CYTOKINE SIGNAL TRANSDUCTION IN RETINAL DEVELOPMENT

描述（改编自申请人的摘要）：本项目的长期目标 研究的目的是阐明细胞外部信号 影响视网膜祖细胞命运选择和分化 正常发育，从而为预防和治疗提供依据 视网膜疾病。本研究的重点是探讨甜菜碱信号通路 小鼠视网膜中的信号通路。睫状神经营养因子（CNTF）是睫状神经营养因子家族的一员， 细胞因子超家族对神经元发育具有多种作用， 生存在发育中的视网膜中，CNTF深刻地影响光感受器， 双极和无长突细胞分化以及促进神经节细胞 轴突生长和存活。此外，CNTF可防止视网膜感光细胞 由突变或光诱导损伤引起的细胞变性。尽管有这些 作用，CNTF在发育或成熟视网膜中的作用机制不是 明白细胞因子信号通常通过膜受体介导 激活细胞Jak酪氨酸激酶和STAT转录因子。 然而，视网膜中介导这种变化的特定信号成分， CNTF的作用，以及直接对CNTF反应的视网膜细胞类型 信号，尚未被表征。因此，建议进行实验， 生物化学鉴定激活的Jak激酶和STAT转录因子 通过免疫沉淀和Western印迹法检测CNTF对小鼠视网膜发育的影响 视网膜细胞提取物的印迹分析。分布和细胞位置 视网膜中的CNTF受体、Jak蛋白激酶和STAT因子将 通过免疫细胞化学来确定视网膜细胞类型， 对CNTF或CNTF样细胞因子有反应。此外，细胞类型特异性 STAT转录因子的激活将通过免疫荧光法测定 显微镜下观察用CNTF处理的分离细胞和外植体培养物， 识别活化（磷酸化）STAT因子的抗体组合 和不同的视网膜细胞类型。最后，生物学功能的 视网膜神经发生过程中的细胞因子信号通路将通过以下方法进行检查： 干扰Jak和STAT信号分子的活性。胚胎和 出生后的视网膜将在体内和体外被鼠逆转录病毒感染 表达突变型Jak激酶和STAT因子的载体，以及天然存在的 Jak和STAT抑制蛋白。这些扰动对视网膜的影响 将用组织学和免疫细胞化学方法检查发育 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和细胞机制 在哺乳动物视网膜神经发生过程中， 使用CNTF或CNTF样的人视网膜疾病的治疗干预 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.