Division of Molecular Pathology, Netherlands Cancer Institute, Oncode Institute, Plesmanlaan 121, 1066CX, Amsterdam, The Netherlands.
VIB-KULeuven Center for Cancer Biology, Herestraat 49, 3000, Leuven, Belgium.
J Mammary Gland Biol Neoplasia. 2021 Mar;26(1):9-27. doi: 10.1007/s10911-021-09487-2. Epub 2021 May 4.
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.
小鼠乳腺的结构具有高度动态性，在青春期发育和动情周期驱动的肺泡侧支发芽和退化过程中不断重塑。在这些发育阶段中的每一个阶段，细胞更新都由乳腺上皮细胞的不同亚群驱动。大量先前的研究揭示了每个阶段独特的形态和细胞生物学特征。然而，技术上的缺陷使得无法捕捉到乳腺重塑的动态和单细胞贡献。在这里，我们开发了体内成像策略，以跟踪同一乳腺导管随时间的变化，并从单细胞水平到器官水平量化乳腺生长和重塑的动力学。我们结合使用活体显微镜和遗传报告基因系统，展示了增殖异质性如何在不同发育阶段驱动导管形态发生。为了在细胞水平上可视化青春期生长，我们通过乳腺成像窗口对延伸的终末芽进行了长期延时成像。我们表明，终末芽内的单个细胞非常活跃，并在导管伸长的同时不断交换位置。为了在单细胞水平上可视化成年乳腺的短期重塑，我们在光可转换的 Kikume Green-Red 小鼠和荧光泛素化细胞周期标志物小鼠中进行了多日活体成像。我们证明，在同一微环境中，单个细胞对动情周期驱动的重塑的贡献具有高度的可变性。为了评估这种动态增殖贡献对组织结构长期稳定性的影响，我们开发了一种重复皮肤瓣方法，通过活体显微镜评估乳腺形态，时间跨度长达六个月。有趣的是，与短期动态重塑相反，乳腺的长期形态仍然非常稳定。总之，我们的成像策略工具箱允许将短暂和持续的单细胞动力学识别并映射到乳腺的结构上。
