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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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Epicardial cells from donor hearts do not migrate onto sih recipient hearts. (A) Schematic of our epicardial cell migration assay. Control tcf21:DsRed2 donor hearts (108 hpf) were co-cultured with either control or sih injected cmlc2:GFP recipient hearts (60 hpf). (B, C, D) Epicardial cells from control donors migrate onto control recipient hearts (n = 7). (E, F, G) Epicardial cells from control donors do not migrate onto sih recipient hearts (n = 7).
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Figure 9: Epicardial cells from donor hearts do not migrate onto sih recipient hearts. (A) Schematic of our epicardial cell migration assay. Control tcf21:DsRed2 donor hearts (108 hpf) were co-cultured with either control or sih injected cmlc2:GFP recipient hearts (60 hpf). (B, C, D) Epicardial cells from control donors migrate onto control recipient hearts (n = 7). (E, F, G) Epicardial cells from control donors do not migrate onto sih recipient hearts (n = 7).

Mentions: To further explore the necessity of heartbeat during epicardium formation, we developed an in vitro epicardial migration assay. In this assay, we co-cultured isolated donor hearts carrying tcf21:DsRed2 epicardial cells (108 hpf) with cmlc2:GFP recipient hearts isolated from either control or sih MO embryos. The recipient hearts were isolated earlier than in the previous experiment, at 60 hpf, before epicardial cells were present on the myocardium (Figure 9A). We found that epicardial cells from donor hearts could migrate onto control recipient hearts (Figure 9B, C, and D). However, epicardial cells from donor hearts did not migrate onto recipient sih MO hearts in which the heartbeat was inhibited (Figure 9E, F, G). Together, these findings indicate that the heartbeat itself, independent of its effects on pericardial fluid forces, is necessary for epicardial cell migration.


Multiple modes of proepicardial cell migration require heartbeat.

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

Epicardial cells from donor hearts do not migrate onto sih recipient hearts. (A) Schematic of our epicardial cell migration assay. Control tcf21:DsRed2 donor hearts (108 hpf) were co-cultured with either control or sih injected cmlc2:GFP recipient hearts (60 hpf). (B, C, D) Epicardial cells from control donors migrate onto control recipient hearts (n = 7). (E, F, G) Epicardial cells from control donors do not migrate onto sih recipient hearts (n = 7).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Epicardial cells from donor hearts do not migrate onto sih recipient hearts. (A) Schematic of our epicardial cell migration assay. Control tcf21:DsRed2 donor hearts (108 hpf) were co-cultured with either control or sih injected cmlc2:GFP recipient hearts (60 hpf). (B, C, D) Epicardial cells from control donors migrate onto control recipient hearts (n = 7). (E, F, G) Epicardial cells from control donors do not migrate onto sih recipient hearts (n = 7).
Mentions: To further explore the necessity of heartbeat during epicardium formation, we developed an in vitro epicardial migration assay. In this assay, we co-cultured isolated donor hearts carrying tcf21:DsRed2 epicardial cells (108 hpf) with cmlc2:GFP recipient hearts isolated from either control or sih MO embryos. The recipient hearts were isolated earlier than in the previous experiment, at 60 hpf, before epicardial cells were present on the myocardium (Figure 9A). We found that epicardial cells from donor hearts could migrate onto control recipient hearts (Figure 9B, C, and D). However, epicardial cells from donor hearts did not migrate onto recipient sih MO hearts in which the heartbeat was inhibited (Figure 9E, F, G). Together, these findings indicate that the heartbeat itself, independent of its effects on pericardial fluid forces, is necessary for epicardial cell migration.

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