Regulation of enteroendocrine cell differentiation by Neurogenin 3 gene dosage

描述(申请人提供)：肠内分泌细胞(EECS)是肠内五种上皮细胞之一，由常见的肠道干细胞分化而来。类似于 肠细胞和杯状细胞、肠内分泌细胞每3-5天更新一次。肠道干细胞的自我更新是相当重要的，因为出生后的生长和生存需要肠道激素，而且它们在靶向大脑和胰腺调节食物摄取和促进胰岛素分泌方面发挥着重要作用。将EECS限制在大约1-2%的肠道上皮细胞的动态平衡机制尚不清楚，但可能涉及激活邻近细胞中的Notch。Notch通过抑制表达而抑制分泌谱系细胞的分化，有利于吸收肠细胞的分化 碱性螺旋环状螺旋(BHLH)转录因子。然而，目前尚不清楚Notch如何具体限制EEC的分化，而不是一般地减少分泌谱系细胞的命运。BHLH蛋白Neurogenin3(Neurog3)是EEC规范的早期阶段所必需的。然而，Neurog3+细胞可以产生其他类型的肠道细胞。Neurod是另一种由Neurog3激活的bHLH蛋白，其表达仅限于采用内分泌细胞命运的细胞。该提案的三个目标将研究如何调节Neurog3+细胞的细胞命运，以维持EEC的动态平衡。目的1利用一个或两个Neurog3突变等位基因降低Neurog3蛋白表达的小鼠，结合Neurog3细胞的细胞谱系追踪，研究Neurogenin3基因剂量和表达水平对EEC细胞命运的影响。我们预计，Neurog3蛋白表达的减少将增加Neurog3+细胞交替的非内分泌细胞命运，包括向多能肠干细胞的逆转。目的2研究Neurog3基因剂量对EECS中Notch通路活性的影响，以及限制EECs的Notch配体Jagged2(Jag2)在抑制邻近细胞成为EECS中的功能作用。目标3中提出的研究将探索Neurod作为Neurog3转录效应器的作用。我们将有条件地从Neurog3+细胞中删除Neurod，以确定它是否加强了随Neurog3表达减少而出现的EEC分化减少。然后，我们将确定Neurog3+细胞中Neurod的条件表达是否拯救了Neurog3-/-小鼠受损的EEC分化，以及Neurog3表达细胞中的Neurod条件表达是否促进了EEC的分化。最后，Neurod和激活的Notch都将在Neurog3+细胞中有条件地表达，以确定Notch对EEC细胞命运的强大抑制是否被Neurod拯救，这意味着Notch的作用是由于Neurod的丢失所致。拟议研究的完成将扩大我们对不太丰富的肠道细胞类型如何保持正确比例的了解。

DESCRIPTION (provided by applicant): Enteroendocrine cells (EECs) are one of five epithelial cell lineages in the intestine that arise from a common intestinal stem cell. Similar to enterocytes and goblet cells, enteroendocrine cells turnover every 3-5 days. The self-renewal of EECs from intestinal stem cells is of considerable importance given the requirement of gut hormones for postnatal growth and survival as well as their roles in targeting the brain and pancreas to regulate food intake and enhance insulin secretion. The homeostatic mechanisms that limit EECs to approximately 1-2% of the intestinal epithelium are not known but probably involve activating Notch in neighboring cells. Notch favors the differentiation of absorptive enterocytes while selecting against secretory lineage cell differentiation by inhibiting expression of basic helix loop helix (bHLH) transcription factors. However, it is not know how Notch specifically limits EEC differentiation as opposed to generally reducing secretory lineage cell fate. The bHLH protein Neurogenin3 (Neurog3) is required for the earliest stages of EEC specification. However, Neurog3+ cells can give rise to other intestinal cell types. Expression of NeuroD, another bHLH protein activated by Neurog3, is restricted to cells that adopt an endocrine cell fate. The three aims of the proposal will examine how the cell fate of Neurog3+ cells is regulated to maintain EEC homeostasis. Aim 1 will characterize the effects of Neurogenin3 gene dosage and expression level on EEC cell fate using mice with one or two Neurog3 mutant alleles to reduce Neurog3 protein expression combined with cell lineage tracing of Neurog3 cells. We expect that reduced Neurog3 protein expression will increase alternate nonendocrine cell fates by Neurog3+ cells, including reversion to pluripotent intestinal stem cells. Aim 2 will examine the effects of Neurog3 gene dosage on Notch pathway activity in EECs and will examine the functional role of the Notch ligand, Jagged2 (Jag2), which is restricted to EECs, in inhibiting neighboring cells from becoming EECs. Studies proposed in Aim 3 will explore the role of NeuroD as a transcriptional effector of Neurog3. We will conditionally delete NeuroD from Neurog3+ cells to determine if it potentiates reduced EEC differentiation seen with reduced Neurog3 expression. We will then determine if conditional expression of NeuroD in Neurog3+ cells rescues the impaired EEC differentiation in Neurog3-/- mice and whether EEC differentiation is increased by conditional NeuroD expression in Neurog3 expressing cells. Finally, NeuroD and activated Notch will both be conditionally expressed in Neurog3+ cells to determine if the potent inhibition of EEC cell fate by Notch is rescued by NeuroD, implying that the effects of Notch are result from loss of NeuroD. Completion of the proposed studies will expand our knowledge of how less abundant intestinal cell types are maintained in the correct proportions.