Extended Data Fig. 8: Expression of Nlg2 in astrocytes during the critical period is not required for proper excitatory–inhibitory synapse balance.

a–b′′′, Localization of Nlg2 (magenta) relative to astrocyte membranes (green, mcd8-GFP+) and pre-synapses (blue, Brp+) in control (N = 15) and following astrocyte-specific knockdown of nlg2 at 0 h ALH (N = 18). N represents number of larvae. a–a′′′, Nlg2 colocalized with astrocyte membranes (mcd8-GFP+) and synapses (Brp+) in control. b–b′′′, Nlg2 colocalized with synapses (inset), but was absent from astrocyte membranes in knockdown brains, as evidenced by increased clarity of Nlg2+ synaptic puncta. Scale bar, 2 μm. Insets, 1.5× zoom. Genotype: alrm-gal4, UAS-mcd8::GFP, UAS-RNAi or control. c–d′′′, Representative images showing aCC–RP2 dendrites (magenta) and excitatory post-synapses (cyan, Drep-2::GFP+) relative to all presynapses (orange, Brp+) in control (N = 7) (c–c′′′) versus astrocyte-specific knockdown of nlg2 (N = 9) (d–d′′′). c′′′, d′′′, Note close apposition of pre- and post-synapses (arrowheads). N represents number of larvae, with synapses averaged over 4 hemisegments per brain (A1–A2). Scale bar, 3 μm. Genotype: RN2-lexA, lexAop-myr::tdTomato, lexAop-drep-2::GFP; alrm-gal4, UAS-RNAi or control. e, f, Quantification of normalized total synapse number (P < 0.11) (e) and the ratio of excitatory synapses to total synapses (P < 0.96) (f) revealed no significant differences (Mann–Whitney test, two-sided). Data are mean ± s.d. NS, not significant. N values reflect biological replicates from 2 independent experiments.