Regulation of enteroendocrine cell differentiation by Neurogenin 3 gene dosage

描述（由申请人提供）：肠内分泌细胞（EECs）是肠道中由常见肠道干细胞产生的五种上皮细胞谱系之一。类似于 肠上皮细胞和杯状细胞，肠内分泌细胞每3-5天更新一次。肠干细胞的自我更新是相当重要的，因为出生后生长和存活需要肠道激素，以及它们在靶向大脑和胰腺以调节食物摄入和增强胰岛素分泌方面的作用。将EEC限制在肠上皮的约1-2%的稳态机制尚不清楚，但可能涉及激活邻近细胞中的Notch。Notch有利于吸收性肠上皮细胞的分化，同时通过抑制表达来选择性抑制分泌性谱系细胞的分化 基本螺旋环螺旋（bHLH）转录因子的。然而，不知道Notch如何特异性地限制EEC分化，而不是通常减少分泌谱系细胞的命运。bHLH蛋白Neurogenin 3（Neurog 3）是EEC规范的最早阶段所需的。然而，Neurog 3+细胞可以产生其他肠细胞类型。NeuroD是另一种由Neurog 3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Neurog 3+细胞的细胞命运以维持EEC稳态。目的1将使用具有一个或两个Neurog 3突变等位基因的小鼠来表征Neurog 3基因剂量和表达水平对EEC细胞命运的影响，以降低Neurog 3蛋白表达，并结合Neurog 3细胞的细胞谱系追踪。我们预计降低Neurog 3蛋白表达将增加Neurog 3+细胞的替代非内分泌细胞命运，包括逆转为多能肠干细胞。目的2将检查Neurog 3基因剂量对EECs中Notch途径活性的影响，并检查Notch配体Jagged 2（Jag 2）在抑制邻近细胞成为EECs中的功能作用，该配体仅限于EECs。目标3中提出的研究将探索NeuroD作为Neurog 3的转录效应子的作用。我们将有条件地从Neurog 3+细胞中删除NeuroD，以确定它是否会增强随着Neurog 3表达减少而出现的EEC分化减少。然后，我们将确定Neurog 3+细胞中NeuroD的条件性表达是否挽救Neurog 3-/-小鼠中受损的EEC分化，以及Neurog 3表达细胞中NeuroD的条件性表达是否增加EEC分化。最后，NeuroD和活化的Notch都将在Neurog 3+细胞中有条件地表达，以确定Notch对EEC细胞命运的有效抑制是否被NeuroD拯救，这意味着Notch的作用是由NeuroD的丧失引起的。完成拟议的研究将扩大我们的知识，丰富的肠细胞类型是如何保持在正确的比例。

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.