 395 Weüingtaci Street 395. Ne Wellington OttawaON K1AON4 OttawaON K1AW canada canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Lhrary of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, districbuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur fonnat électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT Screening of a zebrafish genomic library to identify the prornoter elements of the zebrafish ivnr8b gene resulted in the isolation of two clones. p8b-3H and p8b-7A. Southem blot analysis demonstrated that clone p8b- 3H contained the 3' portion of the cDNA. p8b-7A showed only weak hybridization to the wnr8b cDNA used to initially isolate this clone. Sequence analysis confirmed the Southem blot results. A 3' exon of at least 619 bp in length was identified in p8b-3H. A splice site at the 5' end of this exon was identified. 3 kb of intron sequence 5' to this splice site was obtained and no other wnt8b exons were found in p8b-3H. Subcloning of p8b-7A fragments that hybridized weakly to the cDNA and subsequent sequence analysis resulted in identification of only weak sirnilarity between these fragments and zebrafish wnt8b cDNA. Significant sequence similarity was found, however, between this sequenced segment of p8b-7A and two zebrafish mRNAs, plasticin and a receptor tyrosine kinase ligand. AL-1. In a Northem blot analysis the length of the zebrafish wnt8b transcript was determined to be 3.4 kb. Hence the wnr8b transcript contains 2.3 kb of untranslated region, which was not identified in the cDN.4 used to screen the genomic library. Keywords: zebrafish, wnr8b, midbrain-hindbrain boundary, CNS development, genomic structure ACKNOWLEDGEMENTS The greatest thanks goes to Dr. Percival-Smith for taking me into his lab when there was no benefit for him. 1 believe that 1 leamed more in the eight months that 1 was in Tony's lab than 1 could have leamed any where else in two years. Tony helped guide me for my future PhD at the University of Toronto, but he taught me more than just research. 1 want to thank al1 the people that I have worked with in the lab over the years. They have made me laugh and created an environment within the lab that 1 wanted to be a part of day after day. They have been supportive in times of trouble and good friends in every way, both in and out of the lab. Thank you to Rene Harrison, Sara Horst. Robyn Gallardi. Kim McClintock, Magda Paladi, Bmno Reversade, David Skidmore, Jennifer Weber, Bob Argiropoulos, Beata Blachuta, Jodi Lackten. and Jessica Sontrop. Thank you to Cathy Regan and Kellie White for being good friends to me. Friday evenings at Collip and then at the Grad Club are arnong my fondest mernories and regardless of how full my Friday evenings are in the future, they will always be missing something. Oana Marcu and Helen Nichol were both a tremendous help to me in ternis of teaching me what they had already leamed from experience and being mentors to guide me outside of the lab. 1 hope that as a PhD student I can give as much to beginning graduate students as they have given to me. Mary Martin has been a immense source of support for me. 1 would not have received a scholarship if Mary had not been there to help me and let me know what 1 had forgotten. If there was ever anything that needed to be handed in or taken care of 1 knew that I couid count on Mary to instruct me. 1 also want to thank Mary for being a good friend. Thank you to Dr. Kidder for discussing the future with me so that 1 would know that 1 still had one. It was Dr. Kidder's course and teaching that first introduced me to Developmental Biology and encouraged me to pursue graduate studies. I would like to thank Dr. Milligan for al1 her help with everything concerning fish and for being supportive when 1 came to her as a member of the Graduate Education Cornmittee. 1 would also like to thank Dr. Kohalmi in Plant Sciences. as well as the members of her lab. for the use of and the help with her sequence analysis program. Friendship and support from my fnends outside the department have also been very important to me. They allowed me to leave the troubles of the lab exactly where they should be. Thank you to especially to Vivienne Edwards and Julie Chen. TABLE OF CONTENTS Title Pa,oe ........................................................................................... .i. Certificate of Examination .................................................................. -11... Abstract and Keywords ....................................................................... iii Acknowledgements ............................................................................. lv Table of Contents ............................................................................... ...vi List of Figures ................................................................................. vil1 List of Tables..................................................................................... x List of Abbreviations .......................................................................... xi Chapter 1: Introduction 1 Developmental Biology ...................................................... 1 Advantages of Zebrafish as a Developmental Mode1.............. 1 Early Zebrafish Embryology ........... .... .......................... 3 Zebrafish Genetic Map ....................................................... 1 Neural Developrnent of the Zebrafish .................................. 5 Wnts ............................... .... .......................................... -6 Drosophila wingless ........................................................... 9 Caenorhabditis elegans ...................................................... 15 Xenopus RNA localization ............................................... -16 Wnt Regulation ............................................................... -17 Zebrafish wnt8b ............................................................... 18 Chapter 2: Materials and Methods ...................................................... -21 Library Screen ................................................................ 21 Southern Blotting .................. .. .........................................73 Restriction Enzyme Mapping .............. .. .......................... 24 DNA Sequencing and Analysis .............................. .. ........... 25 RNA Isolation ................................................................. 25 Northern Blot Analysis .................................................... 29 Chapter 3: Results ............................................................................. 30 Isolation of Genomic DNA Encoding wnr8b ....................... 30 Genomic DNA Analysis ................................................... -33 RNA Analysis .................................................................. 61 Chapter 4: Discussion ....................................................................... -70 References. .. ... .. .. .... .. .. .. ........ .. ...,.. ,.,.. ........ .. .. ... ... .. .. .. - .-... -. .. ..-. -76 Curriculum Vitae. .. ... .. ..,... .. .. .. .... ..... .. .. .... ... ... .. .. ...... .. .. .--... .. ... .. .. ... .. -84 vii LIST OF FIGURES Figure 1. A diagrammatic representation of components and events in the Wingless signaling pathway in Drosophila melanoguster ................................................................... 12 Figure 2. Agarose gel electrophoresis of Pst1 digested zebrafish zoenomic clones isolated from )c FIX II genomic library ...... .32 Figure 3. Southern blot illustrating restriction enzyme mapping strategy for p8b-3H using partial digestion- and indirect end labelling.. ................................................................. -35 Figure 4. Restriction enzyme map of p8b-3H.. ................................. .38 Figure 5. Restriction enzyme map of p8b-7A .................................... 40 Figure 6. Agarose gel electrophoresis and Southern blot of p8b-3H and p8b-7A probed with the 3' end of the zebrafish wnt8b cDNA ........................................................................... -43 Figure 7. Agarose gel electrophoresis and Southem blot of p8b-7A probed with the entire zebrafish wnt8b open reading - frame.. ........................................................................... -43 Figure 8. Agarose gel electrophoresis and Southern Blot analysis demonstrating that p8b-3H and p8b-7A are non- overlapping clones ...........................................................48 Figure 9. Schematic representation of subclones