The role of Ldb1 in regulating pancreatic islet cell delamination and differentiation

Ldb1在调节胰岛细胞分层和分化中的作用

• 批准号: 9911402
• 负责人: Eliana Toren
• 金额: $ 4.37万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911402
• 关键词: Appearance; Automobile Driving; B-Lymphocytes; Birth; Blood Glucose; Cell Maturation; Cell physiology; Cells; Characteristics; Chromatin; Chronic; Complex; Confocal Microscopy; Data; Defect; Development; Diabetes Mellitus; Diagnosis; Disease; Duct (organ) structure; Ductal Epithelium; Embryo; Endocrine; Epithelium; Exhibits; Future; Genes; Genetic Transcription; Glucagon; Goals; Hormones; Immunofluorescence Immunologic; In Vitro; Insulin; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans; Knock-out; Knockout Mice; LIM Domain; Life; Mammals; Mediating; Mediator of activation protein; Mesenchyme; Metabolic; Modeling; Mus; Mutant Strains Mice; Neonatal; Non-Insulin-Dependent Diabetes Mellitus; Pancreas; Patients; Phase; Population; Process; Promoter Regions; Protein Binding Domain; Publishing; Quality of life; RNA; Regulation; Regulator Genes; Research; Role; Signal Transduction; Stains; Structure; Structure of beta Cell of islet; Tissues; Training; Transcriptional Regulation; base; blood glucose regulation; cell type; chromatin immunoprecipitation; design; diabetes mellitus therapy; diabetic; endocrine pancreas development; epithelial to mesenchymal transition; experimental study; functional loss; genome-wide; improved; insight; islet; pancreas development; progenitor; stem cell differentiation; transcription factor; transcriptome sequencing

PROJECT SUMMARY Pancreatic islets are comprised of five hormone-secreting cell types and are vital regulators of glucose homeostasis in mammals. Specifically, glucagon producing α-cells and insulin producing b-cells act synchronously to tightly regulate blood glucose and allow for normal metabolic function in tissues. During diabetes, loss of functional b-cell mass leads to severe complications in suffering patients. With diagnoses of diabetes rising at an alarming rate worldwide, key to future diabetes treatments is a greater understanding of the transcriptional complexes mediating not only early b-cell, but whole islet differentiation and formation. The first step of specification of all islet cells is the delamination of endocrine progenitors (marked by Neurogenin3, Ngn3) out of the ductal epithelium through a process known as epithelial to mesenchymal transition (EMT), mediated by the Snail2 transcription factor. This step occurs as early as embryonic day (E)10.5 in mice and is absolutely required for formation of mature islets. Our lab previously showed that a transcriptional co-regulator, LIM domain binding protein 1 (Ldb1) is required for pancreatic development at various stages. In a pancreas-wide Ldb1 knockout, we observed disrupted progenitor pools at E13.5, and a severe loss of Ngn3+ endocrine progenitors at E15.5. Upon birth, these mice lack islet structures, highlighting the importance of Ldb1 in the development of endocrine cells and formation of islet structures. Additionally, these neonatal mutant mice exhibited an abnormal clustering of immature endocrine cells along the ducts. This observation, plus the loss of Ngn3+ cells, suggests that there is a delamination defect during development. For this training plan, I propose to investigate the role of Ldb1 in pancreatic endocrine progenitor delamination and the mechanisms by which Ldb1 complexes impart their effects. My overarching hypothesis is that Ldb1 is required for development of the endocrine progenitor population. In Aim 1, I will determine the contribution of Ldb1 to endocrine progenitor delamination, by assessing canonical EMT markers throughout development, as well as lineage tracing to determine the fate of endocrine progenitors in the absence of Ldb1. In Aim 2, I will characterize Ldb1-mediated regulatory mechanisms, with a focus on Ldb1 regulation of Ngn3 and Snail2. I will use Chromatin ImmunoPreciptation (ChIP)-Seq to determine targets directly bound by Ldb1, and RNA-Seq to gain insight into the genome-wide effects of Ldb1 loss in endocrine progenitors. A greater understanding of transcriptional control of EMT in the developing pancreas gained from this research will provide a deeper understanding of how islets arise, benefiting future diabetes therapies, including directed stem cell differentiation strategies.

项目摘要 胰岛由五种胰岛分泌细胞组成，是葡萄糖的重要调节因子 哺乳动物体内平衡具体来说，产生胰高血糖素的α细胞和产生胰岛素的b细胞起作用， 同时严格调节血糖并允许组织中的正常代谢功能。期间 在糖尿病患者中，功能性B细胞团的丧失导致严重的并发症。诊断为 糖尿病在全球范围内以惊人的速度上升，未来糖尿病治疗的关键是更大的 理解转录复合物不仅介导早期b细胞，而且介导整个胰岛 分化和形成。所有胰岛细胞规格化的第一步是内分泌分层 祖细胞（标记为神经生成素3，Ngn 3）通过称为上皮细胞的过程从导管上皮细胞中分离出来。 间充质转化（EMT），由Snail 2转录因子介导。这一步最早发生在 小鼠胚胎日龄（E）10.5，并且是成熟胰岛形成所绝对需要的。我们的实验室以前 表明转录辅助调节因子LIM结构域结合蛋白1（Ldb 1）是胰腺癌发生所必需的。 在不同的发展阶段。在全胰腺Ldb 1基因敲除中，我们观察到破坏的祖细胞库， E13.5时，Ngn 3+内分泌祖细胞严重丢失。出生时，这些小鼠缺乏胰岛结构， 突出了Ldb 1在内分泌细胞发育和胰岛结构形成中的重要性。 此外，这些新生突变小鼠表现出不成熟内分泌细胞沿着 管道该观察结果加上Ngn 3+细胞的损失表明，在培养过程中存在分层缺陷。 发展对于这个培训计划，我建议研究Ldb 1在胰腺内分泌中的作用， 祖细胞分层和Ldb 1复合物赋予其作用的机制。我 总体假设是Ldb 1是内分泌祖细胞群体发育所必需的。在Aim中 1，我将通过评估典型的EMT来确定Ldb 1对内分泌祖细胞分层的贡献。 在整个发育过程中的标记，以及谱系追踪，以确定内分泌祖细胞在 没有LDB 1。在目标2中，我将描述Ldb 1介导的调节机制，重点是Ldb 1 Ngn 3和Snail 2的调节。我将使用染色质免疫荧光（ChIP）-Seq直接确定目标 结合Ldb 1和RNA-Seq，以深入了解内分泌祖细胞中Ldb 1缺失的全基因组影响。 通过这项研究，我们对胰腺发育过程中EMT的转录调控有了更深入的了解， 将提供更深入的了解胰岛如何产生，有利于未来的糖尿病治疗，包括直接 干细胞分化策略。