Fig. 4

MiR-9 promoted neurogenesis in MHB and hindbrain. Open-book preparations of HH16/17 midbrains. Brain halves (C’,E’) or dorsal midbrain (F’) were electroporated at HH9–10 with miR-9 (a, c-E’) or miR-9-LNAi (f, F′). Transfected cells were immunostained for EGFP (green) and neurones and axons for 3A10 (red). b is a scheme of the specific pattern of neurogenesis in midbrain around HH16/17. Overexpression of miR-9 did not promote ectopic neurogenesis within the midbrain (a, c-E’) but in the MHB (a,c). (c-D’) are magnifications of the MHB area in (a). The white arrowheads in (a) to (d) indicate the posterior border of the mesencephalon and the beginning of the IZ of the MHB. Ectopic miR-9 in the left half of the MHB (a, c) induced ectopic neurones (white arrows), which are not found in the control right half (a, d). Ectopic miR-9 in posterior midbrain (a, c,C’) or in more anterior midbrain regions (e,E’) did not promote early neurogenesis in midbrain. Some of the miR-9-LNAi expressing cells in dorsal midbrain (f-F′) developed axons (arrows). MiR-9-LNAi expressing cells in the MHB did not show long axons (arrowheads in F′). Abbreviations: Di-diencephalon, FP-floor plate, IZ-intermediate zone, Mes-mesencephalon, MHB-mid-hindbrain boundary, MLF-medial longitudinal tract, MTN-mesencephalic trigeminus nucleus, nMLF – nucleus of MLF [82], LLF-lateral longitudinal tract, Rh-rhombencephalon, RP-roof plate