Fig. 5

Characterisation of novel enhancers from the SOX9 genomic region with PRS-associated point mutations: spatial loss of expression.

(A) Regulatory landscape of SOX9, depicting the location and sequence conservation across evolution for the novel SOX9 enhancers. (B) p300-PK19: Lateral and ventral views of F1 dual-fluorescence reporter transgenic embryos at 48 hpf. The Wt(G) allele drives expression in the region around the oral cavity (OC), pharyngeal arches (PA2-5), otic vesicle (OV), ciliary margin zone (CMZ) of the eye, and midbrain (MB). The Mut(A) allele has lost expression in the region around the oral cavity and pharyngeal arches (curved white arrow) and the midbrain (white arrow). (C) hoc-CNE-A: F1 dual-fluorescence reporter transgenic embryos at 72 hpf and 96 hpf. At 72 hpf the Wt(C) allele drives expression in the olfactory placodes (OP), and in the palatoquadrate (PQ), ceratohyal (CH), ceratobranchial (CB) (weak) and hyosymplectic (HS) cartilages. The Mut(T) allele has lost PQ, CH, CB and HS expression; OP expression is retained. At 96 hpf the Wt(C) allele drives expression in PQ, CH, CB. The Mut(T) allele has lost CH and CB expression, but retains reduced PQ expression. (D) RNA in situ hybridisation analysis of zebrafish sox9a at 48–96hpf showing overlap of reporter expression driven by the p300-Pk19 Wt(G) and hoc-CNE-A Wt(C) alleles with the endogenous sox9a expression pattern in the developing jaw. Wt: wild-type; Mut: mutant; hpf: hours post fertilization.