Fig. 3.

Zebrafish astrocyte–like cells respond to Notch inhibition and injury.

(A) Expression levels of indicated genes in qNSCs from DMSO- or LY-treated datasets projected on the integrated embedding of qNSCs. igfbp2a and timp4.3 mark astrocyte-like cells and ascl1a marks preactivated cells and the lack of Notch activity. q1e and q1f are circled. In the LY dataset, but not in control, many cells coexpress astrocyte-like NSC markers and ascl1a, notably in clusters q1e and q1f. (B) Expression levels of ascl1a or ccnd1 in timp4.3^pos cells from DMSO- or LY-treated datasets. (C) High magnification of the pallial ventricular surface in controls (top) or upon a 24-hour LY treatment (bottom) showing expression of timp4.1 (yellow), ascl1a (purple), and ccnd1 (green) (coexpression of ascl1a and ccnd1 appears in cyan, left merged views) revealed by smFISH together with immunohistochemistry for Zo1 (white). Examples of cells with high levels of expression of timp4.3, ascl1a and ccnd1 upon LY are indicated (yellow arrowheads). (D and D’) High magnifications of the pallial ventricular surface close to a lesion (dorsal whole-mount view), processed for immunohistochemistry (ZO1, white; Pcna, red) and smFISH (timp4.3, cyan). Proliferating cells (Pcna^pos) are more numerous neighboring the lesion. (D) all channels; (D’) Zo1 and timp4.3 only. Scale bar, 10 μm. (E) Quantification of timp4.3 expression (number of dots per cell) in Pcna^neg and Pcna^pos cells neighboring the lesion (“lesion proximity”) or far away (“control area”). P values were calculated with a Wilcoxon signed-rank test to avoid inflating sensitivity with a bootstrapped test. Control area: n = 3 brains, 92 cells; lesion proximity: n = 3 brains, 149 cells. timp4.3 levels close to the lesion are decreased, with a significant decrease in Pcna^neg cells. In contrast, Pcna^pos cells tend to express timp4.3 at higher levels close to the lesion than in control cells, but this difference is not statistically significant.