Kessler DS and Melton DA (1995), Induction of dorsal mesoderm by soluble, mature...

XB-ART-19534

Development 1995 Jul 01;1217:2155-64.

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Induction of dorsal mesoderm by soluble, mature Vg1 protein.

Kessler DS , Melton DA .

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Mesoderm induction during Xenopus development has been extensively studied, and two members of the transforming growth factor-beta family, activin beta B and Vg1, have emerged as candidates for a natural inducer of dorsal mesoderm. Heretofore, analysis of Vg1 activity has relied on injection of hybrid Vg1 mRNAs, which have not been shown to direct efficient secretion of ligand and, therefore, the mechanism of mesoderm induction by processed Vg1 protein is unclear. This report describes injection of Xenopus oocytes with a chimeric activin-Vg1 mRNA, encoding the pro-region of activin beta B fused to the mature region of Vg1, resulting in the processing and secretion of mature Vg1. Treatment of animal pole explants with mature Vg1 protein resulted in differentiation of dorsal, but not ventral, mesodermal tissues and dose-dependent activation of both dorsal and ventrolateral mesodermal markers. At high doses, mature Vg1 induced formation of 'embryoids' with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system. Furthermore, truncated forms of the activin and FGF receptors, which block mesoderm induction in the intact embryo, fully inhibited mature Vg1 activity. To examine the mechanism of inhibition, we have performed receptor-binding assays with radiolabeled Vg1. Finally, follistatin, a specific inhibitor of activin beta B which is shown not to block endogenous dorsal mesoderm induction, failed to inhibit Vg1. The results support a role for endogenous Vg1 in dorsal mesoderm induction during Xenopus development.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a bmp2 eef1a2 fst gdf1 gsc myc ncam1 nog tbxt wnt8a

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	Fig. 1. (A) Chimeric Vg1 constructs. Schematic representation of activin bB, BMP2, Vg1, and chimeric BMP2-Vg1 and activin-Vg1. Members of the TGF-b superfamily, these genes contain an signal sequence, pro-region, tetrabasic cleavage site and mature region. Activin bB and BMP2, but not Vg1, form disulfide-linked dimers that are subsequently cleaved, releasing the mature C-terminal peptide as a secreted bioactive dimer. The chimeric constructs, fused four amino acids downstream of the cleavage site, are designed to facilitate processing and secretion of mature Vg1. (B) Western blot analysis of oocyte supernatants. Oocytes were injected with the indicated mRNA and conditioned supernatants (10 ml) analyzed by western blotting of reducing SDS-PAGE using activin-specific (left) or Vg1-specific (right) antisera. Activin bB mRNA directs secretion of mature protein. While Vg1 mRNA directs no secretion and BMP2-Vg1 directs low levels of secretion, activin-Vg1 mRNA results in secretion of abundant mature Vg1 protein.
	Fig. 2. (A) Mature Vg1 induces morphogenetic movements in ectodermal explants. Blastula-stage animal pole explants were treated with 10% supernatant (1/10 dilution) until the gastrula stage and cultured to the neurula stage (stage 15). Mature Vg1 (Vg) results in a dramatic elongation indicative of mesoderm induction (Symes and Smith, 1987). Activin bB (bB) results in similar elongation, while supernatant of uninjected oocytes (CT) have no effect. Scale bar, 300 mm. (B) Dose-dependent induction of mesodermal markers by mature Vg1. Blastulastage animal pole explants were treated with increasing doses of mature Vg1 supernatant (1%, 3%, 10%, 30%), or control (30%) or activin bB supernatant (10%), cultured to the neurula stage and analyzed by RT-PCR. At low doses (1%) the general marker Xbra is induced; at intermediate doses (3%) the ventrolateral marker Xwnt8 and the dorsolateral marker cardiac actin (M. Actin) are induced; and at high doses (10- 30%) the dorsoanterior markers goosecoid (Gsc) and noggin (Nog), and the neural marker NCAM are induced. Activin bB treatment results in a similar response and control supernatants have no effect. EF1a is a loading and reverse transcription control and the embryo and embryo-RT are additional positive and negative controls. (C) Induction of embryoids by mature Vg1. Blastula animal pole explants treated with a high dose of mature Vg1 and cultured to the late tadpole stage (stage 40), differentiated into embryoids displaying a rudimentary axial organization, with anterior-posterior pattern and head structures. The embryoid shown (top right) has a clear head-to-tail pattern and pigmented eye and cement gland. A histological section (bottom right) reveals differentiated notochord (no), somitic muscle (sm), neural tube (nt), eye (e) and cement gland (cg). Treatment with supernatant of uninjected oocytes has no effect and explants form atypical epidermis (left). Scale bar, 150 mm.
	Fig. 3. Mature Vg1 is not bound by the activin type II receptor. Uninjected oocytes, or oocytes expressing a myc-tagged activin type II receptor (XARmyc) were bound with radiolabeled activin bB or mature Vg1, complexes chemically crosslinked, immunoprecipitated with a myc-specific antiserum and visualized by 15% reducing SDSPAGE and fluorography. Co-precipitating activin is released from complexes by reducing the crosslinker, and is therefore resolved as an ~14´103 Mr band. Uninjected oocytes did not bind radiolabeled activin bB (lane 1) and XARmyc-expressing oocytes displayed no binding in the absence of radiolabeled ligand (lane 2). Receptorexpressing oocytes strongly bound activin (lane 3) and this binding was competed by a 10-fold excess of unlabeled activin (lane 4). In contrast, a 10-fold excess of unlabeled mature Vg1 was unable to compete activin binding (lane 5). Furthermore, a 10-fold higher dose of radiolabeled Vg1 than that required for activin binding did not result in detectable binding (lane 6). The higher molecular weight bands (lanes 3, 5) may be due to non-reduced ligand-receptor complexes and co-precipitating unprocessed activin.
	Fig. 4. Follistatin does not inhibit endogenous dorsal mesoderm induction. Both blastomeres of 2-cell-stage embryos were injected with b-galactosidase mRNA (A) or follistatin mRNA (B) (1 ng total mRNA) and cultured to the tadpole stage. Follistatin resulted in a loss of posterior structures with maintenance or enhancement of dorsoanterior structures. Histological analysis reveals the presence of dorsal mesodermal tissues in both b- galactosidase (C)- and follistatin (D)-injected embryos (no, notochord; sm, somitic muscle; nt, neural tube; cg, cement gland). (E) Follistatin does not inhibit expression of mesodermal markers at the gastrula stage. 2-cell-stage embryos were injected with a total of 2 ng of b- galactosidase (bGal) or follistatin (XFS) mRNA, harvested at the mid-gastrula stage (stage 11) and analyzed by RT-PCR. The expression of a general mesodermal marker, brachyury (Xbra), the dorsal markers, goosecoid (Gsc) and noggin (Nog), and a ventrolateral marker, Xwnt8, were unaffected by follistatin. EF1a is a loading and reverse transcription control. Scale bars, 1 mm (A,B) and 0.5 mm (C,D).
	Fig. 5. Follistatin does not inhibit mature Vg1 activity. Blastulastage animal pole explants were treated with control (A,B), activin bB (C,D), or mature Vg1 (E,F) supernatants following mixture and preincubation with control (A,C,E) or follistatin (B,D,F) supernatants. While follistatin fully blocks activin-induced morphogenetic movements, induction by mature Vg1 was unaffected. (G) Follistatin fails to inhibit cardiac actin (M. Actin) induction by mature Vg1. Blastula animal pole explants were treated with mature Vg1 or activin only, or with these supernatants preincubated with follistatin. Upon reaching the neurula stage, samples were analyzed by RT-PCR. While follistatin fully inhibits cardiac actin induction by activin, no effect on mature Vg1 activity is detected. EF1a is a loading and reverse transcription control. Scale bar, 300 mm.
	Fig. 6. Mature Vg1 does not dorsalize gastrula ventral marginal zone explants. Dorsal or ventral marginal zone explants were prepared from blastulae (stage 8) or early gastrulae (stage 10.25) and incubated in supernatants (30%) containing activin, mature Vg1 or noggin, or in a supernatant of uninjected oocytes (control). At the neurula stage, samples were analyzed by RT-PCR. While all three factor induced cardiac actin (M. Actin) in blastula ventral marginal zone explants, only noggin did so in gastrula-stage explants. EF1a is a loading and reverse transcription control.