Extended Data Figure 6: Astrocytic neuroligins control astrocyte morphogenesis in vivo.

a–d, Verification of shRNAs used in vivo. a, Schematic of pLKO.1_hU6 plasmid vector housing the shRNA sequence and EGFP reporter. shRNA constructs targeting both mouse and rat NL1 and NL3 transcripts were obtained from Dharmacon. shNL2 used^40,41 effectively silences both mouse and rat NL2. In this plasmid backbone, the shRNA expression is driven from the human U6 minimal promoter. The commercially available plasmids do not encode a fluorescent protein reporter; therefore, we cloned EGFP under the control of a CAG promoter (see Supplementary Methods). b, shRNA sequences used to silence mouse neuroligins for PALE. Because they are a perfect match with the rat sequences, the same shRNAs also target rat neuroligins. shNL1 and shNL3 targeting vectors were verified here. shNL2 was verified previously^40,41 (and see Extended Data Fig. 4c). c, d, Western blot analysis of lysates from cultured rat (c) or mouse (d) astrocytes transduced with lentiviruses expressing shCtrl or shNLs. The shNLs effectively silenced the expression of endogenous NL1 (left), NL2 (middle) and NL3 (right) in both rat and mouse astrocytes. β-tubulin levels are shown as a loading control. Blots represent one experiment. Similar results were obtained from three separate experiments. e–h, Astrocytic neuroligins control astrocyte morphogenesis in vivo. e, Data from Fig. 3e, f normalized to P7 shCtrl astrocyte NIV values and replotted to determine how neuroligin silencing affects the growth trajectory of astrocyte NIV. shNL2 and shNL3-transfected astrocytes failed to expand their neuropil infiltration from P7 to P21, whereas shCtrl and shNL1-transfected astrocytes displayed robust (~2.5-fold) growth. Three NIV per cell, 10–20 cells per condition, at least three mice per condition. f, Top, representative images of P21 neuroligin-overexpressing PALE astrocytes from L4–5 V1 cortex. The territories of the neuroligin-overexpressing PALE astrocytes were determined in Imaris Bitplane software with a Matlab Xtension. This method identifies the terminal fluorescent points of each astrocyte and connects these points to generate the territory of each cell (red outline). Bottom, representative NIV (magenta) for neuroligin-overexpressing PALE astrocytes. g, Fold change in average territory volume of NL1- or NL2-overexpressing PALE astrocytes normalized to HA–NL1-SWAP. In the brains from three cohorts of seven PALE NL3-overexpressing mice, we were unable to find NL3-overexpressing astrocytes at P21, indicating that NL3-overexpression starting at P1 is not compatible with astrocyte survival and/or maturation. h, Fold change in average NIV of neuroligin-overexpressing PALE astrocytes normalized to HA–NL1-SWAP. Astrocytes might already occupy the available neuropil space; thus, neuroligin overexpression primarily forces astrocytes to expand. Alternatively, each neuroligin might direct astrocyte processes to certain neuronal elements; thus, neuroligin overexpression drives the astrocyte towards such structures, expanding their domains. g, h, Three NIVs per cell (h only), 14–20 cells per condition, 4 mice per condition. ANCOVA (e), one-way ANOVA (g, h). For gel source data, see Supplementary Fig. 1. Data are means ± s.e.m. Scale bars, 10 μm.