Actin dynamics and the Bmp pathway drive apical extrusion of proepicardial cells

ABSTRACT The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.


Movie 3. Convergence of dorsal pericardial cells leads proepicardial cells to extrude apically.
In vivo time-lapse of epi:GFP embryo during 16.5 h starting at 52 hpf. The movie shows a maximum intensity projection of 22 µm. The left panel shows an overview of the ventral and dorsal pericardium (DP). Right panels show a zoomed frame of the time-lapse. An extruding PE cells can be observed. This movie is representative for the event observed in a total of 10 different movies. Scale bar of overview 20 µm and zoom 10 µm. HT, heart tube. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.
Movie 4. Cell divisions in the dorsal pericardium. 3D reconstruction of an in vivo time-lapse of an epi:GFP 35 hpf embryo during 33 h. GFP labels dorsal pericardial cells (DP, grey). The ventral pericardium was digitally extracted (white box) from frame 7 onwards to allow a top view on the DP. Ten cell divisions in the DP are indicated with yellow arrows. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.

Movie 5. Macrophages remove emerging PE cells in the absence of a heartbeat.
Movie of an epi:GFP; mpeg1:mcherry embryo microinjected with a tnnt2 morpholino. One optical section is shown, after digitally fixing the extruded cell in the field of view. White arrowhead indicates extruding proepicardial (PE) cell (green) and magenta arrowhead shows a macrophage (red), which removes the PE cell by phagocytosis. Scale bar: 10 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.

Movie 6. Myosin accumulates in proepicardial cells.
In vivo time-lapse imaging of epi:GFP;myl12.1:mCherry 52-hpf embryo during 7 h. Optical section zoomed view of the proepicardial (PE) region. GFP labels dorsal pericardial (DP) cells (green). myosin12.1-mCherry is shown in red. Note the presence of myosin-positive PE cells and PE cells that are advected in the pericardial cavity. Scale bar: 50 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.
Movie 7. Actin cables in the dorsal pericardium. 3D reconstruction of an in vivo high resolution Z-stack of an epi:GFP;β-actin:LifeAct-RFP embryo at 52 hpf. GFP labels dorsal pericardial (DP) and proepicardial (PE) cells in green. LifeAct-RFP marks F-actin in red. The ventral pericardium was partially removed with a clipping plane in Imaris. An actin cable (arrows) connects DP cells together and with the venous pole (VP) of the heart tube. Scale bar: 10 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.

Movie 8. Actin cables in the dorsal pericardium.
Maximum projection of the in vivo high resolution Zstack shown in movie 7 from an epi:GFP;β-actin:LifeAct-RFP embryo at 52 hpf. GFP-labeled dorsal pericardium (DP) and proepicardial (PE) cells are shown in green. LifeAct-RFP marking F-actin is shown in red. Multiple actin cables are visible in the DP and one (arrows) connects a cluster of DP cells to the venous pole (VP) of the heart tube. This cluster joins a PE cluster closer to the VP and the midline at the end of the acquisition. During acquisition, the cable reduces in length and finally dissolves Scale bar, 20 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.
Movie 9. Actin accumulates at midline, where the PE forms. 3D reconstruction of an in vivo time-lapse imaging of an epi:GFP;β-actin:LifeAct-RFP 52-hpf embryo during 18.5 h. GFP-labeled dorsal pericardium (DP) and proepicardial (PE) cells are shown in green. LifeAct-RFP marking F-actin is shown in red. The ventral pericardium was digitally clipped out from frame 5 onwards to allow a better view on the DP. HT, heart tube. Scale bar: 50 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.

Movie 10. Proepicardial cells accumulate F-Actin.
Optical section of 18 h in vivo time-lapse imaging of an epi:GFP;β-actin:LifeAct-RFP embryo starting at 52 hpf. GFP-labeled dorsal pericardium (DP) and proepicardial (PE) cells are shown in green. LifeAct-RFP marking F-actin is shown in red. Note that a subset of GFP + cells accumulate LifeAct-RFP and, at the end of the video, are released into the pericardial cavity (arrow). The remaining GFP-positve DP cells reveal lower LifeAct-RFP intensity. HT, heart tube. Scale bar: 50 µm. Shown is a representative movie from ≥3 biological and ≥ 2 technical replicates.
Movie 11. Apical extrusion pf PE cells. epi:GFP;β-actin:LifeAct-RFP embryo in vivo time-lapse starting at 52 hpf with drift correction. Top panels show 3D reconstruction, bottom panels show a single 0.320 µm thick optical section. On the right green (epi:GFP) labels dorsal pericardial (DP) and proepicardial (PE) cells in green. LifeAct-RFP marks F-actin in red. On the left, movie in gray-scale, the cell outlines were traced to create masks and each color identifies a cell over time. White arrowheads point to region were a cell extrusion will take place (cell labeled in green). At this site an actin ring becomes visible (red arrowhead). Scale bar, 10 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.

Movie 12. Proepicardial cells are attached to the dorsal pericardium by actin filopodia until release.
Optical section of a 14 h in vivo time-lapse imaging of an epi:GFP;β-actin:LifeAct-RFP embryo starting at 52 hpf. GFP-labeled dorsal pericardium (DP) and proepicardial (PE) cells are shown in green. LifeAct-RFP marking F-actin is shown in red. Arrow in frame 75 points to an actin stalk connecting a PE cell with the DP. Scale bar: 50 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.
Movie 13. 2,3-Butanedione monoxime (BDM) inhibits F-actin accumulation at the dorsal pericardial midline. 3D reconstruction of in vivo time-lapse imaging of an epi:GFP;β-actin:LifeAct-RFP embryo starting at 52 hpf. The embryo was treated with 10 mM butanedione monoxime during acquisition. GFP-labeled dorsal pericardium (DP) and proepicardial (PE) cells are shown in green. LifeAct-RFP marking F-actin is shown in red. The ventral pericardium was digitally clipped out to allow a better view on the DP. On the left merged channels are shown. Scale bar: 50 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.
Movie 15. Segmentation of different PE clusters. 3D reconstruction of an epi:GFP 60 hpf zebrafish embryo. epi:GFP were immunostained for GFP in green, myosin heavy chain (MHC) in red and nuclei were counterstained with DAPI (blue). The ventral pericardium was digitally removed to allow a top view on the DP. avcPE and vpPE clusters were segmented (yellow and white colors, respectively). From second 10 onwards, transversal optical sections through the heart are shown to highlight the morphology of DP vs PE cells. at, atrium; avc, atrio-ventricular canal; DP, dorsal pericardium; PE, proepicardium; v, ventricle; vp, venous pole. Scale bar: 50 µm. Shown is a representative movie from ≥ 3 biological and ≥ 2 technical replicates.