LoxCode in vivo barcoding resolves epiblast clonal fate to fetal organs

Abstract

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.

Amulti-tracklandscape of haematopoiesis informed by cellular barcoding and agent-based modelling

“…It is also important to note that this study employed isolation and transplantation into myeloablated hosts and this is unlikely to reflect steady-state haematopoiesis. Native cellular barcoding technologies are only emerging and population-specific induction of barcode generation does not yet extend beyond Cre-induced models (Feng et al, 2024; Pei et al, 2020; Weber et al, 2023). Even with such models, identifying an appropriate driver for the literature-defined stem and progenitor populations remains contentious.…”

Extraction and quantification of lineage-tracing barcodes with NextClone and CloneDetective

“…Recent advances in lineage-tracing protocols, such as ClonMapper [1], SPLINTR [2], Larry [3] and LoxCode [4], which integrate DNA barcoding with single-cell RNA sequencing (scRNA-seq), have catalysed significant progress in clonal biology. These techniques allow biologists to explicitly trace the ancestry and evolutionary trajectories of individual clones while observing their transcriptional changes over time.…”