Fig. 4. coe2 expression, but not that of asha, ashb and neurod4, is an additional target of Her5 activity at the IZ. Whole-mount in situ hybridisation (except O); dorsal views of the MH area in flat-mounted embryos at the three-somite stage (A-J,N-R), tail bud (M) or 75% epiboly (K,L); anterior towards the top; the markers indicated bottom left; red bracket indicates the IZ. (A-H) Comparison of asha (A,B), ashb (C,D), neurod4 (zath3 in figure) (E,F) and coe2 (G,H) expression upon loss of Her5 activity (B,D,F,H) compared with wild-type siblings (A,C,E,G). coe2 expression is the only target solely repressed by Her5 across the medial IZ (H, blue arrow indicates coe2 induction). (I,J) Comparison of coe2 expression in a pzhsp70:her5 transgenic embryo (J) compared with non-transgenic sibling (I) upon heat-shock (hs) of both embryos at late gastrulation. Note that coe2 expression is repressed upon ectopic her5 expression (weaker staining in J). (K-O) Time-course of her5 and coe2 expression. coe2 expression is initiated at 75% epiboly, thus following her5, across the entire MH area (K, black bracket). Double staining for coe2 and her5 at 75% epiboly (L, both in blue) demonstrates that expression of coe2 and her5 are overlapping across the IZ. coe2 expression is maintained at the IZ until the bud stage (M, blue arrow). Later on, coe2 expression is cleared from the IZ and becomes similar to ngn1 (see O). (P-R) her5 expression at the tail-bud stage in embryos injected with capped RNA encoding a dominant-negative form of Coe2 (Xcoe2DBD) (Dubois et al., 1998) (Q) compared with non-injected siblings (P) demonstrates strong downregulation of her5 expression when Coe2 activity is impaired. This phenotype is rescued upon coinjection of wild-type coe2 RNA (R). IZ, intervening zone; hs, embryo submitted to a 1 hour heat-shock pulse at late gastrulation; r2M, presumptive motorneurons of rhombomere 2; tg, presumptive trigeminal ganglia; vcc, ventrocaudal cluster.