The Hippo pathway effector Taz is required for cell morphogenesis and fertilization in zebrafish.

Hippo signaling is a critical pathway that integrates extrinsic and intrinsic mechanical cues to regulate organ size. Despite its essential role in organogenesis, little is known about its role in cell fate specification and differentiation. Here, we unravel a novel and unexpected role of the Hippo pathway effector Taz (wwtr1) in controlling the size, shape and fate of a unique cell in the zebrafish ovary. We show that wwtr1 mutant females are infertile. In teleosts, fertilization occurs through the micropyle, a funnel-like opening in the chorion, formed by a unique enlarged follicle cell, the micropylar cell (MC). We describe here, for the first time, the mechanism that underlies the differentiation of the MC. Our genetic analyses show that Taz is essential for MC fate acquisition and subsequent micropyle formation in zebrafish. We identify Taz as the first bona fide MC marker and show that Taz is specifically and strongly enriched in the MC precursor. Altogether, we performed the first genetic and molecular characterization of the MC and propose that Taz is a key regulator of MC fate.This article has an associated 'The people behind the papers' interview.

Simple "on-demand" production of bioactive natural products.

Exchange of the native promoter to the arabinose-inducible promoter PBAD was established in entomopathogenic bacteria to silence and/or activate gene clusters involved in natural product biosynthesis. This allowed the "on-demand" production of GameXPeptides, xenoamicins, and the blue pigment indigoidine. The gene clusters for the novel "mevalagmapeptides" and the highly toxic xenorhabdins were identified by this approach.

Mammary epithelial reconstitution with gene-modified stem cells assigns roles to Stat5 in luminal alveolar cell fate decisions, differentiation, involution, and mammary tumor formation.

The mammary gland represents a unique model system to study gene functions in adult stem cells. Mammary stem cells (MaSCs) can regenerate a functional epithelium on transplantation into cleared fat pads. We studied the consequences of distinct genetic modifications of MaSCs on their repopulation and differentiation ability. The reconstitution of ductal trees was used as a stem cell selection procedure and the nearly quantitative lentiviral infection efficiency of the primary mammary epithelial cells (MECs) rendered the enrichment of MaSCs before their transplantation unnecessary. The repopulation frequency of transduced MaSCs was nearly 100% in immunodeficient recipients and the resulting transgenic ducts homogeneously expressed the virally encoded fluorescent marker proteins. Transplantation of a mixture of MECs, expressing different fluorescent proteins, resulted in a distinct pattern of ductal outgrowths originating from a small number of individually transduced MaSCs. We used genetically modified MECs to define multiple functions of Stat5 during mammary gland development and differentiation. Stat5-downregulation in MaSCs did not affect primary ductal outgrowth, but impaired side branching and the emergence of mature alveolar cells from luminal progenitors during pregnancy. Conversely, the expression of a constitutively active variant of Stat5 (cS5-F) caused epithelial hyperproliferation, thickening of the ducts and precocious, functional alveoli formation in virgin mice. Expression of cS5-F also prevented involution and caused the formation of estrogen and progesterone receptor positive (ER(+)PR(+)) adenocarcinomas. The tumors expressed activated Stat5 and Stat3 and contained a small fraction of CD44(+) cells, possibly indicative of cancer stem cells.

Labeling the Micropylar Cell in Zebrafish Whole-Mount and Cryo-sectioned Follicles.

In some animal species, fertilization occurs through a funnel-like canal called the "micropyle." In teleost fishes, the micropyle is formed by a very specialized follicle cell, called the micropylar cell (MC). Very little is known about the mechanisms underlying the specification and differentiation of the MC, a unique cell among hundreds that compose the follicle cell layer. The Hippo pathway effector Taz is essential for this process and is the first reported MC marker. Here, we describe a method to identify and mark the micropylar cell following the immunostaining procedure on cryosections or combining it with the RNA in situ hybridization on whole-mount follicles.

Effects of gonadotrophin releasing hormone analogues on human endometrial stromal cells and embryo invasion in vitro.

BACKGROUND: Gonadotrophin releasing hormone (GnRH) analogues are widely used in IVF programmes as a method of suppressing the luteinizing hormone (LH) surge prior to ovarian stimulation, but their roles outside the pituitary remain relatively unknown. A 2002 Cochrane review (Al-Inany et al. Gonadotrophin-releasing hormone antagonists for assisted conception. Cochrane Database Syst Rev 2006;3:CD001750) described lower pregnancy rates in women administered with GnRH antagonist, compared with those using an agonist, as part of an IVF programme, despite the fact that GnRH antagonist is a more effective repressor of LH. This study aimed to analyse the in-vitro effects of GnRH analogues on the decidualizing endometrium, blastocyst invasion and GnRH receptor expression in fertile women. METHODS: We analysed the in-vitro decidualization capacity of endometrial stromal cells, derived from fertile women during the implantation window, in the presence of GnRH analogues. The influence of GnRH analogues on GnRH receptor expression and blastocyst invasion was assessed by in-vitro assays of biomedical marker secretion, immunoblots and blastocyst attachment to the stromal extracellular matrix. RESULTS: We demonstrate that, at the concentrations and time periods used, GnRH analogues did not significantly influence the extent of decidualization of endometrial stromal cells. In addition, no adverse effect of GnRH analogues was seen on human blastocyst invasion. CONCLUSIONS: We suggest that GnRH analogues affect neither the capacity of the endometrium to support invasion nor the invasive potential of the blastocyst in the early stages of implantation.

Molecular and Cellular Pathogenesis of Endometriosis.

BACKGROUND: A substantial body of studies supports the view that molecular and cellular features of endometriotic lesions differ from those of eutopic endometrium. Apart from that, evidence exists that the eutopic endometrium from pa-tients with endometriosis differs from that of females without endometriosis. OBJECTIVE: Aberrant expression profiles include a number of non-steroid signaling pathways that exert their putative influ-ence on the pathogenesis of endometriosis at least in part via crosstalk(s) with estrogen-mediated mechanisms. A rational to focus research on non-steroid signal pathways is that they might be remunerative targets for the development and selection of novel therapeutics to treat endometriosis possibly without affecting estrogen levels. RESULTS AND CONCLUSION: In this article, we describe molecular and cellular features of endometriotic lesions and focus on the canonical WNT/ß-signaling pathway, a key regulatory system in biology (including stem cell homeostasis) and often in pathophysiological conditions such as endometriosis. Recently emerged novel biological concepts in signal transduction and gene regulation like exosomes and microRNAs are discussed in their putative role in the pathogenesis of endometriosis.

Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile.

Shrew-1, also called AJAP1, is a transmembrane protein associated with E-cadherin-mediated adherence junctions and a putative tumor suppressor. Apart from its interaction with ß-catenin and involvement in E-cadherin internalization, little structure or function information exists. Here we explored shrew-1 expression during postnatal differentiation of mammary gland as a model system. Immunohistological analyses with antibodies against either the extracellular or the cytoplasmic domains of shrew-1 consistently revealed the expression of full-length shrew-1 in myoepithelial cells, but only part of it in luminal cells. While shrew-1 localization remained unaltered in myoepithelial cells, nuclear localization occurred in luminal cells during lactation. Based on these observations, we identified two unknown shrew-1 transcript variants encoding N-terminally truncated proteins. The smallest shrew-1 protein lacks the extracellular domain and is most likely the only variant present in luminal cells. RNA analyses of human tissues confirmed that the novel transcript variants of shrew-1 exist in vivo and exhibit a differential tissue expression profile. We conclude that our findings are essential for the understanding and interpretation of future functional and interactome analyses of shrew-1 variants.

The production of interleukin-11 and decidualization are compromised in endometrial stromal cells derived from patients with infertility.

IL-11 signaling is critical for decidualization of the endometrial stroma in early pregnancy in the mouse. In this study, we investigate the function of IL-11 signaling in cAMP-induced decidualization of human endometrial stromal cells. We show that treatment of endometrial stromal cells with 8-bromo-cAMP (8-Br-cAMP) results in an increase in the levels of secreted IL-11, whereas levels of cell surface IL-11 receptor alpha are similar with or without 8-Br-cAMP treatment. The production of IL-11 correlates with the production of molecular markers of decidualization, prolactin and IGF-binding protein-1. The expression of these markers is inhibited when IL-11 signaling is specifically blocked in decidualizing endometrial stromal cells by the IL-11 antagonist W147A. We demonstrate that 8-Br-cAMP-induced endometrial stromal cells derived from patients with primary infertility produce lower levels of prolactin, IGF-binding protein-1, and IL-11 than cells derived from fertile women. Our results suggest that IL-11 expression is critically important during decidualization in the human endometrium, and that aberrant regulation of endometrial IL-11 production may be associated with some types of infertility.

Stat5 assumes distinct functions in mammary gland development and mammary tumor formation.

The mammary epithelium comprises luminal and basal cells which originate from multipotent mammary stem cells (MaSCs). They form ductal structures embedded in the mammary fat pad in virgin mice and differentiate during pregnancy into alveoli under the control of hormones and growth factors and the activation of specific transcription factors. Genetic manipulations of embryonic stem cells and the derivation of transgenic mice allowed the study of regulatory genes in mammary epithelial cells of particular differentiation states. We describe an alternative approach to investigate stage dependent gene functions in transgenic mammary glands based on ex vivo, genetically manipulated MaSCs and the reconstitution of functional epithelium upon their transplantation into cleared fat pads. Modification of MaSCs with Stat5 suppressing shRNA or a constitutively active variant of Stat5 showed that Stat5 assumes essential roles in alveolar lineage commitment, proliferation, differentiation and survival. Its persistent activation during post-lactational involution causes the formation of non-metastatic adenocarcinomas, resembling the human luminal breast cancer subtype. The tumor cells express estrogen and progesterone receptor (ER+PR+) and activated Stat3 and Stat5. They could become valuable to assess the therapeutic potential of anti-estrogens, aromatase inhibitors and Stat3 and Stat5 inhibition on tumor growth.

The potential of amniotic fluid stem cells for cellular therapy and tissue engineering.

INTRODUCTION: Foetal cells present in amniotic fluid (AF) have been used for many years to perform prenatal genetic screening. Recent reports suggested that these cells might have additional benefits. AF contains, in addition to committed and differentiated cells, a subpopulation with stem cell characteristics. AF-derived stem cells (AFS) have functions found in mesenchymal stem cells, but in addition, exhibit a potent expansion capacity and plasticity. AFS are able to undergo multi-lineage differentiation and produce progeny indicative of all three germ layers. AREAS COVERED: The experimental approaches available to isolate AFS and their potential for tissue engineering, the repair of organs through cell replacement and tissue regeneration. EXPERT OPINION: The deployment of AFS for tissue regeneration offers advantages over the use of embryonic or adult stem cells: i) AF represents a convenient and non-contested source for obtaining stem cells; ii) their derivation is relatively simple and rapid; iii) no feeder layers are required for their cultivation; iv) they display no spontaneous differentiation in culture; and v) their stem cell phenotype is not affected by long-term storage. The application of AFS for tissue replacement therapies in vivo is at a very early stage, but existing studies indicate great potential for clinical use.

Stem cells of the breast and cancer therapy.

Breast cancer remains a significant public health problem despite advances in the understanding of the molecular and cellular events that underlie the disease. Crucial pathways regulating the cell cycle, proliferation and survival of breast cancer cells have been investigated and aberrant components of these pathways have been exploited as new drug targets. However, the mortality from breast cancer is only slowly declining. Recently, a model has been proposed that might explain the heterogeneous biological features of breast cancer cell populations and their differential response to therapeutic agents, which has interesting implications for further progress in therapy. This model links the emergence of breast cancer cells to stem cells and progenitors, an observation originally made in other cancer entities. It hypothesizes that the tumors originate from a small population of undifferentiated cells. These cells can undergo self-renewal and are able to generate a large number of partially differentiated cells, which constitute the bulk of the tumor. These cancer stem cells resemble adult stem and progenitor cells found in the normal breast, but are deregulated in their patterns of proliferation and differentiation. They could originate from normal stem cells or from more differentiated progenitors and lose their normal growth restraints through a series of oncogenic mutations that deregulate a small number of central signaling pathways. If breast cancer really is a stem and progenitor cell disease, this will have important implications for the understanding of the emergence of cancer cells. A combination of the cell-type of origin, stem cells, early or late progenitors and the particular oncogenic mutations acquired could provide a new classification of the different types of breast cancer. These parameters might determine the mechanisms of cancer progression and the responsiveness of patients to drug treatment. Stem cell-specific features could possibly be exploited as innovative drug targets.

Murine amniotic fluid stem cells contribute mesenchymal but not epithelial components to reconstituted mammary ducts.

INTRODUCTION: Amniotic fluid harbors cells indicative of all three germ layers, and pluripotent fetal amniotic fluid stem cells (AFSs) are considered potentially valuable for applications in cellular therapy and tissue engineering. We investigated whether it is possible to direct the cell fate of AFSs in vivo by transplantation experiments into a particular microenvironment, the mammary fat pad. This microenvironment provides the prerequisites to study stem cell function and the communication between mesenchymal and epithelial cells. On clearance of the endogenous epithelium, the ductal tree can be reconstituted by the transfer of exogenously provided mammary stem cells. Analogously, exogenously provided stem cells from other tissues can be investigated for their potential to contribute to mammary gland regeneration. METHODS: We derived pluripotent murine AFSs, measured the expression of stem cell markers, and confirmed their in vitro differentiation potential. AFSs were transplanted into cleared and non cleared fat pads of immunocompromised mice to evaluate their ability to assume particular cell fates under the instructive conditions of the fat-pad microenvironment and the hormonal stimulation during pregnancy. RESULTS: Transplantation of AFSs into cleared fat pads alone or in the presence of exogenous mammary epithelial cells caused their differentiation into stroma and adipocytes and replaced endogenous mesenchymal components surrounding the ducts in co-transplantation experiments. Similarly, transplantation of AFSs into fat pads that had not been previously cleared led to AFS-derived stromal cells surrounding the elongating endogenous ducts. AFSs expressed the marker protein α-SMA, but did not integrate into the myoepithelial cell layer of the ducts in virgin mice. With pregnancy, a small number of AFS-derived cells were present in acinar structures. CONCLUSIONS: Our data demonstrate that the microenvironmental cues of the mammary fat pad cause AFSs to participate in mammary gland regeneration by providing mesenchymal components to emerging glandular structures, but do not incorporate or differentiate into ductal epithelial cells.

Visualization of Stat3 and Stat5 transactivation activity with specific response element dependent reporter constructs integrated into lentiviral gene transfer vectors.

BACKGROUND: Signal transducer and activator of transcription 3 and 5 (Stat3 and Stat5) play important roles in cell differentiation, proliferation, apoptosis and inflammation. They are transiently activated by ligand-receptor interactions in normal cells but are often found to be constitutively active in cancer cells. Analysis of their activation pattern is therefore important for the description of developmental processes and the understanding of cellular transformation. MATERIALS AND METHODS: To visualize Stat3 and Stat5 transactivation activity in different cell types, we designed novel reporter constructs. These constructs comprise Stat3 or Stat5 specific promoter elements and reporter genes encoding ß-galactosidase or fluorescent proteins. These constructs were integrated into lentiviral gene transfer vectors facilitating efficient transduction of most cell types. RESULTS: The lentiviral reporter constructs were used to infect different cell types and their inducibility by activated Stat3 or Stat5 was measured. The Stat3-mCherry reporter was active in transduced tumor cells, which exhibit high levels of phosphorylated Stat3 and it was inducible in HepG2 liver cells by interleukin-6 treatment. The Stat5-LacZ reporter was active in cultured cells upon hormone induction of Stat5 and in primary mammary epithelial cells transplanted into cleared fat pads of mice during late pregnancy. CONCLUSION: These novel reporter constructs are valuable tools to investigate and to distinguish between Stat3 and Stat5 activity in primary cells and cancer cells. They will also be useful in the discovery of drugs targeting Stat3 or Stat5. They can also be employed to generate transgenic mice and track Stat activity during development.

miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development.

MicroRNAs are small noncoding RNAs that carry out post-transcriptional regulation of the expression of their target genes. However, their roles in mammalian organogenesis are only beginning to be understood. Here we show that the microRNA-212/132 family (which comprises miR-212 and miR-132) is indispensable during the development of the mammary glands in mice, particularly for the regulation of the outgrowth of the epithelial ducts. Mammary transplantation experiments revealed that the function of the miR-212/132 family is required in the stroma but not in the epithelia. Both miR-212 and miR-132 are expressed exclusively in mammary stroma and directly target the matrix metalloproteinase MMP-9. In glands that lack miR-212 and miR-132, MMP-9 expression increases and accumulates around the ducts. This may interfere with collagen deposition and lead to hyperactivation of the tumor growth factor-ß signaling pathway, thereby impairing ductal outgrowth. Our results identify the miR-212/132 family as one of the main regulators of the epithelial-stromal interactions that are required for proper pubertal development of the mammary gland.

Endometrial cells from women with endometriosis have increased adhesion and proliferative capacity in response to extracellular matrix components: towards a mechanistic model for endometriosis progression.

BACKGROUND: Endometriosis, classified as the presence of endometrial cells in ectopic sites, is a debilitating disease causing pain and infertility in approximately 10% of women of reproductive age. It is associated with the aberrant expression of extracellular matrix (ECM) components and their receptors, integrins. METHODS: We analysed the expression of integrins in stromal cells derived from peritoneal, ovarian and deeply infiltrating endometriotic lesions and from endometrium from women with and without endometriosis in vitro, using quantitative immunocytochemistry. The adhesive and proliferative capacity of each of the cell types in response to ECM components was assessed by in vitro assays of cell attachment and DNA synthesis. RESULTS: We demonstrate that eutopic and ectopic endometrial stromal cells from women with endometriosis exhibit an aberrant integrin profile in vitro compared with stromal cells derived from healthy controls. In addition, the former display increased adhesion and proliferative capacity in response to specific ECM components. CONCLUSIONS: We propose that the increased adhesive and proliferative potential of cells from endometriotic lesions may be a key feature in the pathogenesis of endometriosis. Furthermore, the elevated responsiveness of eutopic cells from women with endometriosis may contribute to the predisposition of some women to the disease.

Stromal cells from endometriotic lesions and endometrium from women with endometriosis have reduced decidualization capacity.

OBJECTIVE: To evaluate the phenotype, proliferative, and differentiation capacities in vitro of stromal cells derived from peritoneal, ovarian, and deeply infiltrating endometriosis. DESIGN: Experimental study using phase contrast microscopy, immunocytochemistry, and functional bioassays. SETTING: University-based laboratory. PATIENT(S): Women with and without endometriosis undergoing surgery for benign indications. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The stability in vitro of stromal cells derived from peritoneal (n = 18), ovarian (n = 29), and deeply infiltrating (n = 14) endometriotic lesions, as well as endometrium from women with (n = 5) and without endometriosis (n = 5) was evaluated by detection of endometrial markers. The proliferative and differentiation capacity of the cells was assessed by the use of cell doubling estimation and in vitro decidualization assays. RESULT(S): The expression of the progesterone receptor and CD10 in stromal cells derived from the three types of endometriotic lesions is retained in culture up to passage 10. The doubling time of stromal cells from deeply infiltrating lesions is lower than that of endometrial stromal cells. Levels of prolactin and insulin-like growth factor binding protein-1 (IGFBP-1) are reduced in supernatants from stromal cells derived from the three types of lesions and from the endometrium of women with endometriosis. CONCLUSION(S): The peritoneal, ovarian, and deeply infiltrating endometriotic stromal cell lines we describe retain in vivo tissue markers. Loss of differentiation capacity of the endometriotic cell lines and endometrial cells from women with endometriosis may influence the capacity for proliferation and survival of these cells in the ectopic environment.