Conserved signals control self-organization and symmetry breaking of murine bilayered epithelia during development and regeneration.

Organ development relies on molecular cues that guide stem cells to differentiate within precise spatial arrangements. After injury, restoring these patterns is key for regeneration. Yet, how tissue geometry shapes cell fate remains unclear.

Transcriptional landscapes underlying Notch-induced lineage conversion and plasticity of mammary basal cells.

The mammary epithelium derives from multipotent mammary stem cells (MaSCs) that engage into differentiation during embryonic development. However, adult MaSCs maintain the ability to reactivate multipotency in non-physiological contexts. We previously reported that Notch1 activation in committed basal cells triggers a basal-to-luminal cell fate switch in the mouse mammary gland.

Recording Lineage History with Cellular Barcodes in the Mammary Epithelium and in Breast Cancer.

Lineage tracing methods have extensively advanced our understanding of physiological cell behaviour in vivo and in situ and have vastly contributed to decipher the phylogeny and cellular hierarchies during normal and tumour development. In recent years, increasingly complex systems have been developed to track thousands of cells within a given tissue or even entire organisms. Cellular barcoding comprises all techniques designed to genetically label single cells with unique DNA sequences or with a combination of fluorescent proteins, in order to trace their history and lineage production in space and time.

Morphological and cellular effects in Boana faber tadpoles (Anura: Hylidae) exposed to atrazine-based herbicide and glyphosate-based herbicide and their mixtures.

Atrazine and glyphosate are considered some of the main pollutants for aquatic ecosystems, directly and indirectly affecting non-target organisms, such as amphibians. This study aimed to evaluate the sublethal effects of different concentrations of atrazine-based herbicide (ABH) and glyphosate-based herbicide (GBH) commercial formulations, both individually and in a mixture, through toxicity tests on the larval stage of Boana faber. Tadpoles were exposed to concentrations of ABH (2, 9.

15th Annual ENBDC Meeting: How do Cellular Potency, Microenvironment and Natural Rhythms Influence Mammary Gland Biology and Breast Cancer?

Following previous editions, the fifteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) on Methods in Mammary Gland Biology and Breast Cancer was held from the 2nd to the 4th of May in Weggis, Switzerland. Over the course of this meeting, participants followed and discussed presentations from a roster of internationally renowned invited speakers and selected abstracts, complemented with two poster sessions covering exciting unpublished results. The sessions covered projects on normal mammary gland development, breast cancer evolution and metastasis, as well as epigenetic and metabolic regulation of breast cancer.

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion.

Background: The aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell-matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions.

Spatially distinct epithelial and mesenchymal cell subsets along progressive lineage restriction in the branching embryonic mammary gland.

How cells coordinate morphogenetic cues and fate specification during development remains a fundamental question in organogenesis. The mammary gland arises from multipotent stem cells (MaSCs), which are progressively replaced by unipotent progenitors by birth. However, the lack of specific markers for early fate specification has prevented the delineation of the features and spatial localization of MaSC-derived lineage-committed progenitors.

Extracting, filtering and simulating cellular barcodes using CellBarcode tools.

Identifying true DNA cellular barcodes among polymerase chain reaction and sequencing errors is challenging. Current tools are restricted in the diversity of barcode types supported or the analysis strategies implemented. As such, there is a need for more versatile and efficient tools for barcode extraction, as well as for tools to investigate which factors impact barcode detection and which filtering strategies to best apply.

Fibroblast-induced mammary epithelial branching depends on fibroblast contractility.

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching.

Fourteenth Annual ENBDC Workshop: Methods in Mammary Gland Biology and Breast Cancer.

The fourteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) on Methods in Mammary Gland Biology and Breast Cancer was held on April 26th - 29th in Weggis, Switzerland. For the first time, early career researchers organised and took part in an additional ECR workshop on the 26th of April, which was received with great enthusiasm. The topics of the main workshop included mammary branching and morphogenesis, novel experimental systems (model organisms), systemic influences on tumour progression and the tumour microenvironment.

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.

DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age.

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche.

Toxicity and genotoxicity of imidacloprid in the tadpoles of Leptodactylus luctator and Physalaemus cuvieri (Anura: Leptodactylidae).

Imidacloprid is a neonicotinoid insecticide used to control agricultural pests around the world. This pesticide can have adverse effects on non-target organisms, especially in aquatic environments. The present study evaluated the toxicity of an imidacloprid-based insecticide in amphibians, using Leptodactylus luctator and Physalaemus cuvieri tadpoles as study models.

Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme.

Tumours are complex ecosystems composed of different types of cells that communicate and influence each other. While the critical role of stromal cells in affecting tumour growth is well established, the impact of mutant cancer cells on healthy surrounding tissues remains poorly defined. Here, using mouse intestinal organoids, we uncover a paracrine mechanism by which intestinal cancer cells reactivate foetal and regenerative YAP-associated transcriptional programmes in neighbouring wildtype epithelial cells, rendering them adapted to thrive in the tumour context.

In vivo imaging of mammary epithelial cell dynamics in response to lineage-biased Wnt/β-catenin activation.

Real-time in vivo imaging provides an essential window into the spatiotemporal cellular events contributing to tissue development and pathology. By coupling longitudinal intravital imaging with genetic lineage tracing, here we capture the earliest cellular events arising in response to active Wnt/β-catenin signaling and the ensuing impact on the organization and differentiation of the mammary epithelium. This enables us to interrogate how Wnt/β-catenin regulates the dynamics of distinct subpopulations of mammary epithelial cells in vivo and in real time.

Lineage Tracing Methods to Study Mammary Epithelial Hierarchies In Vivo.

Lineage tracing is now considered the gold standard approach to study cellular hierarchies and cell fate in vivo (McKenna and Gagnon, Development 146:dev169730, 2019; Kretzschmar and Watt, Cell 148:33-45, 2012). This type of clonal analysis consists of genetically labeling defined cells and following their destiny and progeny in vivo and in situ.Here we will describe different existing in vivo systems to clonally trace targeted cells and will discuss their respective advantages and inconveniences; we will then provide stepwise instructions for setting up and evaluate lineage tracing experiments, listing the most common downstream analyses and read-out assays.

Longitudinal high-resolution imaging through a flexible intravital imaging window.

Intravital microscopy (IVM) is a powerful technique that enables imaging of internal tissues at (sub)cellular resolutions in living animals. Here, we present a silicone-based imaging window consisting of a fully flexible, sutureless design that is ideally suited for long-term, longitudinal IVM of growing tissues and tumors. Crucially, we show that this window, without any customization, is suitable for numerous anatomical locations in mice using a rapid and standardized implantation procedure.

Morphological, behavioral and genotoxic effects of glyphosate and 2,4-D mixture in tadpoles of two native species of South American amphibians.

Pesticide contamination is an important factor in the global decline of amphibians. The herbicides glyphosate and 2,4-D are the most applied worldwide. These herbicides are often found in surface waters close to agricultural areas.

Notch signalling: sensor and instructor of the microenvironment to coordinate cell fate and organ morphogenesis.

During development, stem cells give rise to specialised cell types in a tightly regulated, spatiotemporal manner to drive the formation of complex three-dimensional tissues. While mechanistic insights into the gene regulatory pathways that guide cell fate choices are emerging, how morphogenetic changes are coordinated with cell fate specification remains a fundamental question in organogenesis and adult tissue homeostasis. The requirement of cell contacts for Notch signalling makes it a central pathway capable of linking dynamic cellular rearrangements during tissue morphogenesis with stem cell function.

Lineage tracing of Notch1-expressing cells in intestinal tumours reveals a distinct population of cancer stem cells.

Colon tumours are hierarchically organized and contain multipotent self-renewing cells, called Cancer Stem Cells (CSCs). We have previously shown that the Notch1 receptor is expressed in Intestinal Stem Cells (ISCs); given the critical role played by Notch signalling in promoting intestinal tumourigenesis, we explored Notch1 expression in tumours. Combining lineage tracing in two tumour models with transcriptomic analyses, we found that Notch1+ tumour cells are undifferentiated, proliferative and capable of indefinite self-renewal and of generating a heterogeneous clonal progeny.

Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.

Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis.

Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis.

Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure.

The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch(+)Apc(+/1638N) mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and β-catenin signalling.

Luminal progenitors restrict their lineage potential during mammary gland development.

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing.