The CPEB translational regulator, Orb, functions together with Par proteins to polarize the Drosophila oocyte

orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. Here, we report that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. We show that repolarization of the oocyte also requires orb and that orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.

Introduction Specification of the anterior-posterior (AP) and dorsal-ventral (DV) axes of the Drosophila embryo depends upon determinants that are localized within the egg during oogenesis [1][2][3][4]. For example, expression of the TGF-α cell signaling molecule Gurken (Grk) at the anterior corner of the oocyte during mid-to-late oogenesis establishes the DV axis of the egg and subsequently the embryo by signaling to the overlying somatic follicle cells [5][6][7][8]. Factors important in determining the AP axis of the embryo are also localized during this same period. Specification of the posterior axis is mediated by oskar (osk) [9,10]. osk mRNA is targeted to the posterior cortex of the oocyte, where it is translated and functions in the assembly of the pole plasm and the anchoring of the mRNA encoding the posterior determinant nanos [11,12]. The anterior axis is specified by the Bicoid transcription factor, and its mRNA is localized to the anterior cortex of the oocyte [13][14][15].
The proper localization of these determinants within the oocyte during mid-to-late oogenesis depends upon the disassembly of the existing microtubule cytoskeleton (MT) during stage 7 of oogenesis and its subsequent repolarization [7,8]. The polarity of the MT network in the period prior to stage 7 is established early in oogenesis when the oocyte is initially specified [16]. A microtubule organizing center (MTOC) is assembled at the oocyte cortex just posterior to the oocyte nucleus and it directs the elaboration of the MT network by anchoring the minus-ends of MTs. As a consequence of this polarization of the oocyte, mRNAs encoding determinants critical for the early stages (stage 1-7) of egg chamber development accumulate at the posterior cortex. One of these is gurken (grk) mRNA. Grk protein translated from this localized message signals to the somatic follicle cells covering the posterior of the egg chamber to specify posterior follicle cell fate (PFC) [7,8]. Subsequently, during stage 7, an unknown signal(s) emanating from the somatic PFCs triggers the repolarization of MT network in the germline. This signal induces the disassembly of the posterior MTOC and the network of MTs extending from the MTOC towards the anterior of the oocyte [7,8,[17][18][19][20]. At the same time, de novo MT assembly is nucleated along the anterior and lateral cortex of the oocyte by a centrosome independent mechanism. This mechanism deploys the tubulin minus-end binding protein Patronin and the actin-MT linker Short Stop (Shot) [21]. Accompanying the repolarization of the MT cytoskeleton, the oocyte nucleus migrates from the posterior end of the oocyte to the anterior corner [22]. grk mRNA also relocates so that it is positioned between the oocyte nucleus and the oocyte cortex. Grk protein expressed from the localized message signals dorsal follicle cell fate and this defines the DV axis of the egg chamber and embryo [6,23].
In addition to Patronin and Shot, the other factors implicated in oocyte repolarization are the Drosophila homologs of the partioning-defective (Par) group genes, par-1, cdc42 and bazooka (baz/par-3) [24][25][26]. These three genes together with par-6 and aPKC are also required for the initial polarization of the stage 1 egg chamber [24][25][26][27][28][29][30]. These proteins generate cellular asymmetries by inhibitory cross-regulatory interactions that impede association with the cell cortex [25,[31][32][33]. During MT repolarization, Par-1 becomes enriched along the posterior cortex of the oocyte [34][35][36]. There is a complementary distribution of Baz, Par-6, aPKC and Cdc42: they are enriched along anterior and anterior-lateral cortex, but not the posterior [24,25,37,38]. The available evidence indicates that the asymmetry in the oocyte generated by the activation of the Par polarity network is upstream of the localization of Shot and Patronin along the anterior and lateral cortex, and thus the Patronin dependent de novo assembly of MTs [21].
In addition to being critical for properly localizing grk, osk and bcd mRNAs, the reorganization of the cytoskeleton also alters the distribution of other mRNAs encoding oocyte-specific proteins. One of these mRNAs is orb, which encodes one of the two fly cytoplasmic polyadenylation element RNA-binding (CPEB) proteins [39,40]. During early stages of oogenesis, orb mRNA is localized at the posterior of the oocyte. After repolarization orb mRNA disappears from the posterior and becomes concentrated along the anterior-lateral margin of the oocyte [41]. While the rearrangement of orb mRNA within the oocyte is clearly downstream of the steps involved in repolarizing the oocyte MT network, the orb gene plays a central role in the initial formation and subsequent development of the oocyte and thus could be an active participant in determining oocyte polarity.
In ovaries, orb expression is restricted to the germline and is required at multiple steps during oogenesis [39,40,42]. In wild type ovaries, a cystoblast, generated by an asymmetric division of a stem cell, undergoes four mitotic divisions with incomplete cytokinesis to produce a 16-cell cyst [1]. In the orb null allele, orb 343 , the last of these mitotic divisions is not completed and the cyst degenerates [40]. While the strong loss-of-function allele, orb 303 , forms a 16-cell cyst, the oocyte is not properly specified and egg chambers contain only nurse cells [40]. Unlike orb 343 and orb 303 , the Orb protein expressed by the hypomorphic orb allele, orb mel , is wild type. Instead, orb mel transcripts are incorrectly spliced generating an mRNA lacking sequences from the 5'UTR [42]. The removal of these 5' sequences alters Orb expression as oogenesis proceeds. Prior to stage 7 the level and localization of Orb in the oocyte is similar to that observed in wild type. However, beginning around stage 7, the amount of Orb drops dramatically and most chambers have little residual protein. As a consequence of this reduction in Orb protein, orb mel females produce eggs that give rise to embryos with a range of phenotypic abnormalities including D-V and A-P patterning defects [42]. These patterning defects arise from a failure in the localization and/or translation of two Orb regulatory targets, grk and osk mRNAs, during mid-to-late oogenesis [43][44][45][46].
grk and osk transcripts are not, however, the only mRNAs that could be subject to orb regulation during oogenesis. Several recent studies have identified many other mRNAs that are Orb associated in vivo [47,48]. Included in this group of potential orb regulatory targets are mRNAs encoding the Par proteins, aPKC, Baz, Par-6 and Cdc42. Moreover, there is evidence connecting the other fly CPEB protein, Orb2, to the functioning of one of the Par family proteins, aPKC, in cell polarization in the embryonic CNS, in testes and in tissue culture cells [49][50][51]. These observations prompted us to ask whether orb impacts the process of repolarization of the oocyte during mid-stages of oogenesis, and conversely whether the Par proteins, and in particular, aPKC, have any effect on orb activity.

orb hypomorphic allelic combinations display defects in oskar mRNA localization
In wild type, osk mRNA is localized in a tight crescent at the posterior pole of the oocyte after repolarization ( Fig 1A) [11,52]. While osk mRNA localization to the posterior is independent of Osk, Osk protein is required to ensure that osk mRNA is properly anchored to the posterior cortex [52]. In osk protein null mutants, osk mRNA is localized at the posterior, but localization is not properly maintained (Fig 1B). While orb is required for osk mRNA translation, it also plays a role in the proper localization of osk message [43,44]. As shown in Fig 1C, in orb mel /orb 303 chambers, the tight localization of osk mRNA at the posterior pole is lost. Instead, osk mRNA puncta are distributed in a halo around the posterior pole while there is a diffuse pattern of mRNA along the anterior margin of the oocyte. As previously reported, even more extreme defects in osk mRNA localization are evident when orb mel is combined with the null allele orb 343 ( Fig 1D) [42,43]. In this allelic combination there is little if any osk mRNA at the posterior.

orb does not function in osk mRNA transport
The osk mRNA localization defects in the hypomorphic orb mutant combinations resemble those in staufen mutants. staufen encodes an RNA-binding protein that co-localizes with osk mRNA throughout oogenesis, and in staufen D3/Df , osk mRNA is partially localized to the posterior and also accumulates at the anterior ( Fig 1E) [11,52,53]. Thus, one explanation for the defects in osk mRNA localization during mid-oogenesis is that orb is also required to transport osk mRNA [53]. To test this possibility we compared the distribution of Orb protein with that of osk mRNA. Prior to stage 7, both osk mRNA and Orb protein are localized at the posterior. When the MT network commences repolarization during stage 7, osk mRNA transiently accumulates in a cloud near the middle of the oocyte (Fig 2A) [54]. If Orb is directly involved in osk mRNA transport, it would be expected to co-localize with osk mRNA in this cloud. However, it does not. Instead, most of the Orb is concentrated in the sub-cortical region at the posterior end of the oocyte and along the lateral margins of the oocyte (Fig 2A). Only later, after osk mRNA is re-localized to the posterior pole (and presumably being translated) does it again overlap with the posterior cap of Orb protein ( Fig 2B). Another orb regulatory target is orb mRNA and its pattern of localization differs from that of osk [55]. In stage 7 chambers, when osk mRNA is in the center of the oocyte, orb mRNA has a circumferential subcortical distribution around the anterior of the oocyte (Fig 2C). This distribution is maintained at later stages ( Fig 2D).
Another indication that orb is not directly involved in osk mRNA transport comes from the effects of grk mutations. In grk 2B/2E12 ovaries, posterior follicle cell (PFC) specification is defective and the oocyte fails to initiate repolarization at stage 7 [7,8]. As a consequence, osk mRNA (S1 Fig), the transport protein Staufen, and MT plus ends become enriched in the center of the oocyte, while bicoid mRNA localizes to both the anterior and posterior of the oocyte [7,8]. In this grk mutant combination Orb protein and also orb mRNA accumulate around the circumference of the oocyte, far from osk mRNA (S1 Fig). orb functions in organizing the oocyte cytoskeleton An alternative explanation for the mislocalization of osk mRNA in orb mel /orb 303 and orb mel / orb 343 egg chambers is that the cytoskeleton is not properly reorganized during repolarization in the absence of normal orb function. This possibility was suggested by the studies of Martin The translational regulator, Orb, functions with Par proteins to polarize the Drosophila oocyte et al. ([56]), who showed that in hypomorphic orb mutant alleles the oocyte MT network is disrupted and there is premature oocyte cytoplasmic streaming. Several approaches were used to confirm and extend their findings. The translational regulator, Orb, functions with Par proteins to polarize the Drosophila oocyte a) Kinesin-β-galactosidase is mislocalized in orb mutant chambers. We used the plusend directed motor Kinesin tagged with β-galactosidase (KZ32) to examine the polarity of the MT network [57]. In control oocytes, Kin::β-gal accumulates with the MT plus-ends at the oocyte posterior in a pattern that resembles osk mRNA (Fig 3A-3C; S2A Fig) [58]. A quite different result is observed in orb mel /orb 303 and orb mel /orb 343 egg chambers (Fig 3B & 3C; S2B  Fig) [56]. Instead of being enriched at the posterior, Kinesin-β-galactosidase is distributed diffusely throughout the oocyte cytoplasm. b) MT network is disrupted in orb mutant chambers. During repolarization, α-tubulin assembles into a network that is in tight association with the anterior and lateral cortical actin ( Fig 3D) [20,59,60]. Martin et al. ([56]) found that this network was partially disrupted in orb mutants. To better understand the role of orb in organizing the MT network, we examined different allelic combinations. We found that the frequency and severity of the disruption correlates with the extent of reduction in orb activity. The least severe effects are observed in orb mel / orb 303 egg chambers (Fig 3G). In this genetic background, the MT network is dissociated from the cortex in only about 15% of stages 9-10 oocytes. Similar disruptions in the cortical association of tubulin have been reported for mutants in the actin nucleators cappuccino (capu) and spire (spir) [61][62][63][64]. The frequency of affected oocytes increases to about 70% when orb mel is combined with the null allele orb 343 (Fig 3E-3G; S2D Fig). In most of the orb mel /orb 343 chambers, MTs are diffusely distributed throughout much of the oocyte with little evidence of cortical association (Fig 3E and 3F and S2D Fig). Finally, we combined orb mel with orb 343 and the dominant negative transgene, HD19. The HD19 transgene expresses a chimeric lacZ-orb 3'UTR mRNA that competes with the endogenous orb mRNA [41,55]. This competition interferes with a positive autoregulatory loop in which orb activates its own expression [55]. In orb mel /orb 343 HD19 ovaries, the MT network phenotypes are fully penetrant ( Fig 3G).
As reported by Martin et al. ([56]), we found that the defects in the cortical association of the MT network are accompanied by premature cytoplasmic streaming. Examples of normal cytoplasmic movements in a stage 9 egg chamber and oocytes undergoing premature streaming when compromised for orb function are shown in supplemental movies (S1-S3 Movies).
c) The oocyte nucleus is mispositioned in orb mutant oocytes. As the MT network repolarizes in wild type egg chambers, the oocyte nucleus migrates from the posterior of the oocyte to the dorsal anterior corner (Fig 3H; S2E Fig) [7,8,22,65,66]. Like the MT network, the association of the oocyte nucleus with the dorsal anterior corner is sensitive to reductions in orb activity. In the weakest allelic combination, orb mel /orb 303 , mispositioned oocyte nuclei are observed in less than 5% of stage 9-11 oocytes ( Fig 3K). When orb mel is combined with the null allele, orb 343 , about 7% of stage 9-11 oocytes have mispositioned nuclei (Fig 3J and 3K; S2F Fig). The frequency of nuclear localization defects increases when orb activity is further compromised (Fig 3K). In orb mel /orb 343 HD19 the oocyte nucleus is incorrectly positioned in over 20% of stage 9-11 egg chambers, while it increases to nearly 40% when Orb is depleted by RNAi (#64002) knockdown (S3 Fig) using a driver (maternal α-tubulin Gal4 #7062) that is active during mid-oogenesis (Fig 3I and 3K) [67].
Are the defects in repolarization due to a failure in grk signaling to posterior follicle cells?
Repolarization of the oocyte during stage 7 is triggered by signals emanating from the somatic posterior follicle cells (PFCs). The production of the repolarization signal depends upon the proper specification of the PFCs and this process is orchestrated by the expression of the Grk ligand at the oocyte posterior [7,8]. Since grk mRNA is a known orb regulatory target, one explanation for the repolarization defects is that the PFCs are not properly specified when orb The translational regulator, Orb, functions with Par proteins to polarize the Drosophila oocyte activity is compromised. To test this possibility we examined the expression of an EGFR dependent enhancer trap, kekkon-lacZ, that is activated in follicle cells by grk signaling [68][69][70][71][72]. As illustrated for two stage 7 egg chambers in S4A and S4B Fig we found that kekkon-lacZ expression in PFCs in orb mel /orb 343 egg chambers resembles that in control egg chambers. This result confirms previous studies which showed that anterior follicle cell fate is not duplicated in orb 343/mel egg chambers [7]. While kekkon-lacZ expression is unaffected in orb mel /orb 343 prior to repolarization, abnormalities are evident at later stages. As shown for a stage 10 orb mel /orb 343 chamber in S4C and S4D Fig, expression of kekkon-lacZ in dorsal follicle cells is severely reduced compared to the control. This is expected since grk signaling to the dorsal follicle cells is known to be disrupted in orb mel /orb 343 ovaries [42,45,46].
Other observations are also consistent with the idea that the defects in MT organization in orb are downstream of both the grk dependent specification of PFCs and of the subsequent repolarization signal from the PFCs to the oocyte. For example, in grk mutants, bicoid mRNA is localized not only along the anterior-lateral margin, but also at the posterior pole [7,8]. In contrast, when orb activity is compromised, localization of bcd mRNAs to the posterior pole is not observed (S5 Fig) [42]. The reason for this difference is that in grk mutants the PFCs fail to signal the disassembly of the MTOC at the posterior of the oocyte, whereas the posterior MTOC is disassembled in orb mutants.

Genetic interactions between orb and the anterior Par genes aPKC and cdc42 disrupt grk signaling to dorsal follicle cells
One explanation for the failure to repolarize the MT cytoskeleton is that orb activity impacts either directly or indirectly the functioning of the Par proteins. In fact, precedence for an orb-Par connection comes from experiments showing that one of the targets for the other fly CPEB protein, orb2, in spermatid cyst polarization and in asymmetric cell division in the embryo is the message encoding the apical Par protein aPKC [49,50]. To explore this idea further we took advantage of the fact orb is weakly haploinsufficient for D-V polarity [55]. About 5% of the eggs laid by orb 343 /+ are ventralized due to defects in translating grk mRNA at the dorsal anterior corner of the oocyte (Fig 4A). The frequency of D-V polarity defects can be enhanced by reducing the activity of other genes that are important for orb function in grk signaling.
We used three different aPKC mutants, a strong allele, k06403, and two hypomorphic alleles, ex48 and ex55, to test for dominant genetic interactions with orb [74,75]. While the frequency of D-V polarity defects in eggs produced by mothers heterozygous for these three aPKC alleles is similar to WT (S1 Table), these mutations substantially enhanced the frequency of D-V polarity defects when trans to orb 343 /+. The weak hypomorphic alleles increase the frequency of ventralized eggs four to five fold (20% and 25%), while the frequency is increased nearly nine fold (44%) by the null allele ( Fig 4A).
To extend this analysis, we also asked whether there are genetic interactions between orb 343 and the cdc42 gene, which, like aPKC, plays an important role in establishing apical cell polarity [26,76]. In orb 343 / cdc42 1 trans-heterozygotes there was modest increase (three-fold) in the frequency of D-V polarity defects (S1 Table), while in orb 343 /cdc42 4 trans-heterozygotes the frequency of D-V polarity defects increased by nearly fifteen fold ( Fig 4A). Consistent with the idea that the effects on grk signaling are related, at least indirectly, to the functioning of the Par proteins in the process of repolarization, we also observed genetic interactions between aPKC k06403 and cdc42 4 . Whereas background levels (~1%) of D-V polarity defects are evident in eggs produced by either aPKC k06403 and cdc42 4 heterozygotes, over 35% of the eggs laid by trans-heterozygous mothers had D-V polarity defects ( Fig 4A).  [73]. V2 is a reduction in the space between dorsal appendages, V3 is a fusion of the dorsal appendages and V4 is the most severe reduction in dorsal appendages. Bottom: Graph shows the percentage of ventralized eggs laid by wild type, orb heterozygotes (343/+), double-heterozygotes between orb, aPKC alleles (ex48, ex55 and k06403), and cdc42 4 , double-heterozygotes between cdc42 4 and aPKC k06403 and triple heterozygotes of orb 343 , aPKC k06403 and cdc42 4 . Frequency of DV polarity defects for heterozygous controls not shown in the graph are listed in S1 Table. n = >400 all genotypes. (B) Top: asterisks show mispositioned oocyte nuclei in aPKC k06403 /+; orb 343/mel (left shows a "float" position and right shows a lateral position). Scale bars 50 microns. Bottom: aPKC k06403 enhances orb mutant defects in oocyte nucleus position. Defects in aPKC k06403 /+ (2%, n = 50), aPKC k06403 /+; orb 343/mel (27%, n = 118) and aPKC k06403 /+; orb mel /HD, orb 343 (50%, n = 100). Scale bars are 50 microns.
We also examined eggs produced by females triply heterozygous for orb 343 , aPKC k06403 and cdc42 4 . In this triple heterozygote about 90% of the eggs have D-V polarity defects ( Fig 4A). As would be predicted, accumulation of Grk protein at the dorsal anterior corner of the oocyte is clearly reduced (S6A- S6C Fig). Interestingly, the oogenesis defects are not restricted to grk translation. S6D-S6F Fig also shows that the localization of osk mRNA at the posterior pole is also reduced compared to control egg chambers.

Genetic interactions between orb and aPKC interfere with positioning of the oocyte nucleus
Further evidence that orb might work in conjunction with aPKC in the process of repolarization comes from analysis of oocyte nucleus positioning in backgrounds simultaneously compromised for both genes. As described above, oocyte nucleus mispositioning is observed in~7% of the orb mel /orb 343 egg chambers. The frequency of a mispositioned oocyte nucleus increases to nearly 25% of the chambers when orb mel /orb 343 females are also heterozygous for aPKC k06403 (Fig 4B). A similar enhancement is observed when aPKC k06403 is combined with orb mel /orb 343 HD19G. In orb mel /orb 343 HD19G chambers about 20% have a mispositioned oocyte nucleus, while the frequency of oocytes with a mispositioned nucleus increases to nearly 50% when the orb mel /orb 343 HD19G females are also heterozygous for aPKC k06403 (Fig 4B). Importantly, aPKC on its own is not haploinsufficient for proper oocyte nucleus migration ( Fig 4B).

Localization of aPKC mRNA within the oocyte depends upon orb
One plausible explanation for the genetic interactions is that one of the orb functions in repolarization is to regulate aPKC mRNA. To explore this possibility, we examined the effects of compromising orb on the pattern of accumulation of aPKC mRNA. While aPKC mRNA is present in both somatic and germline cells in wild type ovaries, the highest concentrations of mRNA in the germarium and in stage 1-7 egg chambers are found in the oocyte (S7A and S7B

Orb binds to aPKC mRNAs
As noted in the introduction, aPKC mRNA is one of several thousand mRNAs that are associated with ectopically expressed Orb2 and Orb in tissue culture cells [48]. To determine if aPKC mRNA is bound by Orb in ovary extracts, we used immunoprecipitation to isolate Orb associated RNAs. After reverse transcription using an oligo dT primer, we used quantitative PCR to assay for specific mRNA species. For the positive control, we used primers for orb-RA 3'UTR which contains four canonical cytoplasmic polyadenylation elements (CPEs: UUUUAU or UUUUAAU). Previous studies have shown that Orb binds to the orb mRNA 3'UTR and positively autoregulates its own expression [55]. There are twelve predicted aPKC mRNA species with six different predicted 3'UTRs. Four of the six predicted 3'UTRs have canonical CPE sequences. One of these, aPKC-RA, has a 3'UTR with three canonical CPEs while the remaining aPKC mRNAs (RD, RF RJ, RK, RL and RM) have overlapping UTRs with 2 canonical CPEs. Fig 5F shows that in ovary extracts both types of aPKC 3'UTRs are enriched in Orb immunoprecipitates.

Distribution of aPKC along the anterior lateral cortex depends upon orb
We next examined the pattern of accumulation of aPKC protein. In wild type stage 10-11 oocytes, aPKC protein is localized to the anterior-lateral cortex where it appears to be in close association with the cortical actin network (Fig 6A) [37]. Except for this cortically localized protein, there is little aPKC elsewhere in the oocyte. Orb is localized just interior to the cortical actin-aPKC layer (Fig 6A). aPKC is also localized along the apical surface of the somatic follicle cells facing the germline, and in confocal images the somatic and oocyte aPKC proteins typically appear as a set of parallel tracks along the anterior-lateral cortex (Fig 6A).
The pattern of aPKC localization in the oocyte is altered when orb activity is compromised. Instead of a tightly organized track coincident with cortical actin, aPKC protein distribution becomes irregular and patchy (Fig 6B). In some regions, there are small gaps (Fig 6B:

Par-1 protein localization is also disrupted
The effects of reducing orb activity on aPKC localization within the oocyte, taken together with the genetic interactions between orb, aPKC and cdc42 indicate that orb is required for the The translational regulator, Orb, functions with Par proteins to polarize the Drosophila oocyte To test this idea, we examined the localization of a Par-1-GFP fusion protein that is expressed in the germline. In control stage 8-11 oocytes, the Par-1-GFP fusion protein localizes along the oocyte cortex and tends to be enriched towards the posterior of the oocyte. Fig 6C and S8 Fig show that like aPKC, Par-1-GFP localization depends upon orb, and is disrupted when orb activity is compromised. The extent of disruption is correlated with the severity of the reduction in orb activity. In orb mel /orb 303 , a small percentage of the chambers have an obvious, but not complete loss of Par-1-GFP association with the oocyte cortex ( Fig 6C). Even more extensive alterations are observed in orb mel /orb 343 and orb mel / orb 343 HD19G chambers. In these genetic backgrounds, more than half of the egg chambers show either a reduction (S8B Fig) or complete loss of cortical Par1-GFP (Fig 6C and S8C Fig).

Cortical Actin cytoskeletal organization requires orb, aPKC and cdc42
In addition to its functions in Par dependent polarity, the apical Par protein Cdc42 can also activate effectors of the actin cytoskeleton (Cip4, WASp and Arp23). Studies by Leibfried et al. ([26]) have shown that one of the important Cdc42 targets during oocyte repolarization is the actin cytoskeleton. When cdc42 activity is compromised, the organization of cortical actin is disrupted. While the apical Par proteins aPKC and Baz are not thought to have a direct role in modeling the actin cytoskeleton, they are required for Cdc42 localization. As a consequence, aPKC and baz mutants have equivalent defects in the anterior lateral cortical domain. For these reasons, we wondered whether orb function might also impact the organization of the cortical actin cytoskeleton during repolarization.
To address this question we examined the cortical actin cytoskeleton in orb mel /orb 343 and in orb RNAi egg chambers. In the experiment in Fig 7, we labeled follicle cells membranes with Cadherin 99C (Cad99C) antibodies, while the actin cytoskeleton was labeled with phalloidin [77]. In wild type, actin is enriched along anterior oocyte margin and the anterior lateral cortex (Fig 7A and 7A') [26]. The tight association of actin along the oocyte cortex seen in wild type chambers is disrupted when orb activity is compromised in either orb mel /orb 343 oocytes (Fig 7B  and 7B') or when orb RNAi is expressed during midstages by a maternal α-tubulin driver (#7062) (Fig 7C and 7C'). In some regions, the actin matrix is displaced from the cortex ( Fig  7B, arrow). In other regions, there are "flares" of actin filaments that extend out from the cortical actin matrix into the ooplasm (Fig 7C', arrow). The matrix can also unravel forming small bubbles (Fig 7C', arrowhead) or even disappear completely (Fig 7B'). These defects could be due to a failure to properly crosslink the cortical actin bundles.
aPKC association with the oocyte cortex is thought to depend upon the integrity of the cortical actin cytoskeleton [26]. This raises the possibility that the defects in aPKC localization in orb mutants might be connected to abnormalities in the cortical actin cytoskeleton. The results shown in Fig 6B indicate that this is likely to be the case. In regions where the cortical actin matrix is disrupted, aPKC association with the cortex is reduced or lost. There seems to be a and Actin along the anterior lateral cortex of the oocyte is irregular (red arrow). Bottom: Another example of aPKC and Actin localization in 343/mel. aPKC association is irregular (blue arrow) or diffuse (blue arrowhead). Correlated with the disruptions in the cortical association of aPKC, the actin filament organization in the cortex is abnormal. Instead of the tight actin network at the cortex, there are filaments extending out into the oocyte cytoplasm as well as regions that lack cortical actin Scale bars 10 microns. (C) GFP antibody staining for a GFP-Par-1-N1S fusion protein expressed under the control of a maternal tubulin promoter. GFP-Par-1 localizes to the oocyte cortex in wild type (not shown) and orb mel /+ (n = 45) egg chambers and is enriched at the posterior (arrow). In contrast, GFP-Par-1 is displaced from the cortex in 343/mel egg chambers. The graph shows the frequency of defects in GFP-Par-1 localization in orb mel /+ (0%, n = 45), orb 303/mel (16%, n = 30), orb 343/mel (50%, n = 60) and orb mel /HD19, orb 343 (53%, n = 38). The intermediate phenotype as described in the graph can be seen in S8 Fig, where there is some patchy cortical enrichment of GFP, and lower levels throughout the oocyte cytoplasm. Scale bars 50 microns.

Patronin and Shot association with the anterior-lateral oocyte cortex is orb dependent
The repolarization of the MT network during mid-oogenesis depends upon the MT binding protein Patronin and its association with the actin-MT linker Shot. Since the integrity of the cortical actin network is disrupted when orb activity is compromised, we wondered whether Patronin and Shot association with the oocyte cortex is also affected. To investigate this possibility, we compared the localization of Shot-YFP expressed from a BAC transgene and YFP-Patronin expressed from a germline specific mattub promoter (Fig 8) ( [21]) in wild type egg chambers and in chambers in which orb activity was knocked down by RNAi.
In wild type stage 9-11 oocytes Shot and Patronin are found associated with the oocyte cortex (Fig 8, S9 and S10 Figs) [21]. In the oocyte, Shot and Patronin are localized in a punctate pattern just underneath the cortical actin network (S9 and S10 Figs). Shot-YFP (S9 Fig) and Patronin-YFP (S10 Fig: expressed as an endogenously tagged protein) also localize to the apical surface of the follicle cells, and these two proteins appear as a parallel track along the lateral surface of the oocyte with the cortical actin network in between. Both Shot-YFP and YFP-Patronin are enriched along the anterior and anterior-lateral cortex, while they are absent from the posterior cortex (Fig 8A-8C; arrowheads). When orb activity is knockdown by RNAi, the association of Shot-YFP and YFP-Patronin with the anterior-lateral cortex of the oocyte is disrupted and much of the protein is instead distributed in the ooplasm (Fig 8B and 8D). Similar, though not quite as severe alterations in the cortical association of Shot-YFP and Patronin-YFP are observed in stage 9-11 orb 343 /orb mel egg chambers (S9B and S9B ' Fig and  S10B-S10B" Fig).

orb and aPKC are functionally interdependent
Par proteins establish and maintain polarity within a cell by both positive and negative crossregulatory interactions. For this reason it seemed possible that aPKC and orb function in the oocyte might be mutually interdependent. To explore this possibility we used the mid-oogenesis GAL4 driver maternal α-tubulin (7063) to express aPKC RNAi (35140). In this background, The translational regulator, Orb, functions with Par proteins to polarize the Drosophila oocyte we observed that the oocyte nucleus is mispositioned in 69% of the stage 9-11 egg chambers when aPKC activity is depleted. Accompanying the oocyte nucleus position defects, Gurken protein is mislocalized with the oocyte nucleus (Fig 9A and 9B). Additionally, there are alterations in the pattern of Orb protein accumulation. Instead of being distributed subcortically along the entire surface of the oocyte, high levels of Orb accumulate at the anterior oocytenurse cell margin (Fig 9C and 9D). There is also a reduction in the posterior cap of osk mRNA compared to wild type (Fig 9E and 9F). Similar effects on the positioning of the oocyte nucleus and the localization of polarity markers (Staufen and Vasa) have been reported for cdc42 [26]. Moreover, like orb and cdc42, the cortical actin network is also perturbed in the aPKC knockdown (Fig 9H and 9H').

aPKC regulates orb
The alterations in the pattern of Orb protein accumulation in the RNAi knockdown experiments prompted us to ask whether aPKC impacts orb autoregulation. Orb promotes its own expression through sequences in the orb mRNA 3'UTR. When the orb 3'UTR is linked to coding sequences for E. coli β-galactosidase in the HD19 transgene, expression of β-galactosidase becomes dependent upon orb activity [55]. S11A Fig shows that β-galactosidase expression from the HD19 (hsp83: lacZ-orb 3'UTR) transgene is also dependent upon aPKC activity. In the aPKC mutant combination, aPKC k06403 /aPKC ex48 , β-galactosidase expression is reduced about two-fold compared to the control (S11B Fig). Like CPEB proteins in other species, orb activity is regulated by phosphorylation [78]. In wild type ovaries, there are multiple phosphorylated isoforms. On standard SDS polyacrylamide gels these different Orb isoforms typically resolve into a closely spaced doublet with the more heavily phosphorylated isoforms migrating more slowly (S11C Fig). In S11C Fig, we compared the relative yield of the upper (more phosphorylated) and lower (less phosphorylated) bands in wild type and aPKC k06403 /aPKC ex48 mutant ovaries. In the aPKC k06403 /aPK-C ex48 the ratio of upper to lower bands is reduced compared to wild type (S11D Fig).

Discussion
Previous studies have implicated orb in the translational regulation of osk and grk in the stages following the repolarization of the MT network [43][44][45]. In addition, the proper localization of these mRNAs also depends orb activity [42][43][44][45]. This observation led to the idea that in addition to controlling translation, orb might also have a role in transport and/or anchoring of these mRNAs once they were properly localized. While our results argue against a direct role in transport, they support the idea that the mislocalization of osk and grk mRNAs when orb activity is compromised during mid-oogenesis arises at least in part because orb is required for the proper organization of both MTs and the cortical actin cytoskeleton.
The reorganization of the MT network after stage 7 is a multistep process. It begins with a signal from the PFCs that induces the disassembly of the MTOC that is located just posterior to the oocyte nucleus. The production of this somatic signal depends upon the proper specification of the PFCs, and PFC specification requires the expression of Grk protein at the posterior pole of oocyte earlier in oogenesis [7,8]. Translation of grk mRNA at the posterior pole during stages 1-7 depends upon orb, and consequently it functions upstream of PFC specification. However, in our experiments orb activity prior to stage 7 is not limiting, and sufficient amounts of Grk are expressed to properly specify PFCs [7]. Thus, the defects that we observe in oocyte repolarization when orb activity is compromised during mid-oogenesis are downstream of both the grk signal to the posterior follicle cells and the signal from the PFCs to the germline that induces MTOC disassembly. Three other findings are consistent with this conclusion. First, when PFCs are not properly specified, the posterior MTOC fails to disassemble [7,8,18]. By contrast, when orb activity is compromised during mid-oogenesis the MTOC dissembles as in wild type. Second, the formation of a non-centrosomal cortical based MT network is initiated along the anterior/lateral margin of the oocyte even in the absence of the PFC signal. This is not true in our experiments; the anterior/lateral MT network is not properly established. Third, in the absence of the PFC signal, Staufen protein, Kinesin-β-gal and osk mRNA concentrate in the center of the oocyte, while bcd mRNA is found not only at the anterior but also at the posterior end of the oocyte. In contrast, in orb mutants, osk and also bcd mRNA accumulate at the anterior of the oocyte, while Kinesin-β-gal is unlocalized.
As the posterior MTOC is disassembled, a MT network emanating from the anterior and anterior-lateral cortex of the oocyte is established. The initiation of this non-centrosomal cortical based MT network is mediated by the spectraplakin, Shot, and the minus-end MT binding protein, Patronin [21]. Shot associates with the actin rich anterior and anterior lateral cortex and recruits Patronin. Patronin then nucleates the assembly of the MT network. Nashchekin et al. ([21]) have shown that proper polarization of the MT network by Shot and Patronin depends upon the Par protein Par-1. By an unknown mechanism, Par-1 blocks Shot association with the actin rich cortex. Since Par-1 is enriched around the posterior cortex of the oocyte, this restricts the de novo assembly of MTs to the anterior and anterior-lateral cortex. While Par-1 is required to exclude Shot from the posterior cortex, the de novo assembly of MTs requires Shot association with the anterior and anterior-lateral cortex. This presumably does not happen when aPKC, cdc42 and/or baz are compromised in the germline because the anterior and anterior-lateral cortical actin network is disrupted.
Our results place orb upstream of Shot and Patronin and suggest that the defects in oocyte MT repolarization likely arise for several reasons. One would be defects in the localization and functioning of the Par gene products. When orb activity is compromised, the association of the Par protein Par-1 with the posterior and aPKC with the anterior-lateral cortex is disrupted. In the absence of proper cortical association, the cross-regulatory interactions between the anterior and posterior Par proteins would be expected to be ineffective. Also consistent with a role for orb in the functioning of the Par proteins in MT repolarization are genetic interactions between orb and genes encoding the anterior Par proteins, aPKC and cdc42. orb is weakly haploinsufficient for the grk signaling pathway, and about 5% of the eggs laid by orb 343 /+ females, are ventralized. This weak haploinsufficiency is enhanced when the orb 343 mutation is trans to mutations in either aPKC or cdc42. For the aPKC null allele, aPKC k06403 , the frequency of ventralized eggs increases to nearly 50%, while about 70% of the eggs laid by females trans-heteozygous for orb 343 and cdc42 4 are ventralized. Moreover, while females heterozygous for either aPKC k06403 or cdc42 4 alone do not lay ventralized eggs, nearly 40% of the eggs laid by females trans-heterozygous for these two mutations are ventralized. As we found for orb, the localization of the oocyte nucleus to the dorsal anterior corner of the oocyte depends upon cdc42 and aPKC. Leibfried et al. ([26]) found that the oocyte nucleus is mispositioned in egg chambers homozygous for cdc42 4 , while we have shown here that the oocyte nucleus is mispositioned when aPKC is knocked down by RNAi. Moreover, the frequency of mispositioned nuclei in orb mel /orb 343 is enhanced when the females are also heterozygous for mutations in aPKC.
At least some of the effects of orb on the Par proteins could be direct. Thus, aPKC mRNAs contain CPEs in their 3'UTRs and we have found that aPKC mRNA is bound by Orb protein The translational regulator, Orb, functions with Par proteins to polarize the Drosophila oocyte in ovary extracts. Moreover, the distribution of aPKC mRNA within the oocyte is altered when orb activity is compromised. Interestingly, mRNAs encoding the three other anterior Par proteins, cdc42, baz, and par-6 also have CPE motifs in their 3'UTRs and are bound by ectopically expressed Orb in tissue culture cells [48]. Thus, the localization and translation of these Par mRNAs could be regulated by or dependent upon orb. In addition, there appears to be a reciprocal relationship between orb and anterior Par proteins. This is suggested by the synergistic genetic interactions between orb and the Par genes encoding aPKC and cdc42. It also fits with our finding that orb autoregulatory activity and the phosphorylation status of Orb are impacted by aPKC depletion.
There are also likely to be indirect effects on the functioning of the Par proteins that in turn perturb the organization of the MT network. For example, Leibfried et al. ([26]) have shown that there is a mutually interdependent relationship between the Par proteins and the actin cytoskeleton. They found that Cdc42 localization along the anterior and anterior-lateral cortex of the oocyte depends upon the integrity of the cortical actin network. Conversely, the assembly of the cortical actin network requires cdc42, aPKC and baz. In fact, one of the more striking phenotypes in orb mutant oocytes is the disorganization of the cortical actin network. As was observed for cdc42 [26], the disruptions in the actin network are accompanied by the mislocalization of aPKC. Given the interdependence of the Par proteins and the actin network the disruption of the actin cytoskeleton in orb mutants could be due to the misexpression of the Par proteins. However, the Par proteins need not be the only or even the key targets for orb regulation of the actin cytoskeleton. For example, the formation of the cortical actin network during mid-oogenesis depends upon two actin nucleators, capu and spir [61][62][63][64]. Mutations in these two genes have a number of phenotypes in common with orb. The actin cytoskeleton is fragmented and this in turn leads to a failure to properly organize the MT network and localize osk and grk mRNAs. Moreover, as has been reported for orb [56], premature cytoplasmic streaming is observed in capu and spir mutant egg chambers. Like the Par proteins, the mRNAs encoding capu and spir are bound by ectopically expressed Orb in tissue culture cells, and thus could be targets for orb regulation. On other the hand, there are some notable differences. In contrast to orb, aPKC and cdc42, capu and spir eggs are dorsalized not ventralized. Additionally, Par-1 localization to the posterior and lateral cortex does not appear to depend upon capu or spir [62] whereas it is disrupted in orb mutant chambers. Moreover, the effects of orb on the actin cytoskeleton need not be limited to these proteins. The mRNA encoding the actin effectors Cip4 and WASp have CPEs in their 3'UTRs and are bound by ectopically expressed Orb in tissue culture cells [48]. Defects in the expression of these proteins would interfere with the remodeling of the anterior/anterior-lateral cortical actin cytoskeleton and consequently disrupt Par dependent MT polarization. Finally, orb could also act downstream of the Par proteins. Like cip4 and WASp mRNAs, the mRNAs encoding the MT assembly factors, shot and patronin, have CPEs in their 3'UTRs and are bound by ectopically expressed Orb in tissue culture cells [48]. Insufficient levels of these factors would be expected to slow or block the de novo assembly of MTs along the anterior-lateral cortex.
Thus, a plausible idea is that the defects in the repolarization of the MT network when orb is depleted during mid-oogenesis are likely the consequence of the cumulative effects of misregulating mRNAs encoding not only Par proteins but also proteins involved in organizing the actin cytoskeleton and assembling MTs. Because the MT and actin cytoskeleton regulators have interdependent functions, even small perturbations in the abundance of multiple players could lead to wide ranging disruptions in cytoskeletal organization. That mRNA localization/ translational regulation might impact the reorganization of the egg chamber after stage 7 at multiple levels is supported by recent studies on egalitarian (egl). Sanghavi et al. ([67]) report that knocking down egl just before the MT network in the egg chamber is repolarized induces many of the same phenotypic abnormalities and disruptions in cytoskeletal organization that we have observed when orb activity is compromised during mid-oogenesis. Egl together with the Bicaudal-D (BicD) protein loads mRNAs onto a Dynein motors [79][80][81]. This mRNA cargo complex is responsible for localizing mRNAs in somatic cells and in developing egg chambers. Like Orb, the Egl-BicD cargo complex interacts with many different mRNA species including orb. For this reason, loss of egl activity is likely to have a global impact on mRNA transport and consequently the localized production of a diverse array of factors needed for the reorganization of the oocyte cytoskeleton during mid-oogenesis.

Eggshell phenotype scoring
Eggs were collected by placing flies of the appropriate genotype into cups and were kept at 18 degrees and given fresh apple juice and yeast paste plates daily. The eggshell phenotypes were scored starting on day 3.

Fluorescence in situ hybridization
osk FISH probes were a gift from Shawn Little at University of Pennsylvania [54].
orb FISH probes were ordered from Biosearch Technologies, and orb probes and aPKCcom FISH probes (from Xu et al. [49]) were coupled to Atto NHS-Ester 565 or 633 (Sigma) and purified using HPLC. Secondary antibodies used were goat anti-mouse IgG Alexa 488, 546 or 647, goat anti-rabbit Alexa 488, 546 or 647 (Molecular Probes). Samples were mounted using aqua polymount (Polysciences) on slides and visualized on a Leica SP5 or Nikon A1 confocal microscope.

Live imaging
Cytoplasmic movements were imaged in live oocytes in halocarbon oil on a Nikon A1 inverted confocal microscope. An image was collected every 5 seconds for at least 2 minutes to visualize cytoplasmic streaming.

Immunoprecipitation and RNA extraction
Mouse anti-Orb (4H8 and 6H4) or mouse antiβ-gal (401A) were coupled to A/G agarose beads (Santa Cruz Biotechnology) by incubating overnight at 4 degrees. 250 females were dissected in ice cold 1xPBS and ovaries were transferred to dry ice while dissecting. RNAsin (Promega) was added to ovaries and they were crushed using a plastic pestle to make a paste. The ovary paste was centrifuged at 3000 rpm for 5 minutes at 4 degrees twice, and the supernatant was saved. Half of the supernatant was added to the Orb antibody coupled with beads, and the other half was added to the control antibody beads. CoIP buffer ( [55]) and RNasin (Promega) was added to IPs, which were left to rotate for 3 hours at 4 degrees. The beads were pelleted by centrifugation and washed with coIP buffer 5 times.
RNA was released from the beads by adding 10 mM HEPES 1% SDS solution and β-mercaptoethanol, and left in a 65 degree water bath for 15 minutes. Phenol followed by phenol chloroform was used for extraction, and the water phase was ethanol precipitated with glycogen added as a carrier. The pellet was dried and then DNAse (Promega) treated.

RT-qPCR
The RNA samples were incubated with oligodT (IDT) at 65 degrees for 10 minutes. AMV reverse transcriptase (Promega) reactions were set up, and for each IP a control reaction was set up without reverse transcriptase. The samples went through the following program for reverse transcription on a PCR machine: 55 degrees for 1 min, 48 degrees for 30 min, 55 degrees for 15 min, 95 degrees for 5 min, hold at 4. For quantitative PCR, Power CybrGreen PCR master mix (Life Technologies) was used. Each qPCR reaction was done in triplicate and the average CT was used. The control samples without reverse transcriptase were also run to confirm the DNase treatment worked. The amplification of target 3'UTRs from the Orb IP were compared to the amplification from the control IP and normalized to a control (RPL32) to calculate ΔΔCT.

Westerns
For the Western blots to measure levels of β-gal expression ovaries were dissected in PBS and frozen on dry ice. Frozen tissue was crushed with a pestle in SDS buffer with urea, boiled and spun down. The extracts were loaded on a 10% SDS-Page gel. Proteins were transferred to a PVDF membrane and the membrane was cut to blot for β-gal and BEAF. For the phosphorylated Orb isoforms, ovaries from two female flies were dissected in 100 ul of 1X PBS. The ovaries were immediately transferred to 40 ul of 2XSDS buffer (100 mM Tris-Cl; 4% SDS, 200 mM DTT and 0.2% bromphenol blue) and boiled. A second set of ovaries were dissected, transferred to the same tube and boiled. The samples were then loaded onto a 7.5% SDS polyacrylamide gel. Image J was used to measure the Orb protein upper:lower band ratio.  Table. Additional ventralized egg counts. Many of the genetic interactions explored in this paper were put into cups at multiple times and have a large "n" for the total number of eggs counted. Fig 4 shows