Hippo Pathway Regulation of Vertebrate Ventricle Morphogenesis and Expansion

在发育过程中，脊椎动物的大脑形成四个被称为脑室的空腔，作为第二循环系统，运输营养物质并从神经细胞中清除废物。脑室系统发育不良所造成的后果凸显了它的重要性。脑裂畸形(以癫痫发作和严重的神经功能障碍为特征)发生在脑裂使脑脊液从脑室漏出时。脑积水(脑室扩大通常是由于液体流动受阻引起的)会导致神经元数量受损和认知障碍。鉴于其关键功能，令人惊讶的是，我们对脊椎动物脑室系统发育的控制机制缺乏完整的了解。在斑马鱼和小鼠的研究基础上，我们知道脑室的形成关键依赖于正确的顶基细胞极性和调节的增殖/凋亡。然而，调控这些过程的信号通路仍然只有部分特征。我们认为，考虑到河马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很好的候选调节器。我们利用TALEN突变创造了一个缺少Taz的斑马鱼品系，Taz是一种受河马信号调节的转录辅助激活因子。斑马鱼taz突变体在脑室形成方面显示出严重的缺陷。在这种令人兴奋的联系的基础上，这项建议将利用以下目标探索河马信号在脑室形成过程中的更广泛功能：*目标1：利用Crispr-Cas9技术创建斑马鱼河马途径突变体，并评估脑室形成过程中的形态表型。这个目标将在yap、mst2、wts1和wts2中产生突变，以评估河马通路中哪些成分调节脑室形成。*目标2：脑室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质和转基因来检测河马突变体顶基极性的缺陷。此外，我们将使用增殖和凋亡细胞的检测来评估突变的细胞数量缺陷。初步证据表明，在Taz突变体中，Wnt和Notch信号发生了变化。因此，我们将在Taz突变体中操纵Wnt和Notch信号，试图定义一个调控后脑室形成的全面信号网络。*目标3：河马信号的靶点仍未完全确定。我们将利用RNA-Seq来鉴定在河马突变体中差异表达的基因。一项初步的RNA-Seq研究表明，Taz和肌动蛋白聚合的调控之间存在联系。我们建议研究可能的Taz效应分子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.**