gurken

Factors affecting Gurken mRNA localization and translation (part 1/2)

Anterior-posterior polarity in Drosophila arises from the movement of the oocyte to the posterior of the egg chamber, and the subsequent acquisition of posterior fate by the adjacent somatic follicle cells. Gurken is necessary in the oocyte and Torpedo/EGF-R in the follicle cells for the induction of posterior fate. The role of Gurken and EGF-R in establishing A-P polarity precedes their role in establishing D-V polarity (González-Reys, 1995).

fs(1)K10 mRNA transport and anterior localization is mediated by a 44 nucleotide stem-loop structure. A similar putative stem-loop structure is found in the 3' untranslated region of Drosophila Orb mRNA, suggesting that the same factors mediate the transport and anterior localization of both K10 and Orb mRNAs. Apart from Orb, the K10 TLS (transport/localization sequence) is not found in any other localized mRNA, raising the possibility that the transport and localization of other mRNAs, e.g., Bicoid, Oskar and Gurken, are mediated by novel sets of cis- and trans-acting factors. K10 TLS overrides the activity of Oskar cis-regulatory elements that mediate the late stage movement of the mRNA to the posterior pole (Serano, 1995a).

The gurken-torpedo/EGF-R pathway also establishes dorsoventral polarity later in oogenesis; Drosophila uses the same germline to soma signaling pathway to determine both embryonic axes (Gonzalez-Reyes, 1995).

A critical step in Drosophila dorsoventral patterning is the movement of Gurken mRNA from the anterior cortex of the oocyte to the oocyte's anteriodorsal corner at stage 8 of oogenesis. Such movement is dependent on fs(1)K10. A direct role has been proposed for fs(1)K10 in the Gurken mRNA localization process (Serano, 1995b).

fs(1)K10 mutant embryos still possess a dorsoventral polarity. However, instead of forming a 90 degree angle, the dorsoventral and the anterior/posterior axes lie parallel to each other. This axis misorientation is partially corrected by decreasing the wild-type grk gene copy number such that embryos issuing from K10/K10; grk/+ females show a variability in their fate map, interpreted as a progressive rotation of dorsoventral axis relative to the unmodified anterior/posterior axis. This rotation is maximal in the K10 embryos, reaching 90 degrees and resulting in the congruence of the two axes. The alteration of the embryonic fate map can be traced back to oogenesis where it correlates with the mislocalization of the GRK transcripts (Haenlin, 1995).

The cytoskeleton is necessary for Gurken mRNA localization. The homeless gene of Drosophila is required for anteroposterior and dorsoventral axis formation during oogenesis. Transport and localization of bicoid and oskar messages during vitellogenic stages are strongly disrupted by homeless mutation, and the distribution and/or quantity of Gurken, Orb, and Fs(1)K10 mRNAs is also affected, but to a lesser degree. Examination of the microtubule structure with anti-alpha-Tubulin antibodies reveals aberrant microtubule organizing center movement and an abnormally dense cytoplasmic microtubule meshwork (Gillespie, 1995).

Strong mutations in the orb gene, an ovarian-specific member of a large family of RNA-binding proteins, arrest oogenesis at a very early stage, even prior to egg chamber formation. However, females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of Oskar and Gurken mRNAs within the oocyte during the later stages of oogenesis (Christerson, 1994).

The D-elg gene encodes an ETS domain transcription factor that functions in Drosophila oogenesis. D-elg belongs to a small group of genes required for the formation of both the anterior/posterior and dorsoventral axes of the egg chamber. During oogenesis in D-elg mutant females, the spatial localization of Oskar and Gurken mRNAs in the oocyte is disrupted and a follicle cell enhancer trap marker identifies dorsoventral polarity defects. Specialized follicle cells, called border cells, fail to migrate from their anterior location to a position adjacent to the developing oocyte. D-elg is expressed in both germline and follicle cells of the ovary. Mutant phenotypes resemble orb mutants (Gajewski, 1995).

A mutant, maelstrom (mael), is described that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, Bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of Gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. mael mutation affects the distribution and dynamics of oocyte microtubules. grk and mael mutants have a defective microtubule cytoskeleton similar to that previously described for the oocyte polarity mutants PKA and mago nashi; however, the grk and mael cytoskeletons are not identical. Both mutants have a high concentration of microtubules at the posterior of the oocyte in stages 8 and 9 when microtubules are normally concentrated at the oocyte anterior. In stage 7 however, mael microtubules are tightly bundled around the cortex, while grk mutants have a more diffuse network. This bundling is similar to the continous subcortical array of microtubules in wild-type stage 10b oocytes. Time-lapse videomicroscopy indicates that the cytoplasm undergoes premature streaming. Posterior localization of mRNA in stage 3-6 oocytes could be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein with a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis (Clegg, 1997).

encore(enc) codes for a novel protein that is involved both in regulating the number of germline mitoses and in the process of oocyte differentation. Mutations in encore result in exactly one extra round of mitosis in the germline. Genetic and molecular studies indicate that this mitotic defect may be mediated through the gene bag-of-marbles. The isolation and characterization of multiple alleles of encore reveal that they are all temperature sensitive for this phenotype. Mutations in encore also affect the process of oocyte differentiation. Egg chambers are produced in which the oocyte nucleus has undergone endoreplication often resulting in the formation of 16 nurse cells. It is argued that these two phenotypes produced by mutations in encore represent two independent requirements for encore during oogenesis (Hawkins, 1996). A third defect, one associated with Gurken (Grk), has been found in encore mutants. Post-transcriptional regulation of Grk protein levels is required for correct oocyte pattern formation. encore is required for the accumulation of Grk protein during oogenesis. Enc regulates Grk post-transcriptionally to ensure adequate levels of signaling for the establishment of the anterior-posterior and dorsal-ventral axes. The extra round of germline mitoses in enc mutants is most likely due to an overproduction of bag-of-marbles mRNA early in oogenesis. In contrast, the ventralization phenotype appears to result from a lack of Gurken protein. Encore could be a protein that regulates RNA function and stability in oogenesis, and thus may be involved in the turn-over of BAM mRNA and the translational control of GRK mRNA (Hawkins, 1996 and 1997).

licorne codes for a MAP kinase kinase exciting the p38 pathway in Drosophila. licorne mutant embryos are defined, for the purpose of this study, as hemipterous;licorne double mutants engineered to express a hemipterous transgene (see Licorne Effects of Mutation for more information about this genotype). In addition to its requirement in AP patterning, lic mutations also affect the DV axis, as evidenced by ventralization of the eggshell. One important event in DV patterning is the correct localization of the Gurken ligand on the future dorsal side of the oocyte, a position that depends on the correct localization of the nucleus in the oocyte. Because mislocalized nuclei was never observed in lic mutant oocytes, lic DV defects are not likely to be the result of inappropriate nucleus migration or microtubule polarization. It was thus asked whether the grk determinant itself might be affected in lic mutants. In situ hybridization using a grk probe did not detect any defect, suggesting that expression and localization of the GRK mRNA are normal in lic mutant oocytes. However, immunostaining of egg chambers using an anti-Grk antibody shows reduction (15%) or mislocalization (~5%) of Grk protein. To further characterize a loss of grk activity in lic oocytes, wild-type and mutant ovaries were stained using a kekkon (kek)-lacZ reporter construct. The kek gene is a target of the Egfr in the follicle cells and thus serves as an indirect and sensitive assay to measure grk activity in the oocyte. In wild-type egg chambers, kek is expressed in dorsal follicle cells in a characteristic graded pattern reflecting both the intensity and localization of the underlying grk signal. In ~50% stage 10 lic mutant egg chambers, kek expression is reduced dramatically, as shown by a reduction in the number of responding follicle cells and a change in the shape of the kek expression domain. In rare cases (<5%), an expansion of the kek signal in more lateral and ventral positions is also observed, an observation that might suggest a partial delocalization of grk activity in the oocyte. Consistent with this result, dorsalization of the chorion is observed in very rare cases. Thus, lic loss of function in the germ line reduces Egfr activity in the dorsal follicle cells, most likely as a result of a reduction of grk activity in the oocyte (Suzanne, 1999).

In Drosophila, dorsoventral polarity is established by the asymmetric positioning of the oocyte nucleus. In egg chambers mutant for cap 'n' collar, the oocyte nucleus migrates correctly from a posterior to an anterior-dorsal position, where it remains during stage 9 of oogenesis. However, at the end of stage 9, the nucleus leaves its anterior position and migrates towards the posterior pole. The mislocalization of the nucleus is accompanied by changes in the microtubule network and a failure to maintain Bicoid and Oskar mRNAs at the anterior and posterior poles, respectively. Gurken mRNA associates with the oocyte nucleus in cap 'n' collar mutants and initially the local secretion of Gurken protein activates the Drosophila EGF receptor in the overlying dorsal follicle cells. However, despite the presence of spatially correct Grk signaling during stage 9, eggs laid by cap 'n' collar females lack dorsoventral polarity. cap 'n' collar mutants, therefore, allow for the study of the influence of Grk signal duration on DV patterning in the follicular epithelium (Guichet, 2001).

cnc is a complex locus coding for three protein isoforms (CncA, CncB, CncC) which share a basic-leucine zipper domain at the carboxy terminus. While CncA and CncC are expressed ubiquitously, CncB is expressed specifically in the head region of early embryos where it is required for the repression of deformed function and the formation of intercalary and labral structures. Double-stranded RNA interference experiments have shown that CncA and CncC are dispensible for embryonic development. The two P-insertions used in this study affect all three isoforms. CncB is not expressed during oogenesis, thus the mutant phenotypes observed are due to a lack of either CncA, CncC, or both isoforms. Judging from their structure, both proteins probably function as transcription factors, as has been demonstrated for CncB and the Cnc homologs of vertebrates and other invertebrates. At present, no genes are known to be regulated by Cnc proteins during oogenesis. However, the cnc phenotype reveals two new aspects as to how DV polarity is established during oogenesis. (1) The initial asymmetric movement of the oocyte nucleus has to be followed by a separate process of stable anchoring of the nucleus at the anterior cortex. (2) An early pulse of asymmetric Egf signaling is insufficient to induce stable DV follicle cell patterning, indeed Egf receptor activation by Gurken has to persist until stage 10A to establish the DV axis of the Drosophila egg (Guichet, 2001).

Orb and K(10)

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, the role of the orb gene in the Drosophila gurken (grk)-epidermal growth factor receptor (Egfr) signaling pathway has been examined. During the pre-vitellogenic stages of oogenesis, the grk-Egfr signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, the grk-Egfr pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. orb functions in both the early and late grk-Egfr signaling pathways, and in each case is required for the localized expression of Grk protein. orb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10) (Chang, 2001).

orb activity is required for early grk-Egfr signaling, since abnormalities in Grk expression are observed in orb³⁴³ and orb³⁰³ ovaries. In the presumed Orb protein null, orb³⁴³, Grk is not detected. In orb³⁰³, Grk expression parallels the aberrant pattern of Orb³⁰³ protein accumulation. In newly formed 16-cell cysts, all germ cells have high levels of the Orb³⁰³ protein. These germ cells also express much higher than normal levels of Grk protein. In older pseudo-egg chambers, both Orb³⁰³ and Grk disappear. These findings argue that the Orb³⁰³ protein inappropriately activates translation of GRK mRNA, and that the mutant Orb protein must be present to sustain Grk expression. Later in oogenesis, after the oocyte nucleus moves from the posterior of the oocyte to the dorsal anterior corner, the grk-Egfr pathway is used to signal dorsal identity to the follicle cells above the oocyte. At this stage orb is required for the proper expression not only of Grk but also of K(10) (Chang, 2001).

How does orb function in regulating translation and localization? Orb homologs in other organisms, the CPEB proteins, interact with elements in the 3' UTRs of masked mRNAs, and activate their translation by a mechanism that is thought to involve polyA addition. Since the translational function of the CPEB proteins is conserved in animals as diverse as clams and mice, it would be reasonable to suppose that the role of the orb gene in the Drosophila grk-Egfr signaling pathway also involves translational activation. Accordingly, the defects in the expression of both Grk and K(10) proteins would arise because wild type orb activity is required to properly regulate the translation of GRK and K(10) mRNAs. In the case of K(10), it seems possible that Orb protein might act directly on the mRNA: (1) K(10) mRNA is associated with Orb protein in an immunoprecipitable complex and (2) K(10) mRNA is mislocalized in orb mutant ovaries (Chang, 2001).

Since translational activation by CPEB proteins in other systems has been tied to polyadenylation, an obvious question is whether the polyA tails of K(10) mRNA are affected in orb mutants. Unfortunately, experiments aimed at testing this point have been inconclusive. Using an anchored-dT RT-PCR procedure, it was found that K(10) mRNA isolated from the strong loss-of-function orb mutant, orb³⁴³, had shorter poly(A) tails than wild type. However, the possibility cannot be excluded that the short poly (A) tails in this mutant arise because K(10) mRNA is targeted for deadenylation in the absence of translation. For orb^mel, the average poly(A) length appeared, at most, to be only marginally shorter than wild type. Of course, since K(10) protein is expressed normally in pre-vitellogenic stages in this mutant, the presence of mRNAs with extended poly(A) tails is not altogether surprising. Further studies will be required to determine whether the mechanism used to promote the translation of K(10) mRNA depends upon polyA addition as is thought to be the case in other organisms (Chang, 2001).

In contrast to K(10), GRK mRNA was not found in Orb immunoprecipitates. Although there are many reasons why an Orb protein:GRK mRNA complex might not be detected, this result forces consideration of the possibility that orb acts on GRK only indirectly. In this case, other mechanisms would have to be proposed to account for the defects in both the localization and translation of GRK mRNA that are observed in orb mutants (Chang, 2001).

It seems possible that the mislocalization of GRK mRNA in the weak hypomorphic orb^mel mutant could arise, at least in part, because the expression of K(10) protein is greatly reduced in stage 8-10 orb^mel chambers. However, since the localization defects in orb^mel are more severe than those seen in K(10) mutants, orb may regulate some other factor in addition to K(10) that helps direct the proper localization of GRK mRNA. An obvious candidate is sqd. Although no alterations in Sqd protein expression could be detected in orb^mel chambers, it should be noted that only one of the three Sqd isoforms seems to be involved in GRK mRNA localization. Consequently, any effects on the expression of this specific isoform could be obscured by the other isoforms (Chang, 2001).

Why is GRK mRNA not properly translated in orb mutant ovaries? Orb protein could be required for the expression of factors that activate translation of GRK mRNA. In orb³⁰³ this factor(s) could be prematurely produced throughout the cyst, leading to the very high levels of unlocalized Grk seen in this mutant. As K(10) and sqd do not seem to function in the localization or translation of GRK mRNA at the posterior of the oocyte in pre-vitellogenic stages, the orb regulatory target(s) early in oogenesis could be different from that used later in DV signaling. Another possibility is that orb regulates the expression of a signal(s) that coordinates the activation of GRK mRNA translation with other events in oogenesis. This function is suggested by the fact that CPEB activity in other organisms helps govern progression through oogenesis and by the finding that grk expression in the DV pathway is sensitive to check points that monitor progression through meiosis. In this case, signals crucial for translation of GRK mRNA might not be produced in the absence of orb activity (Chang, 2001).

The epistatic relationship between orb^mel and K(10) is rather surprising. Since orb is required for the localization and translation of GRK mRNA, it is expected that orb^mel would be epistatic to K(10). However, contrary to this expectation, eggs produced by K(10);orb^mel double mutant females have the dorsalized egg shell phenotype that is characteristic of K(10) mutations, rather than the ventralized phenotype of orb^mel. This result implies that the loss of K(10) function rescues the orb^mel defect in GRK mRNA translation (but not the localization defect). Interestingly, a similar epistatic relationship is found for K(10) and mutations in the spindle (spn) genes. Mutants in the spn genes resemble orb in that GRK mRNA is mislocalized in a K(10)-like pattern but is not properly translated, giving ventralized eggs. Moreover, the defects in GRK mRNA translation in spn mutants can also be rescued by mutations in K(10) and double mutant females produce dorsalized eggs. To explain these findings, it has been postulated that the function of the spn genes is to alleviate K(10)-dependent repression of GRK mRNA translation (Chang, 2001 and references therein).

Although orb could have a similar role in alleviating K(10)-dependent repression of GRK, an alternative (or additional) explanation for the epistatic relationship between orb^mel and K(10) is that K(10) negatively regulates Orb protein expression. This possibility is suggested by the finding that the amount of Orb protein in vitellogenic chambers from the double mutant is close to that seen at equivalent stages in wild-type ovaries. The restoration of near wild-type levels of Orb protein in these orb^mel;K(10) chambers would in turn be expected to produce a concomitant increase in Grk expression, giving the observed gain-of-function phenotype (Chang, 2001).

Complicating the conclusion that K(10) negatively regulates Orb expression is the finding that K(10) protein does not properly accumulate in the oocyte nucleus of vitellogenic orb^mel chambers. One might have expected that this reduction in the level of K(10) protein would alleviate the K(10)-dependent repression of Orb protein expression, leading to an increased accumulation of Orb protein in the orb^mel mutant and a dorsalized (not ventralized) DV phenotype. However, it does not. One explanation for this paradox is that orb^mel is wild type for K(10), whereas this is not the case in the double mutant. In addition, there are no apparent defects in K(10) expression in pre-vitellogenic orb^mel chambers. It is possible that there is sufficient residual K(10) protein remaining at later stages to effectively repress orb, or that K(10) repression of orb is linked to a process that occurs before the time when the accumulation of K(10) protein drops below some critical threshold value in the orb^mel chambers. In this context, it is interesting to note that the most severe defects in both ORB mRNA localization and Orb protein expression in orb^mel occur after the reorganization of the cytoskeleton and the concomitant movement of the oocyte nucleus from the posterior to the anterior of the oocyte. This marks a shift in the localization of orb mRNA and the site of Orb protein synthesis from the posterior of the oocyte to the anterior. Since the expression of K(10) protein before this time is normal in orb^mel ovaries, its possible that K(10) repression may be somehow linked to this spatial transition in orb regulation (Chang, 2001).

Although the K(10) mutation has quite dramatic effects on Orb expression in orb^mel ovaries, there are no obvious changes in Orb expression in K(10) mutant ovaries that are wild type for orb. It seems possible that there may be some special features of the orb^mel mutation that make it especially sensitive to K(10) repression. However, genetic interaction experiments suggest that K(10) also negatively regulates expression of the wild-type orb gene. An important unanswered question is the mechanism of regulation. Here, there is a problem of compartmentalization. For example, since ORB mRNA is thought to be synthesized in nurse cells, K(10) protein is unlikely to influence transcription. Even effects on the localization/translation of orb mRNA must be indirect. Further studies will clearly be required to understand how K(10) regulates orb expression (Chang, 2001).

Kinesin and Dynein

To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the 'anterodorsal corner'). These localizations depend on microtubules that are thought to be organized such that plus end-directed motors can move cargoes, like oskar mRNA, away from the anterior/lateral surfaces and hence toward the posterior pole. Likewise, minus end-directed motors may move cargoes toward anterior destinations. Contradicting this, cytoplasmic Dynein, a minus-end motor, accumulates at the posterior. Disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends (Brendza, 2002).

Examination of the chorions of eggs produced by Khc null germline clones has suggested defects in dorsal-ventral axis formation. Proper dorsal pole specification within the oocyte induces follicle cells to differentiate into a pair of dorsal respiratory appendages near the anterior end of mature eggs. Of 359 eggs from Khc null germline clones, only 1% had normal appendages. Of the remainder, 17% had fused appendages, 26% had a rudimentary dorsal bump, and 56% showed no dorsal material. These phenotypes are completely rescued by a wild-type Khc transgene. These results indicate that germline kinesin I has an important role in dorsal pole specification (Brendza, 2002).

Early steps in dorsal specification occur during stage 7. The posterior microtubule-organizing center (MTOC) disassembles, and the oocyte cortex takes on MTOC activity. Microtubules become particularly abundant at the anterior and anterior margins and are least abundant at the posterior. This suggests an anterior-posterior gradient of cortical microtubule minus ends. The nucleus then shifts from the posterior pole to the anterior margin in a microtubule-dependent manner, and gurken mRNA becomes concentrated around the entire anterior margin. Subsequently, during stages 8–10, gurken disappears from most of the anterior margin and becomes concentrated between the nuclear envelope and the adjacent anterior-lateral cortex (the anterodorsal corner) in a microtubule-dependent manner. Gurken protein is expressed and secreted there, inducing dorsal fates in neighboring follicle cells (Brendza, 2002).

In Khc null stage-8 to -10 oocytes, anti-Gurken immunostaining reveals that anterodorsal accumulation is either weak or absent. Consistent with poor Gurken expression, kekkonI mRNA, which is normally induced in anterodorsal follicle cells by Gurken signaling from the oocyte, is weak or absent. These results indicate that Khc in the germline is required for normal anterodorsal Gurken expression and signaling (Brendza, 2002).

The processes underlying anterodorsal Gurken expression were examined by in situ hybridization and light microscopy. During stages 6–8, gurken mRNA shows a normal transition from localization at the posterior to localization at the anterior margin. The anterior signal in stage 8 appears as a ring in both mutants and controls. However, in stage-9 and -10 mutant oocytes, rather than localizing to the anterodorsal corner, the gurken signal is almost always spread evenly across the anterior in a broad diffuse band that has no ring-like profile. This indicates that kinesin I is critical for normal anterodorsal localization of gurken mRNA. Poor expression of Gurken from the mislocalized mRNA, and the consequent lack of dorsalization, is likely to reflect position-dependent translational repression (Brendza, 2002).

The position of the oocyte nucleus on the anterior margin defines the site of gurken mRNA localization and thus is a critical part of the localization mechanism. Nuclear positioning was defective in about 50% of stage-9 and -10 Khc null oocytes. Nuclei appear to accomplish the initial posterior to anterior shift during stage 7; however, a rigorous assessment of nuclear position is difficult in stage 7 because of the small size of the oocyte. To gain further insight, nuclear positioning was compared in wild-type and Khc null stage-8 to -10 oocytes. Although some nuclei were mispositioned in stage-8 mutants, there was a marked shift away from the anterior margin in stages 9 and 10. While these data do not establish whether or not Khc has a minor role in initial anterior migration, the decline in normal positioning during stages 8–10 suggests that kinesin I does help keep the nucleus at the anterior. The poor retention in Khc mutants may reflect defects in the anchoring of the nucleus to the cortex of the anterior margin. It could also reflect a decline in ongoing anterodorsal forces on the nucleus that may be needed to maintain its normal position. Thus, the mechanism of anterodorsal gurken localization requires proper nuclear positioning, microtubules, and kinesin I (Brendza, 2002).

In summary, the results provide several insights into localization processes during mid-late oogenesis: (1) kinesin I colocalizes at the posterior pole with cytoplasmic dynein; (2) kinesin I is required for the posterior localization of cytoplasmic dynein; (3) kinesin I is required for the dorsal localization of gurken mRNA, and (4) kinesin I contributes to the proper anterior positioning of the oocyte nucleus. A role for kinesin in moving dynein toward the posterior pole provides a solution to the paradox of the accumulation of a minus-end motor in an area thought to be a destination for plus end-directed transport. However, a role for kinesin in anterodorsal localization is surprising because of evidence that minus ends are most concentrated there. In particular, a Nod:ß-galactosidase fusion protein that is targeted to microtubule minus ends accumulates around the nucleus and at the anterior margin during stages 8–10. How might a plus end-directed motor participate in localization toward an area dominated by microtubule minus ends (Brendza, 2002)?

Previous reports and recent results suggest that dorsal pole specification requires the minus end-directed motor, cytoplasmic dynein. Hypomorphic mutations that impair the function of Drosophila Lis1, which is known to be required in various systems for dynein/dynactin function in nuclear migration and other motility processes, can cause ventralization of chorions, mislocalization of the nucleus, and failure of anterodorsal gurken localization. Conditional overexpression of a protein that disrupts the dynein/dynactin complex has been shown to cause equivalent, though more severe, defects in those same dorsal specification processes. The fact that the same dorsal pathway phenotypes are caused by germline Khc disruption suggests that kinesin I and cytoplasmic dynein both are required for nuclear positioning and anterodorsal gurken mRNA localization (Brendza, 2002).

The following model is proposed to explain these results. Dynein, which is synthesized in nurse cells, walks along microtubules from nurse cells through connecting ring canals toward microtubule minus ends at the oocyte posterior until stage 4. After the microtubule cytoskeleton reorganizes during stage 7, concentrating minus ends at the anterior cortex, dynein-generated movements are redirected away from the posterior. This drives the nucleus and gurken mRNA to the anterior margin. Materials like dynein and determinant mRNAs, moved by unknown forces, continue to enter the oocyte from nurse cells through the anterior ring canals. Those that need to be distributed toward the posterior and are too large to diffuse efficiently are moved by kinesin I, either directly or by means of cytoplasmic flows. As the oocyte enlarges during late stages, diffusion of the large cytoplasmic dynein/dynactin complex away from anterior minus ends becomes limiting. Thus, active transport of dynein away from the anterior by kinesin or by kinesin-generated cytoplasmic flows becomes critical. In stage-9 and -10 Khc mutant oocytes, dynein is trapped near minus ends at the anterior cortex. Anterior-directed dynein-based forces that act on gurken mRNA, the nucleus, and/or nuclear anchors are reduced, disrupting their normal positioning mechanisms (Brendza, 2002).

If this dynein recycling model is correct, why does a loss of Khc influence nuclear position and disrupt anterodorsal gurken localization but not other putative dynein functions, such as the anterior localization of bicoid mRNA? As with the initial localization of gurken mRNA, dynein-based forces toward the anterior margin may not be sensitive to poor recycling while the oocyte is small. Subsequent anterior localization of bicoid, as the oocyte enlarges, may be relatively insensitive to a decline in long-range, anterior-directed forces because its requirements for such forces are less than those of the nucleus and gurken mRNA (Brendza, 2002).

In addition to a later role in fostering the dorsal-ventral polarity of the egg chamber, cornichon, gurken, and torpedo also function in an earlier signaling event that establishes posterior follicle cell fates and specifies the anterior-posterior polarity of the egg chamber. Mutations in all three genes prevent the formation of a correctly polarized microtubule cytoskeleton required for proper localization of the anterior and posterior determinants Bicoid and Oskar and for the asymmetric positioning of the oocyte nucleus. cornichon functions in the egg chamber to facilitate Gurken localization, first in posterior terminal follicle cell specification and later in dorsal follicle cell specification. cornichon mutations disrupt the localization of Kinesin, a cytoskeletal motor protein (Roth, 1995).

In Drosophila oocytes, gurken mRNA localization orients the TGF-alpha signal to establish the anteroposterior and dorsoventral axes. The path and mechanism of gurken mRNA localization has been evaluated by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. Evidence is presented suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations (MacDougall, 2003).

The organization of MTs in the oocyte was analyzed with high-resolution imaging of Tau-GFP and Nod-LacZ distributions in the oocyte. A particularly high concentration of MT minus ends is detected at the dorsoanterior corner as well as in the anterior cortex and entire anterior. The presence of an MT network associated with the oocyte nucleus explains why a higher concentration of MTs are found in the anterior than in the posterior, despite the diffuse nature of the MTOC in the oocyte. A distinct network of MTs associated with the oocyte nucleus also explains why, in merlin mutant oocytes, injected grk RNA is observed accumulating at the posterior, where the oocyte nucleus is located. A high concentration of MT minus ends is also detected in an anterior ring in addition to a lower concentration all over the anterior. Considering all these results in the context of the published data on MT distribution in the oocyte leads the authors to propose the following model for MT organization in the oocyte. In addition to MTs with their minus ends at the diffuse anterior MTOC and their plus ends at the posterior, there are some other MTs with their minus ends throughout all parts of the cortex. It is proposed that, in addition to these networks, there is also a distinct network of MTs that are specifically associated with the oocyte nucleus. These MTs form a loose basket surrounding the nucleus and radiate throughout the anterior and partly into the middle of the oocyte. Observations of Tau-GFP suggest that there are many other MTs that are more loosely organized throughout much of the oocyte (MacDougall, 2003).

The organization of MTs proposed provides a good explanation for why the grk particle movements occur in two distinct steps. It is proposed that, during the first step of movement of grk particles to the anterior of the oocyte, the RNA is likely to be moving on MTs whose plus ends are at the posterior of the oocyte and whose minus ends are along the entire anterior. The second step of movement of the particles is likely to occur on the MT network that forms a basket around the nucleus, with the MT minus ends at the dorsoanterior corner and the plus ends extending toward the anterior and, also, partly into the middle of the oocyte. This model for MT organization fits well with the fact that many grk RNA particles were observed to make sharp turns at the anterior, and some in the interior, of the oocyte (MacDougall, 2003).

The model showing that distinct classes of MTs exist within the oocyte begs a question: how does Dynein-dependent transport deliver grk RNA to a very different destination from other RNAs in the oocyte, which may also be transported to the minus ends of MTs by Dynein? It is proposed that different RNAs that are transported to the minus ends of MTs by the same Dynein motors could move on distinct networks of MTs. This would explain why the destination of injected bcd RNA (which is thought to require Dynein for its localization), depends on whether it is preexposed to nurse cell cytoplasm. bcd RNA injected into the oocyte moves to the nearest cortex along MTs whose minus ends are at the cortex. However, bcd RNA that is preexposed to nurse cell cytoplasm is able to move from the posterior to the anterior of the oocyte, apparently in a similar route to that in step 1 of grk localization, which has been defined. Step 2 of grk RNA particle movement is not shared with bcd RNA and could occur along the MT network that is specifically associated with the oocyte nucleus. Interestingly, bcd, but not grk, mRNA localization requires gamma-Tub37C and Dgrip75 (MacDougall, 2003 and references therein).

It is most likely that specific transacting factors that recognize RNA signals are responsible for determining which RNAs use which motors and also which distinct MT network is utilized during the Dynein-dependent transport to different destinations. For example, in nerve cells, the choice of cytoplasmic destination of cargo transported by Kinesin is determined by the presence or absence of a protein called GRIP. Such key transacting factors are likely to also include Squid and K10, since, in mutants of these genes, grk mRNA is localized in the anterior, rather than the dorsoanterior corner. However, in addition to the transacting factors, the different MTs are likely to differ in some way, allowing the different kinds of RNA-motor complexes to distinguish among them. Such differences could include chemical modifications of tubulin or different tubulin isoforms as well as distinct populations of MT-associated proteins (MAPs). It is also possible that alphaTub37C and Dgrip75 could be involved in selectively nucleating a subset of MTs used for bcd, but not grk, mRNA localization (MacDougall, 2003 and references therein).

Dynein-dependent motility of RNA and other cargo to the minus ends of MTs is likely to be a widely deployed mechanism within cells. Selective utilization of different MT networks would provide a nice way to sort different cellular components that are transported by the same Dynein motor to a variety of distinct minus ends in the same cell. Rapid and efficient real-time assays for mRNA localization will allow the definition of cis-acting signals and trans-acting factors that determine which subset of MTs are selected by different RNA cargos that utilize the same motors (MacDougall, 2003).

In the Drosophila oocyte, microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. mRNA localization and nuclear positioning at mid-oogenesis is shown to depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. It is concluded that the microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole (Januschke, 2002).

grk mRNA is produced by both the nurse cells and the oocyte nucleus. After nuclear migration, grk mRNA accumulates briefly along the anterior margin of the oocyte, before it concentrates in a perinuclear position. The anterior localization of grk is not affected when Dynein function is reduced or if Kinesin I function is completely abolished. However, both motors are required to transport grk to the nucleus. It is suggested that grk mRNA is transported toward the minus ends of MTs, which emanate from the nucleus. This would explain the Dynein requirement for grk transport to the nucleus. The role of Kinesin I in anterodorsal grk transport might again reflect the need to retrieve the Dynein motors for renewed cargo loading, as suggested for bcd and the oocyte nucleus (Januschke, 2002).

This model has to assume, however, that Dynein-Dynactin complexes carrying different cargos can distinguish between distinct populations of MTs: Dynein-Dynactin complexes loaded with bcd mRNA should be transported to and remain at anterior cortex, while those loaded with grk mRNA should be subject to a second transport step toward the nucleus. Deletions within the grk 3'UTR allow anterior localization of grk mRNA but prevent its transport to the nucleus. This suggests that specific factors distinguish anterior and anterodorsal transport of grk. The heterogeneous nuclear RNA binding protein (hnRNP) Squid plays a central role in this process. It regulates both grk localization and translation and binds directly to the grk 3'UTR. Squid protein, like grk, appears to be transiently localized along the anterior cortex during the transition from stage 7 to stage 8 (Januschke, 2002).

grk mRNA, though mislocalized, is frequently translated when Kinesin I or Dynein motor activities are impaired. Since grk mRNA is found around the anterior cortex in those cases, Grk secretion should occur around the entire circumference of the oocyte instead of being restricted to the dorsal side. Secreted Grk induces dorsal follicle cell fates. Thus, ectopic secretion should lead to the formation of dorsalized eggs as in squid and fs(1)K10 mutants in which grk mRNA is also mislocalized. However, impaired MT motor activity leads to ventralized eggs and thus to reduced Grk signaling. An analysis of Grk distribution in oocytes shows that, in contrast to wild-type or squid and fs(1)K10, Grk protein is not closely associated with grk mRNA and fails to reach the plasma membrane. Thus, polar transport of Grk protein and exocytosis requires Dynein and Kinesin I activity. This is not surprising, since both motors have been shown to be involved in Golgi dynamics in higher eukaryotes and it has been shown that vesicular trafficking from the Golgi to the plasma membrane requires Kinesin activity (Januschke, 2002).

Interestingly, no requirement has been detected for the two motors in earlier Grk signaling, which induces posterior follicle cells and prevents the formation of a second micropyle at the posterior pole. In the case of Dynamitin overexpression, this might be due once more to residual levels of Dynein function. In the case of Kinesin I, it is assumed that Grk secretion is only impaired, but not entirely blocked. The phenotypic series of grk mutations suggests that minute amounts of secreted Grk are sufficient to induce posterior follicle cells (Januschke, 2002).

Egalitarian, Bicaudal D and Vasa

To determine whether Egalitarian and Bicaudal D directly affect the extent to which OSK mRNA mislocalizes, the distribution of OSK mRNA was examined in BicD-Dominant mutants. Reducing the amount of egl wild-type product decreases ectopic localization of osk to the anterior and increasing the amount of egl wild-type product enhances the mislocalization of OSK to the anterior. Because the effect of BicD-Dominant mutants depends on egl wild type function, it is concluded that egl and BicD act in the same pathway and that the two function in concert to control OSK mRNA localization. It is also thought that Egl and BicD have a role in dorsoventral polarity, as mutation of the two genes reduce the level of Gurken mRNA. Localization of GUR is known to require an intact microtubule cytoskeleton (Mach, 1997).

Localization of cytoplasmic messenger RNA transcripts is widely used to target proteins within cells. For many transcripts, localization depends on cis-acting elements within the transcripts and on microtubule-based motors; however, little is known about other components of the transport machinery or how these components recognize specific RNA cargoes. In Drosophila the same machinery and RNA signals drive specific accumulation of maternal RNAs in the early oocyte and apical transcript localization in blastoderm embryos. It has been demonstrated in vivo that Egalitarian (Egl) and Bicaudal D (BicD), maternal proteins required for oocyte determination, are selectively recruited by, and co-transported with, localizing transcripts in blastoderm embryos; interfering with the activities of Egl and BicD blocks apical localization. It is proposed that Egl and BicD are core components of a selective dynein motor complex that drives transcript localization in a variety of tissues (Bullock, 2001).

During Drosophila oogenesis, specification of the oocyte is associated with selective accumulation of RNA determinants supplied by the neighboring, interconnecting ovarian nurse cells. Subsequently, deposition of mRNA transcripts at selected sites within the oocyte leads to localized translation of the proteins that establish the prospective embryonic body axes. gurken (grk) transcripts reside first posteriorly and then anterodorsally, and sequentially establish the anteroposterior and dorsoventral axes. bicoid (bcd) and oskar (osk) transcripts localize to the anterior and posterior of the oocyte, respectively, to pattern the anteroposterior body axis (Bullock, 2001).

The injection assay was used to investigate whether any maternal transcripts that localize in the oocyte are recognized by the localization machinery of blastoderm embryos. Five such transcripts [bcd, grk, nanos (nos), osk and female sterile (1) K10 (K10)] were tested, and all accumulate in the apical cytoplasm after injection. With the exception of osk transcripts -- only a small proportion of which localize apically -- the efficiency of localization of these transcripts appears indistinguishable from that of pair-rule transcripts. Maternal transcripts also localize apically when zygotically expressed from endogenous transgenes. Preinjection with colcemid severely inhibits apical localization of the injected maternal transcripts, indicating that their localization in blastoderm embryos, like that of the pair-rule transcripts, is dependent on intact microtubules (Bullock, 2001).

The common aspect of maternal RNA localization measured in these experiments is unlikely to be transport within the oocyte, because the maternal transcripts tested are distinctly distributed in late stage oocytes by means of different motors and accessory factors. However, all the transcripts -- with the possible exception of grk -- are synthesized in adjacent nurse cells and reach the oocyte by transport along microtubules. To test whether this process is analogous to apical localization in blastoderm embryos, a bcd transcript was used containing a single nucleotide change (4496G->U). This change prevents early oocyte-specific transport (stages 4-6) without disrupting later (stage 6 onwards) import of transcripts into the oocyte or their subsequent accumulation at the anterior cortex. This mutation inhibits apical bcd localization in blastoderm embryos, suggesting that transcripts localize in this injection assay through the same machinery that transports transcripts into the early oocyte (Bullock, 2001).

These data suggest that components of the blastoderm localization machinery are also likely to function in RNA transport into the early oocyte. Genetic screens for maternal mutations that affect formation of the embryonic axis have identified egl and BicD as genes required for oocyte differentiation and for specific RNA accumulation in the oocyte. However, their exact activities are uncertain. BicD protein includes multiple heptad repeats, which may mediate oligomerization and interactions with other proteins; Egl includes a domain shared with 3'-5' exonucleases. During oogenesis, these two proteins form complexes together and colocalize at the minus ends of microtubules. The integrity of the microtubule cytoskeleton is defective in egl and BicD mutants, which has been proposed to explain subsequent defects in RNA localization. Alternatively, Egl and BicD might act directly in RNA transport. However, evidence that distinguishes between these two possibilities is lacking (Bullock, 2001).

Whether Egl and BicD are present in early embryos was examined. Both proteins are supplied maternally to the embryo. They are noticeably enriched apical to the nuclei at blastoderm stages where they colocalize with dynein heavy chain (Dhc) -- a component of the motor associated with apical transcript transport. Nevertheless, a large proportion of both of the proteins is present in the basal cytoplasm (Bullock, 2001).

Egl/BicD is enriched at sites of RNA localization in both blastoderm embryos and oocytes, presumably as the consequence of protein/RNA co-transport. The complex may have an additional role in anchoring transcripts at their destination. Alternatively, maintenance of localized transcripts might not depend on an independent anchorage step, but result from sustained minus-end-directed transport (Bullock, 2001).

gurken and the I factor retrotransposon RNAs share common localization signals and machinery

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFα signal and forming the primary embryonic axes. gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, that forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further an understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. It is proposed that the transposition of other elements may exploit the host's RNA transport signals and machinery (Van De Bor, 2005).

Retrotransposons are transposable elements whose transposition involves RNA intermediates. The Drosophila I factor, a non-long-terminal-repeat (non-LTR) retrotransposon, is similar to the LINE1 (L1) elements that make up at least 17% of the human genome. Interestingly, its transcript also localizes in the oocyte but its localization signal has been mapped only crudely. The I factor encodes two proteins: a nucleic acid binding protein (ORF1p) and protein encoding domains with predicted endonuclease, reverse transcriptase, and RNaseH activities (ORF2p) . Most strains of D. melanogaster contain about 10 copies of full-length and potentially active I factors in euchromatin [Inducer (I)] and about 30 defective I factors in the pericentromeric heterochromatin [reactive (R)] (Van De Bor, 2005).

I factor transposition occurs at high frequency in the germline of the female progeny of a cross between a reactive female and an inducer male. Such females, known as 'SF' (sterilité femelle) females, have greatly reduced fertility and are said to manifest I-R hybrid dysgenesis. There is an increased frequency of mutations among the progeny of SF females that do survive, and these are thought mostly to be due to I factor insertions or chromosome rearrangements. The exact cause of I factor-induced female sterility is not known (Van De Bor, 2005).

The I factor, like other non-LTR retrotransposons, is believed to transpose by target-primed reverse transcription (TPR), a mechanism in which reverse transcription of the RNA transposition intermediate is primed by a 3′ OH at a break in chromosomal DNA at the site of integration. This is assumed to require entry of the RNA into the nucleus of the cell in which transposition takes place. Indeed, I factor RNA has been detected adjacent to the oocyte nucleus at stages 8 and 9 and in an anterior ring. A 552 bp sequence within the second open reading frame has been shown to be necessary and sufficient for this localization (Seleme Mdel, 2005). This is referred to as the Loc⁺ sequence. The mechanism by which localization of I factor RNA is achieved is not known (Van De Bor, 2005).

This study uncovers a surprising dependence of both grk and I factor transposable element RNAs on shared components of the cellular transport machinery of the oocyte. grk and I factor transcripts contain a small stem loop of common secondary structure, but very limited sequence similarity, which represents a destination consensus signal for targeting RNAs in two steps to the oocyte nucleus by dynein-mediated transport along MTs. I factor transposition causes a grk mislocalization phenotype and subsequent eggshell and embryonic dorsoventral patterning defects, as well as mislocalization of bcd RNA, leading to anteroposterior embryonic axis defects. I factor RNA was also detected within the oocyte nucleus, suggesting that entry into the nucleus is required for transposition and transmission into the germline. These observations provide an explanation for SF (sterilité femelle) female sterility and for the mechanism and route of transposition in the germline. A common principle is proposed that could apply to other transposable elements, namely that selective germline transposition is achieved through intracellular mRNA transport in the oocyte, using the host's machinery followed by import into the oocyte nucleus (Van De Bor, 2005).

Using an in vivo injection assay for dorsoanterior localization of fluorescently tagged RNA, the minimal regions, the GLS (gurken Localization Signal) and ILS (I factor Localization Signal) have been defined as necessary and sufficient for the respective localization of grk and I factor RNA in two steps to a dorsoanterior cap in oocytes. Like endogenous and injected grk RNA, the anterior localization of ILS and GLS RNA depends on MTs and dynein. The GLS and ILS have similar secondary structure but only limited sequence similarity. A novel in vivo competition assay has been developed and was used to show that the GLS, ILS, and full-length grk compete specifically for a transacting factor or factors required for localization. When the I factor is mobilized in the female germline, its transcript can be detected close to the oocyte nucleus; it causes a disruption of grk and bcd mRNA localization and patterning defects in the embryos. These observations indicate that the I factor is highly integrated into the biology of its host, utilizing cellular localization pathways that are of key importance to the development of the fly. Furthermore, they provide a molecular mechanism for the previously unexplained sterility associated with I factor transposition, that has been known for many years (Van De Bor, 2005).

It is now known that the GLS is contained within a 400 bp fragment of the coding region of grk, previously shown to be necessary for grk mRNA localization in transgenes. In these studies, the 3'UTR was also required for localization and the 5'UTR was required for stability and late localization. The data agree with most of these previous results: the dorsoanterior localization of transgenic GLS-GFP RNA becomes diffuse in stage 10, and sequences outside the GLS are required for full efficiency of localization of injected RNA. While the previous studies did not test directly whether part of the coding region including the GLS is sufficient for localization, they did show that the 3'UTR is necessary for the second (dorsoanterior) step of grk RNA localization but not for the first (anterior) step. The slight differences between the prior and current results are probably due to differences in the structure of the transgenes affecting RNA secondary structure and the function of the signals (Van De Bor, 2005 and references therein).

The endogenous I factor RNA is localized in a pattern that overlaps with both endogenous bcd and grk transcripts. The ILS is sufficient to promote a grk-like localization pattern, and the signal that promotes a bcd-like pattern of localization to the endogenous I factor remains to be defined. The GLS is not sufficient to promote all aspects of grk mRNA localization, since the GLS-GFP transgene RNA fails to persist at stage 10 and the signal required for such persistence probably resides in the 5′UTR. It is also possible that there is some degree of redundancy in anterior and dorsoanterior localization signals in grk and I factor transcripts (Van De Bor, 2005).

The results show that the I factor RNA is localized by a dynein- and MT-dependent mechanism, adding to two previously characterized dynein- and MT-dependent RNA transport cargos in the oocyte, namely grk and bcd. It is likely that there are many more RNAs that are transported by dynein in the oocyte, such as K10 and orb. Additional transcripts that localize by a dynein-dependent mechanism may also include the transcripts of other retrotransposons in order to target transposition to the oocyte nucleus, thus ensuring passage through the germline to the next generation. Interestingly, some retroviruses, such as HIV, also require MTs and dynein for their transport to the nucleus. It is proposed that RNA transport may play an important role in the life cycle of other transposable elements and viruses (Van De Bor, 2005).

The competition assay that was developed shows that grk and I factor transcripts share the same localization machinery. Furthermore, a semiquantitative comparison between the levels of endogenous I factor, grk, and bcd RNA, suggests that the I factor RNA is present in excess to the other RNAs, consistent with it competing for factors required for bcd and grk RNA localization. However, the endogenous and injected case could be mechanistically very different. While the injected ILS or GLS RNA are likely to swamp the machinery required for the anterior and second step of grk RNA transport, the endogenous I factor transcript might interfere with a different step such as grk RNA anchoring and/or bind to the same factors at different affinity (Van De Bor, 2005).

The localization signals that were defined are necessary and sufficient for localization, and the injected RNA signal is able to recruit all the factors in the cytoplasm required for their localization. Therefore, the specificity of mRNA localization to the dorsoanterior corner is completely defined by these RNA signals and by the proteins that bind to them in the cytoplasm. These proteins must somehow define which motor the RNP complex binds to and possibly the choice of a subset of MTs that the particular motor-cargo complex moves along. It is anticipated that the protein composition of the I factor and grk RNP complexes are very similar but could be subtly different in either composition or spatial organization of the same factors. An important difference between both RNAs is that the I factor transport appears to require the ORF1 protein. Another difference may be in the factors required to import the I factor mRNA into the oocyte nucleus, a step that is absent in the case of grk. Future biochemical experiments will be required to define the complement of proteins that bind to the GLS, ILS, and K10 localization signal, as well as the composition of the motor complexes and their accessory factors. The great challenge will be to define which of these are required only for general mRNA metabolism and which are involved in defining the specificity of cargo destination (Van De Bor, 2005).

A Dynein-dependent shortcut rapidly delivers axis determination transcripts into the Drosophila oocyte

The primary axes of Drosophila are set up by the localization of transcripts within the oocyte. These mRNAs originate in the nurse cells, but how they move into the oocyte remains poorly understood. This study investigates the path and mechanism of movement of gurken RNA within the nurse cells and towards and through ring canals connecting them to the oocyte. gurken transcripts, but not control transcripts, recruit the cytoplasmic Dynein-associated co-factors Bicaudal D (BicD) and Egalitarian in the nurse cells. gurken RNA requires BicD and Dynein for its transport towards the ring canals, where it accumulates before moving into the oocyte. The results suggest that bicoid and oskar transcripts are also delivered to the oocyte by the same mechanism, which is distinct from cytoplasmic flow. It is proposed that Dynein-mediated transport of specific RNAs along specialized networks of microtubules increases the efficiency of their delivery, over the flow of general cytoplasmic components, into the oocyte (Clark, 2007).

Within nurse cells, a new path of Dynein-dependent transport to the ring canals has been identified that links the nurse cells to the oocyte. grk RNA moves along this route, and the data suggest that bcd and osk transcripts also follow the same path. This intracellular shortcut requires BicD and is distinct from the route taken by general cytoplasmic components and control RNAs, which move into the oocyte less effectively. The data suggest that the distinction between RNA components that follow this direct path and those that do not is the ability to recruit the Dynein-associated co-factors BicD and Egl. It is proposed that Dynein-dependent transport of grk, bcd and osk transcripts towards the ring canals follows a MT network, which is distinct from other networks in the nurse cells (Clark, 2007).

It was not possible to determine the proportion of grk RNA particles that move compared with ones that were stationary because it is hard to distinguish stationary particles from autofluorescence. By contrast, rapidly moving RNA particles of the same intensity are easy to distinguish from background. By showing directly that Dynein is required for the transport of axis specification transcripts from the nurse cells to the oocyte, this work explains previous work on this topic that did not directly address the mechanism of transport from the nurse cells into the oocyte. The results also explain why the movement of bcd and osk mRNA into the oocyte is MT dependent, and why pair-rule transcripts, which are transported in the blastoderm embryo in a MT-dependent manner, by Dynein, are also transported into the oocyte when exogenously expressed in the nurse cells. It is suggested that nurse cell-to-oocyte transport is likely to be a fairly promiscuous transport system that can deliver any transcript that has the capacity for transport by the Dynein motor complex along MTs to their minus ends. It is therefore likely that the Dynein-dependent shortcut is deployed by many other transcripts that are localized in the oocyte during mid-oogenesis, such as orb, K10 and nanos (nos). In fact, given that the oocyte nucleus is largely transcriptionally inactive, it is possible that up to 10% of all transcripts thought to be localized in the oocyte could first be transported by the same Dynein-dependent mechanism into the oocyte (Clark, 2007).

The Dynein-dependent transport route uncovered within the nurse cells is likely to allow transcripts encoding axis specification determinants to be delivered rapidly at key times in oogenesis. In particular, cytoplasmic transport during stages 5-8 is likely to be relatively slow and non-specific, so delivery of transcripts from the nurse cell nuclei to the oocyte cytoplasm is likely to be very slow, if it involves an undirected diffusion-based process. Certainly, osk and bcd mRNA and other transcripts are thought to form large multimeric complexes in the nurse cells, so are unlikely to be easily dispersed within the cytoplasm by free diffusion. osk and grk are transported into the oocyte at the same stages of oogenesis, and both require Bruno and Hrp48 (also known as Hrb27C - FlyBase); however, it is unclear whether they are transported within the same complexes into the oocyte. At stage 10B, the mechanism this paper has described is not required, because the rapid dumping of all of the cytoplasmic contents of the nurse cells into the oocyte occurs. However, by stage 10B, most of the major patterning transcripts have probably been localized in the oocyte (Clark, 2007).

This work does not address directly the speed of passive diffusion of RNA into the oocyte or the mechanism of cytoplasmic flow and dumping in stage 10B. Although Kinesin 1 is required for cytoplasmic movements within the oocyte, it is not required for the general growth of the oocyte or for the presence of mRNAs in the oocyte. These observations suggest that Kinesin 1 is not important for cytoplasmic transport or for specific mRNA transport into the oocyte (Clark, 2007).

The existence of a specific intracellular route for the transport of transcripts in nurse cells adds to existing evidence that there are various minus-end destinations to which different cargos are delivered by Dynein within the same cell. For example, within the oocyte, bcd RNA is transported to the cortex if injected into the oocyte, but to the anterior, after transport into the oocyte, following injection into the nurse cells. grk RNA is transported in two steps, both of which depend on Dynein. The second step is towards the oocyte nucleus, and is unique to grk and I factor RNA, but is not shared with bcd and K10 transcripts, despite the fact that all of these transcripts are probably being transported by Dynein. There are, therefore, likely to be several distinct MT routes along which Dynein can transport cargos within egg chambers. In neurons, choices between distinct MT routes are made by Kinesin-dependent vesicle transport depending on the presence of a specific neurotransmitter-receptor-interacting protein, GRIP1. How Dynein chooses between distinct MT networks is less clear, but could be based on distinct isoforms of the motor complex, on distinct kinds of MTs with different tubulin isoforms, or on their decoration with different MT-associated proteins. In addition, there is evidence that cargos can influence the behaviour of their motor, raising the interesting possibility that cargos could also influence the choice of MT route adopted by their motors. This work suggests that the presence of BicD and Egl could also influence the choice of MT route adopted by motors. Future work, including new approaches for co-visualizing MTs and RNAs in living cells, will be required to distinguish between all of these possible ways of selecting intracellular routes. Whatever the basis of such distinct routes, they are likely to exist for various kinds of molecular motors and to be functionally important for a wide range of tissues and cargos (Clark, 2007).

Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor

Cytoplasmic sorting of mRNAs by microtubule-based transport is widespread, yet very little is known at the molecular level about how specific transcripts are linked to motor complexes. In Drosophila, minus-end-directed transport of developmentally important transcripts by the dynein motor is mediated by seemingly divergent mRNA elements. Evidence is provided that direct recognition of these mRNA localization signals is mediated by the Egalitarian (Egl) protein. Egl and the dynein cofactor Bicaudal-D (BicD) are the only proteins from embryonic extracts that are abundantly and specifically enriched on RNA localization signals from transcripts of gurken, hairy, K10, and the I factor retrotransposon. In vitro assays show that, despite lacking a canonical RNA-binding motif, Egl directly recognizes active localization elements. A physical interaction was revealed between Egl and a conserved domain for cargo recruitment in BicD and data is presented suggesting that Egl participates selectively in BicD-mediated transport of mRNA in vivo. This work leads to the first working model for a complete connection between minus-end-directed mRNA localization signals and microtubules and reveals molecular strategies that are likely to be of general relevance for cargo transport by dynein (Dienstbier, 2009).

Many proteins achieve an asymmetric localization within the cytoplasm through the transport of their mRNAs along the cytoskeleton by molecular motors. Despite the widespread occurrence of mRNA transport, the detailed mechanisms by which specific transcripts are recognized and recruited to motor complexes are poorly understood. One exception is during bud-specific enrichment of mRNAs along actin filaments in the yeast Saccharomyces cerevisiae, where proteins have been identified that can account for a complete link between localizing mRNAs and the cytoskeleton. However, many metazoans rely on microtubules to deliver mRNAs over the requisite longer distances, and mechanistic insights into how these transcripts are linked to motors are relatively sparse (Dienstbier, 2009).

One of the best prospects for elucidating microtubule-based mRNA transport is in the Drosophila syncytial blastoderm embryo, where a pathway for apical localization of a subset of endogenous mRNAs can be accessed by microinjection of in vitro synthesized, fluorescently labeled transcripts. Consistent with the nucleation of the minus ends of the microtubules in the apical cytoplasm, localization of these transcripts is driven by cytoplasmic dynein together with its accessory complex dynactin. Related machinery delivers mRNAs to the minus ends of microtubules in other Drosophila cell types, including oocytes and neuroblasts (Dienstbier, 2009).

The cis-acting RNA elements mediating asymmetric localization by dynein have been studied in detail for seven transcripts (the developmentally important mRNAs bicoid [bcd], fushi tarazu [ftz], gurken [grk], hairy [h], fs(1)K10 [K10], and wingless [wg], and the I Factor retrotransposon RNA) and contain one or more stem-loop structures. These 'localization signals' are necessary for minus-end-directed localization and also sufficient when inserted into heterologous transcripts (Dienstbier, 2009).

The localization signals in the different transcripts do not share significant primary sequence similarity and often have different lengths. This has led to two competing models: the first in which the RNA elements contain cryptic features that associate with a common recognition machinery, and the second in which they are recognized by different proteins, each able to independently provide a link to the dynein complex. It has not been possible to discriminate between these scenarios, because proteins that specifically bind any of these elements and are required for transport have not been identified (Dienstbier, 2009).

In addition to dynein/dynactin, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for targeting of mRNAs to the minus ends of microtubules. Egl and BicD are found in a complex with each other in vivo (together with other copies of themselves), although it is not known whether they interact directly. Egl and BicD also associate with dynein light chain (Dlc) and the dynein/dynactin complex, respectively, and are recruited to injected localizing mRNAs in embryos to bias the net movements of a bidirectional mRNA transport complex apically. Together, these observations have led to a model in which Egl and BicD associate with localization signals and increase the frequency of minus-end-directed dynein/dynactin movements. Because neither Egl nor BicD has a known RNA-binding motif, it has been reasoned that they are recruited to localization signals by intermediary factors that directly contact the message (Dienstbier, 2009 and references therein).

Whether Egl has roles outside of mRNA transport has not been reported, but BicD functions in the transport of a subset of other cargoes for dynein. It has been proposed that the N-terminal two-thirds of mammalian BicD are sufficient for stimulating dynein transport and that the remaining C-terminal sequences (hereafter referred to as the CTD [C-terminal domain]) mediate a link between cargoes and the motor. This is based on the findings that the CTD can be functionally substituted by heterologous motifs for organelle recognition and can bind Rab6, a membrane-linked GTPase that recruits dynein to Golgi vesicles (Dienstbier, 2009).

This study attempts to elucidate the mechanism of linkage of different mRNA localization signals to dynein. The surprising finding is reported that Egl is a selective RNA-binding protein that directly contacts active localization signals. Thus, seemingly divergent mRNA signals are recognized by the same factor. Egl associates with a conserved domain for cargo recruitment in BicD and is selectively required for mRNA transport in vivo. This work provides unique insights into the molecular links between localizing mRNAs and microtubule-based motors, and also sheds light on general mechanisms of cargo transport by dynein (Dienstbier, 2009).

Because of difficulties in finding shared features between dynein-dependent localization signals in different transcripts, it was not known whether dedicated factors are responsible for recognizing each of these elements. This uncertainty has severely restricted the ability to generalize conclusions from studies of localization mechanisms of individual transcripts. This work demonstrates that the same protein, Egl, is capable of specifically contacting minus-end-directed localization signals from multiple different transcripts. This conclusion is supported by the findings that (1) Egl and BicD are the only factors visibly enriched from embryonic extracts on all four localizing elements tested relative to a number of nonlocalizing controls, (2) Egl function in Drosophila is required for BicD-mediated transport of mRNAs and not other cargoes tested, (3) the majority of Egl, but not BicD, in cell extracts is found in a complex whose size is sensitive to Rnase treatment, and (4) recombinant Egl, but not BicD, binds RNA in vitro and is capable of discriminating between active apical localization signals and those containing subtle inactivating mutations (Dienstbier, 2009).

In addition to the four elements tested in this study, Egl is also likely to associate directly with other mRNA localization signals because bcd, ftz, and wg recruit Egl in vivo and depend on its function for minus-end-directed transport. Indeed, Egl binding may be the major, and perhaps only, specific determinant of the activity of an apical localization signal, as all three subtle inactivating mutations that were tested inhibit association of Egl from embryonic extracts (TLS^δbub, TLS^U6C, and hSL1^C15G), and a fourth inactive point mutant (bcdSLV^4496G-U) prevents recruitment of Egl to bcd injected into embryos. Presumably, despite differences in primary sequence composition, all of the characterized localization elements contain cryptic structural features that are recognized by Egl. Elucidating the structural basis of this recognition event will be the goal of future long-term studies (Dienstbier, 2009).

Interestingly, Egl exhibits some affinity for inactive localization elements when expressed recombinantly, as well as in embryonic extracts. Egl may well exhibit greater selectivity for active signals in the appropriate in vivo context. This could be because the composition of in vitro binding buffers is suboptimal. Alternatively, the incorporation of mRNAs into oligomeric particles within the cell may give rise to cooperative interactions between individual Egl and BicD complexes, thereby increasing cargo specificity. Nonetheless, an inherent degree of promiscuity by Egl in vivo would fit with a previous finding that its overexpression in embryos is sufficient to target a small amount of an endogenous nonlocalizing transcript population to the apical cytoplasm (Bullock, 2006) and could also be the basis of repeated emergence of apical localization signals during dipteran evolution (Dienstbier, 2009).

The mRNA elements that direct apical transport in the blastoderm embryo are also capable of mediating localization of transcripts toward the minus ends of microtubules during oogenesis. It is therefore very likely that direct binding of Egl to these stem-loops is also functionally significant during these stages. Indeed, Egl and BicD have been shown to be components of motor complexes that transport grk from the nurse cells into the oocyte. Interestingly, within the oocyte the h and K10 elements are involved in localization to the anterior cortex, whereas those in grk and the I factor are also sufficient for translocation from the anterior to the dorso-anterior corner. Dorsalward movement is presumably due to the binding of the ILS and GLS to oocyte-specific factors in addition to Egl, either sequentially or simultaneously, or by modulating the mode of Egl binding (Dienstbier, 2009).

It has been shown that Egl and BicD are in a complex together in vivo. The current data shows for the first time that Egl, through its N-terminal 79 amino acids, directly interacts with BicD. In addition, Egl also binds Dlc through a consensus light chain-binding site between amino acids 963 and 969 (Navarro, 2004). BicD is able to recruit the dynein/dynactin complex (Hoogenraad, 2003) and Dlc associates with other dynein subunits. Thus, together with evidence for Egl RNA binding through amino acids 1-814, it is now possible to build a working model of a complete link between minus-end-directed mRNA signals and microtubules for the first time (Dienstbier, 2009).

Egl, BicD, and mRNA elements do not appear to be obligatory for particle assembly or bidirectional mRNA motility (Bullock, 2006). Instead, they are likely to be essential parts of a cassette that up-regulates minus-end-directed movement of a generic bidirectional mRNA transport complex. Other RNA-binding factors presumably package both localizing and nonlocalizing RNAs and provide additional links to motors (Dienstbier, 2009).

Within the minus-end regulatory cassette, the role of Egl is probably to recruit both BicD and Dlc to the mRNA to ensure efficient targeting of transcripts to the minus ends of microtubules. The presence of both Egl-interacting partners might be required for the stability of the motor complex. Alternatively, previous observations of the effects of altering protein concentrations on mRNA transport are consistent with Egl-Dlc and Egl-BicD interactions regulating different aspects of motility of the bidirectional motor complex: processivity and switching behavior, respectively (Bullock, 2006). Like Egl, Rab6 is able to associate with both BicD and a Dlc. Association with both BicD and Dlc may therefore be a common strategy used by cargo adaptors to ensure efficient minus-end-directed transport (Dienstbier, 2009).

Binding of both Egl and Rab6 to BicD is sensitive to the same amino acid substitution in the CTD. Egl and Rab6 recognize localizing mRNAs and Golgi vesicles, respectively, raising the possibility that BicD functions in the transport of different cargoes through mutually exclusive association of the CTD with cargo-specific adaptors. It was found that relatively subtle overexpression of Egl not only augments BicD-dependent apical mRNA transport (Bullock, 2006), but also antagonizes BicD function in lipid droplet motility. This implies that, through competition for the BicD CTD, the pathways for microtubule-based transport of different cargoes can be finely balanced. Alteration of the availability of adaptors for BicD is therefore a potentially effective strategy for regulating net sorting of cargoes (Dienstbier, 2009).

Experiments involving the tethering of cargoes to BicD domains also shed light on potential general mechanisms of dynein-based cargo transport. As is the case for mammalian BicD, removal of the CTD of the Drosophila protein stimulates transport by dynein. This situation presumably mimics a version of the full-length protein bound to a cargo adaptor in which an autoinhibitory effect of the C terminus is negated. The N terminus of BicD can efficiently capture dynein/dynactin components from cell extracts, suggesting that this interaction could be entirely sufficient for productive transport. However, the results indicate that, at least in Drosophila, the capacity of BicDδC to mediate net movement of tethered cargoes is dependent on its association with endogenous BicD transport complexes. Such a scenario was not directly tested in the previous mammalian cell assays (Dienstbier, 2009).

In the case of minus-end-directed mRNA transport in flies, the CTD appears to provide an essential link, through Egl, to Dlc. In addition, the CTD can associate with the dynamitin subunit of dynactin. The significance of this interaction was not clear in light of a model in which only the N-terminal sequences of BicD are important for transport by dynein. The finding that the CTD is needed in trans for the activity of BicDδC revives the possibility that the dynamitin interaction is functionally important (Dienstbier, 2009).

The ability of BicDδC::CP, but not BicD::CP, to target heterologous cargoes apically is likely to reflect a role for the CTD in inhibiting association with other copies of BicD. Consistent with this notion, BicDδC::CP accumulates in large, apically enriched puncta, whereas the full-length protein fused to the coat protein fails to form discrete particles and has a uniform distribution. Together with the observation that BicD is able to associate with other copies of itself in vivo, these results imply that dimerization or oligomerization of BicD could be an important step in the activation of transport by cargo binding. Future experiments will be aimed at determining the copy number of components of the transport complex in the presence and absence of a bound consignment (Dienstbier, 2009).

Modulation of gurken translation by insulin and TOR signaling in Drosophila

Localized Gurken (Grk) translation specifies the anterior-posterior and dorsal-ventral axes of the developing Drosophila oocyte; spindle-class females lay ventralized eggs resulting from inefficient grk translation. This phenotype is thought to result from inhibition of the Vasa RNA helicase. In a screen for modifiers of the eggshell phenotype in spn-B flies, a mutation was identified in the lnk gene. lnk mutations restore Grk expression but do not suppress the persistence of double-strand breaks nor other spn-B phenotypes. This suppression does not affect Egfr directly, but rather overcomes the translational block of grk messages seen in spindle mutants. Lnk was recently identified as a component of the insulin/insulin-like growth factor signaling (IIS) and TOR pathway. Interestingly, direct inhibition of TOR with rapamycin in spn-B or vas mutant mothers can also suppress the ventralized eggshell phenotype. When dietary protein is inadequate, reduced IIS-TOR activity inhibits cap-dependent translation by promoting the activity of the translation inhibitor eIF4E-binding protein (4EBP). It is hypothesized that reduced TOR activity promotes grk translation independent of the canonical Vasa- and cap-dependent mechanism. This model might explain how flies can maintain the translation of developmentally important transcripts during periods of nutrient limitation when bulk cap-dependent translation is repressed (Ferguson, 2012).

Reproduction represents a substantial energy investment for an organism. Many studies have shown that ovarian physiology is exquisitely sensitive to nutritional status. Limitation of dietary protein intake results in a dramatic slowing of egg chamber maturation via developmental arrest, programmed cell death, or loss of germline stem cells. Several signaling pathways are integrated to bring about this response including 20-hydroxyecdysone, Juvenile Hormone (JH), and insulin/insulin-like signaling (IIS). IIS is stimulated by protein feeding and is required for oogenesis to progress. The IIS pathway integrates nutritional signals at two distinct points during oogenesis. The first is in region 2A of the germarium where developing germline cysts undergo apoptosis in the absence of a source of maternal dietary protein. The second point of nutritional control is at stage 8 of oogenesis during the onset of vitellogenesis. In the absence of food, egg chambers develop to stage 8, where they are arrested until a favorable food source is located. These two checkpoints represent points at which the energetically expensive process of oogenesis can be halted if insufficient resources are available (Ferguson, 2012 and references therein).

The IIS pathway elicits its effect on Drosophila physiology through several effector pathways, namely the dFOXO transcription factor and the Target of Rapamycin kinase (TOR). IIS inhibits dFOXO activity by promoting its phosphorylation by PKB/Akt and subsequent exclusion from the nucleus. Starvation or mutations in the insulin pathway allow dFOXO to translocate to the nucleus where it directs the transcription of genes that promote longevity, stress resistance, fat storage, and growth attenuation. TOR activity is stimulated by both IIS through the dRheb GTPase and by amino acids via Rag GTPases. When nutrients are plentiful, high TOR activity stimulates the translation of mRNA by phosphorylating S6K which in turn phosphorylates eIF4B and promotes its interaction with eIF3. These steps are critical for recruiting the translation preinitiation complex (PIC) to the m7G cap at the 5’ end of the mRNA. Once bound, the PIC recruits the small ribosomal subunit and proceeds to scan the transcript for an initiating AUG codon. This process requires the activity of the eIF4A RNA helicase. TOR also phosphorylates and inactivates the inhibitory eIF4E binding protein, 4EBP. Starvation inhibits cap- dependent translation through reduced TOR activity. When nutrients are limiting and TOR activity is low, eIF4B is not phosphorylated and can no longer participate in PIC assembly, furthermore 4EBP inhibition is lifted and it proceeds to inhibit cap-recognition by eIF4E. Both activities have the effect of strongly blocking cap-dependent translation initiation when nutrients are scarce. A select few transcripts escape this translational block by upregulating the utilization of an alternative mechanism that relies on an Internal Ribosomal Entry Site (IRES) that obviates the requirement for cap recognition and start codon scanning. The list of transcripts that contain IRES sequences is growing and includes numerous growth factors such as VEGF-A , PDGF2, and IGF-II. A prominent example of IRES-mediated nutritional adaptation is the Drosophila insulin receptor dInR, the translation of which is upregulated in response to starvation as a way to sensitize the cell to insulin when nutrients become available (Ferguson, 2012 and references therein).

Control of translation is vitally important to developmental patterning. The transcripts of many morphogens, including nanos, oskar, and gurken, are co-transcriptionally packaged into silencing particles and transported in a translationally quiescent form. Once localized, this repression is alleviated and translation proceeds in the developmentally appropriate locale. Gurken (Grk) is a TGF-α related ligand for the Drosophila Egfr. Localized translation of the spatially restricted grk transcript results in signaling by germline-derived Grk to the Egfr in the overlying follicle cells. This signal is required to specify the posterior fate in early oogenesis and the dorsal fate during mid oogenesis. Mutations that reduce grk translation are female sterile due to an inability to correctly pattern the developing oocyte and result in concomitant patterning defects in the embryo. grk translation requires the eIF4A-related DEAD-box helicase Vasa (Vas). Mutations in vas are female sterile owing to a failure to specify dorsal structures in the egg shell or posterior structures in the embryo (Ferguson, 2012 and references therein).

Spindle class genes are responsible for repairing DNA double strand breaks (DSBs) that are induced during homologous recombination in Drosophila oogenesis. In wild type females, DSBs are induced in germ line cells entering pachytene in region 2A of the germarium. This process is initiated by the Spo11 homologue Mei-W68 and Mei-P22, a protein that aids in break site selection. These breaks are then repaired by homologous recombination, a process that requires the RAD-51 homologue spindle-B (spn-B). Mutations in spn-B result in an accumulation of unrepaired DSBs that lead to activation of a meiotic checkpoint. The checkpoint is comprised of the ATR homologue mei-41 and the downstream kinase chk-2. Persistent DSBs in spn-B^BU females activate the checkpoint that requires the Mei-41 and Chk2 kinases and leads to inefficient grk translation and ventralized eggshell phenotypes. Checkpoint activation also results in phosphorylation of Vasa, a modification that is thought to inhibit its function. Early in oogenesis, the oocyte nucleus becomes arrested in pachytene and forms a compact structure called the karyosome. The formation of the karyosome is disrupted in spindle-class mutants where the chromatin appears fractured or ellipsoid. Weak grk translation and an inability to properly form the karyosome are both spindle phenotypes that are consistent with reduced Vasa activity (Ferguson, 2012).

This study has identified the SH2B family adaptor gene lnk in a genetic screen for modifiers of the ventralized eggshell phenotype seen in spn-B^BU mutant flies. SH2B proteins are known to regulate intracellular signaling by membrane bound receptor tyrosine kinases (RTKs). SH2Bs can promote signaling by scaffolding downstream effectors to the RTK or mediate proteosomal receptor destruction by recruiting the Cbl ubiquitin ligase. Lnk was recently identified as a positive regulator of the Insulin/Insulin-like Signaling (IIS) pathway that functions at the level of the insulin receptor substrate Chico. This study shows that lnk mutations can promote grk translation and suppress the ventralized eggshell phenotype in a spn-B^BU mutant background. This suppression occurs independent of Vasa activity and does not suppress the karyosome phenotype. No genetic interactions were found with a weak grk allele nor downstream targets of Egfr suggesting that lnk-mediated suppression of spindle phenotypes does not occur by directly modulating Egfr activity. The data suggest that lnk mutations promote grk translation by inhibiting TOR activity as Rapamycin feeding experiments can also suppress the eggshell phenotype of spn-B and vas mutant flies. A model is proposed in which reduced IIS/TOR signaling inhibits cap-dependent translation and promotes utilization of an alternative translation initiation mechanism of the grk mRNA. This mechanism enables flies to faithfully pattern their oocytes when nutrients are scarce (Ferguson, 2012).

This study demonstrates a novel interaction between a meiotic checkpoint, the insulin/insulin- like signaling pathway, and translation of gurken mRNA in Drosophila oogenesis. Mutations in meiotic DNA repair enzymes such as spn-B result in persistent DSBs in early oogenesis that activate an ATR- Chk2-dependent meiotic checkpoint. Checkpoint activation results in phosphorylation of the eIF4A-like RNA helicase Vasa, the activity of which is important for grk translation. In these mutants, low levels of Grk protein are synthesized which is insufficient to pattern the eggshell correctly and results in ventralized eggs. Using forward genetics, an allele was isolated of the insulin receptor adapter, lnk. This mutation can suppress the weak grk translation phenotype and restore normal patterning to eggs laid by spn-B^BU flies. Clonal analysis has shown that lnk mutations reduce IIS in a cell-autonomous manner in the ovary. As in mammals, Drosophila IIS controls the rate of cap-dependent translation initiation in the cell by regulating the activity of the TOR kinase. Rapamycin inhibits TOR activity, and feeding rapamycin can suppress the ventralized eggshell phenotype not only in spn-B^BU females, but also in vasa^PH165 / vas^aRG53 flies. These data suggest an alternative translation initiation mechanism for the grk mRNA by which flies can maintain D/V axis patterning in times of moderate nutrient limitation (Ferguson, 2012).

The discovery that mutations in lnk, a positive regulator of IIS, can suppress the patterning defects in spn-B flies was initially surprising. The eggshell phenotypes of the different genotypes were assessed after keeping the flies on apple or grape juice agar plates on which abundant amounts of yeast paste had been added thus allowing the females to eat a very protein rich diet. A protein rich diet stimulates the activity of the TOR kinase via two mechanisms. Insulin-like peptides (dilps) are secreted into the hemolymph by neuroendocrine cells in response to nutrient availability. This in turn activates the IIS cascade comprised of Chico/Lnk, PI3K, Akt, Tsc1/2, and Rheb which promotes TOR-C1 activity. The second mechanism acts more directly through the levels of intracellular amino acids that are imported in part by the slimfast and pathetic transporters. Both of these mechanisms stimulate TOR-C1 activity which has been shown to promote cap-dependent translation by inhibiting 4EBP sequestration of eIF4E. Therefore, reducing TOR activity either by a mutation in lnk or by addition of rapamycin, would be expected to interfere with cap-dependent translation and therefore further enhance the mutant phenotype. However, in spn-B mutant flies, cap-dependent translation is already inhibited by the activity of the checkpoint, presumably acting via Vasa modification. The fact that a suppression of the ventralized phenotype was observed in lnk mutants indicates that reduction in TOR signaling must activate a second mode of translation that allows Gurken protein to be produced independently of the block in cap-dependent translation (Ferguson, 2012).

Several ovarian phenotypes are shared between mutations in spindle genes and vas mutants, including failure to form a compact karyosome, very weak grk translation, and ventralized eggs. Combined with the reproducible phosphorylation of Vas protein in spindle-class mutants, these phenotypes are consistent with a defect in Vas activity. While the specific effect of this phosphorylation is unknown, Vas serves several functions in cap-dependent translation initiation of grk mRNA. Vasa has been shown to interact with eIF5B and mutations that interfere with this interaction inhibit grk translation. This interaction is thought to facilitate assembly of the 60S ribosomal subunit at the AUG start codon. Furthermore, as a DEAD-box RNA helicase, Vasa may permit the pre-initiation complex to scan the 5’ UTR of grk and negotiate secondary structures that may impede the progress of this complex. IRES sequences adopt strong secondary structures in the 5’ UTR of RNAs that they regulate. If it can be demonstrated in the future that grk possess an IRES sequence, this may explain the requirement for Vasa helicase activity to unwind this structure when translation is initiated from the 5'cap during conditions of adequate nutrient availability. Whether the checkpoint dependent phosphorylation of Vas affects its stability, RNA helicase activity, or its eIF5B interaction, the expected result is a block in cap-dependent translation initiation of grk mRNA and concomitant D/V patterning defects. The observation that grk translation can be induced to occur in spn-B^BU and in vasa^PH165 / vas^aRG53 flies indicates that an alternative mechanism for supporting translation initiation is taking place. Because reduced IIS and TOR activity both block bulk cap-dependent translation initiation through sequestration of eIF4E by 4EBP, yet stimulate IRES activity, it is proposed that the latter may provide an explanation for the results (Ferguson, 2012).

Grk plays a central role in shaping the development of the egg and subsequent embryo. Mutations that disrupt Grk / Egfr signaling during oogenesis result in female sterility. Blocking the translation of this essential morphogen in spindle class mutants that are unable to repair DNA damage is an effective mechanism to prevent the transmission of mutations to the progeny. This reproductive checkpoint is effective when nutrients are abundant, however as this study has demonstrated, the strategy breaks down when IIS/TOR activity is low. Under these conditions, grk can be translated and result in eggs that are patterned correctly, even though the DNA damage and karyosome malformation phenotypes persist. It is proposed that this difference occurs because the DNA-damage checkpoint can only impinge on one of the two mechanisms by which grk translation can be initiated (Ferguson, 2012).

One mechanism by which suppression of the D/V patterning defects of spn-B^BU may occur is through the effects of the additional time that lnk^CR642 egg chambers spend completing oogenesis. While Grk production is reduced in spn-B^BU flies, it is not completely blocked and some Grk protein is made. If the reduced rate of Grk production is integrated over the extended time spent during mid oogenesis, sufficient Grk levels could accumulate and support normal D/V patterning. However, this model is inconsistent with the inability of lnk^CR642 to suppress the ventralized eggs laid by grk^ED22 females. These flies do retain some Grk activity as is evident by the single appendage that is specified, however if the mechanism of suppression were via accumulation, then grk^ED22 should be suppressed by lnk mutations. Therefore, the IRES-dependent model proposed in this study is favored (Ferguson, 2012).

The selective pressure that may have driven the evolution of this bi-modal translation mechanism for grk can be best understood by considering that in wild populations of Drosophila, females feed and oviposit at locations where yeast is abundant. This behavior ensures adequate nutrition to support oogenesis in the female as well as for the developing larvae. If however nutrients become scarce, females adjust the rate of oogenesis to match nutrient availability. In response to complete starvation, egg chambers undergo apoptosis and are reabsorbed, however moderate reductions in IIS slow the rate of oogenesis until an abundant protein source is found. The conserved response to dietary restriction is to repress cap- dependent translation of most cellular transcripts while a select population of RNAs that are essential for survival escape this repression by utilizing a cap-independent IRES mechanism. It is posited that grk may be one such transcript. Oocytes that are in mid development when nutrients are scarce must still be patterned appropriately so that the resulting eggs are fertile. IRES activity may facilitate Grk expression to maintain normal D/V patterning in times of lean whereas when nutrients are abundant, cap-dependent translation predominates (Ferguson, 2012). .

Factors affecting Gurken RNA localization and translation

Continued part 2/2

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