Role of Notch signaling in cell fate decision during gastrulation of Xenopus embryo

Notch信号在神经发生中的作用已有较详细的研究，但在原肠胚胚胎中的Notch信号尚不清楚。1.为了了解Notch信号在原肠胚期的作用，我们利用吗啉寡核苷酸(MO)分析了非洲爪哇无毛抑制因子(XSu(H))的功能。XSu(H)2-MO引起原肠发育异常，导致脊索和体细胞中胚层严重缺陷。对XSU(H)2下游因子的研究表明Xoct25和91的基因表达受XSU(H)2的调控。启动子分析和芯片分析表明XSU(H)2通过直接与启动子上的SPS基序结合来调控Xoct25的基因表达。2.为了了解MasterMind的作用，将Xenopus Mainder Homoloue(XMam1)注射到4细胞期胚胎的腹缘区域。XMam1在预期的腹侧中胚层中的过度表达导致了过度的细胞团。将XMam1-MO注射到预期的背侧中胚层，导致原肠发育缺陷和头部畸形，表明XMam1参与了胚轴的形成。使用截断形式的XMam1和Notch信号抑制剂的实验结果表明，XMam1可以在没有Notch信号的情况下诱导次级轴。为了了解Notch信号在原肠胚发育过程中的作用，利用基因芯片技术对新发现的Notch靶基因进行了分析。筛选确定了11个候选基因。然而，在功能获得实验中，只有一个基因导致原肠发育缺陷。我们将这个新的靶基因命名为非洲爪哇atf1，XATF1。XATF1的显性负性形式导致了Xbrachyury基因的下调和严重的原肠发育缺陷。用吗啉寡核苷酸敲除XSU(H)2后，XSU(H)2产生了类似的缺陷胚胎，但XATF1的联合注射挽救了XSU(H)2。这些结果表明，XSU(H)2和XMam1以Notch依赖和非Notch依赖的方式调控非洲爪哇胚胎原肠形成过程中的细胞命运。

Notch signaling in neurogenesis has been studied in detail, whereas Notch signaling in gastrula embryo remains unknown.1.In order to understand the role of Notch signaling at gastrula stage, we analyzed a function of Xenopus Suppressor of Hairless (XSu(H)) using morpholino oligos (MO). XSu(H)2-MO caused abnormal gastrulation, which resulted in severe defects of the notochord and somitic mesoderm. Studies on the downstream factors of XSu(H)2 showed that gene expression of Xoct25 and 91, Xenopus Oct4 homologues, are regulated by XSu(H)2. Promoter analyses and ChIP assays showed that XSu(H)2 regulates the gene expression of Xoct25 by direct binding to SPS motif on the promoter.2.In order to understand the role of Mastermind, Xenopus mastermind homologue (XMam1) was injected into ventral marginal zone of 4-cell stage blastomeres. The over-expression of XMam1 in prospective ventral mesoderm caused the excessive cell mass. Injection of XMam1-MO into prospective dorsal mesoderm caused defective gastrulation and head deformity, suggesting that XMam1 is involved in the embryonic axis formation. Experimental results using truncated form of XMam1 and Notch signaling inhibitors showed that XMam1 can induce secondary axis without Notch signaling.3. In order to understand the role of Notch signaling at gastrula stage, novel Notch target genes were examined by using microarray analysis. The screening identified 11 candidate genes. However, in gain of function experiment, only one gene caused the defective gastrulation. We designated this novel target gene Xenopus ATF1, XATF1. The dominant negative form of XATF1 caused the down-regulation of Xbrachyury and the severe gastrulation defect. Knockdown of XSu(H)2 using morpholino oligo DNA caused the similar defective embryo, which was rescued by co-injection of XATF1.These results suggest that XSu(H)2 and XMam1 regulate the cell fate in the gastrulation of Xenopus embryo both in Notch-dependent and Notch-independent manner.