Hippo Pathway Regulation of Vertebrate Ventricle Morphogenesis and Expansion

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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.**