Endoderm Formation and Patterning in the Mouse

定形内胚层是早期胚胎的三个初级胚层之一， 3原始肠管，这反过来又有助于各种组织，包括甲状腺，胸腺， 肝脏、肺和消化道。来自定形内胚层的信号也在过程中发挥着重要作用 心脏和前部CNS的诱导和模式化。我们最近发现了Smad 2， TGF β/激活素/nodal信号下游效应子作为内胚层形成的重要调节因子 在老鼠。为了剖析潜在的独特和/或重叠的Smad 2/3活性，Smad 2缺陷 用表达构建体转染的ES细胞将测试它们定殖于细胞的能力。 定形内胚层谱系。表达不同表达的新“敲入”等位基因的功能活性 还将检测内源性Smad 2调控元件控制下的Smad 2/3亚型。到 为了评估TGF β信号在内胚层衍生物形成过程中的作用，我们 将利用Smad 2和Smad 4的条件等位基因与转基因小鼠品系组合 表达Cre在不同的时间和空间域的肠道谱系，包括发展中国家， 肝脏和胰腺锌指转录抑制因子Blimp 1已被证明控制 内胚层与中胚层的青蛙胚胎的命运。我们将生成并分析 小鼠Blimp 1基因座的功能突变，以测试内胚层中可能的保守功能 阵最后，为了鉴定内胚层形成所需的靶基因，我们进行了 使用Smad 2、Smad 4、Foxhl和Mix-1缺陷型ES细胞进行转录谱分析， 在培养中分化成内胚层。本提案中概述的实验旨在提高我们的 了解Smad 2和其他转录介质如何控制细胞命运、增殖和 内胚层细胞谱系的分化。

The definitive endoderm, one of the three primary germ layers of the early embryo gives rise to the 3rimitive gut tube, which in turn contributes to a diverse set of tissues including the thyroid, thymus, liver, lungs and digestive tract. Signals from the definitive endoderm also play essential roles during the induction and patterning of the heart and anterior CNS. We recently identified Smad2, an effector downstream of TGFb/activin/nodal signals as an essential regulator of endoderm formation in the mouse. To dissect potentially unique and/or overlapping Smad2/3 activities, Smad2 deficient ES cells transfected with expression constructs will be tested for their ability to colonize the definitive endoderm lineage. Functional activities of novel "knock-in" alleles expressing different Smad2/3 isoforms under control of endogenous Smad2 regulatory elements will also be tested. To evaluate the roles of TGFb signaling during patterning and formation of endodermal derivatives, we will exploit conditional alleles of Smad2 and Smad4 in combination with transgenic mouse strains expressing Cre in different temporal and spatial domains of the gut lineage, including the developing liver and pancreas. The zinc finger transcriptional repressor Blimp1 has been shown to control endodermal versus mesodermal fates in the frog embryo. We will generate and analyze a loss of function mutation at the mouse Blimp1 locus to test for possibly conserved function in endoderm formation. Finally to identify target genes required for endoderm formation we perform transcriptional profiling using Smad2, Smad4, Foxhl, and Mix-1 deficient ES cells induced to differentiate into endoderm in culture. Experiments outlined in this proposal aim to enhance our knowledge of how Smad2 and other transcriptional mediators control cell fate, proliferation and differentiation of the endodermal cell lineage.