Abstract

BackgroundHeterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty.MethodsThe single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points.ResultsThe mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states.ConclusionsThis extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

Highlights

  • The mammary epithelium undergoes remarkable remodeling during the different stages of postnatal morphogenesis [1, 2]

  • As the developing mammary gland is an ectodermal appendage closely associated with skin, we isolated adjacent skin to provide a control for epithelial purification

  • Comparison of scRNA-seq expression profiles of different adult strains As different mouse strains have been used in the field for scRNA-seq studies, we investigated the level of similarity across them by determining the single cell transcriptomes of adult mammary epithelial cells derived from three different strains: C57BL/6, FVB/N and SWISS (CD1-like)

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Summary

Introduction

The mammary epithelium undergoes remarkable remodeling during the different stages of postnatal morphogenesis [1, 2]. The ductal tree elongates and bifurcates to form an extensive ductal network that fills the mammary fat pad. This process is driven by terminal end buds (TEBs), which are located at the termini of the growing ducts [3]. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty

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