DETERMINATION OF CELL TYPES IN DEVELOPING RETINA

视网膜发育中细胞类型的测定

• 批准号: 3266927
• 负责人: STEVEN C MCLOON
• 金额: $ 11.23万
• 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/3266927
• 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的细胞类型组成。 机制 视网膜细胞类型的多样性是如何发展的尚不清楚。 细胞 在发育中的视网膜在有丝分裂层中进行分裂， 到原始心室 有丝分裂后的细胞从有丝分裂的 视网膜的内表面，在那里它们发展成 各种视网膜细胞类型。 人们普遍认为， 不定型地迁移到一个特定的表型，然后在 迁移，基于各种局部影响，细胞变得定型。 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RA 4的表达。 这 表明细胞在分化之前就已经定型为特定的表型。 留下有丝分裂层。 这个提议将发展这个假设 视网膜细胞在细胞分裂过程中形成特定的表型， 由于与已经存在的细胞相互作用， 差异化。 这一假设将在四个实验中得到验证。 的 第一项研究将证明，分化的细胞决定命运， 未定型细胞的体外培养。 这将涉及培养混合物， 年轻未分化的视网膜细胞和各种年龄的视网膜细胞。 将跟踪年轻细胞的分化。 第二 这项研究将确定细胞分裂是否是可塑性所必需的。 在第一个实验中观察到的细胞表型。 BrdU将用于 标记重组培养物中的分裂细胞。 然后它将 可以确定E4细胞是否 在不同的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 有丝分裂后神经节细胞开始分化。 这将是 在胚胎发育的高峰期， 神经节细胞的诞生来标记分裂的细胞。 BrdU之间的时间 注射和BrdU阳性有丝分裂细胞的第一次出现， 测定 接下来，BrdU注射和第一次注射之间的时间 将确定BrdU阳性/RA 4阳性细胞的出现。 的 这两个时间之间的差近似于最短间隔 有丝分裂和明显的神经节细胞分化之间的联系。 该间隔 将给出一些关于神经节细胞表型是否 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RA 4蛋白的基因 表达式库。 这将允许研究RA 4的调节 基因在未来，这可能最终导致深入了解 神经节细胞表型的调节。