Multiple modes of proepicardial cell migration require heartbeat

Abstract

BackgroundThe outermost layer of the vertebrate heart, the epicardium, forms from a cluster of progenitor cells termed the proepicardium (PE). PE cells migrate onto the myocardium to give rise to the epicardium. Impaired epicardial development has been associated with defects in valve development, cardiomyocyte proliferation and alignment, cardiac conduction system maturation and adult heart regeneration. Zebrafish are an excellent model for studying cardiac development and regeneration; however, little is known about how the zebrafish epicardium forms.ResultsWe report that PE migration occurs through multiple mechanisms and that the zebrafish epicardium is composed of a heterogeneous population of cells. Heterogeneity is first observed within the PE and persists through epicardium formation. Using in vivo imaging, histology and confocal microscopy, we show that PE cells migrate through a cellular bridge that forms between the pericardial mesothelium and the heart. We also observed the formation of PE aggregates on the pericardial surface, which were released into the pericardial cavity. It was previously reported that heartbeat-induced pericardiac fluid advections are necessary for PE cluster formation and subsequent epicardium development. We manipulated heartbeat genetically and pharmacologically and found that PE clusters clearly form in the absence of heartbeat. However, when heartbeat was inhibited the PE failed to migrate to the myocardium and the epicardium did not form. We isolated and cultured hearts with only a few epicardial progenitor cells and found a complete epicardial layer formed. However, pharmacologically inhibiting contraction in culture prevented epicardium formation. Furthermore, we isolated control and silent heart (sih) morpholino (MO) injected hearts prior to epicardium formation (60 hpf) and co-cultured these hearts with “donor” hearts that had an epicardium forming (108 hpf). Epicardial cells from donor hearts migrated on to control but not sih MO injected hearts.ConclusionsEpicardial cells stem from a heterogeneous population of progenitors, suggesting that the progenitors in the PE have distinct identities. PE cells attach to the heart via a cellular bridge and free-floating cell clusters. Pericardiac fluid advections are not necessary for the development of the PE cluster, however heartbeat is required for epicardium formation. Epicardium formation can occur in culture without normal hydrodynamic and hemodynamic forces, but not without contraction.

Proepicardium: Current Understanding of its Structure, Induction, and Fate

“…Heartbeat has been demonstrated to be necessary not only for the movement of single cells and cell clusters but also for their proper formation. Most epicardial cells derive from an AV cluster of the PE (Peralta et al, 2013(Peralta et al, , 2014Plavicki et al, 2014). PE induction and development in the zebrafish is similar to its development in mammalian and avian species.…”

“…Heartbeat has been demonstrated to be necessary not only for the movement of single cells and cell clusters but also for their proper formation. Most epicardial cells derive from an AV cluster of the PE (Peralta et al, , ; Plavicki et al, ).…”

Recent insights on the role and regulation of retinoic acid signaling during epicardial development

“…Epicardial formation and investment requires the pumping action of the heart (Peralta et al, 2013;Plavicki et al, 2014), meanwhile, the epicardium is required for the growth of the myocardial compact zone. Epicardial formation and investment requires the pumping action of the heart (Peralta et al, 2013;Plavicki et al, 2014), meanwhile, the epicardium is required for the growth of the myocardial compact zone.…”

“…Another obstacle in determining the roles of RA in epicardial development is related to the heterogenous nature of the cell populations found within the proepicardium and epicardium (Katz et al, 2012;Plavicki et al, 2014). It is very likely that the effects of RA-signaling on proepicardial maturation, fate specification and determination, epicardial EMT, or EPDC differentiation are very much dependent on the specific epicardial subset involved.…”

“…At E9.5-E10.5 in mice, proepicardial cells delaminate and cover the heart to form the epicardium and subepicardial mesenchyme. Proepicardial cells and resulting cells populating the epicardium and subepicardial space encompass a heterogenous population of cells that have distinct molecular signatures and developmental potential (Katz et al, 2012;Plavicki et al, 2014). Epicardial and subepicardial cells also express retinoid enzymes, DHRS3, RDH10, and CYP26A1 as well as RAR and RXR receptors which mediate epicardial RA signaling, as visualized by the expression of RARE-lacZ transgene (Dolle, 2009;Merki et al, 2005;Wang, Yu, et al, 2018;Zimmer, Zimmer, & Reynolds, 1994).…”