Fig. 3

Attenuation of barx1 expression perturbs viscerocranial morphology and cartilage patterning. Larvae injected with (A) control MO (4 ng), and (B and C) Bx MO (4 ng). Alcian Blue staining of control MO (D) and Bx MO injected (E and F) 120 hpf larvae. Dissection of the first and second arches of control (G) and mildly affected larvae (H). (I and J) Parasagittal sections of 72 hpf embryos. (J) Bx MO (4 ng) injected compared with (I) control MO injected (4 ng). Anterior is to the left; (A–C, G–J) lateral views, (D–F) ventral view. (A–C) Arrows indicate mouth, and arrowheads the pharyngeal arches; (D–F) open arrowheads indicate the ceratohyal cartilage; (G, H) asterisk, perturbed fusion; black arrowhead, joint fusion between Meckel's cartilage (M) and the palatoquadrate (pq); cb, ceratobranchials; ch, ceratohyal; ep, ethmoid plate; hm, hyomandibular; ov, otic vesicle; sy, symplectic element. Scale bars: (A–C) 250 μm; (D–H) 100 μm; (J) 50 μm. (K, L) Effectiveness and specificity of the barx1 (Bx) and splice-acceptor (Sa) morpholinos. (K) Western analysis of in vivo attenuation of barx1:GFP translation in 24 hpf zebrafish embryos. (L) Total RNA extracts (18S and 28S bands shown) used for RT-PCR of barx1. β-actin used as a control. (M) Percentage of affected embryos comparing control MO, Bx MO and Sa MO, and rescue by co-injection of barx1 mRNA with a 5-bp mismatch to the ATG target site. Affected embryos are defined as those exhibiting micrognathia as a result of a reduction or loss of arch cartilage elements, as seen by Alcian Blue staining at 120 hpf. (1) Control MO 4 ng; (2) Bx MO 4 ng; (3) Sa MO 4 ng; (4) Bx MO 2 ng + Sa MO 2 ng. Rescue: (5) gfp mRNA 25 pg; (6) barx1 mRNA 25 pg; (7) Bx MO + Sa MO 2 ng each + barx1 mRNA 25 pg. Number of embryos indicated on top of each bar.