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The Smad proteins have been implicated in the intracellular signaling of transforming growth factor-beta (TGF-beta) ligands. Here we describe the function of Smad5 in early Xenopus development. Misexpression of Smad5 in the embryo causes ventralization and induces ventral mesoderm. Moreover, Smad5 induces epidermis in dissociated ectoderm cells which would otherwise form neural tissue. Both of these activities require Smad4 (DPC4) activity. We propose that Smad5 acts downstream of the BMP4 signaling pathway in Xenopus embryos and directs the formation of ventral mesoderm and epidermis.
FIG. 1. Smad5 ventralizes Xenopus embryos and induces ventral mesoderm. (A) Smad5 ventralizes Xenopus embryos. 2.0 ng of Smad5
RNA was injected into the animal pole of 2-cell stage embryos and developed until sibling embryos reached tadpole (stage 33). Uninjected
sibling embryos are shown as a control. (B) Smad5 induces ventral mesoderm in animal caps. Blastula stage animal caps isolated from
embryos injected with a graded amount of Smad5 RNA were cultured until sibling embryos reached gastrula (stage 11.5) and tadpole
(stage 27). The amount of RNA used is indicated on the figure. (C) Smad5 promotes epidermal induction in dissociated ectoderm cells.
Animal caps obtained in B were dissociated for 5 hr and reaggregated as described under Materials and Methods. The reaggregated cells
were harvested at neurula stage (stage 19) and subjected to RT–PCR. Undiss, undissociated animal caps. 0RT indicates embryos without
reverse transcriptase. Histone H4 is used as a loading control.
FIG. 2. Induction of mesoderm and epidermis by Smad5 requires
Smad4 activity. (A) Smad5 or tSmad4 RNA (1.0 ng) was
injected separately or together into the animal pole of 2-cell embryos. Animal caps from blastulae were cultured and harvested
at gastrula stage. (B) Smad5 RNA (125 or 1000 pg) were injected
alone (lanes 5 and 6) or with 1.0 ng of tSmad4 RNA (lanes 8 and
9) into animal pole of 2-cell embryos. Animal caps were isolated
at blastula stage and dissociated for 5 hr as described under Materials and Methods.
