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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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Normal progression of epicardium formation. Ventral views of zebrafish hearts. (A-F) Epicardial cells are marked with pard3 (EGFP; green) and cardiomyocytes are marked with ALCAM (red). Confocal images from 72-120 hpf are optical slices showing progressive epicardium coverage (white arrows) proceeding across the ventricle (V) and then onto the atrium (A) at 120 hpf. The z-series at one-week shows epicardial cells on the ventricle and atrium, however epicardium coverage is not complete. For all panels, with anterior to the left and BA is bulbus arteriosus. Scale bars = 50 microns. For each time point n = 5.
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Figure 4: Normal progression of epicardium formation. Ventral views of zebrafish hearts. (A-F) Epicardial cells are marked with pard3 (EGFP; green) and cardiomyocytes are marked with ALCAM (red). Confocal images from 72-120 hpf are optical slices showing progressive epicardium coverage (white arrows) proceeding across the ventricle (V) and then onto the atrium (A) at 120 hpf. The z-series at one-week shows epicardial cells on the ventricle and atrium, however epicardium coverage is not complete. For all panels, with anterior to the left and BA is bulbus arteriosus. Scale bars = 50 microns. For each time point n = 5.

Mentions: We followed the path of epicardium development over time using the pard3:EGFP reporter to mark the developing epicardium and ALCAM staining to visualize the underlying myocardium. We consistently found that epicardial progenitors first migrated onto and over the ventricle to form a ventricular epicardium. At 78, 84, and 96 hpf, epicardial cells were only found overlying the ventricle (Figure 4A-D). It was not until 120 hpf that epicardial cells were detected on the atrium (Figure 4E). Epicardial cells were clearly present on both heart chambers by one week; however, even then epicardial coverage was incomplete (Figure 4F). The epicardium continued to mature over the ensuing weeks (see also Figure 3B-C”).


Multiple modes of proepicardial cell migration require heartbeat.

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

Normal progression of epicardium formation. Ventral views of zebrafish hearts. (A-F) Epicardial cells are marked with pard3 (EGFP; green) and cardiomyocytes are marked with ALCAM (red). Confocal images from 72-120 hpf are optical slices showing progressive epicardium coverage (white arrows) proceeding across the ventricle (V) and then onto the atrium (A) at 120 hpf. The z-series at one-week shows epicardial cells on the ventricle and atrium, however epicardium coverage is not complete. For all panels, with anterior to the left and BA is bulbus arteriosus. Scale bars = 50 microns. For each time point n = 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Normal progression of epicardium formation. Ventral views of zebrafish hearts. (A-F) Epicardial cells are marked with pard3 (EGFP; green) and cardiomyocytes are marked with ALCAM (red). Confocal images from 72-120 hpf are optical slices showing progressive epicardium coverage (white arrows) proceeding across the ventricle (V) and then onto the atrium (A) at 120 hpf. The z-series at one-week shows epicardial cells on the ventricle and atrium, however epicardium coverage is not complete. For all panels, with anterior to the left and BA is bulbus arteriosus. Scale bars = 50 microns. For each time point n = 5.
Mentions: We followed the path of epicardium development over time using the pard3:EGFP reporter to mark the developing epicardium and ALCAM staining to visualize the underlying myocardium. We consistently found that epicardial progenitors first migrated onto and over the ventricle to form a ventricular epicardium. At 78, 84, and 96 hpf, epicardial cells were only found overlying the ventricle (Figure 4A-D). It was not until 120 hpf that epicardial cells were detected on the atrium (Figure 4E). Epicardial cells were clearly present on both heart chambers by one week; however, even then epicardial coverage was incomplete (Figure 4F). The epicardium continued to mature over the ensuing weeks (see also Figure 3B-C”).

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