Dev Genes Evol (1996) 206:3–13

© Springer-Verlag 1996

O R I G I NA L A RT I C L E

&roles:Patrick Blader · Uwe Strähle · Philip W. Ingham

Three Wnt genes expressed in a wide variety of tissues during development of the zebrafish, Danio rerio: developmental and evolutionary perspectives &misc:Received 24 October 1995 / Accepted in revised form: 16 January 1996

&p.1:Abstract Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide variety of developmental processes. Here we describe the cloning of three Wnt family members from the zebrafish, Danio rerio, which encode proteins with homology to murine Wnt-2, -4 and -5A/B. The expression patterns of the latter two zebrafish genes, designated ZfWnt4 and ZfWnt5 show considerable similarity with their homologues in other vertebrates; ZfWnt2, however, is expressed in the developing viscera in a pattern distinct from its closest murine homologue. In the light of the similarities and differences in the patterns of expression of these genes relative to their homologues in other vertebrates, we speculate on their possible functions. &kwd:Key words Wnt · Zebrafish · Viscera · Brain · Neural tube · Tailbud · Presomitic mesoderm&bdy:

Introduction Signalling between cells is a fundamental process required in the development of multicellular organisms. Recently, several superfamilies of intercellular signalling molecules, including the Transforming growth factors β (TGF-β), the Fibroblast growth factors (FGF), the Hedgehogs (Hh) and the Wnt families, have been characOriginally submitted to Roux’s Archives of Developmental Biology and accepted by Klaus Sander P. Blader1 · U. Strähle1 · P.W. Ingham Molecular Embryology, Laboratory ICRF Developmental Biology Unit, Department of Zoology, South Parks Road, Oxford, OX1 3PS, U.K. P.W. Ingham (✉) Molecular Embryology Laboratory, Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London, United Kingdom Present address: 1 IGBMC BP163 F-67404 Illkirch Cedex, France&/fn-block:

terised and their importance in development is now well established (for reviews see Jessell and Melton 1992; McMahon 1992; Ingham 1995). Since the isolation of the Wnt-1 gene from mouse, members of this family have been found in a wide variety of higher eukaryotes, ranging from mouse and humans to Xenopus and zebrafish (Gavin et al. 1990; McMahon and McMahon 1989; Christian et al. 1991; Wolda and Moon 1992; Molven et al. 1991; Krauss et al. 1992); Wnt genes are not restricted to vertebrates, however, as members of the family have also been isolated from Drosophila and C. elegans (Baker et al. 1987, Rijsewijk et al. 1987; Shackleford et al. 1993). The spatial and temporal patterns of expression of the vertebrate members of the Wnt family suggest roles for these genes in processes ranging from neural development (Shackleford et al. 1987; Wilkinson et al. 1987) to limb development (Gavin et al. 1990; Dealy et al. 1993; Yang and Niswander 1995; Parr and McMahon 1995) to mesoderm induction and primary axis formation (Christian et al. 1991; Ku and Melton 1993). Indeed, targeted mutations of several members of the murine family of Wnt genes indicate a requirement for these proteins in formation of the midbrain-hindbrain junction and cerebellum, the tailbud and the kidney (McMahon et al. 1990; Thomas and Capecchi 1990; Takada et al. 1994; Stark et al. 1994). The multifaceted potential of these proteins is highlighted further by the range of different phenotype produced by injection of synthetic Wnt mRNAs into early Xenopus embryos, phenotypes that depend on the family member encoded by the synthetic message (Christian et al. 1991; Smith and Harland 1991; Wolda et al. 1993). Finally, variation in the functional capabilities of Wnt family members also differs with respect to their ability to transform mammary cells in culture, again indicating that these proteins are multipotent and suggesting that the variety of functions for the various family members might be controlled by multiple Wnt receptors (Wong et al. 1994). The multifaceted functional potential, the widespread expression and developmental importance of Wnt genes

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makes them attractive molecules for further study. Accordingly, we have isolated three Wnt family members from the zebrafish, Danio rerio with homologies to murine Wnt-2, -4 and -5. Here we describe the cloning of these zebrafish Wnt homologues and their patterns of expression during zebrafish embryogenesis. We discuss the similarities and differences between these expression patterns and those of orthologous genes in various other species and speculate on their possible functions in the developing embryo.

Materials and methods Zebrafish culture Zebrafish (Danio rerio) were maintained at 28.5 °C on a cycle of 14 h light/10 h dark using standard methods (Westerfield 1993). Embryos were obtained by natural spawnings and were staged according to Westerfield (1993). Stages are described in terms of percent epiboly completed or somite number until 18 somites after which stages are given in hours post fertilisation (h); fortuitously, at 28.5 °C 18 h embryos possess 18 somites. Isolation and sequencing of ZfWnt cDNAs Total RNA was isolated from 15–17 h zebrafish embryos as previously described (Chirgwin et al. 1979). The RNA was reverse transcribed using random DNA hexanucleotides following standard protocols (Sambrook et al. 1989). Partial cDNAs were generated by Polymerase Chain Reaction (PCR; Saiki et al. 1988) employing oligonucleotide primers previously described by Gavin et al. (1990). Alternatively, degenerate oligonucleotide primers targeted to homologies between various members of the Wnt4 subfamily were used; the Wnt4 specific primer are as follows: N-term. GATCGGATCCCCNCA(A/G)GGNTT(C/T)CA(A/G) ' TGG C-term. GATCGGATCCAA(A/G)GA(A/G)AA(A/G)TT(C/T)GA (C/T)GG In both cases the PCR conditions used were those described by Gavin et al. (1990). The resultant partial cDNAs were cloned into Bluescript II (Stratagene) by taking advantage of the restriction sites designed into the PCR primers; clones containing fragments of the appropriate sizes were subsequently sequenced by the dideoxy-termination method using Sequenase (USB Inc.) following the manufacturer’s instructions. Three partial cDNA clones encoding proteins with homologies to members of the Wnt protein family were used as probes to screen a cDNA library constructed from 33 h zebrafish embryos (kindly provided by Dr. K. Zinn); the longest positive inserts from this screen were subcloned into Bluescript II (Stratagene). Exonuclease III deletions were made using a commercially available kit (Pharmacia) and used to sequence the first strand of these cDNAs, whereafter specific oligonucleotide primers were used to sequence the opposite DNA strand. The putative translation products encoded by these clones showed a high homology to the murine Wnt2, Wnt4 and Wnt5 proteins. Interestingly, however, initial comparison of the protein encoded by ZfWnt4 with Wnt4 homologues from other species suggested the cysteine residue at position 335 had been substituted for an ariginine. This residue is invariant in all the other Wnt proteins, including those from Drosophila and the nematode C. elegans (Rijsewijk et al. 1987; Baker 1987; Eisenberg et al. 1992; Russell et al. 1992; Shackleford et al. 1993; Graba et al. 1995). To address this anomaly a fragment of genomic DNA was amplified by PCR using primers designed against ZfWnt4 flanking the site of the “mutation”; when sequenced the ampified genomic fragment was found to encode the expected cysteine residue. Whether the “mutation” in ZfWnt4 represents a cloning artefact or a bone fide polymorphism in the strain of zebrafish

Fig. 1 Dendogram showing the alignment of the predicted amino acid sequence of the three zebrafish genes isolated in this study versus the murine Wnt proteins (Gavin et al. 1990). The scale indicates percent homology between the compared sequences. Zf and M indicate zebrafish and murine sequences, respectively&ig.c:/f from which the cDNA library was constructed is not known, but for the purposes of this report a cysteine has been included at this position. All other nucleic acid manipulations were performed following standard procedures (Sambrook et al. 1989). DNA sequences were compiled, amino acidsequences compared and the dendogram (Fig. 1) generated on a UNIX computer using the GCG software package version 8.0. The nucleotide sequences of the cDNA clones described in this paper are deposited in the Gen Bank data base under accession numbers U51266-8. In situ hybridisation Whole mount in situ hybridisation and in situ hybridisation on cryostat sectioned embryos was accomplished as described by Oxtoby and Jowett (1993) and Strähle et al. (1994), respectively. In both cases, digoxygenin (DIG) labelled cRNA probes were synthesised using linearised ZfWnt cDNAs as template by following the manufacturer’s instructions (Boehringer Mannheim). In whole mount stained, late stage embryos in which pigment cells obscured staining or where higher cellular resolution was required, embryos were embedded in wax and sectioned. In these cases embryos were pre-embedded in low melting point agarose prior to wax imbedding to aid orientation of the sections. The agarose blocks were dehydrated and embedded in wax essentially as described by Godsave et al. (1988). Sectioned material was mounted in Permount prior to photographing; whole mount stained embryos were cleared in 99% glycerol prior to photographing.

Results Sequence analysis Using degenerate oligonucleotide primers complementary to conserved regions of the Wnt gene family or to con-

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Fig. 2 Deduced amino acid sequence of the full-length ZfWnt2 cDNA clone aligned with murine and human Wnt2 (Wainwright et al. 1988; McMahon and McMahon 1989). Dashes indicate identities between residues and dots indicate gaps introduced for optimal alignment of the sequences. Conserved cysteine residues are in bold type, the hydrophobic leader sequence is in italic type and potential N-linked glycosylation site is underlined&ig.c:/f

served regions of murine and Xenopus Wnt4, three distinct partial cDNA fragments were amplified and cloned from first strand cDNA. Using these fragments as probes to screen a cDNA library a number of clones were isolated and sequenced. Comparison of the predicted proteins encoded by these longer clones with previously isolated Wnt protein family members from the mouse (Gavin et al. 1990) revealed significant homologies to Wnt2, the Wnt5A/Wnt5B subfamily and Wnt4, respectively (Fig. 1). These cDNAs were designated ZfWnt2, ZfWnt4 and ZfWnt5. ZfWnt2 ZfWnt2 is 1509 base pairs (bp) and contains an open reading frame encoding a protein of 350 amino acids (aa). An in-frame termination site is located in the 166 bp untranslated leader, two “codons” upstream of the predicted initiation methionine. At its 3′ end ZfWnt2 is flanked by 293 bp of noncoding sequence terminating in a 23 nucleotide poly-A tail; 17 bp upstream of the polyA tail is a consensus AAUAAA poly-adenylation signal (data not shown). Like its murine and human homologues, the predicted amino acid sequence of the open reading frame encoded

Fig. 3 Deduced amino acid sequence of the full-length ZfWnt4 cDNA clone aligned with Xenopus and murine Wnt4 (Gavin et al. 1990; McGrew et al. 1992; Yoshioka et al. 1994). Dashes indicate identities between residues and dots indicate gaps introduced for optimal alignment of the sequences. Conserved cysteine residues are in bold type, the hydrophobic leader sequence is in italic type and potential N-linked glycosylation site is underlined&ig.c:/f

by ZfWnt2 contains a 20 aa signal peptide (Fig. 2). Outside the highly diverged signal peptide ZfWnt2 displays 71% aa identity with both the murine and human Wnt2 protein, and except for a 2 aa deletion at position 34 and an 8 aa deletion at position 346 the differences are evenly distributed throughout the ZfWnt2 protein (Fig. 2). All 24 cysteine residues are conserved between the three proteins, as is the putative N-linked glycosylation site at residue 293. ZfWnt4 ZfWnt4 is 1778 bp and contains 359 bp of 5′ noncoding sequence upstream of an open reading frame of 352 amino acids. As with ZfWnt2 there is an in-frame termination codon upstream of the initiation methionine. The 363 bp of A/T rich 3′ noncoding sequence is not terminated in a poly A tail, nor does it contain a consensus poly-adenylation signal (data not shown).

6 Fig. 4 Deduced amino acid sequence of the full-length ZfWnt5 cDNA clone aligned with Axolotl and murine Wnt5 A/B and Xenopus Wnt5A (Gavin et al. 1990; Busse and Séguin 1992; Moon et al. 1993). Dashes indicate identities between residues and dots indicate gaps introduced for optimal alignment of the sequences. Conserved cysteine residues are in bold type, the hydrophobic leader sequence is in italic type and potential Nlinked glycosylation sites are underlined&ig.c:/f

The predicted protein encoded by ZfWnt4 has 87% aa identity to chick Wnt4, 82% to Xenopus Wnt4 and 81% to murine Wnt4, not including the hydrophobic signal sequence (Fig. 3). As with ZfWnt2, the unconserved amino acids are evenly distributed throughout the coding region. The putative N-linked glycosylation site of ZfWnt4, residue 298, is conserved in comparison to the other Wnt4 homologues as are the 24 cysteine residues (Fig. 3). ZfWnt5 ZfWnt5 is 1730 bp and contains an internal fragment that is identical to the previously characterised PCR clone Zwnt [b] (Krauss et al. 1992). The longest open reading frame in the cDNA is 1089 bp and encodes a protein of 363 amino acids with a putative hydrophobic leader sequence of 23 amino acids. A second in-frame methionine at position 5 of the open reading frame could provide an alternative translation initiation site, as both methionines are preceded by favourable Kozak consensus sequences (Kozak 1986). Unlike either ZfWnt2 or ZfWnt4 there is no in-frame termination codon in the 5′ untranslated leader (data not shown). It is possible, therefore, that the initiation methionines designated above are actually internal to the coding region and that the bona fide transcription initiation site is upstream of the 5′ end of the ZfWnt5 cDNA. Alignment of the predicted translation product of ZfWnt5 with other members of the Wnt5 subfamily reveals significant homology with both Wnt5A and Wnt5B except in the hydrophobic leader sequences which, unlike ZfWnt2 and ZfWnt4, differs in length as well as amino acid sequence when compared to the other members

of the subfamily (Fig. 4). If the divergent leader peptides are excluded from comparison, ZfWnt5 displays between 80% and 86% homology to other amino acid sequences of the Wnt5 subfamily; overall, ZfWnt5 is most homologous to Axolotl Wnt5B (86%) and mouse Wnt5B (84%). Consistent with the overall higher conservation between ZfWnt5 and the Wnt5B subfamily, conservation between ZfWnt5 and Wnt5B specific residues is higher than for Wnt5A specific residues (11 versus 4, respectively). The 24 cysteine residues characteristic of Wnt proteins are completely conserved between the various family members as are the three putative N-linked glycosylation sites characteristic of the Wnt5 subfamily (Fig. 4). Spatial expression of ZfWnt2, 4 and 5 during embryogenesis ZfWnt2 Expression of ZfWnt2 is confined to a small region of the developing viscera, being first detected by in situ hybridisation at 30 h (Fig. 5a). At this stage transcripts are present in cells underlying the notochord at the level of the pectoral fin buds. By 48 h expression is detected in cells at the distal tip of an outpocketing of the gut (Fig. 5b); at 72 h this region of expression has decreased and comprises a small number of cells overlying the developing viscera (Fig. 5c). Endodermally derived organs, including the liver, pancreas and gall bladder develop in this region as does the mesodermally derived spleen. Even though the expression of ZfWnt2 was determined on cryostat sectioned tissue, it is difficult to establish whether the endodermally derived cells that constitute

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Fig. 5a–c ZfWnt2 expression in the viscera of organogenesis stage zebrafish embryos. Stained cryostat sections of 30 h (a), 48 h (b) and 72 h (c) are shown accompanied by schematic zebrafish embryos that indicate the location of the photographed regions. Arrowheads indicated the sites of ZfWnt2 expression which are difficult to distinguish from surrounding pigment cells. Bar is 100 µm&ig.c:/f

the walls of the outpocketing express ZfWnt2 or whether the expression is located in mesenchymal cells surrounding the endodermal outpocketing. By 5 days, when it is possible to identify positively individual visceral organs, expression is no longer detectable. ZfWnt4 ZfWnt4 expression is first detectable at the end of gastrulation as a transverse stripe in the anterior neurectoderm. This expression increases during early somitogenesis (Fig. 6a, b). By the 10 somites stage transcripts can be detected in the brain immediately caudal to the eye (Fig. 6c) in a region which, viewed laterally, resembles a trapezoid, being broader dorsally and tapering as it extends ventrally into the diencephalon (data not shown). Expression is still strong in the diencephalon at 18 h by which stage additional, fine stripes of expression are de-

tected dorsally in the hindbrain (Fig. 6d); the location of these fine stripes relative to individual rhombomeres is difficult to determine because of the low level of expression. By 24 h the anterior expression in the brain has retracted to the dorsal midline of the midbrain and diencephalon while expression in the hindbrain shows a rhombomeric periodicity (Fig. 6e). Expression is no longer detected in the forebrain or dorsal hindbrain at 36 h. Expression becomes evident lateral to the hindbrain at 10 somites, appearing as a pair of bilateral stripes that extend from the midbrain/hindbrain junction to the otic vesicle (Fig. 6c). By 18 h this expression becomes irregular and by 24 h has split into two patches, one anterior and one posterior (Fig. 6d and e). This staining would appear to be in an appropriate lateral position to be part of the pronephric system of the embryo; sections indicate, however, that the expression is in the ectoderm and not the underlying mesoderm (data not shown). Furthermore, the expression of ZfWnt4 lies further anterior than the anterior-most expression of pax[zf-b]/Pax-2, which is thought to mark the pronephros (data not shown; Krauss et al. 1991; Püschel et al. 1992). Expression is also detected in a restricted ventrolateral patch of the otic epithelium from 28 h (Fig. 6f) and by 36 h this is the only remaining rostral expression domain. During early somitogenesis ZfWnt4 expression is detected in a pair of bilateral longitudinal stripes, that are

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Fig. 7a–e ZfWnt4 expression in the dorsal neural tube and the floor plate. Whole mount stained embryos at the 2 somite (a), 4 somite (b) and 6 somite (c) stages are shown from the vegetal pole of the embryos with anterior up. Lateral views of whole mount stained and flattened embryos at the 10 somite stage (d) and 24 h (e) are shown with anterior to the left and dorsal up. Arrowheads indicate ZfWnt4 expression in the floor plate. Bar is 280 µm in (a–c), 50 µm in (d) and 200 µm in (e)&ig.c:/f

Fig. 6a–f ZfWnt4 expression in the anterior of the zebrafish embryo. Whole mount stained embryos at the bud stage (a), 3 somites (b), 10 somites (c), 18 h (d), 24 h (e) and 28 h (f) are shown. Arrowheads (d, e) indicate transverse stripes of ZfWnt4 expression in the hindbrain, arrow (f) indicates the anterior-most expression of ZfWnt4 in the floor plate and arrow heads (f) indicate ZfWnt4 expression in the ventro-lateral otic epithelium. Embryos are viewed from the dorsal side with anterior up (a, b, c, d, f) or from the lateral side with anterior up (e). Bar is 50 µm&ig.c:/f

fused in the hindbrain anlage and diverge as they extend caudally along the neural plate (Fig. 7a, b). As somitogenesis continues these stripes fuse at more caudal positions such that by 5–6 somites a single stripe of expression is present along the dorsal neural keel from the hindbrain to a position immediately rostral to the tailbud (Fig. 7a–c). The fusion of these expression domains occurs in an anterior to posterior direction and is consistent with the convergence movements in the neural plate that give rise to the neural keel (Schmitz et al. 1993; Papan and Campos-Ortega 1994). Beginning at the 10 somites stage ZfWnt4 is expressed in the floorplate (Fig. 7d). Formation of the neurocoel, by which the neural rod is transformed into a tube, progresses caudally from the anterior trunk region at 17–18 h (Schmitz et al. 1993) and is accompanied by a decrease in dorsal expression of ZfWnt4 in a rostral to

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caudal fashion such that while expression is still detected dorsally near the tailbud, dorsal expression is no longer present in the anterior trunk at 24 h (Fig. 7e). ZfWnt4 continues to be expressed in the floorplate as its dorsal expression fades. Caudally, floorplate expression extends to a similar level as the remaining dorsal expression in the tail (Fig. 7e); anteriorly, unlike axial and sonic hedgehog, which are expressed along the floorplate from the forebrain/midbrain boundary to the tailbud (Strähle et al. 1993; Krauss et al. 1993), ZfWnt4 expression in the floorplate only extends to a level caudal to the otic vesicles (Fig. 6f). ZfWnt4 expression persists in the floorplate of free swimming fry at 72 h (data not shown). ZfWnt5 Staining of early cleavage stage embryos indicates that ZfWnt5 is maternally deposited in the egg. Strong, ubiquitous expression can be seen in cleavage stage embryos labelled with an antisense but not the sense probe (Fig. 8a and b). Ubiquitous expression is detected throughout gastrulation (Fig. 8c), at the end of which expression is seen to fade. Restricted zygotic expression of ZfWnt5 is first detected in the developing tailbud of early somite stage embryos (Fig. 8d). Also at this stage, weak expression of ZfWnt5 is discernible in the condensing somitic mesoderm. By 10 somites, when the expression in the tailbud has become clearly visible, this weak expression is restricted to the 4–5 posterior-most somites. At 18 h, strong expression is detected in the tailbud and in two to three stripes in the somitic mesoderm immediately rostral to the tailbud (Fig. 8f). Although visible from 18 h, expression of ZfWnt5 in cells corresponding to the future finblade ectoderm along the dorsal and ventral margin of the tailbud is more evident at 24 h (Fig. 8g). The area of expression of ZfWnt5 in the tailbud mesenchyme decreases with the size of the tailbud but the pattern of expression does not change until completion of somitogenesis. In post somitogenesis stages, expression is no longer detected in the mesoderm of the tailbud; very weak expression remains in the ectoderm of the finblade (data not shown). Anteriorly, transcripts are present in tissues ventrolateral to the head from 18 h (Fig. 8e). By 28 h, expression is detected throughout the ventral and lateral aspects of the head, extending posteriorly to the caudal end of the hindbrain (Fig. 9a). This expression corresponds in position to cranial neural crest and paraxial mesoderm which will later migrate to populate the pharyngeal arches. Distinguishing if expression is restricted to one of these two cell types is not possible, however, as it is not detectable before crest cells have migrated from the dorsal aspect of the neural tube and mixed with the paraxial mesoderm. At 48 h transcripts can be detected in the mesenchyme of the pharyngeal arches (Fig. 9b). Expression persists in cells in the arches after the bones of the jaw and gills

Fig. 8a–g ZfWnt5 expression in the zebrafish embryos at stages up to 24 h. Expression is detected in 8-cell stage embryos using antisense (a) but not sense (b) probes. While ubiquitous maternal expression is still detected at 80% epiboly (c), zygotic expression becomes visible during somitogenesis (d–g) and is confined to the mesenchyme of the developing tailbud, to the posterior-most condensing somites and to ventrolateral mesenchyme of the head; 5 somite (d), 18 h (e, f) and 24 h (g) embryos are shown. Embryos are oriented animal pole up (a–c), anterior up (d, e) or anterior left and dorsal up (f, g). Arrowheads indicate expression in the mostposterior condensing somites (d, f, g) or in the ventrolateral mesenchyme of the head (e), the arrow (g) indicates expression in the presumptive fin blade ectoderm. Bar is 350 µm in (a–c), 280 µm in (d, g) and 200 µm in (e, f)&ig.c:/f

have differentiated but is not detected in mature chondrocytes (data not shown). From 24 h, cells that will contribute to the pectoral fin stain weakly for ZfWnt5 (data not shown). Initially, transcripts appear uniform over the fin field but by 30 h, as the size of the finbud increases, cells in the ventral half of the bud display an increased level of ZfWnt5 expression relative to those in the dorsal half (Fig. 9c). The ventral restriction of ZfWnt5 expression in the finbud is more apparent at 36 h; at this stage expression can only be seen in the outermost ventral mesenchymal cells and in the fin-fold (Fig. 9d). By 48 h the distal aspect of the pectoral fin has elongated to form the blade of the fin leaving the majority of the mesenchymal component of the pectoral fin lying proximal, adjacent to the main body axis. At this stage ZfWnt5 expression remains re-

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Fig. 9a–e ZfWnt5 expression in the pharyngeal arch mesenchyme and pectoral finbuds. Expression is detected in ‘pouches’ of mesenchyme ventro-lateral to the head at 28 h (a). By 48 h (b) the mesenchyme expression of ZfWnt5 has condensed to become the pharyngeal arches which will give rise to the bones of the jaw and gills (arrow heads). Expression of ZfWnt5 is detected in the ventral mesenchyme, ventral ectoderm and the fin-fold of the finbuds of whole mount stained and setioned embryos at 30 h (c), 36 h (d) and 48 h (e). Whole mounts (a, b) are oriented anterior left; sectioned embryos (c–e) are oriented with the dorsal neural tube up, in this orientation the dorsal side of the finbud is held close to the neural tube and the ventral, ZfWnt5 expressing side of the finbud facing away from the neural tube. Black arrowheads indicate expression in the pharyngeal arches at 28 h (b) and the fin-fold of the pectoral finbuds (b, d, e); white arrowheads (b) indicate condensing pharyngeal arches. Bar is 350 µm in (a), 300 µm in (b) and 200 µm in (c–e)

stricted to the ventral surface mesenchyme and ectoderm (Fig. 9e). As with the jaw and gills, ZfWnt5 expression is excluded from the differentiated chondrocytes.

Discussion In this study we report the isolation of cDNA clones from the zebrafish, Danio rerio, whose putative amino acid sequences display significant homology to the murine Wnt2, 4 and 5A/B proteins. The developmental functions of these zebrafish genes and the biochemical pathways by which they accomplish these functions remain to be determined. Analysis of the expression patterns of these genes suggests, however, that they participate in a wide range of processes including neurogenesis, somitogenesis and organogenesis. Expression of MWnt-2 (murine int-1-related protein, m-irp) is found in the mesoderm of the pericardium, the umbilicus and allantoic mesoderm and ventral lateral mesoderm surrounding the umbilical vein of the developing mouse embryo (McMahon and McMahon 1989), sites which either do not express ZfWnt2 or do not have a directly homologous structure in the zebrafish. Whereas

in mouse the allantois is solely mesodermal, in birds and reptiles it forms as an extension of the gut and includes both mesoderm and endoderm. It is interesting to speculate that the expression of ZfWnt2 associated with an outpocketing of the gut in the viscera of the developing zebrafish embryo may correspond to an allantois-like organ. Although partial cDNA fragments of Xenopus Wnt2 have been isolated, the expression of XWnt2 has not been reported. RNAse protection analysis of Xwnt-2, however, indicates that its temporal expression resembles that of ZfWnt2 (Wolda and Moon 1992). While comparison of the amino acid sequence encoded by ZfWnt4 and ZfWnt5 with Xenopus and mouse shows a higher level of conservation than similar comparisons between members of the Wnt2 subfamily, perhaps more remarkable is the degree of similarity in the spatial and temporal deployment of the Wnt4 and Wnt5 subfamilies in the developing embryos of lower and higher vertebrates (Gavin et al. 1990; McGrew et al. 1992; Dealy et al. 1993; Moon et al. 1993; Parr et al. 1993; Stark et al. 1994). It may be overly simplistic, however, to interpret the conservation of spatial and temporal expression of these two genes between the various species as an indication of functional significance. For example, signals from the floorplate are required for patterning of the various neuronal subpopulations adjacent to it (Yamada et al. 1993). The expression of murine, Xenopus and zebrafish Wnt4 in the floorplate could suggest that Wnt4 protein may play a role in this patterning (Parr et al. 1993; McGrew et al. 1992 and this report); however, no overt phenotype is detected in mouse embryos homozygous for targeted mutations in Wnt4 (Stark et al. 1994). Interestingly, the spatial and temporal expression of chicken Wnt4 is also highly conserved except with respect to the floorplate, which lacks expression (Yoshioka et al. 1994; Hollyday et al. 1995). Whereas conservation of expression does not provide direct evidence of a developmental function for a particular gene product, differences in expression patterns of homologous genes may provide us with insights into differ-

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ences in the developmental strategy employed by different organisms. Wnt4 is expressed during the development of the mouse metanephros and the chick mesonephros and metanephros (Stark et al. 1994). Mouse pups homozygous for targeted mutations of the Wnt-4 gene die within 24 h of birth and display agenic kidneys (Stark et al. 1994). Analysis of the development of the kidneys of these embryos has shown that although initial condensation of the nephric mesenchyme occurs normally the condensations fail to form pretubular aggregates. The lack of expression of ZfWnt4 in the embryonic zebrafish pronephros, as defined by the expression of pax[zf-b]/Pax-2 (Krauss et al. 1991; Püschel et al. 1992) and the ectodermal rather than mesodermal expression of ZfWnt4, is suggestive that, unlike murine Wnt4, ZfWnt4 is not required for the formation of the nephric system in the fish. A more attractive explanation, however, suggests that Wnt4 signalling is required for maturation of the embryonic pronephros, but that the signalling is supplied by the neural tube, which expresses ZfWnt4 and which closely flanks the pronephros. Interestingly, it has long been known that explants of neural tube are capable of inducing condensation of nephric tubules in combination with presumptive nephritic mesenchyme in vitro (Grobstein 1955). Amino acid sequence comparison indicates that ZfWnt5 is more closely related to Wnt5B than to Wnt5A. Conversely, the expression pattern of ZfWnt5 is similar, if not identical to the expression patterns reported for Wnt5A subfamily members (Gavin et al. 1990; Dealy et al. 1993; Moon et al. 1993; Parr et al. 1993). The duplication giving rise to Wnt5A and Wnt5B predates the common ancestor of the teleosts and tetrapods and suggests that the zebrafish should possess a member of both types (Sidow 1992). In the context of the work presented here, however, it is impossible to determine if ZfWnt5 is the orthologue of Wnt5A or Wnt5B. Unlike murine and chick Wnt5A, which display a proximal-distal gradient from low levels proximally to maximal levels distally in both the mesenchyme and ectoderm of the limb (Gavin et al. 1990; Dealy et al. 1993; Parr et al. 1994), ZfWnt5 appears to be uniform in its expression in the ventral mesenchyme and ectoderm of the pectoral fin during development. It has been hypothesised that the graded expression of Wnt5a along the chick limb may correspond to and, therefore, be important for the development of the three proximodistal segments of the limb (Dealy et al. 1993). In light of the apparent uniform expression of ZfWnt5 in the zebrafish pectoral fin one could speculate that as the zebrafish pectoral fin is not segmented in the proximodistal axis the graded expression of ZfWnt5 is not required for the subdivision of the prospective limb. Furthermore, expression of ZfWnt5 in the fin-fold is reminiscent of the expression of wg at the margin of the wingblade in Drosophila. By analogy, the outgrowth of the pectoral fin in the zebrafish may require ZfWnt5 expression in a similar fashion to the outgrowth of the wingblade in Drosophila, a process for which wg is required (Williams et al. 1993). Confirmation of these speculations, however, awaits the disrup-

tion of the gene in mouse or the induction of a ZfWnt5 mutation in the zebrafish. A Wnt5 homologue, DWnt-5, has been characterised and found to be expressed in the limb primordia of the head and thoracic segments of developing Drosophila embryos (Eisenberg et al. 1992; Russell et al. 1992). The expression of DWnt-5 in the limb primordia requires the function of the homeobox gene Distalless, Dll (Eisenberg et al. 1992), of which several zebrafish homologues have also been isolated (Akimenko et al. 1994). Interestingly, the expression of the zebrafish dlx genes, dlx2, dlx3 and dlx4, overlaps extensively with the expression of ZfWnt5 suggesting conservation of the regulatory hierarchy demonstrated in Drosophila. Again, by analogy with Drosophila, ZfWnt genes may act through a feed back loop similar to that in which wg expression maintains en expression and is itself in turn maintained by hedgehog (hh) expressed in the en expressing cells. A vertebrate parallel of this loop has already been demonstrated; mutations of the murine Wnt-1 locus lead to a loss of midbrain and cerebellar structures, a phenotype that correlates with the stepwise loss of engrailed-expressing cells (McMahon et al. 1992). Three engrailed (eng 1–3; Ekker et al. 1992) genes are expressed during zebrafish development, primarily in a nested set at the midbrain/hindbrain boundary but also in specific neurons in the spinal chord, in the ventral ectoderm of the pectoral fin bud, in muscle pioneers in the trunk somites and in specific jaw muscles (Hatta et al. 1990; Hatta et al. 1991; Ekker et al. 1992). Given these domains of expression it is possible that cells that express eng-1, 2 and 3 may interact with cells expressing ZfWnt genes, specifically with cells expressing ZfWnt4 at the forebrain/midbrain junction and ZfWnt5 in the mesenchyme of the fin buds. &p.2:Acknowledgements We thank Dr. Kai Zinn for the library from which the Wnt cDNA clones were isolated, Jenny Corrigan for cutting cryostat sections and Emma Burns and Stephan Massey for keeping the aquarium in running order and the IGBMC photographers for help with the artwork. We should also like to thank our colleagues at the Developmental Biology Unit (DBU) for many helpful discussions. PB was supported by an ICRF bursary and a grant from the Adrian Darby Charitable Trust. US was a Boehringer Ingelheim fellow for the initiation of this project and was subsequently funded by the Deutsche Forschungsgemeinschaft (DFG). This work was supported by the Imperial Cancer Research Fund.

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Note added in proof. During the preparation of this manuscript, the isolation of zebrafish wnt4 was reported (Ungar et al. 1995).

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