Functional divergence of NPR1 paralog in the Japanese eel: Identification and neuroanatomical characterization

Natriuretic peptides (NPs) play critical roles in cardiovascular and osmoregulatory homeostasis via guanylyl cyclase-coupled natriuretic peptide receptors (NPRs). Although teleost fishes possess a diverse NP repertoire, their receptor systems remain incompletely understood. In the present study, we identified and characterized a novel npr1 paralog (npr1a) in the Japanese eel (Anguilla japonica), a representative of early-diverging lineages. Phylogenetic and synteny analyses revealed that eel npr1a and the previously reported npr1b (also known as npra) are teleost-specific paralogs generated by the third round of whole-genome duplication (3R). Functional analysis using COS-7 cells demonstrated distinct ligand responsiveness: Npr1a exhibited preferential responsiveness to VNP with lower signaling efficacy than Npr1b, whereas Npr1b responded broadly to cardiac NPs (eel ANP-NH₂ > VNP > BNP). In situ hybridization chain reaction revealed marked spatial divergence of these receptors in the eel brain: npr1a was predominantly localized in the parvocellular preoptic nucleus (Pp) and anterior tuberal nucleus (NAT), whereas npr1b was specifically expressed in the area postrema (AP). These regions are located at or near blood-brain barrier-deficient areas. Notably, RT-qPCR data showed that npr1b expression was significantly upregulated in seawater-acclimated eels, while npr1a levels remained stable, indicating distinct transcriptional regulation. Collectively, our findings demonstrate 3R-driven subfunctionalization of Npr1 in the Japanese eel and suggest that the retention of vnp in early-diverging lineages may have facilitated lineage-specific functional diversification of NP receptors during teleost evolution.