Cytokine Signal Transduction in Retinal Development

描述（由申请人提供）：本研究的长期目标是阐明细胞外源性信号影响祖细胞命运指定和神经元分化的机制。这项研究的重点是睫状神经营养因子（CNTF）样细胞因子在小鼠视网膜中的作用机制。在发育过程中，CNTF影响感光细胞、双极细胞和Muller胶质细胞的分化;而在成熟视网膜中，CNTF增强视网膜神经节细胞的存活，并在各种哺乳动物模型中防止感光细胞变性。 我们已经证明，CNTF差异激活细胞内信号通路在增殖的祖细胞和有丝分裂后的神经元在新生儿视网膜; CNTF依赖性抑制视杆细胞分化需要STAT功能，而CNTF增强生产的穆勒胶质细胞涉及STAT和ERK的活动。我们假设STAT和ERK信号在视网膜祖细胞和有丝分裂后细胞中发挥不同的作用。为了验证这一假设，我们将使用细胞类型特异性启动子和出生后视网膜中的突变信号蛋白干扰细胞因子信号通路。我们还观察到持续的CNTF信号仅触发瞬时视网膜信号转导，这与细胞因子信号传导抑制剂SOCS 3的上调相关。我们推测SOCS 3对细胞因子信号的负调节对于视网膜的正常发育至关重要。为了阐明SOCS 3的功能，我们将错误表达SOCS 3，并通过RNA干扰介导的基因抑制来抑制SOCS 3的表达。已经表明，病毒介导的 CNTF在rds突变体视网膜中的表达挽救了感光细胞死亡，但导致细胞形态和视网膜功能异常。我们将描述CNTF处理的rds视网膜中的信号事件，并测试光感受器的存活是否由细胞因子激活的Muller胶质细胞介导。我们也开始筛选细胞因子的靶基因在出生后的视网膜微阵列分析。潜在的候选靶基因将通过RT-PCR、原位杂交、Western印迹和启动子序列分析进行验证。STAT转录因子和靶基因启动子之间的相互作用将通过启动子结合试验进行检查。这些拟议的研究将促进我们对正常发育视网膜和感光细胞变性突变模型中细胞因子信号传导机制的理解。

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate mechanisms by which cell-extrinsic signals influence progenitor cell fate specification and neuronal differentiation. The proposed study focuses on mechanisms by which ciliary neurotrophic factor (CNTF)-like cytokines act in the mouse retina. During development, CNTF affects the differentiation of photoreceptor cells, bipolar cells, and Muller glia; while in the mature retina, CNTF enhances retinal ganglion cell survival and prevents photoreceptor degeneration in various mammalian models. We have demonstrated that CNTF differentially activates intracellular signaling pathways in proliferating progenitors and postmitotic neurons in the neonatal retina; CNTF-dependent inhibition of rod photoreceptor differentiation requires STAT function, whereas CNTF-enhanced production of Muller glia involves both STAT and ERK activities. We hypothesize that STAT and ERK signaling play distinct roles in retinal progenitor cells and in postmitotic cells. To test this hypothesis, we will perturb cytokine signaling pathways using cell type-specific promoters and mutant signaling proteins in the postnatal retina. We have also observed that persistent CNTF signals trigger only transient retinal signal transduction, which correlates with an up-regulation of the cytokine signaling inhibitor SOCS3. We hypothesize that the negative regulation of cytokine signals by SOCS3 is critical for proper retinal development. To elucidate the function of SOCS3, we will misexpress SOCS3 and inhibit SOCS3 expression by RNA interference-mediated gene inhibition. It has been shown that viral mediated CNTF expression in the rds mutant retina rescues photoreceptor cell death, but causes abnormal cell morphology and retinal function. We will characterize signaling events in CNTF treated rds retina and test if the survival of photoreceptors is mediated by cytokine activated Muller glia. We have also begun to screen for cytokine target genes in the postnatal retina by microarray analyses. Potential candidate target genes will be validated by RT-PCR, in situ hybridization, Western blot, and promoter sequence analyses. Interactions between STAT transcription factors and target gene promoters will be examined by promoter binding assays. These proposed studies will advance our understanding of cytokine signaling mechanisms in the normal developing retina and in a mutant model of photoreceptor degeneration.