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Signals from the transforming growth factor beta family members are transmitted in the cell through specific receptor-activated Smads and a common partner Smad4. Two Smad4 genes (alpha and beta/10, or smad4 and smad4.2) have been isolated from Xenopus, and conflicting data are reported for Smad4beta/10 actions in mesodermal and neural induction. To further understand the functions of the Smad4s in early frog development, we analyzed their activities in detail. We report that Smad10 is a mutant form of Smad4beta that harbors a missense mutation of a conserved arginine to histidine in the MH1 domain. The mutation results in enhanced association of Smad10 with the nuclear transcription corepressor Ski and leads to its neural inducing activity through inhibition of bone morphogenetic protein (BMP) signaling. In contrast to Smad10, both Smad4alpha and Smad4beta enhanced BMP signals in ectodermal explants. Using antisense morpholino oligonucleotides (MOs) to knockdown endogenous Smad4 protein levels, we discovered that Smad4beta was required for both activin- and BMP-mediated mesodermal induction in animal caps, whereas Smad4alpha affected only the BMP signals. Neither Smad4 was involved directly in neural induction. Expression of Smad4beta-MO in early frog embryos resulted in reduction of mesodermal markers and defects in axial structures, which were rescued by either Smad4alpha or Smad4beta. Smad4alpha-MO induced only minor deficiency at late stages. As Smad4beta, but not Smad4alpha, is expressed at high levels maternally and during early gastrulation, our data suggest that although Smad4alpha and Smad4beta may have similar activities, they are differentially utilized during frog embryogenesis, with only Smad4beta being essential for mesoderm induction.
FIGURE 1.
Both the long and short forms of Smad10 induce neural markers at high doses when they also inhibit BMP signaling. A, Smad10 and its N-terminal 32-amino acid deletion isoform (Smad10δN) both induce neural markers when expressed at high doses. B, Smad10 and Smad10δN inhibit BMP signaling at similar doses as those required for neural induction. The doses of Smad10 RNA were: 0.125, 0.25, 0.5, 1, 2, 4, and 8 ng. BMP2 RNA was injected at 0.25 ng. Animal caps from injected embryos were dissected at blastula stage 9, and RNA was extracted from the caps when sibling embryos reached tailbud stages 28-32.
FIGURE 2.
Smad4α and Smad4β differ from Smad10 in regulation of the TGFβ signals, and a point mutation in the MH1 domain of Smad4β converts it to a Smad10-like molecule. A, sequence difference between Smad4β and Smad10 in the MH1 domain of the molecules. Mutation of the conserved arginine has been identified in human cancer patients. B, Smad4α and Smad4β induce the blood marker globin, whereas Smad10 and Smad4β(R132H) induce the neural marker NRP-1 and the cement gland marker XAG-1. 4 ng of RNAs were used. C, at high doses, Smad4α and Smad4β ventralize mesodermal induction by activin, whereas Smad10 and Smad4β(R132H) inhibit activin and BMP. 2 ng of Smad4/10, 1 pg of activin, and 10 pg of BMP4 RNAs were used. The animal caps were harvested at stages 11 or 32.
FIGURE 3.
Smad10, but not Smad4α or Smad4β, constitutively binds to the transcription corepressor Ski. Two ng of RNAs encoding the tagged proteins were injected into the two-cell stage embryos. Protein extract was made at early gastrula stages and coimmunoprecipitation was performed to examine protein-protein interaction. IP, immunoprecipitation; IB, immunoblotting.
FIGURE 4.
Smad4β is required for activin- and BMP-induced mesoderm formation, whereas Smad4α is required only for BMP-mediated mesoderm induction. Neither Smad4s is involved directly in neural induction. A, the Smad4-MOs were specific in blocking translation only from messenger RNAs that contain the corresponding 5′-UTR sequences. B and C, depletion of Smad4β resulted in inhibition of activin- and BMP-induced marker expression at both gastrula (B) and tadpole (C) stages; but Smad4α-MO only blocked the marker induction by BMP4. Neither MO inhibited neural gene induction by noggin. The dose of the MOs used was 20 ng, and the RNAs were injected at 2.5 pg for activin, 10 pg for BMP4, and 10 pg for noggin. D, rescue of MO repression of marker expression by both Smad4α and Smad4β suggests that the two Smad4s have similar activities. The doses of the MOs used were 20 ng, and the doses of RNAs used were: 1 pg of activin, 10 pg of BMP4, 100 pg of Smad4α and Smad4β. The assays were performed at gastrula (stage 11) or tadpole (stage 32) stages.
FIGURE 5.
Knockdown of endogenous Smad4β protein levels leads to embryos with axial defects. A, injection of total 20 ng of Smad4β-MO into the animal poles of two-cell stage embryos results in reduction of the head structures, malformation of the eyes, and tail defects in the embryos. Smad4α-MO only induced minor defects in the dorsal fin, and a control MO did not induce significant axial defects. B, injection of 20-40 ng of Smad4β-MO into the marginal zone region of two-cell stage embryos induced more severe axial defects, with the morphant embryos showing gastrulation defects and reduced body axis.
FIGURE 6.
Knockdown of Smad4β leads to reduction of mesodermal and neural markers in the morphant embryos. A, injection of 20-40 ng of Smad4β-MO in two-cell stage embryos led to reduction of the mesodermal markers Brachyury (Xbra), Chordin, MyoD, and Wnt8 at gastrula stages, and the reduction was only slightly enhanced by Smad4α-MO. Smad4α-MO alone or the control MO did not affect marker expression. B, at neurula stages, the notochordal expression of Xbra and the paraxial mesodermal gene MyoD were both reduced in Smad4β morphant embryos. Co-injection of Smad4α-MO and Smad4β-MO resulted in further reduction of these markers. The pan-neural marker Sox2 was also down-regulated in embryos expressing Smad4β-MO. Control MO and Smad4α-MO did not significantly inhibit expression of these genes. C, expression of either Smad4α-MO or Smad4β-MO led to reduction of globin expression in the resulting tadpoles. In A, the embryos were viewed from the vegetal pole of gastrula embryos with the dorsal side to the right. In B, the embryos were viewed from the dorsal side with anterior to the left. In C, the embryos were oriented with the anterior to the left and viewed from the lateral side.
FIGURE 7.
Rescue of Smad4β morphant embryos with Smad4α or Smad4β. Both Smad4α and Smad4β (with modified UTR sequences) rescued the marker expression in Smad4β morphant embryos at gastrula (A) and neurula (B) stages. Embryos in A were viewed from the vegetal side with the dorsal quadrant to the right, whereas embryos in B were viewed from the dorsal side with the anterior to the left. 20-40 ng of Smad4β-MO were injected, and 100 pg of Smad4α or Smad4β RNAs were used in the rescue experiments.
FIGURE 8.
Reduction of neural markers in Smad4β morphant embryos is associated with reduction of mesodermal genes. A single blastomere injection of 20 ng of Smad4β-MO with the lineage tracer nuclear β-galactosidase at the two-cell stage resulted in suppression of the muscle marker 12/101 epitope and the neural marker Sox2 on the injected side. Injection of the control MO or Smad4α-MO did not block these markers. The embryos were viewed from the dorsal (a, d, and g) or lateral (b, c, e, f, h, and i) sides. The injected side (labeled with β-galactosidase) was viewed in the right column, and the control uninjected side was viewed in the center column. The left column shows the dorsal view of the embryos with both injected and uninjected sides.
