Endoderm Formation and Patterning in the Mouse

描述(申请人提供)：最终的内胚层，早期胚胎的三个主要胚层之一，形成原始的肠管，进而形成一组不同的组织，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形成过程中也发挥着重要作用。我们最近发现了Smad2，它是TGFb/激活素/结节信号下游的一个效应器，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，将检测Smad2缺陷的ES细胞的表达构建体对其定植最终内胚层谱系的能力。在内源Smad2调控元件的控制下，表达不同Smad2/3亚型的新的“敲入”等位基因的功能活性也将被测试。为了评估TGFb信号在内皮衍生物形成和形成中的作用，我们将利用Smad2和Smad4的条件等位基因与表达Cre的转基因小鼠品系相结合，在肠道谱系的不同时空区域，包括发育中的肝脏和胰腺。锌指转录抑制因子Blimp1已经被证明在青蛙胚胎中控制内胚层和中胚层的命运。我们将产生并分析小鼠Blimp1基因的功能缺失突变，以测试内胚层可能的保守功能 队形。最后，为了确定内胚层形成所需的靶基因，我们使用Smad2、Smad4、Foxh1和Mix-1缺陷的ES细胞在培养中诱导分化为内胚层，进行转录图谱分析。这项提案中概述的实验旨在增强我们对Smad2和其他转录介质如何控制内皮细胞系的细胞命运、增殖和分化的了解。

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.