Understanding the role of minor intron splicing in cortical development

了解小内含子剪接在皮质发育中的作用

基本信息

批准号：
10368061
负责人：
RAHUL N KANADIA
金额：
$ 36.61万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10368061
关键词：
Ablation Affect Apoptosis Birth Brain Caring Cell Cycle Cell Cycle Regulation Cell Death Cell division Cells Cessation of life Competence Complex DNA Sequence Alteration Data Defect Development Disease Electroporation Embryo Excision Fluorescence Gene Expression Gene Expression Regulation Genes Goals Infant Inherited Intellectual functioning disability Introns Knockout Mice Knowledge Life Link Measures Mediating Microcephaly Minor Modeling Molecular Mus Mutation Neurodevelopmental Disorder Neuroglia Neurons Pathogenesis Pathway interactions Phenotype Physiologic pulse Population Predisposition Process Production RNA Splicing Radial Resistance Role Small Nuclear RNA Specificity Speed Spliceosomes Syndrome TP53 gene Testing Tissues ZIKV infection base cell behavior cell type comparative conditional knockout design developmental disease experimental study in utero insight mammalian genome mutant nerve stem cell neurogenesis neuron loss novel osteodysplastic primordial dwarfism postnatal prevent programs severe intellectual disability stem cells transcriptome sequencing

Ablation Affect Apoptosis Birth Brain Caring Cell Cycle Cell Cycle Regulation Cell Death Cell division Cells Cessation of life Competence Complex DNA Sequence Alteration Data Defect Development Disease Electroporation Embryo Excision Fluorescence Gene Expression Gene Expression Regulation Genes Goals Infant Inherited Intellectual functioning disability Introns Knockout Mice Knowledge Life Link Measures Mediating Microcephaly Minor Modeling Molecular Mus Mutation Neurodevelopmental Disorder Neuroglia Neurons Pathogenesis Pathway interactions Phenotype Physiologic pulse Population Predisposition Process Production RNA Splicing Radial Resistance Role Small Nuclear RNA Specificity Speed Spliceosomes Syndrome TP53 gene Testing Tissues ZIKV infection base cell behavior cell type comparative conditional knockout design developmental disease experimental study in utero insight mammalian genome mutant nerve stem cell neurogenesis neuron loss novel osteodysplastic primordial dwarfism postnatal prevent programs severe intellectual disability stem cells transcriptome sequencing

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项目摘要

Microcephaly is a devastating developmental defect that can be caused by inherited mutations such as those that inactivate the minor spliceosome or more recently Zika virus infections. In order to better understand the underlying molecular and cellular defects that cause microcephaly, we must first understand the molecular and cellular changes during normal development. The current proposal extends our finding that inactivation of the minor spliceosome in our U11 conditional knockout (cKO) mouse crossed with Emx1-Cre results in microcephaly observed at birth. We found that the primary cause of the microcephaly is loss of self-amplifying radial glial cells (RGCs) and delayed death of intermediate progenitor cells (IPCs) without the corresponding loss of neurons during early cortical development. Despite the complete loss of NPCs by E14, the developing mutant cortex managed to produce layer IV neurons that are normally born at/after E14. We found that this shift in neuron production is in conjunction with increased neurogenesis measured by EdU pulse/chase experiments. Based on these complex phenotypes, we have proposed three aims to test the hypothesis that minor spliceosome acts in a cell-type specific manner to inform cell cycle regulation, NPC competence, and neuron production. Experiments proposed in aim 1a are designed to elucidate the precise cell-cycle regulation of RGCs and IPCs and whether these two cell-types are undergoing self-amplifying or neurogenic cell divisions. In aim 1b, we explore how the changes in U11-null RGCs and IPCs impact neuron production. In aim 2, the objective is to understand the molecular underpinning of the cell-type specific effect of U11 loss. The one unique identifier of RGCs is that they divide rapidly compared to the IPCs, so the experiments proposed in aim 2a test the hypothesis that cell cycle speed confers susceptibility to loss of U11. Another possibility that the experiments proposed in aim 2b is that each cell-type has a unique signature of minor intron-containing genes (MIGs) that might make cells resistant/susceptible to U1 loss. Finally, RNAseq data showed activation of P53- medated apoptosis pathway and cell cycle defect in the U11-null tissue. The experiments proposed in aim 3a test whether rescuing cell cycle defect would prevent P53-mediated apoptosis or is P53-mediated apoptosis independent of the cell cycle defect. Aim 3b tests the idea that if P53 is ablation, would it rescue cell cycle or cell death and in turn rescue microcephaly. In all, we will find the role of this novel form of gene regulation in cortical development and in turn provide insights into microcephaly observed in diseases such as microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1) and Roifman syndrome that are both caused by defective minor spliceosome.

小头畸形是一种毁灭性的发育缺陷，可能是由遗传突变引起的这会使较小的剪接体或最近的寨卡病毒感染失活。为了更好地了解引起小头畸形的基本分子和细胞缺陷，我们必须首先了解分子和正常发育过程中的细胞变化。当前的提议扩展了我们的发现我们的U11条件敲除（CKO）小鼠与EMX1-CRE交叉的次要剪接体导致出生时观察到的小头畸形。我们发现，小头畸形的主要原因是自我扩增的丧失径向神经胶质细胞（RGC）和中间祖细胞（IPC）的延迟死亡，没有相应早期皮质发育期间神经元的丧失。尽管E14完全损失了NPC，但发展突变皮层设法产生了通常在E14处/之后出生的IV层神经元。我们发现这个神经元产生的转移与通过EDU脉冲/Chase测得的神经发生增加了实验。基于这些复杂的表型，我们提出了三个旨在检验假设的目的较小的剪接体以细胞类型的特定方式起作用，以告知细胞周期调节，NPC能力和神经元产生。 AIM 1A中提出的实验旨在阐明精确的细胞周期调节 RGC和IPC以及这两种细胞类型是否正在进行自我扩增还是神经源细胞部门。在AIM 1B中，我们探讨了U11-NULL RGC和IPC的变化如何影响神经元的产生。目标 2，目的是了解U11损耗的细胞类型特异性效应的分子基础。一个 RGC的唯一标识符是它们与IPC相比迅速划分，因此提出的实验在AIM中提出 2a检验了细胞周期速度赋予U11损失的易感性的假设。另一种可能性 AIM 2B中提出的实验是每个细胞类型具有较小内含子内基因的独特特征（MIG）可能会使细胞具有抗性/易感U1损失。最后，RNASEQ数据显示p53-的激活 U11无效组织中的染色凋亡途径和细胞周期缺陷。 AIM 3A提出的实验测试拯救细胞周期缺陷是否会预防p53介导的细胞凋亡或p53介导的细胞凋亡独立于细胞周期缺陷。 AIM 3B测试了一个想法，即如果p53是消融，它会挽救单元周期还是细胞死亡并救出小头畸形。总的来说，我们将发现这种新型基因调节形式的作用皮质发育和反过小头骨骨出现的原始矮人型1型（MOPD1）和Roifman综合征都是由有缺陷的小剪接体引起。