Fig. 3. Regulation of XPak3 expression in both embryos and animal caps. (A) XPak3 expression is positively regulated by proneural genes and negatively regulated by X-Notch-1. Xenopus albino embryos were injected into one cell at the two-cell stage with synthetic RNA encoding X-Ngnr-1, X-NeuroD, Xebf3 and ICD-Notch, along with LacZ RNA as tracer. Embryos were fixed at the neurula stage, then stained with X-gal (light blue) and analysed by whole-mount in situ hybridization for XPak3 expression (purple). (1) X-Ngnr-1 and (2) X-NeuroD injection results in strong ectopic expression of XPak3 (100 and 96%, n = 76 and 64, respectively). (3) Xebf3 is sufficient to turn on ectopically the expression of XPak3 but in a weak and scattered manner (62%, n = 48). (4 and 5) The activated form of X-Notch-1, ICD-Notch, blocks XPak3 expression (100%, n = 56). (B) X-Ngnr-1 is sufficient to activate XPak3 expression in animal caps. Pigmented embryos were injected with 50 pg of X-Ngnr-1 mRNA. Animal caps were isolated and cultivated to the indicated stages. Total RNAs were prepared from the caps and analysed by RT–PCR for XPak3 expression, using NeuroD, N-tubulin and histone H4 as controls. For each stage, E (embryos), Co (control caps) and Ngn (neurogenin-injected caps) are shown. (C) XPak3 is not induced directly by X-Ngnr-1. Embryos were injected into two blastomeres of the two-cell stage with hGR Ngn. Cycloheximide (CHX) treatment was from stage 11.5 followed by 2.5 h treatment with or without dexamethasone (Dex). Total RNAs were prepared from these caps and analysed by RT–PCR for XPak3, NeuroD and histone H4. (D) XPak3, but not XPak1 and XPak2, is activated by X-Ngnr-1 in animal cap explants. Pigmented embryos were injected with X-Ngnr-1 RNA and/or ICD-Notch RNA and allowed to grow up to stage 9. Animal caps were isolated and cultivated to stage 15. Total RNAs were prepared from the caps and analysed by RT–PCR for the genes indicated. From left to right, the columns represent: water control (H₂O), uninjected control embryos stage 15 (E), uninjected control caps (Co), X-Ngnr-1-injected caps (Ngn), ICD-Notch-injected caps (ICD) and X-Ngnr-1/ICD-Notch-co-injected caps (Ngn + ICD).