Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

诱导心脏发生的早期信号通路

心脏是胚胎发生过程中最早形成的器官 .心脏前体的特化是组织间及细胞与细胞之间相互作用的结果 ,这一过程包含了诱导信号作用的时间和空间完整程序 .以脊椎动物和无脊椎动物作为模式动物 ,总结了在早期心脏发生中发挥重要作用的诱导信号通路 :BMP Dpp ,Wnt Wingless ,FGF及Notch信号通路 ,并阐述了信号通路之间的通讯 (crosstalk)以及信号通路与心脏发生相关的关键转录调节因子之间的协同诱导作用 .     

scalloped functions in a regulatory loop with vestigial and wingless to pattern the Drosophila wing

 The development of the Drosophila wing involves progressive patterning events. In the second larval instar, cells of the wing disc are allotted wing or notum fates by a wingless-mediated process and dorsal or ventral fates by the action of apterous and wingless. Notch-mediated signalling is required for the expression of the genes vestigial and scalloped in the presumptive wing blade. Later, wingless, Notch and cut are involved in cell fate specification along the wing margin. The function of scalloped in this process is not well understood and is the focus of this study. We show that patterning downstream of Notch and wingless pathways is altered in scalloped mutants. Reduction in scalloped expression results in a loss of expression of wing blade- and margin-specific markers. Misexpression of scalloped in the presumptive wing causes misexpression of scalloped, vestigial and wingless reporter genes. However, high levels of scalloped expression have a negative influence on wingless, vestigial and its own expression. Our results demonstrate that scalloped functions in a level-dependent manner in the presumptive wing blade in a loop that involves vestigial and itself. We suggest that wing development requires the regulated expression of scalloped together with vestigial–the ”wing formation” effects of Vestigial in other imaginal discs are probably due to its interaction with the scalloped gene product normally expressed in these discs. Received: 6 May 1998 / Accepted: 22 July 1998     

Calphostin C induces tyrosine dephosphorylation/inactivation of protein kinase Fa/GSK-3α in a pathway independent of tumor promoter phorbol ester-mediated down-regulation of protein kinase C

The signal transduction mechanism of protein kinase FA /GSK-3α by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase Fa /GSK-3α could be tyrosine-dephosphorylated and inactivated to ∼ 10% of control in a concentration-dependent manner by 0.1–10 μM calphostin C (IC₅₀, ∼ 1 μM), as demonstrated by immunoprecipitation of kinase Fa /GSK-3α from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase Fa /GSK-3α immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 μM calphostin C (IC₅₀, ∼ 0.05 μM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase Fa /GSK-3α, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase Fa /GSK-3α in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase Fa /GSK-3α is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA /GSK-3α in tumor cells. © 1996 Wiley-Liss, Inc.     

Endothelial cells promote the proliferation of lymphocytes partly through the Wnt pathway via LEF-1

The function of T cells and B cells is to recognize specific “non-self” antigens, during a process known as antigen presentation. Once they have identified an invader, the cells generate specific responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells. Endothelial cells (ECs) can trigger the activation of T cells through their class I and class II MHC molecules. In this study, we examined the effect of ECs on the proliferation of lymphocytes. We report that the proliferation of T and B cells can be improved by interaction with ECs. LEF-1 is one of the main molecular mediators in this process, and the inhibition of LEF-1 induces apoptosis. These results suggest that LEF-1 modulates positively the proliferation of lymphocytes induced by their interaction with ECs.     

Dysfunction of protein kinase FA/GSK-3α in lymphocytes of patients with schizophrenic disorder

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP. Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA /glycogen synthase kinase-3α (kinase FA /GSK-3α) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA /GSK-3α immunoblotting and kinase activity analysis in an anti-kinase FA /GSK-3α immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA /GSK-3α in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA /GSK-3α activity in the high levels of 14.8 ± 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA /GSK-3α activity in the low levels of 2.8 ± 1.6 units/mg, indicating that the different levels of kinase FA /GSK-3α activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide intial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA /GSK-3α, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes. © 1995 Wiley-Liss, Inc.     

Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of beta-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3beta (GSK-3beta) and inhibition of GSK-3beta attenuated the DIF-1-induced beta-catenin degradation, indicating the involvement of GSK-3beta in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of cyclin D1 promoter activity.     

Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg

 Genetically mosaic flies were constructed which lack a functional decapentaplegic (dpp) or wingless (wg) gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has previously been shown to be transcribed both ventrally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally – a result that was expected, given that wg is only expressed ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications confined to individual leg segments. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg-null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both dpp and wg are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene’s null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another. Received: 25 March 1996 / Accepted: 13 June 1996     

流体剪切力对成骨细胞LEF-1蛋白表达的影响

目的：探讨MC3T3-E1细胞在流体剪切力作用下LEF-1的表达。方法：通过流体剪切加载系统对MC3T3-E1爬片细胞施加12dyn/cm的流体剪切力,分别作用0h,2h,4h,8h,12h,用RT-PCR方法检测细胞受力前后LEF-1 mRNA表达的变化;应用免疫荧光双标记法检测不同时间点流体剪切力作用下MC3T3-E1细胞中的LEF-1 mRNA表达改变。结果：RT-PCR和免疫荧光双标记法的结果表明12dyn/cm 8h流体剪切力作用下的MC3T3-E1细胞LEF-1 mRNA的表达较其它各组明显增强。结论：通过流体剪切力力学刺激,激活了成骨细胞LEF-1/TCF1转录活动,LEF-1 mRNA的表达增强可能是成骨细胞经典Wnt信号通路对剪切应力的应答反应。     

Activation of Akt/GSK-3β signaling pathway is involved in intermedin1-53 protection against myocardial apoptosis induced by ischemia/reperfusion

Intermedin (IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. We investigated the cardioprotective mechanism of IMD_1-53 in the in vivo rat model of myocardial ischemia/reperfusion (I/R) injury and in vitro primary neonatal cardiomyocyte model of hypoxia/reoxygenation (H/R). Myocardial infarct size was measured by 2,3,5-triphenyl tetrazolium chloride staining. Cardiomyocyte viability was determined by trypan blue staining, cell injury by lactate dehydrogenase (LDH) leakage, and cardiomyocyte apoptosis by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay, Hoechst staining, gel electrophoresis and caspase 3 activity. The translocation of mitochondrial cytochrome c of myocardia and expression of apoptosis-related factors Bcl-2 and Bax, phosphorylated Akt and phosphorylated GSK-3β were determined by western blot analysis. IMD_1-53 (20 nmol/kg) limited the myocardial infarct size in rats with I/R; the infarct size was decreased by 54%, the apoptotic index by 30%, and caspase 3 activity by 32%; and the translocation of cytochrome c from mitochondria to cytosol was attenuated. IMD_1-53 increased the mRNA and protein expression of Bcl-2 and ratio of Bcl-2 to Bax by 81 and 261%, respectively. IMD_1-53 (1 × 10⁻⁷ mol/L) inhibited the H/R effect in cardiomyocytes by reducing cell death by 43% and LDH leakage by 16%; diminishing cellular apoptosis; decreasing caspase 3 activity by 50%; and increasing the phosphorylated Akt and GSK-3β by 41 and 90%, respectively. The cytoprotection of IMD_1-53 was abolished with LY294002, a PI3K inhibitor. In conclusion, IMD_1-53 exerts cardioprotective effect against myocardial I/R injury through the activation of the Akt/GSK-3β signaling pathway to inhibit mitochondria-mediated myocardial apoptosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.