Single-cell multiomics identifies Tcf1 and Lef1 as key initiators of early thymic progenitor fate

Bone marrow-derived multipotent hematopoietic progenitors seed the thymus and generate early thymic progenitors (ETPs). However, the factors governing ETP formation remain poorly defined. Using single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), we dissected the heterogeneity of transcriptomic and chromatin accessibility landscapes in murine ETPs. Whereas Tcf1^- ETPs exhibited higher proliferative capacity, Tcf1⁺ ETPs appeared to be immediate, more robust precursors to T lineage-specified early thymocytes. Prethymic ablation of Tcf1 and its homolog Lef1 severely impaired ETP formation in vivo. Whereas ablating Tcf1 alone had limited impact, loss of both Tcf1 and Lef1 impaired transcriptional activation of <i>Notch1</i> and Notch pathway effector molecules, including <i>Hes1</i> and <i>Hhex</i>, accompanied by aberrantly induced B cell and myeloid gene programs. Acute deletion of both factors compromised Notch pathway, glycolysis, and T cell gene programs in emergent ETPs ex vivo. Thus, Tcf1 and Lef1 act upstream of the Notch pathway, functioning as prethymic initiators of ETP fate and intrathymic gatekeepers of ETP identity and T lineage potential.