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Multiple modes of proepicardial cell migration require heartbeat.

Plavicki JS, Hofsteen P, Yue MS, Lanham KA, Peterson RE, Heideman W - BMC Dev. Biol. (2014)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, 777 Highland Avenue, Madison, WI 53705-2222, USA. plavicki@wisc.edu.

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.

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Inhibiting heartbeat impairs expansion of the epicardium. Lateral confocal images of zebrafish hearts at 120 hpf with anterior to the left. The epicardial marker, pard3, is green and ALCAM (cardiomyocytes) is red. Arrows in panels A and C indicate epicardial cells on the ventricle (n = 7 per group). (A) Control. (B) BDM added at 48 hpf and maintained to the end of the experiments. (C) BDM added at 72hpf and maintained to the end of the experiments. For all panels, V is ventricle; A, Atrium. Scale bars = 50 microns.
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Figure 6: Inhibiting heartbeat impairs expansion of the epicardium. Lateral confocal images of zebrafish hearts at 120 hpf with anterior to the left. The epicardial marker, pard3, is green and ALCAM (cardiomyocytes) is red. Arrows in panels A and C indicate epicardial cells on the ventricle (n = 7 per group). (A) Control. (B) BDM added at 48 hpf and maintained to the end of the experiments. (C) BDM added at 72hpf and maintained to the end of the experiments. For all panels, V is ventricle; A, Atrium. Scale bars = 50 microns.

Mentions: We repeated the sih MO injection experiment with embryos from a second epicardial reporter line, pard3:EGFP. Again, at 96 hpf pard3:EGFP + epicardial cells were easily detected on the ventricles of control hearts, but never on the hearts of sih morphants. As with the previous experiment, we often observed what appeared to be small PE-like clusters in the pericardial cavity (arrowhead in Figure 5I) and near the venous pole (not shown). We did not observe the formation of a PE bridge in sih morphants, but did observe incidences where free-floating aggregates were present in the pericardial cavity.Using BDM, we impaired heartbeat during different stages of epicardial development (Figure 6). BDM treatment from 48-120 hpf also blocked epicardium formation (Figure 6B). If we waited until 72 hpf to add BDM, we found some epicardial cells on the ventricle at 120 hpf (Figure 6C); however, epicardium formation was incomplete and epicardial cells were not detected on the atrium. This suggests that inhibiting heartbeat with BDM halted expansion of the epicardial layer.


Multiple modes of proepicardial cell migration require heartbeat.

Plavicki JS, Hofsteen P, Yue MS, Lanham KA, Peterson RE, Heideman W - BMC Dev. Biol. (2014)

Inhibiting heartbeat impairs expansion of the epicardium. Lateral confocal images of zebrafish hearts at 120 hpf with anterior to the left. The epicardial marker, pard3, is green and ALCAM (cardiomyocytes) is red. Arrows in panels A and C indicate epicardial cells on the ventricle (n = 7 per group). (A) Control. (B) BDM added at 48 hpf and maintained to the end of the experiments. (C) BDM added at 72hpf and maintained to the end of the experiments. For all panels, V is ventricle; A, Atrium. Scale bars = 50 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4048602&req=5

Figure 6: Inhibiting heartbeat impairs expansion of the epicardium. Lateral confocal images of zebrafish hearts at 120 hpf with anterior to the left. The epicardial marker, pard3, is green and ALCAM (cardiomyocytes) is red. Arrows in panels A and C indicate epicardial cells on the ventricle (n = 7 per group). (A) Control. (B) BDM added at 48 hpf and maintained to the end of the experiments. (C) BDM added at 72hpf and maintained to the end of the experiments. For all panels, V is ventricle; A, Atrium. Scale bars = 50 microns.
Mentions: We repeated the sih MO injection experiment with embryos from a second epicardial reporter line, pard3:EGFP. Again, at 96 hpf pard3:EGFP + epicardial cells were easily detected on the ventricles of control hearts, but never on the hearts of sih morphants. As with the previous experiment, we often observed what appeared to be small PE-like clusters in the pericardial cavity (arrowhead in Figure 5I) and near the venous pole (not shown). We did not observe the formation of a PE bridge in sih morphants, but did observe incidences where free-floating aggregates were present in the pericardial cavity.Using BDM, we impaired heartbeat during different stages of epicardial development (Figure 6). BDM treatment from 48-120 hpf also blocked epicardium formation (Figure 6B). If we waited until 72 hpf to add BDM, we found some epicardial cells on the ventricle at 120 hpf (Figure 6C); however, epicardium formation was incomplete and epicardial cells were not detected on the atrium. This suggests that inhibiting heartbeat with BDM halted expansion of the epicardial layer.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, 777 Highland Avenue, Madison, WI 53705-2222, USA. plavicki@wisc.edu.

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.

Show MeSH
Related in: MedlinePlus