LoxCode in vivo barcoding resolves epiblast clonal fate to fetal organs

Abstract Contribution of the mammalian epiblast 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. However, much remains to be learned 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 E5.5 pre-gastrulation embryos were barcoded in utero and assessed in bulk via PCR or via single-cell RNA sequencing for their contribution to a comprehensive range of tissues and cell types in the E12.5 organogenesis-stage embryo. While a few typically large clones contributed to a diverse range of cell types of multiple germ layer derivatives, many clones displayed 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. In the context of a stochastic agent-based model of tissue development, clonal fate biases could be explained by early differentiation events occurring shortly after barcoding, and clonal similarities between tissues arose as a consequence of shared differentiation paths. At the single-cell level, clones exhibited heterogeneity in terms of tissue contributions, gene expression profiles, and in some instances left-right asymmetries and/or anterior-posterior segregation. 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.