(A) Representative images of larval peripheral nerves stained for the nerve membrane marker HRP. Repo-GAL4, a pan-glial driver, was used to express LexA RNAi (control; abbreviated as Repo>), SIK3 RNAi (Repo>SIK3 RNAi), PKA-R1 RNAi (Repo>PKA-R1 RNAi), or a UAS-PKA-C1 transgene (Repo>PKA-C1). PKA hyperactivity, induced by either glial overexpression of its catalytic subunit (PKA-C1) or knockdown of a regulatory subunit (PKA-R1), causes localized nerve swellings (arrow) that resemble defects caused by loss of SIK3 from glia. Control larvae do not display swellings. Scale bars 20 μm. (B) Quantification of nerve swellings in (A). n ≥ 30. One-way ANOVA with Tukey's multiple comparisons; NS = not significant, p>0.05. (C) Quantification of nerve swellings per animal in control, larvae with glial expression of constitutively activated Gαs protein, HDAC4 RNAi, or co-expression of HDAC4 RNAi and Gαs. Constitutively activated Gαs in glia (Repo>Gsα. Q215L) causes nerve swellings that resemble (A); swellings are suppressed by loss of HDAC4 from glia (Repo>Gsα. Q215L, HDAC4 RNAi). Glial knockdown of HDAC4 (Repo>HDAC4 RNAi) does not result in nerve swellings. n ≥ 30. One-way ANOVA with Tukey's multiple comparisons; ****, p<0.0001. (D) Representative images of larval peripheral nerves demonstrating the effects of Gαs and PKA activation on HDAC4 localization in glia. Left: glial nuclei (green) and HDAC4 (red). Right: grayscale images show HDAC4 staining; glial nuclei are outlined. Scale bars 15 μm. (E) Quantification of nucleo:cytoplasmic ratio of HDAC4 for genotypes in (D). n ≥ 20. Data are presented as fold changes relative to Repo>HDAC4. One-way ANOVA with Tukey's multiple comparisons; ****, p<0.0001. (F) Quantification of number of nerve swellings per animal for genotypes in (D). n ≥ 20. One-way ANOVA with Tukey's multiple comparisons; ****, p<0.0001. Data are mean ± SEM.