Extended Data Figure 7: NL2 protein is expressed in astrocytes and is required for neuropil infiltration in vivo.

a–c, NL2 protein staining in NL2 PALE HET and NL2 PALE KO astrocytes. a, Representative high-magnification Airyscan confocal images of P21 td-Tomato/Cre⁺ PALE astrocytes from Nlgn2^f/+ and Nlgn2^f/f (bottom) mice immunostained using an antibody against the intracellular domain of NL2. Left, single optical section of td-Tomato/Cre⁺ astrocytes depicting NL2 staining (green) and td-Tomato/Cre⁺ astrocyte (red). Middle, zoomed-in view of boxed region from left. Right, representative Imaris 3D reconstructed surface renderings of the co-localized NL2 (green) and td-Tomato/Cre⁺ (red) signals in PALE NL2 HET and NL2 KO astrocytes (NL2 signal outside the td-Tomato/Cre⁺ cell is not surface rendered). b, Quantification of average NL2 puncta volume inside td-Tomato/Cre⁺ NIV of PALE NL2 HET and NL2 KO astrocytes. c, Fold change in NL2 puncta volume per td-Tomato/Cre⁺ volume, normalized to NL2 HET. b, c, Two NIVs per image, three cells per mouse, three mice per genotype. d–f, shNL2 has no off-target effects in astrocytes in vivo. d, Approach to test the specificity of shNL2 in vivo. pCAG–Cre plasmid was injected with or without the shNL2 plasmid into P1 Nlgn2^f/+ or Nlgn2^f/f pups that also contained two copies of the RTM reporter. Animals were killed at P7 for astrocyte morphological analysis. e, Representative images of P7 td-Tomato/Cre⁺ astrocytes (red) or co-expressing shNL2 (green and red, appears as yellow) from PALE NL2 HET and NL2 KO mice. Representative NIVs are shown below each astrocyte (magenta). f, Fold change in average NIV of P7 td-Tomato/Cre⁺ PALE NL2 HET and NL2 KO astrocytes with or without shNL2, normalized to td-Tomato/Cre⁺ PALE NL2 HET. Three NIVs per cell, 16 cells per condition, at least 2 mice per genotype. One-tailed t-test (b, c), one-way ANOVA (f). Data are means ± s.e.m. Scale bars, 10 μm.