Morpholinos for splice modificatio

Morpholinos for splice modification


Repression of the vertebrate organizer by Wnt8 is mediated by Vent and Vox
Marie-Christine Ramel, Arne C. Lekven


Dorsoventral (DV) patterning of vertebrate embryos requires the concerted action of the Bone Morphogenetic Protein (BMP) and Wnt signaling pathways. In contrast to our understanding of the role of BMP in establishing ventral fates, our understanding of the role of Wnts in ventralizing embryos is less complete. Wnt8 is required for ventral patterning in both Xenopus and zebrafish; however, its mechanism of action remains unclear. We have used the zebrafish to address the requirement for Wnt8 in restricting the size of the dorsal organizer. Epistasis experiments suggest that Wnt8 achieves this restriction by regulating the early expression of the transcriptional repressors Vent and Vox. Our data show that vent and vox are direct transcriptional targets of Wnt8/β-catenin. Additionally, we show that Wnt8 and Bmp2b co-regulate vent and vox in a dynamic fashion. Thus, whereas both Wnt8 and zygotic BMP are ventralizing agents that regulate common target genes, their temporally different modes of action are necessary to pattern the embryo harmoniously along its DV axis.


Formation of the vertebrate embryonic axes requires Wnt signaling at two points: after fertilization, to establish a dorsal signaling center, and during gastrulation, to pattern and specify ventral fates (for reviews, see De Robertis et al., 2000; Schier, 2001). Although canonical Wnt/β-catenin signaling is involved in both processes, it is triggered differently in each case. Specification of the dorsal signaling center appears to be a ligand-independent mechanism involving the accumulation of β-catenin, the nuclear effector of Wnt signaling, in dorsal nuclei (Larabell et al., 1997; Kelly et al., 2000; Schier, 2001). Accumulation of nuclear β-catenin leads to the formation of the Niewkoop center, which induces the dorsal mesodermal structure known as Spemann's Organizer (known as the `shield' in zebrafish or the `node' in the mouse) (for a review, see Moon and Kimelman, 1998). After the establishment of the dorsoventral (DV) axis, Wnt/β-catenin activity stimulated by the ligand Wnt8 is required to antagonize the organizer; thus, zebrafish wnt8 mutants, or Xenopus embryos expressing a dominant-negative Xwnt8, display enlarged organizers and concomitant loss of posterior and ventral tissues (Hoppler et al., 1996; Lekven et al., 2001). Because proteins secreted by the organizer are known to be required for head formation and embryonic patterning (for a review, see De Robertis et al., 2000), understanding the mechanisms that limit organizer expansion is crucial for understanding embryonic patterning.

The organizer influences DV patterning through its secretion of BMP inhibitors such as Chordin (Chd) or Noggin (De Robertis et al., 2000). However, BMP also exerts its own effect on the organizer. The Xvent ventral homeobox genes were identified as transcriptional targets of BMP in Xenopus, and were shown to repress organizer gene expression on the ventral side of the embryo (Gawantka et al., 1995; Onichtchouk et al., 1996; Onichtchouk et al., 1998; Melby et al., 1999; Lee et al., 2002). Indeed, Xvents repress the transcription of targets such as chd and goosecoid (gsc) (Onichtchouk et al., 1996; Melby et al., 1999; Trindade et al., 1999). Analysis of the Xvent1b and Xvent2b promoters revealed the presence of consensus Lef/Tcf binding sites (Friedle and Knöchel, 2002). In addition, the Xvent1b promoter is responsive to zygotic Wnt activity, suggesting that the expression of Xvent genes in general may be under the control of Wnt8 (Friedle and Knöchel, 2002). In support of this, Hoppler and Moon found that overexpression of dn-Xwnt8 leads to the reduction of both Xvent1 and Xvent2 expression in Xenopus (Hoppler and Moon, 1998). Thus, these studies suggest that the expression of transcriptional repressors required to restrict organizer gene expression may be under the concerted control of both the BMP and Wnt pathways.

Genetic analysis of zebrafish vent (also known as vega2, similar to Xvent1) and vox (also known as vega1, similar to Xvent2) showed that the proteins encoded by these genes function as redundant transcriptional repressors (Kawahara et al., 2000; Melby et al., 2000; Imai et al., 2001). Zebrafish embryos homozygous for a chromosomal deficiency of the closely linked vent and vox loci show an expansion of organizer gene expression and severe DV patterning defects (Imai et al., 2001). Further epistatic analysis suggested that the primary role of Vent and Vox is to modulate BMP inhibitors secreted by the organizer (Imai et al., 2001). vent and vox are known BMP transcriptional targets in zebrafish as well, but their dependency on BMP signaling starts at around 70-75% epiboly (Kawahara et al., 2000; Melby et al., 2000). As a result, zygotic BMP mutants do not have expanded organizers as vent/vox mutants do at shield stage (Mullins et al., 1996; Miller-Bertoglio, 1997; Imai et al., 2001). To date, only two zebrafish zygotic mutants are known to display significantly expanded organizers: vent/vox mutants and wnt8 mutants. These data suggest that the relationship between BMP, Wnt8 and Vent/Vox is an important one for organizer regulation, the nature of which has been unclear but has been suggested to be complex (Hoppler and Moon, 1998; Marom et al., 1999).

We have used a loss-of-function approach in zebrafish to study the relationship between Wnt8, zygotic BMP and Vent/Vox regulation and activity, in order to understand the mechanism by which Wnt8 antagonizes the organizer. Our results suggest that Wnt8 directly regulates the transcriptional levels of vent and vox, and that the maintenance of high levels of vent or vox is required for the repression of organizer genes on the ventral side of the embryo. Furthermore, we provide evidence that Vent and Vox are absolutely essential to mediate the organizer repression activity of Wnt8. We also show that organizer repression and the maintenance of ventrolateral mesoderm fates appear to be independent events. Finally, we show that the early regulation of both vent and vox is under Wnt8 and BMP control, but that Wnt8 is the primary regulator; that is, at the onset of gastrulation, the requirement for BMP is only revealed in the absence of Wnt8. Zygotic BMP becomes the primary regulator of vent (but not vox) transcription during mid to late gastrulation. Therefore, Wnt8 and BMP contribute to the repression of the organizer, which will, as a consequence, regulate the distribution of Wnt and BMP inhibitors.

Materials and methods

Fish maintenance and genetics

Animals were maintained as described (Westerfield, 2000). Embryos were staged according to Kimmel et al. (Kimmel et al., 1995). Our wild-type strain is AB. Mutants used were Df(LG14)wnt8w8 (Lekven et al., 2001), DfST7 (Imai et al., 2001) and swrTC300(Mullins et al., 1996). Results from wnt8 or vent;vox deficiency mutants were confirmed with morpholinos (MOs).

In situ hybridization

In situ hybridizations were performed as described (Oxtoby and Jowett, 1993). Probes used were gsc (Stachel et al., 1993), chd (Miller-Bertoglio et al., 1997), wnt8 ORF1 and wnt8 ORF1+ORF2 (Lekven et al., 2001), eve1 (Joly et al., 1993), vent/vox (Melby et al., 2000), bmp2b (Kishimoto et al., 1997), opl (Grinblat et al., 1998), pax2a (Krauss et al., 1991) and tbx6 (Hug et al., 1997).

Genotyping of embryos

wnt8 mutants were genotyped as described (Lekven et al., 2001). vent;vox mutants were genotyped using vox R1 (5′-GATATTGCACACCAGCGTGA-3′) and vox L1 (5′-GTTCCAGAACCGAAGGATGA-3′) primers. swr mutants were genotyped as described (Wagner and Mullins, 2002). Embryos were classified according to their phenotype, photographed and genotyped. For wnt8;swr double mutants, at least 85 embryos from an intercross were examined in the same fashion.

Embryo microinjection, morpholinos, constructs

MOs (Genetools, LLC), RNA or DNA were injected into one- to four-cell stage embryos. Approximately 3 nL was injected per embryo. Capped mRNAs were synthesized using mMESSAGE mMACHINE (Ambion) and diluted in water. MOs were diluted in Danieau's buffer as recommended (Genetools). wnt8 MOs (targeting ORF1 and ORF2), and vent and vox MOs, have been described (Lekven et al., 2001; Imai et al., 2001). GR-LEFΔN-βCTA RNA was injected at 300 ng/μL into one-cell stage embryos. Embryos were dechorionated manually in fish water (Westerfield, 2000) prior to treatment. Dexamethasone (DEX; Sigma) treatments were performed for one hour at 1, 2, 3, 4 or 5 hours post-fertilization (HPF). DEX (100 mM stock solution in 100% ethanol) was used at a final concentration of 10 mM in 0.3×Danieau's solution. Treated embryos were fixed at 6 HPF. For the Cycloheximide (CHX; Calbiochem) treatment, embryos were first injected with GR-LEFΔN-βCTA RNA then treated with CHX (10 μg/mL), with or without DEX. For vent induction analysis, n(CHX)=37 and 55, n(DEX)=44, 37 and 11, and n(CHX+DEX)=28, 34 and 28, where n=total number of embryos analyzed in each experiment. For vox induction, n(CHX)=16, 17 and 12, n(DEX)=5, 12 and 20, and n(CHX+DEX)=9, 14 and 19. As a control for CHX treatments, uninjected embryos were treated with CHX from 1.5 HPF to sphere stage, then fixed and stained for gsc (Leung et al., 2003). No treated embryos expressed gsc (n=34). The χ2 test was used to determine statistical significance.


Zebrafish wnt8 and vent;vox mutants have expanded organizers, swr mutants do not

Although BMP and Wnt8 both are described as `ventralizing agents' (i.e. overexpression leads to a shift in mesodermal fates), they play non-equivalent roles in DV patterning. To illustrate this, we compared the expression of DV markers in wnt8 (Df w8) (Lekven et al., 2001), vent vox (Df st7) (Imai et al., 2001) and bmp2b (swrtc300) (Mullins et al., 1996) mutants.

In zebrafish, wnt8 contains two open reading frames (ORF1 and ORF2) (Lekven et al., 2001). The two Wnt8 proteins were shown to function redundantly in anteroposterior (AP) and DV patterning, as the Df w8 phenotype is phenocopied only by co-injection of both ORF1 and ORF2 MOs (Lekven et al., 2001). Similarly, the Df st7 phenotype is phenocopied by the co-injection of vent and vox MOs (Imai et al., 2001).

Expression analysis of the dorsal markers chd, gsc, floating head (flh) and dharma (bozozok) at shield stage shows that they are expanded ventrally in wnt8 mutants (Fig. 1B,F) (Lekven et al., 2001) (and data not shown) as well as in vent;vox mutants (Fig. 1C, inset, and Fig. 1G) (Imai et al., 2001). swr mutants, however, do not exhibit a similar expansion at shield stage (Fig. 1D,H) (Mullins et al., 1996; Miller-Bertoglio et al., 1997). Importantly, the expansion of dorsal markers is stronger in vent;vox mutants than in wnt8 mutants. For instance, gsc encircles the margin of vent;vox mutants (Fig. 1C, inset) but extends over a ∼90° arc in wnt8 embryos at the same stage (Fig. 1B). This comparative analysis shows that Wnt8 and Vent/Vox, but not BMP, are normally required ventrally during gastrulation to restrict the size of the organizer, which is in agreement with previous reports (Mullins et al., 1996; Miller-Bertoglio et al., 1997; Imai et al., 2001; Lekven et al., 2001).

Fig. 1.

The wnt8 phenotype is similar to the vent;vox and swr phenotypes. (A,B,D) Double in situ hybridization for eve1 and gsc. (C) eve1 expression, inset shows gsc. Note strongly reduced eve1 in wnt8 and swr mutants but only slightly reduced eve1 in vent;vox mutants. Arrowheads indicate the width of gsc expression (note circumferential gsc in C, inset). (E-H) In situ hybridization for chd (domain width indicated by arrowheads). Note expansion in both wnt8 and vent;vox mutants, but not swr mutants. All embryos are at shield stage. Animal view, dorsal right.

The expanded organizer phenotype is first observed in wnt8 embryos at 40% epiboly (discussed below), a developmental timepoint when convergence movements have not yet started (Kimmel et al., 1995). Thus, the expansion of dorsal markers in these backgrounds must reflect a change in fate rather than an alteration of cell movements.

Wnt8 is also required to promote ventral fates. eve1, a ventral mesodermal marker, is reduced in wnt8 mutants (Fig. 1B). It is similarly reduced in swr mutants (Fig. 1D) (Mullins et al., 1996). By contrast, eve1 is less reduced in vent;vox mutants (Fig. 1C) than in wnt8 and swr mutants (Fig. 1B,D), despite the fact that the dorsal markers gsc (Fig. 1C, inset) or chd (Fig. 1G) encircle the margin of the same embryos. Hence, Wnt8 and BMP are required in the ventral mesoderm for the maintenance of eve1, a ventral-specific gene, and this function is separable from repression of the organizer.

Wnt8 regulates vent and vox mRNA levels

Because Wnt8 and Vent/Vox share the function of repressing dorsal genes, we analyzed their epistatic relationship. We first examined vent and vox mRNA levels in wild-type versus wnt8 backgrounds (Fig. 2). In zebrafish, vent is expressed at the mesodermal margin during gastrulation, whereas vox displays both ventral mesoderm and ectoderm expression (Melby et al., 2000).

Fig. 2.

vent and vox mRNA levels are reduced in wnt8 mutants. In situ hybridization for vent (A-F), vox (G-L) or chd (M-R). Embryo genotypes are indicated above each column; stages are also indicated. At 30% epiboly, vent expression is reduced in wnt8 mutants/morphants (arrows in B,C). vox is reduced at 40% epiboly (arrows in H,I), corresponding to increased chd expression (arrowheads in N,O). Both vent (E,F) and vox (K,L) expression are reduced in shield stage wnt8 mutants/morphants. Animal view, dorsal right.

Starting at 30% epiboly (late blastula), the accumulation of vent at the margin is visibly weaker in wnt8 mutants or morphants than in wild type (Fig. 2A-C). We did not detect any differences in vent expression at earlier stages (data not shown). vox expression is not visibly different in wnt8 mutants at 30% epiboly (data not shown), but is reduced in the margin of wnt8 mutants/morphants at 40% epiboly (Fig. 2G-I).

To determine the correspondence between vent and vox reduction and the onset of an observable phenotype in wnt8 mutants, we examined chd expression at these early stages. At 30% epiboly, no visible difference in the chd expression domain was observed in wnt8 mutants (data not shown), but we did detect an expansion of chd expression at 40% epiboly, the timepoint at which both vent and vox are reduced in wnt8 embryos (Fig. 2M-O). Hence, our results suggest that a reduction in both vent and vox levels may be required to observe the expanded organizer phenotype at 40% epiboly, which is consistent with Vent and Vox functioning redundantly (Imai et al., 2001).

During the rest of gastrulation, vent and vox mRNA levels stay reduced in wnt8 mutants/morphants compared with in wild type (Fig. 2D-F,J-L; data not shown). By comparison, vent and vox levels are unchanged in swr mutants at shield stage (Kawahara et al., 2000; Melby et al., 2000), which explains the lack of an organizer phenotype (Mullins et al., 1996; Miller-Bertoglio et al., 1997). Indeed, Bmp2b is only required at mid to late gastrulation for the maintenance of vent and ectodermal vox expression (Melby et al., 2000). Therefore, Wnt8 regulation of vent and vox starts at the blastula/gastrula transition (30/40% epiboly), whereas Bmp2b regulation of these genes occurs later (70% epiboly).

To test the reciprocal possibility of wnt8 being regulated by Vent and Vox, we looked at the expression of wnt8 in vent;vox mutants (Fig. 3). As zebrafish wnt8 produces transcripts for both protein coding regions, we used probes to detect either the ORF1/ORF2 bicistronic transcript (ORF1), or both the bicistronic transcript and the ORF2 transcript (ORF1+ORF2) (Lekven et al., 2001). No differences from wild-type expression were observed in 30% or 40% epiboly vent;vox mutants (Fig. 3A,B,G,H). Because vent;vox mutants are affected prior to 30% epiboly (Imai et al., 2001), this suggests that a change in wnt8 expression is not responsible for the vent;vox mutant phenotype. The dorsal domain lacking ORF1 expression is slightly expanded in vent;vox mutants at shield stage (Fig. 3C,D; confirmed with MOs) and is more pronounced at 75% epiboly (Fig. 3F). Although there is an observable difference dorsally, ORF1 levels ventrally seem to be unaffected in vent;vox mutants (Fig. 3C-F), suggesting that the reduction in dorsal wnt8 ORF1 expression is an indirect consequence of an enlarged organizer. Analysis of ORF2 expression at later stages revealed that it is not affected by the loss of Vent and Vox (Fig. 3I-L). This is not unexpected as wnt8 ORF2 accumulates dorsally during gastrulation (Fig. 3K) and is therefore insensitive to molecules present in the organizer. Thus, only wnt8 ORF1 expression depends on Vent and Vox, but this dependency is restricted dorsally and may be indirect. By comparison, wnt8 ORF2 expression does not depend on Vent and Vox.

Fig. 3.

Wnt8 ORF1 and ORF2 expression in vent;vox mutants. In situ hybridization for wnt8 ORF1 (A-F) and wnt8 ORF1+ORF2 (G-L). Genotypes are indicated above each column; stages are also indicated. Arrowheads indicate the dorsal limit of wnt8 expression. Note the slight decrease in ORF1 dorsally in shield stage vent;vox mutants (C,D), and the broadened dorsal clearing of wnt8 ORF1 expression at 75% epiboly (F). wnt8 ORF2 expression is not affected. (A-D,G-J) Animal view, dorsal right. (E,F,K,L) Vegetal view, dorsal right.

Wnt8 functions through β-catenin to regulate vent and vox transcription

The above data show that Wnt8/β-catenin is necessary to maintain normal vent and vox expression. To test whether Wnt8 is sufficient to induce vent and vox, we injected Wnt8 ORF1 or ORF2 expression plasmids into wild-type embryos and assayed vent and vox expression by in situ hybridization at shield stage. In both cases, ectopic domains were observed in the animal ectoderm region and/or dorsal mesoderm, where vent and vox are normally absent (Table 1, and data not shown). To confirm that canonical Wnt signaling was involved in vent and vox regulation, we modulated β-catenin activity using a hormone inducible β-cat/Lef fusion protein (GR-LEFΔN-βCTA) (Domingos et al., 2001). The GR-LEFΔN-βCTA protein contains the human glucocorticoid receptor domain fused to the DNA-binding domain of murine LEF and the transactivation domain of murine β-catenin. Addition of the hormone dexamethasone (DEX) leads to the nuclear translocation of the fusion protein and toβ -catenin/Lef-induced transcription, thus allowing controlled induction of Wnt signaling (Domingos et al., 2001). Addition of DEX for a one-hour period at 1, 2, 3, 4 or 5 HPF led to ectopic vent and vox expression in a proportion of injected embryos (∼50-70% of embryos; Fig. 4A, panels b,d; data not shown). Consistent with the role of β-catenin in organizer induction, ectopic gsc was observed in a proportion of embryos treated at 1, 2 or 3 HPF, but not at later timepoints (data not shown).

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Table 1.

Injection of either wnt8 ORF induces ectopic vent and vox expression

Fig. 4.

vent and vox are direct transcriptional targets of Wnt8/β-catenin signaling. (A) vent and vox expression in control (a,c) or treated (b,d) embryos. Arrows in panels b and d indicate ectopic expression upon induction of GR-LEFΔN-βCTA with DEX. (B) Percentage of embryos displaying ectopic vent or vox domains (y-axis) upon treatment with CHX alone, DEX alone, or CHX+DEX (x-axis). The control bar represents embryos injected with GR-LEFΔN-βCTA and treated with ethanol (n=109 for vent, n=177 for vox). Error bars represent s.e.m. When performing the χ2 test on DEX versus DEX+CHX means, P>0.05 for both vent and vox, meaning that the difference between the means is not statistically significant.

Although our results suggest that Wnt8/β-catenin regulates vent and vox transcription, it is unclear whether this is direct (through β-catenin/Lef-induced transcription) or indirect (through the synthesis of an intermediate transcriptional regulator). Interestingly, the genomic region upstream of zebrafish vox contains consensus Lef/Tcf binding sites consistent with Wnt regulation of vox transcription (our own observations, and D. Kimelman, personal communication). To address this, we used cycloheximide (CHX) to test whether protein synthesis is required for the induction of ectopic vent or vox by GR-LEFΔN-βCTA. Treatment of GR-LEFΔN-βCTA-injected embryos with DEX at 5 HPF results in ectopic vent or vox RNA expression in 49% and 62.1% of embryos, respectively (Fig. 4B). Addition of CHX simultaneously with DEX did not result in a statistically different number of embryos with ectopic vent and vox domains (72.2% and 59.5%; Fig. 4B), indicating that GR-LEFΔN-βCTA activation of vent and vox does not require de novo protein synthesis. Thus, our results suggest that vent and vox are direct transcriptional targets of Wnt8/β-catenin signaling.

Wnt8 repression of the organizer requires Vent/Vox

As vent and vox transcription is regulated by Wnt8, we hypothesized that Vent and Vox function downstream of Wnt8 to repress dorsal genes, and that the wnt8 organizer phenotype is due to reduced vent and vox levels. If this is correct, injection of vent or vox RNA or DNA into wnt8 mutants would suppress the expanded organizer phenotype. We first established amounts of injected Vox or Vent that are sufficient to reduce the expression of dorsal markers (gsc, chd, flh) in wild-type embryos (Fig. 5A, panels a,c; data not shown). When injected into wnt8 mutants, Vox was able to reduce the expression of dorsal genes (Fig. 5A, compare panels b and d; Table 2). Similar results were obtained with either DNA or RNA injection for both vent and vox (Table 2, and data not shown). Thus, Vent and Vox expression can bypass wnt8 loss-of-function in repressing organizer genes, thus supporting the placement of vent and vox genetically downstream of wnt8. These results suggest that the difference in severity of the wnt8 and vent;vox organizer phenotypes (see Fig. 1) could be explained by residual Vent and Vox activity in wnt8 mutants. In agreement with this, further reduction of Vent and Vox in wnt8 mutants by injection of sub-maximal concentrations of vent and vox MOs enhances the severity of the wnt8 phenotype (Fig. 5B).

Fig. 5.

The wnt8 expanded organizer phenotype is due to reduced vent and vox expression. (A) Rescue of wnt8 mutants by Vox. gsc expression (bracket) in wild-type (a,c) or wnt8 (b,d) embryos, uninjected (a,b) or injected (c,d) with a vox expression plasmid. Some isolated lateral cells still express gsc in injected wnt8 embryos because of the mosaic expression of Vox (panel d, arrow). Embryos shown are at 70% epiboly, dorsal view. (B) Reduction of Vent/Vox enhances the wnt8 organizer phenotype. Graph shows the percentage of embryos belonging to a specific phenotypic class. Class I, wild-type chd expression; class II to IV, increasingly expanded chd expression. 100% of wild-type and wnt8 embryos belong to class I and class II, respectively. Upon injection of vent+vox MOs, most wild-type embryos belong to class II (96.5%, n=85), whereas wnt8 embryos belong to both classes III and IV (60.7% and 39.3%, respectively, n=28). Embryos shown at the bottom of the graph are at shield stage, animal view, dorsal right.

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Table 2.

Increased Vent/Vox expression in wnt8 mutants leads to the repression of dorsal genes

While Vent and Vox can bypass Wnt8 to repress organizer genes, we wished to assess whether Wnt8 requires Vent and Vox to repress the organizer. If Vent and Vox are essential for this Wnt8 function, then Wnt8/β-catenin activity should be ineffective in their absence. In support of this, vent;vox mutants express nearly normal levels of wnt8 mRNA (see Fig. 3), hence the expansion of the organizer in vent;vox mutants occurs in the presence of wnt8 transcripts.

To confirm that the wnt8 transcripts in vent;vox mutants produce functional proteins, we used two assays of Wnt8 function. First, we examined the expression of the Wnt/β-catenin activity reporter TOPdGFP (Dorsky et al., 2002; Lewis et al., 2004). We analyzed the expression of TOPdGFP mRNA at 100% epiboly in embryos homozygous for the transgene after injection of wnt8 or vent+vox MOs (Fig. 6A-D). As expected, and confirming previous results (Phillips et al., 2004), wnt8 MOs severely reduce TOPdGFP expression in 90% of injected embryos to almost undetectable levels (n=20; Fig. 6B). In vent/vox morphants, three phenotypic classes were observed: the first class displayed wild-type TOPdGFP expression (50%, n=22; Fig. 6C); the second class showed moderate reduction in TOPdGFP (14%, not shown); and the third class displayed a stonger reduction in staining (36%; Fig. 6D), but this class had significantly more TOPdGFP expression than wnt8 morphants (compare Fig. 6D to Fig. 6B). As a control for the strength of the vent+vox MO injections, a sample of the injected embryos was examined at 24 HPF and all showed a strong vent/vox loss-of-function phenotype (n=23) (Imai et al., 2001). Thus, TOPdGFP is a reporter of Wnt8 activity and is still expressed in vent+vox morphants. Reduced levels of TOPdGFP expression in some vent+vox morphants could reflect the fact that expression of the Wnt antagonists Dickkopf 1 and Frzb is significantly expanded (Imai et al., 2001) (and our own observations).

Fig. 6.

Wnt8 requires Vent and Vox to repress dorsal genes. (A-D) GFP in situ hybridization to embryos homozygous for the TOPdGFP transgene. (E-H) opl, pax2a and tbx6 in situ hybridization. Genotype/treatment is indicated above each panel. (A) TOPdGFP is expressed in the mesoderm. In wnt8 morphants (B), TOPdGFP is barely detectable (arrow). vent+vox MO-injected embryos display mostly wild-type TOPdGFP expression (C), but some display somewhat reduced expression (D, arrows). Arrowheads in A-D indicate the AP extent of the TOPdGFP positive domain. (E) In wild type, opl and pax2a expression domains in relation to tbx6 indicate normal neural posteriorization. In wnt8 morphants, opl is expanded posteriorly, pax2a is delayed and tbx6 is reduced (F). vent;vox mutants (G) do not display a strong AP defect, and ventral tbx6 staining is as strong as in wild-type embryos. Reducing Wnt8 in vent;vox mutants (H) results in decreased tbx6 and pax2a expression. The distance between the arrowheads in F, G and H show the degree of posteriorization. Embryos shown are at ∼100% epiboly, lateral view, dorsal right.

To confirm that expressed Wnt8 actively patterns vent;vox mutants, we analyzed AP neural patterning, a function known to require Wnt8 (Lekven et al., 2001; Erter et al., 2001). To assess the AP phenotype of vent;vox mutants, a combination of three probes was used: opl (anterior neuroectoderm), pax2a (midbrain-hindbrain border) and tbx6 (posterior non-axial mesoderm). In wnt8 mutants or morphants, AP patterning is severely disrupted at 90%-100% epiboly: the opl domain is expanded along the AP axis, pax2a expression is delayed and tbx6 expression is strongly reduced (Fig. 6F). By comparison, vent;vox mutants have only mildly affected AP patterning illustrated by a slight posterior shift of the opl and pax2a domain away from the animal pole, but the distance between opl or pax2a and tbx6 is significantly greater than in wnt8 morphants (Fig. 6G, compare with Fig. 6F). As expected, the expanded organizer of vent;vox mutants results in an enlarged dorsal clearing of tbx6 expression, whereas the levels of tbx6 ventrally are relatively unaffected (Fig. 6G, compare with Fig. 6E). As tbx6 expression depends on Wnt8, our results do not support an absence of Wnt8/β-catenin activity in vent;vox mutants. Furthermore, reducing Wnt8 translation in vent;vox mutants results in an additive phenotype. opl extends ventrally, as in vent;vox mutants, whereas pax2a and tbx6 expression is severely reduced, as in wnt8 mutants (Fig. 6H). Taken together, these results show that Wnt8 expression and patterning activity does not depend on Vent and Vox, with the significant exception that Wnt8 is unable to repress organizer genes when Vent and Vox are absent.

To further show that Wnt8 requires Vent and Vox in organizer repression, we tested whether exogenous Wnt8 can repress organizer genes in vent;vox mutants. We injected a wnt8 ORF1 expression plasmid (20 ng/μL) into one-cell stage vent;vox mutants and assayed gsc expression at shield stage. No injected vent;vox mutant embryos (n=25; genotyped by PCR) displayed reduced gsc expression, although this treatment did result in decreased gsc expression in wild-type siblings (n=54). As a control, we checked that the injected wnt8 DNA was sufficient to induce ectopic vent and vox expression in wild-type embryos (64% ectopic expression for vent, n=25; 42.8% ectopic expression for vox, n=35). Thus, repression of the organizer by exogenous Wnt8 requires Vent or Vox.

Our results show that in the absence of Vent and Vox, wnt8 is expressed and is active, as assayed by TOPdGFP reporter expression, tbx6 expression and embryonic AP patterning. Furthermore, ectopic Wnt8 cannot repress gsc in vent;vox mutants. These data strongly support a linear model in which Wnt8 acts directly upstream of Vent and Vox to repress the organizer.

Both Wnt8 and Bmp2b are required at different timepoints for the maintenance of vent and vox

Two pathways are required for the maintenance of vent and vox expression in zebrafish: the zygotic BMP pathway (Melby et al., 2000; Imai et al., 2001) and the Wnt pathway (this work). To understand the combined regulation of vent and vox during gastrulation by the Wnt8 and BMP pathways, we analyzed the phenotype of wnt8;swr double mutants (Fig. 7). Using swr (bmp2b) mutants is sufficient to assess the influence of zygotic BMP signaling, as it was previously shown that loss of Bmp2b produces a zygotic bmp null phenotype (Schmid et al., 2000). The requirement for both BMP and Wnt8 inputs towards vent and vox expression would be revealed if wnt8;swr double mutants exhibit a phenotype similar to the vent;vox phenotype. We found that gsc and chd are expressed in a broader domain around the mesodermal margin in shield stage wnt8;swr double mutants compared with either single mutant (Fig. 7B, compare with Fig. 1; data not shown), and thus they phenocopy vent;vox mutants (Fig. 7A). The same results were obtained when using the wnt8 deficiency or wnt8 MO knockdown (Fig. 7G), confirming the specificity of the interaction.

Fig. 7.

Wnt8 and zygotic BMP both regulate vent and vox, but do so differently. In situ hybridization for gsc (A,B,F,G), vent (C-E), vox (H-J), wnt8 (K,L) and bmp2b (M-O). Genotypes/treatments are indicated above each panel. Note circumferential gsc in vent;vox (A) and wnt8;swr (B,G) double mutants/morphants, and the strong reduction of vent (E) and vox (J) in wnt8;swr. wnt8 is still expressed in swr mutants (L), and bmp2b is still expressed in wnt8 mutants/morphants (N,O). Arrowheads in M-O indicate the dorsal limits of mesodermal bmp2b, which is shifted slightly ventrally in wnt8 mutants/morphants (N,O). Embryos shown are at shield stage, animal view, dorsal right.

As wnt8;swr double mutants display the same expanded organizer phenotype as vent;vox mutants at shield stage, we expected vent and vox mRNAs to be either absent or strongly reduced. We found both vent and mesodermal vox to be strongly reduced but not completely absent in shield stage wnt8;swr double mutants (Fig. 7E,J). Both vent and vox transcripts are not detectable in the mesoderm of later stage wnt8;swr double mutants (data not shown).

The fact that double mutants appear to be worse than wnt8 or swr single mutants suggests that Wnt8 and BMP function in parallel to regulate vent and vox. Consistent with this, bmp2b expression in wnt8 mutants/morphants is close to wild type (Fig. 7M-O), and wnt8 expression in swr mutants is normal at shield stage (Fig. 7K,L). Hence, both Wnt8 and Bmp2b are early regulators of vent and vox, but Wnt8 has a more prominent role until mid-gastrula stages.


To understand the DV phenotype of wnt8 mutants, we have analyzed the interaction of Wnt8, BMP, Vent and Vox. We found that the levels of both repressors are lower in wnt8 embryos at 40% epiboly when the expanded organizer phenotype initiates (Fig. 8). Consistent with a direct role for Wnt8 in vent/vox regulation, an inducible Lef/β-catenin fusion protein induces ectopic vent and vox transcription in the absence of new protein synthesis. Vent and Vox can repress organizer genes in the absence of Wnt8, suggesting that a simple linear pathway connects Wnt8/β-catenin with Vent/Vox-dependent organizer repression. In support of this, Wnt8 is unable to repress the organizer in the absence of Vent and Vox, although it is able to induce a Wnt reporter gene and to function in AP patterning. In addition, exogenous Wnt8 cannot repress gsc in vent;vox mutants. Finally, vent and vox regulation is under the control of both Wnt8 and zygotic BMP (Fig. 8), although Wnt8 is the primary regulator during early- to mid-gastrula stages.

Fig. 8.

Regulation of vent and vox by Wnt8 and zygotic BMP. (A) vent and vox are induced around MBT by an unknown factor. (B) At 40% epiboly, Wnt8 is required to maintain high levels of vent and mesodermal vox expression. (C) At 70% epiboly, in addition to Wnt8, zygotic BMP is required to maintain vent expression. BMP is also required for ectodermal vox expression. Thicker arrows represent stronger regulatory connections, as vent and ectodermal vox expression is absent in zygotic BMP mutants at this stage, whereas vent and mesodermal vox expression are only reduced in wnt8 mutants.

vent and vox are transcriptional targets of Wnt8/β-catenin signaling

Although it is not known what induces vent and vox, our data show that Wnt8 regulates their early transcriptional maintenance. What is unclear is which Lef or Tcf proteins are involved in Wnt8-mediated transcriptional regulation. Studies in Xenopus suggest that Lef1 and not Tcf3 may mediate Xwnt8 function (Roel et al., 2002), but this has not yet been addressed in zebrafish.

Interestingly, it has recently been observed that overexpression of a conditional dominant repressor form of Tcf (hs-ΔTcf) leads to a more severe phenotype than the loss of Wnt8 (Lewis et al., 2004). Lewis et al. found that gsc expression encircles the margin of transgenic hs-ΔTcf embryos heat-shocked at 4 HPF, a phenotype similar to vent;vox or wnt8;swr double mutants. Why would overexpression of a dominant-negative Tcf produce a more severe phenotype than loss of Wnt8 signaling? This could be explained if ΔTcf not only abolishes Wnt8 function but also prevents other factors from positively regulating vent and vox. One such factor could be the Smads that mediate Bmp2b function, as we have shown that zygotic BMP signaling is essential for maintaining vent and vox expression in the absence of Wnt8. In other words, ΔTcf may prevent Smad-dependent regulation of vent and vox.

Regulation of vent and vox by Wnt8: comparison between zebrafish and Xenopus

The transcriptional regulation of Xvent genes has been studied quite extensively in Xenopus, where most were found to be direct targets of Bmp4 signaling (Rastegar et al., 1999; Henningfeld et al., 2000; Henningfeld et al., 2002; Lee et al., 2002). However, the analysis of their regulation by Xwnt8 is less complete. It was found that zygotic Wnt signaling is necessary and sufficient for Xvent1 and Xvent2 expression (Hoppler and Moon, 1998; Marom et al., 1999), in agreement with our findings for zebrafish Wnt8. Analysis of Xenopus embryos overexpressing dominant-negative Xvent1 and Xvent2 revealed that Xwnt8 expression is not affected by the loss of Xvent activity (Onichtchouk et al., 1998). Again, our data agree as wnt8 is expressed in vent;vox mutants. The inability of Xwnt8 to rescue the dominant-negative Xvent phenotype was interpreted to mean that Xwnt8 functions in a different pathway than Bmp4/Xvent (Onichtchouk et al., 1998). However, we propose that, as in zebrafish, Xwnt8 functions upstream of Xvent genes, and that apparent differences between our model and Xenopus models may be due to the different experimental approaches. For example, concomitant reduction of Xwnt8, and Xvent1 and Xvent2, activities using dominant-negative proteins results in a more severe phenotype than reducing Xvent1 and Xvent2 alone (Onichtchouk et al., 1998). This is also what we observed when injecting vent and vox MOs in a wnt8 background. Thus, our results agree with data obtained in Xenopus, although our interpretation of the Wnt8/Vent/Vox relationship is somewhat different.

Wnt8 and zygotic BMP are required during gastrulation to maintain vent and vox expression at different timepoints

Our results show that both Wnt8 and Bmp2b (hence zygotic BMP) are required to maintain vent and vox levels during gastrulation, but that Wnt8 regulation of those genes occurs earlier at the blastula/gastrula transition (Fig. 8). The lack of an expanded organizer in swr mutants can be explained by the late regulation of vent and vox by zygotic BMP after the organizer has been formed. In addition, mesodermal vox levels are unchanged in swr mutants (only ectodermal vox levels are reduced at 70%) (Melby et al., 2000). Hence, mesodermal Vox can repress dorsal genes in swr mutants. Consistent with this, injection of vox MO in swr mutants results in expanded gsc expression at 70% epiboly (M.-C.R. and A.C.L., unpublished).

There are two known BMP signaling pathways in Xenopus and zebrafish (Dale and Jones, 1999; Wilm and Solnica-Krezel, 2003). In zebrafish, the maternal BMP pathway is thought to establish ventral identity in a manner analogous to the establishment of a dorsal axis by maternal β-catenin activity (Kramer et al., 2002; Sidi et al., 2003). Understanding the regulation of Wnt8 by maternal and zygotic BMP may explain apparently contradictory results from Xenopus and zebrafish. For instance, whereas it was found that regulation of zebrafish vent and vox by zygotic BMP occurs at mid to late gastrulation (Melby et al., 2000), Xenopus Xvent2 regulation by BMP signaling occurs during early gastrulation (stage 10.5) (Ladher et al., 1996). Xvent2 regulation was observed in embryos overexpressing a truncated Bmp2/4 receptor that does not distinguish between Bmp2 or Bmp4 ligands (Suzuki et al., 1994). However, Bmp2 is both maternally provided and zygotically expressed (Dale and Jones, 1999). It has therefore been suggested that Xvent2 expression may be under the influence of a maternal BMP signal (Ladher et al., 1996). Interestingly, the use of the same BMP-knockdown approach also results in decreased Xwnt8 expression (Schmidt et al., 1995; Hoppler and Moon, 1998). In zebrafish, it has been reported that loss of maternal BMP (Radar) signaling does not interfere with the induction of vent and vox at MBT (Sidi et al., 2003), although embryos homozygous for maternal smad5 display slightly expanded gsc and chd expression (Kramer et al., 2002). Thus, the elucidation of the relationship between Wnt8 and maternal or zygotic BMP in zebrafish using a loss-of-function approach may address whether the regulation of vent and vox is fundamentally different between zebrafish and Xenopus.


We thank Gerri Buckles for invaluable support, William Talbot for the Dfst7 line, Nobue Itasaki for GR-LEFΔN-βCTA, David Kimelman for the vent and vox constructs, Richard Dorsky for the TOPdGFPreporter line, and Bruce Riley and Bryan Phillips for discussion and critical reading of the manuscript. This work was supported in part by a Beginner Grant in Aid from the American Heart Association, Texas Affiliate (no. 0365081Y).


    • Accepted May 12, 2004.


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