Craniofacial bone and cartilage abnormalities in clcn7 morphants. (A) Craniofacial phenotype comparison between control and clcn7 morphants. Cranial cartilages (blue) and mineralized bones (red) were stained with alcian blue and alizarin red in control and clcn7 morphants at 5 dpf, respectively. (A-C) Compared to WT and Control embryos, clcn7 morphants showed more malformed craniofacial skeletons, and clcn7 mRNA rescued the abnormalities in craniofacial region. (D, E) Abnormal ceratohyal (Ch) angle and altered location of otolith in clcn7 morphants. (F)Quantitative analysis of a series of changes of phenotypic indexes. clcn7 morphants showed obvious changes in the pattern of craniofacial structure. (G) WISH analysis showed the abolished expression of sp7 and col10a1 in cranial skeleton, most notably in the parasphenoid (Ps) and opercle (Op) bones in clcn7 morphants at 3 dpf. (H) Q-PCR analysis confirmed the reduced level of sp7 and col10a1. For A-E, WT n=300, Control MO n=320, clcn7 MO n=300, clcn7 MO + mRNA n=182. For H: Control MO n=50, clcn7 MO n=50. The red arrow indicates abnormal Ch angle, and the white arrow points to abnormal otolith. Ch: ceratohyal. M: Meckel's. Op: opercle. Pq: palatoquadrate. Ps: parasphenoid. WT: wide type. Error bar represents the SD. The experiment was repeated at least thrice with the same conditions. * p<0.05, ** p<0.01, *** p<0.001. Scale bars: 100 μm.
