The Nodal Signaling Pathway In Embryonic Development

我们正在进行的Nodal信号传导分析涉及使用条件诱变：我们已经产生了一个条件性的“floxed”等位基因，该等位基因与在早期胚胎的各种谱系中表达Cre重组酶的转基因系结合使用。我们实验室对Nodal的其他工作是解决其表达的表观遗传调控。组蛋白修饰被认为是一种独立于其在染色质过程中的作用的表观遗传密码，组蛋白H3上的三甲基化赖氨酸4（H3 K4 me 3）主要存在于活性启动子区域，三甲基化赖氨酸27（H3 K27 me 3）标记基因被Polycomb蛋白抑制。这两种修饰的存在下，在胚胎干细胞（ES）的发育重要基因的启动子最近已被建议作为一个表观遗传信号标记这些基因的转录激活后，在发展。我们已经发现了将Nodal-Smad 2/3途径与Polycomb去阻遏联系起来的证据。我们已经发现，Nodal和Brachyury（中胚层形成中Wnt/β-连环蛋白信号传导的靶标，但我们现在已经鉴定为Nodal途径的直接靶标）都受到Polycomb抑制的调节。两种基因都被Polycomb蛋白结合，并且在Nodal信号传导不存在的情况下或在Brachyury的情况下在Nodal和Wnt信号不存在的情况下显示H3 K27 me 3抑制性组蛋白标记。我们进一步发现，Nodal-Smad 2/3信号通路直接与H3 K27 me 3去甲基化酶Jmjd 3结合，以抵消靶基因座处的Polycomb阻遏。Smads 2/3与Jmjd 3之间的物理相互作用以及Jmjd 3向靶基因的募集依赖于主动信号传导。然而，在不存在Polycomb功能的情况下，靶基因座独立于Nodal信号传导而表达。这些结果将Polycomb去抑制鉴定为Nodal-Smad 2/3通路的新功能，并表明这可能是干细胞和早期胚胎中的主要作用。

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.