Hey1-depleted RGs lose stemness characters. (A-D) Effect of Hey1 abrogation on Sox2 expression in adult RGs. (A) Experimental scheme: fluorescein-tagged MOs (control MO or hey1 splice MO) are electroporated into the pallial VZ and Sox2 expression is analysed after 3 days. (B,C) Examples of electroporated VZ double-immunostained for Gs (white) and Sox2 (magenta), with MO-containing cells in green. Colour-coded arrows indicate the different cell types (see D). (D) Quantification of the proportion of Sox2-positive (blue) and -negative (green) RGs within MO-electroporated cells. (E-J) Effect of Hey1 abrogation on RG reactivation potential. (E) Experimental scheme: fluorescein-tagged MOs are electroporated into the pallial VZ and LY411575 (or the vehicle DMSO) is applied into the swimming water between 2 and 4 days post-electroporation. RG proliferation is analysed at 4 days. (F-I) Representative examples of whole-mount electroporated/LY-treated brains double immunoprocessed for Gs (white) and PCNA (magenta). (J) Quantification of the proportions of the different cell types (colour-code indicated in E). LY treatment induces RG activation (decrease in the proportion of qRGs, increase in the proportion of aRGs) upon electroporation of the control MO, but is without effect when Hey1 function is abrogated. Number of cells counted per brain: 99-293 for control MO treated with DMSO; 153-298 for splice MO treated with DMSO; 98-262 for control MO treated with LY; and 151-353 for splice MO treated with LY. n=3-5 brains per condition. *P<0.05, **P<0.01, ***P<0.001, all pairwise comparisons were adjusted for multiple comparisons following the Holm's procedure. Scale bars: 10 µm in B,C; 70 µm in F-I.
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