Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways

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1 Development 121, (1995) Printed in Great Britain The Company of Biologists Limited Zebrafish wnt8 and wnt8b share a common activity but are involved in distinct developmental pathways Gregory M. Kelly 1, Penny Greenstein 2, Deniz F. Erezyilmaz 1 and Randall T. Moon 1 1 Howard Hughes Medical Institute, Department of Pharmacology, and 2 Division of Medical Genetics, University of Washington School of Medicine, Seattle, Washington 98195, USA SUMMARY The specification of the vertebrate body plan is dependent on numerous signaling molecules, including members of the Wnt family. We have identified two zebrafish wnt8 paralogs related to Xwnt-8B and Xwnt-8, respectively. A RT-PCR assay demonstrated that wnt8 is expressed maternally, with transcripts detected throughout embryogenesis, whereas wnt8b transcripts were first detected during late gastrulation. The wnt8 transcripts at 50% epiboly are spatially restricted to those cells at the blastoderm margin, overlying gsc-expressing cells in the axial hypoblast. During late gastrulation, wnt8 was no longer detected in the marginal cells at the dorsal midline and by mid-segmentation, transcripts were found in the presumptive tail bud. In contrast, wnt8b expression is spatially restricted to prospective neuroepithelium, and later to neural-specific structures. Overexpression of both wnts results in two major phenotypes: radialized embryos and embryos with anterior defects. These phenotypes were preceded by significant changes in the spatial expression patterns of gsc and ntl transcripts, reminiscent of activities of Xwnt-8 in Xenopus, and consistent with a role for wnt8 in the specification or patterning of mesoderm. Key words: Danio rerio, wnt, krox20, gsc, pax, snail1, spadetail, cyclops, mesoderm induction, pattern formation INTRODUCTION The events leading to the ultimate specification of the vertebrate body plan are dependent on the interaction between numerous signaling molecules from several multigene families, including the Wnts (reviewed by McMahon, 1992; Moon, 1993). The developmentally regulated Wnt genes encode secreted, glycosylated proteins that act at the cell surface and/or in the extracellular matrix to mediate cell-cell signaling (Bradley and Brown, 1990; Papkoff and Schryver, 1990; Parkin et al., 1993). The majority of Wnt genes are expressed in discrete, sometimes overlapping regions in the developing central nervous system (CNS) (reviewed by McMahon, 1992; Moon, 1993), yet expression is not entirely restricted to neural structures. For example, Xwnt-8 is expressed in the marginal zone and vegetal hemisphere of the Xenopus embryo, with the exception of cells in the Spemann organizer field (Christian et al., 1991; Smith and Harland, 1991; Christian and Moon, 1993). In vivo assays have led to the suggestion that there may be at least two functionally distinct classes of Wnt activity (Moon et al., 1993a; Wong et al., 1994; Du et al., 1995). We have identified two genes in zebrafish that are related to Xenopus Xwnt-8, and which are expressed in unique non-overlapping patterns during embryogenesis. This expression is perturbed in spadetail (spt) and cyclops (cyc) mutants. Genes closely related to Xwnt-8 have been isolated from Xenopus (Xwnt-8B, Wolda and Moon, 1992), and from chicken (Cwnt- 8C, Hume and Dodd, 1993; Cwnt-8B, A. McMahon, personal communication), but none have been reported with the unique pattern of expression in the CNS that we report for wnt8b. To begin to investigate the activities and functions of each zebrafish wnt in embryogenesis, we injected synthetic wnt8 and wnt8b mrna in both zebrafish and Xenopus embryos. Overexpression in zebrafish leads to changes in expression of gsc and ntl in the gastrula, and subsequently to anterior defects or radialized or bustled embryos. MATERIALS AND METHODS Cloning and sequencing wnt8 and wnt8b As screening zebrafish cdna libraries with two partial length (450 bp) zebrafish wnt cdnas with predicted amino acid sequences related to Xenopus Xwnt-8 (Christian et al., 1991) and Xwnt-8B (Wolda and Moon, 1992) failed to yield positive clones, the 5 AmpliFINDER kit for the rapid amplification of cdna ends (RACE) (Clontech, Palo Alto, CA), was then used to clone the complete 5 coding sequences of these Xwnt-8 paralogs. To obtain the coding sequence at the carboxy terminus of the zebrafish wnt8 gene, we screened a zebrafish gastrula λ-zap cdna library (a gift from K. Helde and D. Grunwald, University of Utah School of Medicine). Positive clones were isolated and the rescued phagemids were sequenced from both directions. Sequence at the carboxy terminus of wnt8b was obtained from a zebrafish wnt cdna (a gift from R. Riggleman, Washington State University), containing partially overlapping sequence with our original zebrafish wnt8b PCR clone. In summary, amino acids 1 to

2 1788 G. M. Kelly and others 298 of wnt8, and 1 to 265 of wnt8b were from PCR products, and the remainder of the coding sequences were from λ clones. RT-PCR Total RNA from embryos at select developmental stages was isolated using the TRIzol reagent (Gibco BRL, Gaithersburg, MD). Firststrand cdna was synthesized (Life Science, Inc., St. Petersburg, FL) using total RNA from approximately 2.5 embryos. Controls omitted the reverse transcriptase enzyme. For PCR, 1 µl of first strand cdna from each stage was used in a standard PCR reaction buffer containing 1.5 mm MgCl 2 and 1 µm of each forward and reverse oligonucleotide primer. The primers used were: wnt8, forward, 5 -CAAGCAAGGAAGTTGGAGATGG-3, reverse, 5 -CGCATT- TGACTGTGCAGCAC-3 wnt8b, forward, 5 -CGGGATCCTCTC- TCAGGATGTTC-3, reverse, 5 -CACTGCTGGAGTAGATC-3. The PCR conditions were: 1 cycle of 95 C-5 minutes, 50 C-5 minutes and 72 C-30 minutes; 35 cycles of 95 C-1 minute, 50 C-2 minutes and 72 C-4 minutes; and a final extension at 72 C for 16 minutes. In a separate experiment, aliquots of the PCR mixture were removed after 36, 40 and 44 cycles to determine that the fragments being generated were in the exponential phase of amplification. Similarly, as a PCR control to ensure that the bands being amplified corresponded to their specific genes, the pt 7Ts-wnt8 or pt 7Ts-wnt8b cdnas (see below) were used as templates with their respective primers in the PCR. These controls were then co-electrophoresed with samples from the developmental series. The constitutively expressed zebrafish max gene (Schreiber-Agus et al., 1993), was used as a PCR control. The max primers used were: forward, 5 -GCCGAAGAAT- GAGCGACAAC-3, reverse, 5 -CTGCTGTGTGTGTGGTTTTTC- 3. All samples were electrophoresed on 5% nondenaturing polyacrylamide gels, then exposed to X-ray film. Whole-mount in situ hybridization Procedures for hybridizing and visualizing digoxigenin (DIG)-labeled RNA probes was reported previously (Kelly et al., 1993). The pgem- 4Z-wnt8b and pgem-4z-wnt8 cdnas (described above) were used as templates to generate DIG-labeled probes. Zebrafish pax2 (Krauss et al., 1991a), snail1 (Thisse et al., 1993) and krox20 (Oxtoby and Jowett, 1993) were cloned using the PCR and oligonucleotide primers flanking the predicted coding sequences. Zebrafish gsc and ntl cdnas were generously provided by J. Postlethwait, University of Oregon and by S. Schulte-Merker, NIMR, London, respectively. After visualizing the probes using the NBT-BCIP color reaction, embryos were fixed in methanol, placed in 2:1 benzyl benzoate/benzyl alcohol and examined with Nomarski Differential Interference Contrast optics. Expression analysis Coding sequences were cloned into the RNA expression vector pt 7Ts (a gift from P. Krieg, University of Texas, Austin), or into the DNA expression construct pcs2+ (a gift from D. Turner, R. Rupp, J. Lee and H. Weintraub, Fred Hutchinson Cancer Research Center, Seattle). The mmessage mmachine kit (Ambion, Austin TX), was used to transcribe synthetic capped RNA. Synthetic wnt8, wnt8b, or prolactin RNAs were injected into Xenopus embryos as outlined by Moon and Christian (1989). Similarly, wnt8, wnt8b and β-galactosidase RNAs or CS2+wnt8b DNA, were injected into 1- to 4-cell-stage zebrafish embryos as outlined in Westerfield (1989). Embryos were cultured in Instant Ocean saline then fixed at select developmental stages. RESULTS Cloning of zebrafish wnt8 and wnt8b We have isolated two zebrafish genes related to Xenopus Xwnt- 8 (and chicken Cwnt-8C) and to Xenopus Xwnt-8B, respectively (Fig. 1). The overall degree of similarity and identity Fig. 1. Alignment of the deduced amino acid sequences of the fulllength wnt8 and wnt8b cdna clones. Gaps were introduced for optimal alignment. In the alignment, solid bars between residues indicates identity, and two dots represents a change to a similar amino acid. Asterisks denote the positions of the conserved cysteine residues. between wnt8, wnt8b, Xwnt-8 (Christian et al., 1991), Xwnt-8B (Wolda et al., 1992), Cwnt-8C (Hume and Dodd, 1993) and Cwnt-8B (A. McMahon, personal communication) is outlined in Table 1. Sequences analyzed by the maximum-likelihood method of phylogenetic inference indicate that wnt8 and Cwnt- 8C are homologs of Xwnt-8, whereas wnt8b is a distinct gene most closely related to Xwnt-8B (A. Sidow, personal communication, see Sidow, 1992). Temporal expression of wnt8 and wnt8b during embryogenesis The embryonic expression of wnt8 and wnt8b was determined by a RT-PCR assay (Fig. 2), with comparable results from two separate sets of RNA. wnt8 is expressed maternally since the expected 334 nucleotide band amplified from 2- to 4-cell cdna was detected only in the presence of reverse transcriptase (Fig. 2A). At the 128- to 256-cell stage, at a time prior to midblastula transition (Kane and Kimmel, 1993), there is an Table 1. Amino acid comparisons between zebrafish wnt8 and wnt8b and other Wnt-8 paralogs Zebrafish wnt8 Zebrafish wnt8b Xenopus Xwnt-8 73(83) 65(77) Xenopus Xwnt-8B* 61(74) 85(95) Chicken Cwnt-8B* 1 61(74) 88(94) Chicken Cwnt-8C 73(83) 69(79) Zebrafish wnt (78) *Partial sequence. 1 A. McMahon, personal communication. Numbers represent the percent identities. Percent similarities are indicated in parentheses.

3 wnt8 and wnt8b expression in zebrafish embryos 1789 Fig. 2. RT-PCR analyses of the temporal expression patterns of wnt8, wnt8b and max during normal embryogenesis. RT-PCR was performed on cdnas synthesized from RNA isolated from select developmental stages. Lanes designated plus or minus refer to the presence or absence (control) of reverse transcriptase in the firststrand cdna synthesis. (A) wnt8 expression, first detected at the 2- to 4-cell stage and corresponding to the presence of maternal transcripts, continues throughout embryogenesis. (B) wnt8b transcripts are not maternal and, unlike wnt8, they are first detected at a time corresponding to the late gastrula stage. (C) The relative consistency in the max signals throughout embryogenesis indicates that approximate equivalent amounts of cdnas were being assayed in the wnt8 and wnt8b PCR analyses. apparent decline in wnt8 expression. Following midblastula transition, wnt8 expression increases and transcripts are detected throughout gastrulation and segmentation and through 24 hours postfertilization. In contrast, wnt8b expression is first detected in the late gastrula stage, at approximately 8-9 hours postfertilization (Fig. 2B), and persists throughout segmentation. The increase in wnt8b expression by the 12- to 16-somite stage is followed by a sharp decrease at the prim (primordium of the lateral line) 3-5 stage (Fig. 2B). The temporal expression pattern of the constitutively expressed max gene is illustrated in Fig. 2C. Spatial expression of wnt8 and wnt8b during normal embryogenesis wnt8 At 40% epiboly no wnt8 was detected by in situ hybridization but by 50% epiboly, the onset of gastrulation (Westerfield, 1989), wnt8 was observed in cells at the blastoderm margin (Fig. 3A). With the morphological appearance of the embryonic shield, which is the site of the organizer (Stachel et al., 1993), wnt8 continues to be expressed around the margin of the embryo. At approximately 75-80% epiboly (8-8.5 hours), wnt8 is no longer detected in the dorsal midline (triangles, Fig. 3B). Dorsally, at approximately 90-95% epiboly or hours, the wnt8 signal has become enriched on either side of the paraxial mesoderm (open arrows, Fig. 3C). The loss of the wnt8 signal on the ventral side and the reduction of the signal in more lateral regions is apparent at late gastrulation (black arrows, Fig. 3C). wnt8 continues to be expressed in a dorsolateral semicircle around the margin during yolk plug closure. At early segmentation (approximately hours), the wnt8 signal is prominent in the caudal axial and paraxial mesoderm surrounding the presumptive tailbud (open arrow, Fig. 3D). wnt8 is also detected in the caudal and lateral margin of the coalescing embryonic shield (black arrows, Fig. 3D). With the continued convergence of cells toward the dorsal midline, the expression of wnt8 in embryos at the 1- to 2- somite stage (approximately hours), remains prominent in the presumptive tail bud (open arrow, Fig. 3E) and to a lesser extent in the lateral and caudal areas surrounding it (black arrow, Fig. 3E). In addition, we were able to detect a weak wnt8 signal in adaxial cells (see Thisse et al., 1993), but this was only in the extreme caudal-most region of the embryo (Fig. 3E). By mid-segmentation, hours, wnt8 is detected in the tip of the tailbud (Fig. 3F). To determine the relationship between the wnt8-expressing cells in the margin and cells in the organizer, it was first necessary to identify the cells that will contribute to the shield. Embryos at mid-to-late gastrula stages were hybridized to both antisense wnt8 and gsc probes. During early gastrulation, gsc expression appears as an arc in cells near the margin on the prospective dorsal side of the embryo (Stachel et al., 1993). In double-labeled embryos, wnt8 is seen in cells more ventral but remaining juxtaposed to those that have involuted and now express gsc (data not shown), thus indicating that the wnt8 signal is confined to marginal cells that have not involuted. As the gsc pattern continues to narrow as cells migrate toward the animal pole, the wnt8 signal is still evident around the blastoderm margin, albeit the expression on the extreme ventral side had began to diminish (data not shown). This pattern remains until approximately 75-80% epiboly, when the gsc-positive cells in the anterior shield of the axial hypoblast are now at their most anterior position (see also Stachel et al., 1993). At this time, there is a clear distinction between the wnt8 and gsc signals and more important, wnt8 is no longer detected in the dorsal midline (open arrows, Fig. 3G). The absence of the wnt8 signal overlying the axial mesoderm is evident in Fig. 3H (arrows). The expression pattern of wnt8 in the lateral and caudal regions of the embryo at hours (Fig. 3D) is similar to the pax2 expression pattern first reported by Krauss et al. (1991a,b). The expression pattern of pax2 in the caudal region of an embryo at hours is illustrated in Fig. 3I. Note that, although the patterns are similar between pax2 (Fig. 3I) and wnt8 (Fig. 3D), the pax2 signal continues in more rostral regions. Similarly, wnt8 expression throughout embryogenesis parallels very closely that demonstrated by snail1 (see Thisse et al., 1993; Hammerschmidt and Nüsslein-Volhard, 1993). During mid-to-late gastrulation, snail1 expression in the embryonic shield is down-regulated prior to wnt8. Unlike wnt8, snail1 expression is seen in more rostral positions particularly along the trunk (data not shown). At early segmentation, the expression patterns of snail1 (Fig. 3J) and wnt8 (Fig. 3E) are similar in the tailbud yet the snail1 signal clearly exists in more rostral regions. wnt8b wnt8b exhibits a spatial pattern of expression distinct from that

4 1790 G. M. Kelly and others Fig. 3. Whole-mount in situ hybridization analyses of the spatial localization of wnt8 during early gastrulation to midsegmentation. (A) Embryo at 50% epiboly illustrating the wnt8 signal in cells at the blastoderm margin; animal pole is to the top. (B) Dorsal view (animal pole to the top), of a whole-mount embryo at approximately 75-80% epiboly. Note the absence of the wnt8 signal in the dorsal midline (triangles). (C) Vegetal pole view of a whole-mount embryo at late gastrulation (90-95% epiboly); dorsal is to the top. The wnt8 signal, no longer evident on the ventral side and downregulated on the lateral sides (black arrows), becomes enriched on either side of the dorsal axial mesoderm (open arrows). (D) During early segmentation (approximately hours), the wnt8 signal, seen in this dorsal view of a whole-mount embryo where anterior is to the top, is confined to the lateral and caudal margin of the embryonic shield (black arrows), and in the area surrounding the presumptive tailbud (open arrow). (E) Vegetal pole view illustrating the wnt8 pattern of expression at the posterior end of an embryo at the 1- to 2-somite stage. Note that the signal is now restricted to the tailbud and the cells immediately adjacent to it (open arrow). (F) At hours, the wnt8 signal is obvious at the tip of the tail. (G) Dorsal view of a whole-mount embryo at approximately 80% epiboly, illustrating the expression pattern of wnt8 and gsc. The expression of gsc, in cells of the anterior embryonic shield (es), delineates the wnt8- negative area corresponding to the axial hypoblast (open arrows). (H) Higher magnification view from the animal pole clearly demonstrates that by late gastrulation, wnt8 is no longer expressed in cells in the dorsal midline (triangles). (I) Dorsal view with anterior to the top of a whole-mount embryo stained for pax2 expression. Note that the pax2 signals in the lateral and caudal margins of the shield (arrows), are similar to those in a comparable stage embryo stained for wnt8 (Fig. 3D). (J) The expression pattern of snail1 in a whole-mount embryo at a stage comparable to the one in Fig. 2E. Although wnt8 (Fig. 3E) and snail1 (Fig. 3J) are expressed in the tailbud, only snail1 is expressed in the more anterior regions flanking the midline axis. Abbreviations: a, axial mesoderm; n, notochord; tb, tailbud. Scale bar (A-E, G-J), 55 µm; (F), 45 µm. of wnt8. Whole-mount in situ hybridization demonstrates that cells in the prospective neuroepithelium begin to express wnt8b by the mid-to-late gastrula stage (Fig. 4A). The signal, which appears initially in two stripes but not connected over the dorsal midline, is in the area corresponding to the future midbrain-hindbrain boundary (MHB) (Kelly and Moon, 1995). By the 2- to 3-somite stage, the extent of the apposing wnt8b signals has been reduced toward the dorsal midline and are now joined as one continuous band (large arrow, Fig. 4B). Signals flanking the midline and continuous with the bulk of the wnt8b signal at the prospective MHB extend in a caudal direction (arrowheads, Fig. 4B). By mid-segmentation, the wnt8b signal at the MHB has narrowed along the transverse axis into a prominent stripe (Fig. 4C). The signal at the MHB extends for a short distance in a rostral direction, over the dorsal midbrain. In addition, wnt8b is also detected in the forebrain, specifically at the forebrain-midbrain boundary, the epiphysis (e, Fig. 4C) and in the ventral floor (asterisk, Fig. 4C). Three additional areas of wnt8b expression are detected in the hindbrain (rhombomeres (r) 1, 3, 5, Fig. 4C). In the lateral view of the whole mount in Fig. 4C, note that, in the two caudal-most hindbrain stripes, the majority of the wnt8b signal appears to be in the dorsal region of the keel. The area in the middle of the keel is devoid of any signal. In a dorsal view at the same stage (Fig. 4D), it is evident that the wnt8b signals in r3 and r5 are more distinct than that in r1.

5 wnt8 and wnt8b expression in zebrafish embryos 1791 Fig. 4. The spatial localization of wnt8b transcripts during normal zebrafish embryogenesis. All embryos are oriented anterior to the top and dorsal facing up, except C, which is a lateral view of the embryo in D. (A) At approximately 90% epiboly, wnt8b signals appear in two wing-like patterns flanking the dorsal midline. (B) At the 2- to 3- somite stage, the wnt8b signal has joined over the dorsal midline (large arrow), and is continuous with signals at the lateral edge of the body axis (small arrows). (C) Lateral view of an embryo at approximately 16 hours, illustrating the wnt8b signals at the future midbrain-hindbrain boundary, the epiphysis, the ventral floor of the forebrain (arrow from asterisk), and in rhombomeres (r) r1, 3 and 5. (D) Dorsal view of the whole mount in C, demonstrates that wnt8b expression is contiguous over the entire width of the rhombomere. wnt8b expression in the forebrain is not obvious in C due to the curvature of the embryo, but close examination in these more rostral regions (E) reveals that wnt8b is expressed at the forebrain-midbrain border and in a discrete layer of the eye (arrows). (F) At approximately 19 hours, wnt8b signals are also detected in a limited region of the optic stalk and in the ventral forebrain (arrow from asterisk). (G) Whole-mount embryo having been hybridized to both the wnt8b and krox20 antisense DIG-labeled RNA probes. The wnt8b signals are confined to the prospective rostral neuroepithelium, in the area corresponding to the future midbrain-hindbrain boundary, and the two thin stripes denoted by the open arrows represent the krox20 expression in prospective r3. (H) To compare directly the relationship between the expression of wnt8b and krox20 in specific rhombomeres, embryos were hybridized simultaneously to wnt8b and krox20 antisense probes. Only the characteristically weak wnt8b signal in r1 can be detected in the double in situ; signals in r3 and r5 are not resolved due to the robust krox20 signal. (I) Dorsal view of a whole-mount embryo at a stage comparable to the embryo in F, illustrating the prominent pax2 expression in the optic stalk and at the midbrain-hindbrain boundary. Abbreviations: e, epiphysis; fmb, forebrain-midbrain boundary; mhb, midbrain-hindbrain boundary; os, optic stalk; r, rhombomeres. Scale bar (A,B), 55 µm; (C-F, H-I), 45 µm; (G), 65 µm. The rostral extent of the wnt8b signal in embryos at 16 hours is shown in detail in Fig. 4E. The most obvious features are the large wnt8b-positive regions at the forebrain-midbrain boundary (FMB), the MHB and in the retinal layer of the developing eye (arrows, Fig. 4E). By approximately 19 hours and with the appearance of the brain ventricles, the wnt8b signal clearly delineates the MHB (Fig. 4F). In more anterior regions, wnt8b continues to be expressed at the FMB, in the ventral floor of the forebrain (arrow from asterisk, Fig. 4F), and now in the optic stalk. In 26 hour embryos, wnt8b continues to be expressed in the forebrain and in the caudal midbrain (data not shown). In the hindbrain, the expression continues in r3 and r5 but not in r1, possibly due to the presence of the hindbrain ventricle (data not shown). By hours, no expression was detected by whole-mount in situ hybridization. Specific markers were used to help orient the expression of wnt8b. The expression of wnt8b at the future MHB in the midto-late gastrula precedes the onset of krox20 expression in r3

6 1792 G. M. Kelly and others and r5 (data not shown). Double in situ hybridization analysis with wnt8b and krox20 probes shows the relationship between wnt8b at the prospective MHB and krox20 (open arrows, Fig. 4G) in the rostral neuroepithelium, specifically r3 (Oxtoby and Jowett, 1993). The fact that wnt8b expression demarcates the future MHB before krox20 is detected in r3 and r5 suggests that the specification of the rostral CNS proceeds in an anteroposterior orientation. To test whether wnt8b was expressed in specific rhombomeres, embryos at similar stages were hybridized to both wnt8b and krox20 probes, the latter gene of which is known to localize to r3 and r5 (Oxtoby and Jowett, 1993). Since each probe alone hybridizes to r3 and r5, and since Fig. 4H demonstrates that wnt8b and krox20 probes together specifically label r3 and 5 (arrrows), then the two genes must be co-expressed. Interestingly, the wnt8b pattern in the optic stalk (Fig. 4F) and at the MHB is comparable to but not identical to that seen for pax2 (Fig. 4I), as the pax2 signal extends the entire length of the optic stalk (previously reported by Krauss et al., 1991a; Mikkola et al., 1992; Püschel et al., 1992). wnt gene expression in spadetail and cyclops mutants spadetail We examined the expression of wnt8 in spt mutants (Fig. 5), which exhibit perturbed mesoderm migration (Kimmel et al., 1989; Ho and Kane, 1990), to test the prediction that wnt8 expression would be affected. No appreciable difference between the staining pattern of wnt8 compared to snail1 could be detected in wild-type or spt embryos at 50% epiboly (data not shown; see also Thisse et al., 1993). During late gastrulation and concurrent with the loss of the wnt8 signal in the dorsal midline, it became apparent which embryos were normal and which ones exhibited the characteristic spt defects (see Kimmel et al., 1989; Ho and Kane, 1990). An obvious difference in spt embryos at approximately 70-80% epiboly was the increase in the space between the wnt8 signals flanking the dorsal midline (data not shown). By comparison, Thisse et al. (1993) reported that snail1, which normally borders the lateral edges of the embryonic shield during late gastrulation, is absent in spt embryos. The grossly enlarged tailbud, which is known to contain paraxial mesoderm that failed to converge properly during gastrulation, is characteristic of the spt phenotype (Kimmel et al., 1989; Ho and Kane, 1990). At hours, a prominent wnt8 (Fig. 5A) and snail (Fig. 5B) signal was detected in the enlarged tailbud of spt mutants. cyclops The cyc mutation is known to affect ventral midline axial structures and embryos lack a normal floor plate and much of the ventral forebrain (Hatta et al., 1991). No changes in either the wnt8b or krox20 expression patterns were seen in cyc embryos at late gastrula (data not shown). By approximately 18 hours and with the cyc phenotype morphologically visible, we were able to detect changes in the wnt8b expression pattern. The normal ventral limit of the wnt8b signal at the FMB was reduced in cyc mutants and the signal that is present in the optic stalk of the wild-type normal embryos (lower arrow, Fig. 5C) is absent in the cyc mutants (arrow from asterisk, Fig. 5D). Staining in the epiphysis is unaffected by the cyc mutation (arrow, Fig. 5D). Although the expression of wnt8b and pax2 overlaps significantly in the optic stalk (described above), they do not overlap directly since in cyc mutants the wnt8b signal in the optic stalk is completely absent (arrow from asterisk, Fig. 5D), whereas pax2 expression is affected but not abolished (see Hatta et al., 1994). No visible effects on the expression of either wnt8b or krox20 due to cyc mutation were detected posterior to the MHB (data not shown). Ectopic expression of wnt8 and wnt8b Injection of low doses of RNA and constitutive promoter constructs lead to anterior defects We were next interested in testing whether overexpression of Fig. 5. Expression pattern of wnt8 (A) and snail1 (B) in the tailbud of spadetail mutant embryos, and wnt8b in the rostral CNS of wild-type (C) and cyclops b16 mutant (D) embryos. Anterior is to the left and dorsal is to the top in all photomicrographs. (A) For wnt8, the increase in the relative intensity of the signal compared with that in a wild-type embryo (Fig. 3F), can be attributed to the snail1-positive cells (Fig. 5B) that failed to converge properly during gastrulation. (C) In wild-type embryos at approximately 24 hours postfertilization, wnt8b is expressed in the epiphysis (top arrow), the optic stalk (lower arrow), the ventral floor of the forebrain, just caudal to the optic stalk signal, and at the midbrain and hindbrain junction. (D) In homozygous cyc / mutants, wnt8b expression is unaffected in the epiphysis (top arrow) and at the boundary behind midbrain and hindbrain. Note the loss of the signal in the optic stalk (arrow from asterisk) and in the ventral floor of the forebrain just caudal to it. Abbreviations: h, hindbrain; m, midbrain; n, notochord. Scale bar (A,B), 45 µm; (C,D), 55 µm.

7 wnt8 and wnt8b expression in zebrafish embryos 1793 wnt8 or wnt8b would affect early zebrafish development. Phenotypes resulting from the injection of wnt8, wnt8b and β-galactosidase RNA or wnt8b expressed from a DNA construct into 2- to 4-cell embryos are summarized in Table 2. When either 0.01 or 0.1 ng of wnt8 or wnt8b RNA was injected into zebrafish embryos, the primary defect, observed in 55-79% of embryos, was in dorsoanterior structures (Table 2). The majority of these embryos failed to develop either one or both eyes (open arrow, Fig. 6C), and there was a loss of the prominent midbrain-hindbrain boundary compared to uninjected embryos (MHB, Fig. 6A), or to embryos injected with 1 ng of β-galactosidase RNA (MHB, Fig. 6B). In addition, eyes that did develop in the wnt8 or wnt8b injected embryos were smaller than those in uninjected (Fig. 6A) or β-galactosidase-injected controls (Fig. 6B) (data not shown). Despite the obvious perturbations in the fore-, mid- and anterior hindbrain structures, the otocysts (O, Fig. 6C), which form at the level of r5, developed normally (see O, Fig. 6A, B). The loss or reduction in the size of the eyes attributed to perturbations in forebrain structures was observed in 75% of embryos injected with a plasmid driving wnt8b expression from a constitutive promoter, and is significantly greater than was observed in embryos injected with a control plasmid (12%; Table 2). Injection of higher doses of RNA leads to bustled and radialized embryos The most prominent phenotypes (60-68%; Table 2) associated with injecting 0.2 ng of either wnt8 or wnt8b RNA into zebrafish embryos can be grouped in a continuum of bustled to radialized phenotypes previously described for embryos treated with lithium (see Stachel et al., 1993). Embryos treated with these higher doses of wnt8 and wnt8b appear to have a single often broader axis, they lack anterior structures including eyes, and they develop in a twisted fashion above the yolk (arrows, Fig. 6F). These embryos do form a recognizable single notochord exhibiting the characteristic vacuolated arrangement (data not shown). The loss of eyes and defects in the developing forebrain described above was also observed at the higher doses of Fig. 6. Morphological comparisons between uninjected, β-galactosidase-rna-injected and wnt8-rna-injected zebrafish embryos at approximately 30 hours postfertilization. Embryos in (A-C) are oriented anterior to the top and dorsal to the left. (A) The prominent eye, otocyst and midbrain-hindbrain boundary are evident in this lateral view of an uninjected embryo. (B) Note the apparent normal morphology of these structures in a lateral view of an embryo having been injected with approximately 1 ng of β-galactosidase RNA. (C) In embryos injected with approximately 0.1 ng of wnt8 RNA, the region where the eye should have formed (arrow) and the regions flanking the midbrain-hindbrain boundary are noticeably affected, but the otocyst appears to have developed normally. (D) Dorsal view of an uninjected embryo at approximately 30 hours, illustrating the spinal cord oriented in an anterior (top) to posterior (bottom) direction. (E) Dorsal view of an embryo having been injected with approximately 0.2 ng of wnt8 RNA, illustrating that a single axis forms even though there were defects apparent in the mid- and forebrain similar to those seen in C, but out of the plane of focus. The spinal cord in these wnt8-injected embryos is morphologically similar to that seen in the uninjected control (D). (F) Lateral view of an embryo having been injected with approximately 0.2 ng of wnt8 RNA and examined at 18 hours of development. The majority of this embryo has developed as an amorphous mass above the yolk (arrows). Despite the few anatomically visible landmarks, and although it is out of the plane of focus, there is a morphologically recognizable notochord exhibiting the characteristic vacuolated arrangement (see Westerfield, 1989). Abbreviations: mhb, midbrain-hindbrain boundary; o, otocyst; sc, spinal cord; y, yolk. Scale bar (A, B), 55 µm; (C), 40 µm; (D-F), 65 µm.

8 1794 G. M. Kelly and others Table 2. Effects of wnt8 and wnt8b overexpression in zebrafish embryos* Injection Dose β- CS2-βgalactosidase wnt8 wnt8b wnt8 wnt8b wnt8 wnt8b galactosidase CS2-wnt8b Phenotype (1 ng) (0.01 ng) (0.01 ng) (0.1 ng) (0.1 ng) (0.2 ng) (0.2 ng) ( ) ( ) Normal 98% (42) 39% (36) 19% (36) 8% (64) 2% (127) 16% (19) 5% (60) 81% (43) 25% (40) Bustled/radialized defects 1 0 3% 14% 22% 19% 68% 60% 0 0 Forebrain defects % 67% 70% 79% 16% 35% 12% 75% Unknown defects 3 2% 3% % 0 Broadened gsc pattern 4 11% (91) 20% (86) nd 36% (87) nd 73% (55) nd 0 (13) 16% (25) Broadened ntl pattern 4 10% (108) 19% (82) nd 21% (87) nd 87% (47) nd nd nd Abnormal notochord 5 0 (17) nd nd 10% (21) 4% (23) nd nd 0 (25) 23% (56) *Scored after hours postfertilization. 0.2 ng of the β-galactosidase plasmid, or 0.05 ng of the wnt8b plasmid, was injected per embryo. nd: not determined. (n): number in brackets represents sample size. 1 As previously reported by Stachel et al. (1993), there is a continuum of phenotypes, beginning with the bustled phenotype where embryos exhibit a single axis, vacuolated notochord and normal dorsal anterior structures, to the radialized phenotype, where development proceeds but the embryo has no distinguishable axis, anterior structures, or distinct single notochord. 2 Forebrain defects with no apparent accompanying axial defect. These phenotypes are associated with the loss of one or both eyes, reduced eye cup size and/or loss of lens. Stachel et al. (1993) reported a similar category of defects associated with lithium perturbation where eye development was preferentially affected by the treatment. 3 Defects attributed to injection artifacts, whereby the trunk of the embryo is split on either side of the yolk. 4 The pattern of expression is either broadened laterally in the margin or extended out from the dorsal midline. 5 The morphological appearance of the notochord was assayed by Tor 70 monoclonal antibody staining. injected RNA, though at lower frequency (16-35%; Table 2). Interestingly, in this group of embryos, which still display an axis (with the majority of embryos displaying the bustled to radialized phenotype), neither wnt8 nor wnt8b induced a secondary axis, and posterior regions, including the spinal cord (SC, Fig. 6E) appeared similar to uninjected controls (Fig. 6D). Perturbation of ntl and gsc expression by wnt8 To understand further how the overexpression of wnt8 and wnt8b may lead to the above phenotypes during early development, it seemed reasonable to postulate that overexpression may affect the expression of mesodermal genes, given that wnt8 is expressed in future mesoderm and overexpression causes defects involving mesoderm and axis formation. Therefore, 1- to 4-cell embryos were injected with wnt8 RNA then assayed at the onset of gastrulation by in situ hybridization, scoring for ectopic or broadened domains of expression of ntl and gsc. Fig. 7 illustrates differences in gsc (Fig. 7A-D) and ntl (Fig. 7E-H) expression in wnt8 versus β-galactosidaseinjected embryos at 55-60% epiboly. The most obvious difference in gsc expression arising from the overexpression of wnt8 was the broadening of the signal at the germ ring (arrows, Fig. 7C) and the loss of the highly restricted pattern in the shield observed in β-galactosidase-injected controls (arrow, Fig. 7A). In a whole-mount view from the animal pole of wnt8-injected embryos, this broadened gsc expression is continuous around the circumference of the embryo (arrows, Fig. 7D) and, unlike controls (Fig. 7B), there is no obvious localized expression in the shield. The extent of the ntl signal in the germ ring was also altered by the overexpression of wnt8 (Fig. 7G) when compared to control embryos (Fig. 7E). The ntl signal in the involuted cells at the shield, as evident in control embryos (open arrows, Fig. 7E, F), cannot be resolved in wnt8-injected embryos due to the overall increase in ntl expression (Fig. 7G, H). In addition, a ntl signal is also seen in cells of the enveloping layer (open arrows, Fig. 7G), which is normal (see Schulte- Merker et al., 1992). However, and as seen more clearly in a view from the animal pole, it would appear that staining in these cells has increased in the wnt8-injected embryos (open arrows, Fig. 7H). Injection of wnt8 RNA led to changes in gsc and ntl expression in a dose-dependent manner (Table 2). At the lowest doses of wnt8 RNA (0.01 ng), about 20% of embryos exhibited changes in ntl and gsc expression, significantly above the effects of injection of 100-fold greater amounts of β-galactosidase RNA (Table 2). Similarly, expression of wnt8b from the injected constitutive promoter construct (CS2-wnt8b, Table 2) resulted in changes in gsc expression in a small percentage of embryos. At higher doses of wnt8 RNA (0.1 ng), 36% of embryos now exhibited overt changes in gsc expression, whereas expression of ntl, which is more difficult to score, was affected to a lesser extent (Table 2). Injecting approximately 0.2 ng of wnt8 RNA leads to a 2- to 4-fold increase over endogenous wnt8 RNA levels at 30% epiboly as determined by RT-PCR (data not shown) and has a dramatic affect on expression of gsc (73% of embryos, Table 2) and ntl (87% of embryos, Table 2) relative to controls (10-11% of embryos, Table 2, see also Fig. 7). Many of these embryos developed with bustled to radialized phenotypes as in Fig. 6F. Injection of zebrafish wnts into Xenopus embryos We were surprised that the overexpression of wnt8 or wnt8b in zebrafish did not induce a secondary axis as reported for the overexpression of Xwnt-8 in Xenopus (Christian et al., 1991; Smith and Harland, 1991; Sokol et al., 1991; Moon, 1993). Thus, to test whether zebrafish wnt8 and wnt8b activated a different pathway than Xwnt-8 or if zebrafish and Xenopus responded differently to the activation of a common pathway, we injected zebrafish wnt8 and wnt8b into Xenopus embryos. Injected embryos were then scored for the presence of a secondary dorsal axis, as reported for Xwnt-8 (reviewed in Moon et al, 1993b). Results from the injection of either wnt8

9 wnt8 and wnt8b expression in zebrafish embryos 1795 Fig. 7. The spatial localization of gsc and ntl transcripts in β-galactosidase-rna-injected and wnt8-rna-injected embryos at 55-60% epiboly. Embryos in A, C, E and G are oriented animal pole to the top, whereas embryos in B, D, F and H are viewed from the animal pole. (A,B) gsc expression in embryos injected with 1 ng of β-galactosidase RNA. gsc expression is restricted to the shield (arrow) in these embryos. (C,D) gsc expression in embryos injected with approximately 0.5 ng of wnt8 RNA. Note that the gsc signal has expanded in thickness at the germ ring (arrows) and there is no readily identifiable shield. The increased gsc signal in the germ ring can occur in a uniform manner around the entire circumference as in C, or as seen in a view from the animal pole of another early gastrula embryo (D), it may appear intermittently in a random fashion (arrows). (E,F) The spatial pattern of ntl expression in embryos injected with approximately 1 ng of β- galactosidase RNA. The ntl signals are detected in the germ ring (arrows) and in cells that have involuted in the embryonic shield (open arrow). (G,H) The spatial pattern of ntl expression in embryos injected with approximately 0.5 ng of wnt8 RNA, illustrating the increase in the width of the ntl expression in the germ ring (arrows). In addition, the ntl signal in cells ventral to the germ ring in the enveloping layer (open arrows), has increased over those cells in β- galactosidase injected (F) or uninjected controls (data not shown). This apparent increase in the signal is not restricted to any one region since there are numerous cells around the entire germ ring expressing ntl (open arrows, H). Scale bar (A-H), 65 µm. or wnt8b into Xenopus embryos clearly demonstrate that both Wnts induce secondary axes (Fig. 8B,C), and examination at later stages reveals that these are complete axes (data not shown). Embryos injected with synthetic prolactin RNA developed normally (Fig. 8A). DISCUSSION With the expectation that studies of wnts in zebrafish embryos will lead to analyses not feasible in other vertebrate embryos, we have initiated an investigation of the expression and activities of zebrafish wnt8 and wnt8b during early development. As discussed below, we have unexpectedly found that zebrafish express a maternal wnt8 that becomes expressed in an area in the embryo, and with an activity, consistent with a role in mesoderm formation. Moreover, we report that while the highly related gene wnt8b has a striking pattern of expression in the CNS, it nevertheless appears to have an activity related to wnt8. These data contribute to the idea that functionally equivalent wnts may be involved in diverse processes in the formation of both mesoderm and neural tissue, with the spatial and temporal regulation of expression of these putative signaling molecules, rather than ligand specificity, serving to regulate wnt functions. Patterns of expression of wnt8 in developing mesoderm We found that wnt8 is a maternal transcript which is subsequently expressed in future mesoderm. The spatially restricted pattern of expression of zebrafish wnt8 in the margin of the pregastrula embryo closely parallels that reported for other

10 1796 G. M. Kelly and others regions fated to give rise to mesoderm in both Xenopus and zebrafish appear to express either gsc or wnt8, and in Xenopus this may involve an active role for gsc in the negative regulation of Xwnt-8 (Christian and Moon, 1993). Patterns of expression and potential roles of wnt8b in the developing nervous system A series of inductive events are responsible for generating the regional anteroposterior differentiation of the neuroepithelium (reviewed in Keynes and Lumsden, 1990). During late gastrulation, wnt8b is detected in prospective neuroepithelial cells believed to be the progenitors of those that later localize to the MHB. In fact, the anteroposterior axis of the brain is already determined by the position of the cells in the early gastrula by fate map analysis (Kimmel et al., 1990). Unexpectedly, the early spatial expression pattern of wnt8b is remarkably similar to the patterns exhibited by wnt1 (Kelly and Moon, 1995), pax2 (Krauss et al., 1991a,b; Mikkola et al., 1992; Püschel et al., 1992) and eng2 (Fjose et al., 1992). This is noteworthy given the fact that all of these genes, which later co-localize to the MHB, belong to gene families that exhibit a high degree of conservation to the Drosophila segmentation genes (reviewed in Hooper and Scott, 1992; Ingham and Martinez Arias, 1992). Expression of wnt8b in the hindbrain is highly restricted to r1, 3 and 5, which is notable since krox20 expression is also restricted to r3 and r5 (see Oxtoby and Jowett, 1993). Since krox20 is first detected during late gastrulation and precedes the appearance of wnt8b in r3 and r5, it seems quite unlikely that the rhombomeric patterning is established by wnt8b. One speculation is that wnt8b might have a role in assigning particular developmental identities to specific regions or subpopulations of neurons in the differentiating rhombomeres, though loss-of-function data are required to test this idea. Fig. 8. Ectopic expression of prolactin, zebrafish wnt8 and wnt8b in Xenopus embryos. (A) Embryos injected with prolactin RNA appear normal with a single axis (arrow), whereas those injected with either wnt8 (B) or wnt8b (C) exhibit two axes (arrows). genes, including eve1 (Joly et al., 1993), Zf-cad1 (Joly et al., 1992), ntl (Schulte-Merker et al., 1992, 1994), and snail1 (Thisse et al., 1993; Hammerschmidt and Nüsslein-Volhard, 1993), which are likely to have roles in generating embryonic pattern. Consistent with our interpretation of the expression of wnt8 in mesoderm, in spadetail mutants exhibiting perturbed mesodermal cell migration, wnt8 expression is localized in the affected mesodermal cells. Since we have established that zebrafish wnt8 is the homolog of Xenopus Xwnt-8 (and analysis by A. Sidow, personal communication), it is useful to compare their patterns of expression. In zebrafish, wnt8 is detected in a homogenous fashion in all non-involuted cells of the margin, including those directly overlying the gsc-expressing cells in the shield (see Stachel et al., 1993 regarding gsc). In contrast, Xwnt-8 is not expressed around the entire cirumference of the marginal zone of Xenopus gastrulae; it is absent from the gsc-positve cells of the Spemann organizer field (Christian and Moon, 1993). Thus, Ectopic expression of wnt8 and wnt8b Phenotypes resulting from the overexpression of wnt8 and wnt8b in zebrafish embryos fit into two general categories: (1) an apparent hyperdorsalization of development yielding bustled/radialized embryos and (2) perturbations in eye development and loss of dorsoanterior structures. These phenotypes are remarkably similar to those produced by treatment of zebrafish embryos with lithium (Stachel et al., 1993). This further supports speculation that this class of wnt activity has the capacity to modulate IP 3 levels (Moon et al., 1993b), and establishes wnts as a candidate class of endogenous signaling molecules, which may explain the data of the LiCl studies in zebrafish. Analysis of bustled/radialized embryos In zebrafish embryos injected with a range of doses of wnt8 RNA (current study) or treated with LiCl (Stachel et al., 1993), substantial changes in gsc expression are detected by in situ hybridization. The broadened and ectopic domains of expression of gsc in the present study are also similar to those seen following the injection of Wnt-1 (Christian and Moon, 1993) or Xwnt-8 (Steinbeisser et al., 1993) RNA into ventral vegetal blastomeres of cleavage-stage Xenopus embryos, suggesting that this class of wnt activity has the conserved ability to modulate gsc. Significantly, Xwnt-8 in Xenopus is expressed too late to normally play a role in modulating gsc expression, whereas in zebrafish wnt8 is maternal, and is thus a potential

11 wnt8 and wnt8b expression in zebrafish embryos 1797 endogenous modulator of gsc, though this awaits loss-offunction data. An apparent distinction between zebrafish and Xenopus is that overexpression of wnt8 or treatment with LiCl leads to a duplication of the embryonic axis in Xenopus (reviewed by Christian and Moon, 1993) but not in zebrafish. That zebrafish wnt8 has not simply lost through divergent evolution the ability to induce secondary axes was established by our finding that both zebrafish wnt8 and wnt8b cause duplication of the axis after overexpression in Xenopus embryos. That zebrafish embryos do have an ability to respond to signals that lead to a duplication of the axis is evident from data showing that mouse nodal induces ectopic expression of gsc, and induces an axis duplication (Toyama et al., 1995). The observation that ectopic wnt8 and nodal both induce gsc, but only nodal leads to high frequency duplication of the axis, is unlikely to be due to technical differences in the injection protocols, as when we have injected wnt8 RNA into 8-cell or older embryos as in Toyama et al. (1995) we still do not observe axial duplications as monitored by in situ hybridization with ntl, even though we do observe occasional ectopic ntl staining (data not shown). As Toyama et al. (1995) do not report the LiCl-like phenotypes we observe with wnt8 it is possible that there are differences between the responses of zebrafish embryos to ectopic nodal and wnt8. However, we prefer to emphasize the similar effects of these putative signaling molecules on the expression of molecular markers rather than apparent differences in an endpoint phenotype, which may involve unsuspected and complex downstream consequences. This is particularly important in comparing our data to Toyama et al. (1995) because simply the cell autonomy of the responses to the injected RNAs or differences in the extent of localization of the RNAs might affect the phenotypes, even though both wnt8 and nodal induce gsc. Supporting this caveat, it is notable that injection of Xwnt-8 into Xenopus eggs at multiple sites, or injection of high amounts into one site, often leads to highly dorsalized embryos as we report in zebrafish, rather than promoting formation of multiple axes. In light of these considerations, the most interesting observation is that both maternal wnt8 (current study), and a yet to be described endogenous nodal (Toyama et al., 1995), are candidates for playing roles in formation and patterning of mesoderm in zebrafish. Also regarding the early effects of overexpression of Wnts, it is noteworthy that, when increases in gsc and ntl expression are observed, it is noted only in cells in or near the germ ring, where these genes are normally expressed. Since analysis of β- galactosidase-injected embryos reveals expression of β-galactosidase throughout the embryos (data not shown), it is clear that expression of wnt8 or wnt8b by themselves is not sufficient for an increase in expression in gsc or ntl. One potential explanation for this localized increase in gsc and ntl expression in response to injected wnt RNA comes from related studies in Xenopus, where early signaling by injected Xwnt-8 acts to modify the responsiveness of the cells to endogenous mesoderm-inducing growth factors of the FGF and TGF-beta families (reviewed by Kimelman et al., 1992; Moon et al., 1993b). In Xenopus, the activities of these mesoderm-inducing growth factors are localized to the vegetal and marginal zone regions. Whether this activity is localized to comparable regions in zebrafish remains largely unknown, but it has been argued that injection of Xwnt-8 mrna in Xenopus, and monitoring expression of other mesodermal genes, can be employed to map the spatial activity of mesoderm-inducing growth factors, which synergize with the Xwnt-8 to change gene expression (Sokol, 1993). We speculate that overexpression of wnt8 or wnt8b in zebrafish embryos leads to a localized increase in gsc and ntl expression solely in or near the germ ring, despite broader expression of the ectopic Wnt throughout the embryo because, as in Xenopus, the overexpressed Wnt must synergize with localized endogenous mesoderm-inducing growth factors, in order to alter expression of mesodermal genes. Analysis of dorsoanterior and eye defects Defects in the forebrain and eye occur in response to ectopic wnt expression, that closely resembles the phenotypes arising from treatment of embryos with LiCl at or around mid blastula transition (Stachel et al., 1993). Since this phenotype is obtained in embryos injected with wnt8b under the control of a promoter, which is expected to be transcribed around midblastula transition (Kane and Kimmel, 1993), the most likely explanation for the forebrain and eye defects is that the ectopic wnt, like LiCl, is affecting wnt-sensitive developmental processes after mid-blastula transition. What is less clear is why the lowest doses of injected wnt8 and wnt8b RNA also primarily lead to the forebrain and eye defects. It is possible that the lowest doses of injected RNAs lead to an insufficient level of protein at early developmental stages to promote the bustled/radialized phenotype, though both gsc and ntl are affected in some gastrula-stage embryos. Thereafter, the low dose of RNA produces sufficient wnt protein to affect wntsensitive developmental processes after the axis has been specified, leading to the forebrain and eye defects. Supporting the idea that the forebrain and eye defects arise by affecting wnt-sensitive developmental processes after mid blastula transition, in Xenopus embryos both Xwnt-8 (reviewed by Moon et al., 1993b) and lithium (Kao and Elinson, 1986, 1989) can mimic the signals responsible for the early specification of dorsoanterior structures, but treatment at midblastula transition results in a loss of anterior structures (Yamaguchi and Shinagawa, 1989). Speculations on the normal roles of wnt8 and wnt8b in zebrafish We speculate that maternal wnt8 is one of multiple factors that are involved in the specification of the spatial patterns of expression of gsc and other regulatory genes, leading to the establishment of the embryonic axis. Based on the pattern of expression of wnt8 during gastrulation in zebrafish, and considering functional studies of zygotic Xwnt-8 in Xenopus (Christian and Moon, 1993), we speculate that zygotically expressed wnt8 may be involved in specifying or patterning ventral mesoderm. However, other roles may exist, given the notable expression in the tip of the tail. While these speculations might be useful in formulating hypotheses and experiments, we emphasize that loss-of-function data are required for determining the embryonic requirements for both maternal and zygotic wnts. The common phenotypes arising from our overexpression analyses indicate that both zebrafish wnt8 and wnt8b may act on a common signaling pathway, despite obvious differences in their spatial patterns of expression. Thus, we speculate that

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