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被深入研究，但Notch信号在原肠胚发育中的作用尚不清楚。1.为了了解Notch信号在原肠胚发育中的作用，我们利用morpholino oligos（MO）分析了非洲爪蟾无毛抑制因子（XSu（H））的功能。XSu（H）2-MO引起原肠胚形成异常，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，Xoct 25和Xoct 91（非洲爪蟾Oct 4同源物）的基因表达受XSu（H）2调控。启动子分析和ChIP分析表明XSu（H）2通过与启动子上的SPS基序直接结合来调控Xoct 25基因的表达。XMam 1在前腹中胚层中的过表达导致了细胞团的过多。将XMam 1-MO注射到预期的背侧中胚层中导致原肠胚形成缺陷和头部畸形，表明XMam 1参与了胚胎轴的形成。使用截短形式的XMam 1和Notch信号传导抑制剂的实验结果表明，XMam 1可以在没有Notch信号传导的情况下诱导次级轴.为了了解Notch信号在原肠胚阶段的作用，通过使用微阵列分析来检查新的Notch靶基因。筛选确定了11个候选基因。但在功能获得实验中，只有一个基因导致了原肠胚形成缺陷。我们将这个新的靶基因命名为Xenopus ATF 1，XATF 1。XATF 1的显性负性形式导致Xbrachyury的下调和严重的原肠胚形成缺陷。用morpholino oligo DNA敲除XSu（H）2基因，也可导致类似的缺陷胚胎，而XATF 1基因则可挽救该缺陷胚胎。这些结果表明，XSu（H）2和XMam 1在非洲爪蟾胚胎原肠胚形成过程中均以Notch依赖和非Notch依赖的方式调控细胞命运。

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.