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: 10026175
• 负责人: Eliana Toren
• 金额: $ 4.48万
• 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/10026175
• 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; Epithelial; 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; conditional knockout; 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.

项目概要 胰岛由五种激素分泌细胞类型组成，是葡萄糖的重要调节因子 哺乳动物体内的稳态。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 同步严格调节血糖并允许组织正常代谢功能。期间 糖尿病，功能性 B 细胞群的丧失会导致患者出现严重的并发症。诊断为 糖尿病在全球范围内以惊人的速度增长，未来糖尿病治疗的关键是更大的 了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。所有胰岛细胞分化的第一步是内分泌细胞的分层 祖细胞（以 Neurogenin3、Ngn3 标记）通过称为上皮细胞的过程从导管上皮中分离出来 间充质转化 (EMT)，由 Snail2 转录因子介导。此步骤最早发生于 小鼠胚胎日 (E)10.5，是成熟胰岛形成所必需的。我们实验室以前 表明转录共调节因子 LIM 结构域结合蛋白 1 (Ldb1) 是胰腺 各个阶段的发展。在全胰腺 Ldb1 敲除中，我们观察到祖细胞池被破坏 E13.5，Ngn3+内分泌祖细胞在E15.5严重丧失。这些小鼠出生后缺乏胰岛结构， 强调了 Ldb1 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未成熟内分泌细胞的异常聚集。 管道。这一观察结果，加上 Ngn3+ 细胞的损失，表明在 发展。对于这个训练计划，我建议研究Ldb1在胰腺内分泌中的作用 祖细胞分层以及 Ldb1 复合物发挥其作用的机制。我的 总体假设是 Ldb1 是内分泌祖细胞群发育所必需的。瞄准 1，我将通过评估典型的EMT来确定Ldb1对内分泌祖细胞分层的贡献 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的命运 Ldb1 缺失。在目标 2 中，我将描述 Ldb1 介导的调控机制，重点是 Ldb1 Ngn3 和 Snail2 的调节。我将使用染色质免疫沉淀 (ChIP)-Seq 直接确定目标 通过 Ldb1 和 RNA-Seq 结合，深入了解 Ldb1 丢失对内分泌祖细胞的全基因组影响。 从这项研究中获得了对发育中胰腺中 EMT 转录控制的更深入的了解 将提供对胰岛如何产生的更深入的了解，有利于未来的糖尿病治疗，包括定向治疗 干细胞分化策略。