FGF controls epithelial-mesenchymal transitions during gastrulation by regulating cell division and apicobasal polarity.
ABSTRACT: To support tissue and organ development, cells transition between epithelial and mesenchymal states. Here, we have investigated how mesoderm cells change state in Drosophila embryos and whether fibroblast growth factor (FGF) signaling plays a role. During gastrulation, presumptive mesoderm cells invaginate, undergo an epithelial-to-mesenchymal state transition (EMT) and migrate upon the ectoderm. Our data show that EMT is a prolonged process in which adherens junctions progressively decrease in number throughout the migration of mesoderm cells. FGF influences adherens junction number and promotes mesoderm cell division, which we propose decreases cell-cell attachments to support slow EMT while retaining collective cell movement. We also found that, at the completion of migration, cells form a monolayer and undergo a reverse mesenchymal-to-epithelial transition (MET). FGF activity leads to accumulation of β-integrin Myospheroid basally and cell polarity factor Bazooka apically within mesoderm cells, thereby reestablishing apicobasal cell polarity in an epithelialized state in which cells express both E-Cadherin and N-Cadherin. In summary, FGF plays a dynamic role in supporting mesoderm cell development to ensure collective mesoderm cell movements, as well as proper differentiation of mesoderm cell types.
Project description:Tissue morphogenesis requires control over cell shape changes and rearrangements. In the Drosophila mesoderm, linked epithelial cells apically constrict, without cell extrusion or intercalation, to fold the epithelium into a tube that will then undergo epithelial-to-mesenchymal transition (EMT). Apical constriction drives tissue folding or cell extrusion in different contexts, but the mechanisms that dictate the specific outcomes are poorly understood. Using live imaging, we found that Abelson (Abl) tyrosine kinase depletion causes apically constricting cells to undergo aberrant basal cell extrusion and cell intercalation. abl depletion disrupted apical-basal polarity and adherens junction organization in mesoderm cells, suggesting that extruding cells undergo premature EMT. The polarity loss was associated with abnormal basolateral contractile actomyosin and Enabled (Ena) accumulation. Depletion of the Abl effector Enabled (Ena) in abl-depleted embryos suppressed the abl phenotype, consistent with cell extrusion resulting from misregulated ena Our work provides new insight into how Abl loss and Ena misregulation promote cell extrusion and EMT.
Project description:Fibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. We show here that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. It is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. We show that both movements are important for layer formation and present evidence suggesting that they are controlled by genetically distinct mechanisms.
Project description:Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.
Project description:FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.We find that pharmacological inhibition of FGFR activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of E-cadherin. E-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. FGFR inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. This includes members of the FGF, NOTCH, EPH, PDGF, and canonical and non-canonical WNT pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. SNAI2 expression in the primitive streak and mesoderm is not altered by FGFR inhibition, but is downregulated only in the preingression epiblast region with no significant effect on E-cadherin. Furthermore, over expression of SNAIL has no discernable effect on E-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. FGFR activity modulates distinct downstream pathways including RAS/MAPK and PI3K/AKT. Pharmacological inhibition of MEK or AKT indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. FGFR activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including RHOA, the non-canonical WNT pathway, PDGF signalling and the cell adhesion protein N-cadherin.In chicken embryos, FGF signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in E-cadherin expression or protein localization. The positive and negative effects on large groups of genes by pharmacological inhibition of FGF signalling, including major signalling pathways and transcription factor families, indicates that the FGF pathway is a focal point of regulation during gastrulation in chicken.
Project description:We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.
Project description:The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-? induced EMT and that long-term exposure to TGF-? elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-? induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-?-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor ?EF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-? and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-? and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.
Project description:Animal embryogenesis requires a precise coordination between morphogenesis and cell fate specification. During mesoderm induction, mesodermal fate acquisition is tightly coordinated with the morphogenetic process of epithelial-to-mesenchymal transition (EMT). In zebrafish, cells exist transiently in a partial EMT state during mesoderm induction. Here, we show that cells expressing the transcription factor Sox2 are held in the partial EMT state, stopping them from completing the EMT and joining the mesoderm. This is critical for preventing the formation of ectopic neural tissue. The mechanism involves synergy between Sox2 and the mesoderm-inducing canonical Wnt signaling pathway. When Wnt signaling is inhibited in Sox2-expressing cells trapped in the partial EMT, cells exit into the mesodermal territory but form an ectopic spinal cord instead of mesoderm. Our work identifies a critical developmental checkpoint that ensures that morphogenetic movements establishing the mesodermal germ layer are accompanied by robust mesodermal cell fate acquisition.
Project description:Epithelial-mesenchymal transition (EMT) is an essential morphogenetic process during embryonic development. It can be induced in vitro by hepatocyte growth factor/scatter factor (HGF/SF), or by FGF-1 in our NBT-II cell model for EMT. We tested for a central role in EMT of a zinc-finger protein called Slug. Slug mRNA and protein levels were increased transiently in FGF-1-treated NBT-II cells. Transient or stable transfection of Slug cDNA in NBT-II cells resulted in a striking disappearance of the desmosomal markers desmoplakin and desmoglein from cell-cell contact areas, mimicking the initial steps of FGF-1 or HGF/SF- induced EMT. Stable transfectant cells expressed Slug protein and were less epithelial, with increased cell spreading and cell-cell separation in subconfluent cultures. Interestingly, NBT-II cells transfected with antisense Slug cDNA were able to resist EMT induction by FGF-1 or even HGF/SF. This antisense effect was suppressed by retransfection with Slug sense cDNA. Our results indicate that Slug induces the first phase of growth factor-induced EMT, including desmosome dissociation, cell spreading, and initiation of cell separation. Moreover, the antisense inhibition experiments suggest that Slug is also necessary for EMT.
Project description:Epithelial-mesenchymal transition (EMT) or its reverse process mesenchymal-epithelial transition (MET) occurs in multiple physiological and pathological processes. However, whether an entire EMT-MET process exists and the potential function during human hematopoiesis remain largely elusive. Utilizing human pluripotent stem cell (hPSC)-based systems, it is discovered that while EMT occurs at the onset of human hematopoietic differentiation, MET is not detected subsequently during differentiation. Instead, a biphasic activation of mesenchymal genes during hematopoietic differentiation of hPSCs is observed. The expression of mesenchymal genes is upregulated during the fate switch from pluripotency to the mesoderm, sustained at the hemogenic endothelium (HE) stage, and attenuated during hemogenic endothelial cell (HEP) differentiation to hematopoietic progenitor cells (HPCs). A similar expression pattern of mesenchymal genes is also observed during human and murine hematopoietic development in vivo. Wnt signaling and its downstream gene SNAI1 mediate the up-regulation of mesenchymal genes and initiation of mesoderm induction from pluripotency. Inhibition of transforming growth factor-? (TGF-?) signaling and downregulation of HAND1, a downstream gene of TGF-?, are required for the downregulation of mesenchymal genes and the capacity of HEPs to generate HPCs. These results suggest that the biphasic regulation of mesenchymal genes is an essential mechanism during human hematopoiesis.
Project description:The oral, cervical, and genital mucosa, covered by stratified squamous epithelia with polarized organization and strong tight and adherens junctions, play a critical role in preventing transmission of viral pathogens, including human immunodeficiency virus (HIV). HIV-1 interaction with mucosal epithelial cells may depolarize epithelia and disrupt their tight and adherens junctions; however, the molecular mechanism of HIV-induced epithelial disruption has not been completely understood. We showed that prolonged interaction of cell-free HIV-1 virions, and viral envelope and transactivator proteins gp120 and tat, respectively, with tonsil, cervical, and foreskin epithelial cells induces an epithelial-mesenchymal transition (EMT). EMT is an epigenetic process leading to the disruption of mucosal epithelia and allowing the paracellular spread of viral and other pathogens. Interaction of cell-free virions and gp120 and tat proteins with epithelial cells substantially reduced E-cadherin expression and activated vimentin and N-cadherin expression, which are well-known mesenchymal markers. HIV gp120- and tat-induced EMT was mediated by SMAD2 phosphorylation and activation of transcription factors Slug, Snail, Twist1 and ZEB1. Activation of TGF-? and MAPK signaling by gp120, tat, and cell-free HIV virions revealed the critical roles of these signaling pathways in EMT induction. gp120- and tat-induced EMT cells were highly migratory via collagen-coated membranes, which is one of the main features of mesenchymal cells. Inhibitors of TGF-?1 and MAPK signaling reduced HIV-induced EMT, suggesting that inactivation of these signaling pathways may restore the normal barrier function of mucosal epithelia.