Cell-fate of gastrointestinal endocrine cells

描述（申请人提供）：胃肠道内分泌细胞的分化取决于基本螺旋环螺旋（bHLH）转录因子的表达和功能。Notch信号通过抑制bHLH蛋白的功能，限制肠、胃和胰腺中能够接受内分泌细胞命运的细胞数量。本研究的总体目标是确定内分泌祖细胞在分化的哪个阶段可以恢复到非内分泌细胞的命运，确定发育中的祖细胞在什么时候对notch信号的抑制作用保持敏感，并确定在肠内分泌细胞分化的不同阶段激活的基因。计划了三个具体目标。第一个目标将在转基因小鼠中使用基于Cre/Iox的方法来检查表达bHLH蛋白的胃肠道内分泌前体细胞的细胞命运。第二个目的是确定GI内分泌祖细胞受notch及其相关蛋白Deltex-1调控时的发育背景。为此，notch 1的激活形式将在转基因小鼠内分泌分化的不同阶段有条件地表达，以确定notch信号可以抑制前体细胞分化的阶段。Deltex-1的作用也将在转基因小鼠中进行研究，以确定该蛋白在胃肠道中是notch的增强剂还是拮抗剂。第三个目标是确定小肠细胞从隐窝内分泌前体分化到绒毛内终末分化的肠内分泌细胞时的差异表达基因。利用激光显微解剖收集不同发育阶段的肠内分泌前体，提取RNA，制备探针，对高密度微阵列进行查询。预计这些研究将为胃肠道内分泌细胞的命运决定、notch信号调节胃肠道内分泌分化的机制以及内胚层内分泌分化的调控基因提供重要的新见解。了解前体细胞如何分化为胃肠道内分泌细胞可能会对治疗糖尿病等疾病的潜在新疗法产生影响。来自这个项目的信息可能有助于未来治疗的发展，包括操纵干细胞或诱导其他器官转分化为能够产生胰岛素或其他激素的细胞。

DESCRIPTION (provided by applicant): Differentiation of gastrointestinal endocrine cells depends on the expression and function of basic helix loop helix (bHLH) transcription factors. Notch signaling restricts the number of cells able to adopt an endocrine cell fate in the intestine, stomach, and pancreas by inhibiting the function of bHLH proteins. The overall goal of the proposed research is to determine at what stage of differentiation endocrine progenitor cells can revert to non endocrine cell fates, to determine when in development progenitor cells remain sensitive to the inhibitory effects of notch signaling, and to identify genes activated at different stages of enteroendocrine cell differentiation. Three specific aims are planned. The first aim will use a Cre/Iox based approach in transgenic mice to examine the cell fate of gastrointestinal endocrine precursor cells expressing bHLH proteins. The second aim will determine the developmental context when GI endocrine progenitors are regulated by notch and its associated protein, Deltex-1. For this aim, an activated form of notch 1 will be conditionally expressed at different stages of endocrine differentiation in transgenic mice to determine the stage when precursor cell differentiation can be inhibited by notch signals. The effects of Deltex-1 will also be examined in transgenic mice to determine whether this protein functions as a potentiator or antagonist of notch in the gastrointestinal tract. The third aim will identify genes differentially expressed in cells of the small intestine as they differentiate from endocrine precursors in crypts to terminally differentiated enteroendocrine cells in the villi. Enteroendocrine precursors at different developmental stages will be collected by laser microdissection for RNA extraction and preparation of probes to interrogate high-density microarrays. It is anticipated that these studies will provide important new insights regarding cell fate determination of GI endocrine cells, the mechanism of notch signaling in regulating endocrine differentiation in the GI tract, and genes that may regulate endodermal endocrine differentiation. Understanding how precursor cells become committed to differentiate into gastrointestinal endocrine cells may have an impact on potential new therapies for treating diseases like diabetes mellitus. Information from this project may contribute to the development of future treatments that involve manipulating stem cells or inducing other organs to transdifferentiate into cells capable of producing insulin or other hormones.