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Fig. 5 grhl2b cooperates with a novel direct transcriptional target, spec1, to regulate MHB morphogenesis. (A) ChIP from embryos expressing FLAG-tagged grhl2b (+) or non-tagged grhl2b (–) using anti-FLAG antibody and primers to three predicted grhl2b-binding sites in the spec1 promoter. Data are shown as the mean fold-enrichment±s.e.m. (Q-RT-PCR) from three independent experiments with at least five embryos per group. (B,C) Loss of MHB folding in spec1 morphants at 26 hpf. (D,E) Expression of MHB markers (eng2a, pax2a, arrows) persists in spec1 morphants at 26 hpf. (F) Q-RT-PCR of spec1 expression in the MHB of grhl2b morphants. The MHB was dissected from five embryos per group, and the data are presented as the mean±s.d. (G,H) Cooperativity between grhl2b and spec1 ATG-blocking MOs in inducing aberrant MHB folding (G, dorsal view), and mid/hindbrain ventricularisation (H, lateral view). (I,J) spec1 mRNA expression at the MHB is restored following rescue of grhl2b morphants by injection of Grhl2 mRNA (I), and is significantly upregulated in wild-type embryos overexpressing grhl2b mRNA (J).