An integrated single-cell analysis of human adrenal cortex development

Abstract

The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress.

Keywords: Development; Embryonic development; Endocrinology; Genetic diseases; Transcription.

Figure 1. Study design, adrenal development, and transcriptome analysis.

(A) Overview of the study design for generating bulk transcriptomes (bulk RNA-Seq), single-cell mRNA transcriptomes (scRNA-Seq), spatial transcriptomics, micro-CT (micro-focus computed tomography), and histology/immunohistochemistry. Stages are shown as weeks (w) and days (d) postconception (pc). (B) Growth curve of the adrenal gland between 7 weeks postconception and 2 days (7wpc+2d) and 20wpc (n = 36). Data for single glands are shown. (C) Photographs of adrenal glands (10% formalin) at 6wpc+6d (left, scale bar: 300 μm) and 16wpc (right, scale bar: 3 mm) to show marked growth and anatomical changes. (D) Micro-CT surface image of the adrenal gland at 17wpc showing the anterior sulcus and vascularization (maximum dimension, 15 mm). (E) Principal component analysis (PCA) of bulk transcriptome data for adrenal glands at 7wpc (n = 8), 8wpc (n = 8), 9wpc (n = 8), and 11.5wpc (n = 8) and control tissues (n = 14, from 8 different tissues) as indicated. (F) Volcano plot showing differential gene expression of genes in the bulk transcriptome adrenal gland data set (total n = 32) compared with controls (n = 14). Selected highly differentially expressed adrenal genes are indicated. (G) Uniform manifold approximation and projection (UMAP) of scRNA-Seq transcriptome data from 4 adrenal glands (6w, 6wpc+6d, 8wpc+5d, 19w) with the major different cell populations annotated (6wpc, n = 3,047 cells; 6wpc+6d, n = 2,650 cells; 8wpc+5d, n = 23,313 cells; 19wpc, n = 15,348 cells). (H) Relative proportion of mesenchyme and vascular endothelial cells in the adrenal gland at each time point studied. (I) Feature plot showing expression of FLT1 (encoding vascular endothelial growth factor receptor 1, VEGFR1) in the vascular endothelial cluster (see G for annotation). (J) Micro-CT (17wpc) to show the extensive surface vascular network on the inferior surface of the gland (maximum dimension, 15 mm).

Figure 2. Adrenal cortex zonation and proliferation.

(A) Dot plot to show the most highly differentially expressed genes in the adrenal cortex single-cell transcriptome (scRNA-Seq) compared with other cells in the adrenal gland. (B) Histology of the human fetal adrenal gland at 8.5wpc (H&E staining). Scale: 400 μm. DZ, definitive zone; FZ, fetal zone. (C–E) The developing DZ shown by NOV (also known as CCN3) expression using a single-cell mRNA transcriptome UMAP (C), spatial transcriptomic spot plot (7wpc+4d, darker red shows higher expression) (D) and immunohistochemistry (11wpc; scale: 50 μm) (E). Integrated data from samples at all 4 time points are shown. (F–H) The developing FZ shown by SULT2A1 expression using a single-cell mRNA transcriptome UMAP (F), spatial transcriptomic spot plot (7wpc+4d) (G) and immunohistochemistry (11wpc; scale: 50 μm) (H). (I) Integrated UMAP showing cell cycle states. (J) IHC of fetal adrenal gland showing Ki-67 expression as a marker of cell proliferation at different ages. Scales: all 100 μm. (K) Relative proportion of cells in each cell cycle state at each time point.

Figure 3. Expression of classic steroidogenic pathway genes during human adrenal development.

(A) Time series bulk RNA-Seq expression (normalized counts) of the melanocortin-2 receptor gene (MC2R), encoding the adrenocorticotropin (ACTH) receptor (n = 8 at each stage). Violin plots show the median values (horizontal bars), outliers, and the distribution of upper and lower interquartile ranges (boxes). (B) Time series bulk RNA-Seq expression of the gene encoding steroidogenic acute regulatory protein (STAR) (n = 8 at each stage). (C) UMAP of adrenal cortex clusters used for subsequent analysis. DZ, definitive zone; FZ, fetal zone. (D) Graphical representation of the “classic” steroidogenic pathway showing the key genetic components leading to the synthesis of mineralocorticoids (e.g., aldosterone), glucocorticoids (e.g., cortisol), and androgens (e.g., DHEA, androstenedione, testosterone). Feature plots showing the expression of key genes in the adrenal cortex clusters at different time points are shown. ACTH, adrenocorticotropin; AR, androgen receptor; CYB5A, cytochrome 5A; CYP11A1, P450 side-chain cleavage enzyme; CYP11B1, 11β-hydroxylase type 1; CYP11B2, aldosterone synthase; CYP17A1, 17α-hydroxylase/17,20-lyase; CYP21A2, 21-hydroxylase; DHEA(S), dehydroepiandrosterone (sulfate); DHT, dihydrotestosterone; HSD3B2, 3β-hydroxysteroid dehydrogenase type 2; MC2R, melanocortin-2 receptor (ACTHR); MRAP, MC2R accessory protein; POR, P450 oxidoreductase. (E) Heatmap of scRNA-Seq expression of HSD3B2, CYP21A2, CYP11B1, and CYP11B2 at different ages in the adrenal cortex clusters. (F) Scatterplots of expression of HSD3B2 in individual cortex single cells (scRNA-Seq) compared with CYP21A2 at 3 different time points (6wpc, 8wpc+5d, 19wpc). (G) Scatterplots of expression of HSD3B2 in individual cortex single cells (scRNA-Seq) compared with CYP11B2 at 3 different time points (6wpc, 8wpc+5d, 19wpc).

Figure 4. Expression of transcription factors during human adrenal cortex development.

(A) Venn diagram showing the overlap of differentially expressed transcription factors in the scRNA-Seq data set at each age. Differential expression was defined as being enriched in the adrenal cortex cluster compared with all other clusters in the whole adrenal sample at each age (log₂FC > 0.25, padj < 0.05). A core group of 17 transcription factors common to all ages was identified. (B) Heatmap showing relative expression of these 17 transcription factors at each age in the scRNA-Seq data set. (C) Feature plots showing expression of these 17 transcription factors in adrenal cortex clusters (for annotation, see Figure 3C). (D) Spatial transcriptomic spot plot expression of the key nuclear receptors, NR0B1 (also known as DAX-1) and NR5A1 (also known as steroidogenic factor-1, SF-1) at 7wpc+4d. (E) Scatterplots of expression of NR5A1 in individual adrenal cortex single cells (scRNA-Seq) compared with NR0B1 (6wpc, 6wpc+5d, 19wpc).

Figure 5. HOPX is a potentially novel DZ factor.

(A) HOPX expression (normalized counts) in the human developing adrenal gland (combined adrenal gland samples, bulk RNA-Seq, n = 32) compared with controls (n = 14). (B) Feature plot of HOPX expression in the adrenal cortex clusters (for annotation, see Figure 1G). (C) Spatial transcriptomic spot plot showing DZ expression of HOPX at 7wpc+4d. Msc, mesenchyme. (D) Immunohistochemistry showing expression of HOPX in the DZ at late 6wpc between the layer of outer Msc and inner adrenal FZ. Scale: 20 μm. (E) Immunohistochemistry showing representative DZ expression of HOPX at each stage. Scales: all 50 μm. (F) Feature plots of HOPX expression in the adrenal cortex cluster at 2 different ages (8wpc+5d, 19wpc) compared with the DZ marker NOV. (G) UMAP of key cortex clusters at 19wpc. (H) Dot plot of the top differentially expressed genes in cluster 0 compared with other clusters at 19wpc. (I) Dual-labeled IHC of HOPX expression (brown) and NOV (magenta). Scales: 250 μm, upper panel; 50 μm, center panel; 20 μm, lower panel.

Figure 6. Potential bidirectional signaling interactions between the mesenchyme cluster and adrenal cortex.

Data (scRNA-Seq) shown at 6wpc+6d. (A) UMAP of the mesenchyme and adrenal cortex clusters demonstrating the potential “bridge” between the 2 populations of cells. Subclusters used for cell-cell communication analysis are shown. (B) Single-cell velocity estimates overlaid on the UMAP of mesenchyme–adrenal cortex clusters (RNA velocity). (C) Potential ligand-receptor interactions for key subclusters in the mesenchyme (clusters 3, 4) and adrenal cortex (cluster 0), using CellPhoneDB. (D) Feature plot showing expression of IGF2 (encoding ligand) and expression of IGF1R (encoding cognate receptor). (E) Feature plot showing expression of DLK1 (encoding ligand) and expression of Notch2 (encoding receptor) (see also Supplemental Figure 19). (F) Feature plot showing expression of CXCL12 (encoding ligand) and expression of CXCR4 (encoding receptor). (G) Feature plot showing expression of RSPO3 (encoding ligand) and expression of LGR4 (encoding receptor). (H) Spatial transcriptomic spot plot (7wpc+4d) of DLK1 in the definitive DZ and FZ of the adrenal gland, with weaker expression in the mesenchyme (Msc)/subcapsular region. (I) Spatial transcriptomic spot plot (7wpc+4d) of CXCL12, strongest in the Msc/subcapsular region of the adrenal gland. (J) Spatial transcriptomic spot plot (7wpc+4d) of RSPO3 in the Msc (arrowheads)/subcapsular region of the adrenal gland and LGR4 in the adrenal cortex. IGF2, insulin-like growth factor 2; RSPO3, R-Spondin 3; LGR4, leucine-rich repeat-containing G protein–coupled receptor 4.

Figure 7. Imprinted genes in human adrenal development.

(A) Venn diagram showing the 15 imprinted genes (non-placental-specific) that are differentially expressed in the adrenal cortex cluster (bulk RNA-Seq adrenal > control, log₂FC > 1.5, padj < 0.05). (B) Violin plots (normalized counts) of bulk RNA-Seq expression of several key imprinted genes in the adrenal gland (n = 32) compared with control tissues (n = 14). (C) Dot plot of key differentially expressed imprinted genes in different scRNA-Seq clusters of the developing human adrenal gland. (D) Violin plots showing time series bulk RNA-Seq expression of key imprinted factors in the developing human adrenal gland (n = 8 at each stage). (E) Heatmap of the expression of key imprinted genes in different clusters of the adrenal cortex at 19wpc (see Figure 6F). (F) UMAP of adrenal cortex subclusters at 19wpc. (G) Spatial transcriptomic spot plot (7wpc+4d) of paternally expressed gene 3 (PEG3) showing strong expression, especially in the central FZ. (H) Feature plots of 6 key paternally expressed (maternally imprinted) genes in the adrenal cortex (19wpc). (I) Feature plot of 3 key maternally expressed (paternally imprinted) genes in the adrenal cortex (19wpc).

Figure 8. Expression of genes enriched in the adult adrenal gland and in monogenic causes of PAI.

(A) Dot plot showing fetal adrenal gland expression of genes with the highest “tissue specificity score” (enriched expression) in the adult adrenal gland, as defined in the Human Protein Atlas (https://www.proteinatlas.org). VE, vascular endothelium; SCPs, Schwann cell precursors. (B) Dot plot showing the expression of genes associated with monogenic causes of primary adrenal (glucocorticoid) insufficiency (PAI) in the adrenal cortex and other adrenal clusters during development (see UMAP, A). Dev, developmental disorders; ACTH, ACTH resistance; Std, steroidogenic disorders; Ox. st, oxidative stress; Metab, metabolic disorders. (C) Feature plot for expression of nicotinamide nucleotide transhydrogenase (NNT) and sphingosine-1-phosphate lyase 1 (SGPL1). (D) Dot plot of the expression of PAI-causing genes proposed to be involved in adrenal growth and cell division in different cell cycle phases (S phase, G2M, G1). (E) Expression of mini-chromosome maintenance complex component 4 (MCM4) and cyclin-dependent kinase inhibitor 1C (CDKN1C) in the integrated adrenal cortex cluster with cycling cells included (see Figure 2I). (F) Age at presentation with adrenal insufficiency of children and young people with selected monogenic causes of PAI.