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A co-culture assay of embryonic zebrafish hearts to assess migration of epicardial cells in vitro.

Yue MS, Plavicki JS, Li XY, Peterson RE, Heideman W - BMC Dev. Biol. (2015)

Bottom Line: In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo.We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression.This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Environmental Toxicology Center, University of Wisconsin, 1300 University Avenue, Madison, WI, 53706, USA. msyue@wisc.edu.

ABSTRACT

Background: The vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium. The epicardium is vital for heart development and function, and forms from epicardial progenitor cells (EPCs), which migrate to the myocardium during early development. Disruptions in EPC migration and epicardium formation result in a number of cardiac malformations, many of which resemble congenital heart diseases in humans. Hence, it is important to understand the mechanisms that influence EPC migration and spreading in the developing heart. In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo.

Results: Here we describe a novel in vitro co-culture assay for assessing epicardial cell migration using embryonic zebrafish hearts. We isolated donor hearts from embryonic zebrafish carrying an epicardial-specific fluorescent reporter after epicardial cells were present on the heart. These were co-cultured with recipient hearts expressing a myocardial-specific fluorescent reporter, isolated prior to EPC migration. Using this method, we can clearly visualize the movement of epicardial cells from the donor heart onto the myocardium of the recipient heart. We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression.

Conclusions: We present a method to assess the migration of epicardial cells in an in vitro assay, wherein the migration of epicardial cells from a donor heart onto the myocardium of a recipient heart in co-culture is monitored and scored. The donor and recipient hearts can be independently manipulated, using either genetic tools or pharmacological agents. This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.

No MeSH data available.


Related in: MedlinePlus

Migration of epicardial cells from donor hearts onto control, sih, or tbx5 MO recipient hearts. a-c Fluorescence images taken on Days 1, 3, 5, and 7 in culture show progression of epicardial cell migration. Red (tcf21:DsRed2) shows epicardial cells from the donor, green (cmlc2:EGFP) shows recipient myocardial cells. a Migration of donor epicardial cells onto a control MO recipient heart is apparent by Day 5 in culture. The merged red-on-green signal, appearing yellow, is significantly noticeable by Day 7. There does not appear to be any significant migration of donor epicardial cells onto either the sih MO recipient heart (b) or the tbx5 MO heart (c) throughout the 7 days in culture. d-f Confocal microscopy images of donor/recipient heart samples after 7 days in culture. Red indicates tcf21:DsRed2 donor epicardial cells, green indicates cmlc2:EGFP recipient myocardial cells, blue indicates DNA (DAPI stain). d Confocal microscopy verifies the presence of donor epicardial cells that have migrated onto the control MO recipient myocardium (yellow arrows). In contrast, there does not seem be any donor epicardial cells on the sih MO recipient heart (e) or the tbx5 MO recipient heart (f), which is consistent with the epifluorescence images. In this figure, single hearts were followed throughout the 7-day time course in panels a-c, and were then collected to produce the confocal images shown in panels d-f. Scale bars in all images represent 100 μm
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Fig2: Migration of epicardial cells from donor hearts onto control, sih, or tbx5 MO recipient hearts. a-c Fluorescence images taken on Days 1, 3, 5, and 7 in culture show progression of epicardial cell migration. Red (tcf21:DsRed2) shows epicardial cells from the donor, green (cmlc2:EGFP) shows recipient myocardial cells. a Migration of donor epicardial cells onto a control MO recipient heart is apparent by Day 5 in culture. The merged red-on-green signal, appearing yellow, is significantly noticeable by Day 7. There does not appear to be any significant migration of donor epicardial cells onto either the sih MO recipient heart (b) or the tbx5 MO heart (c) throughout the 7 days in culture. d-f Confocal microscopy images of donor/recipient heart samples after 7 days in culture. Red indicates tcf21:DsRed2 donor epicardial cells, green indicates cmlc2:EGFP recipient myocardial cells, blue indicates DNA (DAPI stain). d Confocal microscopy verifies the presence of donor epicardial cells that have migrated onto the control MO recipient myocardium (yellow arrows). In contrast, there does not seem be any donor epicardial cells on the sih MO recipient heart (e) or the tbx5 MO recipient heart (f), which is consistent with the epifluorescence images. In this figure, single hearts were followed throughout the 7-day time course in panels a-c, and were then collected to produce the confocal images shown in panels d-f. Scale bars in all images represent 100 μm

Mentions: Scoring for epicardial cell migration was assessed by the increasing overlap between red epicardial signal (tcf21+) and green myocardial signal (cmlc2+) over time (Fig. 2a-c). In control experiments, epicardial cell migration was observed in 12 of 13 samples (controls for sih MO cohort) and 17 of 19 samples (controls for tbx5 MO cohort). In general, epicardial cells from control samples showed clear signs of migration onto recipient myocardia between Day 4 and 5 (Figs. 2a, and 3). This was reflected in the scoring: average migration scores for control samples were 3.615 (SEM ±0.385) for the sih MO cohort, and 3.737 (SEM ±0.445) for the tbx5 MO cohort (Fig. 3). In contrast, no migration was observed at all in 5 of 14 samples in the sih MO group. If migration occurred it was minimal and significantly delayed, beginning in most cases between Day 6 and 7 (Fig. 2b). This was reflected in a significantly lower average migration score of 1.786 (SEM ±0.435) (Fig. 3). Similarly, there was no migration in 6 of 14 samples in the tbx5 MO group. Again, in those cases in which migration occurred the area of overlap was small and migration was significantly delayed, beginning in most cases on Day 6, with an average migration score of 2.000 (SEM ±0.584) (Fig. 3).Fig. 2


A co-culture assay of embryonic zebrafish hearts to assess migration of epicardial cells in vitro.

Yue MS, Plavicki JS, Li XY, Peterson RE, Heideman W - BMC Dev. Biol. (2015)

Migration of epicardial cells from donor hearts onto control, sih, or tbx5 MO recipient hearts. a-c Fluorescence images taken on Days 1, 3, 5, and 7 in culture show progression of epicardial cell migration. Red (tcf21:DsRed2) shows epicardial cells from the donor, green (cmlc2:EGFP) shows recipient myocardial cells. a Migration of donor epicardial cells onto a control MO recipient heart is apparent by Day 5 in culture. The merged red-on-green signal, appearing yellow, is significantly noticeable by Day 7. There does not appear to be any significant migration of donor epicardial cells onto either the sih MO recipient heart (b) or the tbx5 MO heart (c) throughout the 7 days in culture. d-f Confocal microscopy images of donor/recipient heart samples after 7 days in culture. Red indicates tcf21:DsRed2 donor epicardial cells, green indicates cmlc2:EGFP recipient myocardial cells, blue indicates DNA (DAPI stain). d Confocal microscopy verifies the presence of donor epicardial cells that have migrated onto the control MO recipient myocardium (yellow arrows). In contrast, there does not seem be any donor epicardial cells on the sih MO recipient heart (e) or the tbx5 MO recipient heart (f), which is consistent with the epifluorescence images. In this figure, single hearts were followed throughout the 7-day time course in panels a-c, and were then collected to produce the confocal images shown in panels d-f. Scale bars in all images represent 100 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696273&req=5

Fig2: Migration of epicardial cells from donor hearts onto control, sih, or tbx5 MO recipient hearts. a-c Fluorescence images taken on Days 1, 3, 5, and 7 in culture show progression of epicardial cell migration. Red (tcf21:DsRed2) shows epicardial cells from the donor, green (cmlc2:EGFP) shows recipient myocardial cells. a Migration of donor epicardial cells onto a control MO recipient heart is apparent by Day 5 in culture. The merged red-on-green signal, appearing yellow, is significantly noticeable by Day 7. There does not appear to be any significant migration of donor epicardial cells onto either the sih MO recipient heart (b) or the tbx5 MO heart (c) throughout the 7 days in culture. d-f Confocal microscopy images of donor/recipient heart samples after 7 days in culture. Red indicates tcf21:DsRed2 donor epicardial cells, green indicates cmlc2:EGFP recipient myocardial cells, blue indicates DNA (DAPI stain). d Confocal microscopy verifies the presence of donor epicardial cells that have migrated onto the control MO recipient myocardium (yellow arrows). In contrast, there does not seem be any donor epicardial cells on the sih MO recipient heart (e) or the tbx5 MO recipient heart (f), which is consistent with the epifluorescence images. In this figure, single hearts were followed throughout the 7-day time course in panels a-c, and were then collected to produce the confocal images shown in panels d-f. Scale bars in all images represent 100 μm
Mentions: Scoring for epicardial cell migration was assessed by the increasing overlap between red epicardial signal (tcf21+) and green myocardial signal (cmlc2+) over time (Fig. 2a-c). In control experiments, epicardial cell migration was observed in 12 of 13 samples (controls for sih MO cohort) and 17 of 19 samples (controls for tbx5 MO cohort). In general, epicardial cells from control samples showed clear signs of migration onto recipient myocardia between Day 4 and 5 (Figs. 2a, and 3). This was reflected in the scoring: average migration scores for control samples were 3.615 (SEM ±0.385) for the sih MO cohort, and 3.737 (SEM ±0.445) for the tbx5 MO cohort (Fig. 3). In contrast, no migration was observed at all in 5 of 14 samples in the sih MO group. If migration occurred it was minimal and significantly delayed, beginning in most cases between Day 6 and 7 (Fig. 2b). This was reflected in a significantly lower average migration score of 1.786 (SEM ±0.435) (Fig. 3). Similarly, there was no migration in 6 of 14 samples in the tbx5 MO group. Again, in those cases in which migration occurred the area of overlap was small and migration was significantly delayed, beginning in most cases on Day 6, with an average migration score of 2.000 (SEM ±0.584) (Fig. 3).Fig. 2

Bottom Line: In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo.We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression.This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Environmental Toxicology Center, University of Wisconsin, 1300 University Avenue, Madison, WI, 53706, USA. msyue@wisc.edu.

ABSTRACT

Background: The vertebrate heart consists of three cell layers: the innermost endothelium, the contractile myocardium and the outermost epicardium. The epicardium is vital for heart development and function, and forms from epicardial progenitor cells (EPCs), which migrate to the myocardium during early development. Disruptions in EPC migration and epicardium formation result in a number of cardiac malformations, many of which resemble congenital heart diseases in humans. Hence, it is important to understand the mechanisms that influence EPC migration and spreading in the developing heart. In vitro approaches heretofore have been limited to monolayer epicardial cell cultures, which may not fully capture the complex interactions that can occur between epicardial and myocardial cells in vivo.

Results: Here we describe a novel in vitro co-culture assay for assessing epicardial cell migration using embryonic zebrafish hearts. We isolated donor hearts from embryonic zebrafish carrying an epicardial-specific fluorescent reporter after epicardial cells were present on the heart. These were co-cultured with recipient hearts expressing a myocardial-specific fluorescent reporter, isolated prior to EPC migration. Using this method, we can clearly visualize the movement of epicardial cells from the donor heart onto the myocardium of the recipient heart. We demonstrate the utility of this method by showing that epicardial cell migration is significantly delayed or absent when myocardial cells lack contractility and when myocardial cells are deficient in tbx5 expression.

Conclusions: We present a method to assess the migration of epicardial cells in an in vitro assay, wherein the migration of epicardial cells from a donor heart onto the myocardium of a recipient heart in co-culture is monitored and scored. The donor and recipient hearts can be independently manipulated, using either genetic tools or pharmacological agents. This allows flexibility in experimental design for determining the role that target genes/signaling pathways in specific cell types may have on epicardial cell migration.

No MeSH data available.


Related in: MedlinePlus