The extracellular matrix protein agrin is essential for epicardial epithelial-to-mesenchymal transition during heart development

Figures

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  Fig. 1.. The developing epicardium has active regions of EMT and exhibits morphological heterogeneity. (A-H) The surfaces of E13.5 and E14.5 hearts viewed by SEM. (A) The ventral aspect of an E13.5 heart. (B) Magnified view of the boxed area in A. (C,D): Magnified views of the boxed areas in B. (E) The dorsal aspect of an E14.5 heart. (F) Magnified view of the boxed area in E. (G) Magnified view of the boxed area in F. (H) Magnified view of the boxed area in G. Note the distinct cell morphologies, including flat and tightly connected cells (arrow in D,G) and cells detached from each other suggesting undergoing EMT (arrow in D,H). (I-R) Whole-mount immunofluorescence staining for the epicardium markers WT1 (red) and podoplanin (PDPN, green) and DAPI (blue) in an E13.5 heart (ventral aspect). (J-M) Magnified view of the boxed area in I at the ventricular septation. (O-R) Magnified view of the boxed area in I on the ventricle wall. (S-W) Whole-mount immunofluorescence staining for WT1 (red), podoplanin (green) and DAPI (blue) in an E14.5 heart (dorsal aspect). (T-W) Magnified view of the boxed area in S near the ventricle septum. LV, left ventricle; OFT, outflow tract; RV, right ventricle.…

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  Fig. 2.. Active EMT regions within the embryonic epicardium are associated with ECM components. (A) Immunofluorescence staining of a sagittal section of an E10.5 heart for WT1 (green) and integrin α4 (magenta). (B,C) Immunofluorescence staining of a sagittal section of an E13.5 heart for WT1 (white), integrin α4 (green) and DAPI (blue) showing clusters of WT1+ cells migrating into the myocardium and downregulated integrin α4 around these cells (arrows in B), and WT1+ cells localizing within the epicardium (arrowhead in B) (C) Magnified view of the boxed area in B. (D-G) Immunofluorescence staining for laminin (LMN; red), WT1 (white) and DAPI (blue) on serial sections from an E14.5 heart, near the ventral surface (D,E) and near the dorsal-ventral midline (F,G). (E) Magnified view of the boxed area in D. (G) Magnified view of the boxed area in F. Arrowheads in G indicate continuous LMN in basal membrane. (H) Immunofluorescence staining of an adjacent section to that shown in D (upper panels) and to that shown in F (lower panels) for integrin β1 (green) and fibronectin (FN; white). Scale bars: 50 μm (A); 100 μm (B,C); 50 μm (D-H).

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  Fig. 3.. Localization of agrin in the developing epicardium. (A,H,O) SEM showing the area near the interventricular septum of E13.5, E14.5 and E16.5 hearts, respectively. (B-G,I-N,P-U) Whole-mount immunofluorescence staining of E13.5, E14.5 and E16.5 heart for podoplanin (PDPN, green), WT1 (red), agrin (white) and DAPI (blue). (C,J,Q) Magnified views of the boxed areas in B,I,P. Agrin localizes among epicardial cells in close proximity to podoplanin (C,F, arrows). The punctate localization of agrin increased at E14.5 (J,M) and decreased at E16.5 (Q,T, arrows). LV, left ventricle; RV, right ventricle. Scale bars: 50 μm.

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  Fig. 4.. Loss of agrin results in developmental defects specifically in the epicardium. (A-D) Epicardial cells on the surface of E14.5 (A) and E16.5 (C) littermate controls (CO) and agrin KO hearts (B,D) hearts under SEM. Arrows in A-D indicate protrusions along cell-cell borders. (E,F) Immunofluorescence staining for WT1 (red) in E14.5 heart sections of the littermate control and agrin KO. (G,H) Magnified views of the boxed areas in E and F. (I) Quantification of WT1+ cells in the epicardium of control and KO hearts (percentage of WT1+ nuclei among epicardial cells). (J) Quantification of the number of WT1+ cells in a designated myocardium area. Data represent mean±s.e.m. n=3 hearts per group. (K,L) Immunofluorescence staining for podoplanin (PDPN, green) in E14.5 control (K) and agrin KO (L) sections. (M,N) Whole-mount immunofluorescence staining of E16.5 control (M) and agrin KO (N) hearts for podoplanin (green) labeling cardiac lymphatic vessels and epicardial cells. (O,P) Magnified views of the boxed areas in M and N. Note the downregulated epicardial podoplanin in agrin KO hearts. (Q,R) Whole-mount immunofluorescence staining for WT1 (red) of the same hearts shown in M and N at the…

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  Fig. 5.. Deletion of agrin compromises ECM deposition and dystroglycan aggregation in embryonic hearts. Immunofluorescence staining of integrin α4 (A,B), integrin β1 (C,D), collagen I (E,F), laminin (G,H) and β-catenin (I,J) in serial sections of embryonic hearts of E14.5 littermate control (CO; A,C,E,G,I) and agrin KO (B,D,F,H,J). Note that images in C and E are from the same region of a control section, and images in D and F are from the same region of an agrin KO section. Insets show magnifications of the respective boxed areas. (K,L) Quantification of mean intensity of collagen I (K) and laminin (L) in embryonic heart sections of E14.5 controls and agrin KO. Data represent mean±s.e.m. n=3 hearts per group. P-values were calculated using an unpaired, two-tailed Student's t-test. (M-P) Wholemount immunofluorescence staining of DAG1 to show localization of dystroglycan (DAG1) in the epicardium of littermate control (M,O) and agrin KO (N,P) hearts. (O,P) Magnified views of the boxed areas in M and N. Arrows in M,O,P indicate DAG1 puncta. (Q,R) Immunofluorescence staining of WT1 in the same regions as shown in O and P showing downregulation of WT1 in E16.5 agrin KO (R) compared with the…

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  Fig. 6.. Agrin promotes EMT by enhancing pFAK and decreasing β-catenin. (A) Schematic showing the differentiation protocol of human epicardial cells (hEPDCs) from hESCs. d, day. (B) Treatment of human epicardial-like cells with TGFβ and agrin resulted in enhancement of stress fibers (phalloidin, green) and pFAK (white). (C) Immunofluorescence staining for stress fibers (phalloidin, green) and β-catenin (white) on human epicardial-like cells treated with agrin or blocking antibody against integrin α4 and integrin β1. (D) Addition of agrin enhanced cell size, indicating EMT. Data represent mean±s.e.m. n=3 treatments per group. P-value was calculated using one-way ANOVA among all the groups (the P value at the top). Two-tailed Student's t-tests were used to examine differences between groups (all the other P values). Scale bars: 50 μm.

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  Fig. 7.. Agrin aggregates DAG1 to the Golgi apparatus in epicardial cells. (A) Immunofluorescence staining for DAG1 (green) and DAPI (blue) of human epicardial-like cells treated with agrin or blocking antibodies. Compared with the negative DMSO control, DAG1 was detected in proximity of nuclei when 200 ng/ml agrin (Agrin) was added. Blocking agrin with agrin blocking antibody (Agrin Ab) resulted in dispersal of dystroglycan (arrow). Treatment with blocking antibody against DAG1 (DAG1Ab) also resulted in dystroglycan dispersal (arrow). These effects were partially rescued by addition of agrin (DAG1Ab Agrin and Agrin Ab Agrin). TGFβ treatment did not aggregate dystroglycan to proximal nuclei. (B-D) Immunofluorescence staining with DAG1 antibody (red) and GM130 antibody (white) showing that agrin aggregates dystroglyan to the Golgi apparatus in epicardial cells. Addition of agrin caused aggregation of dystroglycan (arrows in C), which overlapped with the Golgi apparatus labeled with GM130 (white). TGFβ did not aggregate dystroglycan (D). (E-H) Wholemount immunofluorescence staining in E14.5 hearts showing that dystroglycan in epicardial cells aggregated in Golgi apparatus labeled with…