The role of PIAS3 in retinal development

描述(由申请人提供)：项目摘要：我们的目标是确定哺乳动物杆状感光细胞发育和生存所需的分子网络。我们发现激活的STAT3蛋白抑制因子(PIAS3)在发育中的杆状感受器中强烈表达，它是一种转录辅助调节因子和位点特异性的SUMOylase。我们观察到，PIAS3过表达导致杆状感光细胞数量增加，而PIAS3基因敲除会产生过量的Muller神经胶质细胞。我们还鉴定了两个转录因子CRX和Nr2e3为PIAS3相互作用蛋白。因此，我们假设PIAS3通过与光感受器特异的转录因子相互作用，在杆状细胞的发育和存活中发挥关键作用。我们建议通过一系列实验来检验这一假设。 首先，我们将使用荧光原位杂交和免疫细胞化学来确定PIAS3是否在发育中的杆状感光细胞中表达，而在发育中的视锥细胞和有丝分裂前体中缺失。 其次，根据我们的初步数据，我们假设PIAS3既调节分化的光感受器的命运，调节视紫红质的表达动力学，也调节新分化的光感受器的维持。为了解决这些问题，我们将使用一组分子标记对PIAS3功能的得失进行详细的分析。我们将使用基于BrdU的出生测年来确定PIAS3功能的获得/丧失是否影响视紫红质的表达动力学，并使用基于视紫红质的表达结构来确定PIAS3功能的获得/丧失是否对视网膜和分化的光感受器的发育有明显的影响。 最后，我们将探讨PIAS3在视网膜发育中的作用机制。我们推测PIAS3的作用部分是由CRX、Nr2e3和可能的STAT3介导的。我们还假设PIAS3直接调控许多视杆细胞、视锥细胞和可能的穆勒胶质细胞特异基因的转录。我们测试了PIAS3在体内是否与CRX、Nr2e3或STAT3相互作用，以及这些因素是否介导了PIAS3在视网膜发育中的作用。接下来，我们将确定PIAS3的哪些结构域是其在视网膜中活动所必需的，以及这些结构域是否也是与CRX和Nr2e3相互作用所必需的。最后，我们将确定PIAS3是否直接调控杆、锥和穆勒特异基因的表达，以及这是否需要CRX和Nr2e3。 相关性：中枢神经系统中细胞规范的分子基础知之甚少，这些研究将提供对这一过程的机械性洞察。此外，杆状富含转录因子的突变经常导致感光细胞退化和失明，我们预计这可能也适用于PIAS3。

DESCRIPTION (provided by applicant): Project summary: We aim to identify the molecular network required for development and survival of mammalian rod photoreceptors. We identified Protein Inhibitor of Activated STAT3 (PIAS3), a transcription co-regulator and site-specific SUMOylase as being strongly expressed in developing rod photoreceptors. We observed that Pias3 overexpression gives rise to an increase in rod photoreceptors, while PIAS3 knockdown produces excess Muller glia. We also identified two transcription factors, Crx and Nr2e3, as Pias3 interacting proteins. We thus hypothesize that PIAS3 plays a critical role in rod development and survival by interacting with photoreceptor-specific transcription factors. We propose to test this hypothesis with a series of experiments. First, we will use fluorescent in situ hybridization and immunocytochemistry to determine if Pias3 is expressed in developing rod photoreceptors and absent from developing cones and mitotic progenitors. Second, based on our preliminary data, we hypothesize that PIAS3 regulates both the fates of differentiating photoreceptors, the kinetics of rhodopsin expression, and the maintenance of newly differentiated photoreceptors. To address these questions, we will perform a detailed analysis of the effects of gain/loss of function of Pias3 using a panel of molecular markers. We will use BrdU-based birthdating to determine whether gain/loss of function of PIAS3 influences the kinetics of rhodopsin expression, and use rhodopsin-based expression constructs to determine whether gain/loss of Pias3 function has distinct effects in developing retina and differentiated photoreceptors. Finally, we will explore the mechanism by which Pias3 acts in retinal development. We hypothesize that the effects of PIAS3 are in part mediated by Crx, Nr2e3 and possibly Stat3. We also hypothesize that Pias3 directly regulates transcription of many rod, cone and possibly Muller glia-specific genes. We test whether PIAS3 interacts with Crx, Nr2e3 or Stat3 in vivo, and whether these factors mediate the effects of PIAS3 in developing retina. Following on from this, we will determine which domains of PIAS3 are required for its activity in the retina, and whether these are also required for interaction with Crx and Nr2e3. Finally, we will determine whether PIAS3 directly regulates expression of rod, cone and Muller-specific genes, and if this requires Crx and Nr2e3. Relevance: The molecular basis of cell specification in the central nervous system is poorly understood, and these studies will provide mechanistic insight into this process. Moreover, mutations in rod-enriched transcription factors very often lead to photoreceptor degeneration and blindness, and we anticipate that this may also hold for Pias3.