Neural crest cell markers sox9a and sox10 are expressed in trunk and cranial neural crest cells in APAP-treated embryos. (A–K) Lateral views (A,B,G,H) or ventral views (C–F,I–K) of embryos processed for sox9a (A–F) or sox10 (G–K) immunohistochemistry. sox9a-positive trunk NC cells can be seen migrating ventrally in both untreated (A) and APAP-treated (B) embryos at 32 hpf (white arrowheads). In the head at 48 hpf (C) and 72 hpf (D), sox9a labels NC-derived prechondrogenic cells. In APAP-treated embryos (E,F), sox9a expression is reduced due to missing viscerocranium/pharyngeal arch-derived cartilage types. At 32 hpf, sox10-positive trunk NC cells migrate ventrally in streams in untreated embryos ((G), white arrowheads). In APAP-treated embryos (H), sox10-positive cells are reduced in number but follow similar migration paths (white arrowheads). In the head at 48 hpf (I), sox10 is expressed along the midline of the neurocranium, verified by comparison with negative control embryos in which the same secondary antibody (but no primary antibody) was applied (J). In APAP-treated embryos, sox10 expression is similar but more robust, with enhanced expression along the yolk sac (white arrowhead). (L) An overview of a basic gene regulatory network in NC-derived cartilage precursor cells in chick (from Suzuki 2006). Red lines indicate interactions that have not been established in zebrafish. In early NC cells, Sox9 induces Sox10 expression, which in turn downregulates Sox9. In cartilage precursors, Sox10 expression is downregulated causing the recovery of Sox9 expression. Together, both Sox9 and Sox10 positively regulate Col2a1 expression, a marker for differentiating chondrocytes. Abbreviations: ceratobranchial cartilages, cb; ventral ceratohyal, ch; ethmoid plate, ep; dorsal hyosymplectic, dh; Meckel’s cartilage, mc; palatoquadrate, pq; trabecular, tr. Scale bar (in (K)) for (A–K), 200 µm.
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