The midbrain-hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis.
Basal constriction occurs at the zebrafish midbrain–hindbrain boundary constriction (MHBC) and is likely a widespread morphogenetic mechanism. 3D reconstruction demonstrates that MHBC cells are wedge-shaped, and initially constrict basally, with subsequent apical expansion. wnt5b is expressed in the MHB and is required for basal constriction. Consistent with a requirement for this pathway, expression of dominant negative Gsk3β overcomes wnt5b knockdown. Immunostaining identifies focal adhesion kinase (Fak) as active in the MHB region, and knockdown demonstrates Fak is a regulator of basal constriction. Tissue specific knockdown further indicates that Fak functions cell autonomously within the MHBC. Fak acts downstream of wnt5b, suggesting that Wnt5b signals locally as an early step in basal constriction and acts together with more widespread Fak activation. This study delineates signaling pathways that regulate basal constriction during brain morphogenesis.
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