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

Overview

1. Institute of Marine & Environmental Technology, Department of Marine Biotechnology, University of Maryland Baltimore County, Baltimore, Maryland, USA
2. Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel
3. Department of Microbiology and Immunology, University of Maryland Baltimore, Baltimore, Maryland, USA

Abstract

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: Gnrh3cyp1a, Gnrh3nrg1, and Gnrh3npffl/prl2. Gnrh3npffl/prl2 was further clustered into two subpopulations, Gnrh3npffl/prl2 and Gnrh3npffl, which localized in the olfactory bulbs/terminal nerve and telencephalon, whereas Gnrh3cyp1a and Gnrh3nrg1 were detected in distinct cells within the olfactory epithelium. Trajectory inference and gene ontology enrichment analyses were consistent with a putative transcriptomic continuum from Gnrh3cyp1a to Gnrh3npffl/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.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Data

Datasets cited

• geo:GSE309816, at NCBI GEO; found in “DATA AVAILABILITY STATEMENT”

Data availability statement

The data that support the findings of this study are openly available in GEO at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE309816, reference number GSE309816 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE309816). The snRNA‐seq datasets generated in this study have been deposited at the NCBI's Gene Expression Omnibus under accession number GSE309816 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE309816). The datasets generated during this study are currently under private access. They will be made publicly available in GEO immediately upon acceptance and publication of the manuscript, superseding the current embargo date (July 30, 2026). For peer review, the data may be accessed using the secure token ankhocqejzqtnwr through the GEO private review link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE309816. No custom code was generated for this study. All analyses were conducted using publicly available software packages. Further requests are available from the corresponding authors upon reasonable request.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Version 2, 28 September 2026

• Publisher: n/a → Wiley

Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 5 authors, 4 keywords, 9 MeSH terms, 2 funders, 54 references, 1 RRID.

Cite

This paper

Sun, Y., Golan, M., Zohar, Y., Fan, X., & Zmora, N. (2026). Single-nuclei RNA sequencing reveals heterogeneity within developing GnRH3 neurons in zebrafish. Journal of neuroendocrinology, 38(7), e70230. https://doi.org/10.1111/jne.70230

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