. There are no modifications to the original Summary/Abstract section. In the adrenal, aldosterone is produced in the zona glomerulosa (ZG), cortisol in the zona fasciculata (ZF) and adrenal androgens, including dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), in the zona reticularis (ZR). The cortical zones are thought to be maintained by centripetal ‘streaming’ of the outer proliferative subcapsular progenitor ZG cells to aldosterone-producing ZG cells to glucocorticoid-producing ZF cells, and in humans to androgen-producing ZR cells. This proposal focuses on defining the least understood processes within adrenal zonation. Specific Aim 1 will test the hypothesis that angiotensin II (AngII) regulates mouse adrenal progenitor cell differentiation to aldosterone-producing ZG cells. Rationale: The ZG houses (i) an undifferentiated progenitor pool under the control of Wnt signaling that exclusively expresses Sonic hedgehog (SHH), and (ii) an organized group of cells that produce aldosterone under the control of AngII that exclusively expresses aldosterone synthase (AS). The mechanisms whereby progenitor cells (SHH+/AS-) give rise to differentiated ZG cells (SHH-/AS+) is unknown. We will determine 1) if AngII mediates the homeostatic lineage conversion of SHH+/AS- progenitor cells into differentiated SHH-/AS+ aldosterone-producing cells of the ZG; and 2) if AngII does so by reorganization of the chromatin landscape in the progenitor cells. Approach: We will use lineage tracing transgenic mouse models and techniques that include single cell multi-omics (RNAseq+ATACseq) and cut-and-tag sequencing to dissect these processes. Specific Aim 2 will define the mechanisms controlling conversion of human ZF cells to DHEAS-producing ZR cells. Rationale: The molecular mechanisms controlling ZR production of DHEAS are unknown. The ZF expression of the steroidogenic enzyme HSD3B2 allows for the production of cortisol. However, the complete lack of HSD3B2 in the ZR and the presence of the enzyme SULT2A1 is a critical factor for the ZR-specific synthesis of DHEAS. We will test the function of two previously unstudied adrenal nuclear receptor signaling pathways and probe the hypotheses that (i) ZR activation of the transcriptional repressor Rev-ERB blocks HSD3B2 transcription in the ZR cells, which facilitates DHEAS biosynthesis; and (ii) ZR activation of vitamin D3 receptor by locally produced ligands activates SULT2A1 transcription, which is instrumental for DHEAS production. Approach: Mice do not have a functional ZR, do not produce DHEAS and, therefore, cannot be used to study adrenal DHEAS production. We will use human adrenal tissue and cells in combination with single cell multi-omics, spatial transcriptomics and in situ high-resolution mass spectrometry imaging to define the role of the Rev-ERB and vitamin D3 nuclear hormone receptors in the transcriptional programming leading to conversion of a ZF to ZR DHEAS-producing phenotype. Significance and Impa...