Role of Intracellular Calcium Release in Hedgehog Growth Factor Signaling

描述（由申请人提供）：Hedgehog （Hh）生长因子信号是胚胎发生过程中调节组织模式的主要途径之一，在脊椎动物中因其在神经系统和肢体发育中的重要作用而闻名。即使是Hh信号的轻微扰动也与人类的发育和认知缺陷有关。此外，Hh还在调节成体干细胞的生长和分化中起作用，因此Hh通路信号的缺陷与许多类型的癌症有因果关系，包括基底细胞癌、胰腺癌、肠癌和神经系统癌。了解Hh信号的需求和调控对人类健康的维持和治疗方法的发展具有重要意义。我们最近发现了一个意想不到的和以前未描述的对Hh信号的细胞反应的要求。在Ryanodine受体细胞内钙释放通道（CRC）缺乏正常功能的情况下，几种已知直接依赖Hh信号的细胞类型在斑马鱼胚胎中无法产生。在缺乏CRC功能的胚胎中，正常感知和响应Hh信号的组织前体细胞不能上调包括补丁在内的基因，而这些基因是已知的Hh反应途径的最早标记。由于初步研究结果表明，是应答细胞需要CRC活性，并且这些细胞在没有CRC活性的情况下不会死亡，我们假设细胞需要调节钙动员来感知或执行对Hh生长因子的反应。由于这些发现是新颖的，并且没有被任何Hh通路功能的主流模型所预测，并且由于Hh通路信号的调节对人类健康如此重要，我们寻求使用R21探索性/发展研究资助计划（“高风险-高影响”）机制来进一步定义细胞内钙动员在Hedgehog信号传导中的作用。首先，我们将通过测量被阻断的CRC活性对多个Hh依赖组织发育的影响，来确定对CRC功能的需求是否是斑马鱼胚胎Hh信号反应的一般特征。其次，我们将确定hh信号和/或hh接收细胞是否需要CRC功能。第三，我们将开始确定钙动员在Hh信号转导途径中的具体作用。这些实验的结果将确定CRC活性是否需要传输、接收或响应Hh信号，从而形成我们未来研究方向的基础。

DESCRIPTION (provided by applicant): Hedgehog (Hh) growth factor signaling is one of the principal pathways regulating tissue patterning in embryogenesis, best known in vertebrates for its essential roles in nervous system and limb development. Even modest perturbations in Hh signaling have been linked to developmental and cognitive defects in humans. Moreover, Hh also functions in regulating growth and differentiation of adult stem cells, so that defects in Hh pathway signaling have been causally linked to many types of cancers, including Basal Cell Carcinoma, and cancers of the pancreas, intestine, and nervous system. Understanding the requirements for and regulation of Hh signaling has important significance for the maintenance of human health and development of therapies. We have recently discovered an unexpected and previously undescribed requirement for the cellular response to Hh signaling. In the absence of normal function of the Ryanodine Receptor intracellular Calcium Release Channel (CRC), several cell types whose development is known to depend directly on Hh signaling in the zebrafish embryo fail to arise. In embryos deficient in CRC function, tissue precursor cells that normally sense and respond to Hh signals fail to upregulate genes, including patched, that are the earliest known markers of the Hh response pathway. As preliminary findings indicate that it is the responding cells that require CRC activity and that these cells do not die in the absence of CRC activity, we hypothesize that regulated calcium mobilization is required for cells to sense or to execute the response to the Hh growth factor. Because these findings are novel and are not anticipated by any prevailing model of Hh pathway function, and because the regulation of Hh pathway signaling is so important for human health, we seek to use the R21 Exploratory/Developmental Research Grant Program ("high risk - high impact") mechanism to further define the role of intracellular calcium mobilization in Hedgehog signaling. First, we will determine whether the requirement for CRC function is a general feature of the response to Hh signaling in the zebrafish embryo by measuring the effects of blocked CRC activity on the development of multiple Hh-dependent tissues. Second, we will determine whether Hh-signaling and/or Hh-receiving cells require CRC function. Third, we will begin to identify the specific role of calcium mobilization within the Hh signal transduction pathway. Results from these experiments will establish whether CRC activity is needed to transmit, receive, or respond to Hh signaling, and thus will form the basis of our future research directions. PUBLIC HEALTH RELEVANCE: Hedgehog (Hh) growth factor signaling is one of the principal intercellular signaling pathways controlling cell growth and differentiation. Defects in the regulation of Hh signaling contribute to both cancers and birth defects in humans. Understanding the requirements for and regulation of Hh signaling has important significance for the maintenance of human health and the development of disease therapies. This proposal investigates how the controlled release and mobilization of calcium within the cell contributes to the regulation of Hh signaling.