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Fig. 3 Nkx2.7 is necessary for branchiomeric muscle morphogenesis. A Schematic of the BMs derived from PA1 (green) and PA2 (orange) including intermandibularis anterior (ima), intermandibularis posterior (imp), hyohyals (hh), and interhyals (ih). The heart, delineated in grey, has two chambers: ventricle (V) and atrium (A). Confocal images of MF20 (magenta) and Elnb (green) immunofluorescence designates BMs and smooth muscle of the outflow tract, respectively, of wild-type (n = 16) (B), nkx2.5−/− (n = 11) (C), nkx2.7−/− (n = 16) (D), and nkx2.5−/−;nkx2.7−/− (n = 5) (E) embryos. The extraocular muscles include the inferior oblique (io), inferior rectus (ir), and the adductor mandibulae (am) along with an additional PA-derived cephalic muscle, the sternohyoideus (sh). nkx2.5−/−;nkx2.7−/− embryos exhibit exacerbation of the PA2-derived BMs defects in comparison to nkx2.7−/− embryos, implicating partially redundant functions of nkx2.7 and nkx2.5. Ventral views, anterior to the top. Scale, 100 μm. Two-color fluorescent ISH of wild-type embryos illuminates overlapping expression of nkx2.7 (green) and nkx2.5 (magenta) in PA1-5 at 26 hpf (n = 12) (F). Higher magnification panels represent PA1 and PA2 domains (white dotted lines) in a merged (G) and single channel maximum intensity (H, I) projections. Lateral views, anterior to the left. Scale, 100 μm. J Quantification of the number of BMs derived from PAs in wild-type (n = 16), nkx2.5−/− (n = 11), nkx2.7−/− (n = 16), and nkx2.5−/−;nkx2.7−/− (n = 5) embryos. Mean and standard error of each data set are shown. Unpaired, two-tailed t-test yields statistically significant differences between wild-type and nkx2.7−/− (p < 0.0001), wild-type and nkx2.5−/−;nkx2.7−/− (p < 0.0001), and nkx2.7−/− and nkx2.5−/−;nkx2.7−/− (p = 0.0024) embryos.