Fig. 3. Her5 is necessary and sufficient to control IZ formation. (A-C) Reliability of the hsp-dependent expression system. (A,B) Embryos from a cross between parents heterozygous for the hsp-her5 transgene probed for her5 expression (in situ hybridization) before (A) and after (B) a 1- hour heat-shock. While no ectopic expression of her5 is detected without heat-shock, ectopic her5 expression is ubiquitously induced upon heat-shock (white arrows indicate endogenous her5 expression at the MHB, black arrows indicate hsp-driven ubiquitous expression). (C) Stability of the induced her5 mRNA upon heat-shock, determined by whole-mount in situ hybridization (in percentage of the estimated intensity of staining that immediately follows a 0.5-hour heat-shock pulse). Induced mRNAs become undetectable within 1.5 hours following the end of the heat-shock. (D-H,N,O) Ectopic expression of Her5 inhibits ngn1 expression in the vcc and presumptive r2MN. Whole-mount in situ hybridization or immunocytochemistry with the markers indicated (bottom left, color coded) on transgenic embryos (tg) (E,F,H,O) and their wild-type siblings (wt) (D,G,N) at the 3-somite (D-F), 20-somite (G,H), and 36 hpf (N,O) stages, following a 1-hour heat-shock at late gastrulation (hs). D-F and N,O are dorsal views of the MH area in flat-mounted embryos, anterior to the top; G,H are sagittal views of the head, anterior to left; the insets show unperturbed ngn1 expression in the spinal cord. The misexpression of her5 during late gastrulation inhibits ngn1 expression in the vcc and r2MN at the 3- somite stage (white asterisks in E). Non-heat-shocked transgenics display a ngn1 profile indistinguishable from non-transgenic controls (not shown). This effect is maintained until at least the 20-somite stage (H), and is rescued upon injection of MO^tg, a morpholino oligonucleotide selective of the transgene (F). At 24 hpf, the number of nMLF neurons (brown arrows), which derive at least in part from the vcc, is also significantly reduced in hsp-her5 transgenics (O) (red arrow to her5 expression at the MH junction). (I) Genotyping results to identify transgenic embryos in H (PCR for the transgene). Lane 1: embryo H, lane 2: embryo G, lane 3: negative control, lane 4: positive control. An identical procedure was used to identify embryos in N,O. (J-M,P,Q) The inhibition of Her5 activity leads to the differentiation of ectopic neurons in place of the IZ. J,K: dorsal views of the MH area in flat-mounted embryos at the 3-somite stage, anterior to the top, probed for ngn1 expression following injection of MO^her5, a morpholino selective of endogenous her5 (K), compared to non-injected wild-type control embryos (J). Note that the vcc and r2MN clusters are bridged (double arrow), while other neuronal populations (e.g.r4MN, arrowhead) are unaffected. (L,M) TUNEL assay in wild-type (L) and MO^her5-injected (M) embryos shows that injections are not followed by increased apoptosis in the MH area (bar). (P,Q) At 36 hpf, an ectopic HNK1-positive neuronal cluster (brown arrows) lies across the MH junction (identified by her5 expression, blue arrow) upon MO^her5 injection. Note reduced her5 levels at the MHB in Q (compared with P), a late event suggesting indirect positive autoregulation of her5 expression. IZ, intervening zone; MH, midbrain-hindbrain domain; nMLF, nucleus of the medial longitudinal fascicle; r2MN, motorneurons of rhombomere 2; r4MN, motorneurons of rhombomere 4; vcc, ventrocaudal cluster.