Functional spatio-temporal transcriptome analysis of enriched cell populations during postnatal mammary gland development

Abstract

Postnatal development of the mammary gland is a complex, highly specialized process characterized by massive cell proliferation and tissue remodeling to ensure milk production and excretion after parturition. Many of the genes that regulate this process have been identified and their expression extensively characterized at all major postnatal developmental stages. Until recently, transcriptional profiling during pregnancy and lactation has been performed using whole mammary glands, and thus, most likely the findings mainly represent the transcriptome of the dominant cell type (luminal epithelial cells) yielding a superficial analysis of the functions of mammary gland components. The basal epithelial cells represent a generally overlooked population of the breast tissue. Their major function during lactation is to contract to eject milk through the ducts. Recent studies emphasize the importance of basal cells in regulating branching morphogenesis, epithelial cell differentiation and for their innate tumor suppressor activity. Yet, many of the molecular mechanisms regulating basal cell function during development remain largely unknown. To characterize the molecular mechanisms governing cell proliferation, morphogenesis and differentiation during this process, we combined fluorescence activated cell sorting (FAGS) with RNA-sequencing to profile the transcriptome of enriched luminal and basal epithelial cells during all stages of post-natal mammary gland development. We used this dataset to (i) validate our novel approach for enriching these cell populations based on known cell subtype-specific markers; (ii) identify functional spatio-temporal expression differences; and (iii) expand upon a pathway known to be essential for the contractile function in the basal cells. The spatio-temporal analysis confirmed interactions of genes from known pathways, but also revealed a new gene set to build upon these known pathways. The results uncovered an unprecedented complexity of basal cells in terms of dynamic gene expression changes and allowed for the identification of novel pathways associated with basal epithelial cell function during all stages of pregnancy, lactation and involution. Overall, these findings provide an improved model for basal cell responses during mammary gland development, reveal genes that may be essential to lactogenesis and provide novel markers for functional validation in normal mammary gland development and breast tumorigenesis.