The role of PIAS3 in retinal development

项目概述：我们的目标是确定发展和生存所需的分子网络 哺乳动物的杆状感光器。我们鉴定了激活的STATS蛋白抑制因子(PIASS)，一种转录 在发育中的杆状光感受器中强烈表达的协同调节因子和位点特异性的Sumoylase。 我们观察到PIAS3的过度表达引起杆状感光细胞的增加，而PIAS3 击倒会产生多余的穆勒神经胶质细胞。我们还鉴定了两个转录因子CRX和Nr2e3，它们是 PIASS相互作用蛋白。因此我们假设PIAS3在杆状细胞的发育和发育中起关键作用 通过与光感受器特异的转录因子相互作用存活。我们建议检验这一假设 进行了一系列的实验。 首先，我们将使用荧光原位杂交和免疫细胞化学来确定PiasS是否 在发育中的杆状感光器中表达，在发育中的锥体和有丝分裂前体细胞中不表达。 其次，根据我们的初步数据，我们假设PIASS调节着 光感受器的分化、视紫红质表达的动力学和新的 分化的光感受器。为了解决这些问题，我们将对影响进行详细分析 使用一组分子标记研究PIASS功能的得失。我们将使用基于BrdU的出生日期来 确定PIASS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来确定PiasS功能的得失是否具有明显的影响 在发育视网膜和分化的光感受器方面。 最后，我们将探讨PiasS在视网膜发育中的作用机制。我们假设 PIASS的作用部分是由CRX、Nr2e3和可能的STATs介导的。我们还假设PiasS 直接调节许多视杆细胞、视锥细胞和可能的穆勒胶质细胞特异基因的转录。我们测试是否 PIASS在体内与CRX、Nr2e3或STATS相互作用，以及这些因素是否介导了PIASS在体内的作用 正在发育中的视网膜。接下来，我们将确定ITS需要哪些PIASS域 视网膜中的活动，以及这些是否也是与CRX和Nr2e3相互作用所必需的。最后，我们会 确定PIASS是否直接调节视杆细胞、视锥细胞和穆勒特异基因的表达，以及是否如此 需要CRX和Nr2e3。 相关性：中枢神经系统中细胞规格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，杆状富集型的突变 转录因子经常导致光感受器退化和失明，我们预计这 可能也适用于PiasS。

Project summary: We aim to identify the molecular network required for development and survival of mammalian rod photoreceptors. We identified Protein Inhibitor of Activated STATS (PIASS), 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 PiasS 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 PiasS is expressed in developing rod photoreceptors and absent from developing cones and mitotic progenitors. Second, based on our preliminary data, we hypothesize that PIASS 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 PiasS using a panel of molecular markers. We will use BrdU-based birthdating to determine whether gain/loss of function of PIASS influences the kinetics of rhodopsin expression, and use rhodopsin-based expression constructs to determine whether gain/loss of PiasS function has distinct effects in developing retina and differentiated photoreceptors. Finally, we will explore the mechanism by which PiasS acts in retinal development. We hypothesize that the effects of PIASS are in part mediated by Crx, Nr2e3 and possibly StatS. We also hypothesize that PiasS directly regulates transcription of many rod, cone and possibly Muller glia-specific genes. We test whether PIASS interacts with Crx, Nr2e3 or StatS in vivo, and whether these factors mediate the effects of PIASS in developing retina. Following on from this, we will determine which domains of PIASS 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 PIASS 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 PiasS.