title: Multiple modes of proepicardial cell migration require heartbeat.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Epicardial 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.
authors.affiliation: Department of Pharmaceutical Sciences, 777 Highland Avenue, Madison, WI 53705-2222, USA. plavicki@wisc.edu.
authors.forename: Jessica S
authors.initials: JS
authors.lastname: Plavicki
authors.forename: Peter
authors.initials: P
authors.lastname: Hofsteen
authors.forename: Monica S
authors.initials: MS
authors.lastname: Yue
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Lanham
authors.forename: Richard E
authors.initials: RE
authors.lastname: Peterson
authors.forename: Warren
authors.initials: W
authors.lastname: Heideman
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002465:Cell Movement; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006339:Heart Rate / Q000502:physiology; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D038081:Organogenesis; D010496:Pericardium / Q000166:cytology* / Q000196:embryology / Q000502:physiology; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors; D046509:Tissue Culture Techniques; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism / Q000502:physiology
chemical_list: D008164:Luminescent Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins