Endoderm Formation and Patterning in the Mouse

描述（由申请人提供）：最终的内胚层，早期胚胎的三个主要胚层之一，产生原始的肠管，这反过来又有助于多种组织的形成，包括甲状腺，胸腺，肝脏，肺和消化道。来自最终内胚层的信号在心脏和前中枢神经系统的诱导和模式中也起着重要作用。我们最近发现Smad2是TGFb/激活素/节点信号的下游效应因子，是小鼠内胚层形成的重要调节因子。为了分析潜在的独特和/或重叠的Smad2/3活性，将测试转染表达构建的Smad2缺陷ES细胞定植最终内胚层谱系的能力。在内源性Smad2调控元件的控制下，表达不同Smad2/3亚型的新型“敲入”等位基因的功能活性也将被测试。为了评估TGFb信号在内胚层衍生物的模式和形成过程中的作用，我们将利用Smad2和Smad4的条件等位基因与表达Cre的转基因小鼠菌株在肠道谱系的不同时间和空间域中的结合，包括发育中的肝脏和胰腺。锌指转录抑制因子Blimp1已被证明可以控制青蛙胚胎的内胚层和中胚层命运。我们将生成并分析小鼠Blimp1位点的功能缺失突变，以测试内胚层中可能的保守功能

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.