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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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Heartbeat is not required for PE cluster formation, but is necessary for epicardium development. Lateral views of zebrafish hearts with anterior to the left. (A-C) Brightfield micrographs showing hearts from control (A), sih MO (B), and BDM-treated (C) fish at 72 hpf. The PE clusters are pseudo colored purple and indicated by arrows (n = 10 per group). (D and E) Epifluorescence images showing hearts from control (D) and sih MO-treated (E) fish at 72 hpf, using the tcf21:DsRed2 reporter to reveal PE clusters (arrows) (n = 15 per group). The pericardial space is outlined with a dashed line. (F-I) Confocal images of embryos treated with control and sih-MO collected at 96 hpf (n = 12 per group). (F and G)tcf21:DsRed2 is red and ALCAM is green. (H and I)pard3:EGFP is green and ALCAM is red. Arrows indicate epicardial cells developing across the ventricle. Arrows indicate small PE clusters expressing tcf21 or pard3. For all panels, V is ventricle; A, Atrium; BA, bulbus arteriosus. Scale bars = 50 microns.
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Figure 5: Heartbeat is not required for PE cluster formation, but is necessary for epicardium development. Lateral views of zebrafish hearts with anterior to the left. (A-C) Brightfield micrographs showing hearts from control (A), sih MO (B), and BDM-treated (C) fish at 72 hpf. The PE clusters are pseudo colored purple and indicated by arrows (n = 10 per group). (D and E) Epifluorescence images showing hearts from control (D) and sih MO-treated (E) fish at 72 hpf, using the tcf21:DsRed2 reporter to reveal PE clusters (arrows) (n = 15 per group). The pericardial space is outlined with a dashed line. (F-I) Confocal images of embryos treated with control and sih-MO collected at 96 hpf (n = 12 per group). (F and G)tcf21:DsRed2 is red and ALCAM is green. (H and I)pard3:EGFP is green and ALCAM is red. Arrows indicate epicardial cells developing across the ventricle. Arrows indicate small PE clusters expressing tcf21 or pard3. For all panels, V is ventricle; A, Atrium; BA, bulbus arteriosus. Scale bars = 50 microns.

Mentions: We first examined PE development in sih MO and BDM treated larvae at 72 hpf. Brightfield images clearly show PE clusters in control, sih MO-injected and BDM-treated larvae (Figure 5A-C), indicating that inhibiting heartbeat did not prevent PE development. We examined 10 individuals for each condition, and observed a PE in 10/10 fish for the control, sih MO, and BDM groups. Furthermore, if BDM was present during the period in which the PE cluster forms (24-72 hpf), and then removed afterwards, the epicardium appeared normal at 120 hpf (not shown).


Multiple modes of proepicardial cell migration require heartbeat.

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

Heartbeat is not required for PE cluster formation, but is necessary for epicardium development. Lateral views of zebrafish hearts with anterior to the left. (A-C) Brightfield micrographs showing hearts from control (A), sih MO (B), and BDM-treated (C) fish at 72 hpf. The PE clusters are pseudo colored purple and indicated by arrows (n = 10 per group). (D and E) Epifluorescence images showing hearts from control (D) and sih MO-treated (E) fish at 72 hpf, using the tcf21:DsRed2 reporter to reveal PE clusters (arrows) (n = 15 per group). The pericardial space is outlined with a dashed line. (F-I) Confocal images of embryos treated with control and sih-MO collected at 96 hpf (n = 12 per group). (F and G)tcf21:DsRed2 is red and ALCAM is green. (H and I)pard3:EGFP is green and ALCAM is red. Arrows indicate epicardial cells developing across the ventricle. Arrows indicate small PE clusters expressing tcf21 or pard3. For all panels, V is ventricle; A, Atrium; BA, bulbus arteriosus. 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 5: Heartbeat is not required for PE cluster formation, but is necessary for epicardium development. Lateral views of zebrafish hearts with anterior to the left. (A-C) Brightfield micrographs showing hearts from control (A), sih MO (B), and BDM-treated (C) fish at 72 hpf. The PE clusters are pseudo colored purple and indicated by arrows (n = 10 per group). (D and E) Epifluorescence images showing hearts from control (D) and sih MO-treated (E) fish at 72 hpf, using the tcf21:DsRed2 reporter to reveal PE clusters (arrows) (n = 15 per group). The pericardial space is outlined with a dashed line. (F-I) Confocal images of embryos treated with control and sih-MO collected at 96 hpf (n = 12 per group). (F and G)tcf21:DsRed2 is red and ALCAM is green. (H and I)pard3:EGFP is green and ALCAM is red. Arrows indicate epicardial cells developing across the ventricle. Arrows indicate small PE clusters expressing tcf21 or pard3. For all panels, V is ventricle; A, Atrium; BA, bulbus arteriosus. Scale bars = 50 microns.
Mentions: We first examined PE development in sih MO and BDM treated larvae at 72 hpf. Brightfield images clearly show PE clusters in control, sih MO-injected and BDM-treated larvae (Figure 5A-C), indicating that inhibiting heartbeat did not prevent PE development. We examined 10 individuals for each condition, and observed a PE in 10/10 fish for the control, sih MO, and BDM groups. Furthermore, if BDM was present during the period in which the PE cluster forms (24-72 hpf), and then removed afterwards, the epicardium appeared normal at 120 hpf (not shown).

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