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

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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 from p8b-3H and the sequencing strategy ...... -50

Figure 10. Sequence of the subclones from p8b-3H demonstrating - overlap with a 3' portion of the zebrafish wnt8b cDNA ...... 9

Figure 1 1. Comparison of p8b-3H and the zebrafish wnt8b cDNA sequence demonstrating splice site consensus sequence ...... 55

Figure 12. Schematic representation of subclones from p8b-7A and the sequencing strategy ...... -58

S.. Vlll Figure 13. Sequence of subcloned fragment of p8b-7A ...... 60

Figure 14. Sequence of the subclones from p8b-7A demonstrating weak similarity with the zebrafish wnt8b cDNA...... 63

Figure 15. Sequence of the subclones from p8b-7A showing similarity with zebrafish plasticin and a zebrafish tyrosine kinase receptor ligand, AL- 1 genes...... -66

Figure 16. Denaturing agarose gel electrophoresis and Northern blot analysis to determine the length of the zebrafish wnt8b transcript ...... 69

Figure 17. Schematic surnrnary of the data obtained through sequence and Northern blot analysis ...... --74 LIST OF TABLES

Table 1. A surnrnary of the characteristics of molecules known to function in Wingless signaling pathway ...... 14

Table 7. Sequence of primers designed for sequencing of subclones of p8b-3H and p8b-7A ...... 27 LIST OF ABBREVIATIONS a rrn armadillo gene ARM armadillo protein PTrCP B-transducin repeat containing protein RNA cDNA complementary DNA Ce-wnt Caenorhabditis elegans wnt gene cmk2 casien kinase 2 mRNA CK2 Casein kinase 2 DFZ2 Drosophila Frizzled 2 protein DSH Dishevelled protein Dwnt Drosophila wnt gene efla elongation factor la: mRNA en engrailed gene EN Engrailed protein GSK-3 glycogen synthase kinase 3 HBS 1 homeodomain binding site 1 LEF- 1 lymphoid enhancer-binding factor MHB midbrain-hindbrain boundary 112 0tn -more mesoderm gene ORF open reading frame PAN Pangolin PKC Protein kinase C porc porcupine gene PORC Porcupine protein SSLPs single sequence length polymorphisms TCF T cell factor VegemlI gene wingless gene wingless phenotype Wingless protein Wnt gene farnily Wnt protein wn I zebrafish wnt gene Xdd l dominant negative allele of Xenopus dishevelled Xdsh Xenopus dishevelled gene Xgsk-3 Xenopus glycogen synthase kinase 3 gene Xgsk-3 Xenopus glycogen synthase kinase 3 protein X wnt Xenopus wnt gene zw3 Zeste-white3 kinase

xii CHAPTER 1 INTRODUCTION

1.1 DEVELOPMENTAL BIOLOGY The process of creating an entire organism, whether insect. fish or man, from one cell, requires a vast amount of regulation. How does this initial ce11 give rise to such a large number of highly varied ce11 types'? How do these cells arrange themselves spatially to form an organism? Some developmental questions can be answered by observing embryological processes such as the formation of the central nervous system. A me understanding of these events, however. can only be gained when the mechanisms governing these processes are uncovered.

1.2 ADVANTAGES OF ZEBRAFISH AS A DEVELOPMENTAL MODEL Zebrafish is a convenient, relatively inexpensive model system for the study of developrnental biolopy for a number of reasons. Being vertebrates themselves, experimental observations made with zebrafish cmbe compared to those in other vertebrates. As Jones and Huffman (1957) point out. this would not be possible with other organisms classically used as developmental models, such as sea urchins. Zebrafish are small and easy to breed. They have a relatively short life cycle, being sexually mature at 100 days (Skidmore, 1965). A zebrafish colony can be maintained without much concern paid to water hardness, temperature and pH, and although not exempt, zebrafish have been found to be relatively resistant to some common fish diseases (Piron, 1978). As a model in developmental biology, or any discipline utilizing embryos, one advantage of zebraf5sh over other fish systems is the large quantity of embryos that are easily procured throughout the year (Roosen- Runge, 1938). These embryos are easily manipulated (Jones and Huffman. 1957), do not have extensive adhesive properties (Skidrnore, 1965). contain a relatively mal1 amount of yolk (Roosen-Runge, 1938) and at the same tirne are optically transparent. The oviparous nature of zebrafish allow embryos to be examined throughout development (Jones and Huffman. 1957). which is very rapid with hatching occumng in 60 hours at 270C (Roosen-Runge, 1938). The mature zebrafish egg is initially white and nearly opaque but it becomes transparent approximately five minutes after fertilization (Roosen- Runge, 1938). Given the relative transparency of the embryo, cellular and matornical events have been studied in great detail for over forty years (Jones and Huffman, 1957). The free-swimming fry are small enough to allow investigators to house several hundred in a standard forty-litre aquarium. Furthemore, these fry have behavioral and morphological properties of the adult zebrafish. The adult properties of these fry and the fact that many cm be housed together allows for early and easy identification of mutants in large scale mutagenesis screens (Streisinger et al., 198 1). Another important factor to consider with regards to the mutagenesis screens is the fact that the relatively broad thermal tolerance of the zebrafkh allows the possibility of isolating temperature sensitive mutants (Streisinger et al., 198 1 ). Incidentally. details of two large mutagenesis screens were recently reported (see Developrnent. vol 123, December 1996). 1.3 EARLY ZEBRAFISH EMBRYOLOGY As with many biological studies, the characterization of developmental abnormalities completely hinges on a fm understanding of the series of events underlying normal development. This is evident in past teratological studies but also holds me, and must be considered, in the contemporary overexpression/misexpression investigations and in the cataloguing of the recently identified plethora of mutations (Development v 123). Early work in outlining normal developmental processes has been an essential basis for such studies. Roosen-Runge ( 1938) made extensive observations of the initial events occumng directly after fertilization. They reported movements within the ce11 and changes in diameter, viscosity and surface tension: therefore, they have provided an essential introduction to the development of the zebrafish, or at least an extensive insight into the first few minutes in the life of the zebrafish embryo. Despite the number of publications following that of Roosen-Runge ( 1938), it was not until 1958 that Hisaoka and Battle supplemented these characterizations by outlining overall zebrafish embryogenesis. Hisaoka and Battle ( 1958) provided the time-table of normal developrnent and hence the basis for experimental studies. by dividing normal development into 25 stages. Their descriptions include time intervals of stages at 260C, measurements, cleavage angles and structures specific to certain stages selected for identification purposes. In 1960 Hisaoka and Firlit reported details of zebrafish development beginning at stage 2, the one ce11 blastodisc. Their paper not only describes how cytoplasmic strearning is responsible for moving cytoplasm to the animal pole where the blastoderm foms, but also confms and extends results presented by Hisaoka and Battle (1958). One feature which makes the Hisaoka and Firlit ( 1960) investigation unique from earlier characterization is the inclusion of stained, sectioned material of various embryonic stages. The Hisaoka and Battle staging series has since been replaced by a more complete series detailed by Kimmel et al. ( 1995).

1.4 ZEBMFISH GENETIC MAP With the description of zebrafish embryogenesis complete there was a firm basis for studying abnormal developrnent. Research by Streisinger el al. (198 1) opened up the genetic analysis of zebrafish development. More specifically, Streisinger and colleagues outlined a procedure whereby an egg, fertilized by UV irradiated sperm, is prevented from dividing using pressure or heat. Since these eggs duplicate their own genetic material, a hornozygous embryo is created containing only the materna1 genetic complement. Interestingly, this procedure was devised to study neuronal development but obviously has more widespread applications. More recent mutant screens occupy an entire issue of Development (v. 123, December 1996). Genetic maps are useful for linking known genes to mutant phenotypes. For such positional cloning to be successful, however, a complete genetic map must be available. Mapping projects by Johnson er al. (1994), Postlethwait er al. (1994) and Johnson et al. (1996) have resulted in a map consisting of 25 linkage groups, the haploid number of zebrafkh. More recently, Knapik et al. (1996) constnicted a reference cross DNA panel using simple sequence length polymorphisms (SSLPs) against which any cross can now be mapped. Although recent screens have produced mutants for ear development (Malicki et al., 1996), locomotion behavior (Granato et al., 1996). pigmentation (Odenthal et al., 1996) and fin development (van Eeden et al.. 1996), 1 will concentrate on mutants affecting neural development. As with any process, to understand the alteration of this process a complete understanding of normal neural development is essential.

1.5 NEURAL DEVELOPMENT OF THE ZEBRAFISH Zebrafish development can be loosely divided into a series of key developmental periods: zygotic, cleavage, blastula, gastrula, segmentation. pharyngula, hatching and early larval. By 90% epiboly, approximately nine hours after fertilization, the neural plate appears as a thickening of the epiblast in the dorsal, anterior region of the embryo. This thickening of the neural plate, arising from the cells assuming a columnar structure. extends dong the dorsal axis reaching the posterior of the embryo by the end of coastrulation (Kimmel et al., 1995). Neural tube formation in the zebr~shthen occurs through secondary neurulation, a feature unique to teleost embryos. In prirnary neumlation, the neural tube foms when the neural plate folds into a hollow tube containing the neurocoele. During secondary neumlation, the neural plate indents to form the neural keel which then folds to produce a solid neural rod. Starting at approximately 18 hours postfertilization the neural tube foms by cavitation, where the neural rod is hollowed to form the neurocoele beginning at the floor plate on the ventral surface. At this time, the apical surface of the original neural plate epithelium becomes the inner surface of the neural tube and the cells dong the center of the plate become the ventral portion of the neural tube (Kirnmel et al., 1995). One striking feature of neural tube formation in the zebrafish is that by approximately 18 hours postfertilization, before cavitation has begun. ten swellings appear at the anterior of the neural rod. These swellings represent morphological landmarks prior to the appearance of the neurocoele. The two most anterior swellings are the diencephalon and the telencephalon, which together make up the forebrain, or prosencephalon. The third swelling is the midbrain segment or mesencephalon. The rernaining seven swellings, rhombomeres (r 1-r7), make up the hindbrain or rhombencephalon. One other distinguishing feature is the cerebellar primordia which will form at the midbrain-hindbrain boundary (MHB); the MHB is the constriction between the midbrain swelling and r1 (Kimmel et al., 1995). Understanding normal neural development in zebrafish requires the characterization of molecular events as well as morphological events. A nurnber of genes including wnt,par. eph (Macdonald et al.. 1994) and engrailed (Joyner, 1996) are involved in the development of the zebrafish central nervous system but 1 will concentrate on the zebrafish wnt genes.

1.6 WNTS int- 1 (reclassified as Wnt- 1) is a proto-oncogene initially identified through its involvement in the formation of virally induced mouse rnammary tumors (Nusse and Varmus, 1982) and is one of the prototypes of a large family of related genes. Members of this Wnt gene family exist in man, mouse, Xenopus, zebrafish, Drosophila and Caenorhabditis elegans. Interestingly, the Wnt- l orthologue in Drosophila is wingless, a segment polarity gene instrumental in patteming embryonic and larval development (Siegfried and Pemmon, 1994). Further evidence for the evolutionary conservation of the Wnt farnily is obtained by comparing the amino acid identity arnong Wnt family members. Certain Wnts in two different species often have a higher amino acid identity than two Wnts within the same species. This interspecific identity suggests the presence of classes of homologous Wnts between species which have resulted from the duplication of common ancestral Wnt-like genes (Nusse and Varmus, 1992). Al1 Wnts encode polypeptides of approximately 350-380 amino acids which have one or more N-linked glycosylation sites, a highly hydrophobic amino terminus signal sequence (McMahon et. al, 1992) and over 100 conserved amino acid residues. Although this basic outline of similatities exists, there are some important differences, such as the addition of arnino or carboxy terminal domains on certain Wnts (Nusse and Varmus, 1992). The C. elegans wrzts, Ce-wntl and Ce-wnt2, and Drosophila wnt5 contain an insertion in the centre of the gene at the sarne location indicating that there is evolutionary conservation of these distinctive characteristics (Shackleford et al.. 1993). The invariant hallmark feature of the Wnts is the positions of 33 cysteine residues in all family members (Nusse and Varmus, 1992). The importance of this cysteine conservation is demonstrated by a mutation of one cysteine codon of Wnt-I resulting in loss of transforming activity and the ability to duplicate the axis in early Xenopus embryos. The high degree of conservation of these cysteine residues also suggests an important role for disulfide bridges in the function of the Wnt molecule (McMahon et. ai. 1992). In addition, the majority of Wnts, including zebrafïsh wntl (Molven et al., 199 1), mouse Wntl (van Ooyen and Nusse, 1984), and mouse wnt3 (Roelink et al., 1990), have sirnilar structural organization?each containing three introns. Exceptions to this mie include Ce-wntl with eight introns (Shackleford et al., 1993) and Drosophila wingless with four introns (Uzrolygi et al., 1988). Finally Wnts are al1 expressed in a spatially and tempordly restncted fashion dunng embryogenesis; these expression patterns are also highly conserved (Christian et. al, 199 1). The amino acid sequence of al1 Wnts indicates that they comprise a family of secreted, cell-ce11 signaling molecules (McMahon er al., 1992). The significance of this family of signaling molecules is apparent from the loss or misexpression of various family members. In mouse, the absence of a functional Wnt- 1 gene is associated with the absence of the midbrain and some of the rostral hindbrain (McMahon and Bradley, 1990; Thomas and Capecchi, 1990). Loss of expression experiments using antisense oligonucleotides specific to Wnt-3Acaused hypoplasia of the midbrain and forebrain and lateral outpocketings of the spinal cord (Augustine et. al. 1993). Loss-of-function mutations of mouse Wnt2 result in defects of the placenta and circulatory system (Monkley et aL, 1996). In gain-of-function studies, the ectopic expression of Wnt- l in the developing mouse limb bud results in a congenital limb malformation of distal tnincations, skeletal fusion and webbing between digits (Zakany and Duboule, 1993). In Xenopus, ectopic expression of mouse Wnt-1 in early embryos leads to bifurcation of the antenor neural plate, an expansion of the posterior neural plate and duplication of axial mesodemal structures (McMahon and Moon. 1989). Ectopic expression of wnt4 in zebrafïsh embryos results in cyclopis. abnormal folding of the brain, dorsal localization of the telencephalon and other central nervous system abnormalities (Ungar et. al. 1995). Despite the phenotypic affects resulting from gain- and loss-of-function studies in vertebrate systems, we are only begiming to understand how Wnts act in signal transduction processes associated with development. The inability to isolate active Wnt proteins has been a hindrance to understanding the mechanism of Wnt signaling in vertebrates (Moon, 1993). Fominately, a c'oreat deal of genetic evidence from Drosophila has paved the way to functionally dissecting the Wnt signaling pathway (Siegfried and Pemmon. 1994).

1.7 DROSPHILA WZNGLESS Drosophila contain a series of Wnt homologues wingless, (wg) DwnrZ D1vnt3 and Dwnr5, with wingless being the most studied (McMahon et. al. 1992). Wingless (WG), the Drosophila homologue of the mouse Wnt 1. shows funetional equivalence as both WG and Wnt l transform mouse mamary epithelial cells (Ramakrishna and Brown, 1993). In Drosophila. WG is a secreted glycoprotein which signals through a paracrine mechanism (van den Heuvel et. al, 1989). Although WG signaling cmresult in both short range and long range effects (Zecca et al., 1996), one well characterized example of WG signaling is the short range effect involved in segmentation in the Drosophila embryo. In the Drosophila embryo. wg expression appears in a suipe of cells that is directly anterior to cells expressing another segment polarity gene engrailed, en. This pattern of expression of wg and en is essential for establishing and maintaining the borders between adjacent segments. wg also functions in ce11 fate determination, causing diversification of ce11 types in the embryonic epidermis, neuroblast differentiation, ce11 proliferation, axis specification and imaginal disc development (Siegfried and Pemmon, 1994). The pattern of juxtaposed wg and en expression is a feedback mechanism that maintains the pattern of wg and en expression. wg expression reinforces en expression in adjacent cells and vice versa. Components of this signal transduction pathway from wg to en have been elucidated through characterization of other Drosophila segmentation mutants (Figure 1, Table 1). Porcupine (PORC) is a transmembrane protein localized to the endoplasmic reticulum. porc mutant alleles cause a phenotype similar to wg and results in WG being restncted to the ce11 expressing it. PORC, therefore, is likely involved in the processing of WG for secretion (Kadowaki et al., 1996). WG interaction with the receiving ce11 is modulated through proteoglycans located at the surface of the receiving ce11 (Reichsman et al.. 1996). A Drosophila homologue of UDP- a4ucose dehydrogenase, identified independently as sugarless, suppenkasprr or kiwi, gives a mutant phenotype sirnilar to wg. The affect the mutation of this gene has on the WG signaling pathway suggests an important role for glycosarninoglycans and proteoglycans, possibly by affecting Wg diffusion (Haerry et al., 1997; Binari et al., 1997; Hacker et al.. 1997). DFZ2, a member of the Drosophila farnily of frizzled proteins, is the receptor for the WG protein (Bhanot et al.. 1996). WG bound to the frizzled receptor results in the inhibition of Zeste-white 3 (ZW3) kinase, through Dishevelled (DSH), Protein kinase C and Casein kinase 2 (Cook et al., 1996). This inhibition of ZW3 causes an increase in the stability of Armadillo (ARM) and also modifies the subcellular localization of ARM (Pemmon and Siegfried, 1994). With inhibition of ZW3, ARM is now localized to the nucleus. In the nucleus ARM interacts with Drosophila Pangolin (PAN). It is interesting to note that WG signaling can also result in changes in ce11 adhesion (Peifer et al., 1993) and that ARM and p-catenin are components of adhesion junctions (Moon et al., 1993a).

1.8 Caenorhabditis elegans Further support for the role of Wnts in signaling pathways cornes from characterization of one of the C. elegans wnr genes, lin-44. lin-44 is Figure 1. A diagrammatic representation of components and events in the Wingless signaling pathway of Drosophila melanogaster. Translation of the wingless (wg) results in the protein Wingless (WG) which is modified by Porcupine (PORC) located on the endoplasmic reticulum. Sugarless/Kiwi/Suppenkasperaffect the Wingless pathway through the synthesis of ECM molecules. Upon binding of WG to the Drosophila friuled receptor (DFZZ), Zeste-white 3 (ZW3) is inhibited through a mechanism involving a Protein kinase C (PKC), Dishevelled (DSH) and Casein kinase 2 (CU).Subsequently, ZW3 cannot inhibit Armadillo (ARM) through phosphorylation. In its dephosphorylated state ARM is more stable and can be translocated to the nucleus where, with Pangolin (PAN) it cm affect the transcription of genes such as engrailed (en). Arrows indicate positive regulation and flathead arrows indicate negative regulation (after Siegfried and Pemmon, 1994). -@ Sugarless Kiwi 1PKC /ZW~ Suppen kasper 1 ARM Table 1. A summary of the characteristics of molecules known to function in the Wingless signaling pathway (after Moon et al., 1997). se&eted glycoprotein extrace Mar associated with ECM and ce11 surface localizes to ce11 surface

Porcupine transmembrane protein endoplasmic re ticulum likely involved in processing WG for of signding ce11 secretion

S ugarless homologue of vertebrate affects ECM Kiwi UDP-glucose dehydrogenase Suppenkasper may synthesize proteoglycans which affect Wingless signaling

Friuled transmembrane protein plasma membrane receptor of receiving ce11

Casein kinase 2 associates with and phosphorylates cytoplasm Dishevelled

Dishevelled phosphoprotein inner plasma membrane cytoplasm

Protein kinase C . serinelthreonine kinase inner plasma membrane responsible for inhibition of ZW3 cytoplasm nucleus

serinehhreonine kinase cytoplasm phosphorylates ARM to decrease activity

ArrnadilIo phos phoprotein - inner plasma membrane ce11 adhesion molecule cytoplasm nucleus

PangoIin HMG-box transcription factor cytoplasm binds with ARM to alter gene nucleus regulation expressed in hypodermal cells at the tip of the tail. lin-44 activity is required for proper polarity of the cells anterior to these hypodermal cells. Thus. lin- 44 activity is another example of Wnt signaling affecting ce11 polarity. The interaction of LIN-44 with cells directly anterior to the hypodermal cells responsible for secreting LIN-44 also reinforces the observation that Wnt signaling often occurs over a very small distance of a few cells. Consistent with examples of Wnt activity in ce11 fate determination is the finding that lin-44 mutants, dong with altered polarity, have an altered specification of the P 13 ce11 fate (Herman et al.. 1995). Another C. elegans gene, lin-17,encodes a receptor homologous to the Drosophila Frizzled. As LIN- 17 is required for regulating the polarity of cells arising from asymmetric ce11 divisions similar to LIN-44 and Drosophila Frizzled is a WG receptor. it appears that LIN- 17 is the receptor for LIN44 and these elements of the Drosophila pathway are conserved in C. rlegans (Sawa et al.. 1996). Another study isolated five genes, termed moin for more -soderm. that act in a Wnt-like pathway involved in gut differentiation. mom-1 encodes a protein similar to the Drosophila porc involved in processing of Wnt proteins; mom-2 encodes a Wnt-like protein; mom-3 activity is required in the signaling cell; mom-4 activity is required in the receiving cell; and mom-5 encodes a Frizzled-like receptor. Other factors include a P-catenin homologue, WRM- 1, and the TCF (T ce11 factor) homologue, POP- 1. Analysis of how these activities are required for gut differentiation has shown that these activities are required differently from their Drosophila counter parts. In accepted rnodels Wnt signaling results in an inhibition of glycogen synthase kinase 3 (GSK-3) from interacting with and phosphorylating p-catenin. The dephosphorylated P-catenin interacts with TCFLEF- 1 proteins which are translocated to the nucleus to activate other

Eoenes. In C. elegans, the binding of WRM- 1 inactivates POP- 1. Thus. although certain molecules and interactions within the Wnt pathway may be conserved. there exist variations in how those components are required in the pathway (Han, 1997; Thorpe et al., 1997; Rocheleau et al., 1997).

1.9 Xenopus RNA LOCALIZATION Early development in Xenopus gives interesting clues to the involvement of Wnts in early patterning events and demonstrates the evolutionary conservation of the Wnt signaling pathway. An ectopic expression system in Xenopus has been used to classify many Wnts into three functional categories based on their ability to alter mesodemal expression and duplicate the embryonic axis (Moon, 1993; Moon et. al. 1993a; Moon et. al, 1993~). The formation of the dorsoanterior axis in Xenopus is a major example of the involvement of the Wnt signaling pathway in early Xenopus development. Xwntll RNA and Vgl RNA are localized to the vegetal cortex in the Xenopus oocyte and either of these RNAs is capable of forming a dorsoanterior axis (Elinson, 1997). Many other RNAs such as PrCP (P tramducin repeat containing protein) (Elinson et al., 1993), Xcatl, and Xcat2 (Hudson et a., 1996) are localized to the vegetal cortex but do not possess this mis-forming ability. pcatenin, Xdsh and Xgsk-3, homologues of Drosophila am , dsh and Zw-3 respectively, have also been found to play an important role in this axis inducing ability (Yost et al., 1996). The role of p-catenin is downstrearn of Xwnt 11 as ody pcatenin RNA, not Wnr RNA, can rescue a mutant with altered dorsal mesodermd induction due to the overexpression of cadherins or injection of antisense p-catenin (Heasman et al., 1994). Xddl, a dominant negative allele of Xdsh, caused a decreased induction of secondary axes in Xenopus embryos by Xwnt8 but had no effect on induction of secondary axes by pcatenin (Sokol, 1996). As in the WG signaling pathway, therefore, Xdsh is downstream of Xwnt8 but upstream of p-catenin. Xgsk-3 contains an amino terminal phosphorylation site which is necessary for phosphorylation of pcatenin in vivo. Although not evidence for a direct interaction, this suggests that a pathway conserved from Drosophila is essential for the early mis inducing ability of the vegetal cortex in the Xenopus oocyte (Yost et al., 1996). Other support for P- catenin in Xenopus and ARM in Drosophila acting in a Wnt pathway similarly is that not only does p-catenin induce axis formation but it also increases gap junctional communication like certain Wnts (Guger and Gumbiner, 1995). Also, nuclear accumulation of P-catenin results from ectopic Xwnt8 expression or through decreased Xgsk-3 activity analagous to the nuclear localization of ARM as a result of WG signaling (Larabel1 et al.. 1997).

1.10 WNT REGULATION To fully understand Wnt signaling pathways it is necessary to elucidate the factors that affect Wnr expression and those factors whose expression is altered by the Wnt signals. In mouse, the Wnt- 1 promoter contains a G/C nch area where the Wnt- l inducing factor binds, influencing Wnt-1 regulation (St-Arnaud and Moir, 1993). Another sequence important for mouse Wntl regulation is a homeodomain binding site, HBS 1, that is found in the Wntl enhancer. Removai of this element results in ectopic expression of Wntl in the forebrain. It is, therefore, thought that two horneodomain proteins, D1x2 and Emx2, which are expressed in the forebrain and bind to HBS 1, may be responsible for repression of Wntl expression in the forebrain in normal rnice (Iler et al.. 1995). In Xenopcls a 320 bp promoter region for Xwntl was found to be sufficient to confer Xwntl like expression of a reporter gene. Of five protein binding sites present here only one, containing SV40 GT-1 and GT-II motifs, was determined to be essential for the promoter activity (Gao et a!., 1994). In the Wnt5a gene in humans, homeodornain binding sites have been found in intron sequences. Three such intron sequences were found to contain numerous binding sites for Msx- 1 using immunoprecipitation studies (Iler and Abate-Shen, 1996). In regards to genes known to be regulated by Wnt signaling,Wnt- 1 can directly or indirectly regulate chick Engrailed-2 in the met-mesencephalon (Baily-Cuif et al., 19%). In tissue culture, cells overexpressing Wnt- 1 exhibit an increase and altered distribution of plakoglobin and cadherin, two of many proteins associated with modulating cell-ce11 adhesion (Bradley et al.. 1993). Xwntl and Xwnr-Sc have been found to activate XEn-I which is interesting when compared to mouse Wnt-UEn interactions (Koster et al.. 1996). In zebrafish wntl and par;! are involved in the formation of the MHB. Ectopically expressing par2 results in a ventral expansion of the wnt 1 signal. Although pax2 affects wnt 1 expression, pax2 alone cannot induce expression of wnt 1 at ectopic sites, indicating that other factors present at the MHB are necessary to upregulate the expression of wntl (Kelly and Moon, 1995; Fjose, 1994).

1.1 1 ZEBRAFISH wnt8b The Wnt pathways make important developmental decisions. For example, in zebrafish ivntl (Kelly and Moon, 1995). wnt 4 (Ungar et al.. 1995),wnt 8b (Kelly et al., 1995) and wntlOa (Kelly et al., 1993) are each involved in neural development. My research has focused on zebrafish wnt8b which is thought to be involved in establishing distinct boundaries in the central nervous system. wnt8b is fxst expressed in presumptive neuroepithelium, believed to be the progenitors of cells which later form the MHB. Initially wnt8b expression appears as two stripes that are separated over the midline of the embryo which then converge to transverse the midline. Later, rt*nr8b is found in the forebrain, specifically at the forebrain- midbrain boundary, at the MHB and in three discrete stripes of cells in the hindbrain. This expression in the hindbrain demarcates rhombomere (hindbrain segments) 1, 3 and 5; a unique feature for a vertebrate Wnt expression pattern (Kelly et al., 1995). In Drosophila, wingless is expressed in every segment and is necessary for the establishment and maintenance of segmental boundaries. The role of ivingless in the establishment of segmental borders and the expression of zebrafish wnt8b at specific boundaries of the central nervous system suggests that wnt8b may have a role in establishing boundaries in the developing zebrafish central nervous system. To elucidate the role that wnt8b plays in establishing and rnaintaining boundaries within the developing zebrafïsh central nervous system, 1 wanted to overexpress wnt8b in its normal expression domain and assay for the effect of overexpression on central nervous system development. The first step was to identify a zebrafish wnt8b promoter. A zebrafish genomic library was screened using a wnt8b cDNA clone to isolate genornic fragments encoding the 5' end of the zebrafish wnt8b gene. Two distinct clones were isolated from the genomic library and analyzed in more detail. Sequencing of clone p8b-3H resulted in identification of one 3' exon of at least 619 bp in length. p8b-3H also contained 3 kb of intron sequence that was 5' to this exon. No other wnt8b exons were identified in p8b-3H or the other clone, p8b-7A. CHAPTER 2

MATERIALS AND METHODS

2.1 LIBRARY SCREEN One million clones from a zebrafish adult male/female genomic library in Lambda Fix II (Stratagene) were screened to isolate the genomic sequence of the zf wnt8b gene. Lambda Fix II phage and XL-1 Blues cells were incubated at 370C for 15 min, added to 8 ml of top agar (NZY broth and 0.7% agarose) and poured on NZY plates. Plates were incubated overnight at 370C and resultant plaqucs were transferred to nitrocellulose . The DECAprime II labelling kit (Ambion) and 32~-~CTP(Amersham) were used to random prime label a 600 bp Pst1 fragment at the 3' end of the zebrafish wnt8b cDNA to high specific activity. Filter hybridizations were carried out in 5X SSC, 1X Denhardt's, 0.2% SDS and 100 pg/ml of denatured, sheared hemng sperm DNA. Washes were done at moderate stringency of 1X SSC and 0.1% SDS at 550C. Initial screening resulted in 13 positive clones and, subsequently, seven positive clones were identified through secondary screening, with hybridizations as above but with higher stringency washes of 0.2X SSC and 0.1% SDS. Five of these clones, 8b- 1. 8b-2, 8b-3, 8b-4 and 8b-7, were purified and analyzed further. Phage DNA was purified as follows. Approximately 1 X 106 plaque forming units were incubated for 15 min at 370C with 150 pl of XL-1 Blues that had been grown in 0.2% maltose and 10 mM MgS04- Cells and phage were added to 8 ml of top agar (NZY broth and 0.7% agarose) and poured on extremely moist, 150 mm NZY plates. Plates were cultured top-side up overnight at 370C in a humid environment. The plates were then kept at 40C for 30 min. 10 ml of SM buffer (100 mM NaCl, 8.1 mM MgSO4- 7H20,50 mM Tris-Cl pH 5.0,O.O 1% gelatin) was added to each plate with occasional gentle mixing. RNase A and DNase 1 were added to a final concentration of 10 pg/ml and incubated for 30 min at 200C. 58.4 g/L of NaCl was added to these samples and rnixed gently. 100 g/L of PEG 8000 was then added and the samples were rnixed gently on a tube rocker for 10 min at room temperature. Sarnples were then kept at 40C overnight and centrifuged at 5 000 Xg for 10 min. Pellets were pently resuspended in 6 ml of 10 mM Tris-CI (pH8.0)/10 mM MgC12. Phage DNA was phenoVchloroform extracted, ethanol precipitated and resuspended in 1 ml of sterile dH-O. Through restriction enzyme digest analysis four of these clones, 8b- 1, 8b-2, 8b-3, and 8b-4, were determined to be identical. 8b-7 was deterrnined to contain a zebrdsh genomic fragment distinct from the other four clones analyzed. Zebrafish genomic DNA of approximately 12 kb was subsequently isolated from the Lambda Fix II vector (Stratagene) for clones 8b-3 and 8b-7 using Nd. pSK (Stratagene) was also digested with Nor1 and dephosphorylated using 10 units of calf intestinal alkaline phosphatase (NEB). Genomic inserts were ligated into linearized pSK with 1 unit of T4 DNA ligase (Gibco BRL) and transforrned into DHIOB cells through electroporation. Cloning of 8b-3 into pSK resulted in two positive clones, p8b-3H and p8b-31, with the zebrafish genomic insert in opposite orientations within the vector. Cloning of 8b-7 also resulted in two positive clones, p8b-7A and p8b-7E, both in the same orientation.

2.2 SOUTHERN BLOTTING Southem blots were carried out using p8b-3H, p8b-7A and p8b-7E constructs digested with restriction enzymes EcoRI, PstI, Sad, XbaI and XhoI. Digested fragments were electrophoresed on a 1% agarose gel, denatured, neutralized and blotted on a nitrocellulose filter ovemight in 20X SSC. Southern blots were crosslinked using a UV crosslinker (Stratagene) and were probed with a 5' fragment of the zebrafish wnt8b cDNA. This 5' EcoRVSacI fragment contained a small portion of the 5' untranslated region and 300 base pairs of the coding region. This approximately 250 base pair long fragment was random primed with the DECAprime II labelling kit (Ambion) and 32~-~CTP(Amersham). 5' Southem blots were hybridized ovemight at 550C in hybridization buffer (5X SSC, 1X Denhardt's, 0.2% SDS and 100 pg/ml of denatured, sheared hemng sperm DNA). Washes were done at high stringency, 0.1X SSC and 0.1% SDS at 550C. and exposed to X-ray film with intensifying screen at -8OoC for 36 hours. Identical digests of p8b-3H and p8b-7A were blotted as above and probed with a 3' fragment of the zebrafish wnt8b cDNA. This 3' Pst1 fragment contained approxirnately 600 base pairs of the open reading frarne and a portion of the 3' untranslated region. The 3' zebrafish wnt8b cDNA sequence was random primed with the DECAprime labelling kit (Arnbion) and 32~-~CTP(Amersham). 3' Southern Blots were hybridized overnight at 60oC in hybridization buffer. Washes were done at high stringency, 0. 1X SSC and 0.1 % SDS at 650C and exposed to X-ray film at -800C for 24 hours. A Southem blot was also done using above enzymes, as well as BarnHi, HindIII, KpnI, ScaI and XmnI, to digest clones p8b-3H and p8b-7A. The entire coding region of the cDNA was labelled with the Random Pnmers DNA Labelling System (Gibco BRL) and 32~-~CTP(Amersharn). The blot was hybridized ovemight at 550C in 6X SSC, 5X Denhardts, 0.4% SDS and 100 pg/ml denatured, sheared herring sperm DNA. The blot was washed to a stringency of 0.2X SSC and 0.1 % SDS at 680C and exposed to X-ray film at room temperature for 24 hours. To determine if 8b-3 and 8b-7 were overlapping clones two Southem blots were done using fragments of the genomic clones at one end. A 1.8 kb fragment from the end of 8b-3 was used as a probe for 8b-7 and a 1.7 kb fragment from 8b-7 was used as a probe for 8b-3.

2.3 RESTRICTION ENZYME MAPPING Clone 8b-3 was linearized using Kpn 1 and clone 8b-7 was linearized using Xho 1. Partial digests were then carried out on these linearized clones. 8b-3 was partially digested with EcoEU, PstI, SacI, XbaI and XhoI. 8b-7 was partially digested with BamHI, EcoRI, HindIII, KpnI, PstI, SacI and XbaI. Partial digests were carried out using 03, lu or 2u of enzyme incubated at 370C for 1 hour. Complete digests using these enzymes were carried out using 1Ou of enzyme at 370C for 2 hours. Partial and complete digests were run on a 1% agarose gel and Southern blotted using rnethods stated above. A 1045 nucleotide PvuI fragment from pSK, spanning nucleotides 1 to 500 and 24 16 to 296 1, was used to make a random primed probe with a random primer DNA labelling kit (Gibco BRL) and 32~-~CTP(Amersharn). This probe was designed to label one end of the linearized p8b-3H and p8b-7A. The band from a complete digest present on the Southem blot would, therefore, represent the fragment which contains pSK. Bands of increasing size present in partial digests on the Southern blot would represent this pSK- containing fragment plus successive fragments in the linearized clones. Thus the order in which these successive fragments occurred, relative to the pSK-containing fragment, could be determined by the sizes of bands in the partial digests. The position of restriction enzyme sites were conflrmed using double digests of some of these enzymes. Restriction enzyme mapping of ScaI and XmnI sites was done by double digests with enzymes already mapped.

2.4 DNA SEQUENCING AND ANALYSIS Using Southem blotting results, fragments which hybridized to the cDNA probe were subcloned into pSK and transformed into HB 10 1 cells (Gibco BRL). Two overlapping subclones were constructed frorn both p8b- 3H and p8b-7A. T3 and T7 primers present in pSK were used for sequencing. To obtain double stranded sequence, primers were designed (Table 2), and used as shown in the sequencing strategies (Figure 9 and Figure 12). Double stranded sequence was obtained from The John P. Robarts Research Institute (University of Western Ontario) and GenAIyTiC (University of Guelph) DNA sequencing facilities and analyzed using DNAstar sequence analysis prograrn. Homologies of sequences were analyzed through best fit and gap analysis on GCG Wisconsin Sequence Analysis Package Version 8.1-UNIX and by searching the NCBI database (Altschul et al.. 1990).

2.5 RNA ISOLATION Zebrafish were kept on a 12 hour light- 12 hour dark schedule. Zebrafish were placed in spawning chambers, ten to 20 zebrafish per charnber, at the beginning of the light cycle each day. Embryos were collected for one hour and then placed in a 250C incubator for 24 hours. The developmental stages of these embryos varied from approximately the 14-somite stage to the 26-somite stage. Embryos were frozen in liquid nitrogen and stored at -800C. Table 2. Sequences of primers used for sequencing of subclones of p8b- 3H and p8b-7A. Sequencing primers were used according to the sequencing strategy shown in Figure 9 and Figure 12. Primer Usai For: Name Primer Seauence

Sequencing A 5' TAG GTA GTï TCC CAC ïTC T 3' I B 5' AGA AGT GGG AAA mACCT A 3' I E 5' TGT TCT TiT TAA ATG TGT GTG 3' I F - 5' CAC ACA CAT T'TA AAA AGA ACA 3' N2 5' GGT AGT GTC GTG TiT AAA GC 3'

M 5' GAA CAA GCA TGG AAT TGA TC 3' I hi1 2 5' GCT GïTTGT TCA AAC TGC TC 3' N 5' GGT ?TA CAA CTC TGT TCC C 3'

5' ?TA ACC AAA CIT AïT îTC GGA 3'

1 7A 5' CAG TGT AGA AAA TCA TCT C?T 3'

7B 5' GGA ATG TGA TIT AAT TTC MG3'

7C 5' GAA ïTC GAT AAT TCT CGG A 3' Frozen embryos were ground using a mortar and pestle. RNA was extracted from the frozen embryos using approximately 1 ml of TRIzol Reagent (Gibco BRL) per 0.1 ml of ground embryos. Briefly. ground embryos were thoroughly homogenized in TRIzol reagent in a glas homogenizer and incubated for 5 min at room temperature. Chloroform. 0.2 ml per 1 ml of TRIzol reagent used, was added to the sample, shaken vigorously for 15 seconds and incubated for 3 min at room temperature. Samples were centrifuged for 15 min at 12 000 Xg at 40C. The aqueous phase was removed and RNA was precipitated by adding 0.5 ml of isopropanol per 1 ml of TRIzol reagent used. Sarnples were incubated for 10 min at room temperature and centrifuged for 10 min at 12 000 Xg at 40C. Pellets were washed with 70% ethanol, dried bnefly under vacuum and resuspended in DEPC treated dH20. Isolated RNA was then stored at -8OOC. From total RNA, poly(A)+RNA was isolated using oligo d(T) cellulose (Pharmacia). The oligo d(T) cellulose column was packed using 100 mg of cellulose in elution buffer (10 mM Tris-CI (pH7.6), 1 mM EDTA (pH 8.0),0.05% SDS) and washed with ten column volumes of 1X column loading buffer (20 mM Tris-Cl (pH 7.6), 0.5 M NaCl, 1 mM EDTA (pH $.O), 0.1% sodium lauryl sarcosinate). Total RNA samples were heated to 630C for 5 minutes and an equai volume of 2X column loading buffer (40 mM Tris-Cl (pH 7.6), 1.0 M NaCl, 2 mM EDTA (pH 8.0), 0.2% sodium lauryl sarcosinate) was added before putting the sarnple into the column. Eluate was collected and one column volume of 1X column loading buffer was added and the eluate was collected again. This eluate was heated to 650C for 5 minutes and reapplied to the column. The colurnn was washed thoroughly with ten column volumes of 1X column loading buffer. The poly (A)+ RNA was eluted using 3 volumes of elution buffer and collected in aliquots of approximateiy 250 pl and ethanol precipitated.

2.6 NORTHERN BLOT ANALYSIS For Northem blots an RNA standard (Gibco BRL), 5 yg of total RNA from zebrafish, 12 pg of poly (A)+ RNA were resuspended in 4.5 pi of DEPC treated dH20 and added to 2.0 pl of 5X formaldehyde gel-running buffer (0.1 M MOPS (pH 7.0), 40 mM sodium acetate and 5 mM EDTA (pH 8.0)). Samples were incubated at 650C for 15 min and placed on ice. RNA was electrophoresed in 1X formaldehyde gel-running buffer, on a 1.5% agarose gel. containing 6.6% formaldehyde and 1X formaldehyde gel- running buffer. Lanes containing the RNA standard and total RNA were separated and soaked in RNase free water to remove formaldehyde then stained with ethidium bromide. Lanes containing poly (A)+ RNA were soaked in RNase free water to remove formaldehyde, soaked in 20X SSC and blotted for 24 hours in 20X SSC. The blot was baked for three hours in a vacuum and probed with a 600 bp fragment of zebrafish wnt8b cDNA that was random primed using a n~uick~rirneKit (Pharmacia B iotech) and 32~ dCTP (Amersham). The blot was hybridized ovemight at 600C in 6X SSC, 5X Denhardts, 0.4% SDS and 100 pg/ml denatured, sheared hemng sperm DNA. Washes were done to a stringency of 0.2X SSC and 0.1 % SDS at 620C and exposed to X-ray film at room temperature for one week. CHAPTER 3

RESULTS

3.1 ISOLATION OF GENOMIC DNA ENCODING wnt8b The initial purpose of this study was to isolate zebrafish genomic sequences containing a wnt8b promoter. Screening of a zebrafish genomic library with a probe from the zebrafish ivnt8b cDNA, resulted in the identification of thirteen positive clones. These clones were rescreened at higher stringency resulting in the isolation of seven positive clones, 8b- 1 through 8b-7. DNA was prepared from five of these positive phage clones and they were digested with restriction enzymes to determine if these clones contained sirnilar fragments of zebrafish genornic DNA. The restriction enzyme Pst1 proved particularly useful in this analysis, as it generated a large number of fragments. As Figure 2 illustrates, clones 8b- 1 and 8b-3 have identical banding patterns when the phage/genomic DNA was digested with PstI. Pst1 digestion of clones 8b-2 and 8b-4 also gave this banding pattem (data not shown). Pst1 digestion of phage/genomic DNA from clone 8b-7 resulted in a distinct pattem of fragments (Figure Z), possessing 1.8 kb. 2.1 kb. 2.5 kb, 8.1 kb and 10 kb fragments not present in 8b- 1 and 8b-3. The NotI DNA fragments from 8b-3 and 8b-7, containing the entire eoenomic insert isolated from h vector DNA. were subcloned into pSK. This subcloning resulted in two subclones from 8b-3, p8b-3H and p8b-31. A series of enzyme digests of p8b-3H and p8b-31 contained the genornic fragment in alternate orientations. The subcloning of 8b-7 also resulted in two subclones p8b-7A and p8b-7E which, upon similar enzyme digestion resulted in the same series of fragments, indicating that these two clones had Figure 2. Agarose gel electrophoresis of PstI digested zebrafish genomic clones isolated from h FIX II genornic library. PsrI fragments, sizes 1.9 kb, 2.2 kb, 2.8 kb, 3.0 kb, 12 kb, and 15 kb, of clones 8b-1 (1) and 8b-3 (3) are identical whereas PstI digestion of 8b-7 (7) produces 1.8 kb, 2.1 kb, 2.5 kb, 8.1 and 10 kb fragments which are not present in 8b-1 and 8b-3. Arrows indicate PstI fragments present in 8b-7 but not present in 8b-1 and Sb-3. the genomic fragment inserted in the same orientation.

3.2 GENOMIC DNA ANALYSIS As an initial step in the characterization of the genomic region contained in these two clones, a restriction map was generated and Southem blots were done to locate the regions that hybridized to the wnt8b cDNA. Figure 3 is p8b-3H digested with XhoI to illustrate the method through which the restriction maps were generated. Complete digests of p8b-3H with XhoI results in three fragments: 2.5,5.2, and 8.1 kb. These three fragments were ordered in a partial restriction enzyme digest using indirect end labelling of the products. p8b-3H was first linearized with KpnI, which cuts in the multiple cloning site and provides a reference point for building the map. A radiolabelled probe was made from pSK DNA from nucleotide 1 to 500 and 24 16 to 296 1. This probe does not overlap the multiple cloning site. In a complete digest, therefore, only the fragment that includes pSK should hybridize with this probe. In a partial digest only this fragment, and other fragments containing this fragment, will hybridize with the probe. In Figure 3 the lane with the complete digest shows a 5.2 kb band hybridizing with the probe. In the partial digest lanes, we see three bands: 5.2, 7.7 and 15.8 kb. The 5.2 band, therefore, is the 5.2 XhoI fragment which includes pSK. The 7.7 kb band is the 5.2 kb fragment plus the 2.5 kb XhoI fragment. Finally, the 15.8 band is al1 three fragments. Therefore, from the reference point at the KpnI site in the multiple cloning site, the order of the XhoI bands is 5.2 -2.5-8.1 kb. Similar partial digests were done using p8b-3H linearized with KpnI. to create a cornmon reference site, and partially digested with EcoRI, PstI, Sad,and XbaI. A restriction map was created in like fashion for p8b-7A Figure 3. Southern blot illustrating restriction enzyme mapping strategy for p8b-3H using partial digestion and indirect end labelling. O u represents a lane containing p8b-3H which has been linearized with KpnI and not further digested. 0.5 u, 1 u and 2 u contain p8b-3h linearized with KpnI and digested with XhoI for one hour with 0.5 units, 1 unit and 2 units of enzyme respectively. 10 u contains p8b-3H linearized with KpnI and digested for two hours with 10 units of XhoI. The Southern blot was probed with a 1045 bp fragment of pSK which had been labelled by random priming. When no XhoI was added. the probe hybridized with the linearized p8b-3H clone. When completely digested with 10 u only the XhoI fragment containing pSK hybridized with the probe. In lanes 0.5 u, 1 u and 2 u there are three bands hybridizing with the probe representing the 5.2 kb fragment containing pSK, a 7.7 kb fragment consisting of the 5.2 pSK containing fragment and a 2.5 kb fragment and linearized p8b-3H. using XhoI to linearize the clone and partial digests with BamHI. EcoRI. HindIII, KpnI, Pd, SacI and XbaI . ScaI and XmnI sites were added to these restriction maps by analysis of double restriction enzyme digests using previously mapped enzymes. The information obtained from these Southern blots and double digests is summarized in Figure 4 and Figure 5. Subsequent to restriction enzyme mapping, Southem blots were performed to determine which fragments contained zebrafish wnt8b cDNA sequence and should, therefore, be subcloned for sequencing. A Southern blot of EcoEU, PstI, SacI, XbaI and XhoI digested DNA of both p8b-3H and p8b-7A was hybrïdized with a 600 bp fragment from the 3' region of the zebrafish wnr8b cDNA. Sirnilar blots were prepared with p8b-3H . p8b-7A and p-8b-7E and were probed with a 250 bp fragment located at the 5' end of the cDNA. As Figure 6 illustrates, the 4.2 kb EcoRI fragment, 4.8 kb Pst1 fragment, 7.2 kb SacI fragment, 11 kb XbaI fragment and 8 kb XhoI fragment of p8b-3H showed hybridization with the 3' probe. In reference to the restriction map (Figure 4) of p8b-3H, these Southern blot results showed that the area frorn the EcoRI site at 1 1.5 kb to the PstI site at 13.7 kb contained i.vnt8b cDNA sequence. This fragment was isolated and subcloned into pSK for sequence analysis. There was no hybridization obsewed between this 3' wnt8b probe and the p8b-7A digests shown in Figure 6B. In similar blots probed with a 250 bp fragment at the 5' end of the open reading frame (ORF) of wnt8b, only fragments containing vector sequences of p8b-7A hybrïdized with this probe (data not shown). In the p8b-3H blot with this probe al1 fragments showed non specific hybridization, suggesting that p8b-3H may contain only 3' sequence (data not shown). Because clone 7 was isolated with a wnt86 cDNA probe, a blot was Figure 4. Restriction enzyme rnap of p8b-3H. This map was constructed using Southern blot analysis of complete and partial digests of p8b-3H similar to that presented in Figure 3. The enzymes EcoRI, PstI, Sad, XbaI and XhoI were mapped using this strategy. The KpnI site on the left was used as a reference point. Map positions of ScaI were determined with double restriction enzyme digests. pSK is shown as a thick black line. Fragments that hybridized with the probe of wnt8b cDNA are indicated by the stippled box.

Figure 5. Restriction enzyme map of p8b-7A. This map was constnicted using Southem blot analysis of complete and partial digests of p8b-7A similar to that presented in Figure 3. The enzymes BarnHI, EcoRI, HindIII, KpnI, PstI, Sad, and XbaI were mapped using this strategy. The site XhoI on the left was used as a reference point. Map positions of ScaI and XmnI were determined with double restriction enzyme digests. pSK is shown as a thick black Iine. Fragments that hybridized with the probe of wnt8b cDNA are indicated by the stippled box.

Figure 6. Agarose gel electrophoresis and Southem blot of p8b-3H and p8b-7A probed with the 3' end of the zebrafish wnt8b cDNA. Figure A is an agarose gel electrophoresis of p8b-3H and p8b-7A either uncut or digested with EcoRI, PstI, SacI, XbaI and XhoI. Figure B is the Southern blot of the gel shown in Figure A. The probe used for the Southern blot was a 600 bp fragment at the 3' end of the zebrafish wnt8b cDNA that was random prime labelled. The blot demonstrates hybridization of this 3' cDNA fragment with the 4.2 kb EcoRI fragment, 4.8 kb PstI fragment, 7.2 kb SacI fragment, 1 I kb XbaI fragment and the 8 kb XhoI fragment. No hybridization was observed between the 3' cDNA probe and p8b-7A.

probed with the entire ORF of the wnt8b cDNA to determine which fragments of p8b-7A hybridized to the zebrafish i.vntBb cDNA. The result of BarnHI, EcoRI, HindIII, KpnI, PstI, Sad, ScaI and XbaI digests being probed with the full ORF was hybridization of the probe to a 4 kb BamHI fragment, 6.8 kb EcoRI fragment, 3.6 kb HindIII fragment, 4.2 kb KpnI fragment, 3 kb PstI fragment, 3 kb Sac1 fragment, 2.4 kb and 3.6 kb ScaI fragments and 7 kb XbaI fragment (Figure 7). As with the Southem blot of p8b-7A digests probed with the 5' probe, each of these fragments corresponds to the fragment containhg pSK, hence, vector contamination was considered to be responsible for these hybridizations. This was especially true of the 3 kb PstI fragment and the 3 kb Sac1 fragment as these each contain almost only vector DNA sequence. There were, however. a couple of bands that showed very weak hybridization that did not correspond to vector fragments. These fragments represented the 2.2 kb DNA fragment seen with KpnI and Pst1 digests (shown by arrows). In reference to the restriction map of p8b-7A (Figure 5) these fragments overlap for 1.2 kb. The fragment from the PstI site at 5.3 kb to the Sac1 site at 7.2 kb was subcloned for DNA sequencing of this region to determine if any zebrafish ivnt8b cDNA sequence was present. The presence of similar sized DNA fragments in PstI digestion of 8b- 3 and 8b-7 (Figure 2) was an initial indication that although they represented distinct clones isolated from the genomic library, these two clones may contain an overlapping region. Hence, the assumption was made that if one contained more 3' sequence then the other would contain the 5' sequence of the gene. Information from the restriction maps, however, suggests that there is no overlapping area, between these two clones. As Southem blot results indicated that p8b-3H contained definite 3' coding region but no Figure 7. Agarose gel electrophoresis and Southern blot of p8b-7A probed with the entire zebrafhh wnt8b open reading frame. Figure A is an agarose gel electrophoresis of p8b-7A digested with BamHI. EcoRI, HNidIII, KpnI, PstI, Sad, ScaI and XbaI. Figure B is the Southem blot of the gel shown in Figure A. The fragments that show strong hybridization with the probe were determined to be pieces containing vector sequence. In the case of the lanes containing PstI and Sad digests the 3 kb fragment hybridizes with the probe, which are almost completely vector. In the lanes containing KpnI and PsrI digests however, there are areas (indicated by the arrows) which show a very weak hybridization with the probe. specific 5' coding region and that p8b-7A had fragments that showed only weak hybridization with the cDNA, it was uncertain whether p8b-7A contained wnt8b sequence at dl. A Southem blot was done to detennine whether p8b-3H and p8b-7A contained any overlapping fragments at ail. In this blot a series of digests of p8b-3H and linearized p8b-7A were probed with a 1.7 kb fragment from the end of the p8b-7A clone. The fragment used for the probe was adjacent to the p8b-7A area which showed the weak hybridization to the wnt8b cDNA. A sirnilar blot was done with a series of digests of p8b-7A and linearized p8b-3H and probed with a 1.8 kb fragment from the end of the p8b-3H clone. The fragment was adjacent to the area which hybridizes with the 3' cDNA probe. As seen in Figure 8 the p8b-3H probe hybridized solely with the linearized p8b-3H and the p8b-7A probe hybridized solely with the linearized p8b-7A. Thus p8b-7A contained no fragments that overlapped with the area of p8b-3H that was determined to contain the 3' region of the zebrafkh wnt86 gene and p8b-3H contained no fragments that overlapped with the area of p8b-7A that showed very weak hybridization with the zebrafish wnt8b cDNA. The subcloned EcoRUPstI fragment of p8b-3H was sequenced using primer sequences present in the vector and primers designed to complete the sequence, Table 2. The sequencing strategy for this subclone, p6 1B, is illustrated in Figure 9. The sequence obtained from p6 1B (Figure 10) demonstrated that sequence adjacent to the EcoRI site of this fragment was identical to the zebrafish wnt8b cDNA and stopped at the end of the known sequence. Within this sequence a splice junction was found (Figure 1 1). The site at which the zebrafish wnt8b 3' sequence and p8b-3H sequence differed extensively, showed homology to the splice site consensus Figure 8. Agarose gel electrophoresis and Southern blot analysis demonstrating that p8b-3H and p8b-7A are non-overlapping clones. In Figure A the agarose gel contains BamHI, EcoRI, HindIII, KpnI, NotI. Psrl, Sad, Scal XbaI and XmnI digests of p8b-7A and linearized p8b-3h. as well as EcoRI, K'nI, NotI, Pstl, Sacl, Scal XbaI, XhoI and XmnI digested p8b-3H and linearized p8b-7A. Figure B contains two separate Southern blots of the above gel. The half of the gel with digests of p8b-7A was probed with a fragment of p8b-3H and the other half with digests of p8b- 3H was probed with a fragment of p8b-7A. The blots demonstrate that the p8b-3H probe hybridized with only the linearized p8b-3H and the p8b-7A probe hybridized with only the linearized p8b-7A.

Figure 9. Schernatic representation of subclones from p8b-3H and the sequencing strategy. Two subcloned fragments were used to obtain sequence for the area of p8b-3H that hybridized with the probe from the 3' of the zebrafish wnt86 cDNA. p3- 12 and p61 B. Letters above the arrows indicate the primer that was used to obtain the sequence. Arrows indicate the direction of sequence obtained.

Figure 10. Sequence of the subclones from p8b-3H demonstrating overlap with a 3' portion of the zebrafish wnt8b cDNA. The underlined portion is the sequence from p8b-3H which is homologous to the cDNA sequence from bp 547 to 1185. TTCACTTCCT CCTCAATGGA CTTGCCACTG GGCCTGCCCA TGACAGAAAA CATGTTGACA ATGGCTATGC AAAATAAATA CACCCCTTTC CTGCGCCGAA CAATCCGAGC TCGATGTGAG ACCCTGATTC TGCGTCTTAC GCCAAGCGAA TGAGAAAAGA GGCGTTCCTT TTTAAAGGAC AGCTGTTTGT TTCAGCCGAA GTCAGGAATT ATTGTACGAC CACCAAG~AGAGAAAAGC CCAGACTTGA ACCTTGAACC GCTTCCTCTT AACTCGCAGT TCTTTTCTGT GTGTTTAAGT GGATGTATAG GGCGCCTACC TACTMCTA TTCTGGTTTA CAACTCTGTT CCCATGATAA TCCAATAGGT AAAAACTCAA AGCAAGCCTA AGAATGATCA AAATAATGTC ACGGTTTAAA TACAACACAA GTGCTTTAAA CACGACACTA CCATTTAACA TGGTACACAT CCAATTGACT AGTAATTATT TAACTGGTTA CACTTAGTTT CTCTCTTTAA AATTTTACTT GAAATGTGGC CTTTTTAAAC AATAAAAATA TTAAAATATT CTGTTTTAAT CCAGTCAATA TTGCTGACTC AATGTTGATT TGAAAATGAC ACTCACTGTT TAAAGTATAG TTTTGGAAAG TACTAACATG TCTTAACAAT AAAGTAATGT CTTCTTAACT TAGCCCAAGT AATAACAGTA GTTCATTCAT TCATTCATTC ATTCATTCAT TTTCCTTTGG CTCAGTCTCT TTATTCATTA GGGGTCACCA CAGCAGAATG AACCGCCAAC TTTTTCAGTA TATGTTTTAC GCAGCGGATG CCTATTCAGC TGCAACCCAG CACTGGGAAA CACCCATACA CTTTTGCATT CACACACATA CATTATGGCC AATTCAGATT ACTTAGTTCA CATATAGTGC ATGTTTTTGG ACTCTGGGGG AAACCCACGC GAACATGGGG AGAACATGCA AACTCCACAC AGAAATGCCA ACTTATCCAG CCGGGGCTAA AATTAATGAC CTACATGCTT TGAGGTGACA GTGCTAACCA CTGAGCCATC GTTTCACCCC TATTTTTGCT CATCTAACCT TTTAATAATT GCTGTTTTTA CAGTTTATGT CCATTGCCCA ATAACAAAAA TTGAAATAAA AGTTTAAATG TGAGCAAAAA GGAAAAATTT CAGTAAATAA ATTCACAAAA ATGTCAAAAG AATAAAATTA AAATCCAACT GACTCACAAA CTGCACTCAA AAAAATGTCA TGTGCTGTTT GTTCAAACTG CTCATTTAAA TAAACAAATA ATTCTTACGT TTTTTGGGTC AACTCAATTG TTTTATGTTC GGTTGGAATA TTTGATCAAT TCCATGCTTG TTCAATAAAA AAGGTCAGAA GAGGGTTTTT CTTTTATGTC AAAAATATTT GTAATTAATT GGATTCmT AFATAATTAG ATTGACAAAA TGCAATGCAA AAGTCTCAAT GCAATGCAAA AGTTACATTC AGGAGGTGCG CATTAACTTT CATTTCCTGA TTCGAGCTTT TATACGCAGC TGATAATAAC AGATGGAGTT TAGTGAAATT CGTCACTTGT CTCAGTCATC ATTCCTCAGT CTTCCATTGG CCGCACCCAT ACATACATCC TCTTTTTCTA CTAATTCTCT GCACAACGTC AAAAGCACAA GACTTCCCAA CTGC-TGT ATTGTGTCCA TTTTCAACCC ATCTCTGCTC ACAGGAAGTT TTGCTGCAGA GCTTGGGTTA TTTTAGACAA TCAGGCTTTT GCATTTCTTT CAGCTTCATC TCCTTAGCAA AAGTGCTTCA TTAGTATGCA AAGCATATCA CATCCGAGM TAAGTTTGGT TAATATATAT CACTTTAAGC AATACAAATA CAATTGTTTA ATGAAAAGAT ACAAAAATAA TAAAACTATA TATTCATTTT CCCCAACAGT TCAATCCATT TAAATTTGTA AAAACTAATA AGCTAACTTA ATTCCTTCAT GTTGTCCCAA TACTAGGCAT GGGCCTGAAT AAGATTCTGA CGGTATGATA ACCTAAGTAA AAATATCAGG ATTTCATAAT ATCATGGTAT TGTAATTATT TCTCTAAAAT ATGTTCTTTT TAAATGTGTG TGTAAAAAAA AACATCAACT CTTTCCCCAT TCAACATGGA ATATATTTTT CTAACATTTA AAATATTTTG AAACCATAAA TGTCAAGCAA ACATTAAATG ACATCTGCTC TCTTAATTCA ATTTAAATTA ATTATTTCAT TAATTAAAAA AAAAACACAG ATTTCCTTTT AAATWGAA GGCATCTTTG GATATTTTTC ATTTCTTTGA ATATAAAGTG AAGCCACTTC ATTGTTCAGC TCCTCAGCAT GCTGGATGTT AGTTTATTAG AGATTTGTTT TTCAGATGTT TCCTGAATCA TGGACTGACC CAGTAGCTTG CGATGTAAAG GGTCCTTAAA AAAAACACAT AAAAAAACAA ACAAAACTTA CAGATACATT AGGGAACGGT ATAAAGGAAA AATGTAACAG TGTTWCC TTGACTTTTC AAAACCACGG TAAACCTTAA AACCGTTTAT CAATTGTGTA GGACTCAACA TTTTTTAGTA GATCAAGTTT TATCTTTACA GACAGACTAA GTTGATATTT CCTTTATAAA TCCATAACTC CAGTCATCCG TGTCACACAA ATCGTTCTGA TATGCTAAAT TGGTGCTTGA CCAACATTTC TTGTTAACAT CAATTTTGAA AAACAATCTT ACACAGAGTG AATAAGAGGA CAGCTGTTTA TTTCATCATT CATTTCATTA TTTCATCAGT CAGGAATTAT TTCACCCCCA GACTTGAACC TTGAACGGCA CCCTCATCAC TCACCAAATG GATGCATACG GTGCCTGCTA TTGTGCACAT TGTATATTCC AAGTTTGAAT TAGAAAGTTT TGAAATGAGA ATGTCTGCCT CCAGAACTGA GCATGAAGAG TTCTTTAACA TGAGATCCTC TCTTCAACTA GGCAGTGAAA GGAACCATGC AGAGGACATG CAAGTGCCAT GGAGTGTCTG GCAGCTGCAC CACTCAGACC TGCTTGGCTA CAGTTGCCCG AGTTTCGAGA AGTGGGAAAC TACCTAAAAG AGAAGTACCA CCGGGCGGTG AAGGTGGATC TGCTGCGCGG TGCAGGGAAC AGCGCTGCCA GCCGTGGAGC CATTGCTGM ACCTTCAACT CCATTTCACG GAAAGAGîTG GTGCATTTGG AGGATTCTCC AGACTACTGC TTGGAGAACC GCACTCTAGG CTTGCCAGGT ACTGAAGGGC GCGAGTGTTT GAGGAAGGGC AAGAATCTGA GTMTGGGA GAAGCGCAGC TGTAAGCGGC TGTGTGGAGA CTGCGGTTTG GCTGTGGAGG AGCGCAGGGC CGAAACCGTG TCTAGCTGTA ACTGCAAGTT CCACTGGTGT TGTGCTGTGA AGTGCGAGCA Gc TCAAGAATGA CAATGCCAGC CGGAGGAAAA GCTATCGGTT GAAGAAGAAG CACTAGGAGA TATTCTAAAC TGTTCGCTAT GGTGGTTTAG TTCAGCTTGT TTGGTCTAAA Figure 11. Cornparison of p8b-3H sequence and the zebrafish wnt8b cDNA sequence demonstrating splice site consensus sequences. Figure A shows the consensus sequence for a splice site. (0 indicates the location of the splice event. Figure B shows the zebrafish wnt8b cDNA from nucleotides 506 to 555. Nucleotides 544 to 546, in bold, represent the consensus sequence AAG for the donor site of an exon. Nucleotide 547, in bold, represents the consensus sequence G at the acceptor site of an exon. Figure C shows the p8b-3H sequence frorn nucleotides 3001 to 3 100 and its region of overlap with the zebrafish wnt8b cDNA. Nucleotides 3026 to 3035. underlined, represent the string of pyrimidines within the acceptor site of an intron. Nucleotides 3040 and 3041. in bold, represent the consensus sequence AG at the acceptor site of the intron. Nucleotide 3042, in bold. represents the consensus sequence G at the acceptor site of the exon. 5' CAG/GU~AGU- intron - YNNAG/G A G

506 GAGCCGCCAT GAACCTGCAC AACAACGAAG TAGGACGCAA G/G

GCATGAAGAG TTCTTTAACA TGAGATCCTC TCTTCAACTA G/GCAGTGAAA

GCAGTGAAA

3051 GGAACCATGC AGAGGACATG CAAGTGCCAT GGAGTGTCTG GCAGCTGCAC

1111111111 1111111111 1111111111 IIIIIIIIII 1111111111

556 GGAACCATGC AGAGGACATG CAAGTGCCAT GGAGTGTCTG GCAGCTGCAC sequence (C/A)AG/GU(A/G)AGU - intron - YNNAG/G. Figure I l shows that nucleotides 544-546 of the cDNA sequence have the consensus sequence for a donor site of an exon and the subsequent nucleotide. at 547. is the conserved G for an acceptor site of an exon. In the p8b-3H sequence nucleotides 3026 to 3035 are the conserved string of pyrimidines and nucleotides 3040 and 3041 are the AG of the acceptor site of the intron. The following G at 3042 is the conserved G at the acceptor site of the exon. As the 3' end of the sequence ended at the EcoRI site at 1 1.5 kb. the 5' end must be in the direction of the Pst1 site at 13.7. Within the sequence obtained another exon was not found. The entire 4 kb EcoRI fragment was subcloned, producing p3- 12, and sequenced using a primer created from p6 1 B sequence to continue in the 5' direction of the gene. Further sequence for this clone was obtained using primers M, M2, N and N2; that sequence is shown in Figure 9. The complete sequence obtained through the sequencing strategy in Figure 9 is given in Figure 10. This sequence contains a 6 19 bp 3' exon of zebrafish wnt8b just beyond the stop site and 304 1 bp of intron sequence 5' to this exon. These genomic clones do not, however, contain any further homology to the zebrafish wnr8b cDNA. The presence of 3' but not 5' wnt8b sequence was, therefore, consistent with the Southern blot results for p8b- 3H. Figure 12 illustrates the sequence strategy used to obtain sequence from p8b-7A. Sequence of the subcloned fragment from p8b-7A (Figure 13) did not contain any zebrafish wnt8b cDNA sequence. The weak binding demonstrated in the Southern blots of p8b-7A digests (Figure 7), therefore, was not a 5' exon. To determine why this clone was isolated from the zebrafish genomic library in the screen and why the sequenced area of p8b- Figure 12. Schernatic representation of subclones from p8b-7A and the sequencing strategy. Two subcloned fragments were used to obtain sequence for the area of p8b-7A that showed weak hybridization with the probe of the zebrafish wnt8b cDNA, p5 and p44. Letters above the arrows indicate the primer that was used to obtain the sequence. Arrows indicate the direction of sequence obtained.

Figure 13. Sequence of subcloned fragments of p8b-7A. AAGTTTGTGT GTGTTTGTTG TGCCTCAGAG GAGCTAGTTA TCTACCGCTT GCAGGCCGGT TCCTCTCGAC CTCCCCCGGG CTGAGTGGCC CCGCGTAATT GAGTGCCACC TGAACGGTTG TTGTGMGG TGGGTTATCT GCCCACTTTA ATCCCACAGG AGGACAGCAC AAACCCTGAG GACTAAGAGC CCAGTCTGTG ACACAIlAGTA TTGTGGGAAG AGTCACATGG GAACACATCT TTGAAGGCTT TAATGACTTG TGGTACCTAG AGATGGCTGA ACTCATTATA TTTGTTTTTC TTGTCCACAA GACTGTTCTG TAAATTAGTC ACATGAGTAA AAGGATAATG TAAAACATGG GGWCGCT ATAAGACATT TTTGAAACCA GCGTAATAGT TTACATCTGT GCATCAGTAT GTTTGTGTTA AGTATGAGTG GATTAATAAA AATAAGTTTA GAGTTCATGT GAATCAGTTC AAATGATTAT ATGAAAAAGA ATGAAAGTTG TTCCGAGAAT TATCGAATTC CTGCAGCCCG GGGGATCCAA T'T'ACAAACAC TAATGAATAA TTTTTCCACA CATTTCTAAA CATATTAGTT TTAATAACCA ATTTCTAATG ACTGATTTCT TTTATCTTTG TCATATGTTT TACTAGATAT TTGTCAAAAT GCTAGTATTC AGCTTAAAGT GCAATTGAAA GGTTAAACTT GGTTAATTAG GTTAACTAGG GTAATTAGGC AAATCATTGT ATAACAGTGC TTTATTCTGT AGAATATACA GTAGTGTAAG ACGGCTMTA ATATCGACCT TWTGGTT ATAAAAATGC TTGTATTCAA GCAGAAAAAA ACMCAAAT AAGACTTTCT CCAGAAGAAA AAATATTATA GGAAATACTG TGGAAAAATT CCTTGCTCCG TTCAACATAA TTTGAGAAAT TTTAAAATAC TAATAATTTT GACTTAAACT GTATATATAC AGTGTAGAAA ATCATCTCTT GAAATACTTA TTTGTTATTC AGCTTGAAAT TAAATCACAT TCCAAATAAC TTTTTCAGTC TTTGAAATGT TTCTATAATC GTCTCCTAAC CTTGCAACAA CATTAACCTG AAGGTCTTTT AllAAGCTTGA GTGTAATGTG AATATGTAAA GGTTTTGATT TTCACTGTAT GATGATTTTC TATAGGCACT ATATACATAC TAGATTTATT TATTAGTACT GCTTGTAfLAT GTATTGCATG TGTTTAAGCA TAGTTTATAT AAATATTTGC AATTAAAAAT AATTTATAAA TGAAGTAACC TATTTGCGGT AGAGAAAATG TAAATGTTTT ACATTTTAGA GTAATTATAT TTTGAAAAGG TAIUATTATT AGTATTATTA TCAGTAGTAT ATTAAATGTG ATATTTAATG TAATATCAAT TATAACGCAA TACTACTAGT ACATACAATA GAAATCAAAA TAATATGTTT GTTAAGAAAA TGTACACTCA CCGGACACTT TATTAGGTAC ACCTTAATAG TACTGGATTG GACCCAATTT TGGTTTCAAA ACTGCCTTAA TCCTTCGAGG CATTGA 7A showed weak hybridization, a gap analysis of these sequences was done. As is shown in Figure 14. there is approximately 38 percent similarity and identity between the area sequenced and the zebrafish wnt8b cDNA. As the p8b-7A sequence contained only weak wnt8b similarities. the NCBI database was searched in order to determine if there were any other significant sirnilarities. This search resulted in the identification of significant homologies which are shown in Figure 15. In Figure 15A similarities between nucleotides 597-9 15 of p8b-7A and nucleotides 1652- 1937 of the zebrafish plasticin mRNA are shown. Fragments of p8b-7A from nt 597-694. 707-784-791-829 and 852-9 15 have 64,66,74 and 85 percent identity, respectively, with the plasticin mRNA. In Figure 15B similarities between nt 568-769 of p8b-7A sequence and nt 1469-1650 of the zebrafish tyrosine kinase receptor ligand, AL- 1 are shown. The fragments of p8b-7A from 568-623,649-720 and 73 1-769 show 83,77 and 92 percent identity, respectively, with the AL-1. Figure 15A also shows that the sequences from p8b-7A are arranged in the same order, 5' to 3', as the corresponding sequences from the zebrafish plasticin mRNA. The same is true for p8b-7A and AL-1 sequence similarities in Figure 15B. The sequence from p8b-7A showing these sequence similarities was translated in dl six reading frames. The largest ORF sequence was 80 residues. This amino acid sequence was used to search the NCBI database but did not result in any significant sirnilarities.

3.3 RNA ANALYSIS In order to further characterize the structure of the zebrafish wnt8b bOene, both a Northem blot analysis and a primer extension analysis were perforrned. The RNA used in each analysis was from the 14-somite stage to Figure 14. Sequence of the subclones from p8b-7A showing weak similarity with the zebrafish wnt8b cDNA. The top sequence is the zebrdsh wnt8b cDNA and the bottom sequence is the sequence from subclones of p8b-7A. This sequence comparison was done using GCG with default parameters for a gap comparison. The sequences shown have approximately 38 percent similarity and 38 percent identity. GGTGAAGAAAAGTCTCCCTCTCTCAGGATGTTCATGCATTTGGAGGTTTA Il i l II I Il I I I II I I I GCTTTAATGACTTGTGGTACCTAGAGATGGCTGAACTCATTATATTTGTT

TT ...... ATTACGCTTTCATCTTGATGGCTCACATGAAGACTTGCTG 1 l I I 11 I I I II IIi11111 I f TTTCTTGTCCACAAGACTGTTCTGTAAATTAGTCACATGAGTAAAAGGAT

CGGTTGGTCAGTGAATAATTTCCTGATGACCGCGCCAAAGGCCTACTTGA I II II II I I II III ~TGTAAAACATGGGGAAAACGCTATAAGACATTTTTGAAACCAGCGTAA

TCTACTCCAGCAGTGTAGCCGCAGGGGGGGGGGGGGG..AGCTCAGAGTGGGATTG I I~~IIIIII:II I I 111111 I TAGTTTACATCTGTGCATCANTATGTTTGTGTTAAGTATGAGTGGATTAA

AAGAGTGCAAGTATCAGTTTGCGTGGGACCGCTGGAAGTGCCCGGAGA.. I I I I llll III 1 I I Il I I TAAAAATAAGTTTAGAGTTCATGTGAATCAGTTCAAATGATTATATGAAA

...... GGGCCCTTCAGCTCTCCACCCACAGCGGACTC I I 1 1 111 I I Il UGAATG-MiAGTTGTTCCGAGAATTATCGAATTCCTGCAGCCCGGGGGAT

AGTCATGTATACCCTCACCAGGAACTGTAGCCTTGGTGACTTTGATAACT III t III 1 I I 1 11111 1 1 AGTTTTAATAACCAATTTCTMTGACTGATTTCTTTTATCTTTGTCATAT

GTGGATGTGATGATACCCGm4CGGCCAGCGAGGTGGTCAAGGGTGGCTA 1 I t II III II Il 1 l II I I GTTTTACTAGATATTTGTCAAAATGCTAGTATTCAGCTTAJU4GTGCMTT

GTTTGTGGATGCGCTGGAAACCGGACAGGATGCACGAG.. ..CCGCCATG III 1111 I I IlI II Il II 1 TTGTATAACAGTGCTTTATTCTGTAGAATATACAGTAGTGTAAGACGGCT

.AACCTGCACAACAACGAAGTAGGACGCAAGGCAGTGAAAGGAACCATGCA 1 I 1 II II 1 I I l III I I AATAATATCGACCTTAAAATGGTTATAAAAATGCTTGTATTCAAGCAGAA

GAGGACATGCAAGTGCCATGAGTGTCTGGCAGCTGCACCACTCAGACCT I Ill Ill I I I I 1 II I MCAAACAAATAAGACTTTCTCCAGAAGAAMLUTATTATAGGAAAT 617 GCTG-GCTACAGTTGCCCGAGTTTCGAGAAGTGGGAAACTACCTAAAAGA 665 111 1 IlIl l I I I Il II 901 ACTGTGGAAAAATTCCTTGCTCCGTTCAACATAATTTGAGAAATTTTAAA 950

666 GAAGTACCACCGGGCGGTGM4GGTGGATCTGTCGCGCGGCA 715 I II 1 III IIIIII II Il 1 951 ATACTAATAATTTTGACTTAAACTGTATATATACAGTGTAGNU.ATCATC 1000

716 GCGCTGCCAGCCGTGGAGCCATT.GCTGAAACCTTCAACTCCATTTCACG 764 I I I Il 1 I II II IlII II 1001 TCTTGAUiTACTTATTTGTTATTCAGCTTGAAATTAAATCACATTCCAAA 1050

765 GAAAGACCTGGTGCATTTGGAGGATTCTCCAGACTACTGCTAGCC 814 II I l II I Il111 1 I II It f 1051 TAACTTTTTCAGTCTTTGAAATGTTTCTATAATCGTCTCCTAACCTTGCA 1100

615 GCACTCTAGGCTTGCCAGTGACTGAAGGGCGCGAGTGT.TTGA 863 II I I II Il I II II llllll 11 I 1 I 1101 ACAACATTMCCTGAAGGTCTTTTAAAAGCTTGAGTGTAATGTGA?4 1150

864 CAAGAATCTGAGTElAATGGGAGAAGCGCACGTGTAAGCGGCTGTGTGGAG 913 II i t 1 II II 11111II 1151 TAAAGGTTTTGATTTTCACTGTATGATGATTTTCTATAGGTATATAC 1200

914 ACTGCGGTTTGGCTGTTGAGGAGCGCAGCGCAGGGCCGAAACCGTGCTAGCTGT 963 l 1 II I 1 II 1 II I t 1 Ill I 1201 ATACTAGATTTATTTATTAGTACTGCTTGTATTGCATGTGTT 1248

964 AACTGCAAGTTCCACTGGTGTTGTGCTGTGAAGTGCGAGCATCGC 1013 1 IlII I 1111 1 II 1 1 f 1249 T~ULGCATAGTTTATATAAATATTTGCAATTAA...... AAATAATTTAT 1291

10i4 MCTGTCAC.VUiGTACTACTGTGTGAAGAGAGAAGC~CGGGTCA. 1057 II II II II I I II 1 I I III I Il 12 9 2 FLAATGAAGTAACCTATTTGCGGTAGAGAAUTGTAAATGTTTTACATTTT 13 4 1

1058 AGAATGACAATGCCAGCCGGAGGAAAAGCTATCGGTTGAACAC 1107 III 1 I II III Ill 111 Il I I II I 1342 AGAGTMTTATATTTTGAAAAGGTAAAATTATTAGTATTATTCAGTAG 1391 il08 TAGAAGATAT ...... TCT~UULCTGTTCGCTTTTTTAGTTCAGCT 1151 Il I I II l Ill I I l II I I I I II 1392 TATATTAAATGTGATATTTAATGTAATATCAATTATAACGCMTACTACT 1441

1152 TGTTTGGTCTAAAGAATTCGAGTCGACCTGCAGG...... 1185 II I I IlII II I I II I 1442 AGTACATACAATAGAAATCAAAATAATATGTTTGTTAAGATGTACAC 1491

1017 TGTCACAAAGTACTACTGTGTGAAGAG 1043 il IIIIIII II 1111 111111 203 TGACACMG... TATTGTGGGAAGAG 226 Figure 15. Sequence of the subclones from p8b-7A showing similarity with zebrafish plasticin and a zebrafkh tyrosine kinase ligand, AL- I genes. Through a blast search of the NCBI database the following sirnilarities were found. Figure A demonstrates the similar areas between the zebrafish plasticin gene and sequence from p8b-7A. Similarities for these segments range from 64 to 85 percent identity. Figure 15A also shows that these sequences with similarity are arranged in the same order, frorn 5' to 3', in both p8b-7A and plasticin. Figure B demonstrates the sirnilar areas between the zebrafish tyrosine kinase ligand, AL-1, gene and sequence from p8b-7A. Similarities for these segments range from 77 to 92 percent identity. Figure 15B also shows that each of these sequences with similarity are arranged in the same order, from 5' to 3', in both p8b-7A and AL-1. 597 TATTAGTTTTAATAACCAATTTCTAATGACTGATTTCTTTTATCTTTGTCATATTTTA 656 l I I II Illllll llll I I II l I I I Il III 1 1652 TCTCATTTCTAATAACTGATTTATTTTATCTTTGTCATGATGACAGCACATCATATGA 1711

657 CTAGATATTTGTCAAAATGCTAGTAAAGT 694 1111111111 1111 11 111Il111llllI111 1 1712 CTAGATATTTTTCAAGATAGTAGTATTCAGCTTT?' 1749

707 TTMCTTGGTTAATTAGGTTAACTAGGGTAATTAGGCAAATCATTGTATAACAGTGCTT 766 II I III I I I IIIIIIIII llllllll 11 Illlllllllll 1 I 1737 TTCAGCTTAAATTAAAGGCTTA?iCTAGGTTAGGATTTG 1796

767 TATTCTGTAGAATATACA 784 II I t III Il l 1797 TACTGGAGACAATCTAAA 1814

791 TAAGACGGCTAATMTATCGACCTTAAAATGGTTAAA 829 III I Illllilllll I Illlltl Ill 111 1817 TAATATTGCTAATAATATTGGGCTTAAAACAATT- 1855

852 mCAAACAAATAAGACTTTCTCCAGAAGAAAAAATATTATAGGAAATACTGTGGAAAA 911 III 1111I11 IIIIIIIIIIIIIIIII Illllllllllllllllll1111 111 1874 AEIATACAAC-TCAGACTTTCTCCAWGACAAAATATTATAGGAAATACTGTGAGAAA 1933

912 ATTC 915 I I 1934 CTCC 1937

568 TGMTAATTTTTCCACACATTTCTAAACATATTAGTTTTMTMCCMTTTCTMT 623 i 1111 l1111 IIlIIItlIIIIIIlll II 111111111 Illllllll 1469 TAAATATGTTTTCAACACATTTCTAAACATAACAGCTTTAT 1524

649 ATGTTTTACTAGATATTTGTCAAAATGCTAGTATTCAGCTTGTGCTTTT 708 11 111 Il II1 1111 Ill 11 llllllllllllllllllll III Illll I 1537 ATATTTGACCAGAGATTTATCAGGATACTAGTATTCAGCTTWGTGACATTTMaCT 1596

709 AAACTTGGTTAA 720 II 1 II Ill 15 9 7 TAATTAGGGTAA 1 60 8

731 TAGGGTAATTAGGCMTCATTGTATAACAGTGCTTTAT 769 IlIIIIIIIIIIIIII Illlllllllllllll III 1 1612 TAGGGTAATTAGGCAAGTCATTGTATAACAGTGGTTTGT 1650 the 26-somite stage. when wnt8b is maximally expressed. In the Northern blot (Figure 16B), a probe made from the entire ORF of zebrafish wnt8b hybridized with a 3.4 kb transcnpt present in the poly (A+) RNA lane. This transcript is, therefore, significiuitly longer than the 1073 bp wnt8b ORF. Although 12 pg of poly (A+) RNA was used for the blot and after a week of exposure to film with an intensifying screen, only a weak signal was observed. This weak signal suggests that the zebrafish wnt8b mRNA is expressed at a low level. Figure 16. Denatunng agarose gel electrophoresis and Northem blot analysis to determine the lenpth of the zebrafish wnt86 transcript. Figure A shows an ethidium stained agarose gel contain RNA standards and 5 pg of total RNA from zebrafish embryos, 14 somite to 26 somite stage. Figure B is a Northem blot showing one lane in which 12 pg of poly (A+) RNA was probed with the full length zebrafish wnr8b ORF. The hybridizing band demonstrating hybridization represents a length of 3.4 kb.

CHAPTER 4

DISCUSSION

The long term goal of this study was detemine the role that rvnt8b plays in establishing and maintaining boundaries within the developing zebrafish central nervous system. This would be achieved by specifically overexpressing rvnt8b in its normal expression domain, and assaying for the effect of overexpression on central nervous system development. This long term goal required the initial isolation of the wnt86 promoter. As only a cDNA encoding wt8b had been previously identified (Kelly et al.. 1995). 1 screened a zebrafish genomic library with a wnt8b cDNA probe to isolate the genomic DNA that encompassed the wnt8b transcription unit. 1 isolated a number of positive clones. Four of the five clones characterized were the same. Two distinct genomic clones were analyzed in more detail. Clone p8b-3H contained a 3' exon of at least 619 bp in length. p8b-3H dso contained 3 kb of intron sequence that was 5' to this exon. Clone p8b-7A contained no zebrafish wnt8b exons. Characteristically, Wnt

3oenes contain four exons (Nusse and Varmus, 1992). p8b-3H and p8b-7A. however, provided incomplete information for cornparison of the genomic structure of zebrafish wnt8b with other Wnt genes. The clone, p8b-3H, also contains sequence 3' to the stop site. This sequence, however, could not be aligned because the cDNA sequence ends immediately 3' to the ORF stop site. To determine the genomic structure of the zebrafish wnt8b gene, a more complete cDNA would be needed. Initial screening for the zebrafish wnt8b cDNA was a library screen with srnall zebrafish wnt fragments used as a probe, and then RACE amplification to obtain 5' regions (Kelly et al., 1995). The 5' fragment obtained through RACE amplification overlapped the previously identified 3' fragment. The entire zebrafish wnt8b ORF was present within these two overlapping fragments. cDNA library screening may be more successful now as the entire coding region could be used as a probe to isolate a cDNA to the complete transcript of the zebraf5sh wnt8b gene. Also. the cDNA library initially screened was made to mRNA extracted from zebrafish gastrula (Kelly et al., 1995). Analysis of wnt8b expression has shown that it is first expressed in late gastrula stages. A more complete wnt8b cDNA rnight be isolated from a library made to mRNA from late gastrula to late sornite stage embryos. The length of the zebrafish wnt8b transcript was determined to be 3.4 kb (Figure 16). Thus, the hi11 length transcript contains 1073 bases of ORF and approximately 2.3 kb of untranslated region. As well, 12 pg of poly (.A+) mRNA when probed with the full length ORF resulted in only a very weak signai after a week of exposure to X-ray film with an intensifying screen. This suggests that zebrafish i.vnt8b is expressed at a low level. The RNA used for the Northem blot analysis was extracted from 14-somite to 26-sornite stage. kvnt8b expression reaches its maximum at the 12-somite to 16-somite stage and decreases between 26-somite stage and pec fin stage (Kelly et al., 1995). The RNA used, therefore, was isolated from the period of highest expression of wnt8b. Thus, even when zebrafish wnt8b is being maximally expressed, it is still expressed at a low level. Initial primer extension experiments to determine the position of the 5' end were unsuccessful (data not shown). The potential reasons for this are suggested from the result of the Northern analysis. First, there is 2.3 kb of untranslated region and, although the amount of 5' to 3' untranslated region was not determined, it is possible that there is a large 5' untranslated region such that primer extension did not reach the end. Alternately, the expression of wnt8b may be below the sensitivity of primer extension analysis. The isolation of a complete cDNA sequence would answer some of these concerns. The data obtained on the genomic structure of the zebrafish wnt8b

5oene is summarized in Figure 17. The initial objective of obtaining the wnt8b promoter was unsuccessful. 1 have only isolated a genomic fragment containing a 3' exon. Further analysis of the genomic structure of the zebrafish wnt8b gene and isolation of the promoter entails the rescreening of zebrafish genomic and cDNA libraries. It is obvious that a complete. or more complete, cDNA needs to be isolated. Subsequently, two approaches could be taken for the rescreening of the genomic library. If the full length cDNA were available then a portion of the 5' untranslated region could be used to screen the genornic library. Altemately. the 5' portion of the intron sequence identified from p8b-3H could be used in a chromosome walk (Horecka 1995). Although sequence analysis of p8b-7A did not result in identification of any strong sequence homologies with zebrafish wnt8b. searches of the NCBI database did result in sorne interesting similarities (Figure 14). Strong identities were found with zebrafish plasticin and a receptor tyrosine kinase ligand, AL- 1. mRNA. Though sequence similarities were found, translation of p8b-7A sequence in al1 six reading frames, in the regions of similarity, and subsequent searches of the NCBI database with these ORFs, did not result in any significant similarities. Genetic maps are useful for linking known genes to mutant phenotypes (Collins, 1992). Single sequence length polymorphisms that Figure 17. Schematic summary of the data obtained through sequence and Northem blot analysis. Stipled boxes indicate open reading frarne sequences. Question marks indicate unknown length. Broken arrow indicates genomic sequence available to compare to cDNA sequence. have been arnplified from intron sequences in zebrafish have recently been used to consuuct a reference cross DNA panel usehl for such positional cloning strategies (Knapik et al., 1996). Thus, intron sequences identified in this genornic characterization could be used to map wnt8b on to the current zebrafish genetic map. Initial attempts were made to find polymorphic sequences within the isolated intron sequence of the zebrafish wnt8b gene to map it on to the existing genetic map. A number of primer pairs were designed which arnplified 200 to 300 bp fragments within the 3' intron of zebrafish wnt8b. These primers were used to amplify sequences from the eaenomic DNA of two parent fish and a number of their offspring. Although polymerase chain reaction amplification of these intron sequences was successful, no length polymporphisms were detected. During the course of this search for polymorphisms with which to map this locus, the zebrafish wnr8b gene was mapped to linkage group 14 (A. Schier, personal communication). REFERENCES

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