RESUMEN

Quantitative live imaging is a valuable tool that offers insights into cellular dynamics. However, many fundamental biological processes are incompatible with current live imaging modalities. Drosophila oogenesis is a well-studied system that has provided molecular insights into a range of cellular and developmental processes. The length of the oogenesis coupled with the requirement for inputs from multiple tissues has made long-term culture challenging. Here, we have developed Bellymount-Pulsed Tracking (Bellymount-PT), which allows continuous, non-invasive live imaging of Drosophila oogenesis inside the female abdomen for up to 16 hours. Bellymount-PT improves upon the existing Bellymount technique by adding pulsed anesthesia with periods of feeding that support the long-term survival of flies during imaging. Using Bellymount-PT we measure key events of oogenesis including egg chamber growth, yolk uptake, and transfer of specific proteins to the oocyte during nurse cell dumping with high spatiotemporal precision within the abdomen of a live female.

RESUMEN

The thirteenth annual workshop of the European Network for Breast Development and Cancer (ENBDC) Laboratories Annual Workshop took place on the 28-30 April 2022 in Weggis, Switzerland and focused on methods in mammary gland biology and breast cancer. Sixty scientists participated in the ENBDC annual workshop which had not been held in person since 2019 due to the global COVID-19 pandemic. Topics spanned the mammary gland biology field, ranging from lactation biology and embryonic development, single cell sequencing of the human breast, and stunning cutting-edge imaging of the mouse mammary gland and human breast as well as breast cancer research topics including invasive progression of the pre-invasive DCIS stage, metabolic determinants of endocrine therapy resistance, models for lobular breast cancer, and how mutational landscapes of normal breast during age and pregnancy determine cancer risk. The latest findings from participating researchers were presented through oral presentations and poster sessions and included plenty of unpublished work.

Asunto(s)

RESUMEN

Solar ultraviolet radiation (UVR) is a major source of skin damage, resulting in inflammation, premature ageing, and cancer. While several UVR-induced changes, including extracellular matrix reorganisation and epidermal DNA damage, have been documented, the role of different fibroblast lineages and their communication with immune cells has not been explored. We show that acute and chronic UVR exposure led to selective loss of fibroblasts from the upper dermis in human and mouse skin. Lineage tracing and in vivo live imaging revealed that repair following acute UVR is predominantly mediated by papillary fibroblast proliferation and fibroblast reorganisation occurs with minimal migration. In contrast, chronic UVR exposure led to a permanent loss of papillary fibroblasts, with expansion of fibroblast membrane protrusions partially compensating for the reduction in cell number. Although UVR strongly activated Wnt signalling in skin, stimulation of fibroblast proliferation by epidermal ß-catenin stabilisation did not enhance papillary dermis repair. Acute UVR triggered an infiltrate of neutrophils and T cell subpopulations and increased pro-inflammatory prostaglandin signalling in skin. Depletion of CD4- and CD8-positive cells resulted in increased papillary fibroblast depletion, which correlated with an increase in DNA damage, pro-inflammatory prostaglandins, and reduction in fibroblast proliferation. Conversely, topical COX-2 inhibition prevented fibroblast depletion and neutrophil infiltration after UVR. We conclude that loss of papillary fibroblasts is primarily induced by a deregulated inflammatory response, with infiltrating T cells supporting fibroblast survival upon UVR-induced environmental stress.

Asunto(s)

RESUMEN

The twelfth annual workshop of the European Network for Breast Development and Cancer focused on methods in mammary gland biology and breast cancer, was scheduled to take place on March 26-28, 2020, in Weggis, Switzerland. Due to the COVID-19 pandemic, the meeting was rescheduled twice and eventually happened as a virtual meeting on April 22 and 23, 2021. The main topics of the meeting were branching and development of the mammary gland, tumor microenvironment, circulating tumor cells, tumor dormancy and breast cancer metastasis. Novel and unpublished findings related to these topics were presented, with a particular focus on the methods used to obtain them. Virtual poster sessions were a success, with many constructive and fruitful interactions between researchers and covered many areas of mammary gland biology and breast cancer.

Asunto(s)

RESUMEN

Epithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell-cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT-and the reverse mesenchymal-epithelial transition (MET)-are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell-cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos-gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos-that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer's vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET. Video Abstract.

Asunto(s)

RESUMEN

BACKGROUND & AIMS: The reason why small intestinal cancer is rarer than colorectal cancer is not clear. We hypothesized that intraepithelial lymphocytes (IELs), which are enriched in the small intestine, are the closest immune cells to epithelial cells, exclude tumor cells via cell-to-cell contact. METHODS: We developed DPE-green fluorescent protein (DPE-GFP) × adenomatous polyposis coli; multiple intestinal neoplasia (APCmin ) mice, which is a T-cell-reporter mouse with spontaneous intestinal tumors. We visualized the dynamics of IELs in the intestinal tumor microenvironment and the interaction between IELs and epithelial cells, and the roles of cell-to-cell contact in anti-intestinal tumor immunity using a novel in vivo live-imaging system and a novel in vitro co-culture system. RESULTS: In the small intestinal tumor microenvironment, T-cell movement was restricted around blood vessels and the frequency of interaction between IELs and epithelial cells was reduced. Genetic deletion of CD103 decreased the frequency of interaction between IELs and epithelial cells, and increased the number of small intestinal tumors. In the co-culture system, wild-type IELs expanded and infiltrated to intestinal tumor organoids from APCmin mice and reduced the viability of them, which was cell-to-cell contact and CD103 dependent. CONCLUSIONS: The abundance of IELs in the small intestine may contribute to a low number of tumors, although this system may not work in the colon because of the sparseness of IELs. Strategies to increase the number of IELs in the colon or enhance cell-to-cell contact between IELs and epithelial cells may be effective for the prevention of intestinal tumors in patients with a high cancer risk.

Asunto(s)

RESUMEN

The study of pathological processes is often limited to in vitro or ex vivo assays, while understanding pathogenesis of an infectious disease requires in vivo analysis. The use of pathogens, genetically modified to express with luminescent enzymes, combined to charge-coupled device (CCD) cameras, constitutes a major technological advance for assessing the course of infection in an intact, living host in real time and in a noninvasive way. This technology, also called bioluminescence imaging, detects the photons emitted from biological sources of light through animal tissues. Here, we describe the method we developed to monitor leptospirosis in a mouse model, by following in a spatiotemporal scale, the dissemination and spread of leptospires. These bacteria have been genetically modified to express the firefly luciferase, which produces light in the presence of the substrate D-luciferin. This useful and accessible technology facilitates the study of the kinetics of blood and tissue dissemination of live leptospires, and the pharmacological impact of treatments and host directed therapeutics.

Asunto(s)

RESUMEN

Visualization of dynamic cellular activity has greatly expanded our understanding of brain function. Recently, there has been an increasing number of studies imaging rodent brain activity in real time. However, traditional in vivo calcium imaging technology has been limited to superficial brain structures. Because the trigeminal ganglion (TG) is located deep within the cranial cavity of mice, few studies have been able to access to it. To circumvent this limitation, overlying brain tissue must be removed to expose the TG so that optical recording can access deep brain neural ensembles. This unit describes a procedure for conducting non-survival surgery to visualize the TG in live mice. Obtaining large ensembles of direct, real-time readouts of sensory neuron signaling, providing temporal and spatial information across the TG, will help to define the cellular basis of orofacial somatic sensing and pain perception. © 2019 by John Wiley & Sons, Inc.

Asunto(s)