DETERMINATION OF CELL TYPES IN DEVELOPING RETINA

视网膜发育中细胞类型的测定

• 批准号: 2163136
• 负责人: STEVEN C MCLOON
• 金额: $ 12.58万
• 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/2163136
• 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。 机制 这种视网膜细胞类型的多样性是如何形成的尚不清楚。 细胞 在发育中的视网膜中，邻近的有丝分裂层经历分裂 至原始心室。 有丝分裂后细胞从有丝分裂期迁移 一层朝向视网膜的内表面，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 未承诺地迁移到特定的表型，然后在结束时 迁移，基于各种局部影响，细胞变得定向。 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 表明细胞在发生之前就已形成特定的表型 留下有丝分裂层。 该提案将提出假设 视网膜细胞在细胞生长过程中变得特定的表型 分裂是与已经分裂的细胞相互作用的结果 差异化。 该假设将通过四个实验进行检验。 这 第一项研究将证明分化的细胞决定命运 体外未定型细胞。 这将涉及培养 年轻的未分化视网膜细胞和各个年龄段的视网膜细胞。 年轻细胞的分化将随之而来。 第二个 研究将确定细胞的可塑性是否需要细胞分裂 在第一个实验中观察到的细胞表型。 BrdU 将用于 标记重新聚集培养物中的分裂细胞。 那么它将是 可以确定 E4 细胞是否有 在不同的培养环境中分化成神经节细胞 经历了文化上的分裂。 第三项研究将确定多久 有丝分裂后神经节细胞开始分化。 这将是 通过在高峰期将BrdU注射到胚胎中来完成 神经节细胞的诞生是为了标记分裂细胞。 BrdU 之间的时间 注射后首次出现 BrdU 阳性有丝分裂细胞 决定。 接下来是 BrdU 注射和第一次注射之间的时间 将确定 BrdU 阳性/RA4 阳性细胞的出现。 这 这两个时间之间的差值近似于最短间隔 有丝分裂和明显的神经节细胞分化之间。 这个区间 将给出一些关于神经节细胞表型是否是 在细胞分裂期间或之后诱导。 第四项研究将克隆 并使用 cDNA 表征编码 RA4 蛋白的基因 表达库。 这将有助于研究 RA4 的监管 未来的基因，这可能最终导致对 神经节细胞表型的调节。