In utero transduction resolves gut cell lineages and enables conditional gene perturbation in the developing enteric nervous system

Background and AimsUnderstanding the etiology of gastrointestinal malfunction requires insights into how heterogeneous gut cell lineages arise during development and the identification of genes controlling these processes. While in utero genetic manipulation has advanced knowledge in other organs, the gut and its intrinsic enteric nervous system (ENS) remain difficult to target. We aimed to establish an approach to resolve precise lineage relationships and achieve conditional gene perturbation of developing gut cell types in vivo. MethodsWe performed ultrasound-guided in utero nano-injection of lentiviral vectors into the mouse amniotic cavity at embryonic day 7.5. By combining lentiviral DNA barcoding with single cell RNA-sequencing, we reconstructed clonal relationships between distinct gut cell types. We used Cre-dependent lentiviral vectors in transgenic mice to achieve cell type-specific expression and tested whether gene overexpression can direct developmental processes. ResultsLentiviral delivery efficiently targeted gut-innervating ganglia and all major gut cell types, including neural, epithelial, immune, and mesenchymal populations. Barcode-based lineage tracing revealed clonal relationships within and across gut cell types. Notably, specialized mesenchymal populations, including pericytes, mesothelial cells, and interstitial cells of Cajal (ICCs) were each linked to different fibroblast subpopulations. Regional analyses demonstrated early establishment of anterior-posterior identity of mesenchyme, whereas ENS and immune cells exhibited long-range clonal dispersion. Cre-dependent targeting enabled selective gene expression in ENS progenitors or neurons, and temporally controlled overexpression of Ascl1 promoted neuronal differentiation. ConclusionsIn utero lentiviral transduction expands experimental access to the developing gastrointestinal tract, enabling lineage-resolved analysis and conditional gene manipulation of gut cell types, including the ENS. This approach enables mechanistic investigation of gut organogenesis and offers a framework to study developmental origins of disease.