Hdac1 is required to establish and maintain stripes of erm expression within the centres of hindbrain rhombomeres 2-7. In situ hybridisation analysis of erm expression in (A,C,E,G,I,K) wild-type sibling and (B,D,F,H,J,L) homozygous hdac1 mutant embryos, at 21 hpf (A-D), 24 hpf (E-J) and 31 hpf (K,L). Lateral views show significant reduction of erm expression in the hindbrain of hdac1 mutant embryos (B,F) compared to wild-type siblings (A,E). Dorsal views of hindbrain are shown in panels C,D,G,H. Transverse sections through rhombomere 5 are shown in panels I,J,K,L. The hindbrain of wild-type sibling embryos (C,G,I,K) exhibits robust stripes of erm expression at 21 and 24 hpf that are restricted to rhombomere centres (C, G). Both the ventricular zone and mantle region of the hindbrain express erm at 24 hpf, but by 31 hpf erm expression is restricted to the ventricular zone. In hdac1 mutant embryos erm is expressed at 21hpf in a weak, more diffuse domain that encompasses rhombomeres 4, 5 and 6. By 24hpf, erm expression is extinguished in all rhombomeres of the hdac1 mutant hindbrain apart from rhombomere 4.

Restriction of erm expression to narrow stripes at the centres of rhombomeres 2-7 is dependent on Notch signaling. In situ hybridisation analysis of erm expression in (A,C,E,G,I) wild-type sibling and (B,D,F,H,J) homozygous mind bomb mutant embryos, at 24 hpf (A,B), 27 hpf (C,D,G,H) and 30 hpf (E,F,I,J). Dorsal views of hindbrain are shown in panels A-F. Transverse sections through rhombomere 5 are shown in panels G-J. The hindbrain of wild-type sibling embryos (A,C,E) exhibits narrow stripes of erm expression at 24, 27 and 30 hpf that are restricted to rhombomere centres. However, in 24, 27 and 30 hpf mind bomb mutant embryos, erm is expressed in much broader domains within rhombomeres (B,D,F), forming a near-continuous domain of erm expression that extends across rhombomeres 2-7. In transverse sections through the hindbrain of 27 and 30 hpf mind bomb mutant embryos (H,J), a recognisable ventricular zone cannot be distinguished and erm expression is restricted to a central territory within hindbrain tissue, being most intense at the midline. By contrast, in age-matched wild-type sibling embryos (G,I), erm expression is restricted to the clearly recognisable ventricular zone.

Hdac1 acts epistatically and in opposition to the inhibitory effect of Notch signalling on erm transcription in the hindbrain ventricular zone. In situ hybridisation analysis of erm expression in (A,E,I) wild-type sibling embyos injected with the hdac1 mismatch control morpholino, (B,F,J) wild-type sibling embryos injected with the hdac1ATG1 morpholino, (C,G,K) mind bomb mutant embryos injected with the hdac1 mismatch control morpholino, and (D,H,L) mind bomb mutant embryos injected with the hdac1ATG1 morpholino. Panels show lateral views (A-D), dorsal views of hindbrain (E-H), and transverse sections through rhombomere 5 (I-L) of 27 hpf embryos. Expression of erm is almost completely extinguished in the hindbrain of both hdac1ATG1 morphant embryos (B,F,J) and mind bomb mutants microinjected with the hdac1ATG1 morpholino (D,H,L), whereas the erm expression domain is expanded in the hindbrain of mind bomb mutants (C,G,K), and remains restricted to stripes at rhombomere centres in wild-type siblings microinjected with the hdac1 mismatch control morpholino (A,E,I).

Expression of fgf20a in the hindbrain is restricted to rhombomere centres by the opposing actions of Hdac1 and Notch signalling. In situ hybridisation analysis of fgf20a expression in (A,C) wild-type sibling, (B) hdac1 mutant and (D) mind bomb mutant embryos at 27hpf (A-D). Dorsal views of hindbrain are shown in each panel. Wild-type sibling embryos (A,C) exhibit weak, narrow stripes of fgf20a expression in the hindbrain at 27 hpf that are localised to rhombomere centres (signals in rhombomeres 4, 5 and 6 are indicated by red arrowheads). fgf20a expression is extinguished in the hindbrain of hdac1 mutant embryos (B). By contrast, mind bomb mutant embryos exhibit increased and ectopic expression of fgf20a in a broad domain within the hindbrain that encompasses multiple adjacent rhombomeres (D).