Fig. 4

RNA-Seq Analysis Reveals Erythropoietin Pathway as a Potential Clozapine Target

(A) The volcano plot of log2-fold change of gene expression in the adult fish brain between the treatments of clozapine and DMSO in slc6a3−/− allele1. The clozapine highly upregulated genes (epoa, epor, and nt5c2l1) are indicated on the graph.

(B) Bar plot of gene expression of epoa, epor, and nt5c2l1 by RNA-seq analysis. ****p < 0.0001, ***p < 0.001. n = 8 for all. Error bar = standard deviation. Significance test: Wilcoxon-Mann-Whitney test.

(C) The construct maps for over-expression of epoa in neurons with the tetracycline-controlled transcriptional activation system.

(D and E) The dorsal view of the green channel (D) and bright field (E) of a slc6a3^{−/−_allele1}; Tg(aTub:iTTA; TetO:EpoA-2a-GFP) fish at 5 days post-fertilization under a fluorescent dissecting microscope. The brain area (indicated by green fluorescence in D) is outlined by a dotted line. Scale bar, 250 μm.

(F) Whole-brain qRT-PCR at 2 months post-fertilization confirms the upregulation of epoa in the slc6a3^{−/−_allele1};Tg(aTub:iTTA; TetO:EpoA-2a-GFP) fish. Error bar, standard deviation. n = 6 for all conditions. ***p < 0.001, **p < 0.01, N.S., no significance. Significance test, Wilcoxon-Mann-Whitney test.

(G and H) The quantification of The quantification of average swimming height (G) and duration of “digging” feature (H) of slc6a3^{−/−_allele1}; Tg(aTub:iTTA; TetO:EpoA-2a-GFP) compared to slc6a3^{−/−_allele1} and wild-type at 2 months post-fertilization.

n_WT = 40, n_{slc6a3−/−_allele1_} = 20, n_{slc6a3−/−_allele1; tg(aTub:eopa)} = 20. ^∗∗∗∗p < 0.0001, ^∗p < 0.05, N.S., no significance. Significance test: one-way ANOVA Kruskal-Wallis test.