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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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Heterogeneous tcf21 expression within the developing epicardium. Confocal images of the developing zebrafish epicardium. (A-A”) Lateral view of a 1-week pard3:EGFP; tcf21:DsRed2 heart (n = 10). (A) Epicardial cells are marked with pard3:EGFP (green). (A’) Immunostaining for DsRed2 (red). (A”) Merge of A and A’ with DAPI staining (nuclei; blue). Arrows indicate pard3+/tcf21- epicardial cells. (B-B’) Ventral view of a 2-week cmlc2:EGFP; tcf21:DsRed2 heart (n = 5). (B) Epicardial cells are marked with immunostaining for DsRed2 (red). (B’) cmlc2:EGFP; tcf21:DsRed2 heart with DAPI staining (nuclei; blue). tcf21-/DAPI + epicardial cells (arrows) are seen overlying the myocardium. (C-C”) Ventricular epicardium from a 6-week old zebrafish heart (n = 5). (C) Epicardial cells are marked with pard3:EGFP (green). (C’) Immunostaining for DsRed2 (red). (C”) Merge of C and C’. For all panels: V is ventricle, BA is bulbus arteriosus. Scale bars = 50 microns.
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Figure 3: Heterogeneous tcf21 expression within the developing epicardium. Confocal images of the developing zebrafish epicardium. (A-A”) Lateral view of a 1-week pard3:EGFP; tcf21:DsRed2 heart (n = 10). (A) Epicardial cells are marked with pard3:EGFP (green). (A’) Immunostaining for DsRed2 (red). (A”) Merge of A and A’ with DAPI staining (nuclei; blue). Arrows indicate pard3+/tcf21- epicardial cells. (B-B’) Ventral view of a 2-week cmlc2:EGFP; tcf21:DsRed2 heart (n = 5). (B) Epicardial cells are marked with immunostaining for DsRed2 (red). (B’) cmlc2:EGFP; tcf21:DsRed2 heart with DAPI staining (nuclei; blue). tcf21-/DAPI + epicardial cells (arrows) are seen overlying the myocardium. (C-C”) Ventricular epicardium from a 6-week old zebrafish heart (n = 5). (C) Epicardial cells are marked with pard3:EGFP (green). (C’) Immunostaining for DsRed2 (red). (C”) Merge of C and C’. For all panels: V is ventricle, BA is bulbus arteriosus. Scale bars = 50 microns.

Mentions: Heterogeneous tcf21 expression was also found in the epicardium at later stages of development. At 1-week post fertilization (wpf), distinct sections of the epicardium, while clearly marked by the epicardial reporter pard3:EGFP, lacked tcf21 expression (Figure 3A-A”). These tcf21- regions of epicardium persisted over time. Continuous regions of tcf21- cells on the heart surface could be seen covering the trabeculated myocardium at 2 wpf (Figure 3B-B’) and at 6 wpf (Figure 3C-C”). We observed similar results using another known epicardial marker, tbx18[18,25,26]. Again, while some epicardial cells showed strong expression of tbx18, others had weak expression or lacked tbx18 expression completely (Additional file 2: Figure S1). Based on the observed tcf21 and tbx18 expression patterns in the juvenile epicardium, we conclude that the developing epicardium is composed of a heterogeneous population of cells.


Multiple modes of proepicardial cell migration require heartbeat.

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

Heterogeneous tcf21 expression within the developing epicardium. Confocal images of the developing zebrafish epicardium. (A-A”) Lateral view of a 1-week pard3:EGFP; tcf21:DsRed2 heart (n = 10). (A) Epicardial cells are marked with pard3:EGFP (green). (A’) Immunostaining for DsRed2 (red). (A”) Merge of A and A’ with DAPI staining (nuclei; blue). Arrows indicate pard3+/tcf21- epicardial cells. (B-B’) Ventral view of a 2-week cmlc2:EGFP; tcf21:DsRed2 heart (n = 5). (B) Epicardial cells are marked with immunostaining for DsRed2 (red). (B’) cmlc2:EGFP; tcf21:DsRed2 heart with DAPI staining (nuclei; blue). tcf21-/DAPI + epicardial cells (arrows) are seen overlying the myocardium. (C-C”) Ventricular epicardium from a 6-week old zebrafish heart (n = 5). (C) Epicardial cells are marked with pard3:EGFP (green). (C’) Immunostaining for DsRed2 (red). (C”) Merge of C and C’. For all panels: V is ventricle, BA is bulbus arteriosus. Scale bars = 50 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Heterogeneous tcf21 expression within the developing epicardium. Confocal images of the developing zebrafish epicardium. (A-A”) Lateral view of a 1-week pard3:EGFP; tcf21:DsRed2 heart (n = 10). (A) Epicardial cells are marked with pard3:EGFP (green). (A’) Immunostaining for DsRed2 (red). (A”) Merge of A and A’ with DAPI staining (nuclei; blue). Arrows indicate pard3+/tcf21- epicardial cells. (B-B’) Ventral view of a 2-week cmlc2:EGFP; tcf21:DsRed2 heart (n = 5). (B) Epicardial cells are marked with immunostaining for DsRed2 (red). (B’) cmlc2:EGFP; tcf21:DsRed2 heart with DAPI staining (nuclei; blue). tcf21-/DAPI + epicardial cells (arrows) are seen overlying the myocardium. (C-C”) Ventricular epicardium from a 6-week old zebrafish heart (n = 5). (C) Epicardial cells are marked with pard3:EGFP (green). (C’) Immunostaining for DsRed2 (red). (C”) Merge of C and C’. For all panels: V is ventricle, BA is bulbus arteriosus. Scale bars = 50 microns.
Mentions: Heterogeneous tcf21 expression was also found in the epicardium at later stages of development. At 1-week post fertilization (wpf), distinct sections of the epicardium, while clearly marked by the epicardial reporter pard3:EGFP, lacked tcf21 expression (Figure 3A-A”). These tcf21- regions of epicardium persisted over time. Continuous regions of tcf21- cells on the heart surface could be seen covering the trabeculated myocardium at 2 wpf (Figure 3B-B’) and at 6 wpf (Figure 3C-C”). We observed similar results using another known epicardial marker, tbx18[18,25,26]. Again, while some epicardial cells showed strong expression of tbx18, others had weak expression or lacked tbx18 expression completely (Additional file 2: Figure S1). Based on the observed tcf21 and tbx18 expression patterns in the juvenile epicardium, we conclude that the developing epicardium is composed of a heterogeneous population of cells.

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