(A) Quantification of transcriptional changes calculated by RNAseq for four genes (CREG, Ect3, PEPCK2 and Cyp6a23) in CHD8/+; PPP2R5D/+ double heterozygous mutant versus wild-type. (B) Quantification of the transcriptional changes for the same genes in (B) by qPCR. (C) Schematic of the Drosophila CREG locus. The positions of two transposon insertion mutations are shown (red triangles). (D) Average CREG transcript levels calculated by qPCR are shown for the indicated genotypes E) Representative EPSP and mEPSP traces for indicated genotypes. (F), Bar graph (right) shows percent change in mEPSP (black filled) and quantal content (no fill) (+ / - PhTx). Sample size indicated as (–PhTx/+PhTx): wild type n = 17/15; CHD8/+; PPP2R5D/+ n = 11/14; CregM1 n = 8/7; CregM1/+ n = 8/8; CHD8/+; PPP2R5D/+; CregM1/+ n = 8/10; CHD8/+; PPP2R5D/+; CregM2/+ n = 8/8. (G) Representative electron microscopy images of individual active zones from indicated genotypes (double heterozygous mutant at left, triple heterozygous mutant at right). Scale bar:100 nm. (H) Individual data points (vesicle size) shown for indicated genotypes. (I) Cumulative probability distribution of vesicle size for genotypes shown in (H). Sample sizes for (H, I): Animal number: wild type N = 2, CHD8/+; PPP2R5D/+ N = 3; CHD8/+; PPP2R5D/+; CregM1/+ N = 3. Active zone number: in same genotypic order n = 12, n = 14, n = 12; Vesicle number n = 97, n = 89, n = 112. (J) Scatter plots of quantal content (y axis) versus mEPSP amplitude (x axis) for wild type (left), OK371-Gal4 > UAS Creg (middle, red) and Tub-Gal4 >UAS Creg (right, blue). Fits as indicated. R2 values as indicated (calculated based on linear fit). (K) Percent change in mEPSP (gray bars) and quantal content (red bars) in presence of PhTx compared to baseline. Sample sizes as in (F), wild type n = 8/6; OK371-Gal4 > UAS) Creg n = 14/12; Tub-Gal4 >UAS) Creg n = 11/11. n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001.