bioRxiv2026
How diverse cell lineages emerge and are genetically regulated during organogenesis are central questions in understanding the developmental origins of disease. However, the mouse gut, including its intrinsic enteric nervous system (ENS) derived from migratory neural crest, has remained difficult to experimentally target. Here, we introduce an in utero lentiviral nano-injection strategy that enables early and efficient access to progenitor cells of all major cell types within the developing gut as well as gut-innervating ganglia. Leveraging this approach in combination with DNA barcoding and single cell transcriptomics, we resolve clonal relationships in all gut lineages, including epithelial, neural, immune, and mesenchymal cell types. Clonal coupling between distinct subsets of fibroblasts and either pericytes, mesothelial cells, or interstitial cells of Cajal, suggested a developmental logic whereby the mesenchymal compartment arises from a set of fate-biased progenitors. Yet, mesenchymal regionalization along the anterior-posterior axis establishes early, whereas the ENS displays broad clonal dispersion across gut regions and acquires subsequent regional identities. We further adapted the platform for temporally controlled cell-type specific gene manipulation and, as a proof-of-principle, show that induced expression of the proneural factor Ascl1 biases ENS progenitor cells toward neuronal differentiation. Together, this work provides insights into refined spatiotemporal lineage relationships within a multigerm-layer organ and establishes a broadly applicable in vivo framework for probing gene function during gastrointestinal and neural crest development. SIGNIFICANCEThe gastrointestinal tract comprises diverse cell types originating from all three germ layers and includes the neural crest-derived enteric nervous system (ENS). Progress in defining these lineages and their gene regulation is challenged by the limited experimental access to the developing gut. Here, we establish in utero lentiviral transduction as an efficient approach to resolve clonal lineages and address gene functions in defined gut cell types. We show that mesenchyme assumes positional allocation early and differentiates through fate-restricted progenitors, linking specialized mesenchymal cell types to different fibroblasts. In contrast, the ENS differentiates stochastically and acquires late regional identities. Our study reveals fundamental principles of multi-lineage organogenesis and provides a framework to dissect the contribution of developmental programs to visceral dysfunction.