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MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)., Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region.

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.

Summary: Evidence suggests that a stem-cell-driven differentiation hierarchy maintains the dynamic thymic epithelial cell (TEC) network that governs T lymphocyte development. The identification of TEC stem/progenitor cells has been a major focus in the field, and several candidates with contrasting phenotypes have been described. We sought to determine the provenance and function of the only population reported to exhibit TEC stem cell properties in the adult, a Foxn1− EpCAM− cell that generates so-called thymospheres. We provide evidence that the thymosphere-forming cell (TSFC) is not a TEC stem cell but can incorporate bystander TECs into thymospheres, providing an explanation for the epithelial activity ascribed to these structures. TSFCs were found to share a phenotype, transcriptional profile, and developmental origin with thymic fibroblasts and can generate adipocytes. In summary, this study redefines the nature of bipotent TEC stem/progenitor cells in the adult thymus and highlights a potentially important mesenchymal progenitor population. : The phenotype of putative bipotent epithelial stem/progenitor cells in the adult thymus has been controversial. Sheridan et al. find that a prominent candidate, the FoxN1−EpCAM− sphere-forming cell, harbors no epithelial stem/progenitor capacity and present evidence that they are a unique intrathymic mesenchymal stromal/stem cell population with adipocyte differentiation capacity. Keywords: thymus, thymic epithelial cell, stem cell, progenitor, thymosphere, MSC, mesenchyme, adipocyte

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation., LUBAC is a ubiquitin ligase complex of HOIL-1, HOIP and SHARPIN important for signal transduction of a range of stimuli. Here the authors define the function of all three LUBAC components in T cell development and homeostasis.

SUMMARYWe have isolated a mouse strain with a single missense mutation in the gene encoding MLKL, the essential effector of necroptotic cell death. The resulting substitution lies within the two-helix ‘brace’ and confers constitutive, RIPK3 independent, killing activity to MLKL. Mice homozygous forMlklD139Vdevelop lethal inflammation within days of birth, implicating the salivary glands and pericardium as hotspots for necroptosis and inflammatory infiltration. The normal development ofMlklD139Vhomozygotes until birth, and the absence of any overt phenotype in heterozygotes provides importantin vivoprecedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common humanMLKLpolymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, CRMO.

Motivation Gene set enrichment (GSE) analysis allows researchers to efficiently extract biological insight from long lists of differentially expressed genes by interrogating them at a systems level. In recent years, there has been a proliferation of GSE analysis methods and hence it has become increasingly difficult for researchers to select an optimal GSE tool based on their particular dataset. Moreover, the majority of GSE analysis methods do not allow researchers to simultaneously compare gene set level results between multiple experimental conditions. Results The ensemble of genes set enrichment analyses (EGSEA) is a method developed for RNA-sequencing data that combines results from twelve algorithms and calculates collective gene set scores to improve the biological relevance of the highest ranked gene sets. EGSEA’s gene set database contains around 25 000 gene sets from sixteen collections. It has multiple visualization capabilities that allow researchers to view gene sets at various levels of granularity. EGSEA has been tested on simulated data and on a number of human and mouse datasets and, based on biologists’ feedback, consistently outperforms the individual tools that have been combined. Our evaluation demonstrates the superiority of the ensemble approach for GSE analysis, and its utility to effectively and efficiently extrapolate biological functions and potential involvement in disease processes from lists of differentially regulated genes. Availability and Implementation EGSEA is available as an R package at http://www.bioconductor.org/packages/EGSEA/. The gene sets collections are available in the R package EGSEAdata from http://www.bioconductor.org/packages/EGSEAdata/. Supplementary information Supplementary data are available at Bioinformatics online.

T-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.

SummaryThe mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland.

Isolation of mammary epithelial subpopulations, including stem and progenitor cells, has become a standard technique in recent years. However, a number of methods and approaches for this have developed and the relative benefits of the different approaches, and the reason for their development, have not always been clear. Here, three of the leading laboratories working on the separation of mammary cell subpopulations have summarised their methods, highlighted their differences and similarities and also discussed the reasoning behind the approaches they have taken. This article will assist workers establishing mammary cell separation protocols in their laboratories to make informed choices about the methods they should use.

Background The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. Results Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. Conclusions Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0064-6) contains supplementary material, which is available to authorized users.

The ovarian hormones oestrogen and progesterone profoundly influence breast cancer risk, underpinning the benefit of endocrine therapies in the treatment of breast cancer. Modulation of their effects through ovarian ablation or chemoprevention strategies also significantly decreases breast cancer incidence. Conversely, there is an increased risk of breast cancer associated with pregnancy in the short term. The cellular mechanisms underlying these observations, however, are poorly defined. Here we demonstrate that mouse mammary stem cells (MaSCs) are highly responsive to steroid hormone signalling, despite lacking the oestrogen and progesterone receptors. Ovariectomy markedly diminished MaSC number and outgrowth potential in vivo, whereas MaSC activity increased in mice treated with oestrogen plus progesterone. Notably, even three weeks of treatment with the aromatase inhibitor letrozole was sufficient to reduce the MaSC pool. In contrast, pregnancy led to a transient 11-fold increase in MaSC numbers, probably mediated through paracrine signalling from RANK ligand. The augmented MaSC pool indicates a cellular basis for the short-term increase in breast cancer incidence that accompanies pregnancy. These findings further indicate that breast cancer chemoprevention may be achieved, in part, through suppression of MaSC function.

Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.

SummaryElucidating the mechanisms underlying hematopoietic stem cell (HSC) specification and expansion in the embryo has been hampered by the lack of analytical cell culture systems that recapitulate in vivo development. Here, we describe an ex vivo model that facilitates a rapid and robust emergence of multipotent long-term repopulating HSCs in the embryonic AGM region. Because this method includes a cell dissociation step prior to reconstruction of a three-dimensional functional tissue and preserves both stromal and hematopoietic elements, it allowed us to identify the direct ancestry of the rapidly expanding HSC pool. We demonstrate that extensive generation of definitive HSCs in the AGM occurs predominantly through the acquisition of stem characteristics by the VE-cadherin+CD45+ population.

Background The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation. Methods MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells. Results Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity. Conclusions This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1187-z) contains supplementary material, which is available to authorized users.

T cell development depends critically on several distinct thymic epithelial cell types that are organized into two main compartments: cortex and medulla. The prevailing hypothesis suggests that these derive from ectoderm and endoderm, respectively. Here we show that lineage analysis provides no evidence for an ectodermal contribution to the thymic rudiment. We further demonstrate, via ectopic transplantation, that isolated pharyngeal endoderm can generate a functional thymus containing organized cortical and medullary regions and that this capacity is not potentiated by the presence of pharyngeal ectoderm. These data establish that the cortical and medullary thymic epithelial compartments derive from a single germ layer, the endoderm, thus refuting the 'dual-origin' model of thymic epithelial ontogeny.

Polarity coordinates cell movement, differentiation, proliferation and apoptosis to build and maintain complex epithelial tissues such as the mammary gland. Loss of polarity and the deregulation of these processes are critical events in malignant progression but precisely how and at which stage polarity loss impacts on mammary development and tumourigenesis is unclear. Scrib is a core polarity regulator and tumour suppressor gene however to date our understanding of Scrib function in the mammary gland has been limited to cell culture and transplantation studies of cell lines. Utilizing a conditional mouse model of Scrib loss we report for the first time that Scrib is essential for mammary duct morphogenesis, mammary progenitor cell fate and maintenance, and we demonstrate a critical and specific role for Scribble in the control of the early steps of breast cancer progression. In particular, Scrib-deficiency significantly induced Fra1 expression and basal progenitor clonogenicity, which resulted in fully penetrant ductal hyperplasia characterized by high cell turnover, MAPK hyperactivity, frank polarity loss with mixing of apical and basolateral membrane constituents and expansion of atypical luminal cells. We also show for the first time a role for Scribble in mammalian spindle orientation with the onset of mammary hyperplasia being associated with aberrant luminal cell spindle orientation and a failure to apoptose during the final stage of duct tubulogenesis. Restoring MAPK/Fra1 to baseline levels prevented Scrib-hyperplasia, whereas persistent Scrib deficiency induced alveolar hyperplasia and increased the incidence, onset and grade of mammary tumours. These findings, based on a definitive genetic mouse model provide fundamental insights into mammary duct maturation and homeostasis and reveal that Scrib loss activates a MAPK/Fra1 pathway that alters mammary progenitor activity to drive premalignancy and accelerate tumour progression., Author Summary Polarity allows the specialization of cell function and is required to coordinate cell movements, differentiation, proliferation and apoptosis to build and maintain complex tissues such as the mammary gland. Disruption of polarity is a diagnostic criterion of cancer, but exactly how deregulation of core polarity genes contribute to cancer and at which stage polarity loss promotes breast cancer development in vivo is still poorly understood. To address this directly, we deleted the core polarity gene Scrib specifically in the mouse mammary gland. Scrib loss resulted in loss of tissue architecture and duct hyperplasia in mature but not pubescent mice. Onset of hyperplasia was associated with defective spindle orientations, a failure to apoptose and was sustained by high cell turnover and Ras/Erk/Fra1 MAPK pathway activation. Scrib deficiency activated progenitors and resulted in the excess growth of atypical luminal cells and the development of ductal and alveolar hyperplasia. Overall these mice exhibited an increased incidence, onset and grade of mammary tumours. These studies provide a definitive demonstration of the critical role played by core polarity genes in maintaining mammary epithelial integrity in vivo. This mouse model is a valuable tool for understanding the role of polarity in mammary development and the most initial stages of breast cancer.

Cellular reaggregation methods are commonly used to generate tissue organoids for use in biological studies. Using a modified method termed "compaction reaggregation," it is possible to establish reaggregates of reproducible size from defined input cell numbers with ease and without specialist equipment. Importantly, this method is suitable for the study of tissues that have proved refractory to reaggregation by other methods. With the option of juxtaposing cell populations, this method is useful for studies of tissue organization and structure.

Reinforcing the ‘gauntlet’ of thymic negative selection via exosomal transfer of self-antigens

Vertical Cavity Surface Emitting Laser (VCSEL)-based data links are attractive due to their low-power dissipation and low-cost manufacturability. This chapter reviews the foundations for this technology, as well as the device and module design challenges of extending the data rate beyond the current level. We begin with a review of data communications from the business perspective, and continue with a brief discussion of the current and future standards. This is followed by a survey of recent advances in VCSELs, including data links operating at 28 Gb/s. We review the recent efforts on ultra-fast data links and discuss the advantages of the different approaches. We also examine key design aspects of optical transceiver modules and we focus our discussion on novel applications in high-performance computing using both multi-mode and single-mode fiber optics. We highlight the importance of the device/component-level and system-level modeling and show some modeling examples with comparison to measured data. We conclude with a comparison of the VCSEL-based data links with other competing technologies, including silicon photonics and short-cavity edge emitting lasers.

The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development., Author Summary The thymus is the specialized organ responsible for generating T cells, which are required to regulate and effect immune responses. The unique functions of the thymus are mediated by a diverse array of specialized epithelial cells found only within this organ. These specialized, functionally mature thymic epithelial cells are generated from immature epithelial progenitor cells present in the fetal and adult thymus through a highly regulated process, termed differentiation, that is tightly controlled by specific genes. Foxn1, a protein that is expressed in thymic epithelial cells, is a transcription factor—a protein that regulates how other genes are expressed. Here, we have investigated the role of Foxn1 in generating mature thymic epithelial cells from immature progenitors. We find that Foxn1 is required throughout this process, from the onset of differentiation in progenitor thymic epithelial cells in the developing fetus to the final differentiation steps through which thymic epithelial cells mature to acquire their full functionality. We further find that Foxn1 controls the expression of a variety of genes with different functions in thymic epithelial cells. Overall, our study defines the role of Foxn1 in thymus development at the cellular level and provides insight into how it mediates these functions.

Cellular reaggregation methods have been used to generate in vitro organotypic cultures as a means to elucidate the cellular and molecular requirements of organogenesis. However, reproducibility from experiment to experiment has remained problematic and furthermore, current protocols do not support reaggregation of many important tissues. Here, using the thymus as a model organ, we present a novel reaggregation method termed “compaction reaggregation” that offers improved kinetics of reaggregation and greatly improved efficiency. Using compaction reaggregation we have been able to reaggregate the aorta-gonad- mesonephros region, a tissue that previously proved refractory to commonly used reaggregation methods, enabling the study of hematopoietic stem cell emergence and expansion. Additionally, compaction reaggregation permits the juxtaposition of different cell layers within the aggregated structure thus providing the means to study inductive interactions between different cell populations in vitro. genesis 47:346–351, 2009. © 2009 Wiley-Liss, Inc.

Pooled library sequencing screens that perturb gene function in a high-throughput manner are becoming increasingly popular in functional genomics research. Irrespective of the mechanism by which loss of function is achieved, via either RNA interference using short hairpin RNAs (shRNAs) or genetic mutation using single guide RNAs (sgRNAs) with the CRISPR-Cas9 system, there is a need to establish optimal analysis tools to handle such data. Our open-source processing pipeline in edgeR provides a complete analysis solution for screen data, that begins with the raw sequence reads and ends with a ranked list of candidate genes for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (samples in the columns, genes in the rows) with options for allowing mismatches and small shifts in sequence position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or generalized linear modeling that can handle complex experimental designs. A detailed users’ guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from http://www.bioconductor.org.

Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26eYfp reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.

Introduction: MicroRNAs (miRNAs) have been implicated in governing lineage specification and differentiation in multiple organs; however, little is known about their specific roles in mammopoiesis. We have determined the global miRNA expression profiles of functionally distinct epithelial subpopulations in mouse and human mammary tissue, and compared these to their cognate transcriptomes and epigenomes. Finally, the human miRNA signatures were used to interrogate the different subtypes of breast cancer, with a view to determining miRNA networks deregulated during oncogenesis. Methods: RNA from sorted mouse and human mammary cell subpopulations was subjected to miRNA expression analysis using the TaqMan MicroRNA Array. Differentially expressed (DE) miRNAs were correlated with gene expression and histone methylation profiles. Analysis of miRNA signatures of the intrinsic subtypes of breast cancer in The Cancer Genome Atlas (TCGA) database versus those of normal human epithelial subpopulations was performed. Results: Unique miRNA signatures characterized each subset (mammary stem cell (MaSC)/basal, luminal progenitor, mature luminal, stromal), with a high degree of conservation across species. Comparison of miRNA and transcriptome profiles for the epithelial subtypes revealed an inverse relationship and pinpointed key developmental genes. Interestingly, expression of the primate-specific miRNA cluster (19q13.4) was found to be restricted to the MaSC/basal subset. Comparative analysis of miRNA signatures with H3 lysine modification maps of the different epithelial subsets revealed a tight correlation between active or repressive marks for the top DE miRNAs, including derepression of miRNAs in Ezh2-deficient cellular subsets. Interrogation of TCGA-identified miRNA profiles with the miRNA signatures of different human subsets revealed specific relationships. Conclusions: The derivation of global miRNA expression profiles for the different mammary subpopulations provides a comprehensive resource for understanding the interplay between miRNA networks and target gene expression. These data have highlighted lineage-specific miRNAs and potential miRNA-mRNA networks, some of which are disrupted in neoplasia. Furthermore, our findings suggest that key developmental miRNAs are regulated by global changes in histone modification, thus linking the mammary epigenome with genome-wide changes in the expression of genes and miRNAs. Comparative miRNA signature analyses between normal breast epithelial cells and breast tumors confirmed an important linkage between luminal progenitor cells and basal-like tumors. Conclusions: The derivation of global miRNA expression profiles for the different mammary subpopulations provides a comprehensive resource for understanding the interplay between miRNA networks and target gene expression. These data have highlighted lineage-specific miRNAs and potential miRNA–mRNA networks, some of which are disrupted in neoplasia. Furthermore, our findings suggest that key developmental miRNAs are regulated by global changes in histone modification, thus linking the mammary epigenome with genome-wide changes in the expression of genes and miRNAs. Comparative miRNA signature analyses between normal breast epithelial cells and breast tumors confirmed an important linkage between luminal progenitor cells and basal-like tumors. Refereed/Peer-reviewed