LoxCode in vivo clonal barcoding resolves mammalian epiblast contribution to fetal organs

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. E5.5 pre-gastrulation embryos were barcoded in utero and epiblast clones later assessed for their contribution to a wide range of tissues and cell types in the E12.5 organogenesis-stage embryo. While a few epiblast clones contributed broadly to most tissues and cell types of the three germ layers, many clones were lineage biased towards either blood, ectoderm lineages, mesenchymal tissues or limbs. In addition to lineage bias, most epiblast clones were differentially fated for tissue types or descendants in tissues compartments across the body axes. Using a stochastic agent-based model of embryogenesis and LoxCode barcoding, we inferred and experimentally validated predicted cell fate biases as well as clonal compositions across tissues that are consistent with events of lineage segregation and shared trajectory of lineage differentiation. Our study has demonstrated the power of LoxCode barcoding in investigating multi-modal clonal fate at high throughput, thus enabling an in-depth interrogation of the clonal contribution of E5.5 epiblast cells to fetal tissues, organs and body parts.