Investigating crosstalk between Taz and Wnt in the hindbrain ventricle

The brain ventricle system (BVS), composed of several cavities and the cerebral spinal fluid within, requires the complex regulation of signaling pathways to properly form. Molecular analyses and genetic screens have pinpointed key regulators of BVS formation, but the mechanism by which they interact and are regulated remains poorly understood. Our laboratory has identified a novel role for Taz, a co-transcriptional regulator of the Hippo signaling pathway, in BVS development. To generate taz mutants, Transcription Activator-Like Effector Nucleases were used. We utilized transgenic zebrafish lines and fluorescence microscopy to visualize neural structure and signalling pathway outputs. In situ hybridization, immunohistochemistry, mRNA injection and Western blots were used for gene expression, protein localization and quantification studies. Pharmacologic modulation of WNT and NOTCH pathways were used to examine signalling. Zebrafish taz mutant analyses showed a decreased hindbrain ventricle size as a result of ventricle midline separation failure. At locations of defective midline separation, we also observed aberrant cytoskeletal organization. Literature suggested an interaction between Wnt and Hippo signaling, where TAZ/YAP stability is regulated by Wnt activity. Our studies have shown that in wild-type embryos, Taz protein localizes to hindbrain segment boundaries, a domain of active Wnt signaling. In addition, our studies have shown that inhibition of Wnt signaling results in loss of intersegmental stabilization of Taz, suggesting Wnt-dependent stabilization. Similarly, we have shown that overexpression of the Wnt pathway component, Axin1, results in increased Taz protein, suggesting a direct interaction. In addition to Wnt involvement, preliminary studies have suggested interactions between Taz and Notch signaling pathway components. In taz mutants, there is a strong downregulation of the Notch modulator rfng at the segmental boundaries. Taken together, our research results indicate that Hippo signaling lies at the interface between Wnt and Notch signaling pathways and facilitates the crosstalk of three signaling pathways.