Direct Inhibition of Pumilo Activity by Bam and Bgcn in Drosophila Germ Line Stem Cell Differentiation

The fate of stem cells is intricately regulated by numerous extrinsic and intrinsic factors that promote maintenance or differentiation. The RNA-binding translational repressor Pumilio (Pum) in conjunction with Nanos (Nos) is required for self-renewal, whereas Bam (bag-of-marbles) and Bgcn (benign gonial cell neoplasm) promote differentiation of germ line stem cells in the Drosophila ovary. Genetic analysis suggests that Bam and Bgcn antagonize Pum/Nos function to promote differentiation; however, the molecular basis of this epistatic relationship is currently unknown. Here, we show that Bam and Bgcn inhibit Pum function through direct binding. We identified a ternary complex involving Bam, Bgcn, and Pum in which Bam, but not Bgcn, directly interacts with Pum, and this interaction is greatly increased by the presence of Bgcn. In a heterologous reporter assay to monitor Pum activity, Bam, but not Bgcn, inhibits Pum activity. Notably, the N-terminal region of Pum, which lacks the C-terminal RNA-binding Puf domain, mediates both the ternary protein interaction and the Bam inhibition of Pum function. These studies suggest that, in cystoblasts, Bam and Bgcn may directly inhibit Pum/Nos activity to promote differentiation of germ line stem cells.     

Sex-lethal Facilitates the Transition From Germline Stem Cell to Committed Daughter Cell in the Drosophila Ovary

In Drosophila, the female-specific SEX-LETHAL (SXL) protein is required for oogenesis, but how Sxl interfaces with the genetic circuitry controlling oogenesis remains unknown. Here we use an allele of sans fille (snf) that specifically eliminates SXL protein in germ cells to carry out a detailed genetic and cell biological analysis of the resulting ovarian tumor phenotype. We find that tumor growth requires both Cyclin B and zero population growth, demonstrating that these mutant cells retain at least some of the essential growth-control mechanisms used by wild-type germ cells. Using a series of molecular markers, we establish that while the tumor often contains at least one apparently bona fide germline stem cell, the majority of cells exhibit an intermediate fate between a stem cell and its daughter cell fated to differentiate. In addition, snf tumors misexpress a select group of testis-enriched markers, which, remarkably, are also misexpressed in ovarian tumors that arise from the loss of bag of marbles (bam). Results of genetic epistasis experiments further reveal that bam's differentiation-promoting function depends on Sxl. Together these data demonstrate a novel role for Sxl in the lineage progression from stem cell to committed daughter cell and suggest a model in which Sxl partners with bam to facilitate this transition.     

A Progressive Somatic Cell Niche Regulates Germline Cyst Differentiation in the Drosophila Ovary

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Genetic Characterization of Small Ovaries, a Gene Required in the Soma for the Development of the Drosophila Ovary and the Female Germline

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.     

gone early,a Novel Germline Factor,Ensures the Proper Size of the Stem Cell Precursor Pool in the Drosophila Ovary

In order to sustain lifelong production of gametes, many animals have evolved a stem cell–based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell–cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.     

Germline hypermutability in Drosophila and its relation to hybrid dysgenesis and cytotype

In its hypermutable state, an unstable singed allele, sn^w, mutates in the germline to two other alleleic forms at a total frequency usually between 40 and 60%. In its stable state, the mutation rate of sn^w is essentially zero. Its state depends on an extrachromosomal condition indistinguishable from a property called cytotype previously studied as a component of hybrid dysgenesis. Of the two known systems of hybrid dysgenesis, denoted P-M and I-R, sn^w hypermutability is determined by the P-M system and appears to be independent of the I-R system. Cytotype, as defined by the control of sn^w mutability, is self-reproducing in the cytoplasm or nucleoplasm of the germline through at least two generations. However, it is not entirely autonomous, being ultimately determined by the chromosomes after sufficiently many generations of backcrossing. This combination of chromosomal and extrachromosomal transmission agrees well with previous studies on cytotype. Temperature differences have little effect on the mean mutation rates, but they have a pronounced effect on the intrinsic variance among individuals. The latter effect suggests that high temperatures reduce germ-cell survival during the development of dysgenic flies. Chromosomal rearrangements produce no apparent effects on the behavior of sn^w. Hypermutability is thought to be caused by the excision or other alteration of an inserted genetic element in the sn^w gene. This element might be a copy of the "P factor," which is though to be a mobile sequence capable of causing female sterility and other dysgenic traits in the P-M system.     

Selection and Maintenance of Sexual Identity in the Drosophila Germline

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.     

Germline transformation of the spotted wing drosophilid, Drosophila suzukii, with a piggyBac transposon vector

Drosophila suzukii is a pest of small fruits in many parts of the world, whose management is limited to cultural practices and the use of insecticides. Here we describe a method to genetically manipulate this species in the first step to create female lethality strains useful for the sterile insect technique method of population suppression. This was achieved by the germ-line transformation of D. suzukii with a piggyBac transposon vector having a female-specific lethality effector construct. This can be used in a tetracycline-suppressible conditional gene expression system, when crossed to a suitable tet-transactivator strain. Transformation occurred efficiently, at a frequency of 16 % per fertile G0 embryo injected with vector and helper transposase plasmids. The vector was marked for transformant selection with the polyubiquitin-regulated EGFP fluorescent protein, and contains the attP landing site and heterospecific lox recombination sites for post-integration modification of the transgene vector. The 3xP3-AmCyan fluorescent protein marker was inserted within the lox sites to follow a possible recombinase-mediated cassette exchange, that would allow subsequent improvement of the transgenic strain by immobilization of the vector and introduction of new marker cassettes.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 –differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 –differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.     

在小鼠早期胚胎发育和种系中特异表达的转录因子OCT4

在小鼠早期胚胎发育和种系中特异表达的转录因子ＯＣＴ４童英尚克刚（北京大学生命科学学院,北京１００８７１）ＯＣＴ４：ａＴｒａｎｓｃｒｉｐｔｉｏｎＦａｃｔｏｒＥｘｐｒｅｓｅｄＳｐｅｃｉｆｉｃａｌｙｉｎＭｏｕｓｅＥａｒｌｙＤｅｖｅｌｏｐｍｅｎｔａｎｄＧｅｒ...     

A Heterodimer of Thioredoxin and IB2 Cooperates with Sec18p (NSF) to Promote Yeast Vacuole Inheritance

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as I^B₂, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts. Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B. Both Sec18p and LMA1 act at an early stage of the in vitro reaction. Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p. Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1. The action of LMA1 and Sec18p is thus coupled and ordered. These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.     

Nucleotide Variation and Conservation at the Dpp Locus, a Gene Controlling Early Development in Drosophila

A study of polymorphism and species divergence of the dpp gene of Drosophila has been made. Eighteen lines from a population of D. melanogaster were sequenced for 5200 bp of the Hin region of the gene, coding for the dpp polypeptide. A comparison was made with sequence from D. simulans. Ninety-six silent polymorphisms and three amino acid replacement polymorphisms were found. The overall silent polymorphism (0.0247) is low, but haplotype diversity (0.0066 for effectively silent sites and 0.0054 for all sites) is in the range found for enzyme loci. Amino acid variation is absent in the N-terminal signal peptide, the C-terminal TGF-β peptide and in the N-terminal half of the pro-protein region. At the nucleotide level there is strong conservation in the middle half of the large intron and in the 3' untranslated sequence of the last exon. The 3' untranslated conservation, which is perfect for 110 bp among all the divergent species, is unexplained. There is strong positive linkage disequilibrium among polymorphic sites, with stretches of apparent gene conversion among originally divergent sequences. The population apparently is a migration mixture of divergent clades.     

Stand Still, a Drosophila Gene Involved in the Female Germline for Proper Survival, Sex Determination and Differentiation

We identified a new gene, stand still (stil), required in the female germline for proper survival, sex determination and differentiation. Three strong loss-of-function alleles were isolated. The strongest phenotype exhibited by ovaries dissected from adult females is the complete absence of germ cells. In other ovaries, the few surviving germ cells frequently show a morphology typical of primary spermatocytes. still is not required either for fly viability or for male germline development. The gene was cloned and found to encode a novel protein. still is strongly expressed in the female germ cells. Using P[stil(+)] transgenes, we show that stil and a closely localized gene are involved in the modification of the ovarian phenotypes of the dominant alleles of ovo caused by heterozygosity of region 49 A-D. The similarity of the mutant phenotypes of stil to that of otu and ovo suggests that the three genes function in a common or in parallel pathways necessary in the female germline for its survival, sex determination and differentiation.     

Drosophila Lipid Droplets Buffer the H2Av Supply to Protect Early Embryonic Development

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Heat Shock Factor Hsf1 Cooperates with ErbB2 (Her2/Neu) Protein to Promote Mammary Tumorigenesis and Metastasis

ErbB2/Neu oncogene is overexpressed in 25% of invasive/metastatic breast cancers. We have found that deletion of heat shock factor Hsf1 in mice overexpressing ErbB2/Neu significantly reduces mammary tumorigenesis and metastasis. Hsf1^+/−ErbB2/Neu⁺ tumors exhibit reduced cellular proliferative and invasive properties associated with reduced activated ERK1/2 and reduced epithelial-mesenchymal transition (EMT). Hsf1^+/+Neu⁺ mammary epithelial cells exposed to TGFβ show high levels of ERK1/2 activity and EMT; this is associated with reduced expression of E-cadherin and increased expression of Slug and vimentin, a mesenchymal marker. In contrast, Hsf1^−/−Neu⁺ or Hsf1^+/+Neu⁺ cells do not exhibit activated ERK1/2 and show reduced EMT in the presence of TGFβ. The ineffective activation of the RAS/RAF/MEK/ERK1/2 signaling pathway in cells with reduced levels of HSF1 is due to the low levels of HSP90 in complex with RAF1 that are required for RAF1 stability and maturation. These results indicate a powerful inhibitory effect conferred by HSF1 downstream target genes in the inhibition of ErbB2-induced breast cancers in the absence of the Hsf1 gene.     

NOV/CCN3 Induces Adhesion of Muscle Skeletal Cells and Cooperates with FGF2 and IGF-1 to Promote Proliferation and Survival

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin β1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.