XB-ART-14441Dev Biol August 15, 1998; 200 (2): 260-8.
We report the isolation and characterization of a new inhibitory Smad in Xenopus, which we have designated as Xenopus Smad7. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-beta-like signals is mediated by receptor ALK-4 and we show that Smad7 blocks signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway.
PubMed ID: 9705232
Article link: Dev Biol
Genes referenced: acta4 actc1 actl6a acvr1b alk bmp4 cer1 gsc krt12.4 smad7 tbxt tgfb1 ventx2.1
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|FIG. 1. Structure of Xenopus Smad7. Alignment of the predicted amino acid sequences of human Smad7 and Xenopus Smad7. Identical residues are shaded in dark. Mad homology domains, MH1 and MH2 are indicated by arrows.|
|FIG. 2. Localization of Smad7 during development. (Left) RNA isolated from embryos at various stages as indicated in the figure was analyzed by RT–PCR for the expression of Smad7 transcripts. (Right) Blastula-stage embryos dissected into different regions and cultured until equivalent of early gastrula stage. AP, animal pole; M/V, marginal zone and vegetal region; D, dorsal half; V, ventral half; E, whole embryo; 2RT, whole embryo extracted RNA in the absence of reverse transcriptase in the RT–PCR reaction.|
|FIG. 3. Smad7 blocks ALK-4-mediated mesoderm induction in explant assays. (A) Animal pole explants from embryos injected with 500 pg of CA-ALK-4 alone and coinjected with Smad7 (amounts as indicated) were analyzed by RT–PCR at stage 11 for the expression of mesoderm-specific markers expressed at gastrula stages. EF1-a transcripts used as an internal standard. Cer, cerberus; Gsc, goosecoid; Xbra, brachyury. 2RT lane not shown for this experiment. (B) Animal pole explants from embryos injected with 500 pg of ALK-4 alone and coinjected with Smad7 (amounts as indicated) were analyzed by RT–PCR at stage 17 for the expression of mesoderm and ectoderm differentiated markers. NCAM, neuralspecific marker; muscle actin, somitic mesoderm marker; 2RT, whole embryo extracted RNA in the absence of reverse transcriptase in the RT–PCR reaction.|
|FIG. 4. Smad7 activates the default neural pathway. (A) Explants from embryos injected with Smad7 (amounts indicated) alone and analyzed for the expression of differentiated markers for all germ layers. EF1-a transcripts used as an internal standard. NCAM, neuralspecific marker; edd, endodermin—panendoderm marker; XAG, cement gland marker; muscle actin, somitic mesoderm marker; epidermal keratin, epidermis marker; Xbra, Xenopus brachyury, panmesoderm marker. (B) Smad7 blocks BMP-4-mediated induction of mesoderm. Embryos were injected with 500 pg of BMP-4 RNA alone or coinjected with 500 pg of BMP-4 RNA and Smad7 RNA (amounts indicated). Explants at blastula stage were cultured until gastrula stage and analyzed for the expression of mesoderm markers by RT–PCR. 2RT, whole embryo-extracted RNA in the absence of reverse transcriptase in the RT–PCR reaction.|
|FIG. 5. Smad7 has similar effects on Smad1 and Smad2 activities. (Left) Animal pole explants from embryos injected with 1 ng of Smad1 alone and coinjected with Smad7 (amounts as indicated in ng) were analyzed by RT–PCR at stage 17 for the expression of globin, a ventral mesoderm marker. (Right) Animal pole explants from embryos injected with 1 ng of Smad1 alone and coinjected with Smad7 (amounts as indicated in ng) were analyzed by RT– PCR at stage 17 for the expression of muscle actin, somitic mesoderm marker. NCAM, neural-specific marker used as a positive control for Smad7; 2RT, whole embryo-extracted RNA in the absence of reverse transcriptase in the RT–PCR reaction.|
|FIG. 6. (Top) Smad7 blocks activation of mesoderm markers in the embryo. Albino embryos were injected with 25 pg of Smad7 RNA in the marginal zone of both blastomeres at the two-cell stage. The embryos were fixed at gastrula stage 10. One-half and whole mount in situ hybridization was performed using Gsc or Vox antisense probes. (A) Uninjected embryo—Gsc is expressed in the dorsal lip. (B) Embryo injected with Smad7—Gsc is no longer detected (n 5 22). (C) Uninjected embryo—Vox is expressed in the ventral mesoderm and excluded from the dorsal lip. (D) Embryo injected with Smad7—Vox is no longer detected (n 5 16).|
|FIG. 7. (Bottom) Smad7 blocks mesoderm formation and affects body axis in the embryo. Embryos injected with 25 pg of Smad7 RNA. (A) Uninjected embryos. (B) Histological section of uninjected embryos. (C) Embryos injected in a dorsal blastomeres at four-cell stage (n 5 37, 21). (D) Histological section of dorsally injected Smad7 embryos—note the lack of dorsal mesodermal tissue, an enlarged cement gland, and the presence of a neural tube. (E) Embryos injected with Smad7 RNA in ventral blastomeres at four-cell stage (n 5 28)—note incomplete secondary axes. (F) Histological section of embryos injected with Smad7 ventrally—note that the secondary axis does not contain any notochord or somitic mesoderm. cg, cement gland; nt, neural tube; s, somites; ov, otic vessicle; nt, notochord.|