The cooperation between RNA binding proteins ZFP36L1 and ZFP36L2 controls thymic epithelial cell differentiation to promote thymus function.

The thymus generates immunocompetent T cells, but its function declines with age. Thymic activity depends on the proper differentiation of thymic epithelial cells (TECs), which establish unique niches for T-cell development. However, the molecular mechanisms governing the differentiation of functionally diverse cortical (c) and medullary (m) TEC subsets from common progenitors remain poorly understood. Employing dual conditional knockout (dcKO) and lineage-tracing mouse models, we demonstrate that the RNA-binding proteins ZFP36L1 and ZFP36L2 cooperatively maintain a functional TEC microenvironment. Deletion of Zfp36l1 and Zfp36l2 in TEC-specific dcKO mouse models resulted in similarly pronounced and early-onset thymic hypoplasia, with a significant reduction in both TEC and thymocyte cellularity. Mechanistically, single-cell transcriptomics showed significant alterations in the composition and transcriptional landscape of cTECs, mTECs, and thymic mimetic cells of dcKO thymus. The erosion of the TEC compartment, together with the accumulation of age- associated TECs suggested impaired differentiation and maintenance of mature TEC lineages from their progenitors. Fate-mapping analysis revealed that dual deficiency in Zfp36l1 and Zfp36l2 disrupted the canonical developmental trajectory from b5t-expressing TEC progenitors into mature cTEC and mTEC lineages. Our findings uncover a cooperative role for ZFP36L1 and ZFP36L2 in orchestrating TEC lineage differentiation and determining thymic function under physiological conditions.