Specialized boundaries form between the rhombomeres in the hindbrain and provide signaling cues to the surrounding tissues. We are investigating the role of rhombomere boundaries in the morphogenesis of the hindbrain ventricle in zebrafish. The ventricular system forms a specialized neural circulatory system and aberrant ventricle development correlates with hydrocephalus and neurodevelopmental disorders such as schizophrenia and autism. Initial patterning of the neural tube enables the correct positioning of the ventricles. Characteristic morphogenesis and cell proliferation then shapes the ventricles, while the initial secretion of embryonic cerebrospinal fluid inflates the ventricles. The Grinblat and Wilkinson laboratories have provided evidence that Zic1 and Zic4 from the roof plate activate Notch signaling via rfng, which subsequently drives Wnt signaling at rhombomere boundaries. Our research goal is to identify novel signaling pathways that regulate hindbrain ventricle formation. By screening for pathway components with the correct spatio-temporal expression, we determined that the Hippo signaling pathway transcriptional co-activator taz, is expressed in the hindbrain roof plate. Using TALEN mutagenesis, we created mutants with a 29 bp deletion, resulting in a loss of all functional domains of the Taz protein. Taz-/- mutants display midline separation defects in the hindbrain ventricle. This phenotype is apparent by 22hpf and persists through 36hpf indicating defects in the initial shaping of the brain and ventricle inflation. Examination of genes identified by the Sive lab to be required for ventricle morphogenesis (ppp1r12a, mpp5a and crb2a) showed no alterations in expression. Therefore, we hypothesized that taz may regulate rhombomere boundary signals independent of other known roof plate derived cues in order to drive ventricle morphogenesis. Examination of roof plate signals, including the zic family of genes, revealed no alterations in expression, however taz-/- mutants fail to express both wnt1 and rfng at rhombomere boundaries. Treatment with a pharmacological inhibitor of Wnt signaling (XAV939) results in defective ventricle morphogenesis. To determine if rfng is responsible for the expression of wnt1 at rhombomere boundaries, we used a pharmacological inhibitor of Notch signaling (DAPT) and assayed for wnt1 expression. DAPT treatment did not affect wnt1 expression at rhombomere boundaries, although it did perturb ventricle morphogenesis. This is the first evidence of Hippo signaling involvement in ventricle development, and supports a model in which Taz-dependent activation of Wnt and Notch signaling pathways at rhombomere boundaries regulates hindbrain ventricle formation.