An integrated roadmap of human fetal adrenal gland development

Valle Ignacio del; Matthew Young; Gerda Kildisiute; Olumide Ogunbiyi; Federica Buonocore; Ian Simcock; Eleonora Khabirova; Berta Crespo; Nadjeda Moreno; Tony Brooks; Paola Niola; Katherine Swarbrick; Jenifer Suntharalingham; Sinead McGlacken-Byrne; Owen Arthurs; Sam Behjati; John Achermann

Introduction: The human adrenal gland originates from the adrenogonadal primordium at around 4 weeks post conception (wpc) and undergoes marked developmental changes throughout the first half of pregnancy. Several key aspects of adrenal maturation are well-established, such as the formation of a large inner fetal zone (FZ) and synthesis of dehydroepiandrosterone, but many other processes contributing to adrenal gland development and function in humans are still unknown at an anatomical and molecular level. Further insight into human adrenal development has implications of understanding postnatal stress responses, stem cell origin and clinical conditions resulting in primary adrenal insufficiency (PAI).

Methods: In order to develop an integrated map of human adrenal gland development, we combined single cell RNA-sequencing (scRNA-seq) (n=4; 6wpc-19wpc; c.44,000 cells) (10X Genomics), bulk RNA-seq (n=32; 7-11.5wpc, plus 8 controls), spatial transcriptomics (7wpc+4d) (10X Visium), immunohistochemistry (IHC) (n=4 stages), micro-focus computed tomography (microCT) imaging and multiomic-ATAC sequencing (10X genomics) across a key time course between 6-20wpc.

Results: Using this integrated dataset we have elucidated several key aspects of adrenal gland developmental, including: 1) rapid adrenal growth and remodelling, and the generation of fetal adrenal growth curves; 2) marked vascularisation (using surface imaging) and important drivers of vascular endothelial development (e.g., VEGFR1); 3) more marked cell proliferation in the outer definitive zone (DZ) (using cell cycle markers and IHC for KI67) with an early central trajectory of cell differentiation; 4) steroidogenic pathways favouring androgen synthesis in the central fetal zone (FZ), but with DZ capacity to synthesise cortisol and aldosterone with time; 5) the identification of “core” transcriptional regulators (n=17) (e.g. SF-1/NR5A1, DAX-1/NR0B1) and localisation of a novel cell regulator, HOPX, to the outer DZ; 6) early cell trajectories between the mesenchyme and adrenal cortex, with ligand-receptor interactions (e.g., RSPO3/LGR4) (CellPhoneDB); 7) an enrichment of growth-promoting imprinted genes (e.g. IGF2, PEG3), especially paternally-expressed genes in the rapidly-expanding FZ; and 8) a potential relation between the specificity of gene expression and the age of onset of primary adrenal insufficiency in infants and children with adrenal disorders. Several key elements linked to adrenal tumour biology were also identified.

Conclusion: These findings reveal new aspects of human adrenal development and have clinical implications for understanding PAI and related postnatal adrenal disorders, such as steroid biosynthesis and the ability of the extreme preterm baby to respond to neonatal stress.

ESPE Abstracts

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