Extended Data Figure 4. NL CAMs control neuron-induced astrocyte morphogenesis.

a-c, Specificity of shRNAs against NL sequences. a, Schematic representation of EGFP-expressing shRNA plasmids used to silence rat NLs in vitro. Sequences of shRNAs used on rat astrocytes against NL1, NL2, and NL3, as well as a scrambled shNL1 sequence used as a control (shCtrl). Rat-specific shRNAs against NL1, NL2, and NL3 were previously verified for efficiency and specificity by^42,43 shNL2 sequence matches both rat and mouse NL2 sequences and effectively silences NL2 expression in cells from both species (see Extended Data Fig. 6c-d). b, Western blot analyses of cell lysates from HEK293 cells transfected with shCtrl or shrtNL1 with various HA-tagged NL1 constructs. Full length NL1 (HA-NL1) is effectively silenced by the shrtNL1, whereas NL1 shRNA-resistant mutant (HA-NL1-RM) and NL1-SWAP are not silenced by shrtNL1. Note: NL1-SWAP runs smaller than full-length NL1 (previously published in²³). c, Western blot analyses of cell lysates from HEK293 cells transfected with shCtrl or shNL2 with HA-tagged NL2 or HA-tagged NL2-RM. Full length NL2 (HA-NL2) is effectively silenced by the shNL2, whereas NL2-RM is not. b, c, Antibodies against HA were used to detect the expression of NLs and the lysates were blotted for using an EGFP-specific antibody to verify transfection with shRNA plasmids. d-k, NLs play significant roles in controlling neuron-induced astrocyte morphogenesis. d, Sholl quantification of astrocyte complexity of NL1, NL2, NL3, or NL1/2/3 silenced astrocytes cultured on cortical neurons (not visible, compare with Fig. 2a-e). Data represent 1 experiment with 3 biological replicates. Similar results were obtained in 3 independent experiments. n>25cells/condition/experiment. e, Representative images of shNL-transfected astrocytes (green) cultured with MeOH-fixed neurons (not visible). f, Sholl quantification of astrocyte complexity of NL-silenced astrocytes cultured on MeOH-fixed neurons. Data represent 1 experiment with 3 biological replicates. Similar results were obtained in 2 independent experiments. n>20 cells/condition/experiment. g, Representative images of transfected astrocytes (green) with shCtrl or an shRNA against EphrinA3 (EFNA3) in co-culture with neurons (not visible). EFNA3 is a CAM expressed in astrocytes that regulates astrocyte-synapse interactions in the hippocampus²². h, Sholl quantification of astrocyte complexity in shCtrl and shEFNA3-transfected astrocytes in co-culture with neurons. Data represent 1 experiment with 3 biological replicates. Similar results were obtained in 2 independent experiments. n>20cells/condition/experiment. i, Cartoon representation of NL1 domain structure. NLs are type-I transmembrane proteins with a large N-terminal extracellular domain (ECD). NL1-ECD contains a cholinesterase (ChoE)-like domain that interacts with Nrxβs transcellularly. In a previous study, a chimera of NL1 (NL1-SWAP) was created in which the ChoE-like domain is swapped for the ChoE sequence. This chimera is efficiently trafficked to the cell surface, but fails to interact transcellularly with Nrx-βs²³. j, Representative images of astrocytes over-expressing EGFP (green) and HA-tagged NLs (red) in co-culture with neurons (not visible). NL over-expressing astrocytes do not show any reductions in neuron-induced astrocyte morphogenesis. k, Sholl quantification of astrocyte complexity when over-expressing HA tagged-NLs. Data represent 1 experiment with 3 biological replicates. Similar results were obtained in 2 independent experiments. n>20 cells/condition/experiment. ANCOVA (d, f, h, k). For gel source data, see Supplementary Figure 1. Data are means ± s.e.m. Scale bars, 10 μm.