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Fig. 3. Blocking both Smad1 and Smad2 signals leads to efficient neural induction in Xenopus ventral ectoderm. (A) At neurula stages, the truncated activin receptor tActRIB did not induce and tBMPRIA only weakly induced the neural markers Sox2 and Sox3. When the two truncated receptors were co-expressed, the neural markers were induced strongly to a level similar to that induced by the truncated type II receptor tActRIIB. One nanogram of each RNA was used. (B) Co-expression of tActRIB (1 ng) with Smad6 (0.1 ng) or VP16-Msx1 (0.1 ng) in ventral animal cells led to induction of the neural markers Sox2 and Sox3 in ventral ectoderm of frog neurulae. |
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Fig. 2. Neural induction by inhibitors of Smad1 and Smad2 signaling occurs in the absence of the mesoderm in Xenopus. Double in situ hybridizations showed that neural marker induction in embryos injected with tActRIIB, Smad7 or Ski occurred in the absence of the mesodermal markers Chordin and MyoD. The red speckled staining is from the injected lineage tracer. |
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Fig. 1. Neural induction in Xenopus ventral ectoderm by ectopic expression of inhibitors of TGFβ signals. RNAs encoding transmembrane (1 ng tBMPRIA and tActRIIB), cytoplasmic (0.1 ng Smad6 and Smad7) or nuclear (0.1 ng VP16-Msx1 and Ski) inhibitors of TGFβ signaling were co-injected with nβGal (0.1 ng RNA) into one ventral animal blastomere of 32- to 64-cell stage embryos. The embryos were analyzed by Red-Gal staining (red speckled stain) and in situ hybridization of the neural (Sox2 and Sox3), neural crest (Slug) and epidermal (XK70) markers at neurula stages. Inhibitors of both Smad1 and Smad2 signaling (tActRIIB, Smad7 and Ski) induced neural markers efficiently. Among the specific inhibitors of Smad1 signaling, tBMPRIA induced Sox2 and Sox3 weakly, whereas Smad6 and VP16-Msx1 were ineffective in inducing neural markers. |
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Fig. 7. Activation of Smad2 signaling converts neural tissue to neural crest and mesodermal tissues in Xenopus. (A) In the absence of DEX, leaky GR-Smad2 activity was sufficient for neural crest induction, but not sufficient for inhibition of neural markers or induction of mesodermal genes. Activation of GR-Smad2 by DEX (2 μM) at mid-gastrula stages led to inhibition of Sox2 and Sox3 and simultaneous induction of the mesodermal markers MyoD and Chordin in the neural plate (seen more clearly in Fig. 7B and Fig. 8). GR-Smad2 RNA (0.1-0.2 ng) was used. The embryos were orientated with the head toward the left and viewed from the dorsal side. (B) Induction of mesodermal markers by activated GR-Smad2 occurred in the neural plate, as shown in transversely bisected (top) or sectioned (bottom) embryos. |
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Fig. 8. The ability to inhibit neural markers by activated Smad2 attenuates during gastrulation. Treatment of Xenopus embryos expressing GR-Smad2 with DEX at different stages during gastrulation showed that activated GR-Smad2 lost its neural inhibitory activity by the end of gastrulation, at which stages it also failed to induce mesoderm in the neural plate. All the embryos were viewed from the dorsal side with the anterior to the left. |
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Display additional annotations [+]
| Gene |
Clone |
Species |
Stages |
Anatomy |
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twist1
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xenopus
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NF stage 28
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neural crest
,
branchial arch
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head
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otx2
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xenopus
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NF stage 28
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olfactory placode
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brain
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eye
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lens placode
,
head
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myod1
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xenopus
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NF stage 28
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presomitic mesoderm
,
somite
,
muscle
,
trunk somite
,
trunk
,
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Fig. 10. Activation of Smad2 at gastrula stages in the neural plate leads to defective neural development in Xenopus. (A) Activation of GR-Smad2 (0.2-0.5 ng) at mid-gastrula stages (stage 11) in the neural tissue induced neural defects in frog tadpoles. Embryos showed reduced head structures and malformed or missing eyes. Embryos without DEX treatment developed normally. (B) Histological analyses indicated that neural development at both anterior (upper panels) and posterior (lower panels) trunk levels was defective when Smad2 signaling was activated. The neural tube was disrupted and ectopic notochord (yellow arrowhead) and mesenchyme (red arrowhead) were observed in the neural derivatives. (C) In situ hybridization demonstrated that Sox2 was reduced and split from the midline and Otx2 was reduced, but the neural crest marker Twist and the muscle marker MyoD were unaffected. All embryos were viewed from the lateral side with the anterior to the left, except the second column of Sox2 in panel C, which was viewed from the dorsal direction. |
