Caveolae Protect Notochord Cells against Catastrophic Mechanical Failure during Development

The embryonic notochord is a flexible structure present during development that serves as scaffold for formation of the vertebrate spine. This rod-like organ is thought to have evolved in non-vertebrate chordates to facilitate locomotion by providing a rigid but flexible midline structure against which the axial muscles can contract. This hydrostatic skeleton'' is exposed to a variety of mechanical forces during oscillation of the body. There is evidence that caveolae, submicroscopic cup-shaped plasma membrane pits, can buffer tension in cells that undergo high levels of mechanical stress. Indeed, caveolae are particularly abundant in the embryonic notochord. In this study, we used the CRISPR/Cas9 system to generate a mutant zebrafish line lacking Cavin1b, a coat protein required for caveola formation. Our cavin1b(-/-) zebrafish line exhibits reduced locomotor capacity and prominent notochord lesions characterized by necrotic, damaged, and membrane-permeable cells. Notochord diameter and body length are reduced, but remarkably, the mutants recover and are homozygous viable. By manipulating mechanical stress using a number of different assays, we show that progression of lesion severity in the mutant notochord is directly dependent on locomotion. We also demonstrate changes in caveola morphology in vivo in response to mechanical stress. Finally, induction of a catastrophic collapse of live cavin1b(-/-) mutant notochord cells provides the first real-time observation of caveolae mediating cellular mechano-protection.