during stage 9 of oogenesis , oskar mrna localizes to the posterior of the drosophila oocyte , where it nucleates the pole plasm , which contains the determinants that specify the germ cells and abdomen at the posterior of the embryo [ 13 ] .
this localization depends on the rna - binding proteins staufen and hrp48 and on the exon - junction - complex components mago nashi , y14 , eif4aiii , and barentsz [ 814 ] .
in addition , the posterior accumulation of oskar mrna requires microtubules and the plus - end - directed microtubule motor protein , kinesin , suggesting that it is transported by kinesin toward microtubule plus ends at the posterior pole [ 4 , 15 ] . as part of a project to visualize the transport of oskar mrna
, we used the uas - gal4 expression system to overexpress oskar mrna in the female germline .
although increased amounts of oskar mrna are localized to the oocyte posterior in these uas - osk females , a significant proportion of the mrna is found in an ectopic dot in the middle of the oocyte in all stage 9 egg chambers ( 62/62 ) and more than half of those at stage 10a ( 80/150 ) ( figures 1a1d ) .
the mislocalization of oskar mrna to the center of the oocyte is reminiscent of the localization of oskar mrna at stage 8 , suggesting that excess oskar mrna saturates the transport pathway , causing a delay in its translocation to the posterior pole .
we therefore examined the localization of other components of the oskar mrna localization complex that are required for its transport to the posterior .
barentsz , mago nashi , staufen , and the kinesin heavy chain all localize to the ectopic  dot in the middle of the oskar - overexpressing oocytes , as well as to the posterior pole ( figures 1g1n ) .
thus , none of these components appears to be limiting for transport to the posterior .
oskar mrna is normally translationally repressed until it reaches the posterior pole , but overexpression of the oskar 3 untranslated region ( utr ) causes its premature translation .
we therefore examined whether this was also the case in the egg chambers overexpressing full - length oskar rna .
oskar protein starts to accumulate at the posterior pole of wild - type egg chambers at stage 9 ( figures 1o and 1p ) . in uas - osk
egg chambers , robust oskar expression is already visible in the ectopic dot at stage 8 ( figure  1q ) .
thus , overexpressed oskar mrna is not translationally repressed , even though it has not reached the posterior .
furthermore , the ectopic oskar protein recruits vasa , indicating that the premature translation of oskar initiates the formation of the pole plasm in the middle of the oocyte ( figures 1r1 t ) . to test whether oskar protein is required for the mislocalization of oskar mrna
, we generated a second construct , uas - oskar - stop , in which we introduced a stop codon at position k178 , which is equivalent to the mutation in the oskar protein - null allele oskar ( figures 1e and 1f ) .
when oskar - stop mrna is overexpressed , it rarely localizes to the middle of the oocyte ( 13/79 at stage 9 ; 11/96 at stage 10a ) , although it is overexpressed at similar levels to the wild - type uas - oskar mrna ( figure  s1e in the supplemental data online ) .
thus , the formation of the ectopic dot seems to be mainly caused by oskar protein , rather than saturation of the localization pathway .
the expression of the uas - oskar - stop construct induces the premature translation of endogenous oskar mrna ( see below ) , and this may account for the low penetrance of oskar mrna mislocalization to the middle of these oocytes . whereas oskar mutants block abdomen formation , extra copies of oskar cause the opposite bicaudal phenotype , in which the head and thorax are replaced by a mirror - image copy of the abdomen .
although uas - osk females produce some bicaudal embryos , the vast majority show a more extreme phenotype , in which the gut and terminal structures are expanded at the expense of the cuticle ( figures s1a  s1d , table s1 ) .
this is most probably because oskar mrna is very highly expressed in uas - osk ovaries ( equivalent to at least ten copies of oskar ; see figure  s1e ) , leading to oskar and nanos overexpression , which causes an expansion of the expression domains of the terminal - gap genes , tailless and huckebein ( data not shown ) .
surprisingly , uas - oskar - stop gives rise to embryonic phenotypes similar to those of uas - oskar , even though it can not produce functional oskar protein ( figure  s1d and table s1 ) .
a weaker bicaudal phenotype has been observed when the oskar-3utr is overexpressed , leading to the proposal that this rna activates the translation of endogenous oskar mrna by titrating out translational repressors . to test
whether this is  also the case for uas - oskar - stop , we expressed the transgene in females that were transheterozygous for two oskar protein - null mutations ( oskar / oskar ) .
the resulting embryos showed a typical posterior - group phenotype that was indistinguishable from oskar / oskar alone ( table s1 ) .
thus , uas - oskar - stop produces a bicaudal phenotype by activating the translation of endogenous wild - type oskar mrna . at stage 7 of oogenesis , a signal from the posterior follicle cells induces a reorganization of the oocyte microtubule cytoskeleton , so that microtubules are then nucleated from the anterior and lateral cortex [ 2125 ] .
the microtubule plus ends appear to localize to the middle of the oocyte during stages 7 and 8 , as revealed by the localization of oskar mrna , staufen , and kin:-gal ( a marker for the plus ends of the microtubules ) [ 4 , 17 , 26 ] .
this organization is only temporary , however , and plus - end markers start to accumulate at the posterior cortex at the beginning of stage 9 , coincident with the onset of oskar mrna localization to the posterior .
the mislocalization of oskar mrna to the center of uas - oskar oocytes resembles the polarity phenotype produced by par-1 hypomorphic mutant combinations , in which some of the microtubule plus ends localize to the middle of the oocyte , rather than the posterior pole [ 5 , 6 ] .
we therefore examined the localization of the plus - end marker kinesin--gal in uas - osk oocytes .
kinesin--gal always localizes to an ectopic site in the center of these oocytes ( 18/18 ) , as well as to the posterior , and colocalizes with staufen , a marker for oskar mrna ( figures 2a2f ) .
the dynein / dynactin motor complex provides another marker for the polarity of the microtubules because it is localized by kinesin to the posterior of the oocyte independently of oskar mrna [ 17 , 27 , 28 ] .
this can be visualized with gfp - dynamitin , a subunit of the dynactin complex ( figures 2g2i ) .
gfp - dynamitin is mislocalized with staufen to the ectopic dot in the middle of almost all oskar - overexpressing oocytes ( 16/18 ) ( figures 2j and 2k ) .
thus , two markers for microtubule plus ends colocalize with ectopic oskar in the middle of the oocyte .
the ectopic oskar does not appear to interfere with the organization of the microtubule minus ends , or with minus - end - directed processes .
the overall morphology of the microtubules is not affected when visualized with tubulin antibodies ( not shown ) or with a gfp - tagged version of the microtubule - binding protein tau ( figures s2c and s2d ) .
in addition , the oocyte nucleus always migrates normally to the dorsal - anterior corner of the oocyte , and bcd mrna shows a wild - type localization to the anterior cortex ( figures s2a and s2b ) . surprisingly , some gurken mrna is often mislocalized to the posterior of uas - oskar oocytes .
it is not translated there , however , because gurken protein is only found at the correct location adjacent to the oocyte nucleus ( figures s2e  s2i ) .
this phenotype requires high levels of oskar protein because gurken mrna is not mislocalized in uas - oskar - stop oocytes .
these results suggest that prematurely translated oskar protein and/or downstream components of the pole plasm somehow sequester gurken mrna and take it with them to the posterior pole .
the results above indicate that oskar protein recruits microtubule plus ends when overexpressed , raising the possibility that oskar normally plays a role in the recruitment of plus ends to the posterior in wild - type oocytes . to test this hypothesis ,
we re - examined the organization of the microtubule cytoskeleton in the absence of oskar protein and in mutants that affect oskar mrna localization  and translation .
the plus - end marker kin:-gal localizes to a tight posterior crescent in most wild - type stage 9 oocytes ( 88% , n = 58 ) , with 12% displaying a weaker enrichment at the posterior pole ( figure  3a , table s2 ) . in an oskar protein
null , however , only 16% of the oocytes show a robust posterior localization of kin:-gal , whereas 72% show a weak posterior enrichment , and 12% show no localization to the posterior at all ( figure  3d ) .
we also examined germline clones of btz and khc , which completely disrupt oskar mrna localization [ 12 , 15 ] . in these cases ,
the distribution of kin:-gal in the mutant egg chambers can be directly compared with its localization in the heterozygous egg chambers from the same ovaries , which are stained under identical conditions . in each case , the mutant egg chambers show a highly penetrant reduction or loss of the posterior localization of kin:-gal compared to the nonmutant controls ( figures 3g3o , table s2 ) .
these results are in good agreement with those of brendza et al ,  who noted that kin:-gal is not detectably localized in a significant fraction of khc mutant oocytes .
thus , the posterior recruitment of plus ends is strongly impaired when oskar protein is absent from the posterior pole .
the mechanism that recruits microtubule plus ends to the posterior pole at stage 9 is not well understood , but depends on the function of the conserved polarity kinase par-1 [ 5 , 6 ] .
in contrast to the wild - type , microtubules are nucleated all around the oocyte cortex of par-1 mutants , and the plus ends are focused at the center instead of the posterior pole . as a consequence ,
oskar mrna is mislocalized to the middle of the oocyte , and the resulting embryos lack an abdomen and germline .
par-1 plays a similar role in organizing the microtubule cytoskeleton in both drosophila and mammalian epithelial cells , indicating that one of its major functions  is in the establishment of polarized microtubule arrays [ 3032 ] .
the par-1n1 short and long isoforms appear to play the major role in the polarization of the oocyte cytoskeleton because only these fully rescue the polarity defects of par-1 hypomorphs , and gfp - tagged par-1n1s and l are recruited to the posterior cortex of the oocyte at stage 7 , making them the earliest markers for polarization of the anterior - posterior axis .
this localization is more cortical than that of the pole plasm and is not disrupted in staufen , mago nashi , and kinesin heavy chain mutants , indicating that it is independent of oskar mrna localization .
antibody stainings fail to detect the cortical par-1 , perhaps because it is of too low abundance , but recognize a second population of par-1 , which colocalizes to the posterior with the pole plasm [ 5 , 6 ] .
this localization is disrupted in oskar and staufen mutants , but not vasa mutants , suggesting that par-1 is recruited by oskar protein .
these results suggest that oskar overexpression may interfere with polarity by recruiting par-1 to the center of the oocyte . in support of this view
, antibody stainings revealed a strong localization of par-1 to the dot in the middle of the oocyte in uas - osk - overexpressing flies ( 100% , n = 16 ; figures 4a4f ) .
gfp - par-1n1s does not colocalize with ectopic oskar in the center of the oocyte , however , consistent with the previous observation that this isoform is recruited to the posterior cortex , but not the pole plasm .
we also tested whether uas - oskar enhances the phenotype of a weak par-1 mutant combination .
flies transheterozygous for the two hypomorphic par-1 alleles , par-1 and par-1 , have mild polarity defects , in which oskar mrna and staufen protein are mislocalized to the center of the oocyte in 56% ( n = 136 ) of egg chambers ( figures 4g4j , table s3 ) . despite this mislocalization , stau is also visibly enriched at the posterior pole of the oocyte in the majority of cases ( 88% ) .
in contrast , par-1/par-1 oocytes that also overexpress oskar mrna show a much stronger phenotype : stau protein is visibly enriched at the posterior pole of the oocyte in only 27% ( n = 37 ) of these egg chambers and is mislocalized all around the oocyte cortex in most cases ( figure  4j ; table s3 ) .
in addition , these flies do not lay any eggs because of these severe defects in oogenesis .
thus , oskar overexpression strongly enhances the phenotype of a weak par-1 mutant combination , most probably because ectopic oskar protein recruits some of the limited amount of par-1 away from the posterior to the oocyte center .
although oskar mrna localization has always been presumed to be downstream of the polarization of the oocyte microtubule cytoskeleton , here we show through both gain- and loss - of - function experiments that it also plays an active role in this process , through the recruitment of microtubule plus ends by oskar protein .
par-1 is a good candidate to be the effector of oskar function in plus - end recruitment because it is required for the polarization of the microtubule cytoskeleton and is recruited by oskar protein to the posterior of the oocyte .
furthermore , the polarity phenotype of par-1 hypomorphs is strongly enhanced by oskar mrna overexpression , suggesting that par-1 and oskar function in the same pathway to regulate the microtubules .
on the basis of our results , we would like to suggest a revised model for how the polarity of the oocyte is established ( figure  s3 ) .
the polarization of the oocyte is initiated by the posterior - follicle - cell signal , which induces the localization of the par-1n1 isoforms to the posterior cortex .
this localized par-1 then recruits or stabilizes some microtubule plus ends at the posterior cortex , leading to the kinesin - dependent transport of a small amount of oskar mrna from the center of the oocyte to the posterior pole .
once oskar mrna reaches the posterior , its translational repression is relieved , and  the resulting oskar protein recruits another population of par-1 to the posterior .
this par-1 can then recruit further microtubule plus ends , which in turn direct the posterior localization of more oskar mrna .
thus , oskar protein initiates a positive feedback loop that amplifies the polarization of the microtubule cytoskeleton , leading to an increase in the localization of its own mrna .
the microtubule polarity is not reinforced in oskar protein - null mutants or in mutants that disrupt the localization of translation of oskar mrna , and this results in only a partial polarization of the microtubule cytoskeleton . on the other hand ,
oskar mrna overexpression and premature translation in the middle of the oocyte initiates the positive feedback loop in the wrong place . as a consequence , par-1 recruits some of the microtubule plus ends to the center of the oocyte and away from the posterior pole .
this therefore generates a second positive feedback loop that further reinforces the posterior localization of par-1 , oskar protein , and microtubule plus ends .
for example , the polarization of migrating neutrophils depends on the local enrichment of phosphatidylinositol 3,4,5 triphosphate ( ptdinsp3 ) at the leading edge of the cell , and this is amplified by a rho - dependent positive feedback loop , in which ptdinsp3 recruits ptdins-3-oh kinase to the leading edge , which in turn generates more ptdinsp3 
 .
the bud site is defined by the localization of activated cdc42-gtp , and this localization induces the assembly of actin cables that extend into the cytoplasm .
this signal is then reinforced by the transport of more cdc42-gtp along the actin cables to the bud site , where it can induce the assembly of further actin cables .
this mechanism is enhanced by a second feedback loop , in which cdc42-gtp recruits the adaptor protein , bem1 , which binds cdc24 , which activates cdc42 [ 36 , 37 ] .
our results add a third example of the use of multiple feedback loops to reinforce an initially weak cell polarity , and they provide the first case where this amplification involves the microtubule cytoskeleton rather than actin .
 summarythe localization of oskar mrna to the posterior of the drosophila oocyte defines the site of assembly of the  pole plasm , which contains the abdominal and germline determinants [ 13 ] . 
 oskar mrna localization requires the polarization of the microtubule cytoskeleton , which depends on the recruitment of par-1 to the posterior cortex in response to a signal from the follicle cells , where it induces an enrichment of microtubule plus ends [ 47 ] . here 
 , we show that overexpressed oskar mrna localizes to the middle of the oocyte , as well as the posterior . 
 this ectopic localization depends on the premature translation of oskar protein , which recruits par-1 and microtubule - plus - end markers to the oocyte center instead of the posterior pole , indicating that oskar regulates the polarity of the cytoskeleton . 
 oskar also plays a role in the normal polarization of the oocyte ; mutants that disrupt oskar mrna localization or translation strongly reduce the posterior recruitment of microtubule plus ends .  
 thus , oskar mrna localization is required to stabilize and amplify microtubule polarity , generating a positive feedback loop in which oskar recruits par-1 to the posterior to increase the microtubule cytoskeleton 's polarization , which in turn directs the localization of more oskar mrna . 
Results and Discussion