Single-Cell Analysis Reveals Regulatory Gene Expression Dynamics Leading to Lineage Commitment in Early T Cell Development.

Abstract

Intrathymic Tcell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing Tcell genes, but the underlying steps have remained obscure. Single-cellprofiling was used to define the order of regulatory changes, employing single-cell RNA sequencing (scRNA-seq) for full-transcriptome analysis, plus sequential multiplexed single-molecule fluorescent in situ hybridization (seqFISH) to quantitate functionally important transcripts in intrathymic precursors. Single-cell cloning verified high Tcell precursor frequency among theimmunophenotypically defined "early Tcell precursor" (ETP) population; a discrete committed granulocyte precursor subset was also distinguished. We established regulatory phenotypes of sequential ETP subsets, confirmed initial co-expression of progenitor with Tcell specification genes, defined stage-specific relationships between cell cycle and differentiation, and generated a pseudotime model from ETP to T lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to Tcell specification.