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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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Ongoing PE cluster formation. Ventral (A) and lateral (B-D) views of hearts from tcf21:DsRed2 larvae. PE and epicardial cells are marked with immunostaining for DsRed2 (red) and cardiomyocytes are marked with ALCAM (green). Nuclei are stained with DAPI (blue) in panel D. (A) Confocal z-stack of heart at 74 hpf. Epicardial cells have attached to the ventricle and additional PE clusters (arrows) are forming. A PE aggregate (arrowhead) that has been released into the pericardial cavity is located near the atrioventricular (AV) junction (n = 10). (B) Confocal z-stack of a ventricle at 84 hpf. Epicardial cells are established on the ventricle. PE cells clustered on the pericardial wall projecting towards the heart (n = 10). (C) A single optical slice taken from the z-stack, showing the persisting PE cluster (arrow). (C) PE cell aggregates (white arrows) in the pericardial cavity at 120 hpf (n = 7). For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.
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Figure 2: Ongoing PE cluster formation. Ventral (A) and lateral (B-D) views of hearts from tcf21:DsRed2 larvae. PE and epicardial cells are marked with immunostaining for DsRed2 (red) and cardiomyocytes are marked with ALCAM (green). Nuclei are stained with DAPI (blue) in panel D. (A) Confocal z-stack of heart at 74 hpf. Epicardial cells have attached to the ventricle and additional PE clusters (arrows) are forming. A PE aggregate (arrowhead) that has been released into the pericardial cavity is located near the atrioventricular (AV) junction (n = 10). (B) Confocal z-stack of a ventricle at 84 hpf. Epicardial cells are established on the ventricle. PE cells clustered on the pericardial wall projecting towards the heart (n = 10). (C) A single optical slice taken from the z-stack, showing the persisting PE cluster (arrow). (C) PE cell aggregates (white arrows) in the pericardial cavity at 120 hpf (n = 7). For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.

Mentions: By 84 hpf, after the initial establishment of epicardial cells on the ventricle, we found that tcf21+ cells were still present on the pericardial wall near the AV junction protruding towards the heart (Figure 2B and C). In addition to the PE cluster at the AV junction, we consistently observed tcf21+ PE clusters that formed near the venous pole as well as additional smaller clusters forming on the pericardial wall closer to the ventricle (Figure 2A). We frequently observed tcf21+ cells or cell aggregates moving within the pericardial space. Clusters of tcf21+ cells were observed on the pericardial wall and within the pericardial space from 74 hpf (Figure 2A) to 120 hpf (Figure 2C). Together, our results provide support for both cellular bridge and floating aggregate models of PE 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)

Ongoing PE cluster formation. Ventral (A) and lateral (B-D) views of hearts from tcf21:DsRed2 larvae. PE and epicardial cells are marked with immunostaining for DsRed2 (red) and cardiomyocytes are marked with ALCAM (green). Nuclei are stained with DAPI (blue) in panel D. (A) Confocal z-stack of heart at 74 hpf. Epicardial cells have attached to the ventricle and additional PE clusters (arrows) are forming. A PE aggregate (arrowhead) that has been released into the pericardial cavity is located near the atrioventricular (AV) junction (n = 10). (B) Confocal z-stack of a ventricle at 84 hpf. Epicardial cells are established on the ventricle. PE cells clustered on the pericardial wall projecting towards the heart (n = 10). (C) A single optical slice taken from the z-stack, showing the persisting PE cluster (arrow). (C) PE cell aggregates (white arrows) in the pericardial cavity at 120 hpf (n = 7). 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 2: Ongoing PE cluster formation. Ventral (A) and lateral (B-D) views of hearts from tcf21:DsRed2 larvae. PE and epicardial cells are marked with immunostaining for DsRed2 (red) and cardiomyocytes are marked with ALCAM (green). Nuclei are stained with DAPI (blue) in panel D. (A) Confocal z-stack of heart at 74 hpf. Epicardial cells have attached to the ventricle and additional PE clusters (arrows) are forming. A PE aggregate (arrowhead) that has been released into the pericardial cavity is located near the atrioventricular (AV) junction (n = 10). (B) Confocal z-stack of a ventricle at 84 hpf. Epicardial cells are established on the ventricle. PE cells clustered on the pericardial wall projecting towards the heart (n = 10). (C) A single optical slice taken from the z-stack, showing the persisting PE cluster (arrow). (C) PE cell aggregates (white arrows) in the pericardial cavity at 120 hpf (n = 7). For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.
Mentions: By 84 hpf, after the initial establishment of epicardial cells on the ventricle, we found that tcf21+ cells were still present on the pericardial wall near the AV junction protruding towards the heart (Figure 2B and C). In addition to the PE cluster at the AV junction, we consistently observed tcf21+ PE clusters that formed near the venous pole as well as additional smaller clusters forming on the pericardial wall closer to the ventricle (Figure 2A). We frequently observed tcf21+ cells or cell aggregates moving within the pericardial space. Clusters of tcf21+ cells were observed on the pericardial wall and within the pericardial space from 74 hpf (Figure 2A) to 120 hpf (Figure 2C). Together, our results provide support for both cellular bridge and floating aggregate models of PE 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