The Nodal Signaling Pathway In Embryonic Development

我们正在进行的Nodal信号分析涉及到条件突变的使用：我们已经产生了一个条件“开花”等位基因，它与表达Cre重组酶的转基因品系一起在早期胚胎的不同谱系中使用。我们实验室关于Nodal的其他工作正在解决其表达的表观遗传调控问题。组蛋白修饰被认为是一种独立于它们在染色质过程中所起作用的表观遗传密码，组蛋白H3上的三甲基化赖氨酸4(H3K4me3)主要存在于活性启动子区域，三甲基化赖氨酸27(H3K27me3)标记基因被多梳蛋白阻遏。最近，胚胎干细胞中发育重要基因启动子中这两种修饰的存在被认为是一种表观遗传信号，标志着这些基因在发育后期的转录激活。我们已经发现了Nodal-Smad2/3通路与Polycomb抑制有关的证据。我们已经发现，在中胚层形成中，Wnt/β-catenin信号的靶标Nodal和brachyury都受到Polycomb抑制的调节，但我们现在已经确定它们也是Nodal途径的直接靶标。这两个基因都被多梳蛋白结合，并在没有Nodal信号的情况下显示H3K27me3抑制性组蛋白标记，或者在短缩的情况下显示H3K27me3抑制的组蛋白标记。我们进一步发现，Nodal-Smad2/3信号通路直接与H3K27me3去甲基酶Jmjd3结合，在靶基因座上抵消多聚簇的抑制。Smads2/3与Jmjd3之间的物理相互作用以及Jmjd3对靶基因的募集依赖于主动信号。然而，在没有Polycomb函数的情况下，靶基因的表达与Nodal信号无关。这些结果表明，Polycomb去抑制是Nodal-Smad2/3通路的一种新功能，并表明这可能是干细胞和早期胚胎的主要作用。

Our ongoing analysis of Nodal signaling involves the use of conditional mutagenesis: we have generated a conditional "floxed" allele, which is being used in conjunction with transgenic lines expressing Cre recombinase in various lineages of the early embryo. Additional work on Nodal in our lab is addressing the epigenetic regulation of its expression. Histone modifications have been proposed to function as an epigenetic code independent from their role in ongoing chromatin processes, with trimethylated lysine 4 on histone H3 (H3K4me3) mainly found in active promoter regions and trimethylated lysine 27 (H3K27me3) marking genes for repression by Polycomb proteins. The presence of both these modifications in promoters of developmentally important genes in embryonic stem (ES) cells recently has been suggested to function as an epigenetic signal marking these genes for transcriptional activation later in development. We have found evidence connecting the Nodal-Smad2/3 pathway with Polycomb de-repression. We have found that Nodal and Brachyury, a target of Wnt/beta-catenin signaling in mesoderm formation but which we have now identified as a direct target also of the Nodal pathway, are both regulated by Polycomb repression. Both genes are bound by Polycomb proteins and show H3K27me3 repressive histone marks in the absence of Nodal signaling, or in the absence of Nodal and Wnt signals in the case of Brachyury. We further found that the Nodal-Smad2/3 signaling pathway directly engages the H3K27me3 demethylase Jmjd3 to counteract Polycomb repression at target loci. Physical interaction between Smads2/3 with Jmjd3, and recruitment of Jmjd3 to target genes is dependent on active signaling. In the absence of Polycomb function, however, target loci are expressed independent of Nodal signaling. These results identify Polycomb de-repression as a novel function of the Nodal-Smad2/3 pathway, and suggest this may be a primary role in stem cells and perhaps the early embryo.