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视杆细胞光感受器的发育和生存所需的分子网络。我们鉴定了活化STAT 3的蛋白抑制剂（PIAS 3），其是一种转录辅助调节剂和位点特异性SUMO酶，在发育中的视杆细胞中强烈表达。我们观察到PIAS 3过表达引起视杆细胞感光细胞的增加，而PIAS 3敲低产生过量的Muller神经胶质。我们还确定了两个转录因子，Crx和Nr 2 e3，作为Pias 3相互作用蛋白。因此，我们假设PIAS 3通过与光感受器特异性转录因子相互作用在视杆细胞发育和存活中起着关键作用。我们建议用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，以确定是否Pias 3在发展中的视杆细胞的光感受器和发展中的视锥细胞和有丝分裂祖细胞缺席表达。 其次，基于我们的初步数据，我们假设PIAS 3调节分化的光感受器的命运、视紫红质表达的动力学以及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物对Pias 3功能获得/丧失的影响进行详细分析。我们将使用基于BrdU的出生日期来确定PIAS 3功能的获得/丧失是否影响视紫红质表达的动力学，并使用基于视紫红质的表达构建体来确定PIAS 3功能的获得/丧失是否在发育中的视网膜和分化的光感受器中具有不同的影响。 最后，我们将探讨Pias 3在视网膜发育中的作用机制。我们推测PIAS 3的作用部分由Crx、Nr 2 e3和可能的Stat 3介导。我们还假设，Pias 3直接调节许多杆，锥和可能穆勒胶质细胞特异性基因的转录。我们测试PIAS 3是否与Crx，Nr 2 e3或Stat 3在体内相互作用，以及这些因子是否介导PIAS 3在发育中的视网膜中的作用。接下来，我们将确定PIAS 3的哪些结构域是其在视网膜中的活性所必需的，以及这些结构域是否也是与Crx和Nr 2 e3相互作用所必需的。最后，我们将确定PIAS 3是否直接调节视杆细胞、视锥细胞和Muller细胞特异性基因的表达，以及这是否需要Crx和Nr 2 e3。 相关性：人们对中枢神经系统细胞特化的分子基础知之甚少，这些研究将为这一过程提供机制见解。此外，富含视杆细胞的转录因子的突变经常导致感光细胞变性和失明，我们预计这也可能适用于Pias 3。

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.