Spatial Transcriptomics Reveal Potential Sex Differences in Supraoptic Nucleus Gene Expression of Adult Rats

Date

2022

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Nguyen, Dianna H.
Phillips, Nicole
Cunningham, Joseph

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Abstract

Purpose: The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that play a key role in the regulation of fluid and electrolyte homeostasis. Although there are many well-known sexually dimorphic regions of the hypothalamus, little is known about possible sex differences in gene expression in the SON. Our study aims to address this knowledge gap by leveraging spatially-resolved transcriptomics to better visualize gene expression profiles of cells in the SON of male and female rats and gain insight on their physiological functions without sacrificing morphological context. Methods: Visium Spatial Gene Expression (10x Genomics) was used to obtain spatially-resolved gene expression data for the SON of adult male (n=4) and female (n=4) Sprague-Dawley rats. Briefly, each brain was sectioned at 10µm thickness to collect coronal sections (~4x4mm) containing the SON and other brain structures. Each section was then mounted on the capture areas of Visium slides containing probes that bind mRNA. Next, the sections underwent the following workflow: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics' Space Ranger and Loupe Browser applications and other bioinformatic tools. Results: Gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. From the list of significant genes after performing differential expression analysis on the SON region via Loupe Browser, 22 genes (e.g., Avp and Oxt) were common to both sexes, 24 genes were unique to the females, and no genes were unique to the males. Gene Ontology (GO) Enrichment and pathway analyses revealed GO terms and pathways related to: 1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females and 2) endomembrane system and glycerophospholipid metabolism pathway for the significant genes unique to females, as some examples. Further interrogation of the significant genes with Ingenuity Pathway Analysis showed some overlapping networks and common upstream regulators; however, differences between the male and female groups were also identified in upstream regulators, such as Creb, Pka, and LEPR unique to the males and insulin, Cdkal1, and HIF1A unique to the females. Conclusions: These spatially-resolved transcriptomic data suggest potential sex differences in SON gene expression that may be associated with basic endomembrane structure and function and phospholipid metabolism/signaling. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.

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Research Appreciation Day Award Winner - 2022 School of Biomedical Sciences, Department of Physiology & Anatomy-Integrative Physiology Program - 1st Place

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