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Published October 23, 2019 | Accepted Version + Supplemental Material
Journal Article Open

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


Intrathymic T cell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing T cell genes, but the underlying steps have remained obscure. Single-cell profiling 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 T cell precursor frequency among the immunophenotypically defined "early T cell 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 T cell 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 T cell specification.

Additional Information

© 2019 Elsevier Inc. Received 6 May 2019, Revised 10 August 2019, Accepted 18 September 2019, Available online 16 October 2019. We thank Jeff Park, Paul Rivaud, and Sisi Chen from the Caltech Single Cell Profiling and Engineering Center for help with the 10X Genomics samples; Andres Collazo and the Biological Imaging facility of Caltech for clonal live imaging support; Sean Upchurch and Diane Trout for C1 bioinformatic support; and members of the Rothenberg, Wold, and Cai labs for advice. We also thank Rochelle Diamond and members of the Caltech Flow Cytometry Facility for sorting, Ingrid Soto for mouse care, Igor Antoshechkin and Vijaya Kumar of the Caltech Jacobs Genomics Facility, and Xiwei Wu and the Integrative Genomic Core of City of Hope for Smartseq2 and bulk RNA sequencing. Support for this project came from USPHS grants (R01HL119102 and R01HD076915) to E.V.R., The Beckman Institute at Caltech for support of all the Caltech facilities, the Biology and Biological Engineering Division Bowes Leadership Chair Fund, the Louis A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, and the Albert Billings Ruddock Professorship to E.V.R. Author Contributions: W.Z. carried out most of the research and analysis and wrote the paper. M.A.Y. carried out and supervised research and wrote the paper. B.A.W. did C1 experiments, and J.Y. supported seqFISH work. B.J.W. and L.C. guided and supported research and edited the paper. E.V.R. supervised research, designed the project, and wrote the paper. Declaration of Interests: L.C. is a co-founder and B.J.W. is a consultant of Spatial Genomics Inc. The authors declare no other competing interests.

Attached Files

Accepted Version - nihms-1545118.pdf

Supplemental Material - 1-s2.0-S2405471219303163-mmc1.pdf

Supplemental Material - 1-s2.0-S2405471219303163-mmc2.xlsx

Supplemental Material - 1-s2.0-S2405471219303163-mmc3.xlsx

Supplemental Material - 1-s2.0-S2405471219303163-mmc4.xlsx

Supplemental Material - 1-s2.0-S2405471219303163-mmc5.xlsx

Supplemental Material - 1-s2.0-S2405471219303163-mmc6.xlsx

Supplemental Material - 1-s2.0-S2405471219303163-mmc7.xlsx


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August 22, 2023
October 18, 2023