Xenbase Image ID: 135809

Figure 2.  Smad6 induces secondary axes and neuralizes ectoderm in Xenopus. (A) Smad6 overexpression induces the formation of a secondary axis. Smad6 RNA (0.5–4 ng) was coinjected with nuclear β-galactosidase(β-gal)RNA (100 pg) into the ventral vegetal blastomeres of eight cell stage embryos. Unlike control embryos injected with β-gal alone (bottom right), Smad6 coinjected embryos develop an ectopic dorsal axis (left and top right). β-Galactosidase staining of the injected embryos reveals that the progeny of the injected blastomere directly contribute to the ectopic axis. Dorsal atleft; anterior at top. Lateral at right; anterior atleft. (B) Smad6 does not affect formation of the primary dorsal axis. Smad6 RNA (1–4 ng) was injected into the dorsal marginal zone of four cell stage embryos. Compared to control embryos (left panel), Smad6-injected tadpoles display enlarged heads and cyclopia, but have normal body axes (right). (C) Secondary axis formation by the activin signaling molecule Smad2 is enhanced by coinjection of Smad6.Smad2 RNA (1 ng), Smad6 RNA (1 ng), or both RNAs (1 ng each) were injected into ventral vegetal blastomeres of eight cell stage embryos. Coinjection of Smad2 and Smad6produces secondary axes (red arrows) that are more complete than those obtained by injection of either Smad alone. Anterior is attop. (D) Smad6 neuralizes ectodermal explants. Smad6 and Smad6 C-domain RNAs were injected at the indicated amount in the animal pole of two-cell stage embryos. At the blastula stage, animal caps were dissected and cultured in saline solution. At the tailbud stages (stage 22), total RNA was harvested and analyzed by RT–PCR for the presence of the indicated transcripts. Full-length human Smad6 (0.5 and 4 ng) induces NRP-1, a pan–neural marker, andXAG-1, a marker of cement gland. Cement gland is induced efficiently even at the lower dose; induction of neural tissue requires a higher dose of Smad6. The isolated C-domain RNA is a more potent inducer of both XAG-1 and NRP-1 than full-length Smad6. Neither construct induced muscle actin, a marker of dorsal (paraxial) mesoderm. EF-1α, ubiquitously expressed, is a loading control. RNA from whole embryos (Embryo) provides the positive control. The RT lane is identical to the embryo lane, except that reverse transcriptase was omitted. (E) Smad6 interferes with blood induction by a constitutively active BMP receptor. BMPR-IB(QD) RNA (1–1000 pg) was injected either alone or together with Smad6 RNA (250 pg) in the animal pole.BMPR-IB(QD)-injected ectodermal explants show induction ofglobin at stage 30, and this response is blocked by coexpressed Smad6. Image published in: Hata A et al. (1998) Copyright © 1998. Image reproduced on with permission of the Publisher, Cold Spring Harbor Laboratory Press. This is an Open Access article. Larger Image Printer Friendly View

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