Regulation of enteroendocrine cell differentiation by Neurogenin 3 gene dosage

Neurogenin 3 基因剂量对肠内分泌细胞分化的调节

• 批准号: 8848376
• 负责人: ANDREW B. LEITER
• 金额: $ 33.5万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8848376
• 关键词: Adopted; Alleles; American; BHLH Protein; Brain; Cell Count; Cell Differentiation process; Cell Lineage; Cells; Desire for food; Diabetes Mellitus; Disease; Duct (organ) structure; Eating; Effector Cell; Endocrine; Enterocytes; Enteroendocrine Cell; Epithelial Cells; Gene Dosage; Gene Expression; Goblet Cells; Growth; Health; High Prevalence; Homeostasis; Hormones; Hunger; Intestines; Knowledge; Lateral; Ligands; Mediating; Mus; Obesity; Organ; Pancreas; Pathway interactions; Play; Production; Regulation; Role; Severities; Staging; Stem cells; cell type; insulin secretion; interest; intestinal epithelium; mouse Neurog3 protein; mutant; notch protein; postnatal; prevent; protein expression; self-renewal

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.

描述（由申请人提供）：肠内分泌细胞（EEC）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。考虑到出生后生长和生存所需的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中的作用，来自肠道干细胞的 EEC 的自我更新具有相当重要的意义。将 EEC 限制在肠上皮约 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 有利于吸收性肠细胞的分化，同时通过抑制表达来选择反对分泌谱系细胞的分化 碱性螺旋环螺旋（bHLH）转录因子。然而，目前尚不清楚 Notch 如何具体限制 EEC 分化，而不是普遍减少分泌谱系细胞的命运。 bHLH 蛋白 Neurogenin3 (Neurog3) 是 EEC 规范最早阶段所必需的。然而，Neurog3+ 细胞可以产生其他肠道细胞类型。 NeuroD（另一种由 Neurog3 激活的 bHLH 蛋白）的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 稳态。目标 1 将使用具有一或两个 Neurog3 突变等位基因的小鼠来降低 Neurog3 蛋白表达，并结合 Neurog3 细胞的细胞谱系追踪，来表征 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+ 细胞的替代非内分泌细胞命运，包括逆转为多能肠干细胞。目标 2 将检查 Neurog3 基因剂量对 EEC 中 Notch 通路活性的影响，并将检查仅限于 EEC 的 Notch 配体 Jagged2 (Jag2) 在抑制邻近细胞变成 EEC 方面的功能作用。目标 3 中提出的研究将探索 NeuroD 作为 Neurog3 转录效应子的作用。我们将有条件地从 Neurog3+ 细胞中删除 NeuroD，以确定它是否会增强因 Neurog3 表达减少而导致的 EEC 分化减少。然后，我们将确定 Neurog3+ 细胞中 NeuroD 的条件表达是否可以挽救 Neurog3-/- 小鼠中受损的 EEC 分化，以及 Neurog3 表达细胞中的条件 NeuroD 表达是否会增加 EEC 分化。最后，NeuroD 和激活的 Notch 都将在 Neurog3+ 细胞中条件表达，以确定 Notch 对 EEC 细胞命运的有效抑制是否能被 NeuroD 挽救，这意味着 Notch 的作用是由 NeuroD 缺失造成的。完成拟议的研究将扩大我们对如何将数量较少的肠道细胞类型维持在正确比例的认识。