Northern analysis detected a transcript of 1.5 kb for vls expressed in ovaries, early embryos and adult females, but absent from pooled larval instars and adult males. In situ hybridization to OreR ovaries with a vls probe detected signal throughout the germ cell cytoplasm from early oogenesis onwards. The signal showed no specific localization pattern. Surprisingly, an equally strong signal was detected in vlsPG65/RB71 ovaries, and only in the vlsnull ovaries the signal is at background levels (Cavey, 2005).

On Western blots, polyclonal anti-Vls antibodies do not detect any Vls in vlsnull, vlsPG65, vlsRB71 and vlsHC33 ovary extracts. This shows that the antibody specifically recognizes the Vls protein and that the EMS mutants do not make significant levels of stable full length Vls. However, it is not known which epitopes are recognized by the polyclonal antibody, it is still possible that the EMS alleles produce truncated forms of Vls. In wild-type flies, Vls is abundant in ovaries, early embryos and adult females, but reduced in adult males. The fact that it is present in ovaries and in 0- to 1-hour-old embryos indicates that Vls is a maternally provided protein and this is consistent with the maternal-effect phenotype of vls mutants (Cavey, 2005).

Comparing OreR and control vlsnull ovaries stained with anti-Vls antibodies reveals Vls signal at low levels along parts of the oocyte cortex of wild-type stage 10 egg chambers, and also a stronger signal in the nurse cells, where it appears to be concentrated in nuage. Because anti-Vls antibodies do not work well for immunostaining, transgenic flies were generated that express the fusion gene vls-eGFP. The P[w+ vls-eGFP] transgene rescues the female sterile phenotype of vlsnull mutants, proving that Vls-eGFP possesses vls+ activity. Vls-eGFP localization in vlsnull background is indistinguishable from that in wild-type background. Vls-eGFP signal is cytoplasmic and stronger in the germline than in somatic cells, but in contrast to the immunostaining, specific localization patterns of Vls-eGFP were usually not observed. Only in the germarium was Vls-eGFP ocassionaly observed concentrating in perinuclear aggregates that disappear by stage 2 of oogenesis. At later stages, Vls-eGFP signal is uniformly distributed in the nurse cells and oocyte, as well as in young embryos, with no particular enrichment at the posterior or inside the pole cells (Cavey, 2005).

Western blot analysis of Vls as well as localization studies of Vls-eGFP in other posterior group mutants (osk, vas, tud, gus and orb) and in grk failed to identify potential upstream factors of vls that could control its expression levels, potential post-translational modifications, or its spatiotemporal distribution patterns. Together with the uniform distribution pattern of Vls-eGFP, this argues that Vls may act as a co-factor in the posterior pathway (Cavey, 2005).

To determine the distribution of Vls during oogenesis, the localization of HA-Vls protein was examined using anti-HA antibodies. Immunostaining of vls3; P{HA-vls} egg chambers revealed that Vls accumulates at the posterior pole of the oocyte in stage 10 egg chambers and in the pole plasm of early syncytial embryos. Vls is also found in pole cells and in migrating primordial germ cells. During oogenesis, Vls decorates a region surrounding the nurse cell nuclei, which bears strong similarity to the nuage. By contrast, immunostaining of wild type ovaries using anti-HA antibodies displayed only an overall residual staining, These data show that Vls is a component of both the nuage and the pole plasm (Anne, 2005).

Effects of Mutation or Deletion
Cytoplasm at the posterior pole of the early Drosophila embryo, known as polar plasm, serves as a source of information necessary for germ cell determination and for specification of the abdominal region. Likely candidates for cytoplasmic elements important in one or both of these processes are polar granules, organelles concentrated in the cortical cytoplasm of the posterior pole. Females homozygous for any one of the maternal-effect mutations, tudor, oskar, staufen, vasa, or valois give rise to embryos that lack localized polar granules, fail to form the germ cell lineage and have abdominal segment deletions. Using antibodies against a polar granule component, the Vasa protein, it was fou.nd that Vasa synthesis or localization is affected by these mutations. In vasa mutants, synthesis of Vasa protein is absent or severely restricted. In oskar and staufen mutant females, vasa synthesis appears normal, but the Vasa protein is not localized. In tudor and valois mutant females, Vasa is localized to the posterior pole of oocytes, but this localization is lost following egg activation. In addition to the posterior localized Vasa, there is a low level of Vasa distributed throughout the embryo. A function for this distributed Vasa is postulated based on the observation that embryos from Bicaudal-D mothers, in which abdominal determinants are incorrectly localized to the anterior pole, do not show any ectopic Vasa localization, though abdomen development at the anterior end depends on the amount of Vasa protein in the embryo (Hay, 1990).

The protein product of the Drosophila maternal-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-cell-specific antigen in seven other Drosophila species. Of six other maternal-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that Vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene (Lasko, 1990).

A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. Genetic as well as cytoplasmic transfer experiments were used to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. It is suggested that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants (Lehmann, 1991).

valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

Df(2L)be408 removes part of the barr gene, and the entire coding sequences of chk2 and CG10728. This deficiency does not complement vlsEMS alleles, indicating that one of these three transcription units corresponds to vls. A barr+ transgene rescues the barr but not the vls phenotypes. Similarly, a transgene containing chk2 alone does not rescue the vls phenotypes either. By contrast, a transgene containing a wild-type copy of CG10728 was able to rescue the maternal-effect lethal phenotype of chk2null CG10728null double mutants and the grandchildless phenotype associated with vlsEMS/Df(2L)be408 mutants. These results strongly suggest that CG10728 corresponds to vls. Indeed, sequencing this genomic region in the three EMS alleles vlsPG65, vlsRB71 and vlsHC33 finds a single nucleotide substitution in each of them, resulting in premature stop codons in the predicted open reading frame (ORF) of CG10728 (Fig. 1). This identifies CG10728 as vls and Df(2L)be408 / Df(2L)pr2b, P[w+, barr+] constitutes a true null mutant for vls and chk2. Since the chk2+ construct does not rescue any of the vls phenotypes, but the vls+ transgene rescues all of them, this mutant is referred to as vlsnull (Cavey, 2005).

Interestingly, chk2 and vls are encoded by opposite strands and cDNA sequence data shows that their 3'UTRs are complementary over 127 nucleotides. chk2 is translationally repressed by orb during oogenesis, and because translational control often relies on the binding of trans-acting factors to sequences in the 3'UTR of mRNAs, it was of interest to know whether vls could also play a role in chk2 translational control. Indeed, Chk2 levels increase about 6-fold in vlsPG65/HC33 and vlsPG65/RB71 ovaries compared with wild type and this is close to the 10-fold upregulation reported for orb mutants. This indicates that vls is also involved in the regulation of Chk2 levels. However, orb does not simply function to control Vls levels because these are normal in orb mutants (Cavey, 2005).

To investigate further the position of vls in the pathway, the distribution of posterior products was examined in vlsnull ovaries. osk mRNA is efficiently localized at the posterior of vlsnull mutant oocytes, consistent with previous reports for embryos from vlsPE36 mothers (Ephrussi, 1991). Osk protein accumulates at the posterior pole of the oocyte during stages 8-10. During this phase, similar patterns are observed in wild type and vls mutants. However, at later stages (stage 11), Osk levels at the posterior seem somewhat reduced in vlsnull oocytes compared with OreR and vlsnull vls+, and often no signal for Osk was detected in vlsnull oocytes. This reduction of Osk levels at the posterior is also observed in hemizygous vlsPG65, albeit to a lesser extent (Cavey, 2005).

Confirming these observations, Western blot analyses revealed lower levels of Osk in vlsnull mutants and vlsPG65 hemizygotes compared with OreR and vlsnull vls+ ovary extracts, and vlsnull mutants again show a stronger reduction than do vlsPG65 hemizygote. A similar decrease of Osk levels has been reported previously for vlsPE36/RB71. The Long and Short Osk isoforms are affected differently in vls mutants. Whereas Long Osk is only slightly reduced, the Short, indispensable form of Osk, is strongly reduced in vlsnull and vlsPG65 hemizygotes. Moreover, an isoform-specific reduction of the hyperphosphorylated (upper) Short Osk was observed compared with the hypophosphorylated (lower) form in vls mutants. This difference is more clearly seen in hemizygous vlsPG65 than in vlsnull, since the hypophosphorylated form of Short Osk is also practically undetectable in vlsnull. This effect had been described previously for vas mutants. It is noted that vls mutants cause a clear reduction of Long Osk compared with vas mutants, which have relatively normal levels. Furthermore, tud1 mutant extracts contain lower levels of both isoforms of Short Osk with no isoform-specific reduction, and it seems that the Long isoform might also be slightly reduced although it did not appear to be affected in an earlier study (Cavey, 2005).

Taken together, anti-Osk immunostaining and western analyses suggest that vls is required for normal levels of Osk to accumulate at the posterior pole while the pole plasm is assembling. Starting around stage 11, Osk signal progressively disappears from the posterior in the absence of vls function, and by later stages, Osk accumulation at the posterior is probably greatly reduced, explaining the drastic reduction of overall Osk levels observed on western blots (Cavey, 2005).

Vasa protein is the next factor in the posterior pathway to localize to the posterior end of the oocyte after osk mRNA and protein. This osk-dependent Vasa localization remains stable at the posterior pole during the early stages of embryogenesis and Vasa is later incorporated into pole cells. In vlsnull ovaries and in embryos from vlsnull mothers, anti-Vasa antibody staining showed very little or no accumulation of Vasa at the posterior end. This observation was further confirmed by analyzing the distribution of Vas-eGFP in vlsnull and vlsPG65 ovaries and embryos. Although the early localization pattern of Vasa in nuage of the mutant nurse cells is normal, the posterior localization in stage 10 oocytes is not observed in the null mutants, and appears very weak in vlsPG65 hemizygotes (Cavey, 2005).

Later in development, Vas-eGFP signal is detected at the posterior end and then inside the pole cells of embryos from wild-type and vlsnull vls+ mothers, but not from vlsPG65 hemizygotes and vlsnull mothers. These results contrast with previous reports where Vasa localization defects in vls mutants (vlsPE36 and vlsRB71) were observed only slightly after fertilization, before pole cell formation (Hay, 1990; Lasko, 1990). The data for the vlsnull and vlsPG65 alleles implicate vls in the late localization or anchoring of Vasa to the posterior cortex during oogenesis (Cavey, 2005).

Distribution of pole plasm and nuage components in vls egg chambers

Embryos produced by vls females fail to form pole cells at the posterior pole (Schüpbach, 1986. To check whether pole cell formation strictly depends on vls activity, an osk-bcd 3'UTR transgene was introduced into vls1 and vls3 backgrounds, and no evidence was found for ectopic formation of pole cells at the anterior pole, although residual Vas staining could be detected this region of the egg. This result concurs with the suppressor effect of vls1 on ectopic pole cell formation observed after overexpression of osk (Smith, 1992), but contrasts the data using an osk-bcd 3'UTR transgene expressed in vls2/Df(2R)TW2 flies (Ephrussi, 1992). From these data, it is concluded that vls function is essential for pole cell formation (Anne, 2005).

To determine vls hierarchical position among known components of the pole plasm, the distribution of Osk, Vas and Tud was investigated in vls1 and vls3 egg chambers. Use of specific antibodies against Osk revealed that Osk is normally localized at the posterior pole from stage 9 onwards egg chambers in vls. However, it was often noticed that the amount of Osk protein was reduced in vls in comparison with wild type. To determine whether vls regulates Osk protein accumulation, the amount of Osk protein produced in wild-type and vls ovaries was examined by immunoblotting. It was found that the level of Osk short form is markedly reduced in vls in comparison with that in wild type. However, the phosphorylation of this isoform seems unaffected in vls. In addition, it was noticed that the slower migrating form of the short Osk protein is particularly reduced in abundance in aub protein extract, suggesting a defective phosphorylation in aub ovaries. This result extends observations of (Wilson, 1996), showing a strongly reduced level of short Osk in aub ovaries and may explain the aub requirement for osk activity induced anteriorly by the OB1 osk-bcd 3'UTR transgene. Examination of Vas distribution showed a normal localization at the posterior pole in stage 10 vls egg chambers, albeit at a reduced level compared with wild-type egg chambers (Anne, 2005).

Checking the distribution of Tud during oogenesis revealed that Tud was absent from the posterior pole of vls mutant oocytes in stage 10 egg chambers, indicating that Vls may play a role in pole plasm accumulation of Tud. Since Tud is also a component of the nuage encircling nurse cell nuclei, this structure was looked at by examining the distribution of three components of the nuage including Tud, Vas and Maelstrom (Mael). The use of a transgene expressing a GFP-Vas fusion protein associated with the vls chromosome revealed that GFP-Vas was normally localized in the nuage of vls nurse cells, albeit at a lesser degree compared with wild type. Examination of Mael showed that this protein is also present in the nuage, although its distribution diverges from the wild-type pattern. It was found that Mael is concentrated in brighter spots in vls than in wild type at the periphery of the nurse cell nuclei. By contrast, no Tud was detected in the nuage of vls nurse cells, although Tud accumulates in vls oocytes and transiently localizes at their anterior margin. These findings indicate that: (1) vls acts downstream of Vas for the recruitment of Tud in the nuage; and (2) events occurring in the nuage are dispensable for the transport of Tud from the nurse cells to the oocyte but are required for Tud recruitment in the pole plasm (Anne, 2005).

Examination of vls egg chambers revealed also a modification in the structure of the oocyte nucleus in which the karyosome is fragmented into two fuzzy spots instead of forming a single compact dot in wild-type nuclei. A similar phenotype has been described for spindle and vas mutants. Since vls oocytes undergo normal meiosis and support normal embryonic development, this finding suggests that Vls may exert a dispensable function on the karyosome organization (Anne, 2005).


Reference names in red indicate recommended papers.

Anne, J. and Mechler, B. M. (2005). Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuleen. Development 132(9): 2167-77. 15800004

Anne, J. et al. (2007). Arginine methyltransferase Capsuléen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila. Development 134: 137-146. Medline abstract: 17164419

Anne, J. (2010). Arginine methylation of SmB is required for Drosophila germ cell development. Development 137(17): 2819-28. PubMed Citation: 20659974

Arkov, A. L., Wang, J.-Y. S., Ramos, A. and Lehmann, R. (2006). The role of Tudor domains in germline development and polar granule architecture. Development 133: 4053-4062. Medline abstract: 16971472

Bardsley, A., McDonald, K. and Boswell, R. E. (1993). Distribution of Tudor protein in the Drosophila embryo suggests separation of functions based on site of localization. Development 119: 207-219. 8275857

Branscombe, T. L., Frankel, A., Lee, J.-H., Cook, J. R., Yang, Z.-h., Pestka, S. and Clarke, S. (2001). PRMT5 (Janus kinase-binding protein 1) catalyzes the formation of symmetric dimethylarginine residues in proteins. J. Biol. Chem. 276: 32971-32976. Medline abstract: 11413150

Cavey, M., Hijal, S., Zhang, X. and Suter, B. (2005). Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation. Development 132(3):459-68. 15634703

Callebaut, I. and Mornon, J.-P. (1997). The human EBNA-2 coactivator p100: multidomain organization and relationship to the staphylococcal nuclease fold and to the tudor protein involved in Drosophila melanogaster development. Biochem. J. 321: 125-132. 9003410

Cook, J. R., Lee, J. H., Yang, Z. H., Krause, C. D., Herth, N., Hoffmann, R. and Pestka, S. (2006). FBXO11/PRMT9, a new protein arginine methyltransferase, symmetrically dimethylates arginine residues. Biochem. Biophys. Res. Commun. 342: 472-481. Medline abstract: 16487488

Côté, J. and Richard, S. (2005). Tudor domains bind symmetrical dimethylated arginines. J. Biol. Chem. 280: 28476-28483. Medline abstract: 15955813

Ephrussi, A., Dickinson, L. K. and Lehmann, R. (1991). Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66: 37-50. PubMed Citation: 2070417

Ephrussi, A. and Lehmann, R. (1992). Induction of germ cell formation by oskar. Nature 358: 387-392. 1641021

Friesen, W. J., Wyce, A., Paushkin, S., Abel, L., Rappsilber, J., Mann, M. and Dreyfuss, G. (2002). A novel WD repeat protein component of the methylosome binds Sm proteins. J. Biol.Chem. 277: 8243-8247. 11756452

Gonsalvez, G. B., Rajendra, T. K., Tian, L. and Matera, A. G. (2006). The Sm protein methyltransferase, dart5, is essential for germ-cell specification and maintenance. Curr. Biol. 16: 1077-1089. Medline abstract: 16753561

Gonsalvez G. B., et al. (2007). Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins. J. Cell Biol. 178: 733-740. PubMed Citation: 17709427

Hay, B., Jan, L. Y. and Jan, Y. N. (1990). Localization of vasa, a component of Drosophila polar granules, in maternal-effect mutants that alter embryonic anteroposterior polarity. Development 109: 425-433. 2119289

Khusial, P. R., Vaidya, K. and Zieve, G. W. (2005). The symmetrical dimethylarginine post-translational modification of the SmD3 protein is not required for snRNP assembly and nuclear transport. Biochem. Biophys. Res. Commun. 337: 1119-1124. Medline abstract: 16236255

Lasko, P. F. and Ashburner, M. (1990). Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development. Genes Dev. 4: 905-921. 2384213

Lee, J. H., Cook, J. R., Pollack, B. P., Kinzy, T. G., Norris, D. and Pestka, S. (2000). Hsl7p, the yeast homologue of human JBP1, is a protein methyltransferase. Biochem. Biophys. Res. Commun. 274: 105-111. Medline abstract: 10903903

Lee, J. H., Cook, J. R., Yang, Z. H., Mirochnitchenko, O., Gunderson, S. I., Felix, A. M., Herth, N., Hoffmann, R. and Pestka, S. (2005). PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine. J. Biol. Chem. 280: 3656-3664. Medline abstract: 15494416

Lehmann, R. and Nusslein-Volhard, C. (1991). The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo. Development 112: 679-691. 1935684

Licciardo, P., Amente, S., Ruggiero, L., Monti, M., Pucci, P., Lania, L. and Majello, B. (2003). The FCP1 phosphatase interacts with RNA polymerase II and with MEP50 a component of the methylosome complex involved in the assembly of snRNP. Nucleic Acids Res. 31(3): 999-1005. 12560496

Pollack, B. P., Kotenko, S. V., He, W., Izotova, L. S., Barnoski, B. L. and Pestka, S. (1999). The human homologue of the yeast proteins Skb1 and Hls7p interacts with Jak kinases and contains protein methyltransferase activity. J. Biol. Chem. 274: 31531-31542. Medline abstract: 10531356

Ponting, C. P. (1997). Tudor domains in proteins that interact with RNA. Trends Biochem. Sci. 22: 51-52. 9048482

Schupbach, T. and Wieschaus, E. (1986). Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo. Roux's Arch. Dev. Biol. 195: 302-317

Smith, J. L., Wilson, J. E. and Macdonald, P. M. (1992). Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos. Cell 70: 849-859. 1516136

Snee, M. J. and Macdonald, P. M. (2004). Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components. J. Cell Sci. 117: 2109-2120. 15090597

Wilson, J. E., Connell, J. E. and Macdonald, P. M. (1996). aubergine enhances oskar translation in the Drosophila ovary. Development 122: 1631-1639. 8625849

valois: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 February 2011

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