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Fig. 5 Craniofacial cartilage development requires nkx2.7 expression. A Schematic illustrates the cartilage elements derived from PA1 (green) and PA2 (orange) along with associated morphometric parameters (gray dotted lines) measured in panels E and F. Cartilages, such as ceratobranchial elements, derived from PA3-7 are noted in gray. Dissected Alcian Blue staining in wild-type (n = 21) (B), nkx2.5−/− (n = 11) (C), and nkx2.7−/− (n = 15) (D) demonstrate shorter and wider orientation of the PA1- and PA2-derived cartilages in nkx2.7−/− compared to wild-type and nkx2.5−/− embryos. Viscerocranium cartilage elements are labeled as follows: Meckel’s (M), palatoquadrate (Pq), ceratohyal (Ch), hyosymplectic (Hs), basihyal (Bh), basibranchial (Bb), and ceratobranchial (Cb). Ventral views, anterior to the top. Scale, 100 μm. E Morphometric analysis of the cartilage segments reveals statistically significant decrease in lengths between wild-type (n = 21), nkx2.5−/− (n = 11), and nkx2.7−/− (n = 15) embryos in M (p < 0.0001), Ch (p < 0.0001), and Pq (p < 0.0001), employing an unpaired, two-tailed t-test. Mean and standard error of each data set are shown. F Measurement of the M-Pq angle delineates a statistically significant difference between wild-type (n = 14) and nkx2.7−/− (n = 20) embryos with no difference detected when comparing wild-type and nkx2.5−/− (n = 13) embryos. Unpaired, two-tailed t-test is used to demonstrate mean and standard error of each data set (p < 0.0001 between wild-type and nkx2.7−/− embryos). G Schematic illustrates a lateral view (anterior to the left) of cartilage elements and joint articulations contributing to the lower jaw at 96 hpf. H–J Visualization of viscerocranium following Alcian Blue and Alizarin Red staining at 7 dpf reveals aggravation of the craniofacial abnormalities in nkx2.7−/− embryos. Craniofacial skeletal elements identified as follows: maxilla bone (mx), entopterygoid (en), branchiostegal ray (br), and opercle (op). Ventral views (anterior to the top) illuminate widening and shortening of the lower jaw in nkx2.7−/− (n = 7) (J) compared with wild-type (n = 35) (H) and nkx2.5−/− (n = 16) (I) embryos. Scale, 100 μm. K Quantification of the statistically significant decline in br and op bones when evaluating wild-type and nkx2.5−/− compared with nkx2.7−/− embryos using an unpaired, two-tailed t-test (br, p < 0.0001; op, p = 0.0019). The number of samples for each genotype is noted above. Mean and standard error of each data set are shown. L–N Lateral views (anterior to the left) of dissected wild-type (n = 9) (L), nkx2.5−/− (n = 7), and nkx2.7−/− (n = 8) (N) embryos following Alcian Blue staining. PA2 jaw joint fusion with thickening of the connection between the ceratohyal and the hyosymplectic cartilages is visible in the loss-of-function model. Scale, 100 μm. O Quantification of the statistically significant enlargement in the Ch width at the juncture of the PA2 joint in nkx2.7−/− compared to wild-type embryos employing an unpaired, two-tailed t-test (p < 0.0001). The number of samples for each genotype is noted above. Mean and standard error of each data set are shown.