Fig. 7.

nr2f1b is sufficient to promote resistance to Notch pathway inhibition.

(A) Schematic of the experiment. After plasmid electroporations and a 3-day rest (dpe, days postelectroporation), fish were treated with LY for 1 day and euthanized for quantification of ascl1a expression. (B) Schematic of the approach to quantify ascl1a molecules (magenta dots) in electroporated cells (green). NSC cell bodies can differ from the shape of their apical membrane, precluding reliable automated quantification of signal with Zo1. However, the slight leakiness of nlsGFP fills the cell body, which can be segmented. Spots falling into the circumscribed volume are assigned to the cell in an unbiased way by detecting an elbow in the plot of pixel intensities in the ascl1a channel. (C) Example of a cell with a complex morphology in which ascl1a dots were semi-automatically segmented. The first two images on top and the first image on the bottom show dorsal views of how the cell body extends beyond the Zo1^pos apical membrane and is surrounded in ascl1a dots. The third image on top shows an orthogonal view from the parenchyma displaying the complex morphology of the cell and the position of numerous ascl1a dots around it. The two last images on the bottom show the electroporated cells with only segmented ascl1a dots highlighted. Scale bars, 5 μm. (D) Example images of electroporated cells with pCMV:nr2f1b-P2A-nlsGFP or pCMV:nlsGFP after 24 hours of LY treatment. Arrows point to electroporated NSCs. Scale bars, 10 μm. (E and F) Violin plots of the number of ascl1a dots per cell in cells electroporated with pCMV:nr2f1b-P2A-nlsGFP or pCMV:nlsGFP across the pallium (E) and when separating the rostral and caudal pallial domains (F) after the experimental scheme in (A). Statistics: unpaired two-sample Wilcoxon test; n = 3 brains per condition, 201 cells in total. Overall: P value = 2.2 × 10⁻¹⁶; anterior: P value = 2.205 × 10⁻¹¹; posterior: P value = 2.771 × 10⁻¹¹.