Wnt2b Is Essential for Adrenocortical Progenitor Cell Fate and Zona Glomerulosa Identity in Vivo

Dysregulation of normal adrenal structure and function contributes to a spectrum of diseases from hypoplasia to cancer. Peripheral adrenocortical progenitor cells in the zona glomerulosa (zG) centripetally migrate and differentiate to replenish steroidogenic cells of the zG and the inner cortex over time. Both the fate of progenitor cells and aldosterone production by steroidogenic cells in the zG are regulated by Wnt/β-catenin signaling, but the cell-specific effects of individual WNT ligands in the adrenal cortex are not fully understood. To further characterize Wnt signaling components crucial for progenitor cell fate and zG identity, we analyzed mouse adrenals using single molecule in situ hybridization, which revealed the previously unknown expression of Wnt2b exclusively in the adrenal capsule. Wnt2b is co-expressed in the capsule with the Wnt signaling potentiator Rspo3, the loss of which causes zG depletion and reduced adrenal size in mice. Therefore, we hypothesized that capsular WNT2B activates Wnt signaling in the underlying zG to maintain the undifferentiated state of progenitor cells. To define the role of WNT2B in these processes, we first generated whole body Wnt2b knockout (KO) mice, which exhibit complete zG loss, as defined by known markers of zG identity (β-catenin and DAB2). To more fully determine the mechanism by which Wnt2b deletion results in zG loss, we crossed Wnt2b-floxed and capsule-specific Gli1-CreERT2 mice to generate a Wnt2b conditional knockout (cKO) model and study the effects of Wnt2b loss on the zG during homeostasis of the adult adrenal cortex. Gli1-CreERT2 activation by tamoxifen in 6-week-old mice significantly decreased Wnt2b expression and resulted in a lower adrenal-to-body weight ratio in Wnt2b cKOs compared to controls four weeks later. Adrenocortical proliferation (Ki67) was also significantly decreased in Wnt2b cKO mice, suggesting that WNT2B may promote progenitor cell self-renewal. To characterize the consequences of WNT2B loss on canonical Wnt signaling, we assessed activation of β-catenin, the primary Wnt signaling effector. High β-catenin activity in the zG observed in wild-type mice was disrupted in Wnt2b cKO mice, together with markedly reduced expression of adrenocortical Wnt target genes Axin2 and Wnt4. In addition, Wnt2b loss resulted in downregulation of steroidogenic genes Cyp11b2 and Hsd3b6. Together, these data reveal that capsule-derived WNT2B is required for zG differentiation and maintenance, potentially through activating adrenocortical Wnt/β-catenin signaling and downstream target gene expression involved in both progenitor cell fate and steroid-producing cell function. Studies to more fully elucidate the dynamic effects of WNT2B on the adrenal zG are ongoing as they have important implications for adrenal homeostasis and disease, including both primary adrenal failure and neoplasia.