IMPE Abstracts

The adrenal cortex undergoes constant cellular turnover: capsular stem cells give rise to cortical progenitor and differentiated steroidogenic cells of the zona glomerulosa (zG). Despite knowledge of their existence, little is known about these progenitor populations and have not previously been identified in single cell RNA sequencing (scRNA-Seq) transcriptomic analysis. We postulated that additional ACTH stimulation in the context of 21-hydroxylase deficiency may affect the progenitor population. Using 21-hydroxylase deficiency as a model for excessive ACTH stimulation, the aim of this study was to compare the adrenal single cell transcriptome in wild-type and 21-hydroxylase deficient mice and to identify potential adrenocortical progenitor cells. Adrenal glands were harvested from wild-type and 21-hydroxylase deficient mice, snap frozen, and dissociated into single nuclei. The scRNA-Seq library was prepared using 10x Chromium technology and sequenced by the Illumina NovaSeq platform. The transcriptome was mapped to the mouse mm10 reference genome using the CellRangerpipeline, followed by scRNA-Seq analysis using the Seurat v4.0 package. Cell types were annotated using a combination of the Clustifyr package and manual annotation using gene expression patterns. The transcriptome was analysed for 4153 and 4141 individual cell nuclei in the wild-type female and male samples respectively. The scRNA-Seq clustering pattern revealed 16 unique clusters by the Clustifyr package, two of which were subsequently manually divided (clusters 8 and 13). Adrenocortical cells made up 56% (n=2313) and 58% (n=2399) of cells in the female and male samples respectively. There were 3 zona glomerulosa and 4 zona fasciculata clusters, and one that had gene markers from both zones which was considered transitional. The progenitor population did not form a distinct cluster. However, cells co-expressing Nr5a1(encodes SF1 protein), Shhand Ctnnb1that did not express Cyp11b1or Cyp11b2(markers of differentiated adrenocortical cells) were detected with most located in cluster 0 (zona glomerulosa), consistent with SF1+/SHH+ progenitors. There were 14 and 8 cells detected in the female and male cluster 0 cell populations, respectively. Other genes that were highly expressed by the progenitors included Bcat1(essential for cell growth), Bicc1(regulation of gene expression during cell differentiation), Daam2(nervous system development and regulation of Wnt signalling), Dzip3(developmental gene regulation), and Ly6d(B-cell progenitor differentiation). In conclusion, potential adrenocortical progenitors were identified using single cell transcriptomic analysis. Further analysis to confirm their identity is required. The transcriptomic analysis of 21-hydroxylase deficient adrenals is underway.