Fig. 2

Morpholino-knockdown ofhdac4results in facial shortening and defects in palatal skeleton cartilage. (A) Splice-blocking morpholinos MO1 and MO2 target exon-9 and exon-10 of hdac4 mRNA. (B) Western blot shows down-regulation of Hdac4 protein in extracts of MO-injected embryos and larvae at 24 hpf and 6 dpf. Co-injection of MO1 (12 mg/ml) + MO2 (2 mg/ml) showed greater down-regulation of protein levels than injection of either MO alone. (C-F) Whole-mount images of living embryos at 7 dpf. C and D are lateral views with anterior towards the left and dorsal upwards, E and F are dorsal views with anterior upwards and dorsal is facing. (C and E) In uninjected fish, the face projects anterior to the eyes (indicated by red arrow in C). (D and E) The anterior projection of the face is lacking in MO-injected fish (indicated by red arrows in C and D). (G-K) Ventral view of Alcian Blue (cartilage) and Alizarin Red (bone) stained palatal skeletons, flat-mounted at 6 dpf. Anterior is upwards. (G) Uninjected fish have a normal palatal skeleton with trabeculae (tb), an ethmoid plate (ep), trabeculae communis (tc), and parachordal cartilage (pa). (H-J) MO-injected fish have a variety of palatal skeletal defects including shortened or narrowing of the ethmoid plate (H-K), holes in the ethmoid plate (H), clefts (I and K), and weak or absent trabeculae communii (H-K). C-F: scale bar = 250 μm, G-K: scale bar = 100 μm.