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.

Whole-Mount In Situ Hybridization for Detection of Migrating Zebrafish Endodermal Cells.

One of the most important events in early vertebrate development is the formation and positioning of the endoderm, the embryonic progenitor cell population that gives rise to the internal organs. Recent years have seen renewed interest in the mechanisms underlying the specification and migration of endodermal progenitor cells. The zebrafish is a well-established, accessible, and powerful model to study this cell population. Zebrafish endodermal cells are specified around 4 h after fertilization and subsequently migrate as evenly spaced single cells in a stereotypical manner in the next 6 h. Given the large numbers of fertilized eggs that can be obtained from a single breeding pair and the ease of chemical and genetic perturbations, the zebrafish is an excellent model to study mechanisms underlying endoderm specification and migration. An easy approach to visualizing and quantitating endodermal cells and their migratory routes is by whole-mount in situ hybridization (WISH) on fixed embryos, collected in time series. This chapter provides basic information on the organization and staging of the embryos, with an emphasis on the migrating endodermal cell population. In addition, optimized protocols for the isolation and fixation of staged embryos are provided as well as detailed probe synthesis and WISH protocols, specific for migrating endoderm. Finally, details are provided on how to approach these experiments quantitatively, and some common pitfalls are discussed.

The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

Long-Range Signaling Activation and Local Inhibition Separate the Mesoderm and Endoderm Lineages.

Specification of the three germ layers by graded Nodal signaling has long been seen as a paradigm for patterning through a single morphogen gradient. However, by exploiting the unique properties of the zebrafish embryo to capture the dynamics of signaling and cell fate allocation, we now demonstrate that Nodal functions in an incoherent feedforward loop, together with Fgf, to determine the pattern of endoderm and mesoderm specification. We show that Nodal induces long-range Fgf signaling while simultaneously inducing the cell-autonomous Fgf signaling inhibitor Dusp4 within the first two cell tiers from the margin. The consequent attenuation of Fgf signaling in these cells allows specification of endoderm progenitors, while the cells further from the margin, which receive Nodal and/or Fgf signaling, are specified as mesoderm. This elegant model demonstrates the necessity of feedforward and feedback interactions between multiple signaling pathways for providing cells with temporal and positional information.

A Temporal Window for Signal Activation Dictates the Dimensions of a Nodal Signaling Domain.

Morphogen signaling is critical for the growth and patterning of tissues in embryos and adults, but how morphogen signaling gradients are generated in tissues remains controversial. The morphogen Nodal was proposed to form a long-range signaling gradient via a reaction-diffusion system, on the basis of differential diffusion rates of Nodal and its antagonist Lefty. Here we use a specific zebrafish Nodal biosensor combined with immunofluorescence for phosphorylated Smad2 to demonstrate that endogenous Nodal is unlikely to diffuse over a long range. Instead, short-range Nodal signaling activation in a temporal window is sufficient to determine the dimensions of the Nodal signaling domain. The size of this temporal window is set by the differentially timed production of Nodal and Lefty, which arises mainly from repression of Lefty translation by the microRNA miR-430. Thus, temporal information is transformed into spatial information to define the dimensions of the Nodal signaling domain and, consequently, to specify mesendoderm.

Epigenetic regulation of galectin-3 expression by ß1 integrins promotes cell adhesion and migration.

Introduction of the integrin ß1- but not the ß3-subunit in GE11 cells induces an epithelial-to-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the ß1-induced phenotype. We examined 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by ß1 but not by ß3. Using ß1-ß3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of ß1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of ß1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances ß1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes ß1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and ß1 integrins. In conclusion, we identify a functional feedback-loop between ß1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.

Lysyl oxidase-like 3b is critical for cartilage maturation during zebrafish craniofacial development.

Vertebrate craniofacial development requires coordinated morphogenetic interactions between the extracellular matrix (ECM) and the differentiating chondrocytes essential for cartilage formation. Recent studies reveal a critical role for specific lysyl oxidases in ECM integrity required for embryonic development. We now demonstrate that loxl3b is abundantly expressed within the head mesenchyme of the zebrafish and is critically important for maturation of neural crest derived cartilage elements. Histological and ultrastructural analyses of cartilage elements in loxl3b morphant embryos reveal abnormal maturation of cartilage and altered chondrocyte morphology. Spatiotemporal analysis of craniofacial markers in loxl3b morphant embryos shows that cranial neural crest cells migrate normally into the developing pharyngeal arches but that differentiation and condensation markers are aberrantly expressed. We further show that the loxl3b morphant phenotype is not due to P53 mediated cell death but likely to be due to reduced chondrogenic progenitor cell proliferation within the pharyngeal arches. Taken together, these data demonstrate a novel role for loxl3b in the maturation of craniofacial cartilage and can provide new insight into the specific genetic factors important in the pathogenesis of craniofacial birth defects.

Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity.

Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

Transcriptional changes in somatic cells recovered from embryonic stem-somatic heterokaryons.

Following fusion, embryonic stem cells (ESCs) are capable of reprogramming somatic cells in cell hybrids. It has also been shown that transcriptional changes can occur in a heterokaryon, without nuclear hybridization. However, it is unclear whether these changes can be sustained after the removal of the dominant nucleus. In this study, we analyze the changes in embryonic stem (ES)-somatic heterokaryons following the removal of the ESCs nucleus. We also show that after ES-somatic cell fusion using tetraploid ESCs, a heterokaryon can be reverted to an autologous diploid state by differential enucleation of the denser tetraploid ES nucleus. To recover somatic cells from ES-somatic heterokaryons, we fused tetraploid ESCs containing the thymidine kinase (TK) suicide gene with mesenchymal stem cells containing a green fluorescent protein (GFP) transgene under the control of the OCT4 promoter. Following post-fusion enucleation (PFE), negative selection against the tetraploid ES genome was achieved using ganciclovir. The resulting GFP-positive clones were analyzed and shown to have undergone changes in growth characteristics, alkaline phosphatase activity, and gene expression using RT-PCR and microarray analysis. These results demonstrate that a change in transcriptional expression can be detected in somatic cells after the removal of the ES nucleus from ES-somatic heterokaryons.

Microarray analysis reveals a mechanism of phenolic polybrominated diphenylether toxicity in zebrafish.

Polybrominated diphenylethers (PBDEs) are ubiquitous in the environment, with the lower brominated congener 2,2',4,4'-tetrabromodiphenylether (BDE47) among the most prevalent. The phenolic PBDE, 6-hydroxy-BDE47 (6-OH-BDE47) is both an important metabolite formed by in vivo metabolism of BDE47 and a natural product produced by marine organisms such as algae. Although this compound has been detected in humans and wildlife, including fish, virtually nothing is known of its in vivo toxicity. Here we report that 6-OH-BDE47 is acutely toxic in developing and adult zebrafish at concentrations in the nanomolar (nM) range. To identify possible mechanisms of toxicity, we used microarray analysis as a diagnostic tool. Zebrafish embryonic fibroblast (PAC2) cells were exposed to 6-OH-BDE47, BDE47, and the methoxylated metabolite 6-MeO-BDE47. These experiments revealed that 6-OH-BDE47 alters the expression of genes involved in proton transport and carbohydrate metabolism. These findings, combined with the acute toxicity, suggested that 6-OH-BDE47 causes disruption of oxidative phosphorylation (OXPHOS).Therefore, we further investigated the effect of 6-OH-BDE47 on OXPHOS in zebrafish mitochondria. Results show unequivocally that this compound is a potent uncoupler of OXPHOS and is an inhibitor of complex II of the electron transport chain. This study provides the first evidence of the in vivo toxicity and an important potential mechanism of toxicity of an environmentally relevant phenolic PBDE of both anthropogenic and natural origin. The results of this study emphasize the need for further investigation on the presence and toxicity of this class of polybrominated compounds.

A novel method for somatic cell nuclear transfer to mouse embryonic stem cells.

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.