Figure 4. Analyses of Dd2-specific open chromatin region (OCR) clusters reveal specialized synaptic gene transcription.

(A) Gene Ontology (GO) analysis of OCR clusters. GO terms related to axon guidance and neurogenesis (top) and synaptic signaling (bottom) are shown (see also Figure 4—figure supplement 1 for other enriched GO terms). Size and color of circles indicate fold enrichment and false discovery rate (FDR), respectively. Circle was plotted only if FDR is lower than 0.01. GO terms of axon guidance and neurogenesis are enriched with pallial clusters (OCR C9 and 12), Dd2 cluster (OCR C6), subpallial cluster (OCR C1, 3, 4, 5), and a common cluster (OCR C14). On the other hand, synaptic genes are enriched in OCR C6. (B) GO analysis of the genes preferentially expressed in Dd2. Top 10 significantly enriched terms are shown. Black bars indicate the fold enrichment and gray bars show the log p-value. GO terms related to synaptic signaling are highlighted with triangles. (C) Anti-synapsin immunohistochemistry on the medaka larvae and adult telencephalon. Blue: Hoechst; red: anti-synapsin signals. In larvae, strong signals were detected in the olfactory bulbs, Dc, Dm4, and Dd2. In the adult telencephalon, the signals were broadly detected and strongly detected in Dd2a. Scale bar in larvae: 50um, scale bar in adult: 200um.

Figure 4—figure supplement 1. Gene Ontology (GO) term enrichment analysis on open chromatin region (OCR) clusters.

Full list of GO terms enriched in OCR cluster-target genes in Figure 3D. In OCR C8, 10, 11, no GO term was enriched. Size and color of circles indicate fold enrichment and false discovery rate (FDR), respectively. Circle was plotted only if FDR is lower than 0.01. OCR C14, 15 were identified as common open peaks among the telencephalon, which included many metabolic and biosynthesis processes (OCR C 15), and developmental signaling pathways that might reflect the adult neurogenesis process (OCR C 14). Also, one of the subpallial OCR clusters (OCR C5) was enriched with neuropeptide signaling, neural differentiation, and vascular processes, which is consistent with previous avian studies that show neuropeptide expression in the subpallium (Abellán and Medina, 2009; Arieli et al., 1996).

Figure 4—figure supplement 2. RNA-seq and ATAC-seq analysis on Dd2.

(A) Procedure for RNA-seq analysis. After making 130-um-thick slices, Dd2 (Dd, white lines), the pallium except Dd (D, cyan lines), and subpallium (V, yellow lines) were dissected based on the DsRed signals in the Tg (HuC-DsRed) line. Scale bar: 150 um. (B) Principal component analysis (PCA) of the result of RNA-sequencing (30 fish were used for one replicate and two replicates were made). (C) Synaptic genes differentially expressed and actively regulated in Dd2 from other regions. The number of synaptic genes with significantly high or low expression in the sample Dd2 detected by RNA-seq, and the number of synaptic genes targeted by OCR C6 are shown. By ATAC-seq, we identified genes actively downregulated. (D) Maximum projection image of anti-PSD95 immunostaining on larval medaka (left) and dissected adult medaka (right) telencephalon. Blue: Hoechst signal; red: PSD95 signal. In larvae, PSD95 signals were detected in olfactory epithelium, olfactory bulb, Dc, retinotectal projection, and both Dd2a and Dd2p. Noisy signals were detected in the skin. In the adult telencephalon, PSD95 signals were broadly detected but we observed strong signals in Dl, Dd2a, and Dm4. Scale bar in larvae: 50um, scale bar in adult: 250um. (E) Maximum projection image of anti-synapsin immunostaining on larval zebrafish. Both PSD95 and synapsin were detected in the olfactory bulb, Dc, and retinotectal projection. Noisy signals were detected in the skin in anti-PSD95 immunostaining. No strong signal in the dorsal telencephalon was observed. Scale bar: 50um.