Spatio-DARLIN enables robust and efficient in situ lineage tracing in mice at single-cell resolution.

Spatially resolved lineage tracing is essential for understanding how clonal relationships shape tissue architecture. However, such an approach has not been established in mice across different tissues. Here we present Spatio-DARLIN, a versatile method that integrates the high-diversity DARLIN lineage-tracing mouse with sequencing-based spatial transcriptomics. Through a dedicated computational pipeline, Spatio-DARLIN achieves accurate clonal mapping at single-cell resolution and recovers reliable lineage information from ~25-50% of cells in the intestine and brain. Spatio-DARLIN identified stereotyped clonal patterns in the intestinal epithelium and revealed clonal dynamics that were consistent with stem-cell neutral drift. In the brain, we uncovered greater clonal expansion of radial glial cells in the cortex and hippocampus during development than in other regions. Moreover, our data strongly suggested that neuronal progenitors across different nuclei in the hypothalamus were already spatially prepatterned by embryonic day E10. Spatio-DARLIN enables high-resolution study of clonal architecture, expansion and migration across diverse tissues in situ.