Intercellular Signalling Pathways Regulate Zebrafish Hindbrain Development

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Introduction: Our laboratory uses zebrafish hindbrain development as a model for understanding how cells undergo complex morphogenetic rearrangements to shape tissues within the head. The hindbrain is a transiently segmented portion of the central nervous system that lies anterior to the spinal cord and posterior to the midbrain. A subpopulation of hindbrain cells delaminate from the neural tube. These cells, known as cranial neural crest, subsequently migrate to create the jaw cartilage elements. Cells that remain within the hindbrain must undergo equally complex changes in cell shape to inflate the fourth brain ventricle, a cavity that functions as a secondary neural circulatory system. Both jaw cartilage and hindbrain ventricle formation require cells to develop precise cell polarity, undergo cell shape changes, and express the precise set of genes necessary for cellular behaviour. How are such processes regulated? We have recently identified two key cell signalling pathway components that regulate jaw cartilage formation and brain ventricle morphogenesis, respectively. Project 1: Regulation of neural crest cells by Bone Morphogenetic Protein 3 (BMP3). Neural crest cells delaminate from the dorsal hindbrain and migrate laterally  and differentiate to form the jaw cartilage elements. We generated a zebrafish bmp3 mutant using Crispr-Cas9 and this mutant displays profound alterations to jaw formation. Our initial goal will be to generate a Smad3 reporter line to determine the precise temporal and spatial parameters of Bmp3 signalling. We will then perform confocal imaging to study the rearrangement of cells within cartilage and how this is altered in bmp3 mutants. Finally, we will undertake transcriptomic analyses of bmp3 mutants to define the molecular mechanisms by which this pathway controls cartilage development. Project 2: Regulation of brain ventricle morphogenesis by Taz. The brain ventricular system consists of a series of fluid-filled cavities that function as a secondary neural circulatory system. The hindbrain ventricle forms as tissues separate along the midline and subsequently inflate the cavity via secretion of cerebrospinal fluid. In an effort to understand Hippo signalling in neural development, we generated taz mutant zebrafish and found that such mutants display a significantly smaller brain ventricle with a defect in midline separation. Taz protein is stabilized by crosstalk from the Wnt signalling pathway and the two pathways coordinately regulate polarity of cells located at hindbrain segment boundaries. We propose a model in which Taz regulates hindbrain rhombomere boundary cell identity. To test this model, we plan to create additional mutants in the Hippo kinase cascade and perform a systematic analysis of gene expression in hindbrain segment boundary cells. Finally, we will determine how Taz-dependent transcripts function in regulating hindbrain boundary cell identity and brain ventricle formation.