Endoderm Formation and Patterning in the Mouse

描述（由申请人提供）：定形内胚层是早期胚胎的三个主要胚层之一，产生原肠管，而原肠管又有助于形成各种组织，包括甲状腺、胸腺、肝脏、肺和消化道。来自定形内胚层的信号在心脏和前部CNS的诱导和模式化过程中也起重要作用。我们最近发现Smad 2是TGFb/激活素/节点信号下游的效应子，是小鼠内胚层形成的重要调节因子。为了剖析潜在独特和/或重叠的Smad 2/3活性，将测试用表达构建体转染的Smad 2缺陷型ES细胞定殖定形内胚层谱系的能力。还将测试在内源性Smad 2调节元件控制下表达不同Smad 2/3同种型的新型“敲入”等位基因的功能活性。为了评估TGF β信号传导在内胚层衍生物的形成和形成过程中的作用，我们将利用Smad 2和Smad 4的条件等位基因与在肠道谱系的不同时间和空间域（包括发育中的肝脏和胰腺）中表达Cre的转基因小鼠品系组合。锌指转录抑制因子Blimp 1已被证明控制青蛙胚胎中的内胚层与中胚层命运。我们将在小鼠Blimp 1基因座产生并分析功能缺失突变，以测试内胚层中可能保守的功能。 阵最后，为了鉴定内胚层形成所需的靶基因，我们使用Smad 2、Smad 4、Foxh 1和Mix-1缺陷的ES细胞进行转录谱分析，所述ES细胞在培养物中被诱导分化成内胚层。本提案中概述的实验旨在提高我们对Smad 2和其他转录介质如何控制内胚层细胞系的细胞命运、增殖和分化的认识。

DESCRIPTION (provided by applicant): The definitive endoderm, one of the three primary germ layers of the early embryo gives rise to the primitive 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, Foxh1, 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.