Figure 1. Knock-down of egr1 severely affects head cartilage formation at 4 dpf.

(A–I) Head cartilages were stained with Alcian Blue in morpholino treated larvae at 4 dpf; ventral (A–C) and lateral (D,E) views are shown. (A,D) control 8 ng MOcon treated larvea. (B) 8 ng translation MOegr1 injected larveas display an absence of ceratobranchials and a reduction of size and mis-shaping of pharyngeal cartilage compared to controls (A); (C,E) 4ng splicing MOegr1 injected embryos display similar cartilage defects than 8 ng translation MOegr1 (B). (F) Ectopic expression of Egr1 does not significantly affect cartilage development. (G) Rescue of 8 ng MOegr1 tr treated larvae restores all cartilaginous elements of the viscerocranium. (H) A complete restoration of all cartilage elements is obtained by rescuing 4 ng splicing MOegr1 injected larvae. Meckel’s cartilage (m), palatoquadrate (pq), ceratohyal (ch) and hyosymplectic (hs), ceratobranchials 1 to 5 (cb1-5). (I) Agarose gel electrophoresis analysis of RT-PCR products from mRNA of injected embryos: 1) control mRNA; 2) mRNA of embryos injected with MOcon 4ng and without reverse transcriptase; 3) mRNA of embryos injected with MOegr1 spl 4 ng and without reverse transcriptase; 4) cDNA of embryos injected with MOcon 4 ng. Presence of a band at 269 bp, intron has been spliced properly; 5) cDNA of embryos injected with MOegr1 spl 4 ng. Presence of a band at 966 bp indicating that intron has not been properly spliced. However a residual band at 269 bp reveals that the mRNA has been partially spliced; 6) and 7) cDNA of MOcon 4 ng and MOegr1 spl 4 ng injected embryos that have not undergone the PCR step; 8) molecular weight marker.