Vinculin b deficiency causes epicardial hyperplasia and coronary vessel disorganization in zebrafish

Abstract

Coronary vessel development is a highly coordinated process during heart formation. Abnormal development and dysfunction of the coronary network are contributory factors in the majority of heart disease. Understanding the molecular mechanisms that regulate coronary vessel formation is crucial for preventing and treating the disease. We report a zebrafish gene-trap vinculin b (vclb) mutant that displays abnormal coronary vessel development among multiple cardiac defects. The mutant shows overproliferation of epicardiumderived cells and disorganization of coronary vessels, and they eventually die off at juvenile stages. Mechanistically, Vclb deficiency results in the release of another cytoskeletal protein, paxillin, from the Vclb complex and the upregulation of ERK and FAK phosphorylation in epicardium and endocardium, causing disorganization of endothelial cells and pericytes during coronary vessel development. By contrast, cardiac muscle development is relatively normal, probably owing to redundancy with Vcla, a vinculin paralog that is expressed in the myocardium but not epicardium. Together, our results reveal a previously unappreciated function of vinculin in epicardium and endocardium and reinforce the notion that well-balanced FAK activity is essential for coronary vessel development.

Zygotic vinculin is not essential for embryonic development in zebrafish

“…Remarkably, during fin clipping no vcla -/- vclb -/- fish were detected (172 adults screened out of three different pairings), while the remaining vcla -/- and vcla -/- vclb +/- siblings roughly show a Mendelian distribution. This result corroborates a recent study in which loss of vclb alone leads to lethality during juvenile stages [ 40 ]. Taken together, the data indicate that vinculin is not essential for early development of the zebrafish embryo.…”

“…This extra exon is indeed annotated for vinculin A in the Zv9 genome database, but full annotation of the vinculin B genomic region spanning this exon was missing. We did not detect the extra exon in the cDNA of vinculin A or B extracted from whole 1dpf embryo lysates, corroborating a recent article on vinculin B function in zebrafish [ 40 ].…”

“…In contrast, for vclb a potential truncated protein could start at M26 in exon 1, leaving the possibility for a functional vinculin protein. However, since the double vinculin mutant is lethal after embryonal stages, just as the single vclb mutants recently described [ 40 ], it is unlikely that such a truncated protein is really expressed. Another often-cited problem inherent to these DNA-editing techniques is the possibility of off-target effects [ 68 – 71 ].…”

“…Furthermore, they are fully viable during the first 5 days of development when most tissue and organs have been formed and are functional. Nevertheless, fish depleted from zygotic vinculin do not survive until adulthood, which is likely due to the loss of vclb , which was recently found to cause lethality in juvenile stages associated with defects in the coronary vasculature [ 40 ]. We conclude that vinculin is mostly dispensable for embryonic development in zebrafish, but crucial for adult life.…”

Drosophila vinculin is more harmful when hyperactive than absent, and can circumvent integrin to form adhesion complexes

“…Other tissues appeared normal, and our attempts to identify additional impairment in the athletic abilities of the flies were not successful, so the fly phenotype remains weaker than the phenotypes observed in mice, zebrafish or nematodes lacking vinculin (Barstead and Waterson, 1991; Xu et al, 1998a; Cheng et al, 2016). Redundancy with other adhesion proteins (such as talin; Klapholz et al, 2015) might explain the relatively mild phenotype of this highly conserved protein.…”