A lower in Tyr1162-phosphorylated ErbB4 (pErbB4) in the OT of MOnrg1IIE1-injected embryos (nrg1IIE1) compared to the control MOnrg1IIE15minjected embryos (nrg1IIE15m) at forty 
hpf. Scale bar, ten m. F. Quantification of pErbB4-positive region in the OT for the experiment shown in E (indicate s.e.m P < 0.01 n = 8, 9 for MOnrg1IIE1, MOnrg1IIE15m, respectively). G. A timeline of the injection of hNRG1 proteins into the hindbrain ventricle. MOnrg1IIE1-injected embryos with neurogenic phenotypes were segregated at 47 hpf, and they were subjected to intra-ventricle injection of hNRG1 or the control BSA. These embryos were analyzed at 54 hpf. H. Partial rescue of the defective neurogenesis in MOnrg1IIE1-injected Tg(pou4f1-hsp70l:GFP) embryos at 54 hpf following the injection of hNRG1 proteins, compared to the control BSA injection. Scale bar, 100 m. I. Quantification of pou4f1-hsp70l:GFP intensity in the OT for the experiment shown in H (mean s.e.m. P < 0.0001 n = 17 for MOnrg1IIE1_pre, n = 21 for MOnrg1IIE15m_pre, n = 11 for MOnrg1IIE1_BSA, n = 16 for MOnrg1IIE1_hNRG1, n = 18 for MOnrg1IIE15m_post.)the sub-ventricular zone resumed without precedent cell divisions in the apical ventricular zone, suggesting that cell divisions of sub-ventricular neural progenitor cells per se are dependent on ErbB signaling. The exposure to AG1478 from 25 hpf could circumvent effects of the ErbB kinase inhibitor on early development, which allowed us to observe selective suppression of sub-ventricular cell divisions in AG1478-treated embryos. Actually, severe retardation in development was observed in embryos treated with AG1478 from an early stage of embryogenesis(data not shown). In contrast, because knockdown of NRG1-II not only suppressed cell divisions in the sub-basal/sub-ventricular zone but also affected those in the ventricular zone to some extent, proliferation and differentiation of neural progenitor cells likely require persistent exposure to the NRG1. Despite continuous suppression of cell divisions in the sub-basal/subventricular zone in NRG1-II-knockdown embryos, the number of cell divisions in the ventricular zone was restored at 40 hpf, which implies a possibility that other ErbB ligands, such as other types of NRG1, NRG2, NRG3 and/or HB-EGF might be involved in the restoration. 8201586Membrane-bound isoforms of NRG1 are known to be subject to proteolytic processing within juxtamembrane domains encoded by exons or (ectodomain shedding) [8,9]. Injection of an antisense MO against the transmembrane domain of NRG1 caused inhibitory effects on 853-23-6 neuron generation similar to isoform-specific MOs against NRG1-II, suggesting a requirement of a membrane-bound isoforms of NRG1-II for the neurogenesis. nrg1 was predominantly expressed in the apical region of the optic tectum. Moreover, defective neurogenesis of NRG1-IIknockdown embryos was ameliorated by intra-ventricular injection of soluble human NRG1 proteins. These results imply the involvement of proteolytically processed, soluble NRG1 emanated from radial glial cells or neural progenitor cells in the ventricular zone for the long-range stimulation of neurogenesis.