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腔，称为脑室，作为次级循环系统，运输营养物质并从神经细胞中清除废物。脑室系统的重要性是由其发育不良所造成的后果所突出的。脑裂畸形（以癫痫发作和严重的神经功能缺损为特征）是由于脑裂导致脑脊液从脑室漏出而引起的。脑积水（通常由液体流动受阻引起的脑室扩大）导致神经元数量受损和认知障碍。考虑到它的关键功能，令人惊讶的是，我们缺乏对脊椎动物脑室系统发育控制机制的完整理解。在斑马鱼和小鼠的研究的基础上，我们知道，脑室的形成是严重依赖于正确的apico-basal细胞极性和调节增殖/凋亡。然而，调节这些过程的信号通路仍然只有部分特征。我们推断，Hippo信号，鉴于其在其他组织中调节极性和增殖的作用，将是脑室形成的一个很好的候选调节剂。我们利用TALEN诱变来创建缺乏Taz的斑马鱼品系，Taz是由Hippo信号传导调节的转录共激活因子。斑马鱼taz突变体在脑室形成中表现出严重缺陷。基于这种令人兴奋的联系，该提案将利用以下目标来追求Hippo信号在脑室形成期间的更广泛功能： 目标1：使用Crispr-Cas9技术创建斑马鱼Hippo Pathway突变体并评估脑室形成期间的形态表型。该目标将在雅普、mst 2、wts 1和wts 2中产生突变，以评估Hippo通路的哪些组分调节心室形成。 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研究海马突变体的顶基极性缺陷。此外，我们将使用增殖和凋亡细胞的测定来评估细胞数量缺陷的突变体。初步证据表明，Taz突变体中的Wnt和Notch信号发生了改变。因此，我们将操纵Taz突变体中的Wnt和Notch信号，试图定义一个全面的信号网络调节后脑心室的形成。 目标3：Hippo信号传导的靶点仍不完全表征。我们将利用RNA-Seq来鉴定在Hippo突变体中差异表达的基因。一项初步的RNA-Seq研究暗示了Taz与肌动蛋白聚合调节之间的联系。我们建议研究心室形成过程中假定的Taz效应器的作用。

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.