Characterizing the SREB G protein-coupled receptor family in fish: Brain gene expression and genomic differences in upstream transcription factor binding sites

Abstract

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

Keywords: (GPR27); Brain; DKK3; Phoenixin; SREB1; SREB2 (GPR85); SREB3 (GPR173).

Fig. 1.

Relative mRNA expression normalized to μg input RNA of sreb genes in brain sections of (A) zebrafish (Danio rerio), (B) mummichog (Fundulus heteroclitus), and (C) pufferfish (Dichotomyctere nigroviridis). TEL+OL, HYP+MB, and HB refer to telencephalon + olfactory bulbs, hypothalamic section with midbrain, and hindbrain sections, respectively. Individual hypothalamus, midbrain, and cerebellum sections in pufferfish are indicated by HYP, MB, and CER respectively. Black ‘X’ refers to sreb3b or sreb3a gene absence in zebrafish and mummichog, respectively. Each bar represents the mean ± standard error, and significant differences (p < 0.05) are indicated by different letters.

Fig. 2.

Relative mRNA expression normalized to μg input RNA of smim20 pnx, dkk3a, dpydb, tgfb3, 18S rRNA, and eef1a in brain sections of (A) zebrafish (Danio rerio), (B) mummichog (Fundulus heteroclitus), and (C) pufferfish (Dichotomyctere nigroviridis). For zebrafish and mummichog sections, TEL+OL, HYP+MB, and HB refer to telencephalon + olfactory bulbs, hypothalamus + midbrain, and hindbrain sections, respectively. For pufferfish, HYP, MB, and CER refer to hypothalamic, midbrain, and cerebellum sections, respectively. Each bar represents the mean ± standard error, and significant differences (p < 0.05) are indicated by different letters.

Fig. 3.

Component loading plots and principal component analyses (PCA) for (A) zebrafish (Danio rerio), (B) mummichog (Fundulus heteroclitus), and (C) pufferfish (Dichotomyctere nigroviridis) genes. Black triangles represent each gene in the loading plots, while telencephalon + olfactory bulbs (TEL+OL), hypothalamus and midbrain (HYP+MB), and hindbrain (HB) samples are represented in the zebrafish and mummichog PCAs by blue squares, red diamonds, and yellow circles, respectively. Pufferfish TEL+OL, hypothalamic section (HYP), midbrain (MB), and cerebellum (CER) are represented by blue squares, red diamonds, orange triangles, and yellow circles, respectively.

Fig. 4.

Enrichment analyses in CiiiDER (Gearing et al., 2019) to identify transcription factor binding sites enriched in genomic regions upstream of sreb1 (top left), sreb2 (top right), sreb3a (bottom left), and sreb3b (bottom right). All ray-finned fish species available in Ensembl version 109 were used with 1,500 base pair (bp) upstream regions from each annotated gene start site (see Supplementary Data). Genomic regions for each gene were compared to all others to identify significantly enriched (red circles) or reduced (blue circles) potential sites. Larger circle sizes and darker colors reflect greater significance, and gray circles refer to transcription factor binding sites that are not significantly enriched. Circles with greater average log2 proportion bound values are more ubiquitous in the region. Binding sites with ≥ 15 significance on the −log₁₀(p-value) scale (p < 1e⁻¹⁵) are labeled in parentheses with transcription factor names.