Fig. 6

Irx7 activates and binds in vivo the krox20 cis-regulatory element C. (A–D) Embryos injected with eltC:gfp DNA alone (A, C) or with eltC:gfp DNA and irx7 + meis1.1 RNAs (B, D) and analysed for GFP activity (A, B) or by double krox20/gfp ISH (C, D) at 24 hpf. (E–H) Tg(eltC:gfp) embryos uninjected (E, F), injected with irx7 + meis1.1 RNAs (G) or injected with Mo1b and Mo7 (H) and analysed for GFP activity at 24 hpf (E) or by double krox20/gfp ISH at 3 s (F–H). In embryos shown in panels F–H, gfp (purple) and krox20 (red) expression domains perfectly overlap, resulting in a brown staining. Dotted ovals in panels A, B and E indicate the position of the otic vesicle. Arrows in panels D and G indicate regions of ectopic coexpression of gfp and krox20. (I, J) PCR amplification of DNA fragments from the krox20 locus: element C (- 41 kbp from the krox20 transcription start site), a 422 bp genomic fragment (- 22,4 kbp), and the core promoter (- 114 bp) on DNA obtained by ChIP with an Irx7 antibody on uninjected embryos (I) or with a anti-Myc antibody on meis1.1myc RNA injected embryos (J). Ab: antibody; preI: preimmune serum. Input: DNA before immunoprecipitation. The embryonic stages are indicated at the bottom right in panels A–H and on the left in panels I, J.