Extended Data Figure 5: Astrocytic NL1 interacts with neuronal neurexins to promote astrocyte morphogenesis in vitro.

a–d, Lentiviral knockdown of rat Nrx1 and Nrx2. a, Domain structures of Nrxα and Nrxβs. Nrxα and Nrxβs are expressed by alternative promoters and share the same LNS6 (laminin-α, neurexin and sex hormone-binding globulin 6), transmembrane and intracellular domains. Nrxαs contain five additional LNS domains and three EGF domains in their extracellular region. Gokce et al. generated a lentiviral construct with tandem shRNAs with recognition sequences localized to the LNS6 and TM domains of mouse Nrxs, such that both Nrxα and Nrxβs are silenced²⁴. b, shRNA sequences that silence mouse Nrxα and Nrxβs²⁴ compared to the corresponding rat sequences. Based on sequence homology, we predicted that this lentiviral construct would silence rat Nrx1α/β and Nrx2α/β, but not Nrx3α/β owing to base-pair mismatches (red). c, RT–PCR amplicons from cortical neurons transduced with a scrambled control lentivirus (shScr) or pan-shNrx lentivirus. Specific primers against Nrx1α, Nrx1β, Nrx2α, Nrx2β, and Nrx3 were used to detect the presence and levels of Nrx transcripts. β-Actin was used as a control. As predicted, pan-shNrx²⁴ effectively diminished expression of rat Nrx1 and Nrx2 but not Nrx3. d, Quantification of Nrx cDNA levels from shScr or pan-shNrx-transduced cortical neurons. c, d, Results are from two independent experiments. e–o, Neuronal neurexins are required to promote astrocyte morphogenesis. e, Diagram of astrocyte–neuron co-culture strategy used to test the requirement for neuronal neurexins in regulating astrocyte complexity. Neurons are transduced on DIV2 with a lentivirus expressing EGFP and tandem shRNAs that silence rat Nrx1 and Nrx2 (green). Astrocytes are transfected with shRNA plasmids, which also encode mCherry, on DIV9 and then plated with neurons on DIV11 for 48 h. f, Representative images of shrtNL1 mCherry-transfected astrocytes (red) in co-culture with shNrx1/2 or shScr lentivirus-transduced neurons (green). g, Sholl quantification of astrocyte complexity in shrtNL1-transfected astrocytes in co-culture with shScr or shNrx1/2-transduced neurons. Data represent one experiment with three biological replicates. Similar results were obtained in two independent experiments. n > 25 cells per condition per experiment. h, Schematic of soluble Fc-tagged Nrxβ ectodomains. Recombinant Fc-only and Nrx1β–Fc proteins were previously described⁴⁰. For this study, we generated recombinant constructs to express and purify Nrx2β–Fc and Nrx3β–Fc proteins (see Supplementary Methods). i, Coomassie staining shows the molecular mass and purity of the recombinant Fc-tagged proteins. Note that Nrx2β–Fc stains weakly with Coomassie dye, probably because it has fewer positively charged amino acids compared to Nrx1β–Fc and Nrx3β–Fc. j, Schematic of in vitro experiments testing the requirement for neurexin–neuroligin interactions in the control of astrocyte complexity. shRNA EGFP-transfected astrocytes are plated onto MeOH-fixed neurons in the presence or absence of soluble Fc-tagged Nrxβ-ectodomain proteins or control Fc protein. k, Quantification of complexity of astrocytes grown on MeOH-fixed neurons with or without Fc-tagged proteins added at various concentrations. Total Sholl intersections are plotted as a function of increasing Fc-tagged protein concentration (normalized to 0 nM). In this assay, Fc protein concentrations above 25 nM reduced astrocyte morphogenesis nonspecifically; thus, all experiments were conducted at 25 nM concentration. Data represent one experiment with three biological replicates. Similar results were obtained in two independent experiments. n > 25 cells per condition per experiment. l, Representative images of shCtrl or shrtNL1-transfected astrocytes (green) cultured with MeOH-fixed neurons (not visible) in the presence of 25 nM Fc (control) or 25 nM Nrx1β–Fc, Nrx2β–Fc and Nrx3β–Fc (8.3 nM concentration each, total of 25 nM). m, Sholl quantification of astrocytes cultured on MeOH-fixed neurons with or without recombinant Fc proteins (25 nM total concentration). Data represent one experiment with three biological replicates. Similar results were obtained in two independent experiments. n > 25 cells per condition per experiment. n, Sholl quantification of shCtrl-transfected astrocytes cultured on MeOH-fixed neurons supplemented with recombinant Fc proteins (25 nM total concentration). Data represent one experiment with three biological replicates. Similar results were obtained in two independent experiments. n > 25 cells per condition per experiment. o, Sholl quantification of shrtNL1-transfected astrocytes cultured on MeOH-fixed neurons supplemented with recombinant Fc proteins (25 nM total concentration). Data represent one experiment with three biological replicates. Similar results were obtained in two independent experiments. n > 25 cells per condition per experiment. One-tailed t-test (d), ANCOVA (g, k, m–o). For gel source data, see Supplementary Fig. 1. Data are means ± s.e.m. Scale bars, 10 μm.