Hippo Pathway Regulation of Vertebrate Ventricle Morphogenesis and Expansion

在发育过程中，脊椎动物的大脑形成了四个腔，称为脑室，作为二级循环系统，运输营养物质并从神经细胞中清除废物。心室系统发育不良的后果突出了它的重要性。脑裂症（以癫痫发作和严重的神经功能缺陷为特征）发生时，脑脊液从脑室泄漏。脑积水（脑室增大，通常是由于流体流动受阻）导致神经元数量减少和认知障碍。考虑到脑室系统的关键功能，令人惊讶的是，我们对控制脊椎动物脑室系统发育的机制缺乏完整的了解。根据斑马鱼和小鼠的研究，我们知道脑室的形成严重依赖于正确的顶基底细胞极性和调节的增殖/凋亡。然而，调节这些过程的信号通路仍然只是部分表征。我们推断，鉴于Hippo信号在其他组织中调节极性和增殖的作用，它将是脑室形成的一个极好的候选调节因子。我们利用TALEN诱变技术创造了一个缺乏Taz的斑马鱼品系，Taz是一种由Hippo信号调节的转录共激活因子。斑马鱼taz突变体在脑室形成方面显示出严重的缺陷。基于这一令人兴奋的联系，本提案将通过以下目标探索Hippo信号在脑室形成过程中的更广泛功能：***目标1：使用Crispr-Cas9技术创建斑马鱼Hippo通路突变体并评估脑室形成过程中的形态表型。该目的将创建yap， mst2， wts1和wts2的突变，以评估Hippo途径的哪些成分调节心室形成。***目的2：脑室的形成需要适当的细胞极性、细胞数量和调节的细胞信号。我们将使用极化蛋白和转基因来检测河马突变体的顶基极性缺陷。此外，我们将使用增殖细胞和凋亡细胞的测定来评估突变体在细胞数量上的缺陷。初步证据表明Taz突变体中Wnt和Notch信号发生改变。因此，我们将在Taz突变体中操纵Wnt和Notch信号，试图定义一个调节后脑室形成的综合信号网络。***目的3:Hippo信号传导的靶点尚未完全确定。我们将利用RNA-Seq鉴定河马突变体中差异表达的基因。一项试点RNA-Seq研究暗示了Taz和肌动蛋白聚合调节之间的联系。我们拟研究假定的Taz效应物在脑室形成过程中的作用

During development, the vertebrate brain forms four cavities, known as ventricles, that serve as a secondary circulatory system, transporting nutrients and removing wastes from neural cells. The importance of the ventricular system is highlighted by consequences resulting from its maldevelopment. Schizencephaly (characterized by seizures and severe neurologic deficits) arises when fissures permit cerebrospinal fluid to leak out of the ventricles. Hydrocephalus (enlarged ventricles typically resulting from obstructed fluid flow) results in impaired neuronal number and cognitive impairment. Given its critical function, it is surprising that we lack a complete understanding of the mechanisms that control development of the vertebrate brain ventricular system. On the basis of studies in zebrafish and mouse, we know that brain ventricle formation is critically dependent on correct apico-basal cell polarity and regulated proliferation/apoptosis. Yet, the signaling pathways that regulate these processes remain only partially characterized. We reasoned that Hippo signaling, given its roles in other tissues in regulating polarity and proliferation, would be an excellent candidate regulator of brain ventricle formation. We utilized TALEN mutagenesis to create a zebrafish strain lacking Taz, a transcription co-activator regulated by Hippo signaling. Zebrafish taz mutants display a profound defect in brain ventricle formation. On the basis of this exciting connection, this proposal will pursue the broader functions of Hippo signaling during brain ventricle formation using the following objectives:***Objective 1: Creation of zebrafish Hippo Pathway mutants using Crispr-Cas9 technology and evaluation of morphological phenotypes during brain ventricle formation. This objective will create mutations in yap, mst2, wts1, and wts2 to evaluate which components of Hippo pathway regulate ventricular formation.***Objective 2: Brain ventricle formation requires proper cell polarity, cell number, and regulated cell signaling. We will examine Hippo mutants for defects in apico-basal polarity using polarized proteins and transgenes. Furthermore, we will assess mutants for defects in cell number using assays for both proliferating and apoptotic cells. Preliminary evidence has demonstrated altered Wnt and Notch signaling in Taz mutants. Therefore, we will manipulate Wnt and Notch signaling in Taz mutants in an attempt to define a comprehensive signaling network regulating hindbrain ventricle formation.***Objective 3: The targets of Hippo signaling remain incompletely characterized. We will utilize RNA-Seq to identify genes that are differentially expressed in Hippo mutants. A pilot RNA-Seq study has implicated a link between Taz and regulation of actin polymerization. We propose to study the role of putative Taz effectors during ventricle formation.**