Abstract
The architecture of the mouse mammary gland is highly dynamic and constantly remodeled during pubertal development and estrous cycle-driven sprouting and regression of alveolar side branches. During each of these developmental stages, turnover is driven by distinct subsets of mammary epithelial cells. Extensive previous research has shed light on the unique morphological and cell biological characteristics of each stage. However, technological shortcomings failed to capture the dynamics and single-cell contributions to mammary remodeling. Here, we developed in vivo imaging strategies to follow the same mammary ducts over time and quantify the dynamics of mammary gland growth and remodeling from single-cell level to organ scale. Using a combination of intravital microscopy and genetic reporter systems we show how proliferative heterogeneity drives ductal morphogenesis during different developmental stages. To visualize pubertal growth at the cellular level, we performed long-term time-lapse imaging of extending terminal end buds through a mammary imaging window. We show that single-cells within the terminal end buds are extremely motile and continuously exchange position whilst the duct is elongating. To visualize short-term remodeling in the adult mammary gland at the single cell level, we performed multi-day intravital imaging in photoconvertible Kikume Green–Red mice and fluorescent ubiquitination-based cell cycle indicator mice. We demonstrate that the contribution of single-cells to estrous-driven remodeling is highly variable between cells in the same micro-environment. To assess the effects of this dynamic proliferative contribution on the long-term stability of tissue architecture, we developed a repeated skin flap method to assess mammary gland morphology by intravital microscopy over extended time spans for up to six months. Interestingly, in contrast to the short-term dynamic remodeling, the long-term morphology of the mammary gland remains remarkably stable. Together, our tool box of imaging strategies allows to identify and map transient and continuing dynamics of single cells to the architecture of the mammary gland.
Highlights
The mammary gland is a secretory organ that plays an essential role during the nursing of mammalian offspring, and is organized as a highly branched tree like structure that is1 3 Vol.:(0123456789)Journal of Mammary Gland Biology and Neoplasia (2021) 26:9–27◂Fig. 1 Time-lapse imaging of pubertal mammary gland development. a Cartoon of skin flap imaging workflow in the pubertal mammary gland
To better understand the proliferative heterogeneity at these distinct anatomical locations during the different developmental phases, we developed a toolbox combining intravital microscopy (IVM) techniques with several reporter mouse models to follow proliferation and morphological changes within the mammary gland at different time scales, ranging from a few hours to several months [16]
We established a toolbox for the quantitative analysis of cellular dynamics in the pubertal and adult mammary gland
Summary
The mammary gland is a secretory organ that plays an essential role during the nursing of mammalian offspring, and is organized as a highly branched tree like structure that is1 3 Vol.:(0123456789)Journal of Mammary Gland Biology and Neoplasia (2021) 26:9–27◂Fig. 1 Time-lapse imaging of pubertal mammary gland development. a Cartoon of skin flap imaging workflow in the pubertal mammary gland. To better understand the proliferative heterogeneity at these distinct anatomical locations during the different developmental phases, we developed a toolbox combining intravital microscopy (IVM) techniques with several reporter mouse models to follow proliferation and morphological changes within the mammary gland at different time scales, ranging from a few hours to several months [16]. By using a repeated skin flap approach, we assessed the long-term stability of the mammary gland over multiple weeks to months at the organ level. Together, these diverse IVM techniques provide a comprehensive toolbox to study mammary gland remodeling from the single cell level to the organ scale
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