Petra Klemmt

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, particulary 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-β 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.

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. STEM CELLS 2010;28:928–938

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.

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.

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

IntroductionAmniotic 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.MethodsWe 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.ResultsTransplantation 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.ConclusionsOur 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.

Mammary epithelial and breast cancer stem cells.

The perpetual renewal of tissues and organs is driven by resident stem cells and progenitors. These cells guarantee tissue maintenance and regeneration after injury or involution and most tissues and organs contain small populations of primitive stem cells and progenitors. These cells play a major role in the developing foetus and contribute to the generation of tissues and organs. Stem cells are positioned on top of the cellular hierarchy and give rise to progenitors with more restricted lineage potential. The stem cells can divide and self-renew to generate daughter stem cells or they can differentiate into a variety of mature cell types [1]. The stem cell hypothesis has been extrapolated to tumour tissues and the postulation of stem cells has many attractive conceptual and practical implications. Cancer cells are distinguished by multiple mutations which alter their cell cycle regulation, their sensitivity to apoptotic signals and their lifespan. Since stem cells persist within the organism for long periods of time, they have the potential to accumulate genetic damage and propagate them to their daughter stem cells and their downstream progenitors. Many tumours comprise minor populations of tumour-initiating cells, cells able to reconstitute a tumour upon transplantation; these cells have been called cancer stem cells (CSCs) [2]. CSCs share characteristics of normal stem cells, i.e. they can self-renew and they can give rise to heterogeneous cell populations within a tumour and maintain the tumour mass [3]. They are also more resistant to chemotherapeutic drugs and radiotherapy than their progenitors and thus represent a cellular reservoir for tumour recurrence. CSCs can be derived directly from mutational events in stem cells which confer proliferative properties or they can result from mutations in progenitor cells which provide them with self renewal capacity. Both pathways might be used and result in tumours with different pathological properties [4,5]. The characterisation of the signalling pathways which provide self renewal properties to CSCs and the targeted interference with limiting signal transduction components offer a promising new therapeutic approach for the treatment of cancer.

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/β-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

ABSTRACT 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 β-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. Summary: Transcripts of the tumor suppressor gene SHREW-1 exist in various splice variants in human and mouse encoding proteins with a differential expression and intracellular localization profile.

The Hippo pathway effector Taz is required for cell fate specification and fertilization in zebrafish

In the last decade, Hippo signaling has emerged as a critical pathway integrating extrinsic and intrinsic mechanical cues to regulate cell proliferation and survival, tissue morphology and organ size in vivo. Despite its essential role in organogenesis, surprisingly much less is known about how it connects biomechanical signals to control of cell fate and cell size during development. Here we unravel a novel and unexpected role of the Hippo pathway effector Taz (wwtr1) in the control of cell size and cell fate specification. In teleosts, fertilization occurs through a specific structure at the animal pole, called the micropyle. This opening in the chorion is formed during oogenesis by a specialized somatic follicle cell, the micropylar cell (MC). The MC has a peculiar shape and is much larger than its neighboring follicle cells but the mechanisms underlying its specification and cell shape acquisition are not known. Here we show that Taz is essential for the specification of the MC 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 before the cell can be identified morphologically. Altogether, our genetic data and molecular characterization of the MC lead us to propose that Taz is a key regulator of the MC fate activated by physical cues emanating from the oocyte to initiate the MC morphogenetic program. We describe here for the first time the mechanism underlying the specification of the MC fate.

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

ABSTRACT 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. Highlighted Article: The Hippo pathway effector Taz is required for the formation of the micropylar cell, a unique enlarged follicle cell in the ovary, and is essential for fertilization in zebrafish.