Fig. 7

Inhibition of TGF-β signaling partially rescues craniofacial abnormalities in clcn7 deficient zebrafish. (A-D)Ventral views of 5dpf zebrafish with alcian blue and alizarin red staining. Control zebrafish treated with DMSO (A) or 10 μM SB431542 (C) showed no obvious changes of Ch angle. (B) clcn7 knockdown zebrafish treated with DMSO showed abnormal Ch angle, and 10 μM SB431542 partially rescued Ch angle, showing significant differences (D, I). (E-H) Comparison of embryonic tooth number among controls and clcn7 morphants treated with DMSO or 10 μM SB431542. (J) clcn7 morphants treated with 10 μM SB431542 showed the higher eruption rate of 4V¹ tooth. (K)Schematic correlation between the amount of ClC-7 and craniofacial/tooth phenotypes in humans and zebrafish. ARO showed more and severe craniofacial/tooth phenotypes than ADO. The more severe the craniofacial phenotypes in clcn7 morphants, the lower level of clcn7 and ctsk, which further results in the imbalance between TGF-β and BMP signals, affecting the craniofacial and tooth development. (L) Schematic representation of ClC-7- and CTSK-mediated TGF-β/BMP pathway in zebrafish. clcn7 deficiency led to impaired lysosomal function and reduced CTSK expression. TGF-β-like Smad2 signals are elevated and BMP-like Smad1/5/8 signals are reduced in clcn7 knockdown zebrafish, which may contribute to the imbalance in TGF-β/BMP signaling during craniofacial and tooth development. The experiment was repeated thrice with the same conditions. The data in image J was one of the representative experimental results. For A-J: Control MO DMSO n=210, Control MO SB431542 n=198, clcn7 MO DMSO n=158, clcn7MO SB431542 n=134. Scale bars: 100 μm.