To elucidate which isoforms of NRG1 are involved in the sub-ventricular mitoses of neural progenitor cells, we injected MOs against typeI, II, and III (data not shown)

To elucidate which isoforms of NRG1 are included in the sub-ventricular mitoses of neural progenitor cells, we injected MOs from typeI, II, and III (data not revealed). When antisense MOs from NRG1-II (MOnrg1II MOnrg1IIatg and TCS 401 MOnrg1IIE1) have been injected into Tg(pou4f1-hsp70l:GFP) or in Tg(BAC(neurod:EGFP)8.4neurog1:nRFP) embryos, put up-mitotic neurons had been significantly reduced in the optic tectum, in contrast to these in embryos injected with the handle MOs (MOnrg1IIatg5m and MOnrg1IIE15m Fig 4B and 4C and S5C and S5D Fig phenotype ratios: nrg1IIatg, .seventy nine .04, n = 104, nrg1IIatg5m, .05 .05, n = a hundred and fifteen, p < 0.01 nrg1IIE1, 0.57 0.10, n = 94, nrg1IIE15m, 0.02 0.02, n = 36, p < 0.05), without increase in the number of apoptotic cells (S6 Fig). We confirmed that MOnrg1IIatg specifically suppressed ectopic expression of NRG1-II from an Fig 3. Mitoses generating neurons in the sub-ventricular zone require ErbB signaling. A. A timeline of experiments of AG1478 treatment. Embryos were soaked into 30 M AG1478 solution or the control 2% DMSO from 26 to 45 hpf. Then, they were washed and grown in a fresh medium from 45 hpf. Embryos at 44, 48 and 54 hpf were collected for analyses. See also S3 Movie on the recovery of neurogenesis after removal of AG1478. B. pH3-positive mitotic cells in the OT. Removal of AG1478 at 45 hpf enhanced mitoses in the SVZ at 48 hpf prior to recovery of GFP-expressing neurons in the OT of Tg (pou4f1-hsp70l:GFP) at 54 hpf. Yellow arrows, pH3-positive cells in the SVZ. Scale bar, 10 m. C. Quantification of the number of pH3-positive cells in the VZ (blue) and the SVZ (red) of AG1478-treated embryos and the control (white) embryos (mean s.e.m. P < 0.05, P < 0.001 n = 3, 5, 5 for AG1478, n = 6, 4, 6 for DMSO at 44, 48, 54 hpf, respectively). D. Quantification of GFP intensity in the OT (mean s.e.m. P < 0.05, P < 0.0001 n = 6, 4, 5 for AG1478, n = 6, 4, 6 for DMSO at 44, 48, 54 hpf, respectively). E. Time-lapse imaging of NPCs stochastically labeled by co-injection of UAS:mCherry/UAS:memb:GFP plasmids into Tg(pou4f1-hsp70l:GFPSAGFF(LF)81C) embryos. After the removal of AG1478, two NPCs (1, 2 arrowheads) divided once in the SVZ (asterisk) to generate two daughter cells (a, b), and then, these cells were incorporated into the presumptive neuronal layer (NL, green bar, above the thin dotted line). Note that mitoses7498254 of these NPCs were not preceded by mitoses in the apical VZ and those NPCs began to express cytoplasmic GFP (right). An apical mitosis of another NPC or RGC labeled with membGFP was also observed in this imaging (arrow). Scale bar, 10 m. See also S4 and S5 Movies.Fig 4. Generation of post-mitotic neurons requires NRG1-II. A. MOs against nrg1 used in this study. Domain structures of NRG1 type III isoforms are shown together with the target site (red bar) of each nrg1 MO. B. Decreased neurod:EGFP-positive neurons in MOnrg1IIE1-injected embryos (nrg1IIE1) compared to the control 5 nucleotides-mismatched MOnrg1IIE15m-injected embryos (nrg1IIE15m) of Tg(BAC(neurod:GFP)-8.4neurog1:nRFP) at 50 hpf. Scale bar, 10 m.