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Optic Stalk-Disc Development and Differentiation

视柄盘的发育和分化

基本信息

批准号：
10415746
负责人：
Nadean L Brown
金额：
$ 37.12万
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/10415746
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 Failure Fissural Freezing Gene Expression Genes Genetic Glaucoma Goals 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 Phenotype Research Retina Retinal Diseases Role Side Signal Induction Structure of retinal pigment epithelium Technology Testing Tissues Transgenic Mice Transgenic Organisms Untranslated RNA Visual Fields Visual impairment Visual system structure 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 next generation sequencing novel optic cup optic nerve disorder optic stalk 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.

项目摘要这项建议调查的根本原因，人类眼部疾病使用小鼠所提出的实验将使用复杂的体内条件（cre-12 lox）小鼠遗传学，小鼠转基因，组织学，免疫组织化学，共聚焦显微镜，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，以解决有关视柄-视神经盘的基本机制问题 Pax 2转录因子启动了星形胶质细胞的表达。所有视泡细胞，但逐渐仅限于正在形成的视盘，与其在其他胚胎组织中的作用一致，我们将检验Pax 2 通过与细胞表观遗传学的全球相互作用关闭神经/视网膜祖细胞基因程序，这种活性最初将眼细胞限制为星形胶质细胞祖细胞（APC）的命运，调节其细胞分裂速率，并启动胶质细胞基因表达谱。在目标1中，我们将测试进化上保守的Pax 2非编码序列作为长期寻求的视神经盘神经通过创建一个新的Pax 2-PaxCre驱动程序来增强增强器。此工具将用于有条件地删除 Hes 1的表达，并评估其对视柄发育、APC分化和成熟的影响。对于目标2，我们将利用先前表征的Rax-β Cre BAC转基因小鼠系，Pax 2GFP敲除蛋白和新的Pax 2 floxed等位基因，以跟踪眼 GFP谱系在对照和Pax 2条件突变细胞中的表达。比较Pax 2 E11和E12杂合子和纯合子的基因表达谱突变的眼睛。在这里，我们将使用单细胞RNA测序和越来越多的财富，关于染色质构型和mRNA表达水平的可用信息，小鼠眼细胞的正常发育。