Optic Stalk-Disc Development and Differentiation

视柄盘的发育和分化

基本信息

批准号：
10666461
负责人：
Nadean L Brown
金额：
$ 36.78万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10666461
关键词：
ATAC-seq Address Adopted Adult Alleles Animal Model Aniridia Anophthalmos Astrocytes Automobile Driving Bacterial Artificial Chromosomes Bioinformatics Blindness Brain Cell Differentiation process Cell Lineage Cell division Cells ChIP-seq Characteristics Childhood Choroid Chromatin Chromatin Structure Coloboma Complex Confocal Microscopy Cre driver DNA Data Databases Deformity Development Disease Embryo Embryology Enhancers Epigenetic Process Event Eye Eye Development Eye diseases Failure Freezing Gene Expression Genes Genetic Glaucoma Goals Heterozygote Histology Homeostasis Human Immunohistochemistry In Situ Hybridization Kidney Knock-in Knowledge LoxP-flanked allele Maintenance Microphthalmos Mitotic Modeling Molecular Morphogenesis Mus Mutation Nephrons Nerve Neural Retina Neuroglia Neurons Null Lymphocytes Optic Disk Optic Nerve Optic Neuritis Optic vesicle Optics Organ Organism Pathway interactions Pattern Peptide Initiation Factors Phenotype Pigment Epithelium Recommendation Research Retina Retinal Diseases Role Side Signal Transduction Technology Testing Tissues Transgenic Mice Transgenic Organisms Untranslated RNA Visual Fields Visual System Visual impairment Xenopus astrocyte progenitor cell type complement C2a conditional mutant embryo tissue experimental study hybrid gene in vivo inner ear development insight interstitial cell mRNA Expression malformation mouse development mouse genetics mouse model mutant mutant mouse model neural next generation sequencing novel optic cup optic nerve disorder optic stalk optical disc postnatal progenitor programs recruit retinal progenitor cell single-cell RNA sequencing tool transcription factor

项目摘要

Project Summary This proposal investigates the underlying causes of human ocular diseases using mouse models. Proposed experiments will use complex in vivo conditional (cre-lox) mouse genetics, mouse transgenics, histology, immunohistochemistry, confocal microscopy, in situ hybridization, mouse embryology, single-cell NEXTgen sequencing, bioinformatics, BAC recombineering, qPCR, and PCR technologies to address basic, mechanistic questions about optic stalk-disc development and astrocyte differentiation. The Pax2 transcription factor initiates expression in all optic vesicle cells, but becomes progressively restricted to only the forming optic disc and stalk. Consistent with its role in other embryonic tissues, we will test a hypothesis that Pax2 shuts off neural/retinal progenitor gene programs, via global interactions with cell epigenetic machinery. This activity initially restricts ocular cells to an astrocytic progenitor cell (APC) fate, regulates their rate of cell division, and initiates glial gene expression profiles. In Aim 1, we will test evolutionarily-conserved Pax2 noncoding sequences as long-sought optic disc-nerve enhancer(s) by creating a new Pax2-Cre driver. This tool will be used to conditionally remove Hes1 and assess the consequences to optic stalk development, APC differentiation and mature astrocyte functionality. For Aim 2, we will take advantage of previously characterized Rax-Cre BAC transgenic mouse line, Pax2GFP knock-in and new Pax2 floxed allele to follow the ocular GFP lineages in control and Pax2 conditionally mutant cells. We will also generate and compare the gene expression profiles of Pax2 E11 and E12 heterozygous and homozygous mutant eyes. Here we will use single-cell RNA sequencing and the growing wealth of publicly available information regarding chromatin configurations, and mRNA expression levels during the normal development of mouse ocular cells.

项目概要该提案利用小鼠研究人类眼部疾病的根本原因拟议的实验将使用复杂的体内条件（cre-lox）小鼠遗传学，小鼠转基因、组织学、免疫组织化学、共焦显微镜、原位杂交、小鼠胚胎学、单细胞 NEXTgen 测序、生物信息学、BAC 重组工程、 qPCR 和 PCR 技术可解决有关视柄盘的基本机械问题 Pax2 转录因子在星形胶质细胞的发育和分化中启动表达。所有视囊细胞，但逐渐仅限于正在形成的视神经盘和与其在其他胚胎组织中的作用一致，我们将测试 Pax2 的假设通过与细胞表观遗传的整体相互作用，关闭神经/视网膜祖基因程序这种活动最初将眼细胞限制为星形胶质细胞祖细胞（APC）的命运，在目标 1 中，我们将调节细胞分裂速率并启动神经胶质基因表达谱。测试进化保守的 Pax2 非编码序列作为长期寻找的视盘神经通过创建新的 Pax2-Cre 驱动程序来增强此工具将用于有条件地删除。 Hes1 并评估了视柄发育、APC 分化和成熟的后果。对于目标 2，我们将利用之前表征的 Rax-Cre。 BAC 转基因小鼠系、Pax2GFP 敲入和新的 Pax2 floxed 等位基因遵循眼部我们还将生成对照细胞和 Pax2 条件突变细胞中的 GFP 谱系。比较 Pax2 E11 和 E12 杂合子和纯合子的基因表达谱在这里我们将利用单细胞RNA测序和不断增长的公开财富。有关染色质构型和 mRNA 表达水平的可用信息小鼠眼细胞的正常发育。