Fig 11

(A) Animals triple heterozygous for mef2ca, dlx5a and jag1b were intercrossed, offspring were raised to 6 dpf and fixed and stained with Alcian Blue (cartilage) and Alizarin Red (bone). Genotyped skeletal preparations were scored for penetrance of mef2ca-associated phenotypes. Removing functional copies of jag1b from mef2ca;dlx5a double mutants decreases penetrance of mef2ca-associated phenotypes. Asterisks indicate significance at alpha = 0.05 Fisher’s exact test compared with mef2ca-/-;dlx5a+/+;jag1b+/+. (B) Genotyped mef2ca;dlx5a;jag1b skeletal preparations were flat mounted and imaged. mef2ca mutant associated phenotypes are indicated including ectopic bone (arrowheads), dysmorphic ch (arrows) and ch-to-hm transformation (red arrow). Stick-like opercle bone is indicated with a white arrow. Scale bar: 50 μm. (C) Head expression of Notch target genes was quantified by RT-qPCR in full-sibling dlx5a mutant and wild-type embryos at 48 hpf. Expression in dlx5a mutants was normalized to wild-type sibling expression levels (black line). Asterisks mark genes with expression levels significantly different in dlx5a mutants compared with dlx5a wild types (T-test at alpha = 0.05). Error bars are standard deviation. (D) Spatial her6 expression was monitored by in situ hybridization in wild types and dlx5a mutants. In wild-type embryos her6 is expressed in three discrete patches in arch 2 (arrows). In dlx5a homozygous mutants, her6 expands ventrally resulting in a larger, more continuous expression domain (arrowhead). dlx2a expression was used to delineate pharyngeal arches one (a1) and two (a2) (dashed outlines). her6 was expanded in 0/4 homozygous wild types and 3/3 homozygous mutants Scale bar: 30 μm.