The tear-producing lacrimal gland is a tubular organ that protects and lubricates the ocular surface. The lacrimal gland possesses many features that make it an excellent model in which to investigate tubulogenesis, but the cell types and lineage relationships that drive lacrimal gland formation are unclear. Using single-cell sequencing and other molecular tools, we reveal novel cell identities and epithelial lineage dynamics that underlie lacrimal gland development. We show that the lacrimal gland from its earliest developmental stages is composed of multiple subpopulations of immune, epithelial and mesenchymal cell lineages. The epithelial lineage exhibits the most substantial cellular changes, transitioning through a series of unique transcriptional states to become terminally differentiated acinar, ductal and myoepithelial cells. Furthermore, lineage tracing in postnatal and adult glands provides the first direct evidence of unipotent KRT5+ epithelial cells in the lacrimal gland. Finally, we show conservation of developmental markers between the developing mouse and human lacrimal gland, supporting the use of mice to understand human development. Together, our data reveal crucial features of lacrimal gland development that have broad implications for understanding epithelial organogenesis
