Joseph EM and Melton DA (1998), Mutant Vg1 ligands disrupt endoderm and mesoder...

XB-ART-14741

Development 1998 Jul 01;12514:2677-85. doi: 10.1242/dev.125.14.2677.

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Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos.

Joseph EM , Melton DA .

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The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a agtr1 bmp2 chrd fst gdf1 ncam1 nodal nodal1 nodal3 nodal3.2 not pdx1 serpina1 tbx2 tbxt tgfb1 tgfb2 twist1 ventx1.2 wnt8a

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	Fig. 1. Site-directed mutagenesis used to construct mutations in the mature region of Vg1. (A) Schematic diagram of the mature region of Vg1, based on a figure in Schlunegger and Grutter (1992). The mature protein acts as a dimer connected at the asterisk to the same residue of another subunit. Mutants were designed by presuming that the structure of Vg1 is like of TGFb2, and are numbered accordingly (Daopin et al., 1992; Schlunegger and Grutter, 1992). The mutant changes were: m23/24/25: Glu to Gly/Phe to Tyr/Lys to Gln; m42/43: Asn to Ser/Asn to Ser; m69: Glu to Gly; m69/71: Glu to Gly/Glu to Gly; m77: Cys to Gly; m78: Cys to Ser; m109: Cys to Gly; m111: Cys to Ser; and m109/111: Cys to Gly/Cys to Ser. (B) Animal cap assay. Fertilized Xenopus laevis eggs were injected with 2-4 ng of capped mRNA and animal cap explants were cut from blastula (stage 8) and cultured along with sibling control embryos. (C) RT-PCR analysis of mesodermal markers. Animal caps were injected with 2 ng of various mutant BVg1 mRNAs and analyzed for expression of muscle actin and Xbra, two mesodermal markers, at stage 17. The BVg1 loop mutant m69/71 and cysteine mutants m78, m109 and m109/111 lost the ability to induce muscle actin and Xbra. These results were confirmed by animal cap assays using 4 ng of the mutant mRNAs (data not shown). EF1-a is used as an RT-PCR control for RNA recovery. Other controls include analysis of uninjected explants, sibling-stage whole embryos, and whole-embryo RNA analyzed without reverse transcriptase (RT).
	Fig. 2. Specificity of mutant ligands tested in a coinjection animal cap assay. (A) The ability of mutant Vg1 ligands to block Vg1 signaling was assayed by coinjecting BVg1 mRNA with increasing concentrations of mutated BVg1 mRNAs. Injection of 10 pg BVg1 mRNA induces muscle actin and Xbra in animal caps (assayed by RT-PCR at stage 17). This concentration of BVg1 mRNA was coinjected with a 10-fold, 50-fold and 100-fold excess of different mutated BVg1 mRNAs. The m109/111 mRNA blocked induction of muscle actin and Xbra at a 50-fold and 100-fold excess. Coinjection with a 50-fold excess of the m109/111 BVg1 mRNA also blocked induction by AVg1 mRNA (data not shown). AVg1 is a construct similiar to BVg1 but with the activin pro-region rather than the BMP2 pro-region (Kessler and Melton, 1995). (B) In a similiar experiment, coinjection with a 100-fold excess of the m78 mRNA blocked induction of muscle actin and Xbra. The m69/71 and m109 mutant constructs did not block induction, even at a 100-fold excess (data not shown). (C) The mutants’ specificity was assayed using a coinjection animal cap assay, in which 2 pg of activin bB mRNA (BB) were coinjected with increasing concentrations of mutant BVg1 mRNA. At a 100-fold excess, the m109/111 mRNA did not block induction by activin (assayed by RTPCR at stage 17-25). Even at a 500-fold excess of mutant mRNA, activin signaling was not blocked. A control for the integrity of the mutant mRNA included induction by BVg1 and a block of that induction by a 100-fold excess of the mutant mRNA. (D) In a similiar experiment to that shown in C, activin signaling was not blocked by a 100- fold excess of the m78 mRNA. These data show that the m78 and m109/111 mutants do not block signaling by activin. (E) The mutants’ specificity was further analyzed using a coinjection animal cap assay, in which 500 pg Xenopus nodal-related mRNA (encoding Xnr1, 2 or 4) was coinjected with 2 ng mutant BVg1 mRNA. In this experiment, signaling by the Xenopus nodal-related factors (Xnr1,2,4) was not blocked by the m109/111 mutant mRNA (as assayed by RT-PCR at stage 25). (F) In a similiar experiment to that of E, Xnr signaling was not blocked by the m78 mutant mRNA.
	Fig. 3. Phenotype of whole embryos injected with mutant Vg1 mRNA. Mutant Vg1 mRNA, m109/111, was injected subequatorially at the one-cell stage, such that half of the sample was injected on one side of the embryo (2 ng/10 nl) and the other half on the opposite side. (A) Severely affected tadpole-stage embryos (at stage 35). (A1a) A severely affected embryo injected with m109/111 BVg1 mRNA. (A1b) A severely affected embryo injected with m109/111 AVg1 mRNA. The phenotypes of embryos injected with m78 BVg1 mRNA were not as extreme as those shown here, consistent with the less efficient block of Vg1 signaling by m78 BVg1 mRNA compared to m109/111 BVg1 mRNA (see Figs 2A,B and 4). (A1c) A histological section of the embryo shown in A1b. Note the lack of any histological differentiation of mesoderm or endoderm. (A2) A sibling control of the embryo shown in A1a. (B) Analysis of markers by RT-PCR at stage 35. Severely affected mutant Vg1 embryos, such as those shown in A, do not form dorsal mesoderm (lane 1), and muscle actin and the notochord marker Xnot are not expressed; however, expression of the ventral mesodermal marker aT1-globin is slightly increased relative to EF1-a controls. The neural marker NCAM and the endodermal marker Xlhbox8 are not expressed. Results from sibling control tadpoles are shown in lane 2; lane 3 is a control embryo analyzed without reverse transcriptase. (C) Severely affected neurula-stage embryos (at stage 15). (C1) An embryo injected with the m109/111 BVg1 mRNA construct. (C2) An embryo injected with tAR mRNA. (C3) A sibling-stage control embryo. (D) Analysis of markers by RT-PCR at stage 14/15. Embryos were analyzed at the early neurula stage so that expression of muscle actin could be assayed; both the m109/111 BVg1 and tAR mRNA embryos analyzed here were shown not to express muscle actin (data not shown). Injection of m109/111 BVg1 mRNA blocks expression of Xtwi (Hopwood et al., 1989) but does not block expression of Xbra, Xwnt8 or Xvent-1 in whole embryos (lane 1), whereas injection of tAR mRNA completely blocks expression of all of these mesodermal markers except for Xvent-1, where expression is greatly reduced (lane 2). Lane 3 is an uninjected embryo control and lane 4 is a control embryo treated without reverse transcriptase.
	Fig. 4. Examples of the less severe phenotype of tadpole-stage embryos injected with mutant Vg1 mRNA. (A1) An embryo injected with the m109/111 AVg1 construct; (A2) an embryo injected with the m109/111 BVg1 construct; (A3) an embryo injected with the m78 BVg1 construct; (A4) a sibling stage control embryo. (B) RTPCR analysis of the embryos shown in A. Lane 1 is the embryo shown in A1, lane 2 is the embryo shown in A2, lane 3 is the embryo shown in A3, lane 4 is the sibling-stage control embryo shown in A4, and lane 5 is a no-reverse transcriptase control. These less severely affected embryos lack expression of NCAM, Xlhbox8 and Xnot; they differ from the extreme affected embryos shown in Fig. 3 in that they express muscle actin. This level of msucle actin expression is reduced compared to the level of expression in the sibling-stage control embryo and the level of aT1-globin expression is slightly increased relative to this control.
	Fig. 5. In situ hybridization analysis of mutated Vg1-injected embryos. Embryos were injected at the one-cell stage with m109/111 BVg1 mRNA (as described in the Fig. 3 legend) and whole-mount in situ hybridizations were performed at the early gastrula stage with antisense digoxigenin probes. A representative of each batch of injected embryos is shown, together with a representative of each batch of uninjected control embryos. Injection of mutated Vg1 mRNA did not disrupt expression of Xbra, as it did for expression of follistatin (XFS) at this stage, although there was often a lighter area within the domain of Xbra expression, as shown here. Xnr3 and chordin are both highly expressed in the marginal zone regions of injected embryos; Vox-15 expression is not blocked or expanded in comparison to the uninjected controls (see Discussion).
	Fig. 6. Vegetal pole explant assay for the effects of mutant Vg1 ligand in the absence of mesoderm. The mutant Vg1 effect on the expression of Xlhbox8 was studied by RT-PCR analysis of injected vegetal pole explants (Henry et al., 1996; Gamer and Wright, 1995). 4 ng of mutant Vg1 mRNAs were injected into the vegetal poles of embryos at the one-cell stage. At stage 9, vegetal pole explants were cut and cultured until stage 33-35. These vegetal pole explants lack expression of the mesodermal marker muscle actin. Autonomous expression of Xlhbox8 was shown in uninjected vegetal pole explants (Gamer and Wright, 1995; Henry et al., 1996). Injection of mutant Vg1 mRNA inhibited this autonomous expression of Xlhbox8, without induction of NCAM.