Single-nuclei RNA sequencing reveals heterogeneity within developing GnRH3 neurons in zebrafish

Gonadotropin-releasing hormone (GnRH) is a central regulator of vertebrate reproduction regulating pituitary gonadotropins. In zebrafish, GnRH3 serves as the hypophysiotropic isoform. Its neurons develop by migrating from the nasal placode to the hypothalamus, forming several distinct subpopulations along the migratory path. However, the molecular heterogeneity underlying this migration remains incompletely understood. To delineate the molecular and spatial heterogeneity, we employed single-nuclei RNA sequencing of nuclei enriched based on gnrh3 promoter-driven EGFP fluorescence from 7 dpf Tg(gnrh3:EGFP) zebrafish. This was followed by GnRH3 neurons clustering, differentially expressed genes identification, trajectory inference as a hypothesis-generating framework, and functional analyses. Marker genes were selected and used for validation and to determine the distribution of each subcluster by in situ hybridization (ISH) and immunohistochemistry (IHC) in larval and adult zebrafish. Three subpopulations were identified featuring highly expressed marker genes: Gnrh3^cyp1a, Gnrh3^nrg1, and Gnrh3^npffl/prl2. Gnrh3^npffl/prl2 was further clustered into two subpopulations, Gnrh3^npffl/prl2 and Gnrh3^npffl, which localized in the olfactory bulbs/terminal nerve and telencephalon, whereas Gnrh3^cyp1a and Gnrh3^nrg1 were detected in distinct cells within the olfactory epithelium. Trajectory inference and gene ontology enrichment analyses were consistent with a putative transcriptomic continuum from Gnrh3^cyp1a to Gnrh3^npffl/prl2 states, with the latter subpopulation lacking canonical migratory gene signatures. Protein–protein interaction network analysis revealed interaction relationships among differentially expressed genes, with a GnRH3-associated interaction module further supporting the selection of the above marker genes. Our results support the notion of at least three distinct GnRH3 subpopulations with distinct transcriptomes and characteristics, providing a broader basis for deciphering the diverse roles of GnRH3 in migration and reproduction.