Shah SB et al. (1997), Misexpression of chick Vg1 in the marginal zone...

XB-ART-15810

Development 1997 Dec 01;12424:5127-38. doi: 10.1242/dev.124.24.5127.

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Misexpression of chick Vg1 in the marginal zone induces primitive streak formation.

Shah SB , Skromne I , Hume CR , Kessler DS , Lee KJ , Stern CD , Dodd J .

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In the chick embryo, the primitive streak is the first axial structure to develop. The initiation of primitive streak formation in the posterior area pellucida is influenced by the adjacent posterior marginal zone (PMZ). We show here that chick Vg1 (cVg1), a member of the TGFbeta family of signalling molecules whose homolog in Xenopus is implicated in mesoderm induction, is expressed in the PMZ of prestreak embryos. Ectopic expression of cVg1 protein in the marginal zone chick blastoderms directs the formation of a secondary primitive streak, which subsequently develops into an ectopic embryo. We have used cell marking techniques to show that cells that contribute to the ectopic primitive streak change fate, acquiring two distinct properties of primitive streak cells, defined by gene expression and cell movements. Furthermore, naive epiblast explants exposed to cVg1 protein in vitro acquire axial mesodermal properties. Together, these results show that cVg1 can mediate ectopic axis formation in the chick by inducing new cell fates and they permit the analysis of distinct events that occur during primitive streak formation.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a chrd eef1a2 gdf1 gsc myc nog tbxt tgfb1

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Fig. 4. Mesoderm-inducing activity of COS cell-generated cVg1 protein. (A) Western blot of COS cell-conditioned media probed with anti-Vg1 mAb, FC1.4F6. Lane 1 is a marker ladder. Conditioned media from COS cells transfected with native cVg1, cVg1-myc and dsl-cVg1 were applied to the gel. (B) Induction of early mesoderm markers in Xenopus animal cap explants assessed by RTPCR. 1:200 of affinity-purified cVg1 protein induced expression of brachyury (Xbra) and noggin (Nog) in explants cultured to the late gastrula stage. A 5-fold higher concentration of cVg1 induced expression of the organizer markers goosecoid (Gsc), chordin (Chd) and noggin in addition to brachyury. This activity mimics the profile of Xenopus Vg1 supernatants obtained from oocytes injected with activin-Xenopus Vg1. Control supernatants did not induce mesoderm markers. (C-E) Animal caps cultured to the tadpole stage. (C) Untreated animal caps develop into ciliated epidermis. (D) Caps cultured in the presence of dsl-cVg1-transfected COS cell-conditioned medium, diluted 1:10 undergo elongation movements indicative of dorsal mesoderm induction. (E) Section of cVg1-treated cap labelled with the muscle marker 12/101 indicating skeletal muscle. Scale bar: C, 200 mm; D, 250 mm; E, 30 mm.

Fig. 8. cVg1 is processed and secreted when expressed in Xenopus oocytes. (A) Western blot analysis of conditioned supernatants and cell extracts probed with anti-Xenopus Vg1 MAb, FC.14F6. Native Xenopus Vg1 (Vg1) is not cleaved or secreted into the medium. An activin-Xenopus Vg1 chimera (Activin-Vg1) is cleaved and secreted. cVg1 precursor is abundant within the cell and both precursor and cleaved mature domain are secreted into the supernatant. Expression of dorsalin-cVg1 (dsl-cVg1) chimera leads to enhanced secretion of the mature peptide. Multiple bands ranging from 50 to 70 k Da in the cell pellets lanes are thought to represent unprocessed precursor Vg1 protein carrying different length propeptides and undergoing different amounts of breakdown. (B) Induction of muscle actin in animal caps by oocyte-conditioned supernatants diluted to 10% and 30%. Supernatant conditioned by oocytes expressing native Xenopus Vg1 does not induce muscle actin (M.Actin), but medium conditioned by chimeric activin-Xenopus Vg1 and diluted to 30% does induce expression of muscle actin. CVg1 produced from either native cVg1 or dsl-cVg1 induces muscle actin when supplied as a 10% dilution of supernatant. A 2% dilution of supernatant containing activin induces muscle whereas control supernatant has no effect. EF1a was used as a control for loading and reverse transcription. Embryo and embryo-RT are additional positive and negative controls, respectively.