Fig. 5. The role of iro1 and iro7 in neurogenesis. (A-H) A knock-down of iro1 and iro7 with morpholino antisense oligos MO1 and MO7 reveals that iro7 is essential for expression of the ngn1 in trigeminal ganglia. Expression of ngn1 in trigeminal ganglia (black arrowhead) is similar in uninjected (A) and MO1-injected embryos (B). Injection of MO7 leads to a loss of ngn1 expression in the trigeminal ganglia (C, open arrowhead). The knockdown of iro7 by MO7-injected at the one-cell stage is rescued on one side by co-injection with Niro7 mRNA (D). ngn1 expression in the trigeminal is not lost (red arrowhead) on the side injected with Niro7 and lacZ mRNA, while it is reduced on the side MO7 was injected without Niro7 RNA (open arrowhead). The distribution of co-injected mRNA is visualized with a blue X-gal reaction product. (E-G) Examination of embryos at 24 hpf with an acetylated -tubulin antibody reveals trigeminal neurons (black arrowhead) in uninjected and MO1-injected embryos but not in MO7-injected embryos (G, open arrowhead). (H,I) Expression of ngn1 in trigeminal ganglia in hdl mutants (H, black arrowhead) is expanded to the anterior but its expression is lost in MO7-injected hdl mutants (I, open arrowhead). (J) Antisense morpholinos specifically block iro1 and iro7 translation. Radiolabeled proteins, Iro1 and Iro7, were synthesized simultaneously in vitro in the presence of an increasing log molar ratio (10¹-10⁴) of morpholinos, iro1 (MO1) or iro7 (MO7), and were run out on a SDS-PAGE gel. Lane 1: control, no morpholino. Increasing amounts of MO1 (lane 2-5) and MO7 (lane 6-9) lead to a specific reduction in the synthesis of Iro1 and Iro7 protein, respectively. (K) Structure of two artificial constructs; top, wild-type iro7; middle, VP16-iro7HD, the homeodomain of iro7 was fused to the activator region of VP16 herpes simplex virus (blue box); bottom, En-iro7HD, the homeodomain of iro7 was fused to the Drosophila Engrailed repressor region (red box). Purple box represents acidic region; green represents the Iroquois box. (L-O) Ectopic expression of iro1 and iro7 mRNA induces relatively broad ngn1 expression in the ventral ectoderm (arrowheads in L,M). En-iro7HD mRNA induces ngn1 expression in a salt-and-pepper pattern in the ventral ectoderm (N), while VP16-iro7HD mRNA is more effective at inducing broad ngn1 expression within the neural plate (O). (L-O) Anterior is towards the left, side view. Broken lines show the boundary between neural plate and ventral ectoderm. Embryos are at the three-somite stage. Distribution of ectopic mRNA is marked by red salmon-Gal staining to detect co-injected nuclear ß-galactosidase activity. (P-S) Expression of gata2 is reduced in iro1 (Q), iro7 (R), En-iro7HD (S) mRNA injected embryos when compared with uninjected control embryos (P). Embryos are at tailbud stage and viewed from ventral side.