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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：
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.

项目总结： -- 这项新的提案旨在调查使用小鼠导致人类眼科疾病的潜在原因。模型。他们提议的实验将不会使用体内复杂的基因和条件基因(cre-lox)来研究小鼠的遗传。小鼠转基因、组织学、免疫组织化学、激光共聚焦显微镜、原位杂交等。小鼠胚胎学、单细胞、下一代测序、生物信息学、生物制品和重组工程。定量聚合酶链式反应、基因和非聚合酶链式反应技术旨在解决有关光学秸秆-圆盘的基本、机械性的问题。发育和促进星形胶质细胞的分化。Pax2转录因子在肿瘤细胞中启动其表达。所有的视神经泡都是细胞，但逐渐变得受限，只限于形成视神经的视盘和视神经。 STEK。这与它在其他胚胎组织中的作用是一致的，所以我们将不会测试一个新的假说，即Pax2。通过与细胞和表观遗传学的全球生物相互作用，关闭神经/视网膜神经前体基因检测程序。机械。这一新的活动最初限制了眼部神经干细胞的生长，以决定其命运。调节他们的细胞分裂频率，启动神经胶质细胞基因的表达和图谱。我们的目标是1，我们将继续。测试进化上保守的Pax2非编码序列，作为长期寻找的视盘神经。增强器(S)通过创建一个新的Pax2--CRE驱动程序来实现。这个新工具将被用来有条件地删除。 HES1评估了对光学技术发展的阻碍、对APC的差异化和对其成熟的影响。星形细胞的功能。为了实现Aim 2，我们希望能够充分利用它以前所特有的Rax-CRE。 BAC是转基因小鼠品系，Pax2GFP是转基因小鼠，Pax2是新的Pax2转基因小鼠，新的Pax2等位基因是用来观察眼睛的。 GFP基因谱系在控制细胞中存在，而Pax2基因则有条件地抑制突变的细胞。我们也不会产生肿瘤和肿瘤。比较Pax2、E11和E12杂合子和纯合子的基因表达谱。突变者的眼睛。在这里，我们将继续使用单细胞DNA测序技术，并公开展示人类日益增长的财富。可获得的信息包括核染色质的结构、基因和核糖核酸的表达水平。小鼠眼部干细胞的正常发育过程。