LGR5 regulates sequential tooth development: evidence from single-cell transcriptomics and a gene inactivation model

Tooth replacement in vertebrates depends on the persistence of the dental lamina, yet the molecular mechanisms that determine species-specific regenerative capacity remain poorly understood. Here we combine single-cell transcriptomics, lineage tracing, and genetic inactivation to define the role of LGR5, a canonical epithelial stem cell marker, in sequential molar development. We identify an unrecognized epithelial Lgr5⁺ population in the dental stalk and rudimentary successional dental lamina, together with a distinct mesenchymal Lgr5⁺ progenitor pool outside the tooth germ. Lineage tracing demonstrates that epithelial Lgr5⁺ cells contribute regionally to second molar formation, implicating them in sequential tooth initiation. Loss of Lgr5 disrupts lamina architecture, leading to shortened stalks, reduced SOX2 expression, impaired basement membrane integrity, and altered Wnt signaling, including downregulation of the LGR5-interacting protein PTK7. Organoid assays further show that Lgr5⁺ epithelial cells function as niche stabilizers rather than classical proliferative progenitors, providing structural and signaling support for replacement tooth development. Comparative analysis in diphyodont minipigs reveals conserved Lgr5 expression in non-regressing lamina domains, linking Lgr5 activity with species capable of multiple tooth generations. Together, these results identify Lgr5 as a key regulator of dental lamina stability and sequential tooth development, uncovering molecular mechanisms that couple Wnt signaling with epithelial integrity and establishing a framework for Lgr5-based regenerative strategies in dentistry.