Extended Data Fig. 3: In vivo manipulation of Ccn1 expression in astrocytes.
From: Astrocyte CCN1 stabilizes neural circuits in the adult brain
a. Experimental set-up for critical period overexpression (o/e) of CCN1 or tdTomato (tdT) viral vectors in astrocytes. BZ, binocular zone. b. Representative z-projection of P28 VC expressing tdT (left) or CCN1 (right). Scale bar, 50 µm. c. Left, penetrance of viral vectors quantified as % SOX9+ cells also expressing tdT/HA-tag. Right, specificity quantified as % total cells expressing tdT or CCN1 that are co-stained with SOX9, NEUN or no co-staining (other). tdT, n = 6 mice. CCN1, n = 5 mice. Unpaired T-test on mouse averages. d. Immunocytochemistry (ICC) of cultured astrocytes transfected with pAAV-GFAP-CCN1-HA. Cyan, HA-tag. Scale bar, 100 µm. e. ICC of HEK cells transfected with pAAV-GFAP-CCN1-HA and stained for CCN1 protein and the HA-tag. Scale bar, 100 µm. f. Cropped representative image of immunoblot against CCN1 (top) and β-actin (bottom). All uncropped immunoblots are shown in Supplementary Fig. 1. β-actin loading control and CCN1 immunoblotted on the same gel. g. Quantification of immunoblotted CCN1 band at ~42 kDa. tdT, n = 3 mice. CCN1, n = 6 mice. Unpaired T-test on mouse averages. h. Representative z-projection of VC GFAP in tdT and CCN1 o/e P28 mice. Scale bar, 200 µm. i. GFAP quantified as % total area of section. N = 5 mice/ group. Unpaired T-test on mouse averages. j. Schematic of adult conditional knockout validation experiments. Created in BioRender. Allen Lab (2025) https://BioRender.com/cp7gqpp. k. Representative z-projection of 2 month VC smFISH against Slc1a3 and Ccn1. Scale bar, 100 µm. l. Left, quantification of Ccn1 threshold area per astrocyte. Unpaired T-test done on mouse averages. Right, Ccn1 threshold area per neuron (labeled with Tubb3, bottom). Mann-Whitney U-test on mouse averages. m. Representative image of Ccn1 smFISH in layer 6 VC of adult WT and CCN1 cKO mice. Scale bar, 50 µm. Z-projection. n. Ccn1 threshold area per Tubb3+ cell in layer 6 of VC. Unpaired T-test on mouse averages. l,n. N = 6 mice/ genotype. For all bar graphs, data shown as mean ± SEM with symbols denoting mouse averages.
