CYTOKINE SIGNAL TRANSDUCTION IN RETINAL DEVELOPMENT

描述(改编自申请人的摘要)：该项目的长期目标 研究是为了阐明细胞-外在提示的分子机制 对视网膜祖细胞命运选择和分化的影响 正常发育，从而为防治提供依据 视网膜疾病。建议的研究集中在细胞因子信号转导上。 在小鼠视网膜中的通路。睫状神经营养因子(CNTF) 细胞因子超家族，对神经元发育和 生死存亡。在发育中的视网膜，CNTF深刻地影响光感受器， 双极、无长突细胞分化及促进神经节细胞分化 轴突的生长和存活。此外，CNTF还可以预防视网膜光感受器 由突变或光损伤引起的细胞退化。尽管如此 CNTF在发育中或成熟的视网膜中的作用机制尚不清楚 明白了。细胞因子信号一般是通过膜受体传递的 激活细胞内的Jak酪氨酸激酶和STAT转录因子。 然而，视网膜中的特定信号成分介导了 CNTF的作用，以及对CNTF有直接反应的视网膜细胞类型 信号，还没有被表征。因此，建议进行实验，以 用生化方法鉴定被激活的JAK激酶和STAT转录因子 CNTF在发育中的小鼠视网膜中的免疫沉淀和Western 视网膜细胞提取物的印迹分析。分布和蜂窝位置 CNTF受体、JAK蛋白激酶和STAT因子在视网膜中的作用 通过免疫细胞化学确定视网膜细胞类型， 对CNTF或CNTF样细胞因子有反应。此外，特定于细胞类型的 STAT转录因子的激活将通过免疫荧光法进行检测 CNTF处理分离细胞和外植体培养的显微镜观察 识别激活(磷酸化)状态因子的抗体组合 和不同的视网膜细胞类型。最后，细胞的生物学功能。 视网膜神经发生过程中的细胞因子信号通路将通过 扰乱JAK和STAT信号分子的活性。萌芽状态和 出生后视网膜将在体内和体外感染小鼠逆转录病毒 表达突变的JAK蛋白和STAT因子的载体，以及自然产生的 JAK和STAT抑制蛋白。这些扰动对视网膜的影响 发育将通过组织学和免疫细胞化学方法进行检查。 使用分子和细胞标记。这些拟议的研究将阐明 细胞因子信号转导的生物学功能及细胞机制 在哺乳动物视网膜神经发生过程中，从而为 CNTF或类CNTF对人类视网膜疾病的干预作用 细胞因子。

DESCRIPTION (Adapted from applicant's abstract): The long term goal of this research is to elucidate molecular mechanisms by which cell-extrinsic cues influence retinal progenitor cell fate choices and differentiation during normal development, thereby providing a basis for the prevention and treatment of retinal diseases. The proposed study focuses on the cytokine-signaling pathway in the mouse retina. Ciliary neurotrophic factor (CNTF), a member of the cytokine superfamily, has a variety of effects on neuronal development and survival. In the developing retina, CNTF profoundly affects photoreceptor, bipolar, and amacrine cell differentiation as well as promoting ganglion cell axonal growth and survival. In addition, CNTF prevents retinal photoreceptor cell degeneration caused by mutations or light-induced damages. Despite these effects, the mechanism of CNTF action in the developing or mature retina is not understood. Cytokine signals are generally mediated through membrane receptors that activate cellular Jak tyrosine kinases and STAT transcription factors. However, the specific signaling components in the retina that mediate the effects of CNTF, and the retinal cell types that directly respond to the CNTF signal, have not been characterized. Therefore, experiments are proposed to biochemically identify the Jak kinases and STAT transcription factors activated by CNTF in the developing mouse retina using immunoprecipitation and Western blot analyses of retinal cell extracts. The distribution and cellular location of the CNTF receptor, Jak protein kinases and STAT factors in the retina will be determined by immunocytochemistry to define retinal cell types that can respond to CNTF or CNTF-like cytokines. In addition, cell type-specific activation of STAT transcription factors will be assayed by immunofluorescence microscopy of dissociated cell and explant cultures treated with CNTF using combinations of antibodies recognizing activated (phosphorylated) STAT factors and distinct retinal cell types. Lastly, the biological function of the cytokine signaling pathway during retinal neurogenesis will be examined by perturbing the activity of Jak and STAT signaling molecules. Embryonic and postnatal retinas will be infected in vivo and in vitro with murine retroviral vectors expressing mutant Jak kinases and STAT factors, and naturally-occurring Jak and STAT inhibitor proteins. Effects of these perturbations on retinal development will be examined with histological and immunocytochemical methods using molecular and cellular markers. These proposed studies will elucidate the biological function and cellular mechanisms of cytokine signal transduction during mammalian retinal neurogenesis and thus establish a foundation for therapeutic interventions of human retinal diseases using CNTF or CNTF-like cytokines.