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

Figures

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  Fig. 1.. Constriction of the dorsal pericardium precedes proepicardium formation. (A) Scheme of lateral and ventral views of a 60 hpf zebrafish embryo (top) and heart (bottom). The lateral view is rotated by −90°. (B) 3D ventral view of heart region in an epi:GFP; myl7:mRFP embryo. epi:GFP+ cells (green) are present in the dorsal pericardium (DP) and proepicardium (PE), myl7:mRFP+ cells (red) form the myocardium. 3D projections from in vivo time lapse at different time points are shown. White arrows point to PE cells (see Movie 1). (C) First and last frame of an in vivo time lapse of DP cells in an epi:GFP embryo; the midline is shown by a dashed white line, blue dots indicate tracked cells. Full colored tracks label the first time frame in purple and the last in red. Arrows indicate overall direction of tracked cells. (D) The half-rose diagram shows the movement angles of tracked cells relative to the midline. (E) 3D projection of the divergence field calculated for the time-lapse video above. The calculated divergence values are represented by colors; purple-blue regions represent constriction and red regions represent expansion. Black arrows point to PE cell clusters. ap, arterial…

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  Fig. 2.. Dorsal pericardial cells reduce their area during displacement to the midline. (A) Maximum intensity projections from a time lapse using epi:GFP; βactin:LifeAct-RFPe2212Tg embryos from 48 hpf onwards. LifeAct-RFP (red) signal shows F-actin. (B) Color-coded segmentation of 47 cell shapes from time series in A. Segmentation was repeated every 10 min in 60 subsequent time frames (n=3 animals). White spots indicate the reference point on the midline. Last frame shows white outline for cells not included in the quantification (see Movie 2). (C) The scatter plot shows the area of each cell per time point against the distance to the reference location. Color code represents the initial distance to the reference point. The mean distance is drawn against the distance to reference. (D,E) Graphs describe tracking and analysis of each cell from three datasets, with mean cell area (D) and mean distance of each cell (E) drawn against time. Each dataset is split into three categories according to the initial distance of a cell to the reference point, indicated by the color code. The line style indicates different datasets. (F) Data are sorted into categories by the initial distance to the…

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  Fig. 3.. Collective cell movements and apical extrusion lead to proepicardium delamination. epi:GFP embryo in vivo time lapse. Maximum intensity projection of 22 µm. Left panel, overview of the pericardial cavity at 52 hpf. Right panels, zoomed views of the dorsal pericardium (DP) during proepicardium (PE) formation. Shown are frames of the time lapse from 52 to 67 hpf. Colored arrowheads, emerging PE cells. Colored asterisks, DP cells surrounding PE cells (shown is one out of 10 observed events, see also Movie 3). hpf, hours post-fertilization. Images from one out of 10 embryo from five independent acquisitions. Scale bar: 20 µm; zoomed images, 10 µm.

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  Fig. 4.. Proliferation of dorsal pericardial cells contributes to proepicardium formation. (A) 3D projection of six frames of an epi:GFP embryo in vivo time lapse (see also Movie 4). Division planes indicated with red (45°≤α≤90°) or green (0°≤α≤45°) lines. A representation of the midline and the venous pole is shown in yellow. The distance of the division to the midline is indicated. (B) Scheme showing the principle of measurements for distance and angles of division planes to midline. (C) Number of cell divisions during in vivo time-lapse imaging relative to their distance to midline (three embryos with 20 cell divisions each). (D) Angles of cell division planes and their distance relative to the midline. A-D, data from ≥10 biological and ≥2 technical replicates. (E) Immunostaining for GFP (green), Myosin heavy chain (red) and pH3 (white). DAPI counterstained nuclei (blue). Optical sections of a control heart or embryos treated with aphidilcolin and hydroxyurea (aph/hydrU). Zoomed view of the PE area on the right. Arrowheads, PE cells. (F) Percentage of pH3+ cells in the dorsal pericardium (DP) compared with the PE in control animals. (G) Quantification of total number of pH3+ cells in…

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  Fig. 5.. Heartbeat-induced pericardial fluid advections influence proepicardium localization and survival. (A,D,G) epi:GFP animals immunostained for GFP (green), Myosin heavy chain (MHC) (red) and nuclei counterstained with DAPI (blue). (A) Top panels show a 3D projection of 60 hpf zebrafish hearts. Optical sections of AVC and VP regions in middle panels. Zoomed images (bottom panels) are views of the proepicardium (PE) region. Control siblings were compared with sih embryos. Arrowheads, PE cells. Asterisk indicates expected avcPE location. (B) Quantification of avcPE and vpPE cell numbers in A. (C) Quantification of total PE cell number in A. (A-C) Data from ≥4 biological and ≥4 technical replicates. (D) Optical sections of control or sih embryos at 5 dpf. Arrows, epicardial cells. Asterisks mark the region where the PE was at 60 hpf. (E) Quantification of total PE cell number at 5 dpf. (F) Quantification of epicardial cell number at 5 dpf. E,F, data from ≥4 biological and two technical replicates. (G) Optical sections at 60 hpf of epi:GFP or sih embryos immunostained for L-plastin (white). Zoomed images (bottom panels) are views of the PE region. Arrowheads, PE cells; arrows, L-plastin+…

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  Fig. 6.. Proepicardium formation depends on actomyosin dynamics. (A,B,F) 60 hpf epi:GFP animals immunostained for GFP (green) and nuclei counterstained with DAPI (blue). (A) Myosin heavy chain (MHC) (white), Myosin II-A (red). Upper panel, maximum intensity projection of a heart. The three zoomed images are different views of the proepicardium (PE) region. Yellow arrows, Myosin II-A concentration sites. (B) MHC (white), phalloidin-488 to visualize F-actin (red). Upper panel, maximum intensity projection of a heart. The three zoomed images are different views of the PE region. White arrows, actin concentration sites. Representative of ≥3 biological and three technical replicates. (C,D) Ventral view of the heart region in epi:GFP; LifeAct-RFP embryos, untreated (C) or treated with 2,3-butanedione monoxime (BDM) (D), shown at different time points from an in vivo time lapse. The DP was digitally isolated. The heart tube shape was drawn with a red dashed line. White arrows, sites of actin concentration in DP cells. ≥3 biological replicates from ≥2 experiments. (E) Scheme of actin concentration (red) at DP and PE clusters (green) during PE formation in untreated or BDM-treated embryos. (F) Top…

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  Fig. 7.. Bmp2b rescues proepicardium formation upon Myosin II inhibition. (A,E) epi:GFP embryos immunostained for GFP (green), Myosin heavy chain (MHC) (red) and nuclei counterstained with DAPI (blue). (A) Top panels, 3D projection of a 60 hpf zebrafish heart, middle panels optical section and zoomed images below. Control compared with embryos overexpressing bmp2b with and without 2,3-butanedione monoxime (BDM) or with those overexpressing Noggin 3. Arrowheads, PE cells; arrows, epicardial cells. (B,C) Quantification of PE cell number (B) and avcPE and vpPE cell number (C) in conditions shown in A. (D) Quantification of epicardial cell number at 60 hpf after bmp2b-overexpression versus non-overexpression embryos. (E) Optical sections at 60 hpf showing anti-pSmad1/5 staining. Zoomed views below. Arrowheads, PE cells; asterisks, DP cells. (F) Percentage of pSmad1/5+ cells in the DP compared with the PE in control animals. (G) Total number of pSmad1/5+ cells in the DP at 60 hpf. (H) Optical section from an in vivo time lapse of a Bmp reporter line, BRE:KuO; epi:GFP embryo. Yellow arrowheads, double-positive PE cells. at, atrium; DP, dorsal pericardium; hpf, hours post-fertilization; PE,…

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  Fig. 8.. Bmp2b controls actin rearrangements necessary for proepicardium formation. (A,B,D,F) epi:GFP embryos at 60 hpf immunostained for GFP to detect dorsal pericardial (DP) and PE cells (green) and DAPI-counterstained nuclei (blue). (A) Anti-myosin II-A immunostaining (red). Zoomed view of the PE area on untreated fish compared with those overexpressing bmp2b with and without 2,3-butanedione monoxime (BDM). Yellow arrows, Myosin II-A apico-lateral accumulation in PE cells. (B) F-actin is detected with fluorescently labeled phalloidin (red). Top panels, maximum intensity projection of embryo hearts. Middle panels, optical sections. Bottom panels, zoomed images of PE region. Untreated fish were compared with those overexpressing bmp2b with and without BDM. Arrowheads, PE cluster; arrows, actin concentration sites in the PE cluster. (C) Quantification of actin intensity (arbitrary units) in PE cells from conditions shown in B. (D) Top images, 3D projections of hearts from control or bmp2b-overexpressing fish treated with cytochalsin D (cytD). Immunostained for MHC (red) and phalloidin to detect actin (white). Middle panels, optical sections of the hearts. Bottom panels, zoomed images of…

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  Fig. 9.. Effect of Bmp2b under Myosin II inhibition on dorsal pericardial layer constriction. (A-C) First and last frame of an epi:GFP in vivo time lapse of bmp2b-overexpressing (A), 2-3-butanedione monoxime (BDM)-treated (B) or bmp2b overexpression in BDM-treated larvae (C); midline is shown by a dashed white line. Arrows indicate overall direction of tracked cells. (D) Ventral view of epi:GFP; myl7:mRFP embryo. Shown are 3D projections of untreated, bmp2b-overexpressing, BDM-treated, and bmp2b-overexpression in BDM-treated fish. Bottom images, 3D projection of the divergence field of the DP at 60 hpf; blue regions represent tissue constriction and red-orange regions represent tissue expansion. Arrows, proepicardial cells; dashed lines mark the midline. (E) Mean divergence of tracked cells within the DP. (F) Percentage of cells moving towards or away from the midline. Data are mean±s.d., Kruskal–Wallis test (black labels); two-tailed Student's t-test (red labels).*P<0.05, **P<0.01, ***P<0.001. ap, arterial pole; at, atrium; hpf, hours post-fertilization; v, ventricle; vp, venous pole. Dorsal pericardium (DP) digitally isolated in 3D projections. Representative images from ≥3 biological…