Transcriptomic Analysis of Pubertal and Adult Virgin Mouse Mammary Epithelial and Stromal Cell Populations.

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and fibroblasts. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

A molecular toolbox to study progesterone receptor signaling.

Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells.

Imagine beyond: recent breakthroughs and next challenges in mammary gland biology and breast cancer research.

On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward.

Visualizing WNT signaling in mammalian systems.

WNT/CTNNB1 signaling plays a critical role in the development of all multicellular animals. Here, we include both the embryonic stages, during which tissue morphogenesis takes place, and the postnatal stages of development, during which tissue homeostasis occurs. Thus, embryonic development concerns lineage development and cell fate specification, while postnatal development involves tissue maintenance and regeneration. Multiple tools are available to researchers who want to investigate, and ideally visualize, the dynamic and pleiotropic involvement of WNT/CTNNB1 signaling in these processes. Here, we discuss and evaluate the decisions that researchers need to make in identifying the experimental system and appropriate tools for the specific question they want to address, covering different types of WNT/CTNNB1 reporters in cells and mice. At a molecular level, advanced quantitative imaging techniques can provide spatio-temporal information that cannot be provided by traditional biochemical assays. We therefore also highlight some recent studies to show their potential in deciphering the complex and dynamic mechanisms that drive WNT/CTNNB1 signaling.

Convergent deployment of ancestral functions during the evolution of mammalian flight membranes.

Lateral flight membranes, or patagia, have evolved repeatedly in diverse mammalian lineages. While little is known about patagium development, its recurrent evolution may suggest a shared molecular basis. By combining transcriptomics, developmental experiments, and mouse transgenics, we demonstrate that lateral Wnt5a expression in the marsupial sugar glider (Petaurus breviceps) promotes the differentiation of its patagium primordium. We further show that this function of Wnt5a reprises ancestral roles in skin morphogenesis predating mammalian flight and has been convergently used during patagium evolution in eutherian bats. Moreover, we find that many genes involved in limb development have been redeployed during patagium outgrowth in both the sugar glider and bat. Together, our findings reveal that deeply conserved genetic toolkits contribute to the evolutionary transition to flight in mammals.

Zooming in on the WNT/CTNNB1 Destruction Complex: Functional Mechanistic Details with Implications for Therapeutic Targeting.

WNT/CTNNB1 signaling is crucial for balancing cell proliferation and differentiation in all multicellular animals. CTNNB1 accumulation is the hallmark of WNT/CTNNB1 pathway activation and the key downstream event in both a physiological and an oncogenic context. In the absence of WNT stimulation, the cytoplasmic and nuclear levels of CTNNB1 are kept low because of its sequestration and phosphorylation by the so-called destruction complex, which targets CTNNB1 for proteasomal degradation. In the presence of WNT proteins, or as a result of oncogenic mutations, this process is impaired and CTNNB1 levels become elevated.Here we discuss recent advances in our understanding of destruction complex activity and inactivation, focusing on the individual components and interactions that ultimately control CTNNB1 turnover (in the "WNT off" situation) and stabilization (in the "WNT on" situation). We especially highlight the insights gleaned from recent quantitative, image-based studies, which paint an unprecedentedly detailed picture of the dynamic events that control destruction protein complex composition and function. We argue that these mechanistic details may reveal new opportunities for therapeutic intervention and could result in the destruction complex re-emerging as a target for therapy in cancer.

Quantitative live-cell imaging and computational modeling shed new light on endogenous WNT/CTNNB1 signaling dynamics.

WNT/CTNNB1 signaling regulates tissue development and homeostasis in all multicellular animals, but the underlying molecular mechanism remains incompletely understood. Specifically, quantitative insight into endogenous protein behavior is missing. Here, we combine CRISPR/Cas9-mediated genome editing and quantitative live-cell microscopy to measure the dynamics, diffusion characteristics and absolute concentrations of fluorescently tagged, endogenous CTNNB1 in human cells under both physiological and oncogenic conditions. State-of-the-art imaging reveals that a substantial fraction of CTNNB1 resides in slow-diffusing cytoplasmic complexes, irrespective of the activation status of the pathway. This cytoplasmic CTNNB1 complex undergoes a major reduction in size when WNT/CTNNB1 is (hyper)activated. Based on our biophysical measurements, we build a computational model of WNT/CTNNB1 signaling. Our integrated experimental and computational approach reveals that WNT pathway activation regulates the dynamic distribution of free and complexed CTNNB1 across different subcellular compartments through three regulatory nodes: the destruction complex, nucleocytoplasmic shuttling, and nuclear retention.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

Walking the tight wire between cell adhesion and WNT signalling: a balancing act for ß-catenin.

CTNNB1 (catenin ß-1, also known as ß-catenin) plays a dual role in the cell. It is the key effector of WNT/CTNNB1 signalling, acting as a transcriptional co-activator of TCF/LEF target genes. It is also crucial for cell adhesion and a critical component of cadherin-based adherens junctions. Two functional pools of CTNNB1, a transcriptionally active and an adhesive pool, can therefore be distinguished. Whether cells merely balance the distribution of available CTNNB1 between these functional pools or whether interplay occurs between them has long been studied and debated. While interplay has been indicated upon artificial modulation of cadherin expression levels and during epithelial-mesenchymal transition, it is unclear to what extent CTNNB1 exchange occurs under physiological conditions and in response to WNT stimulation. Here, we review the available evidence for both of these models, discuss how CTNNB1 binding to its many interaction partners is controlled and propose avenues for future studies.

A novel Axin2 knock-in mouse model for visualization and lineage tracing of WNT/CTNNB1 responsive cells.

Wnt signal transduction controls tissue morphogenesis, maintenance and regeneration in all multicellular animals. In mammals, the WNT/CTNNB1 (Wnt/ß-catenin) pathway controls cell proliferation and cell fate decisions before and after birth. It plays a critical role at multiple stages of embryonic development, but also governs stem cell maintenance and homeostasis in adult tissues. However, it remains challenging to monitor endogenous WNT/CTNNB1 signaling dynamics in vivo. Here, we report the generation and characterization of a new knock-in mouse strain that doubles as a fluorescent reporter and lineage tracing driver for WNT/CTNNB1 responsive cells. We introduced a multi-cistronic targeting cassette at the 3' end of the universal WNT/CTNNB1 target gene Axin2. The resulting knock-in allele expresses a bright fluorescent reporter (3xNLS-SGFP2) and a doxycycline-inducible driver for lineage tracing (rtTA3). We show that the Axin2P2A-rtTA3-T2A-3xNLS-SGFP2 strain labels WNT/CTNNB1 responsive cells at multiple anatomical sites during different stages of embryonic and postnatal development. It faithfully reports the subtle and dynamic changes in physiological WNT/CTNNB1 signaling activity that occur in vivo. We expect this mouse strain to be a useful resource for biologists who want to track and trace the location and developmental fate of WNT/CTNNB1 responsive stem cells in different contexts.

Celebrating Discoveries in Wnt Signaling: How One Man Gave Wings to an Entire Field.

This year's Gairdner Foundation Award for Biomedical Research goes to Roel Nusse for his pioneering work on the Wnt signaling pathway and its many roles in development, cancer, and stem cells.

Aberrant WNT/CTNNB1 Signaling as a Therapeutic Target in Human Breast Cancer: Weighing the Evidence.

WNT signaling is crucial for tissue morphogenesis during development in all multicellular animals. After birth, WNT/CTNNB1 responsive stem cells are responsible for tissue homeostasis in various organs and hyperactive WNT/CTNNB1 signaling is observed in many different human cancers. The first link between WNT signaling and breast cancer was established almost 40 years ago, when Wnt1 was identified as a proto-oncogene capable of driving mammary tumor formation in mice. Since that discovery, there has been a dedicated search for aberrant WNT signaling in human breast cancer. However, much debate and controversy persist regarding the importance of WNT signaling for the initiation, progression or maintenance of different breast cancer subtypes. As the first drugs designed to block functional WNT signaling have entered clinical trials, many questions about the role of aberrant WNT signaling in human breast cancer remain. Here, we discuss three major research gaps in this area. First, we still lack a basic understanding of the function of WNT signaling in normal human breast development and physiology. Second, the overall extent and precise effect of (epi)genetic changes affecting the WNT pathway in different breast cancer subtypes are still unknown. Which underlying molecular and cell biological mechanisms are disrupted as a result also awaits further scrutiny. Third, we survey the current status of targeted therapeutics that are aimed at interfering with the WNT pathway in breast cancer patients and highlight the importance and complexity of selecting the subset of patients that may benefit from treatment.

TMEM98 is a negative regulator of FRAT mediated Wnt/ß-catenin signalling.

Wnt/ß-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and in tissue maintenance throughout postnatal life. Whereas the signalling activities of the core Wnt/ß-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/ß-catenin signalling that have been identified to date. Here we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of ß-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/ß-catenin signalling, but also a new biological activity for TMEM98.

How to Use Online Tools to Generate New Hypotheses for Mammary Gland Biology Research: A Case Study for Wnt7b.

An increasing number of '-omics' datasets, generated by labs all across the world, are becoming available. They contain a wealth of data that are largely unexplored. Not every scientist, however, will have access to the required resources and expertise to analyze such data from scratch. Fortunately, a growing number of investigators is dedicating their time and effort to the development of user friendly, online applications that allow researchers to use and investigate these datasets. Here, we will illustrate the usefulness of such an approach. Using regulation of Wnt7b expression as an example, we will highlight a selection of accessible tools and resources that are available to researchers in the area of mammary gland biology. We show how they can be used for in silico analyses of gene regulatory mechanisms, resulting in new hypotheses and providing leads for experimental follow up. We also call out to the mammary gland community to join forces in a coordinated effort to generate and share additional tissue-specific '-omics' datasets and thereby expand the in silico toolbox.

Wnt signalling: conquering complexity.

The history of the Wnt pathway is an adventure that takes us from mice and flies to frogs, zebrafish and beyond, sketching the outlines of a molecular signalling cascade along the way. Here, we specifically highlight the instrumental role that developmental biology has played throughout. We take the reader on a journey, starting with developmental genetics studies that identified some of the main molecular players, through developmental model organisms that helped unravel their biochemical function and cell biological activities. Culminating in complex analyses of stem cell fate and dynamic tissue growth, these efforts beautifully illustrate how different disciplines provided missing pieces of a puzzle. Together, they have shaped our mechanistic understanding of the Wnt pathway as a conserved signalling process in development and disease. Today, researchers are still uncovering additional roles for Wnts and other members of this multifaceted signal transduction pathway, opening up promising new avenues for clinical applications.

Lineage Tracing of Mammary Stem and Progenitor Cells.

Lineage tracing analysis allows mammary epithelial cells to be tracked in their natural environment, thereby revealing cell fate and proliferation choices in the intact tissue. This technique is particularly informative for studying how stem cells build and maintain the mammary epithelium during development and pregnancy. Here we describe two experimental systems based on Cre/loxP technology (CreERT2/loxP and rtTA/tetO-Cre/loxP), which allow the inducible, permanent labeling of mammary epithelial cells following the administration of either tamoxifen or doxycycline.

Complexity galore: 3D cultures, biomechanics and systems medicine at the eighth ENBDC workshop "Methods in Mammary Gland Development and Cancer".

The ENBDC workshop "Methods in Mammary Gland Development and Cancer" is an established international forum to showcase the latest technical advances in the field. The eighth meeting focused on emerging concepts and technologies for studying normal and neoplastic breast development.