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First published online 24 November 2005
doi: 10.1242/dev.02144

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Vg1 is an essential signaling molecule in Xenopus development

Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

^* Author for correspondence (e-mail: heabq9{at}chmcc.org)

Accepted 4 October 2005

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Xenopus Vg1, a transforming growth factor ß (Tgfß) family member, was one of the first maternally localized mRNAs identified in vertebrates. Its restriction to the vegetal pole of the egg made it the ideal candidate to be the mesoderm-inducing signal released by vegetal cells, but its function in vivo has never been resolved. We show that Vg1 is essential for Xenopus embryonic development, and is required for mesoderm induction and for the expression of several key Bmp antagonists. Although the original Vg1 transcript does not rescue Vg1-depleted embryos, we report that a second allele is effective. This work resolves the mystery of Vg1 function, and shows it to be an essential maternal regulator of embryonic patterning.

Key words: Vg1, Tgfß, Xenopus, Antisense, Bmp antagonist, Maternal localization

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Tgfß family member Vg1 was one of the first examples of a specific mRNA localized in eggs (Melton, 1987; Rebagliati et al., 1985). During oogenesis, Vg1 mRNA becomes restricted to the vegetal cytoplasm of the oocyte, and is inherited by the most vegetal cells of the embryo (Weeks and Melton, 1987). The vegetal cells of the blastula have two important functions: they differentiate into the endoderm of the embryo, and they signal to the adjacent marginal (equatorial) region to form the mesoderm (Dale et al., 1985). Vg1 therefore became a strong candidate to be the mesoderm-inducing signal. However, the in vivo function of Vg1 has proven difficult to assess. Whereas the overexpression of other Tgfß family members causes mesoderm formation (Jones et al., 1995; Koster et al., 1991; Smith et al., 1990), Vg1 mRNA does not (Dale et al., 1989; Tannahill and Melton, 1989). Furthermore, endogenous mature Vg1 protein cannot be detected, and although Vg1 pro-protein can easily be detected from the translation of exogenous mRNA, it is not secreted and cleaved (Dale et al., 1989; Tannahill and Melton, 1989). Gain- and loss-of-function experiments have been performed using chimeric Vg1 proteins with either Bmp (bVg1) or activin (aVg1) pro-domains, both of which are cleaved to produce mature Vg1 (Dale et al., 1993; Joseph and Melton, 1998; Kessler and Melton, 1995; Thomsen and Melton, 1993). However, the possibility remains that such chimeric proteins may not reflect endogenous function, particularly as recent studies have shown that the pro-domains of Tgfß family members are important determinants of signaling function (Cui et al., 2001; Jones et al., 1996; Le Good et al., 2005). A loss-of-function experiment was performed in which the Bmp pro-domain was linked to a putative dominant-negative form of the Vg1 mature protein (Joseph and Melton, 1998). This construct blocked the action of overexpressed bVg1, and caused a loss of dorsal mesodermal and dorsal axial structures, and the loss of expression of the endodermal marker Xlhbox8. In the absence of processing of the full-length Vg1, or of detectable levels of the mature form, these facts have generated a long-standing paradox over the in vivo function of this maternally localized mRNA.

More recently, further complications have arisen with the discovery of VegT, a maternally encoded T-box transcription factor whose mRNA is also localized in the vegetal cells of the embryo (Zhang and King, 1996). Depletion of the maternal stockpile of VegT mRNA abrogates formation of the endoderm and the mesoderm (Zhang et al., 1998). VegT activates the transcription of at least six zygotic Tgfß family members (Xanthos et al., 2001). If nodal signaling is blocked, then mesoderm induction is blocked (Agius et al., 2000; Kofron et al., 1999). Any in vivo function of Vg1 must therefore be reconciled with these facts.

Here, we analyse the role of maternal Vg1, and find it is required for Smad2 phosphorylation and for early zygotic gene expression, particularly of anterior mesendodermal genes that encode Bmp and Wnt antagonists, chordin, cerberus, noggin and dickkopf. Embryos depleted of Vg1 develop with delayed gastrulation, and a dose-responsive reduction in anterior and dorsal development. Although the original Vg1 transcript does not rescue Vg1-depleted embryos, we report that a second allele is effective.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Oligonucleotides
Oligonucleotides (oligos) were as follows:

• Vg1A (antisense), 5'-G^*C^*C^*ATGTAACCTTG^*A^*G^*G-3', where phosphorothioate-modified residues are indicated by an asterix; and
  
• Vg1MO (morpholino), 5'-CCACAGTCTCAGCCACACCATACTG-3'.
  

Real-time PCR
Total RNA was prepared using the proteinase K method and RNase-free DNase treatment before cDNA synthesis (Zhang et al., 1998). cDNA was synthesized using oligo dT primers. Random hexamer (R6) primers were used where indicated. Real-time RT-PCR was carried out using the Light Cycler System (Roche), using the primers and cycling conditions described previously (Birsoy et al., 2005; Kofron et al., 1999; Kofron et al., 2004; Zhang et al., 1998).

Oocytes and embryos
Full-grown oocytes were manually defolliculated and cultured in oocyte culture medium (OCM), as described previously (Xanthos et al., 2001). Oocytes were injected vegetally with the antisense oligo (Vg1A) or the morpholino (Vg1MO) and cultured for 48 hours at 18°C before maturation. Control uninjected oocytes were cultured in the same way in each experiment. All of the oocytes were matured by addition of 2 µM progesterone in OCM and cultured for another 12 hours. Control uninjected and oligo-injected oocytes were then labeled with vital dyes and fertilized using the host transfer technique, as described (Zuck, 1998).

Whole-mount in situ hybridization and histology
For whole-mount in situ hybridization with cerberus and chordin probes, gastrula-stage embryos were fixed in MEMFA for two hours. In situ hybridization was performed as described (Harland, 1991).

For histology, tailbud-stage embryos were fixed in Bouin's fixative for three hours, dehydrated and embedded in paraplast, serially sectioned and stained with Hematoxylin and Eosin.

Nieuwkoop assay
Wild-type animal caps dissected at mid-blastula stage were incubated with control or Vg1-depleted vegetal masses, from oocytes injected with 6 ng oligo, dissected at mid-blastula stage. After 1.5 hours of co-culture, caps were separated from the vegetal masses and frozen down when sibling wild-type embryos reached stage 11.

Western blots
Western blots were carried out under reducing conditions, as described (Birsoy et al., 2005). Antibodies used were anti-Vg1 antibody (D5; 1:1000) (Tannahill and Melton, 1989), anti-phospho-Smad1 (Cell Signaling Technology; 1:1000), anti-phospho-Smad2 (Cell Signaling Technology; 1:1000) and total Smad2 (BD Transduction Laboratories; 1:500). -Tubulin (DM1A Neomarkers 1:20,000) was used as a loading control. IPLab Gel software (V. 1.5) was used to quantify protein levels.

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To study Vg1 function, we targeted Vg1 mRNA using an antisense oligo (Zuck, 1998), which depletes Vg1 mRNA in a dose-responsive fashion (Fig. 1A), and results in a dose-dependent reduction in Vg1 protein levels (Fig. 1B). After fertilization, the amount of Vg1 mRNA and protein continues to decline through the gastrula stages, as there is no transcription of zygotic Vg1 at this time (Fig. 1A,B) (see also Rebagliati et al., 1985). Reduction of Vg1 protein below 50% arrests development at the gastrula stage (data not shown). A similar delayed gastrulation phenotype results when Vg1 protein is depleted (Fig. 1B) using a morpholino oligo, Vg1MO (Table 1B).

At the neurula and tailbud stages, Vg1-depleted embryos have different degrees of anteroposterior and dorsoventral axis abnormality (Fig. 1C; Table 1C). Typically, 6 ng of antisense oligo causes embryos to develop with a loss of head structures, whereas lower doses result in stunted embryos. Histological sections of tailbud-stage embryos show that all three germ layers are present in Vg1-depleted embryos; however, an absence of the notochord and fusion of somites in the midline are observed, with much reduced and abnormal neural structures.

Development of the early embryo involves the interplay of at least three signaling pathways; the Wnt signaling pathway (Heasman et al., 1994) activates the expression of the target genes siamois and Xnr3; the VegT pathway activates the expression of endodermal genes and nodal-related proteins (Xanthos et al., 2001), and initiates Smad2 phosphorylation (Lee et al., 2001), and the Bmp pathway activates Smad1 phosphorylation (Graff et al., 1996; Lee et al., 2001). In Vg1-depleted embryos, the expression of Xnr3 and siamois is not significantly altered (Fig. 1D), suggesting that Vg1 is not required for maternal Wnt pathway activation. However, Vg1 is required for Smad2 phosphorylation, as Smad2 phosphorylation is reduced in Vg1-depleted embryos at the late-blastula stage (Fig. 1E; repeated in three experiments). At the early and mid-gastrula stage, the amount of Smad2 phosphorylation in Vg1-depleted embryos increases, but does not reach wild-type levels. This correlates with the timing of the onset of expression of the VegT target gene derrière, which occurs normally in both control and Vg1-depleted embryos (data not shown). By contrast, Bmp signaling via phospho-Smad1 is initially normal in Vg1-depleted embryos, but is reproducibly elevated (in three experiments) as gastrulation proceeds (Fig. 1E; arrowhead). We conclude that Vg1 signaling is required for Smad2 phosphorylation at the late-blastula stage, and to prevent excess Bmp signaling at the gastrula stages.

Because Smad2 phosphorylation is essential for mesoderm induction, we tested whether Vg1 depletion reduces the mesoderm-inducing signals released by vegetal cells at the blastula stage by performing Nieuwkoop assays. Vg1-depleted vegetal masses co-cultured with wild-type animal caps have a reduced mesoderm-inducing ability compared with wild-type vegetal masses, as measured by the induction of the general mesodermal markers Xbra and Fgf8, and the anterior endo-mesodermal marker chordin, in wild type animal caps (Fig. 1F).

View larger version (54K): [in this window] [in a new window]   Fig. 1. Vg1 is required for initiation of Smad2 phosphorylation and head induction. (A) Real-time RT-PCR analysis of oocytes and gastrula-stage embryos shows that maternal Vg1 mRNA is efficiently depleted by the Vg1A oligo (4 ng oligo, 12% of control levels; 6 ng oligo, 5% of control levels) and that no zygotic transcription of Vg1 is detected during gastrulation. (B) Vg1 protein is depleted in a dose-dependent manner by the Vg1A oligo. Oocytes injected with 45 ng of morpholino (Vg1MO; 50% of control levels), and 4 ng or 6 ng of antisense oligo (Vg1A; 70% and 58% of control levels in oocytes, and 61% and 45% of controls at stage 10, respectively) have reduced levels of Vg1 protein. -Tub, -tubulin. (C) Vg1-depleted embryos show a delay in gastrulation in a dose-dependent manner. Whole-mount in situ hybridization with probes specific for cerberus (cerb) and chordin (chd) shows that the expression of cerb and chd is reduced at mid-gastrula stage in a dose-dependent manner. In histological sections of tailbud stages, Vg1-depleted embryos show an absence of notochord and fusion of somites in the midline (arrow). (D) Real-time RT-PCR analysis of stage 10 embryos shows that expression of the ß-catenin/Xtcf3 target genes Xnr3 and siamois is unaffected by Vg1 depletion. (E) Vg1 depletion reduces the phosphorylation of Smad2 (arrow; 34% of control level at stage 9.5) and increases the phosphorylation of Smad1 (arrowhead; 140% of controls at stage 10.5), as analyzed by western blots. (F) Mesoderm-induction activity of Vg1-depleted vegetal masses (Vg1-), from 6 ng Vg1A-injected oocytes, is decreased compared with controls (wt), as determined by Nieuwkoop assays. Real-time RT-PCR analysis shows that Vg1 depletion reduces the induction of Xbra, Fgf8 and chordin in wild-type animal caps. One whole embryo at stage 11 (WE) was used for quantification. (G) Real-time RT-PCR analysis of embryos during gastrula stages shows that Vg1 depletion causes a downregulation of dorsally expressed cerberus, chordin, noggin and dickkopf (dkk), and an upregulation of dorsal marker Xnr1 and ventral marker sizzled, without affecting the levels of Xsox17 and Bmp4.

To confirm that this phenotype is specifically caused by Vg1 depletion, we attempted to rescue Vg1-depleted embryos by reintroducing Vg1 mRNA prior to fertilization. Previous studies in which Vg1 mRNA was overexpressed used an allele, Vg1(Pro), that contains a proline at position 20 from the N terminus of the sequence (asterisk in Fig. 2A) (Dale et al., 1993; Dohrmann et al., 1996; Tannahill and Melton, 1989). This protein is translated in Xenopus embryos but is very inefficiently processed (Fig. 2B) (Dale et al., 1993; Tannahill and Melton, 1989); in addition, it does not have mesoderm-inducing activity, and is unable to rescue Vg1-depleted embryos (data not shown). However, we recently identified a second allele of Vg1, Vg1(Ser) (Birsoy et al., 2005), that is equally represented with Vg1(Pro) in oocyte and gastrulae libraries, and is more efficiently processed than Vg1(Pro) (Fig. 2B). The Xenopus tropicalis and Xenopus borealis Vg1 homologs, also have a serine rather than proline residue at the equivalent position (Fig. 2A). As the antisense oligo Vg1A is complementary to both serine and proline alleles (Fig. 2A), it was expected that it would bind and deplete them both with the same efficiency. We were unable to confirm this, as neither the PCR primers nor the Vg1 antibody can differentiate between the two allelic forms. Unlike Vg1(Pro), Vg1(Ser) mRNA partially rescues the phenotype of Vg1-depleted embryos (Fig. 2C,D; Table 1D,E), Smad2 phosphorylation (Fig. 2C) and the expression of molecular markers (Fig. 2E,F) at the gastrula stage.

Because Vg1(Ser) is able to rescue the phenotype of Vg1-depleted embryos, we reasoned that unlike Vg1(Pro), it should also have an inducing activity when overexpressed in embryos. We find that Vg1(Ser) has some mesoderm-inducing activity in animal caps, whereas Vg1(Pro) does not in the same experiment (data not shown). Fig. 2G shows that when Vg1(Ser) mRNA is overexpressed in the vegetal area, it causes the upregulation of the endodermal and mesodermal zygotic genes chordin, Xnr1, Fgf8 and Xsox17. We compared the inducing activity of Vg1 with another Tgfß family member, Xnr5. Xnr5 is a zygotic gene regulated by VegT, and, like Vg1, is restricted in its expression to vegetal cells (Takahashi et al., 2000). In comparison to Xnr5, 200 pg of Vg1 mRNA induces Fgf8 and Xnr1 to a similar extent as does 40 pg of Xnr5 mRNA at the early gastrula stage. Interestingly, Xnr5 induces Xsox17 and chordin in mid-gastrula stage embryos more effectively than Vg1.

In the current model of axis formation in Xenopus, Wnt target genes are activated on the dorsal side as a result of the cortical rotation and dorsal concentration of localized maternal Wnt11 mRNA at the early cleavage stage (Tao et al., 2005). Because the targets of Vg1 activity, chordin, cerberus and dickkopf, are all first expressed in the dorsal vegetal quadrant of the early gastrula (Bouwmeester et al., 1996; Glinka et al., 1998; Sasai et al., 1994), we tested whether Vg1 is also concentrated in dorsal compared with ventral cells by hemisecting 32-cell stage embryos into dorsal and ventral halves. Fig. 2H shows that both Vg1 mRNA and protein are more concentrated in the dorsal halves than in the ventral halves at the 32-cell stage. Here, protein and mRNA levels are examined in dorsal and ventral halves taken from the same batch of embryos, and the experiment was repeated three times with the same result. The comparison of oligo dT (dT)- versus random hexamer (R6)-primed cDNA shows that the enrichment of Vg1 mRNA on the dorsal side is not due to differential polyadenylation (data not shown).

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Vg1 is required for anterior development
Homologs of Xenopus Vg1 are expressed maternally in zebrafish (DVR1) (Helde and Grunwald, 1993), and at early embryonic stages in chick (Vg1) and mouse (Gdf1) (Lee, 1990; Shah et al., 1997). Although the role of zebrafish DVR1 has not been established, overexpression experiments suggest that chick Vg1 is important in primitive streak formation in chick (Shah et al., 1997; Skromne and Stern, 2002). By contrast, a loss-of-function mutant of mouse Gdf1 has normal germ layer specification, is viable to E14.5 and has left-right axis abnormalities (Rankin et al., 2000; Wall et al., 2000). In Xenopus, previous loss-of-function studies used a dominant-negative approach (Joseph and Melton, 1998). The Vg1 depletion phenotype caused using the antisense oligo approach is less severe than the ventralized phenotype caused by using mutant bVg1 ligands to disrupt Vg1 function (Joseph and Melton, 1998). There are also significant differences in the expression of zygotic genes caused by the two approaches. Here, we see no effect on Xnr3 expression and a severe decrease in chordin expression as a result of Vg1 depletion, whereas Joseph and Melton showed increased Xnr3 and chordin expression (Joseph and Melton, 1998). It is unlikely that the differences are due to different degrees of inactivation of Vg1, as, when Vg1 is depleted to below 50% of wild-type levels, embryos arrest at gastrulation. Also, the antisense depletion reduces chordin expression, whereas the dominant-negative approach increases it. It is possible that the mutant ligands used in the dominant-negative study were not specific for Vg1.

The effect of Vg1 depletion is also less severe than that caused by depletion of the localized maternal transcription factor VegT (Zhang et al., 1998). In Vg1-depleted embryos, derrière and Xnr1, and the endodermal genes Xsox17 and Gata5, continue to be expressed, and Smad2 phosphorylation occurs, albeit at a reduced level; these activities are completely abrogated by VegT depletion (Xanthos et al., 2001; Lee et al., 2001). Why does the presence of maternal Vg1 mRNA not alleviate the VegT-depletion phenotype? The likely explanation is that the VegT phenotype is in fact a compound phenotype, as VegT mRNA depletion causes the mis-localization of Vg1 mRNA, and leads to a reduction in the levels of Vg1 protein (Heasman et al., 2001). In support of this, the injection of a VegT morpholino oligo, which acts not by degrading VegT mRNA, but by blocking translation, does not affect Vg1 mRNA localization, and it causes a less severe phenotype than the regular VegT oligo does (Heasman et al., 2001).

Because Vg1 is the only dorsally localized maternally inherited Tgfß protein, it is likely that it initiates the first activation of Smad2 in the dorsal vegetal quadrant after the mid-blastula transition (MBT) (Lee et al., 2001). Activated Smad2 is known to bind to the maternal transcription factor Foxh1 (Chen et al., 1996), whose depletion also causes the loss of anterior structures (Kofron et al., 2004). In this model, the VegT-target Tgfßs, which are synthesized after MBT, constitute the second wave of signaling activity.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Active Vg1(S) allele rescues the Vg1 depletion phenotype. (A) Alignment of the N-terminal 39 amino acids of the sequences of Vg1 homologs from different Xenopus species. X. laevis, Xl Vg1(S) AY838794 and Xl Vg1(P) BC090232; X. tropicalis, Xt Vg1 AL849026; X. borealis, Xbo Vg1 AF041844. Asterisk indicates serine (S) versus proline (P) residues at position 20. Alignment of antisense oligo (Vg1A) sequence with Xl Vg1(S) and Xl Vg1(P) shows that it recognizes both forms. (B) A comparison of the profiles of Vg1(P) and Vg1(S) in western blots of oocytes and embryos (stage 10) injected with 300 pg of Vg1(P) or Vg1(S) mRNA and probed with the anti-Vg1 antibody D5, showing that the serine allele of Vg1 is more efficiently translated and processed than the proline allele. -Tub, -tubulin. (C) Vg1 depletion (Vg1-) causes a gastrulation delay, which is partially rescued by the re-introduction of Vg1(S) mRNA (200 pg) into Vg1-depleted oocytes. Un, uninjected; Vg1-, 6 ng Vg1A injected. At mid-gastrulation, Smad2 phosphorylation is also partially rescued by Vg1(S) mRNA. -Tub, -tubulin. (D) Vg1 depletion causes axial defects at tailbud stages and this phenotype can be partially rescued by Vg1(S) mRNA (200 pg). Un, uninjected; Vg1-, 6ng Vg1A injected. (E,F) Real-time RT-PCR shows that dorsal [chordin, cerberus, noggin and dickkopf (dkk)] and ventral marker expression (sizzled) can be partially rescued by Vg1(S) mRNA (200 pg). (G) Vegetal injection of Vg1 (200 pg) mRNA into fertilized eggs causes the upregulation of Xnr1 and Fgf8 to levels similar to those observed with Xnr5 (40 pg), and upregulation of Xsox17 and chordin to a lesser extent, during gastrulation. (H) Vg1 mRNA and protein are more abundant dorsally than ventrally at the 32-cell stage. Real-time RT-PCR analysis of wild-type embryos hemisected into dorsal and ventral halves at the 32-cell stage indicates that Vg1 mRNA is enriched in the dorsal halves while levels of VegT mRNA are equal. Western blot analysis of the dorsal and ventral halves at the 32-cell stage from the same experiment shows that Vg1 protein is more abundant in the dorsal halves (88% of control level) than in the ventral halves (64% of control level). mRNA from two whole embryos (WE), four dorsal (D) and four ventral (V) wild-type half embryos was used in the RT-PCR analysis. Four whole embryos (WE), eight dorsal (D) or eight ventral (V) wild-type half embryos were used for western blot analysis. The results were repeated in three separate experiments and a representative set is shown. -Tubulin (-Tub) was used as a loading control.

	ACKNOWLEDGMENTS

 
Many thanks to Dr Dan Kessler for supplying the Vg1 antibody and the Vg1(P) plasmid, and for much advice; to Aaron Zorn and Scott Rankin for the cerberus in situ probe; and to Helbert Puck and Mansoor Haque for technical assistance. This work was supported by NICHD RO1 HD33002.

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