Fig 2: MBNL1 is highly expressed in the endocardial cushions and heart valves. Immunohistochemistry with an anti-MBNL1 antibody was performed on sagittal sections from wild type mouse embryos (a-f) and coronal sections of adult hearts (g). Representative sections from one of four embryos or one of two adult hearts are shown. a Close-up of the heart at E9 stained with the secondary antibody alone is shown as a negative control. b Close-up of the heart at E9 shows expression of MBNL1 in the early myocardium and endocardium of the nascent endocardial cushions just prior to the onset of EMT. c Close-up of the heart at E11.5 shows strong MBNL1 expression in the endocardial cushions of the AVC and OFT. Higher magnification views of the boxed regions in the endocardial cushion (d) and the ventricular wall (e) show intense nuclear staining in the cushion endocardial cells, but more diffuse staining in the myocardium. Intense nuclear MBNL1 staining persists in the fetal valves at E19.5 (f) and mature heart valves (g), while MBNL1 remains more diffusely distributed in the nucleus and cytoplasm in the myocardium. Asterisks highlight accumulations of melanin in the adult valve. h Western blot analysis confirms high MBNL1 expression in the adult heart, and suggests different cardiac tissues may express different MBNL1 isoforms. Protein integrity and loading were assessed by Ponceau S staining. A representative of three independent blots with similar results is shown. Atr = atrium, Vent = ventricle, EC = endocardial cushion, OFT = outflow tract, TC = tricuspid valve, Val = heart valves

Fig 3: Loss of MBNL1 stimulates EMT ex vivo in a stage-specific manner. Images of wild type (a, a’) or Mbnl1 ?E3/?E3 (b, b’) E9 AVC explant cultures were taken in the plane of focus of the surface of the gel (a, b) and beneath the surface of the gel (a’, b’) to show invaded mesenchymal cells. Filled arrowheads indicate cells in the focal plane, whereas open arrowheads indicate the same cells out of focus in a different plane. c AVC regions from E9 or E9.5 wild type or Mbnl1 ?E3/?E3 hearts were explanted onto collagen gels. The number of explants per group is indicated. An asterisk indicates a significant difference from wild type (P = 0.05). d Conditioned supernatants were collected from E9 wild type and Mbnl1 ?E3/?E3 AVC explants and the levels of active TGFß proteins secreted into the media were assayed by ELISA

Fig 4: Mbnl1 E?3/?E3 mice have precocious EMT in the endocardial cushions without a persistent increase in total mesenchyme production. Close-up views of the hearts in sagittal sections of wild type E9.25 (a) and Mbnl1 ?E3/?E3 E9 (b) embryos stained with H&E reveal precocious invasion of mesenchymal cells (red arrowhead) into the AVC cushions of knockout mice. Sections from three individuals were evaluated for each group; representative images are shown. Close-up views of the AVC cushions in adjacent sections from wild type E9.25 (c) and Mbnl1 ?E3/?E3 E9 (d) embryos subjected to immunohistochemistry with an antibody against pSMAD2 reveal high levels of TGFß signaling in only a few cells in wild type cushions (red arrowhead), but nearly all cells in the knockout cushions. H&E stained sagittal sections of wild type (e) and Mbnl1 ?E3/?E3 (f) hearts do not reveal noticeable hypercellularity in the AVC cushions at E10.5. Atr = atrium, vent = ventricle, EC = endocardial cushion, OFT = outflow tract. g The average relative number of mesenchymal cells was determined by counting the cells within the AVC cushions on serial, H&E-stained, sagittal sections of wild type and Mbnl1 ?E3/?E3 E10.5 embryos. Representative three-dimensional reconstructions of AVC cushions from optical sections of wild type (h) and Mbnl1 ?E3/?E3 (i) E10.5 hearts imaged using optical coherence tomography. Videos of the optical sections and reconstructions of these hearts are shown in Additional file 2: Movie S1 and Additional file 3: Movie S2, respectively; still images shown here were rotated to the same viewing angle

Fig 5: Mbnl1 E?3/?E3 mice exhibit ostium secundum atrial septal defects. a The heart of a one year-old adult Mbnl1 ?E3/?E3 mouse is shown with the atrial appendages removed. Patency of the foramen ovale is demonstrated by the unobstructed passage of a coarse bristle. b Excision of the atrial septum from adult hearts shows failure of the foramen ovale to close in some Mbnl1 ?E3/?E3 mice (left), while the foramen ovale is closed but enlarged in others (right). Representative images of the wild type and patent Mbnl1 ?E3/?E3 foramen ovales shown are from 10 month-old mice; the image of the closed, but enlarged, foramen ovale is from a 2.5-month old Mbnl1 ?E3/?E3 mouse. Compared to a wild type counterpart (c), the foramen ovale (bracketed by the green lines) is enlarged in an E19.5 Mbnl1 ?E3/?E3 heart (d) as shown in close-ups of coronal sections stained with Masson’s trichrome. SS = septum secundum, FV = flap valve, FO = foramen ovale