In the adrenal gland, stem and progenitor cells reside in the capsule and the outer part of the cortex, respectively. The newly formed cortical cells from stem/progenitor cells move inward to the corticomedullary boundary during development. Throughout this developmental process, adrenocortical cells express specific marker genes at different stages which lead to concentric zones of the adrenal cortex (zonation). In mice, the majority of the functional cortex are definitive zones whereas cortical cells in the corticomedullary boundary form the X-zone, which is considered the aged cell population in the cortex and undergoes regression over time. Disruption of signaling pathways such as Wnt, Hedgehog and TGF-beta can cause abnormalities of the developmental process of the adrenal cortex, which may lead to malfunction or malignancy of the adrenal gland. The role of Notch, another key signaling pathway, in adrenal development has not yet been fully studied. Notch signaling pathway involves a variety of gene regulatory mechanisms that control cell proliferation, differentiation, and apoptosis. Although key molecules in Notch signaling pathways are expressed both in the adrenal cortex and in the medulla, the inhibition of canonical Notch signaling only leads to minor/limited effects on the function or the structure of the adrenal gland. We confirmed these observations using Sf1-Cre mediated tissue-specific knockout mouse model that lacks Hes1, one of the most common Notch target genes, in cortical cells in the adrenal gland. Immunostaining with proliferation markers (PCNA & Ki67) and marker genes of different cell populations in the adrenal gland (e.g., β-catenin, 20αHSD, tyrosine hydroxylase) showed normal zonation and proliferation patterns. The unaffected adrenal glands in Hes1 conditional knockout mice suggests Notch signaling is not a dominant developmental pathway in the adrenal cortex. Next, to study how overactivation of Notch signaling affects adrenocortical cells, we used Sf1-Cre mice to overexpress Notch intracellular domain (NICD) in cortical cells in the adrenal gland. The over-expression of NICD in the adrenal cortex disrupted adrenal cortex zonation and led to a small, disorganized adrenal. These Sf1-Cre mediated NICD over-overexpressed adrenals had scattered and disorganized medulla irregularly distributed in the margin of the gland. Immunostaining showed clusters of 3βHSD low-expressing cells underneath the capsule partially surrounding the 3βHSD high-expressing cortex. Moreover, X-zone cells (labeled by 20αHSD) were significantly reduced in NICD over-overexpressed adrenals in two weeks old male and female mice. Our data suggest that overactivation of Notch signaling in the adrenal cortex not only disrupts the development of the definitive cortical zones but also affects the aging and the differentiation of fetal cortical cells in mice.

The Sonic Hedgehog (Shh) gene expressed in the subcapsular cortical region of the adrenal gland has been found to play a role in adrenal gland development. The Shh(+) cell population at the fetal stages contributes to different cortical layers in the adrenal gland. However, the (1) capability of these cells after weaning and (2) how soon they can renew the adrenal cortex in the postnatal stages is not fully understood. Here, we conducted a lineage tracing experiment to track Shh(+) cells and cells descended from them in post-weaning mice to ultimately better understand the processes of adrenal cortex renewal and remodeling over time in young adult mice. This experiment used the NuTRAP; Shh-Cre-ERT2 mice as the Shh-reporter mouse model. This tamoxifen-inducible mouse model allows us to specifically target and label Shh-expressing cells and all descendant cells with green fluorescence. Tamoxifen was given at postnatal days (P) 22, P24, and P26 to enable the Cre recombinase activity driven by the Shh promoter. Adrenal glands were then analyzed after two and four months. This lineage tracing experiment found that Shh(+) cells and their descendant cells reached the margin of the Cyp2f2(+) cortical zone in 2 months and the cortical-medullary boundary in 4 months. This finding indicates that the Shh(+) cell population in post-weaning mice can proliferate, differentiate, and eventually renew the entire adrenal cortex over a four-month period of time. Understanding the adrenal cortex's renewal rate helps us design our follow-up study to test the capability of Shh(+) cells in adult mice at different ages and how their potency changes overtime at the 'omic' level. Because this NuTRAP;Shh-Cre-ERT2 mouse model also allows us to isolate cell-type-specific DNA/RNA, we can further decipher the underlying gene/pathways which control this progenitor cell population of the adrenal gland cortex. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 1:12 p.m. - 1:17 p.m.

It is well known that thyroid hormone plays a key role in amphibian metamorphosis and mammalian tissue remodeling. We and others have reported that treatment with thyroid hormone will prevent or postpone the regression of the adrenal inner cortex (X-zone) and leads to X-zone hypertrophy in mice. The expression of thyroid hormone receptor beta 1 (TRβ1) in the adrenal inner cortex indicates that thyroid hormone could elicit its function directly on this cell population. To support this idea, we examined the expression and the function of major thyroid hormone transporters Mct8, Mct10, and Oatp1c1 in mouse adrenal glands. RNA-seq data showed that the expression levels of Mct10 and Oatp1c1 are relatively low in the adrenal gland (with RPKM less than 5 and 1, respectively) whereas Mct8 is the most abundant transporter (with RPKM around 20). Quantitative RT-PCR showed that Mct8 in the adrenal gland has an expression profile similar to X-zone marker genes and TRβ1 throughout developmental stages. LacZ staining showed similarities between Mct8 and TRβ1 expression, with both specifically expressed in the most inner cortex of the adrenal gland next to the medulla. However, by using thyroid hormone (T3) treatment, we found that the T3-mediated effect in the adrenal gland was not fully blocked in Mct8 knock-out mice. The expression of thyroid hormone transporter Mct8 in the adrenal inner cortex supports our claim that transporters mediate the direct action of thyroid hormone in the adrenal inner cortex. The blunted response to thyroid hormone in Mct8 knockout mice suggests that Mct8 is responsible for the thyroid hormone action in the adrenal gland. However, the deletion of Mct8 does not completely block thyroid hormones effect in the adrenal gland. Our finding suggests other thyroid hormone transporter(s) in the adrenal gland are also responsible for thyroid hormone signaling in the adrenal gland.