Cytokine Signal Transduction in Retinal Development

描述(申请人提供)：这项研究的长期目标是阐明细胞-外在信号影响祖细胞命运指定和神经元分化的机制。这项拟议的研究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影响光感受器细胞、双极细胞和Muller胶质细胞的分化；而在成熟的视网膜中，CNTF提高视网膜神经节细胞的存活，并防止各种哺乳动物模型的光感受器退化。 我们已经证明，CNTF差异性地激活了新生视网膜中增殖的前体细胞和有丝分裂后神经元的细胞内信号通路；CNTF依赖的抑制视杆细胞光感受器分化需要STAT功能，而CNTF增强的Muller胶质细胞的产生涉及STAT和ERK两种活性。我们假设STAT和ERK信号在视网膜前体细胞和有丝分裂后细胞中发挥着不同的作用。为了验证这一假设，我们将使用细胞类型特定的启动子和出生后视网膜中的突变信号蛋白来扰乱细胞因子信号通路。我们还观察到，持续的CNTF信号只触发一过性的视网膜信号转导，这与细胞因子信号抑制物SOCS3的上调有关。我们假设SOCS3对细胞因子信号的负调控对视网膜的正常发育至关重要。为了阐明SOCS3的功能，我们将错误表达SOCS3，并通过RNA干扰介导的基因抑制来抑制SOCS3的表达。已有研究表明，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的死亡，但导致了细胞形态和视网膜功能的异常。我们将表征CNTF治疗的视网膜脱离的信号事件，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。我们也已经开始通过微阵列分析来筛选出生后视网膜中的细胞因子靶基因。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来验证。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分析来检验。这些拟议的研究将促进我们对正常发育的视网膜和光感受器变性突变模型中细胞因子信号机制的理解。

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.