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Fig. 5 fbln2 knockout in zebrafish induced malformations of craniofacial cartilage. (A) General phenotype changes of F0 mosaic crispants and F2 homozygous mutants (fbln2−/−) compared with the wild type (magnification factor: 3×, n = 30 per group, bar = 200 μm). Both F0 and fbln2−/− showed undeveloped mandible (red arrow heads), pericardial edema (black arrow heads), and delayed or absent swim bladders (blue arrow heads). (B) Alcian blue staining revealed craniofacial cartilage malformation in F0 crispants and F2 fbln2−/− mutants at 4 dpf (magnification factor: 4×, n = 30 per group, bar = 200 μm). Compared with the wild types, F0 and fbln2−/− mutants showed hypoplastic or absent Meckel cartilages, shorter palatoquadrates, widened angle between ceratohyal cartilages, and malformed or absent ceratobranchial cartilages. (C) Wheat germ agglutinin (WGA) staining of 4 dpf embryos revealed disarranged chondrocytes in mandibular cartilages (magnification factor: 20×, n = 30 per group, bar = 50 μm). The chondrocytes were thin and columnar in wild-type embryos, but were smaller and showed reduced aspect ratio in fbln2−/−. Furthermore, the chondrocytes of fbln2−/− were aligned irregularly instead of the stacked-coins arrangement observed in wild-type embryos. Abbreviations: cb, ceratobranchial; ch, ceratohyal; e, ethmoid; hs, hyosymplectic; m, Meckel's cartilage; pq, palatoquadrate; WT, wild type.