Fig. 6. XHes2 can regulate proneural gene transcription in both neural and non-neural ectoderm. (A-I,K-V) Whole-mount in situ hybridization analysis of neuro-specific gene expression in stage 14 (A-D,F-I) or 20 (E) or 24 (K-V) Xenopus embryos upon injection of XHes2 variants or morpholinos. Black arrowheads, posterior expression domains; black arrows, anterior expression domains; white arrowheads, ectopic expression. (J) Real-time RT-PCR analysis of neuro-specific gene expression in animal cap assays following injection with XHes2-C-VP16. (A,B) XHes2 represses neurogenesis, as assessed by the downregulation of XNgnr1 (89%, n=9) (A) and N-tubulin (88%, n=16) (B). (C-E) Conversely, inhibition of endogenous XHes2 with XHes2-Mo2 extends XNgnr1 expression in the presumptive otic placodal region (D, injection into one of eight cells, 45%, n=42; one of two cells, 62%, n=13). XHes2-Mo1 shows the same phenotype (not shown, one of eight cells, 56%, n=63; one of two cells, 100%, n=14) in contrast to the control Mo (C, one of eight cells, 5%, n=60; one of two cells, 0%, n=37). (E) At some later neurula stage embryos, ectopic patches of XNgnr1 (white arrowhead) appear close to the otic placode region (arrow). Arrows in C,D point to the presumptive otic placodal region. (F-I) Effects of XHes2 variants on proneural gene expression at stage 14. (F,H) XHes2-W does not significantly alter XNgnr1 expression (slightly reduced in 39%, n=13) (F), but still inhibits N-tubulin expression (91%, n=11) (H). (G,I) In stage 14 embryos, XHes2-W-VP16 leads to ectopic XNgnr1 expression (G1, 100%, n=19; G2 and G3 show the non-injected and the injected lateral sides, respectively) but fails to activate N-tubulin expression (I, 100%, n=15). Moreover, transcription of N-tubulin is repressed in its endogenous domains (I). (J) XHes2-C-VP16 induces neuronal differentiation of animal caps. In early caps (stage 13), XHes2-C-VP16 induces the expression of the proneural genes XNgnr1 and NeuroD, while expression of late neuronal markers is low (Xath3) or even undetectable (N-tubulin). From stage 22 onwards, Xath3 and N-tubulin are significantly upregulated. Relative values are given as mean±s.e.m of two independent experiments. (K-P) Effects of XHes2 variants on proneural gene expression at stage 24. In XHes2-W-VP16 overexpressing embryos, an ectopic activation of XNgnr1 (L, 29%, n=14), NeuroD (N, 100%, n=25) and N-tubulin (P, 85%, n=13) is observed. Conversely, expression of XNgnr1 (K, 93%, n=14), NeuroD (M, 100%, n=22) and N-tubulin (O, 85%, n=13) is strongly reduced at this stage following XHes2 injection. (Q-V) XHes2-W-VP16 induces ectopic sensory neurons. The sensory neuron marker Xhox11L2 is ectopically activated by XHes2-W-VP16 (R, 100%, n=13) while being repressed by XHes2 (Q, 100%, n=13). By contrast, transcription of the interneuron and motoneuron markers Pax2 (S,T) and Xlim3 (U,V) is downregulated by both XHes2 (S, 85%, n=26; U, 92%, n=12) and XHes2-W-VP16 (T, 92%, n=24; V, 64%, n=14). Embryo orientation: (A-I) dorsal views, anterior downwards; (K-V) anterior views, dorsal upwards. is, injected side; nis, non-injected side.