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Fig. 5 Her3 and Her9 repress the expression of proneural genes. (A-H) Expression of neurod4 in the wild-type control embryos (A,E), embryos that received an injection of 2 ng her9ATG-MO (H9MO; B,F), mib mutant embryos (C,G) and mib mutant embryos that received an injection of her9ATG-MO (D,H) at the one-somite stage. Dorsal views (A-D) and dorsolateral views (E-H). neurod4 was expressed in the inter-proneuronal domain between the RB neurons and primary interneurons in the her9ATG-MO-injected wild-type and mib mutant embryos, where neurod4 was not expressed in the control embryos. (I-L) The her9-MO-injected embryos had a reduced and narrow fourth ventricle at 30 hours post fertilization (hpf) (J,L), compared with the control embryos (I,K). atoh1 marks neuronal precursor cells located in the subventricular zone of the fourth ventricle. Lateral views (I,J) and dorsal views of the hindbrain region (K,L). (M-N) Expression of neurog1 in 1 ng of control MO (M) and her3-MO1-injected embryos (H3MO; N) at the one-somite stage. Dorsal views for the anterior hindbrain. Ectopic expression of neurog1 was detected only in rhombomeres 2 and 4 of the her3 morphant embryos. (O-R) Her9 functions as a transcriptional repressor. RNAs for Myc-tagged Her9 (Her9MT, 5 pg; O,P), Her9-VP16 fusion protein (Her9VP16, 5 pg; Q) or Myc-tagged Her3 (Her3MT, 20 pg; R) together with 50 pg ß-galactosidase RNA, were injected into one blastomere of two- to four-cell stage embryos. The embryos were fixed at the one-cell stage, and stained with X-gal and the neurod4 probe. In the majority of the her9MT RNA-injected embryos, cells that received her9MT RNA were distributed to only the inter-proneuronal domains (O). However, when the cells were located in the proneuronal domains, the neurod4 expression was reduced or abrogated (P). Expression of Her9VP16 led to an ectopic but spotty expression of neurod4 in the neural plate (Q). Expression of Her3MT led reduction of the neurod4 expression in the proneuronal domains (R). Quantification of the results is shown in Table 1.