Characterization of gene expression signatures for the identification of cellular heterogeneity in the developing mammary gland

The developing mammary gland utilizes several transcription-dependent networks to support multiple cellular identities and differentiation pathways. Recent technological advancements that allow for single-cell profiling of gene expression have provided an initial picture into the cellular heterogeneity within the mammary epithelium across the diverse stages of gland maturation. Still, a deeper dive into extended molecular signatures of all mammary resident cells would improve our understanding of the diversity of mammary epithelial and non-epithelial cellular populations across different tissue developmental stages, mouse strains and mammalian species. Here, we combined differential mammary gland fractionation and transcriptional profiles obtained from FACS-isolated mammary cells to improve our definitions of mammary-resident, cellular identities at the single-cell level. Our approach yielded a series of expression signatures that illustrate the heterogeneity of mammary epithelial cells, specifically those of the luminal fate, and uncovered transcriptional changes to their lineage- defined, cellular states that are induced during gland development. Our analysis also provided molecular signatures that identified non-epithelial mammary cells, including those of adipocytes, fibroblasts and not so abundant immune cells. Lastly, we extended our study to elucidate expression signatures of human, breast-resident cell fates, a strategy that allowed for the cross-species comparison of mammary epithelial identities. Collectively, our approach improved the existing signatures of normal mammary epithelial cells, as well as elucidated the diversity of non-epithelial cells in murine and human breast tissue. Our study provides a useful resource for future studies that use single-cell molecular profiling strategies to understand normal and malignant breast development.