Regulation of Pattern Formation and Gene Amplification During Drosophila Oogenesis by the miR-318 microRNA

Pattern formation during epithelial development requires the coordination of multiple signaling pathways. Here, we investigate the functions of an ovary-enriched miRNA, miR-318, in epithelial development during Drosophila oogenesis. mir-318 maternal loss-of-function mutants were female-sterile and laid eggs with abnormal morphology. Removal of mir-318 disrupted the dorsal–anterior follicle cell patterning, resulting in abnormal dorsal appendages. mir-318 mutant females also produced thin and fragile eggshells due to impaired chorion gene amplification. We provide evidence that the ecdysone signaling pathway activates expression of miR-318 and that miR-318 cooperates with Tramtrack69 to control the switch from endocycling to chorion gene amplification during differentiation of the follicular epithelium. The multiple functions of miR-318 in oogenesis illustrate the importance of miRNAs in maintaining cell fate and in promoting the developmental transition in the female follicular epithelium.     

On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.     

Computational Modeling Reveals that a Combination of Chemotaxis and Differential Adhesion Leads to Robust Cell Sorting during Tissue Patterning

Robust tissue patterning is crucial to many processes during development. The "French Flag" model of patterning, whereby naïve cells in a gradient of diffusible morphogen signal adopt different fates due to exposure to different amounts of morphogen concentration, has been the most widely proposed model for tissue patterning. However, recently, using time-lapse experiments, cell sorting has been found to be an alternative model for tissue patterning in the zebrafish neural tube. But it remains unclear what the sorting mechanism is. In this article, we used computational modeling to show that two mechanisms, chemotaxis and differential adhesion, are needed for robust cell sorting. We assessed the performance of each of the two mechanisms by quantifying the fraction of correct sorting, the fraction of stable clusters formed after correct sorting, the time needed to achieve correct sorting, and the size variations of the cells having different fates. We found that chemotaxis and differential adhesion confer different advantages to the sorting process. Chemotaxis leads to high fraction of correct sorting as individual cells will either migrate towards or away from the source depending on its cell type. However after the cells have sorted correctly, there is no interaction among cells of the same type to stabilize the sorted boundaries, leading to cell clusters that are unstable. On the other hand, differential adhesion results in low fraction of correct clusters that are more stable. In the absence of morphogen gradient noise, a combination of both chemotaxis and differential adhesion yields cell sorting that is both accurate and robust. However, in the presence of gradient noise, the simple combination of chemotaxis and differential adhesion is insufficient for cell sorting; instead, chemotaxis coupled with delayed differential adhesion is required to yield optimal sorting.     

Blockade of T Cell Contact-Activation of Human Monocytes by High-Density Lipoproteins Reveals a New Pattern of Cytokine and Inflammatory Genes

Background

Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact.

Methodology/Principal Findings

To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNγ, CCL2, CCL5, TNF, IL-1β, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1β, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E₂—both involved in inflammatory response—were triggered by T cell contact and inhibited in the presence of HDL.

Conclusions/Significance

These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.     

The Pteridine Pathway in Zebrafish: Regulation and Specification during the Determination of Neural Crest Cell‐Fate

This review describes pteridine biosynthesis and its relation to the differentiation of neural crest derivatives in zebrafish. During the embryonic development of these fish, neural crest precursor cells segregate into neural elements, ectomesenchymal cells and pigment cells; the latter then diversifying into melanophores, iridophores and xanthophores. The differentiation of neural cells, melanophores, and xanthophores is coupled closely with the onset of pteridine synthesis which starts from GTP and is regulated through the control of GTP cyclohydrolase I activity. De novo pteridine synthesis in embryos of this species increases during the first 72‐h postfertilization, producing H₄biopterin, which serves as a cofactor for neurotransmitter synthesis in neural cells and for tyrosine production in melanophores. Thereafter, sepiapterin (6‐lactoyl‐7,8‐dihydropterin) accumulates as yellow pigment in xanthophores, together with 7‐oxobiopterin, isoxanthopterin and 2,4,7‐trioxopteridine. Sepiapterin is the key intermediate in the formation of 7‐oxopteridines, which depends on the availability of enzymes belonging to the xanthine oxidoreductase family. Expression of the GTP cyclohydrolase I gene (gch) is found in neural cells, in melanoblasts and in early xanthophores (xanthoblasts) of early zebrafish embryos but steeply declines in xanthophores by 42‐h postfertilization. The mechanism(s) whereby sepiapterin branches off from the GTP‐H₄biopterin pathway is currently unknown and will require further study. The surge of interest in zebrafish as a model for vertebrate development and its amenability to genetic manipulation provide powerful tools for analysing the functional commitment of neural crest‐derived cells and the regulation of pteridine synthesis in mammals.     

Regulation of Flagellar Morphogenesis by Temperature: Involvement of the Bacterial Cell Surface in the Synthesis of Flagellin and the Flagellum

The induction of beta-glucosidases (EC 3.2.1.21) was studied in Neurospora crassa. Cellobiase was induced by cellobiose, but other inducers had little effect on this enzyme. Cellobiase activity was very low in all stages of the vegetative life cycle in the absence of di-beta-glucoside inducer. Aryl-beta-glucosidase was semiconstitutive at late stages of culture growth prior to conidiation. At early stages, aryl-beta-glucosidase was induced by cellobiose, laminaribiose, and gentiobiose, and weakly induced by galactose, amino sugars, and aryl-beta-glucosides. The induction properties of the beta-glucosidases are compared with those of the other disaccharidases of Neurospora. The induction of beta-glucosidases was inhibited by glucose, 2-deoxy-d-glucose, and sodium acetate. Sodium phosphate concentrations between 0.01 and 0.1 M stimulated induction of both enzymes, while concentrations above 0.1 M were inhibitory. The optimal condition for induction of both beta-glucosidases was pH 6.0. Cellobiase induction was relatively more inhibited than aryl-beta-glucosidase in the range of pH 6.0 to 8.0.     

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development.     

Regulation of Morphogenesis and Biocontrol Properties in Trichoderma virens by a VELVET Protein,Vel1

Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.Trichoderma-based formulation products account for about 60% of the biofungicide market (35). Despite the use of Trichoderma-based biofungicides as an alternative and additive to chemical fungicides, the applications of these preparations are limited because their efficacy is lower than that of fungicides. A lack of understanding of the regulation of biocontrol has limited progress in enhancing the competitiveness of these fungi through genetic manipulation of desired traits. The success of a biocontrol agent also depends on the ability of researchers to develop an effective formulation based on active propagules that survive under the conditions that occur in nature and are effective against the target pathogens. Trichoderma spp. produce two types of propagules, conidia during solid-state fermentation and chlamydospores during liquid fermentation. Both types are used in commercial formulations depending on the growth conditions (17, 35). Thus, understanding how the two sporulation pathways are controlled is critical for obtaining an improved, balanced formulation product. Identification of a global regulator of morphogenesis and biocontrol properties (such as antibiosis and mycoparasitism) would provide an opportunity to manipulate the morphogenetic and antagonistic traits, leading to wider commercial acceptance of Trichoderma spp. in the long run.Trichoderma virens is a commercially formulated biocontrol agent that is effective against soilborne plant pathogens, such as Rhizoctonia solani, Sclerotium rolfsii, and Pythium spp.; its major direct mode of action is antibiosis and mycoparasitism (20, 36). This species has also been used as a model system for studies of biocontrol mechanisms, and the genome has recently been sequenced (http://genome.jgi-psf.org/Trive1). The role of beta-glucanases, chitinases, and proteases in biocontrol has been reported previously (2, 8, 29). Some strains of T. virens (designated Q strains) produce copious amounts of the antibiotic gliotoxin that is involved in biocontrol (10, 12, 39). In an attempt to identify regulators of biocontrol properties, the role of a mitogen-activated protein kinase (MAPK) pathway was studied previously (22, 24). Deletion of the TmkA/Tvk1 MAPK gene resulted in derepressed conidiation and different biocontrol behavior for two strains of T. virens; Mukherjee et al. (24) noted the reduced ability of these mutants to parasitize the sclerotia of S. rolfsii and R. solani, while Mendoza-Mendoza et al. (22) found that deletion of this MAPK gene improved the biocontrol activity of T. virens against R solani and P. ultimum. The production of secondary metabolites was not affected by deletion of this gene. To date, no gene that regulates the balance between conidiation or chlamydospore formation, secondary metabolism, and antagonistic or biocontrol properties has been identified in any Trichoderma sp.The Vel1 VELVET protein has been shown to be a regulator of morphogenesis and secondary metabolism in some filamentous fungi (6). In Aspergillus nidulans, VeA physically interacts with VelB and the regulator of secondary metabolism LaeA to form a complex that regulates secondary metabolism and sexual reproduction (3). Deletion of the VeA gene leads to an increase in asexual development (conidiation in the dark) and reduced biosynthesis of sterigmatocystin (the product of a polyketide synthetase [PKS]) and penicillin (the product of a nonribosomal peptide synthetase [NRPS]), while it reduces and delays sexual reproduction (15, 16). VeA is also required for the production of sclerotia and for aflatoxin biosynthesis in Aspergillus parasiticus (7). Deletion of the VeA gene in Neurospora crassa, like deletion of the VeA gene in A. nidulans, results in deregulated conidiation, while in Acremonium chrysogenum, loss of VeA leads to increased hyphal fragmentation and reduced cephalosporin production (4, 9). Deletion of the VeA gene in Fusarium verticilliodes resulted in a loss of hydrophobicity and an increased macroconidium-to-microconidium ratio; these defects could be restored by growing the organism on osmotically stabilized media (18). The mutants were also defective in production of the mycotoxins fumonisin and fusarin (25).To test the hypothesis that Vel1 is a global regulator of gene expression in T. virens, we examined the functions of Vel1 in this organism by using gene knockout and complementation. Here we report that in addition to a role in conidiation and secondary metabolism, Vel1 also regulates conidiophore aggregation, chlamydosporogenesis, mycoparasitism, and biocontrol efficacy in T. virens. Thus, we identified the first master regulator of morphogenesis and antagonistic properties in this economically important fungus.     

Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.     

Dpysl4 Is Involved in Tooth Germ Morphogenesis through Growth Regulation,Polarization and Differentiation of Dental Epithelial Cells

Dihydropyrimidinase-related protein 4 (Dpysl4) is a known regulator of hippocampal neuron development. Here, we report that Dpysl4 is involved in growth regulation, polarization and differentiation of dental epithelial cells during tooth germ morphogenesis. A reduction in Dpysl4 gene expression in the tooth germ produced a loss of ameloblasts, resulting in the decrease of synthesis and secretion of enamel. The inhibition of Dpysl4 gene expression led to promotion of cell proliferation of inner enamel epithelial cells and inhibition of the differentiation of these cells into pre-ameloblasts, which was confirmed by analyzing cell polarization, columnar cell structure formation and the expression of ameloblast marker genes. By contrast, overexpression of Dpysl4 in dental epithelial cells induces inhibition of growth and increases the expression of the inner enamel epithelial cell marker gene, Msx2. These findings suggest that Dpysl4 plays essential roles in tooth germ morphogenesis through the regulation of dental epithelial cell proliferation, cell polarization and differentiation.     

Death Receptor-Induced Apoptosis Signalling Regulation by Ezrin Is Cell Type Dependent and Occurs in a DISC-Independent Manner in Colon Cancer Cells

Ezrin belongs to the ERM (ezrin-radixin-moesin) protein family and has been demonstrated to regulate early steps of Fas receptor signalling in lymphoid cells, but its contribution to TRAIL-induced cell death regulation in adherent cancer cells remains unknown. In this study we report that regulation of FasL and TRAIL-induced cell death by ezrin is cell type dependant. Ezrin is a positive regulator of apoptosis in T-lymphoma cell line Jurkat, but a negative regulator in colon cancer cells. Using ezrin phosphorylation or actin-binding mutants, we provide evidence that negative regulation of death receptor-induced apoptosis by ezrin occurs in a cytoskeleton- and DISC-independent manner, in colon cancer cells. Remarkably, inhibition of apoptosis induced by these ligands was found to be tightly associated with regulation of ezrin phosphorylation on serine 66, the tumor suppressor gene WWOX and activation of PKA. Deficiency in WWOX expression in the liver cancer SK-HEP1 or the pancreatic Mia PaCa-2 cell lines as well as WWOX silencing or modulation of PKA activation by pharmacological regulators, in the colon cancer cell line SW480, abrogated regulation of TRAIL signalling by ezrin. Altogether our results show that death receptor pro-apoptotic signalling regulation by ezrin can occur downstream of the DISC in colon cancer cells.     

Peri-gastrulation development of the dasyurid marsupial Sminthopsis macroura (stripe-faced dunnart) in vitro and evidence for patterning of the epiblast prior to gastrulation

Marsupials are potentially excellent models for the study of gastrulation because of their superficial embryonic area (EA), post-gastrulation implantation and their potential to provide information about the evolution of gastrulation. Very few studies have examined this developmental period in marsupials. Using an established developmental timetable, peri-gastrula stage Sminthopsis macroura blastocysts were collected and described in detail by observations on live blastocysts and by the use of histological and immunohistochemical techniques on fixed blastocysts. Gastrulation in S. macroura shares several aspects common to that of both eutherian mammals and birds, but in terms of tissue arrangement and conceptus form, is more similar to the chick than other mammals. Two methods of culturing EA explants flat without their shell were devised. The techniques will markedly increase the number of possible experimental manipulations, which previously were limited by the presence of blastocyst investments. Exposure of fractions of explants of round, morphologically uniform pre-gastrula stage EA to growth factors or signaling molecules implicated in vertebrate gastrulation suggests that like the chick and mouse, the marsupial epiblast is patterned prior to gastrulation. Of all factors tested, basic fibroblast growth factor (bFGF) had the most prominent effect, promoting cell differentiation, and possible mesoderm formation. Data from explant culture suggests that similar to the chick and mouse, limited specification precedes the onset of gastrulation.     

Diabetic HDL Is Dysfunctional in Stimulating Endothelial Cell Migration and Proliferation Due to Down Regulation of SR-BI Expression

Background

Diabetic HDL had diminished capacity to stimulate endothelial cell (EC) proliferation, migration, and adhesion to extracellular matrix. The mechanism of such dysfunction is poorly understood and we therefore sought to determine the mechanistic features of diabetic HDL dysfunction.

Methodology/Principal Findings

We found that the dysfunction of diabetic HDL on human umbilical vein endothelial cells (HUVECs) was associated with the down regulation of the HDL receptor protein, SR-BI. Akt-phosphorylation in HUVECs was induced in a biphasic manner by normal HDL. While diabetic HDL induced Akt phosphorylation normally after 20 minutes, the phosphorylation observed 24 hours after diabetic HDL treatment was reduced. To determine the role of SR-BI down regulation on diminished EC responses of diabetic HDL, Mouse aortic endothelial cells (MAECs) were isolated from wild type and SR-BI (−/−) mice, and treated with normal and diabetic HDL. The proliferative and migratory effects of normal HDL on wild type MAECs were greatly diminished in SR-BI (−/−) cells. In contrast, response to diabetic HDL was impaired in both types suggesting diminished effectiveness of diabetic HDL on EC proliferation and migration might be due to the down regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.

Conclusions/Significance

Diabetic HDL was dysfunctional in promoting EC proliferation, migration, and adhesion to matrix which was associated with the down-regulation of SR-BI. Additionally, SR-BI down regulation diminishes diabetic HDL’s capacity to activate Akt chronically.     

卵黄蛋白原1(vtg1)启动子调控绿色荧光蛋白表达的转基因斑马鱼的构建

环境雌激素严重危害人类的健康.为了简便直观地检测水环境中的雌激素污染,构建了一种转基因斑马鱼,在这种鱼的体内,利用卵黄蛋白原1(vitellogenin1,vtg1)的启动子调控报告基因绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)的表达.用1ng/L的17!-炔雌醇(17"-ethynylestradiol,EE2)诱导4天后,仔鱼肝脏中出现绿色荧光.RT-PCR和整体原位杂交实验证实,仔鱼体内EGFP和vtg1的时空表达模式相同.通过在显微镜下观察转基因鱼的绿色荧光,可以直观判断水环境中是否含有雌激素活性物质.该研究为环境雌激素的监测提供了一种简便直观的新型工具.     

Transmission of Surface Pattern Through a Dedifferentiated Stage in the Ciliate Paraurostyla. Evidence from the Analysis of Microtubule and Basal Body Deployment

ABSTRACT. During conjugation of the hypotrich ciliate Paraurostyla weissei , the two partners fuse to form a transient dedifferentiated stage, the zygocyst, which later redifferentiates into a vegetative cell. Immunocytochemical studies have been performed to follow the deployment of microtubules and basal bodies during the entire cycle of conjugation. They show that a superficial lattice persists during the whole zygocyst stage, after most of the infraciliature of the exconjugants has been disassembled. These superficial microtubules display different immunocytochemical properties in the mature zygocyst and during its morphogenesis, suggesting that some transient chemical modifications of the microtubules are associated with the morphogenetic activity. In the zygocyst, the superficial microtubules retain the specific orientation characteristic of the ventral and the dorsal sides of the recipient cell, respectively. In the course of subsequent morphogenesis of the zygocyst, these specific cellular territories differentiate into the ventral and dorsal sides of the new cell. Although our experiments do not resolve the question of whether superficial microtubules play an active or merely a passive role in the transmission of surface pattern, they show that no complete breakdown in cell polarity occurs, even through a profound dedifferentiated stage. Thus, the overall surface pattern appears to be retained, in a simplified form, through the conjugation cycle.