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A brief inhibition of Notch signaling activates qNSCs without depleting them in favor of cycling NSCs. (A and B) Experimental scheme (A) of the treatment of 3mpf adult zebrafish with DMSO or LY for 12 or 24 hours, followed by whole-mount immunohistochemistry (IHC) and in situ hybridization (RNAscope) and cell quantifications. Rostral and caudal areas of the pallial ventricular zone were analyzed, as depicted on the brain cartoon (B) (dorsal view: A, anterior; P, posterior; ob, olfactory bulb; ot, optic tectum). (C to E) Representative images of the pallial ventricular zone (rostral areas) after treatment with DMSO (24 hours) (C) or LY for 12 hours (D) or 24 hours (E). (C’) to (E’) are higher magnifications of the areas boxed in (C) to (E). Whole-mount dorsal (apical) views, anterior left. Zo1 and Sox2 immunostainings (white) are used to identify progenitor cells with apical ventricular contact, Pcna (red) is used to label and count proliferating cells (quantifications in F), and ascl1a RNAScope (magenta) is used to read decreased Notch activity and transition toward preactivation. ascl1a is expressed at higher levels after LY treatment and higher after 24 than after 12 hours of treatment. See also fig. S1F. Scale bars, 8 μm. (F) Quantification of the proportion of cycling cells (Pcnapos) after each treatment in the rostral or caudal part of the dorsal pallium. P values are the result of a Kruskal-Wallis test. Violin plots are built from bootstrapped random sampling of the measured proportions to estimate the distribution. (G) Experimental scheme to generate the scRNAseq dataset under LY treatment. The DMSO control dataset was reported in (16). (H) Low-dimensional embedding of quiescent NSCs (qNSCs) in the control scRNAseq dataset colored by cluster [after (16)]. (I) Low-dimensional embedding of qNSCs in the scRNAseq dataset of LY-treated fish, colored by cluster, matching colors to those from the control dataset based on inferred equivalence.
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