DETERMINATION OF CELL TYPES IN DEVELOPING RETINA

视网膜发育中细胞类型的测定

• 批准号: 3266926
• 负责人: STEVEN C MCLOON
• 金额: $ 12.23万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3266926
• 关键词: cell cell interaction; cell differentiation; cell type; chick embryo; developmental neurobiology; embryo /fetus culture; immunocytochemistry; molecular cloning; monoclonal antibody; neurogenesis; nucleic acid probes; retinal ganglion; tissue /cell culture

The neural retina is composed of seven major cell types. The mechanism by which this diversity of retinal cell types develops is unknown. Cells in the developing retina undergo division in the mitotic layer adjacent to the primitive ventricle. Postmitotic cells migrate from the mitotic layer towards the inner surface of the retina where they develop into the various retinal cell types. It has been generally believed that cells migrate uncommitted to a particular phenotype and then at the end of migration, based on various local influences, the cells become committed. However, we have evidence that cells, at least the retinal ganglion cells, begin to differentiate prior to departure from the mitotic layer based on expression of a ganglion cell specific protein, RA4. This suggests that cells become committed to a particular phenotype prior to leaving the mitotic layer. This proposal will develop the hypothesis that retinal cells become committed to a particular phenotype during cell division as a result of interactions with cells that have already differentiated. This hypothesis will be tested in four experiments. The first study will demonstrate that differentiated cells determine the fate of uncommitted cells in vitro. This will involve culturing a mixture of young undifferentiated retinal cells and retinal cells of various ages. The differentiation of the young cells will be followed. The second study will determine if cell division is required for the plasticity of cell phenotype observed in the first experiment. BrdU will be used to label dividing cells in the reaggregate cultures. It will then be possible to determine whether the E4 cells that did or did not differentiate into ganglion cells in the different culture environments underwent division in culture. The third study will determine how soon after mitosis ganglion cells begin to differentiate. This will be accomplished by injecting BrdU into embryos during the peak period of ganglion cell birth to label dividing cells. The time between BrdU injection and the first appearance of BrdU positive mitotic cells will be determined. Next the time between BrdU injection and the first appearance of BrdU positive / RA4 positive cells will be determined. The difference between these two times approximates the shortest interval between mitosis and overt ganglion cell differentiation. This interval will give some indication as to whether the ganglion cell phenotype is induced during or following cell division. The fourth study will clone and characterize the gene encoding for the RA4 protein using a cDNA expression library. This will allow studies on the regulation of the RA4 gene in the future, which may ultimately lead to an insight into the regulation of the ganglion cell phenotype.

神经视网膜由七种主要细胞类型组成。这一机制 这种视网膜细胞类型的多样性是通过什么方式发展起来的还不清楚。单元格 在发育中的视网膜在邻近的有丝分裂层中经历分裂 连接到原始脑室。有丝分裂后细胞从有丝分裂中迁移 朝向视网膜内表面的一层，在那里它们发育成 各种视网膜细胞类型。人们普遍认为，细胞 不受约束地迁移到特定的表型，然后在 迁移，基于各种局部影响，细胞变得承诺。 然而，我们有证据表明，细胞，至少是视网膜神经节 细胞，在离开有丝分裂层之前就开始分化 基于神经节细胞特异性蛋白RA4的表达。这 表明细胞在与特定的表型结合之前 离开有丝分裂层。这一提议将发展这一假说 视网膜细胞在细胞过程中变得依赖于特定的表型 由于与细胞的相互作用导致的分裂 差异化。这一假设将在四个实验中得到验证。这个 第一项研究将证明分化的细胞决定命运 未定植细胞的体外实验。这将涉及培养一种混合的 未分化的年轻视网膜细胞和不同年龄的视网膜细胞。 幼小细胞的分化将被跟踪。第二 研究将确定细胞分裂是否是细胞可塑性所必需的 第一次实验中观察到细胞表型。BrdU将用于 标记重组培养中的分裂细胞。那么它将会是 有可能确定是否有E4细胞 在不同培养环境中分化为神经节细胞 在文化上经历了分裂。第三项研究将确定多长时间 有丝分裂后，神经节细胞开始分化。这将是 通过在高峰期向胚胎中注射BrdU完成的 神经节细胞诞生来标记分裂细胞。BrdU之间的时间 注射和首次出现BrdU阳性的有丝分裂细胞 下定决心。下一次注射BrdU和第一次注射之间的时间 BrdU阳性/RA4阳性细胞的出现情况将被确定。这个 这两个时间之间的差值接近最短间隔 有丝分裂和神经节细胞分化之间的关系。此时间间隔 将提供一些关于神经节细胞表型是否为 在细胞分裂期间或之后诱导的。第四项研究将克隆 并使用cDNA鉴定编码RA4蛋白的基因 表达式库。这将使研究RA4的调节成为可能。 基因的未来，这最终可能导致对 神经节细胞表型的调控。