Fig. 6

miR-196 regulates pharyngeal arch segmentation via pharyngeal endoderm. (A–C) Zebrafish embryos injected with miR-196 duplex or mir196-Mo were raised to 6 dpf and stained with Alcian blue for cartilage and Alizarin red for bone. (A) Control larvae had seven pharyngeal arches, including Meckels and ceratohyal cartilages (in PA1 and PA2) and five branchial arches (in PA3–7). (B) Animals overexpressing miR-196 lacked one branchial arch. Some animals with four branchial arches (insert) possessed a skeletal element attached to the last arch (arrows), suggesting that the missing arch was the one anterior to the tooth-bearing pharyngeal arch-7. (C) Injection of mir196-Mo resulted in animals with extra branchial arches. Some animals with seven pharyngeal arches (insert) displayed extra skeletal elements attached to pharyngeal arch-7 (arrows), suggesting that the extra arch was the one just anterior to PA7. (D–F) Transgenic animals expressing GFP in the neural crest (green) that were either overexpressing (E) or knocked down (F) for miR-196 showed defective segmentation of endodermal pouches stained with zn-8 antibody (red) and pharyngeal arches (green) consistent with observed alterations in arch numbers. Abbreviations: bh, basihyal; ch, ceratohyal; cl, cleithrum; hs, hyosymplectic; mc, Meckels cartilage; op, opercle; pa, pharyngeal arch; pf, pectoral fin; pq, palatoquadrate; te, teeth.