Fig. 8

(A-D) her3 in situ hybridisation in two-somite embryos. Transcription of her3 is repressed by Gal4 mediated misexpression of notch1a-intra (A) or her4 (B), and by injection of the her4 mRNA (C). her3 expression is also repressed by injection of neurog1 mRNA (D), probably as an indirect consequence of the activation of her4 (Takke et al., 1999). (E,F) her5 in situ hybridisation to two-somite embryos. Transcription is repressed following misexpression of notch1a-intra by the Gal4-UAS technique. Misexpression of her4 represses the transcription of her5 to the same extent. (G) Genotyping of individual embryos by PCR. Embryos that exhibit transcriptional repression of her3 (or of her5) carry both transgenes (hsp::gal4 and UAS:her4, three first lanes). Phenotypically wild-type embryos carry one transgene. (H) Her4 protein can bind to N boxes in the her3 promoter. Lanes 1-3 contained increasing amounts of Her3 protein (2, 3 and 4 µg, respectively) and 8000 cpm of an oligonucleotide derived from the her3 promoter and including the N1 box (Materials and methods). A clear shift in the electrophoretic mobility of the oligonucleotide can be detected. Lanes 4-6 contain 3 µg of Her4 protein, 8000 cpm of the labelled N1 oligonucleotide, and increasing amounts of unlabelled oligonucleotide (1, 2 and 4 pM, respectively). The band shift is competed out. Lanes 7-9 show the same experiment using an oligonucleotide that contains the N2 box (Materials and methods). Binding to the N2 oligonucleotide is weaker. Lanes 10-12 show that, also in this case, binding can be competed with non-radioactive oligonucleotide. Lanes 7-9 and 10-12 contain same amounts of protein and radioactive and non-radioactive DNA as lanes 1-3 and 4-6, respectively. Lanes 13-14 and 15-16 show the effects of mutating either the N1 or the N2 box on the mobility of the labelled probe. Binding to the N1 mutant is still strong. Lanes contain 1 and 5 µg of Her3 protein and 8000 cpm of the labelled oligonucleotides. As a control, lanes 17-20 contain 8000 cpm of the wild-type (17-18) and mutated (19-20) oligonucleotides, without Her4 protein.