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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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Epicardium formation on isolated hearts in vitro. Hearts from cmlc2:EGFP; tcf21:DsRed2 larvae were extracted and placed in culture for 7 days. (A) Representative confocal image of a single ventricle prior at the time of isolation (Day 0; n = 10). (B) Confocal image of a fixed ventricle after 7 days in culture (n = 10). (C) Epifluorescent images tracking epicardium formation in culture. Images shown are from days 1, 3, 5 and 7 (n = 10). tcf21:DsRed2 marks epicardial cells (red) and cmlc2:EGFP marks cardiomyocytes (green). DAPI (DNA) is in blue in A and B. Scale bars = 50 microns.
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Figure 7: Epicardium formation on isolated hearts in vitro. Hearts from cmlc2:EGFP; tcf21:DsRed2 larvae were extracted and placed in culture for 7 days. (A) Representative confocal image of a single ventricle prior at the time of isolation (Day 0; n = 10). (B) Confocal image of a fixed ventricle after 7 days in culture (n = 10). (C) Epifluorescent images tracking epicardium formation in culture. Images shown are from days 1, 3, 5 and 7 (n = 10). tcf21:DsRed2 marks epicardial cells (red) and cmlc2:EGFP marks cardiomyocytes (green). DAPI (DNA) is in blue in A and B. Scale bars = 50 microns.

Mentions: To test whether pericardial fluid forces are necessary to direct epicardium formation, we examined whether epicardial development could occur on isolated hearts in vitro. Isolation of hearts from tcf21:DsRed2 larvae at 74 hpf yielded intact ventricles carrying along 2-4 tcf21+ pioneer epicardial cells (Figure 7A). Placed in culture, these hearts continued to beat and over the next few days developed complete tcf21+ epicardiums in vitro (Figure 7B and C). If the hearts were removed prior to PE migration, 40-48 hpf, with no tcf21+ cells, they continued to beat, but the epicardium did not form (not shown). We confirmed our findings by extracting pard3:EGFP and tbx18:DsRed2 hearts at 74 hpf and following epicardial development in vitro. Again, we observed the formation of a complete epicardial layer on cultured hearts (Additional file 3: Figure S2 A and B). To determine if epicardial cells were dividing in culture, we stained pard3:EGFP cultured hearts for phospho-histone H3 (pH3) and, indeed, found pH3-positive epicardial cells (Additional file 3 Figure S2A).


Multiple modes of proepicardial cell migration require heartbeat.

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

Epicardium formation on isolated hearts in vitro. Hearts from cmlc2:EGFP; tcf21:DsRed2 larvae were extracted and placed in culture for 7 days. (A) Representative confocal image of a single ventricle prior at the time of isolation (Day 0; n = 10). (B) Confocal image of a fixed ventricle after 7 days in culture (n = 10). (C) Epifluorescent images tracking epicardium formation in culture. Images shown are from days 1, 3, 5 and 7 (n = 10). tcf21:DsRed2 marks epicardial cells (red) and cmlc2:EGFP marks cardiomyocytes (green). DAPI (DNA) is in blue in A and B. 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 7: Epicardium formation on isolated hearts in vitro. Hearts from cmlc2:EGFP; tcf21:DsRed2 larvae were extracted and placed in culture for 7 days. (A) Representative confocal image of a single ventricle prior at the time of isolation (Day 0; n = 10). (B) Confocal image of a fixed ventricle after 7 days in culture (n = 10). (C) Epifluorescent images tracking epicardium formation in culture. Images shown are from days 1, 3, 5 and 7 (n = 10). tcf21:DsRed2 marks epicardial cells (red) and cmlc2:EGFP marks cardiomyocytes (green). DAPI (DNA) is in blue in A and B. Scale bars = 50 microns.
Mentions: To test whether pericardial fluid forces are necessary to direct epicardium formation, we examined whether epicardial development could occur on isolated hearts in vitro. Isolation of hearts from tcf21:DsRed2 larvae at 74 hpf yielded intact ventricles carrying along 2-4 tcf21+ pioneer epicardial cells (Figure 7A). Placed in culture, these hearts continued to beat and over the next few days developed complete tcf21+ epicardiums in vitro (Figure 7B and C). If the hearts were removed prior to PE migration, 40-48 hpf, with no tcf21+ cells, they continued to beat, but the epicardium did not form (not shown). We confirmed our findings by extracting pard3:EGFP and tbx18:DsRed2 hearts at 74 hpf and following epicardial development in vitro. Again, we observed the formation of a complete epicardial layer on cultured hearts (Additional file 3: Figure S2 A and B). To determine if epicardial cells were dividing in culture, we stained pard3:EGFP cultured hearts for phospho-histone H3 (pH3) and, indeed, found pH3-positive epicardial cells (Additional file 3 Figure S2A).

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