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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. |
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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. |
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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. |
