eng2a is a direct target gene of grhl2b in the MHB. (A,B) High-powered coronal cross-sections, showing eng2a (A) and grhl2b (arrows) and pax2a (B) expression in the MHB in wild-type embryos at 12 hpf. mhb, midbrain-hindbrain boundary; se, surface ectoderm. (C,D) Coronal sections of MHB shown before laser capture microdissection (C) and highlighting the excised region (D). (E,F) Q-RT-PCR of grhl2b (E) and eng2a (F) from laser capture microdissection MHB tissue, relative to cDNA from pre-zygotic transcription-stage (one-cell) embryos, water and RT-negative controls. (G) Immunoblot of FLAG-tagged grhl2b (arrow) in cell extracts from HEK-293 cells transfected with pCS2+grhl2b-FLAG or the negative control (pCS2+GFP). n/s, non-specific band. (H) ChIP from embryos expressing FLAG-tagged grhl2b (+) or non-tagged grhl2b (–) using anti-FLAG antibody and primers for the two predicted grhl2b-binding sites in the eng2a promoter. Data are shown as the mean fold enrichment±s.e.m. (Q-RT-PCR) from two independent experiments performed with at least five embryos in each group. Negative control regions of the promoter or exon 1 (′–3 kb′ and ′coding′. respectively) show no significant enrichment. (I) Reporter gene assay using a 3 kb region of the eng2a promoter linked to the luciferase gene (pGL3-eng2a-PROM) in 24 hpf embryos co-injected with either MO:control or MO:grhl2b. Data are shown as mean light units±s.e.m. from two independent experiments performed with a minimum of 40 embryos in each group.
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