Dissecting Wnt/β-catenin signaling during gastrulation using RNA interference in mouse embryos
Dissecting Wnt/β-catenin signaling during gastrulation using RNA interference in mouse embryos
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DOI：
10.1242/dev.01842
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发表时间：
2005-06-01
期刊：
影响因子：
4.6
通讯作者：
Rossant, J
中科院分区：
文献类型：
--
作者：
Lickert, H;Cox, B;Rossant, J
在胚胎发育过程中，时间和空间上整合的差异基因调控驱动着发育程序。为了了解原肠形成程序是如何受Wnt/β-catenin信号调节的，我们使用了条件β-catenin突变胚胎的全基因组表达谱。在这些突变体中，已知的Wnt/β-catenin靶基因、其他信号通路的已知成分以及一些未鉴定的基因都下调了。为了进一步缩小差异表达基因的范围，我们使用整体原位筛选将基因表达与Wnt活性的假定结构域相关联。通过这种方法确定了几个潜在的新的靶基因，并利用RNA干扰(RNAi)对两个基因Grsf1和Fragilis2在完全胚胎干细胞来源的胚胎中进行了功能分析。我们发现，编码RNA结合因子Grsf1的基因对于轴向伸长、中/后脑发育和轴向中胚层规范是重要的，而编码跨膜蛋白的Fragilis2调节体节的上皮化和轴旁中胚层的形成。有趣的是，击倒表型概括了Wnt途径突变的几个方面，表明这些基因是下游Wnt反应的组成部分。这种功能基因组学方法允许从假定靶点的大数据集中快速识别胚胎发育的功能重要组成部分。
Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/beta-catenin signaling, we have used genome-wide expression profiling of conditional beta-catenin mutant embryos. Known Wnt/beta-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, we used whole-mount in situ screening to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. We show that the gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and that Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets.