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Fig. 7. Ectopic neurogenesis in both the medial and lateral IZ in b404 deletion mutants results from the deletion of him and her5. (A-D) b404 mutants lack her5 and him expression. Lateral views of whole-mount embryos assayed for him or her5 expression at the 17-somite stage (anterior left, probes indicated bottom left, genotype bottom right). The position of the MHB in mutant embryos is indicated with a red arrowhead. The position of the head, reflecting the delayed convergence and extension problems in the mutant embryo, is indicated with a black arrowhead. (E-G) Ectopic neurogenesis across the LIZ revealed by ngn1 expression (blue) in b404 mutants (F,F') compared to non-mutant siblings (E,E'). In b404 embryos, ectopic ngn1-positive cells are present both in the lateral (blue arrow) and medial (asterisk) IZ (E' and F' are high magnification of the areas boxed in white in E and F, respectively). (G) The number of ngn1-positive cells in the future alar MH (area indicated with white box in E and F) is 40% higher in mutant embryos compared to WT siblings, while other neural plate areas are not affected (e.g. trigeminal ganglia, area boxed in black in E and F). (H) Reintroducing Knypek function in b404 mutants does not alter the IZ neurogenesis phenotype. Expression of ngn1 (blue) in three-somite b404 mutants where Kny function has been restored by kny RNA injection (dorsal views of flat-mounted embryos, anterior left, A' is a high magnification of the area boxed in A). Note that ectopic ngn1 expression both across the MIZ (asterisk) and LIZ (blue arrows) is not altered compared to uninjected b404 mutants (Fig. 7F). (I-J) Restoring Him function at endogenous levels in b404 mutants is sufficient to rescue the LIZ. (J) Crossing the b404 mutation into the her5PAC::egfp background generates b404/b404;her5PAC::egfp embryos where him expression is recovered with endogenous levels and expression pattern (lateral view of a 17-somite embryo, anterior left). (I,I') Expression of ngn1 (blue) in three-somite b404/b404;her5PAC::egfp embryos (dorsal views of flat-mounted embryos, anterior left, I' is a high magnification of the area boxed in I). Note that no ectopic neurogenesis is detectable any longer across the LIZ (white arrowheads), while the MIZ remains bridged by ectopic ngn1-positive cells (asterisk). (K-M). Him and Her5 equally contribute to the total inhibitory activity and one copy of either Him or Her5 is sufficient for formation of the LIZ. Formation of the LIZ in the b404/+; m119/+ embryos is indistinguishable in Him morphants (K,K'), Her5 morphants (L,L') and uninjected embryos (M,M') (white arrowheads). Note that after blocking either Him or Her5 activity ectopic neurogenesis occurs in the MIZ (white asterisk). K',L' and M' are enlargements of boxed area in K, L and M respectively (25 embryos was analyzed for b404/+; m119/+ injected with her5MO^ATG, 27 for b404/+; m119/+ injected with himGrip^ATG and 20 uninjected b404/+; m119/+).

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