Rac1 is essential for basement membrane-dependent epiblast survival.
ABSTRACT: During murine peri-implantation development, the egg cylinder forms from a solid cell mass by the apoptotic removal of inner cells that do not contact the basement membrane (BM) and the selective survival of the epiblast epithelium, which does. The signaling pathways that mediate this fundamental biological process are largely unknown. Here we demonstrate that Rac1 ablation in embryonic stem cell-derived embryoid bodies (EBs) leads to massive apoptosis of epiblast cells in contact with the BM. Expression of wild-type Rac1 in the mutant EBs rescues the BM-contacting epiblast, while expression of a constitutively active Rac1 additionally blocks the apoptosis of inner cells and cavitation, indicating that the spatially regulated activation of Rac1 is required for epithelial cyst formation. We further show that Rac1 is activated through integrin-mediated recruitment of the Crk-DOCK180 complex and mediates BM-dependent epiblast survival through activating the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Our results reveal a signaling cascade triggered by cell-BM interactions essential for epithelial morphogenesis.
Project description:During early embryogenesis, endodermal ?1-laminin expression is required for basement membrane (BM) assembly, promoting conversion of non-polar pluripotent cells into polarized epiblast. The influence of laminin-111 (Lm111) and its integrin and dystroglycan (DG) receptors on epiblast in embryoid bodies (EBs), a model for differentiation of the embryonic plate, was further investigated. Lm111 added to the medium of EBs initiated conversion of inner nonpolar cell to the polarized epiblast epithelium with an exterior-to-central basal-to-apical orientation. Microinjection of Lm111 into EB interiors resulted in an interior BM with complete inversion of cell polarity. Lm111 assembled a BM on integrin-?1 null EBs with induction of polarization at reduced efficiency. ?-Integrin compensation was not detected in these nulls with integrin adaptor proteins failing to assemble. A dimer of laminin LG domains 4-5 (LZE3) engineered to strongly bind to ?-dystroglycan almost completely inhibited laminin accumulation on integrin ?1-null EBs, reducing BM and ablating cell polarization. When Lm111 was incubated with integrin-?1/dystroglycan double-knockout EBs, laminin failed to accumulate on the EBs, the EBs did not differentiate, and the EBs underwent apoptosis. Collectively the findings support the hypotheses that the locus of laminin cell surface assembly can determine the axis of epithelial polarity. This requires integrin- and/or dystroglycan-dependent binding to laminin LG domains with the highest efficiency achieved when both receptors are present. Finally, EBs that cannot assemble a matrix undergo apoptosis.
Project description:To explore genes involved in epiblast development, we have employed whole genome microarray expression profiling as a discovery platform to identify genes. Embryoid body (EB) formation induces embryonic stem (ES) cells to differentiate into germ layers via epiblasts and extra-embryonic tissues, whereas ES cells give rise only to extra-embryonic tissues in monolayer culture. We compared gene expression profiles of ES cells in each differentiation culture methods at day 1-3 or 4 of differentiation. The expression of organizer related genes (Lhx1, Cer1 and Foxa2) was specifically induced at day 3 of EBs, but not in monolayer culture. In epiblast model cells, the overexpression of Lhx1 induced gene expression of organizer and meso-endodermal markers, suggested that Lhx1 was related to epiblast differentiation. Gene expression related epiblast development was measured at undifferentiated state and day 1-3 or 4 of differentiation. Two independent experiments were performed at each time using different people for each experiment.
Project description:Glioblastoma, the most common primary malignant cancer of the brain, is characterized by rapid tumor growth and infiltration of tumor cells throughout the brain. These traits cause glioblastomas to be highly resistant to current therapies with a resultant poor prognosis. Although aberrant oncogenic signaling driven by signature genetic alterations, such as EGF receptor (EGFR) gene amplification and mutation, plays a major role in glioblastoma pathogenesis, the responsible downstream mechanisms remain less clear. Here, we report that EGFRvIII (also known as ΔEGFR and de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in human glioblastoma, promotes tumorigenesis through Src family kinase (SFK)-dependent phosphorylation of Dock180, a guanine nucleotide exchange factor for Rac1. EGFRvIII induces phosphorylation of Dock180 at tyrosine residue 722 (Dock180(Y722)) and stimulates Rac1-signaling, glioblastoma cell survival and migration. Consistent with this being causal, siRNA knockdown of Dock180 or expression of a Dock180(Y722F) mutant inhibits each of these EGFRvIII-stimulated activities. The SFKs, Src, Fyn, and Lyn, induce phosphorylation of Dock180(Y722) and inhibition of these SFKs by pharmacological inhibitors or shRNA depletion markedly attenuates EGFRvIII-induced phosphorylation of Dock180(Y722), Rac1 activity, and glioblastoma cell migration. Finally, phosphorylated Dock180(Y722) is coexpressed with EGFRvIII and phosphorylated Src(Y418) in clinical specimens, and such coexpression correlates with an extremely poor survival in glioblastoma patients. These results suggest that targeting the SFK-p-Dock180(Y722)-Rac1 signaling pathway may offer a novel therapeutic strategy for glioblastomas with EGFRvIII overexpression.
Project description:GATA-6 is a zinc-finger transcription factor essential for early embryogenesis. Ablation of GATA-6 in mice impairs endoderm differentiation and causes apoptosis of epiblast cells. The endoderm defects have been attributed to the loss of HNF4, disabled-2, and GATA-4. However, the mechanisms underlying epiblast apoptosis are unclear. In this study we used mouse embryonic stem cell-derived embryoid bodies (EBs) as a model for peri-implantation development and found that ablation of GATA-6 causes massive apoptosis during EB differentiation. Endoderm grafting experiments and ectopic basement membrane (BM) assembly suggest that both BM and non-BM factors contribute to cell survival. Furthermore, the increased cell death in mutant EBs is accompanied by reduced expression of bone morphogenetic protein 2 (BMP-2). Chromatin immunoprecipitation reveals direct binding of GATA-6 to the Bmp2 promoter. Treatment of the mutant EBs with BMP-2 markedly suppresses apoptosis, whereas stable overexpression of the BMP antagonist noggin or a dominant-negative BMP receptor in normal EBs leads to increased apoptosis. Last, activation of SMAD1/5 by phosphorylation is significantly inhibited in the absence of GATA-6, and this is reversed by exogenous BMP-2. Treatment of normal EBs with SMAD phosphorylation inhibitor increases apoptosis. Collectively these results suggest that GATA-6 promotes cell survival by regulating endoderm expression of BMP-2 and BM during embryonic epithelial morphogenesis.
Project description:CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135- to 145-, 160-, and 180-kDa proteins. The 135- to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.
Project description:Amniote epiblast cells differentiate into mesoderm and endoderm lineages during gastrulation through a process called epithelial-to-mesenchymal transition (EMT). Molecular regulation of gastrulation EMT is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules to the basal cortex via CLIP-associated protein (CLASP), a microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation epiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediated through the CLASP-binding partner LL5. Live-imaging using EB1-enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilized basal microtubules. We further show that Dystroglycan is localized to basolateral membrane in epiblast cells. Basal but not lateral localization of Dystroglycan was regulated by CLASP. We propose that epiblast-BM interaction requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which initiates gastrulation EMT.
Project description:Netrins are prototypical axon guidance cues whose attractive signaling requires the small GTPase Rac1. It remains unclear how Rac1 is regulated in the netrin pathway. DOCK180 is a member of a new family of guanine nucleotide exchange factors for Rho GTPases. Here we provide evidence implicating DOCK180 in netrin signal transduction. Netrin promoted the formation of a protein-protein interaction complex that included DOCK180 and the netrin receptor deleted in colorectal carcinoma (DCC). Inhibition of DOCK180 reduced activation of Rac1 by netrin. Both axon outgrowth and axon attraction induced by netrin were inhibited after DOCK180 knockdown in vertebrate neurons. The in vivo functional role of DOCK180 was demonstrated by its requirement for projection of commissural axons in the neural tube. These findings indicate that netrin stimulation recruits DOCK180 through DCC, which then activates small GTPases, suggesting an essential role for DOCK180 in mediating attractive responses by neurons to netrin-1.
Project description:Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised.
Project description:Hepatocyte growth factor (HGF) is a potent signaling factor that acts on epithelial cells, causing them to dissociate and scatter. This migration is coordinated by a number of small GTPases, such as ARF6 and Rac1. Active ARF6 is required for HGF-stimulated migration and intracellular levels of ARF6-GTP and Rac1-GTP increase following HGF treatment. During migration, cross talk between ARF6 and Rac1 occurs through formation of a multi-protein complex containing the ARF-GEF cytohesin-2, the scaffolding protein GRASP/Tamalin, and the Rac1-GEF Dock180. Previously, the role of ARF6 in this process was unclear. We have now found that ARF6 and ARF1 regulate trafficking of GRASP and Dock180 to the plasma membrane following HGF treatment. Trafficking of GRASP and Dock180 is impaired by blocking ARF6-mediated recycling pathways and is required for HGF-stimulated Rac1 activation. Finally, HGF treatment stimulates association of GRASP and Dock180. Inhibition of ARF6 trafficking pathways traps GRASP and Dock180 as a complex in the cell.
Project description:Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on second-lineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)? pathway. Inhibition of the TGF? pathway from the two-cell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGF? signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGF? pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways--TGF?, GSK3?, and the fibroblast growth factor (FGF)/extracellular signal-regulated kinases (Erk)--significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGF? pathway alone or by combined inhibition of the GSK3? and FGF/Erk pathways only.