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Abstract

Resolving clonal dynamics in vivo is a fundamental challenge in biology, essential for understanding tissue development and homeostasis. Beginning in embryogenesis, individual cells and their clonal progeny undergo tightly coordinated behavior—including proliferation, differentiation, loss, and migration—that collectively establishes and maintains lifelong tissue and organ function. In most human and murine tissues, it is infeasible to observe clones over long periods within their physiological environment. In mice, high-resolution genetic barcoding now enables large-scale tracing of single cells throughout organismal development; however, the resulting barcode readouts typically provide only temporal endpoint snapshots.

Here, to address these limitations, I combined modeling of clonal dynamics with single-cell barcoding performed as a time series of independent experiments. This integrative framework represents clonal dynamics as stochastic processes, capturing both single-cell behavior and population-level differentiation, and enables time-series–based inference to reveal time-resolved clonal dynamics in vivo. In close collaboration with experimental partners, I applied this approach to uncover the developmental origins of tissue-resident macrophages and to infer the clonal dynamics of the male germline.

Tissue-resident macrophages are critical for organ formation and maintenance, yet the timing of lineage specification and the origins of these lineages during development remain controversial. In this work, by applying holistic Polylox barcoding to all embryonic cells at successive developmental stages, progenitor fates were mapped from gastrulation to macrophages in adult organs. Modeling of progenitor expansion and systematic testing of pathways of fate restriction inferred a unified lineage tree describing the clonal dynamics generating macrophage lineages in the brain, liver, lung, and spleen. This model progresses from a common progenitor around E6.5, through partially fate-restricted progenitors at E8.5, to predominantly unilineage fates by E10.5. It further predicted progenitor clone sizes in the liver, which were validated using spatial Polytope fate mapping to visualize local Kupffer cell colonies. Consistent with the lineage tree model, barcode analyses of tissue fragments revealed early tissue residency of all macrophage progenitors, suggesting local organ-specific seeding during organogenesis followed by a largely stationary existence into adulthood.

The germline is a designated lineage of cells that gives rise to sperm and eggs, enabling sexual reproduction and the evolution of species. It originates from a founding group of primordial germ cells (PGCs) and has been intensively studied in mouse models. However, how individual PGCs contribute to lifelong germline development remains unclear—a process that determines the transmission of genetic and epigenetic variability, including de novo mutations, across generations. Here, by integrating time-series Polylox barcoding with quantitative modeling, the clonal dynamics of the germline were reconstructed from the emergence of PGCs at E6.5 through the full lifespan and into the next generation. During an early migratory phase, PGC clones underwent substantial pruning, with ~30% of the founding clones lost, and subsequently expanded to stable but uneven proportions in the gonads, consistent with neutral drift dynamics. After E12.5 in male mice, PGC clones were spatially segregated in the forming testes, producing a mosaic of small, repeated patches in the postnatal seminiferous tubules. Owing to the quasi-one-dimensional tubular geometry in the testes, these clonal patterns remained globally and locally stable throughout life, such that the early-acquired PGC contributions were proportionally mirrored in sperm output and in the next generation.

Taken together, I developed and applied a modeling framework to resolve longitudinal clonal dynamics in vivo by integrating time-series single-cell barcoding data. This integrative approach established a lineage tree underlying macrophage development and uncovered the spatial and lifetime dynamics of the founding clones of the germline. The framework is broadly applicable and may serve as a blueprint for addressing outstanding questions in developmental and stem cell biology.