Clonal analysis of murine development reveals novel positional programs directing lineage progression

Embryonic development is commonly viewed through a tree model of cell differentiation, which does not adequately represent the precise spatial and temporal modulation of cell multipotency underlying morphogenesis. Identifying the mechanisms conditioning and executing these fate decisions is a major aim of developmental biology. Here we develop an integrated approach, combining in vivo single-cell high-throughput clonal lineage tracing with machine learning, to systematically decompose continuous spectra of clonal fate biases in mammalian embryos traced from neurulation to mid-gestation stages. The reconstructed patterns of recurrent clonal variation were used to reveal gene programs driving dynamic biasing and spatiotemporal control over clonal composition, with axial skeletogenesis and peripheral neurogenesis serving as example models of clonally patterned systems. Experimental mosaic combinatorial perturbations targeting the Hedgehog signaling pathway caused the formation of novel clone types, giving proof of concept that custom cell type assemblages could be programmed in vivo starting from a well-defined progenitor cell. Altogether, our work demonstrates an effective practical approach for interrogating programs guiding clonal lineage specification.