ABSTRACT
The mammary gland consists of an adipose tissue that, in a process called branching morphogenesis, is invaded by a ductal epithelial network comprising basal and luminal epithelial cells. Stem and progenitor cells drive mammary growth, and their proliferation is regulated by multiple extracellular cues. One of the key regulatory pathways for these cells is the ß-catenin-dependent, canonical wingless-type MMTV integration site family (WNT) signaling pathway; however, the role of noncanonical WNT signaling within the mammary stem/progenitor system remains elusive. Here, we focused on the noncanonical WNT receptors receptor tyrosine kinase-like orphan receptor 2 (ROR2) and receptor-like tyrosine kinase (RYK) and their activation by WNT5A, one of the hallmark noncanonical WNT ligands, during mammary epithelial growth and branching morphogenesis. We found that WNT5A inhibits mammary branching morphogenesis in vitro and in vivo through the receptor tyrosine kinase ROR2. Unexpectedly, WNT5A was able to enhance mammary epithelial growth, which is in contrast to its next closest relative WNT5B, which potently inhibits mammary stem/progenitor proliferation. We found that RYK, but not ROR2, is necessary for WNT5A-mediated promotion of mammary growth. These findings provide important insight into the biology of noncanonical WNT signaling in adult stem/progenitor cell regulation and development. Future research will determine how these interactions go awry in diseases such as breast cancer.
Subject(s)
Epithelium/metabolism , Mammary Glands, Animal/metabolism , Morphogenesis , Wnt Signaling Pathway , Amino Acid Sequence , Animals , Cell Differentiation/genetics , Cell Proliferation/genetics , Female , Gene Expression Regulation , Mammary Glands, Animal/cytology , Mammary Glands, Animal/growth & development , Mice , Mice, Knockout , Morphogenesis/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Receptors, Wnt/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt-5a Protein/genetics , Wnt-5a Protein/metabolismABSTRACT
Extracellular vesicle (EV) transfer is increasingly recognized as an important mode of intercellular communication by transferring a wide variety of biomolecules between cells. The characterization of in vitro- or ex vivo-isolated EVs has considerably contributed to the understanding of biological functions of EV transfer. However, the study of EV release and uptake in an in vivo setting has remained challenging, because cells that take up EVs could not be discriminated from cells that do not take up EVs. Recently, a technique based on the Cre-loxP system was developed to fluorescently mark Cre-reporter cells that take up EVs released by Cre recombinase-expressing cells in various in vitro and in vivo settings. Here we describe a detailed protocol for the generation of Cre(+) cells and reporter(+) cells, which takes â¼ 6 weeks, and subsequent assays with these lines to study functional EV transfer in in vitro and in vivo (mouse) settings, which take up to â¼ 2 months.
