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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Dev Biol. 2014 Mar 27;390(2):191–207. doi: 10.1016/j.ydbio.2014.03.008

Figure 2. Abnormalities in OFT cushions in Acvr1-cKO.

Figure 2

A-J, E11, E12 H&E; B-E,G-J transverse to OFT: Parallel to the distal OFT, endocardial contour (arrow) smooth in control (A) but irregular (arrows) and cushion continuity poor (arrowhead) in mutant (F). Distally, two cushions (par, sep) present in control (B) leave endocardium fairly close to OFT wall (asterisk), but in mutant (G) one large dysmorphic cushion, with circumferentially oriented cells on left side (arrows), create a greater distance to between wall and endocardium (asterisk) and functional lumen remains only in right anterior position. Mid-OFT, four cushions (par, sep, I, I) in control (C), abnormal cushion size and location (asterisks) in mutant (H). Proximal cushions faces are parallel to flow (line) in control (D), but an abnormal pyramidal cushion contour (arrowheads) in mutants (I). By E12, parietal cushion continuity with AV cushion present (asterisk), wide cushion-free RV outlet (line) anteriorly in control (E) but undersized proximal cushions, no continuity with AV cushion, no cushion-free outlet space (arrow) in mutant (J).

K-S, E11–12, Plxna2 ISH: From both right (K,P) and left (K’,P’) Plxna2-positive cells present in both control (K,K’) and mutant (P,P’) OFT, though staining mid-OFT in mutant (arrows) weaker. Mid-OFT, strong expression in central cells of each main OFT cushion in control (L), faint misplaced expression in mutant (R). By E12, strong expression in condensed central mesenchyme (arrow) at level of valve septation in control (M), faint in aortic trunk (arrowhead); faint in mutant cushion (S, arrows) but strong expression in irregular thickness arterial wall (arrowhead).

N,T, E10, pgal stain, sagittal sections: in Acvr1-cHet Mef2c[AHF]-Cre control, numerous recombined mesenchymal cells (blue arrows) underlie recombined endocardium (N) in proximal cushion, but very few in Acvr1-cKO (T). Unrecombined mesenchyme (white arrows) and endocardium also present.

O,U, E11, transverse distal OFT sections, immunostaining: Upper panels show MF20, a-pSmad1/5/8 immunostaining on sections in which the region including a particular column of SHF-derived cells is indicated by a box (see also Online Figure 4). Lower panels: darkfield and color-inverted images show an enlargement of the equivalent areas in sister sections to those in the upper panels, demonstrating that the population of Isl1-positive, MF20-negative cells there (outlined in color-inverted images) contained more pSmad1/5/8-positive cells in the control than in the mutant sample (quantification shown in bar chart; n=4, +/− SEM, * p<0.01). Examples of Isl1-positive, pSmad1/5/8-negative cells (green arrows); Isl1-negative, pSmad1/5/8-positive cells (red arrows); and Isl1-positive/pSmad1/5/8-positive cells (yellow arrows on darkfield, blue arrows on color-inverted image) identified. Note many pSmad1/5/8-positive mesenchymal cells in both control and mutant sections (red cells in color-inverted images).

AV, atrioventricular cushion; dOFT, distal OFT; I, intercalated cushion; pOFT, proximal OFT; par, parietal cushion; sep, septal cushion. Embryonic axes in F,G,M: A, anterior; D, dorsal; L, left; P, posterior; R, right; V, ventral. Scale bar, 200µm.