Functional role of Ascl1 in the developing spinal cord

Ascl1 在脊髓发育中的功能作用

基本信息

批准号：
8097391
负责人：
Mark Borromeo
金额：
$ 2.88万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8097391
关键词：
Address Autistic Disorder Automobile Driving BHLH Protein Binding Biology Cell Lineage Cell Separation Cells Chromatin Collecting Cell Coupled DNA Data Development Embryo Gene Targeting Generations Genes Genetic Genetic Transcription Genomics Glioblastoma Goals In Situ Hybridization Laboratories Location Malignant Neoplasms Maps Messenger RNA Molecular Genetics Mus Nerve Regeneration Nervous system structure Neural tube Neuroblastoma Neuroglia Neuronal Differentiation Neurons Neurosciences Neurosecretory Systems Oligodendroglia Organism Peripheral Nervous System Population Process Public Health Quality Control RNA Regulatory Element Reporter Research Role Schizophrenia Signal Pathway Site Spatial Distribution Spinal Spinal Cord Staging Stem cells Testing Tissues Work cell type chromatin immunoprecipitation developmental disease genome-wide malignant neurologic neoplasms mutant nerve stem cell nervous system disorder neurodevelopment neurogenesis new therapeutic target progenitor programs relating to nervous system research study stem transcription factor

项目摘要

A fundamental question in developmental neuroscience addresses how an organism acquires a nervous system with the correct composition and spatial distribution of neurons and glia. The basic helix-loop- helix (bHLH) transcription factor Ascl1 is one factor critical for neuronal differentiation and subtype specification of multiple neuronal cell-types throughout the development of the central and peripheral nervous systems. More recently, genetic fate mapping experiments have shown that early embryonic Ascl1 expressing progenitor cells are fated to become only neurons; however, at a later embryonic stage, Ascl1 expressing progenitor cells are fated to become oligodendrocytes. The transcriptional programs regulated by Ascl1 in the generation of these two cell types remain largely unknown. Therefore, the overall goal of this project is to identify the gene networks controlled by Ascl1 during neurogenic and oligodendrogenic stages of the developing spinal cord. To uncover what roll Ascl1 has generating the appropriate number and type of neural cells in the CNS, the following specific aims will be pursued. 1) Determine the genome-wide interactions of Ascl1 with chromatin during neurogenic and oligodendrogenic stages of the developing spinal cord. The cis-regulatory elements under the control of Ascl1 in each temporally distinct progenitors will be identified by biochemically isolating the chromatin bound to Ascl1 using Chromatin Immunoprecipitation followed by the massive parallel sequencing of all bound chromatin (ChIP-seq). Some unique regulatory elements found in each progenitor population will be tested for their activity in driving the appropriate expression of GFP reporter constructs in mice. 2) Identify and compare Ascl1 target genes during neurogenic and oligodendrogenic stages of the developing spinal cord. Ascl1 expressing progenitors from E11.5 and E15.5 neural tubes will be isolated by FACS and their mRNA profile will be characterizes by RNA-seq. A comparison of the expressed genes from each stage will provide information on the cellular content in a lineage restricted neuronal progenitor versus a glial restricted progenitor cell. The RNA-seq data will be coupled with the ChIP-seq data to identify Ascl1 target genes in the two temporally distinct progenitor populations.

发育神经科学中的一个基本问题是，生物体如何获得神经系统的神经系统的正确组成和神经元和神经胶质的空间分布。在整个中枢和周围神经系统的发展中，基本的螺旋环螺旋（BHLH）转录因子ASCL1 ASCL1是多个神经元细胞类型的神经元分化和亚型规范至关重要的因素。最近，遗传命运图映射实验表明，早期的胚胎ASCL1表达祖细胞被命名为仅成为神经元。然而，在后来的胚胎阶段，ASCL1表达祖细胞被命中为少突胶质细胞。由ASCL1在这两种细胞类型的生成中调节的转录程序在很大程度上未知。因此，该项目的总体目标是确定在发育中的脊髓神经源和少突胞源性阶段中ASCL1控制的基因网络。为了发现哪些滚动ASCL1在中枢神经系统中产生适当的神经细胞数量和类型，将追求以下特定目标。 1）确定在发育中的脊髓的神经源和少突dend骨阶段，ASCL1与染色质的全基因组相互作用。在每个时间上不同祖细胞中ASCL1控制下的顺式调节元件将通过生物化学通过染色质免疫沉淀来分离与ASCL1结合的染色质，然后对所有结合的染色质（CHIP-SEQ）进行大规模平行测序。在每个祖细胞种群中发现的一些独特的调节元素将在小鼠中推动GFP报告基因构建体的适当表达方面的活性进行测试。 2）在发育中的脊髓的神经源和少突dend骨阶段中识别和比较ASCL1靶基因。 ASCL1表达E11.5和E15.5神经管的祖细胞将通过FACS分离，其mRNA谱将通过RNA-Seq来表征。对每个阶段表达的基因的比较将提供有关谱系限制神经元祖细胞中细胞含量的信息，而不是神经胶质限制的祖细胞。 RNA-seq数据将与CHIP-SEQ数据耦合，以识别两个时间上不同的祖细胞种群中的ASCL1靶基因。