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T cells are divided into the αβ and γδ lineages. It is currently thought that these lineages differentiate in the thymus from uncommitted progenitor thymocytes. However, in this study we show that the decision to differentiate into one or either lineage is in fact already fixed in these apparent uncommitted progenitors. Using single-cell RNA sequencing, we reassemble de novo a model of early T cell development based on the transcriptional profiles of individual CD4-CD8- double negative and γδ thymocytes. We show that the earliest thymocyte stage, known as CD4-CD8- double negative 1 (DN1), is actually comprised of a mixture of transcriptionally distinct subpopulations. Although not yet expressing definitive markers of αβ and γδ lineages, such as the lineage-defining T cell receptors, specific DN subpopulations exhibit restricted developmental potential for either αβ or γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. Thus, T cell lineage decisions are hardwired from the earliest stages of T cell development.

The αβ and γδ T cell lineages both differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as CD4-CD8- double negative 1 (DN1), which has previously been shown to be a heterogenous mixture of cells. Of these, only the CD117+ fraction has been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αβ and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells may be derived from a subset of CD117- DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed a single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages indeed comprise a transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell responses, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell responses.

How the mammalian epiblast gives rises to fetal organs during embryogenesis has been investigated using reporters of marker genes, or through single cell or spatial RNA sequencing to infer differentiation trajectories, but less is known about the clonal fate of mammalian epiblast cells in vivo. Here we develop a high diversity, high throughput, Cre recombinase-driven DNA LoxCode barcoding technology for in vivo clonal lineage tracing. Using this LoxCode mouse model cells in pre-gastrulation E5.5 embryos were barcoded in utero and assessed un bulk via PCR or via single-cell RNA sequencing for their contribution to a comprehensive collection of tissues and cell types in the E12.5 organogenesis-stage embryo. While few, typically large clones contributed to a diverse range of cell types in multiple germ layer derivatives, many clones exhibited reproducible patterns of lineage restriction. Most prominent were clonal fate biases towards either blood, ectoderm lineages, mesenchymal tissues or limbs, likely reflecting branch points during development. At the single-cell level, clones exhibited heterogeneity in terms of tissue contributions, gene expression profiles, and in some instances left-right and/or anterior-posterior asymmetries. Our study demonstrates the power and versatility of LoxCode barcoding in investigating native clonal fate and provides a deep clonal interrogation of the contribution of the mammalian epiblast to fetal organs.

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