Positional cues underlie cell fate specification during branching morphogenesis of the embryonic mammary epithelium

How cells coordinate morphogenetic cues and fate specification during development is a fundamental question at the basis of tissue formation. Lineage tracing studies have demonstrated that many stratified epithelia, including the mammary gland, first arise from multipotent stem cells, which are progressively replaced by distinct pools of unipotent progenitors that maintain tissue homeostasis postnatally. The lack of specific markers for early fate specification in the mammary gland has prevented the delineation of the features and spatial localization of lineage-committed progenitors that co-exist with multipotent stem cells (MaSCs) during tissue development. Here, using single-cell RNA-sequencing across 4 stages of embryonic development, we reconstructed the differentiation trajectories of multipotent mammary stem cells towards basal and luminal fate. Our data revealed that MaSCs can already be resolved into distinct populations exhibiting lineage commitment at the time coinciding with the first sprouting events of mammary branching morphogenesis (E15.5). By visualizing gene expression across our developmental atlas, we provide novel molecular markers for committed and multipotent MaSCs, and define their spatial distribution within the developing tissue. Furthermore, we show that the mammary embryonic mesenchyme is composed of two spatially-restricted cell populations, representing the sub-epithelial and dermal mesenchyme. Mechanistically, we explored the communication between different subsets of mesenchymal and epithelial cells, using time-lapse analysis of mammary embryonic explant cultures, and reveal that mesenchymal-produced FGF10 accelerates embryonic mammary branching morphogenesis without affecting cell proliferation. Altogether, our data elucidate the spatiotemporal signals underlying lineage specification of multipotent mammary stem cells and uncover the paracrine interactions between epithelial and mesenchymal cells that guide mammary branching morphogenesis.