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INTRODUCTIONThe Tgf� family member Vg1 was one of the first examples of aspecific mRNA localized in eggs (Melton, 1987; Rebagliati et al.,1985). During oogenesis, Vg1 mRNA becomes restricted to thevegetal cytoplasm of the oocyte, and is inherited by the most vegetalcells of the embryo (Weeks and Melton, 1987). The vegetal cells ofthe blastula have two important functions: they differentiate into theendoderm of the embryo, and they signal to the adjacent marginal(equatorial) region to form the mesoderm (Dale et al., 1985). Vg1therefore became a strong candidate to be the mesoderm-inducingsignal. However, the in vivo function of Vg1 has proven difficult toassess. Whereas the overexpression of other Tgf� family memberscauses mesoderm formation (Jones et al., 1995; Koster et al., 1991;Smith et al., 1990), Vg1 mRNA does not (Dale et al., 1989; Tannahilland Melton, 1989). Furthermore, endogenous mature Vg1 proteincannot be detected, and although Vg1 pro-protein can easily bedetected from the translation of exogenous mRNA, it is not secretedand cleaved (Dale et al., 1989; Tannahill and Melton, 1989). Gain-and loss-of-function experiments have been performed usingchimeric 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 remainsthat such chimeric proteins may not reflect endogenous function,particularly as recent studies have shown that the pro-domains ofTgf� family members are important determinants of signalingfunction (Cui et al., 2001; Jones et al., 1996; Le Good et al., 2005).A loss-of-function experiment was performed in which the Bmppro-domain was linked to a putative dominant-negative form of theVg1 mature protein (Joseph and Melton, 1998). This constructblocked the action of overexpressed bVg1, and caused a loss ofdorsal mesodermal and dorsal axial structures, and the loss ofexpression of the endodermal marker Xlhbox8. In the absence ofprocessing of the full-length Vg1, or of detectable levels of themature form, these facts have generated a long-standing paradoxover 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 factorwhose mRNA is also localized in the vegetal cells of the embryo(Zhang and King, 1996). Depletion of the maternal stockpile of VegTmRNA abrogates formation of the endoderm and the mesoderm(Zhang et al., 1998). VegT activates the transcription of at least sixzygotic Tgf� family members (Xanthos et al., 2001). If nodalsignaling is blocked, then mesoderm induction is blocked (Agius etal., 2000; Kofron et al., 1999). Any in vivo function of Vg1 musttherefore be reconciled with these facts.

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

MATERIALS AND METHODSOligonucleotidesOligonucleotides (oligos) were as follows:

Vg1A (antisense), 5�-G*C*C*ATGTAACCTTG*A*G*G-3�, wherephosphorothioate-modified residues are indicated by an asterix; and

Vg1MO (morpholino), 5�-CCACAGTCTCAGCCACACCATACTG-3�.

Real-time PCRTotal RNA was prepared using the proteinase K method and RNase-freeDNase treatment before cDNA synthesis (Zhang et al., 1998). cDNA wassynthesized using oligo dT primers. Random hexamer (R6) primers wereused where indicated. Real-time RT-PCR was carried out using the LightCycler System (Roche), using the primers and cycling conditions describedpreviously (Birsoy et al., 2005; Kofron et al., 1999; Kofron et al., 2004;Zhang et al., 1998).

Oocytes and embryosFull-grown oocytes were manually defolliculated and cultured in oocyteculture medium (OCM), as described previously (Xanthos et al., 2001).Oocytes were injected vegetally with the antisense oligo (Vg1A) or themorpholino (Vg1MO) and cultured for 48 hours at 18°C beforematuration. Control uninjected oocytes were cultured in the same way ineach experiment. All of the oocytes were matured by addition of 2 �Mprogesterone in OCM and cultured for another 12 hours. Controluninjected and oligo-injected oocytes were then labeled with vital dyesand fertilized using the host transfer technique, as described (Zuck,1998).

Vg1 is an essential signaling molecule in XenopusdevelopmentBilge Birsoy, Matt Kofron, Kyle Schaible, Chris Wylie and Janet Heasman*

Xenopus Vg1, a transforming growth factor � (Tgf�) family member, was one of the first maternally localized mRNAs identified invertebrates. Its restriction to the vegetal pole of the egg made it the ideal candidate to be the mesoderm-inducing signal releasedby vegetal cells, but its function in vivo has never been resolved. We show that Vg1 is essential for Xenopus embryonicdevelopment, and is required for mesoderm induction and for the expression of several key Bmp antagonists. Although the originalVg1 transcript does not rescue Vg1-depleted embryos, we report that a second allele is effective. This work resolves the mystery ofVg1 function, and shows it to be an essential maternal regulator of embryonic patterning.

KEY WORDS: Vg1, Tgf�, Xenopus, Antisense, Bmp antagonist, Maternal localization

Development 133, 15-20 doi:10.1242/dev.02144

Division of Developmental Biology, Cincinnati Children’s Hospital ResearchFoundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

*Author for correspondence (e-mail: heabq9@chmcc.org)

Accepted 4 October 2005

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

For histology, tailbud-stage embryos were fixed in Bouin’s fixative forthree hours, dehydrated and embedded in paraplast, serially sectioned andstained with Hematoxylin and Eosin.

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

Western blotsWestern 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 SignalingTechnology; 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 Gelsoftware (V. 1.5) was used to quantify protein levels.

RESULTSTo study Vg1 function, we targeted Vg1 mRNA using an antisenseoligo (Zuck, 1998), which depletes Vg1 mRNA in a dose-responsivefashion (Fig. 1A), and results in a dose-dependent reduction in Vg1protein levels (Fig. 1B). After fertilization, the amount of Vg1mRNA 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 below50% arrests development at the gastrula stage (data not shown). Asimilar delayed gastrulation phenotype results when Vg1 protein isdepleted (Fig. 1B) using a morpholino oligo, Vg1MO (Table 1B).

Vg1-depleted embryos develop normally until the gastrula stage,but then have a severe, dose-dependent abnormality in gastrulationwhen compared with controls, where the timing of blastoporeformation is delayed, and the blastopore remains enlarged at the lategastrula stage (Fig. 1C; Table 1A). Whole-mount in situ hybridization

of Vg1-depleted embryos shows that cerberus and chordin expressionis reduced during gastrulation in a dose-dependent manner (Fig. 1C).

At the neurula and tailbud stages, Vg1-depleted embryos havedifferent degrees of anteroposterior and dorsoventral axisabnormality (Fig. 1C; Table 1C). Typically, 6 ng of antisense oligocauses embryos to develop with a loss of head structures, whereaslower doses result in stunted embryos. Histological sections oftailbud-stage embryos show that all three germ layers are present inVg1-depleted embryos; however, an absence of the notochord andfusion of somites in the midline are observed, with much reducedand abnormal neural structures.

Development of the early embryo involves the interplay of at leastthree signaling pathways; the Wnt signaling pathway (Heasman etal., 1994) activates the expression of the target genes siamois andXnr3; the VegT pathway activates the expression of endodermalgenes and nodal-related proteins (Xanthos et al., 2001), and initiatesSmad2 phosphorylation (Lee et al., 2001), and the Bmp pathwayactivates Smad1 phosphorylation (Graff et al., 1996; Lee et al.,2001). In Vg1-depleted embryos, the expression of Xnr3 andsiamois is not significantly altered (Fig. 1D), suggesting that Vg1 isnot required for maternal Wnt pathway activation. However, Vg1 isrequired for Smad2 phosphorylation, as Smad2 phosphorylation isreduced 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 embryosincreases, but does not reach wild-type levels. This correlates withthe timing of the onset of expression of the VegT target genederrière, which occurs normally in both control and Vg1-depletedembryos (data not shown). By contrast, Bmp signaling via phospho-Smad1 is initially normal in Vg1-depleted embryos, but isreproducibly elevated (in three experiments) as gastrulation proceeds(Fig. 1E; arrowhead). We conclude that Vg1 signaling is required forSmad2 phosphorylation at the late-blastula stage, and to preventexcess Bmp signaling at the gastrula stages.

Because Smad2 phosphorylation is essential for mesoderminduction, we tested whether Vg1 depletion reduces the mesoderm-inducing signals released by vegetal cells at the blastula stage byperforming Nieuwkoop assays. Vg1-depleted vegetal masses co-

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Table 1. Vg1 depletion causes delayed gastrulation and axial defects that are partially rescued by Vg1(S) mRNAGastrula Total number Normal Delayed

A† Uninjected 65 61 (93.8%) 4 (6.2%)Vg1A (4 ng) 57 18 (31.6%) 39 (68.4%)Vg1A (6 ng) 43 5 (11.6%) 38 (88.4%)

B‡ Uninjected 94 90 (95.7%) 4 (4.3%)MO (45 ng) 66 6 (9.1%) 60 (90.9%)

Tailbud Total number Normal Vg1 phenotype*

C§ Uninjected 39 34 (87.2%) 5 (12.8%)Vg1A (4 ng) 24 9 (37.5%) 15 (62.5%)Vg1A (6 ng) 21 0 (0.0%) 21 (100%)

Gastrula Total number Normal Delayed

D† Uninjected 50 48 (96.0%) 2 (4.0%)Vg1A (6 ng) 39 7 (17.9%) 32 (82.1%)

Vg1A+Vg1(S) mRNA 41 23 (56.1%) 18 (43.9%)

Tailbud Total number Normal Vg1 phenotype*

E† Uninjected 92 86 (93.5%) 6 (6.5%)Vg1A (6 ng) 55 0 (0.0%) 55 (100.0%)

Vg1A+Vg1(S) mRNA 46 7 (15.2%) 39 (84.8%)

*The Vg1 phenotype includes loss of head structures and stunted embryos, as shown in Fig. 1C and Fig. 2D.†Composite data from three experiments.‡Composite data from four experiments.§Composite data from two experiments.

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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% ofcontrol levels) and that no zygotic transcription of Vg1 is detected during gastrulation. (B) Vg1 protein is depleted in a dose-dependent manner bythe 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% and58% 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 forcerberus (cerb) and chordin (chd) shows that the expression of cerb and chd is reduced at mid-gastrula stage in a dose-dependent manner. Inhistological 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 �-catenin/Xtcf3 target genes Xnr3 and siamois is unaffected by Vg1depletion. (E) Vg1 depletion reduces the phosphorylation of Smad2 (arrow; 34% of control level at stage 9.5) and increases the phosphorylation ofSmad1 (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 analysisshows that Vg1 depletion reduces the induction of Xbra, Fgf8 and chordin in wild-type animal caps. One whole embryo at stage 11 (WE) was usedfor quantification. (G) Real-time RT-PCR analysis of embryos during gastrula stages shows that Vg1 depletion causes a downregulation of dorsallyexpressed cerberus, chordin, noggin and dickkopf (dkk), and an upregulation of dorsal marker Xnr1 and ventral marker sizzled, without affectingthe levels of Xsox17� and Bmp4.

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cultured with wild-type animal caps have a reduced mesoderm-inducing ability compared with wild-type vegetal masses, asmeasured by the induction of the general mesodermal markers Xbraand Fgf8, and the anterior endo-mesodermal marker chordin, in wildtype animal caps (Fig. 1F).

We reasoned that increased Smad1 phosphorylation in Vg1-depleted embryos may indicate that Vg1 controls the expression ofBmp antagonists. In support of this, the levels of expression ofchordin, noggin and cerberus are reproducibly reduced to 20% orless of control levels in Vg1-depleted embryos (in three experiments)at the gastrula stage (Fig. 1G). Also, because these proteins normallyrestrict the range of activity of Bmp to the posteroventral quadrant(Khokha et al., 2005), their reduction may cause a concomitantincrease in the expression of Bmp target genes. We find that theventrolaterally expressed Bmp-target gene sizzled (Salic et al., 1997)is upregulated in Vg1-depleted embryos at the mid-gastrula stage,whereas the level of Bmp4 mRNA expression is unchanged (Fig. 1G).

To confirm that this phenotype is specifically caused by Vg1depletion, we attempted to rescue Vg1-depleted embryos byreintroducing Vg1 mRNA prior to fertilization. Previous studies inwhich Vg1 mRNA was overexpressed used an allele, Vg1(Pro), thatcontains 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 Xenopusembryos but is very inefficiently processed (Fig. 2B) (Dale et al.,1993; Tannahill and Melton, 1989); in addition, it does not havemesoderm-inducing activity, and is unable to rescue Vg1-depletedembryos (data not shown). However, we recently identified a secondallele of Vg1, Vg1(Ser) (Birsoy et al., 2005), that is equallyrepresented with Vg1(Pro) in oocyte and gastrulae libraries, and ismore efficiently processed than Vg1(Pro) (Fig. 2B). The Xenopustropicalis and Xenopus borealis Vg1 homologs, also have a serinerather than proline residue at the equivalent position (Fig. 2A). As theantisense oligo Vg1A is complementary to both serine and prolinealleles (Fig. 2A), it was expected that it would bind and deplete themboth with the same efficiency. We were unable to confirm this, asneither the PCR primers nor the Vg1 antibody can differentiatebetween the two allelic forms. Unlike Vg1(Pro), Vg1(Ser) mRNApartially rescues the phenotype of Vg1-depleted embryos (Fig. 2C,D;Table 1D,E), Smad2 phosphorylation (Fig. 2C) and the expression ofmolecular 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 alsohave an inducing activity when overexpressed in embryos. We findthat Vg1(Ser) has some mesoderm-inducing activity in animal caps,whereas Vg1(Pro) does not in the same experiment (data notshown). Fig. 2G shows that when Vg1(Ser) mRNA is overexpressedin the vegetal area, it causes the upregulation of the endodermal andmesodermal zygotic genes chordin, Xnr1, Fgf8 and Xsox17�. Wecompared the inducing activity of Vg1 with another Tgf� familymember, Xnr5. Xnr5 is a zygotic gene regulated by VegT, and, likeVg1, is restricted in its expression to vegetal cells (Takahashi et al.,2000). In comparison to Xnr5, 200 pg of Vg1 mRNA induces Fgf8and Xnr1 to a similar extent as does 40 pg of Xnr5 mRNA at theearly gastrula stage. Interestingly, Xnr5 induces Xsox17� andchordin in mid-gastrula stage embryos more effectively than Vg1.

In the current model of axis formation in Xenopus, Wnt targetgenes are activated on the dorsal side as a result of the corticalrotation and dorsal concentration of localized maternal Wnt11mRNA at the early cleavage stage (Tao et al., 2005). Because thetargets of Vg1 activity, chordin, cerberus and dickkopf, are all firstexpressed 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 withventral cells by hemisecting 32-cell stage embryos into dorsal andventral halves. Fig. 2H shows that both Vg1 mRNA and protein aremore concentrated in the dorsal halves than in the ventral halves atthe 32-cell stage. Here, protein and mRNA levels are examined indorsal and ventral halves taken from the same batch of embryos, andthe experiment was repeated three times with the same result. Thecomparison of oligo dT (dT)- versus random hexamer (R6)-primedcDNA shows that the enrichment of Vg1 mRNA on the dorsal sideis not due to differential polyadenylation (data not shown).

DISCUSSIONVg1 is required for anterior developmentHomologs of Xenopus Vg1 are expressed maternally in zebrafish(DVR1) (Helde and Grunwald, 1993), and at early embryonic stagesin 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 inprimitive streak formation in chick (Shah et al., 1997; Skromne andStern, 2002). By contrast, a loss-of-function mutant of mouse Gdf1has normal germ layer specification, is viable to E14.5 and has left-right axis abnormalities (Rankin et al., 2000; Wall et al., 2000). InXenopus, previous loss-of-function studies used a dominant-negativeapproach (Joseph and Melton, 1998). The Vg1 depletion phenotypecaused using the antisense oligo approach is less severe than theventralized phenotype caused by using mutant bVg1 ligands to disruptVg1 function (Joseph and Melton, 1998). There are also significantdifferences in the expression of zygotic genes caused by the twoapproaches. Here, we see no effect on Xnr3 expression and a severedecrease in chordin expression as a result of Vg1 depletion, whereasJoseph and Melton showed increased Xnr3 and chordin expression(Joseph and Melton, 1998). It is unlikely that the differences are dueto different degrees of inactivation of Vg1, as, when Vg1 is depletedto below 50% of wild-type levels, embryos arrest at gastrulation. Also,the antisense depletion reduces chordin expression, whereas thedominant-negative approach increases it. It is possible that the mutantligands used in the dominant-negative study were not specific for Vg1.

The effect of Vg1 depletion is also less severe than that caused bydepletion of the localized maternal transcription factor VegT (Zhanget al., 1998). In Vg1-depleted embryos, derrière and Xnr1, and theendodermal genes Xsox17� and Gata5, continue to be expressed,and Smad2 phosphorylation occurs, albeit at a reduced level; theseactivities are completely abrogated by VegT depletion (Xanthos etal., 2001; Lee et al., 2001). Why does the presence of maternal Vg1mRNA not alleviate the VegT-depletion phenotype? The likelyexplanation is that the VegT phenotype is in fact a compoundphenotype, as VegT mRNA depletion causes the mis-localization ofVg1 mRNA, and leads to a reduction in the levels of Vg1 protein(Heasman et al., 2001). In support of this, the injection of a VegTmorpholino oligo, which acts not by degrading VegT mRNA, but byblocking translation, does not affect Vg1 mRNA localization, and itcauses a less severe phenotype than the regular VegT oligo does(Heasman et al., 2001).

Because Vg1 is the only dorsally localized maternally inheritedTgf� protein, it is likely that it initiates the first activation of Smad2in the dorsal vegetal quadrant after the mid-blastula transition(MBT) (Lee et al., 2001). Activated Smad2 is known to bind to thematernal transcription factor Foxh1 (Chen et al., 1996), whosedepletion also causes the loss of anterior structures (Kofron et al.,2004). In this model, the VegT-target Tgf�s, which are synthesizedafter MBT, constitute the second wave of signaling activity.

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Fig. 2. Active Vg1(S) allele rescues the Vg1 depletion phenotype. (A) Alignment of the N-terminal 39 amino acids of the sequences of Vg1homologs from different Xenopus species. X. laevis, Xl Vg1(S) AY838794 and Xl Vg1(P) BC090232; X. tropicalis, Xt Vg1 AL849026; X. borealis, XboVg1 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 moreefficiently translated and processed than the proline allele. �-Tub, �-tubulin. (C) Vg1 depletion (Vg1-) causes a gastrulation delay, which is partiallyrescued 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 thisphenotype 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) Vegetalinjection 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 thanventrally 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 indicatesthat 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-PCRanalysis. Four whole embryos (WE), eight dorsal (D) or eight ventral (V) wild-type half embryos were used for western blot analysis. The results wererepeated in three separate experiments and a representative set is shown. �-Tubulin (�-Tub) was used as a loading control.

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This study resolves the long-standing paradox over the in vivofunction of Vg1. The original clone, with proline at position 20 of theprodomain, is less efficiently processed than the version of the proteinwith serine at this position. Overexpressed Vg1(Ser) is secreted(Birsoy et al., 2005), whereas Vg1(Pro) is not secreted (Dale et al.,1989; Tannahill and Melton, 1989). The Vg1(Ser) clone is likely tobe a second pseudoallele, resulting from the pseudo-tetraploidy of theXenopus laevis genome, as equal numbers of serine (13 ESTs) andproline (14 ESTs) forms exist in the EST databases, and both X.borealis and X. tropicalis sequences have a serine at this position.Other Vg1-related genes in zebrafish, chick and mouse genomes aredivergent in this region of the prodomain when compared withXenopus Vg1. Although Vg1(Ser) is not an efficiently secretedprotein when compared with Xnr5 (Birsoy et al., 2005), we find that,unlike Vg1(Pro), it does induce mesodermal and endodermal geneexpression when the mRNA is injected into the vegetal area of theXenopus embryo (Fig. 2G). In addition, it has much less inducingactivity when overexpressed in animal caps (data not shown),indicating the importance of correct localization in development.

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 cerberusin situ probe; and to Helbert Puck and Mansoor Haque for technical assistance.This work was supported by NICHD RO1 HD33002.

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RESEARCH ARTICLE Development 133 (1)