ABSTRACT: The complete cell lineage of C. elegans, mapped over four decades ago, was tractable because the animal is small, transparent, and lineage-invariant. Most animals are none of these, having orders of magnitude more cells, being opaque, and developing with substantial stochasticity. Since 2016, genome editing-based lineage tracing has opened the door to dense cell lineage reconstruction in such organisms, but delivering on that promise has proven technically challenging. Here we apply DNA Typewriter, a prime editing-based recorder that writes stochastic symbolic insertions to an engineered genomic TAPE in strictly sequential order, to trace mouse development from zygote (E0) to late organogenesis (E13.5). We introduce constructs encoding a prime editor, prime editing guide RNAs (pegRNAs), and Pol-III-driven circularized TAPE RNA (circTAPE) into wildtype zygotes by pronuclear injection, then assay embryos by single-nucleus (cell) transcriptional profiling (scRNA-seq) with paired circTAPE recovery. From one E13.5 embryo bearing ~7 integrations of a constitutively expressed prime editor and 11 integrations of 6-unit circTAPE, we recover ~1.75M lineage-informative scRNA-seq profiles and reconstruct a time-calibrated, tip-annotated phylogeny of 1,600,000 cells with parsimony-based node support. The first cell division is marked unequivocally, and although the resulting blastomeres contribute asymmetrically to the embryo proper, they are fate-neutral and serve as an internal replicate that reproduces every finding. We observe striking clonal dominance, in that a small number of pre-gastrulation progenitors accounts for a disproportionate fraction of the E13.5 embryo. Fate is progressively and quantifiably restricted along the lineage. At the finest scale, sibling cells share fate far in excess of chance, with endodermal, blood, and neural-crest lineages exhibiting particularly striking clonal coherence, while the recent differentiations of organogenesis are legible in the structure of terminal clades. From clade co-occurrence alone, we reconstruct a dated hierarchy of cell-type relationships that recovers the major progenitor fields and agrees with a hierarchy that we derived independently from a scRNA-seq time-series of mouse development. Finally, by integrating these data with that timeseries, we impute transcriptional states and cell type annotations and for internal nodes of the tree and chart plausible state histories throughout the phylogeny. All data is made freely available, together with NextCell (https://nextcell.pages.dev/), an interactive browser for this annotated cellular phylogeny of mouse development.
