A fatty acid anabolic pathway in specialized-cells sustains a remote signal that controls egg activation in Drosophila.

Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation.

Learning objectives: an epiphany.

A new Bachelor-Master curriculum in Biomedical Sciences was created at the University of Geneva in 2017. As we organized the new curriculum, we discovered the usefulness of learning objectives. This goal-oriented approach of teaching proved essential to determine the overall structure of the teaching program, as well as the content of specific courses, and the nature of the examinations. It led us to include innovative elements in the program, preparing students for real-life situations. Finally, it convinced us to change our role as teachers, in order to engage students in a more active learning relationship.

Human Bone Marrow Contains Mesenchymal Stromal Stem Cells That Differentiate In Vitro into Contractile Myofibroblasts Controlling T Lymphocyte Proliferation.

Mesenchymal stromal stem cells (MSC) that reside in the bone marrow (BM) can be amplified in vitro. In 2-dimension (D) cultures, MSC exhibit a morphology similar to fibroblasts, are able to inhibit T lymphocyte and natural killer cell proliferation, and can be differentiated into adipocytes, chondrocytes, or osteoblasts if exposed to specific media. Here we show that medullar MSC cultured in 2D formed an adherent stroma of cells expressing well-organized microfilaments containing α-smooth muscle actin and nonmuscle myosin heavy chain IIA. MSC could be grown in 3D in collagen membranes generating a structure which, upon exposition to 50 mM KCl or to an alternating electric current, developed a contractile strength that averaged 34 and 45 µN/mm2, respectively. Such mechanical tension was similar in intensity and in duration to that of human placenta and was annihilated by isosorbide dinitrate or 2,3-butanedione monoxime. Membranes devoid of MSC did not exhibit a significant contractility. Moreover, MSC nested in collagen membranes were able to control T lymphocyte proliferation, and differentiated into adipocytes, chondrocytes, or osteoblasts. Our observations show that BM-derived MSC cultured in collagen membranes spontaneously differentiate into contractile myofibroblasts exhibiting unexpected properties in terms of cell differentiation potential and of immunomodulatory function.

Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium.

The key role of the primary cilium in developmental processes is illustrated by ciliopathies resulting from genetic defects of its components. Ciliopathies include a large variety of dysmorphic syndromes that share in common the presence of multiple kidney cysts. These observations suggest that primary cilia may control morphogenetic processes in the developing kidney. In this study, we assessed the role of primary cilium in branching tubulogenesis and/or lumen development using kidney collecting duct-derived mCCDN21 cells that display spontaneous tubulogenic properties when grown in collagen-Matrigel matrix. Tubulogenesis and branching were not altered when cilium body growth was inhibited by Kif3A or Ift88 silencing. In agreement with the absence of a morphogenetic effect, proliferation and wound-healing assay revealed that neither cell proliferation nor migration were altered by cilium body disruption. The absence of cilium following Kif3A or Ift88 silencing in mCCDN21 cells did not alter the initial stages of tubular lumen generation while lumen maturation and enlargement were delayed. This delay in tubular lumen maturation was not observed after Pkd1 knockdown in mCCDN21 cells. The delayed lumen maturation was explained by neither defective secretion or increased reabsorption of luminal fluid. Our results indicate that primary cilia do not control early morphogenetic processes in renal epithelium. Rather, primary cilia modulate tubular lumen maturation and enlargement resulting from luminal fluid accumulation in tubular structures derived from collecting duct cells.

Spatially restricted hyaluronan production by Has2 drives epithelial tubulogenesis in vitro.

Generation of branched tubes from an epithelial bud is a fundamental process in development. We hypothesized that induction of hyaluronan synthase (Has) and production of hyaluronan (HA) drives tubulogenesis in response to morphogenetic cytokines. Treatment of J3B1A mammary cells with transforming growth factor-ß1 or renal MDCK and mCCD-N21 cells with hepatocyte growth factor induced strong and specific expression of Has2. Immunostaining revealed that HA was preferentially produced at the tips of growing tubules. Inhibition of HA production, either by 4-methylumbelliferone (4-MU) or by Has2 mRNA silencing, abrogated tubule formation. HA production by J3B1A and mCCD-N21 cells was associated with sustained activation of ERK and S6 phosphorylation. However, silencing of either CD44 or RHAMM (receptor for HA-mediated motility), the major HA receptors, by RNA interference, did not alter tubulogenesis, suggesting that this process is not receptor-mediated.

Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells.

BACKGROUND: Formation of branching tubes is a fundamental step in the development of glandular organs. To identify extracellular cues that orchestrate epithelial tubulogenesis, we employed an in vitro assay in which EpH4-J3B1A mammary epithelial cells form spheroidal cysts when grown in collagen gels under serum-free conditions, but form branching tubules in the presence of fetal calf serum (FCS). RESULTS: Initial experiments showed that the tubulogenesis-inducing activity of FCS was markedly increased by heating (70 degrees C) or transient acidification to pH3. We therefore hypothesized that the tubulogenic agent was transforming growth factor-beta (TGF-beta), a cytokine that is present in serum in latent form and can be activated by heat or acid treatment. We found indeed that the tubulogenic activity of acidified FCS is abrogated by addition of either SB-431542, a selective inhibitor of the TGF-beta type I receptor, or a neutralizing antibody to TGF-beta-1. On the other hand, addition of low concentrations (20-100 pg/ml) of exogenous TGF-beta-1 recapitulated the effect of acidified FCS in inducing morphogenesis of hollow tubes. In contrast, higher concentrations of TGF-beta-1 induced the formation of thin cellular cords devoid of a detectable lumen. To gain insight into the mechanisms underlying TGF-beta-1-induced tube formation, we assessed the potential role of matrix metalloproteinases (MMPs). By western blot and gelatin zymography, we observed a dose-dependent increase in MMP-9 upon TGF-beta-1 treatment. Tube formation was suppressed by a synthetic broad-spectrum metalloproteinase inhibitor, by recombinant tissue inhibitor of metalloproteinases-2 (TIMP-2) and by a selective inhibitor of MMP-9, indicating that this morphogenetic process requires the activity of MMP-9. CONCLUSION: Altogether, our results provide evidence that, at low concentrations, TGF-beta-1 promotes MMP-dependent branching tubulogenesis by mammary epithelial cells in vitro, and suggest that it plays a similar role during mammary gland development in vivo.

Tumour necrosis factor alpha confers an invasive, transformed phenotype on mammary epithelial cells.

Although loss of cell-cell adhesion and gain of invasive properties play a crucial role in the malignant progression of epithelial tumours, the molecular signals that trigger these processes have not been fully elucidated. In light of the well-established relationship between chronic inflammation and cancer, we hypothesized that pro-inflammatory cytokines disrupt epithelial-cell adhesion and promote cell migration. To test this hypothesis, we used an in vitro model in which 31EG4-2A4 mouse mammary epithelial cells grown in a collagen gel form compact spheroidal colonies. Among the several cytokines examined, tumour necrosis factor alpha (TNF-alpha) caused a pronounced 3D scattering of preformed epithelial-cell colonies and induced 31EG4-2A4 cells grown on top of a collagen gel to invade the underlying matrix. In addition, TNF-alpha abolished contact-mediated inhibition of cell proliferation and stimulated cell growth both in the absence of exogenous mitogens and under anchorage-independent conditions. TNF-alpha induced the expression of matrix metalloproteinase 9 (MMP-9). Addition of the MMP inhibitor BB-94 abrogated TNF-alpha-induced 3D scattering. TNF-alpha also enhanced the attachment of 31EG4-2A4 cells to type-I collagen and markedly increased the expression of the alpha2 integrin subunit. Addition of a blocking antibody to beta1-integrin or of rhodocetin (a specific alpha2beta1 antagonist) to collagen-gel cultures abrogated 3D scattering. Collectively, these results demonstrate an essential role for MMPs and alpha2beta1 integrin in the invasive response of 31EG4-2A4 cells to TNF-alpha. We propose that the biological activities described in this study contribute to the ability of TNF-alpha to promote tumour progression and cancer-cell dissemination.

Inducible expression of Snail selectively increases paracellular ion permeability and differentially modulates tight junction proteins.

Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.

Membrane-type-1 matrix metalloproteinase confers tumorigenicity on nonmalignant epithelial cells.

Overexpression of membrane-type-1 matrix metalloproteinase (MT1-MMP) in tumor cells has previously been shown to enhance tumor growth and metastasis. To establish if MT1-MMP is also able to confer tumorigenicity on nonmalignant epithelial cells, we transfected human MT1-MMP cDNA into Madin-Darby canine kidney (MDCK) cells expressing a tetracycline-repressible transactivator. Induction of MT1-MMP in the absence of doxycycline (Dox) was associated with activation of exogenous MMP-2 as well as with formation of large cysts and increased invasiveness in collagen matrices. Transfected cells were inoculated subcutaneously into two groups of nude mice, one of which received Dox to inhibit expression of MT1-MMP. Formation of tumor xenografts was observed in 11 of 17 mice maintained without Dox, but only in two of nine mice that received Dox (P<0.05). The xenografts were composed of tubular structures interspersed within a highly cellular stroma. The epithelial cells delimiting the lumen were polarized, as indicated by the basolateral distribution of Na,K-ATPase. Despite their differentiated appearance, the tumors lacked a well-defined boundary, and epithelial tubules invaded adjacent muscular layers. These results demonstrate that conditional expression of MT1-MMP in nonmalignant MDCK epithelial cells is by itself sufficient to drive formation of invasive tumors.