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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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PE migration occurs through a cellular bridge to the heart. Lateral views of zebrafish hearts at 72 hpf. (A) Brightfield image of a live heart (n = 10). Arrow indicates the PE. (B) H&E stained section through heart and pericardium (n = 5). Arrow indicates the PE. (C-D) Confocal images of whole-mount fixed zebrafish. Epicardial cells marked with immunostaining for DsRed2 (red), which is driven by the tcf21 promoter. Nuclei are stained with DAPI (blue) and cardiomyocytes are marked with activated cell adhesion molecule (ALCAM; green). (C) The PE, which is outlined, forms a bridge between the ventricle and the pericardial wall (n = 10). (D) Magnified Z-stack projection and orthogonal slice of area boxed in C. Orthogonal slice at line indicated by “x” shows cross-section of cells below the line. White arrows indicate cells within the PE cluster that are not expressing tcf21. For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.
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Figure 1: PE migration occurs through a cellular bridge to the heart. Lateral views of zebrafish hearts at 72 hpf. (A) Brightfield image of a live heart (n = 10). Arrow indicates the PE. (B) H&E stained section through heart and pericardium (n = 5). Arrow indicates the PE. (C-D) Confocal images of whole-mount fixed zebrafish. Epicardial cells marked with immunostaining for DsRed2 (red), which is driven by the tcf21 promoter. Nuclei are stained with DAPI (blue) and cardiomyocytes are marked with activated cell adhesion molecule (ALCAM; green). (C) The PE, which is outlined, forms a bridge between the ventricle and the pericardial wall (n = 10). (D) Magnified Z-stack projection and orthogonal slice of area boxed in C. Orthogonal slice at line indicated by “x” shows cross-section of cells below the line. White arrows indicate cells within the PE cluster that are not expressing tcf21. For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.

Mentions: Consistent with previous findings, the PE could be observed at 50 hpf [1] and steadily increased in size through 72 hpf, a point at which we repeatedly observed PE clusters near the AV junction forming a cellular bridge between the myocardium and pericardium. This was apparent in still images (Figure 1A), live videos (Additional file 1: Video 1), H&E-stained sections (Figure 1B), and confocal images using a tcf21:DsRed2 epicardial cell reporter (Figure 1C-D).


Multiple modes of proepicardial cell migration require heartbeat.

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

PE migration occurs through a cellular bridge to the heart. Lateral views of zebrafish hearts at 72 hpf. (A) Brightfield image of a live heart (n = 10). Arrow indicates the PE. (B) H&E stained section through heart and pericardium (n = 5). Arrow indicates the PE. (C-D) Confocal images of whole-mount fixed zebrafish. Epicardial cells marked with immunostaining for DsRed2 (red), which is driven by the tcf21 promoter. Nuclei are stained with DAPI (blue) and cardiomyocytes are marked with activated cell adhesion molecule (ALCAM; green). (C) The PE, which is outlined, forms a bridge between the ventricle and the pericardial wall (n = 10). (D) Magnified Z-stack projection and orthogonal slice of area boxed in C. Orthogonal slice at line indicated by “x” shows cross-section of cells below the line. White arrows indicate cells within the PE cluster that are not expressing tcf21. For all panels, anterior is to the left and V is ventricle. 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 1: PE migration occurs through a cellular bridge to the heart. Lateral views of zebrafish hearts at 72 hpf. (A) Brightfield image of a live heart (n = 10). Arrow indicates the PE. (B) H&E stained section through heart and pericardium (n = 5). Arrow indicates the PE. (C-D) Confocal images of whole-mount fixed zebrafish. Epicardial cells marked with immunostaining for DsRed2 (red), which is driven by the tcf21 promoter. Nuclei are stained with DAPI (blue) and cardiomyocytes are marked with activated cell adhesion molecule (ALCAM; green). (C) The PE, which is outlined, forms a bridge between the ventricle and the pericardial wall (n = 10). (D) Magnified Z-stack projection and orthogonal slice of area boxed in C. Orthogonal slice at line indicated by “x” shows cross-section of cells below the line. White arrows indicate cells within the PE cluster that are not expressing tcf21. For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.
Mentions: Consistent with previous findings, the PE could be observed at 50 hpf [1] and steadily increased in size through 72 hpf, a point at which we repeatedly observed PE clusters near the AV junction forming a cellular bridge between the myocardium and pericardium. This was apparent in still images (Figure 1A), live videos (Additional file 1: Video 1), H&E-stained sections (Figure 1B), and confocal images using a tcf21:DsRed2 epicardial cell reporter (Figure 1C-D).

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