An Interaction Type of Genetic Screen Reveals a Role of the Rab11 Gene in oskar mRNA Localization in the Developing Drosophila melanogaster Oocyte

Corresponding author: Miklós Erdélyi, Szeged, P.F.521, H-6701, Hungary., erdelyim{at}nucleus.szbk.u-szeged.hu (E-mail)

Communicating editor: T. SCHÜPBACH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization.

THE anterior-posterior and dorsal-ventral axes of the Drosophila embryo originate from the inherent asymmetry of the egg chamber and depend on the localization of specific mRNAs within the developing oocyte. The mRNA localization process requires multiple, reciprocal communication events between the germline and the follicle cells, as well as dynamic changes in the oocyte cytoskeleton (reviewed by RAY and SCHUPBACH 1996 ; LIPSHITZ and SMIBERT 2000 ). In the early stages of egg chamber development, microtubules nucleate from a microtubule-organizing center (MTOC) located at the posterior pole of the oocyte (THEURKAUF et al. 1992 ). In this period, posterior-to-anterior polarity of the microtubules and posterior localization of the nucleus characterize the asymmetry of the oocyte. The oocyte nucleus produces a localized gurken (grk) signal, which induces the adjacent follicle cells to adopt posterior polar follicular cell fate (ROTH et al. 1995 ). Later in oogenesis, at stages 6–7, the posterior polar follicle cells send back a yet unknown signal to the oocyte to repolarize its microtubule cytoskeleton (RUOHOLA et al. 1991 ; GONZALEZ-REYES and ST JOHNSTON 1994 ; GONZALEZ-REYES et al. 1995 ; ROTH et al. 1995 ). The repolarizing signal inactivates the posterior MTOC and simultaneously a diffuse MTOC appears anteriorly, resulting in a reverse microtubule orientation with the plus ends pointing toward the posterior (THEURKAUF et al. 1992 ). Rearrangement of the microtubule cytoskeleton of the oocyte is required for the proper localization of bicoid (bcd) and oskar (osk), the primary determinants of the anterior-posterior axis of the embryo (CLARK et al. 1994 , CLARK et al. 1997 ; LANE and KALDERON 1994 ).

The repolarized microtubule cytoskeleton enables the transport of osk mRNA to the posterior pole of the oocyte by a plus-end-directed motor kinesinI (BRENDZA et al. 2000 ). At stages 8–9, osk RNA is predominantly localized at the posterior pole where it is translated, and Oskar protein directs the assembly of the pole plasm, a specialized cytoplasm that contains factors for abdomen and germ cell development. However, injection of in vitro-synthesized osk transcripts into the stage 10–11 oocytes revealed that the localization of the osk mRNA to the posterior pole proceeds by the cytoplasmic streaming in the later stages as well (GLOTZER et al. 1997 ). In addition to the role of microtubule cytoskeleton, it has been demonstrated that the actin cytoskeleton is essential for maintenance of the localized osk mRNA at the posterior pole of the oocyte (ERDELYI et al. 1995 ; TETZLAFF et al. 1996 ).

A number of genes having a role in pole plasm assembly or function have been identified and collectively named the posterior group of genes. Females homozygous for any of the posterior group genes lay eggs in which abdomen-less and germ cell-less embryos develop. This complex phenotype is also called the posterior phenotype. Classical posterior group genes such as cappuccino, spire, staufen, oskar, vasa, tudor, and valois were isolated in the early maternal effect lethal (MEL) screens reviewed by ST JOHNSTON and NÜSSLEIN-VOLHARD (1992). These genes represent the backbone of a gene hierarchy that localizes pole plasm components including abdominal determinants (nanos and pumilio; WANG and LEHMANN 1991 ; BARKER et al. 1992 ; SONODA and WHARTON 1999 ) and factors needed for primordial germ cell development (germ cell-less, mtlrRNA, and polar granule component RNA; JONGENS et al. 1992 ; NAKAMURA et al. 1996 ; IIDA and KOBAYASHI 1998 ). Other genes of the posterior gene hierarchy like TropomyosinII (TmII), mago nashi, and pipsqueak (BOSWELL et al. 1991 ; ERDELYI et al. 1995 ; HOROWITZ and BERG 1996 ) have been identified, based on the germ cell-less phenotype. However, both MEL and germ cell-less screens have a mutual limitation; i.e., only homozygous viable mutations can be identified. Additional members of the posterior gene hierarchy, especially the genes with pleiotropic effects, have most likely been missed in such screens. Analysis of homozygous germline clones can be employed to reveal the role of pleiotropic genes in pole plasm formation (PERRIMON et al. 1996 ). This type of experiment, however, frequently produces insufficient results since homozygous mutations can result in early defects in the germline development, making analysis of the late phenotypes impossible.

To circumvent the problem of pleiotropic effects, we performed a novel, interaction type of genetic screen for genes involved in osk mRNA localization and pole plasm formation. We developed two sensitized genetic backgrounds using the TmII and osk mutations and used them to screen newly induced EMS mutations for dominant modifiers of the two related posterior phenotypes: lack of an abdomen and germline. This method magnifies the effect of heterozygous mutations on the posterior phenotypes, thus identifying even lethal genes involved in the targeted process. From our screen, we recovered alleles of known posterior group genes, demonstrating the efficacy of our approach. In this report we describe the analysis of 14 E(To) enhancers of the TmII-osk background and, among them, two allelic mutations that identify the Rab11 gene. We show that mutations in Rab11 lead to mislocalization of osk mRNA in the oocyte, but do not affect either localization of grk and bcd mRNAs or communication events between the oocyte and the follicle cells. We show that Rab11, a small GTPase required for targeting of vesicles, is also involved in the regulation of the polarized microtubule cytoskeleton of the developing oocyte.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutant strains:
Flies were maintained on standard yeast-cornmeal medium. Crosses were performed at 25°. Oregon-R flies were used as wild type. TmII^eg9 and TmII^eg20 mutants are excision revertants of the P-element-induced TmII^gs1 mutation that show strong and weak germ cell-less phenotypes, respectively. TmII^el4 is a lethal excision allele of the TmII^gs1 mutation (ERDELYI et al. 1995 ). osk^P187 is a strong hypomorphic P-element-induced allele. The T(2;3) ITS9, Fab7, a synonym of the T(2;3) TSR-7 translocation, is described in SIPOS et al. 1998 . For a description of other mutant strains see LINDSLEY and ZIMM 1992 .

Mutagenesis:
E(To) mutations were induced in an isogenized TmII^eg9 ca mutant strain. Adult TmII^eg9 ca homozygous males were fed a 25 mM EMS solution for 8 hr (LEWIS and BACHER 1968 ). The EMS-treated males were mated with T(2;3) CyO TM6, Ubx/+;Sb females. Individual +; TmII^eg9 ca/T(2;3) CyO TM6, Ubx males, each bearing one mutagenized second and third chromosome, were crossed to st osk⁵⁴ TmII^el4/TM3, Sb Ser females. The fertility of groups of at least five females that could be of either E(To)/+; TmII^eg9 ca/st osk⁵⁴ TmII^el4 or E(To) TmII^eg9 ca/st osk⁵⁴ TmII^el4 genotype was tested from each mutant line. E(To) TmII^eg9/TM3, Sb Ser or E(To)/+, TmII^eg9 ca/TM3, Sb Ser genotype male siblings were collected from lines that showed enhanced sterility. Stable balanced stocks were established using T(2;3) CyO Tm6, Ubx translocated balancer chromosomes. Since in the test generation the segregation of the mutagenized second chromosome was not followed by a dominant selectable marker mutation, four parallel sublines were established from individual males in each selected line. The sublines were tested again by mating males with st osk⁵⁴ TmII^el4/TM3, Sb Ser females. One confirmed subline of each mutant line was kept.

Analysis of E(To) mutants in TmII^eg9/TmII^eg20 sensitized background:
E(To); TmII^eg9 ca /T(2;3) CyO TM6, Ubx or E(To) TmII^eg9 ca /T(2;3) CyO TM6, Ubx females were crossed to TmII^eg20 homozygous males. E(To)/+; TmII^eg9 ca/ TmII^eg20 or E(To) TmII^eg9 ca/TmII^eg20 heterozygous females were mated with wild-type males. Adult progeny were dissected and scored for rudimentary gonad phenotype.

Complementation analyses:
E(To); TmII^eg9 ca /T(2;3) CyO TM6, Ubx or E(To) TmII^eg9 ca /T(2;3) CyO TM6, Ubx females were crossed to males bearing the following known mutant alleles: oskar^P187, nanos^L7, nanos^RD, pumilio⁶⁸⁰, staufen^D3, valois^PG, egalitarian^RC12, tudor^WC8, cappuccino^RK, spire^RP, vasa⁰¹⁴, BicaudalD⁷¹³⁴, BicaudalC^YC33, aubergine^QC42, arrest^PA62, oo18 RNA-binding protein^mel, mago nashi¹, and chicadee^UC57. Trans-heterozygous females were tested for female sterile phenotype and their progeny, if any, for the germ cell-less phenotype. Complementation analyses were also performed among E(To) mutant lines. All the E(To) trans-heterozygous combinations were analyzed for lethal and female sterile phenotypes.

Analysis of mutant phenotypes:
Embryonic cuticle preparations were made as described by WIESCHAUS and NÜSSLEIN-VOLHARD (1986). Eggs from sterile mutant females were collected, dechorionated in 50% Clorox bleach, washed, mounted in Hoyer's lactic acid 1:1 medium, and cleared for 1 day at 60°. The adult germ cell-less phenotype was detected by dissection. Adult progeny of the mutant females were anesthetized, wet in ethanol, and dissected in Drosophila Ringer solution. The germ cell-less type gonads were examined through a dissecting microscope. Adult cuticle preparations were done as follows: Flies were anesthetized and immersed in boiling 10% KOH solution for 5–10 min. Next, the KOH solution was carefully removed and the cuticles were washed three times in boiling water. Cuticles were mounted and cleared for 1 day at 60° in Hoyer's medium. The only structures remaining after this step were those made of adult cuticle.

Mapping of the E(To) mutations:
Mutagenized second and third chromosomes were segregated by the following scheme (* stands for the newly induced mutations). *; * TmII^eg9 ca /T(2;3) CyO TM6, Ubx males were mated with SM6b; MKRS/T(2;3) ITS9, Fab7 females. * /SM6b, * TmII^eg9 ca/MKRS males were crossed to SM6b/Sco and TM3, Sb Ser/Tm6, Tb females. Finally, * /SM6b and * TmII^eg9 ca/TM3, Sb Ser balanced stocks were established. Homozygous phenotypes of both second and third mutagenized chromosomes of each original mutant line were determined.

For meiotic mapping, E(To)/al dp b pr c px sp or E(To)/ru h th st cu sr e ca females were mated with al dp b pr Bl c px sp/SM1 or ru h th st cu sr Pri e ca/TM6, Ubx males, respectively. E(To) recombinant chromosomes were balanced with SM6b or TM3, Sb Ser balancer chromosomes. Homozygous phenotypes of the recombinant lines were analyzed.

Germline mosaic analysis:
Homozygous germline clones of Rab11 alleles were induced using the FLP-DFS technique (CHOU and PERRIMON 1996 ). Rab11^j2D1, Rab11^EP3017, Rab11^93Bi, and Rab11^E(To)11 mutations were recombined to the p{ry + t7.2 = neoFRT} 82B chromosome. y w p{ry + t7.2 = hsFLP}1/w; p{ry + t7.2 = neoFRT} 82B Rab11/ p{ry + t7.2 = neoFRT} 82B p{w + mc + ovoD1-18} females were heat shocked 72 hr after egg laying. Adult mosaic ovaries were dissected and subjected to osk and bcd mRNA in situ hybridization.

mRNA in situ hybridization:
Digoxigenin-labeled DNA probes were synthesized using the Dig DNA labeling kit (Boehringer Mannheim, Wien). The osk DNA probe corresponded to the 2.1-kb SacI fragment of the osk cDNA (EPHRUSSI et al. 1991 ). The bcd probe was generated by labeling a 2.5-kb EcoRI fragment of the bcd cDNA (BERLETH et al. 1988 ). The grk probe corresponded to the XhoI-NotI fragment in the grk cDNA (NEUMAN-SILBERBERG and SCHUPBACH 1993 ). The hybridizations were carried out as described in EPHRUSSI et al. 1991 . Hybridization signals were detected using the Dig detection kit (Boehringer Mannheim).

Kinesin:ß-galactosidase staining:
Ovaries were dissected in PBS with Triton (PBT) solution and then fixed with 2.5% glutaraldehyde in PBS. Next, ovaries were washed two times in PBT and placed into staining buffer [0.05 M K₃(Fe(CN)₆), 0.05 M K₄(Fe(CN)₆) in PBS]. X-gal solution was added to a final concentration of 0.2%. Staining was performed at room temperature overnight. The stained ovaries were washed in PBT three times for 10 min each and mounted in Aqua Poly/Mount medium (Polysciences).

Visualization of Tau:green fluorescent protein in living oocytes:
Anesthetized females were dissected and their ovaries were mounted in Voltalef oil (10S Atochem). The preparation was examined using an inverted confocal microscope (Zeiss AxioVert).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Construction of the sensitized mutant backgrounds:
To isolate new members of the posterior gene group hierarchy we developed an interaction-type genetic screen. We isolated dominant enhancers (E(To)) of an osk- and TmII-based intermediate posterior phenotype, which was generated by decreasing the amount of posteriorly localized osk mRNA. We chose osk and TmII alleles to sensitize the genetic background since a series of alleles of these genes that reduces the amount of the localized osk mRNA to a different extent is available (EPHRUSSI et al. 1991 ; ERDELYI et al. 1995 ; M. ERDÉLYI and A. EPHRUSSI, personal communication). We performed a systematic search for osk-TmII allele combinations that show intermediate phenotypes. We found two sensitive genetic backgrounds by which it was possible to monitor the effect of dominant enhancers on both the abdomen- and the germ cell-less components of the posterior phenotype. The TmII^eg9 ca/st osk⁵⁴ TmII^el4 allele combination resulted in a leaky maternal effect lethality; 66% of the eggs did not develop into viable larvae (Table 1). In 44% of the defective eggs embryogenesis did not commence or was terminated before the time of the cuticle formation while in 56% of the dead eggs we observed embryos showing a characteristic posterior group phenotype with missing abdominal segments. The intermediate penetrance of the posterior phenotype observed in the sensitive background was coupled with a very high expressivity of the phenotype. The average number of the abdominal segments/embryos was reduced from the normal 8 to 0.69 (Table 1). This reflects that the TmII^eg9 ca/st osk⁵⁴ TmII^el4 sensitive background effectively reduced the osk mRNA concentration. Newly induced mutations were first screened in this sensitive background. The second sensitive background was composed of TmII^eg9 and TmII^eg20 alleles and exhibited a germ cell-less phenotype with a penetrance of 57%. We introduced E(To) mutations, identified in the first sensitive background, into the TmII^eg9 ca/TmII^eg20 background and measured their effect on the germ cell-less phenotype.

Recovery of E(To) mutations:
In total, 2625 lines bearing EMS-treated second and third chromosomes were screened for E(To) mutations. The maternal effect lethal phenotype of the E(To)/+; TmII^eg9 ca/st osk⁵⁴ TmII^el4 or E(To) TmII^eg9 ca/st osk⁵⁴ TmII^el4 females (test females afterward) was assessed by counting their hatched and nonhatched eggs. In a primary screen, we collected mutant lines in which the test females showed a significantly more penetrant sterile phenotype than did females of the sensitive background alone. The progeny of the test females could be homozygous for the E(To) mutations, since, for technical reasons, the test females were not collected as virgins in the primary screen. Thus, the possible zygotic embryonic lethality of the E(To) mutations could virtually increase the penetrance of the observed maternal effect phenotypes. To exclude the effect of the zygotic lethality, we tested E(To) mutations when the test females were collected as virgins and mated with wild-type males. In this way we confirmed the presence of E(To) mutations in 14 lines (Table 1). A specific enhancer effect of the E(To) mutations was detected in the sensitive background. E(To) mutations showed no other cuticle defect in the sensitive background other than a variable, but characteristic, posterior phenotype. The expressivity of the enhanced posterior group phenotype was as high as in the control, ranging from 0.41 to 1.1 abdominal segments/embryo on average (Table 1). Test females from only two E(To) mutant lines, E(To)5 and E(To)12, produced unfertilized or early dead eggs at significantly higher rates than in the control (Table 1). Furthermore, E(To) mutations did not decrease the egg-laying capacity of the test females (Table 1).

E(To) mutations were introduced into the TmII^eg9 ca/TmII^eg20 mutant background and the germ cell-less phenotype of the progeny was scored in adults. Seven E(To) mutations [E(To)1, E(To)4, E(To)6, E(To)10, E(To)11, E(To)7, and E(To)14] enhanced the germ cell-less phenotype of the second sensitive mutant background as well, confirming that these mutations are indeed members of the posterior gene hierarchy (Table 1). However, E(To)12 and E(To)13 had an opposite effect on germ cell development: Instead of enhancement, a suppression effect on the germ cell-less phenotype was observed. The penetrance of the germ cell-less phenotype was significantly reduced when E(To)12 and E(To)13 mutations were introduced into the second sensitive background. The effect of these mutations on abdomen and germ cell development was reminiscent of that of nanos. An excess of germ cells was found in the abdomen-less embryos produced by nanos mutant females (SMITH et al. 1992 ; DESHPANDE et al. 1999 ), although it has been shown that the complete nanos null germ cells show migration defects and were lost on their way to the embryonic gonads (KOBAYASHI et al. 1996 ). However, E(To)12 and E(To)13 may act through nanos. We speculate that in the sensitive background E(To)12 and E(To)13 only partially repressed nanos activity resulting in excess of germ cells without migration defect. However, we also find it a plausible explanation that E(To)12 and E(To)13 act on nanos independently.

To measure the efficiency of the mutation isolation, a sample of the EMS-treated third chromosomes was also screened for recessive lethal mutations. Out of 102 assayed chromosomes, 34 were homozygous viable. The Poisson distribution suggested that on average each mutagenized third chromosome carried 0.99 lethal mutations. Since we induced 11 E(To) mutations (see below) and 2599 lethal ones on the 2625 mutagenized third chromosomes, the relative frequency of the E(To) identification vs. lethal mutation identification was 1:236.

Complementation analysis of the E(To) mutations:
The majority of the E(To) mutations complemented each other. Out of 91 trans-combinations, only three noncomplementing pairs of alleles were found. E(To)3 and E(To)11 trans-heterozygotes were lethal and considered allelic (Table 2). The nature of the noncomplementing phenotypes of the E(To)7/E(To)1 and E(To)7/E(To)14 combinations was quite different. In both cases the trans-heterozygous females were sterile and embryos with a posterior phenotype were found in their eggs; however, the E(To)1/E(To)14 combination was completely fertile, indicating that E(To)1, E(To)14, and E(To)7 are not allelic. We concluded that the observed sterility of the E(To)7/E(To)1 and E(To)7/E(To)14 females was due to dominant interactions. The E(To) mutations were induced in the TmII^eg9 mutation bearing chromosomes. Thus, the trans-heterozygous females in the complementation analysis were in fact homozygous for the TmII^eg9 mutation resulting in a weak sensitive genetic background, in which the otherwise small genetic interactions could be enhanced and become visible. The E(To) mutations were also complemented by mutations in known genes (see MATERIALS AND METHODS). This type of complementation analysis fortuitously confirmed that the E(To)1, E(To)14, and E(To)7 mutations were indeed not allelic. Namely, E(To)14 proved to be a vasa allele, while E(To)7 was found to be an osk allele, and E(To)1 complemented both osk and vasa alleles. In addition to finding new osk and vasa alleles among the E(To) mutations, the complementation analysis also revealed that the E(To)2 mutation is a new allele of the mago nashi gene. Taken together, by complementation analysis we found that the 14 E(To) mutations represent 13 genes, from which three are alleles of the already known posterior group genes, while 11 E(To) mutations identify 10 novel genes of the posterior gene hierarchy.

Mapping of the E(To) mutations:
The mutagenized chromosomes were segregated and two sublines that contained only second or third mutant chromosomes were established from each mutant stock. The sublines were assayed for lethal, female sterile, and maternal effect germ cell-less phenotypes. Altogether, 13 lethal, 4 female sterile, and no germ cell-less chromosomes were found. Out of the 14 original mutant stocks, E(To)2, E(To)3, and E(To)11 were found to carry female sterile or lethal mutations on both the second and third chromosomes. To determine which mutant chromosome is responsible for the enhancer phenotype, mutant chromosomes from the sublines were reintroduced into the first sensitive background. By the phenotypic analysis of the trans-heterozygous test females, the E(To) phenotype of the mutants was mapped to the third chromosome in 11 mutant lines, while in 3 lines the E(To) activity was found to be on the second chromosome (Table 1).

We mapped the E(To) mutations on the segregated mutagenized chromosomes by meiotic recombination. Eleven E(To) chromosomes contained only one lethal or sterile mutation. The single mutations were mapped between marker mutations of the mapping chromosomes (Table 1).

Statistical analysis of the mutagenesis experiment suggested that we could have several mutant chromosomes bearing more than one new mutation. According to the mutagenesis control experiment, we induced on average one recessive lethal mutation on each third chromosome. At this rate of mutant induction the Poisson distribution estimated that 33% of the mutant chromosomes should bear two mutations. Accordingly, 3 of the 11 E(To) third mutant chromosomes, E(To)12, E(To)1, and E(To)4, carried two lethal mutations. To determine which mutation was responsible for the E(To) phenotype, sublines were again established from the recombinant chromosomes that contained only one of the lethal mutations and assayed for the presence of the E(To) activity in the first sensitive background. In this way, we could determine that mutations mapping to the st-cu chromosomal region are responsible for the E(To) phenotype in both E(To)1 and E(To)4 (Table 1). On the E(To)12 mutant chromosome, a mutation mapping to the chromosomal region cu-ca was responsible for the enhancer effect (Table 1). Except for the E(To)13 line, the meiotic mapping experiment enabled us to establish "cleaned" E(To) lines that were free of both the second-site and the TmII^eg9 mutations. Further analyses of the E(To) mutations were performed on the cleaned lines.

The E(To)11 mutation was mapped to a narrow chromosomal region by deficiency mapping. E(To)11 lies distal to the sr marker mutation. We tested deficiencies from this region and we found that Df(3R)e-R1 and Df(3R)e-N19 did not complement the E(To)11 lethality. Using three other deficiencies from this region that complemented the lethality, we mapped the E(To)11 mutation to the 93B6-C3 chromosomal region (Fig 1A).

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Cytological localization and genomic organization of the Rab11 gene. (A) The cytological map of the 93 region. Horizontal lines represent the deleted regions. Df(3R)e-R1 and Df(3R)e-N19 do not complement the Rab11 alleles. Df(3R)e-BS2, Df(3R)e-F1, and Df(3R)e-GC14 complement the Rab11 alleles. Vertical dotted lines indicate the Rab11 map position that is 93B6-93C3. (B) Schematic representation of the structure of the Rab11 gene. Open triangles indicate insertion sites of the Rab11j2D1 and the Rab11EP3017 P-element insertions.

Homozygous E(To) phenotypes:
Cleaned sublines, which contained only one mutagenized second or third chromosome and were free of TmII^eg9, were used to analyze the homozygous E(To) phenotypes. In 10 lines, E(To) mutations were associated with lethal phenotypes and only 4 were homozygous viable. As mentioned previously, 3 of the 4 viable lines were alleles of the osk, vasa, and mago nashi genes, and their homozygous phenotypes were not analyzed in detail. The fourth viable mutation, E(To)9, was associated with a visible phenotype. E(To)9 homozygotes had rough eyes and their wing margins were often nicked. E(To)9 homozygous females were sterile and laid eggs with dorsal appendages slightly shorter than the wild type. Since rudimentary dorsal appendages may indicate a defect in the gurken-DER signaling pathway (SCHUPBACH and ROTH 1994 ), which is also involved in osk mRNA localization, we carefully analyzed the phenotype of the E(To)9-derived eggs. However, the rudimentary dorsal appendages were not shifted to the posterior, as is characteristic of gurken-DER-deficient eggs. The dorsal-ventral pattern of the chorion imprints of the E(To)9 eggs was consistently normal, indicating again that the gurken-DER pathway is intact in the mutant. By in situ mRNA hybridization, we found that in E(To)9 homozygous egg chambers grk and osk mRNA localized as in the wild type (data not shown). We infer that the E(To)9 mutation enhances the posterior phenotype of the sensitive background by acting after the gurken signaling and the osk mRNA localization in the process of the pole plasm assembly.

E(To)3 and E(To)11 mutations are alleles of the RAB11 gene:
The E(To)3 and E(To)11 mutations represent a unique complementation group. We decided to analyze this complementation group in detail. The E(To)11 mutation was mapped as a recessive lethal allele in the 93B6-C3 chromosomal region (Fig 1A). Complementation crosses were carried out between mutations in this region and the E(To)11 and E(To)3 alelles. We identified two noncomplementing lethal P-element insertion mutants: l(3)j2D1^j2D1 and EP(3)3017. These P elements identify the Rab11 gene, which encodes a small GTPase involved in the regulation of the cytoplasmic membrane trafficking (Fig 1B). In addition, we discovered that l(3)93Bi (EISENBERG et al. 1990 ), a semilethal EMS-induced mutation, which had not been identified as a Rab11 allele, also failed to complement the E(To)11 and E(To)3 mutations. Thus, the Rab11 complementation group was enlarged by three new Rab11 alleles. The complementation behavior of the five Rab11 alleles is shown in Table 2. Except for Rab11^93Bi trans-heterozygotes, all Rab11 heteroallelic combinations resulted in lethal phenotypes. Rab11^93Bi combinations were viable and heterozygotes showed semilethality coupled with bristle defects of different expressivity, according to which we put the Rab11 alleles in a phenotypic order (Table 2). The bristle defect varied from slight shortening to complete loss of the macrochetes (Fig 2). As we never observed defects in the socket and shaft cell formation, we conclude that Rab11 is not involved in regulation of the cell lineage of the external sense organs, but rather is specific to bristle growth. In addition to the semilethality and bristle defect, viable Rab11 allele combinations resulted in female sterility (Table 2). The Rab11^j2D1/Rab11^93Bi trans-heterozygous combination showed the weakest semisterile phenotype when 7.7% of the laid eggs developed into larvae. Among these escapers we observed 2% with germ cell-less phenotype, which is characteristic for weak mutations of many genes in the posterior gene hierarchy. The egg-laying capacity of the Rab11^j2D1/Rab11^93Bi females was wild type. The Rab11^EP3017/Rab11^93Bi and the Rab11^E(To)11/Rab11^93Bi allele combinations exhibited stronger phenotypes. Trans-heterozygous females were completely sterile and laid fewer eggs than wild-type control animals. Most of their eggs showed a slightly weaker chorion cell-imprint pattern with shortened dorsal appendages and the eggs were frequently collapsed (data not shown). On the basis of their heterozygous sterile phenotypes, we could arrange the Rab11 alleles into the following order: 93Bi < j2D1 < EP3017 = E(To)11 = E(To)3 < Df, which coincides with the order established on the basis of the bristle phenotypes (Table 2).

View larger version (84K): In this window In a new window Download PPT slide   Figure 2. Micrographs of cuticle preparation of a part of the fourth tergit of a wild-type (A) and a Rab11EP3017/Rab1193Bi male (B).

To test the specificity of the E(To) effect of the Rab11 mutations, we introduced the Rab11^j2D1, Rab11^93Bi, and Rab11^EP3017 alleles into the sensitive genetic backgrounds. We found that all these Rab11 alleles, which were isolated in various mutagenesis screens, behaved like the E(To) mutations and dominantly enhanced the penetrance of the abdomen-less and the germ cell-less phenotypes of the genetic backgrounds (data not shown).

RAB11 is involved in osk RNA localization:
Given that two Rab11^E(To) alleles were identified as modifiers of an osk-deficient genetic background, we examined osk mRNA distribution in the viable Rab11 heteroallelic combinations (Fig 3). In Rab11^j2D1/Rab11^93Bi, Rab11^EP3017/Rab11^93Bi, and Rab11^E(To)11/Rab11^93Bi mutant egg chambers, osk mRNA localized normally throughout stage 8 and after stage 10 as followed by in situ mRNA hybridization. At stage 9, however, transient abnormal osk mRNA localization patterns were detected in the mutant egg chambers. Instead of the tight posterior osk mRNA localization, which is characteristic of the wild type, three types of abnormal osk mRNA localization were found in the mutant egg chambers: (1) The strongest observed abnormality was a scattered osk mRNA staining pattern at multiple loci, which concentrated mostly around the posterior pole; (2) frequently, osk mRNA mislocalized to an ectopic site in the center of the oocyte; and (3) we also observed an intermediate phenotype in the mutant egg chambers in which the normal posterior and the mislocalized central osk stainings were simultaneously present (see Table 3 and Fig 3).

View larger version (74K): In this window In a new window Download PPT slide   Figure 3. Micrograph of in situ osk mRNA hybridization of stage 9 wild-type (A) and Rab11j2D1/Rab1193Bi (B–D) egg chambers. Simultaneous normal and centrally mislocalized osk mRNA (B), central (C), and scattered posterior mislocalization of the osk mRNA (D).

The effect of Rab11 mutations on osk localization is germline dependent:
Since osk mRNA localization is dependent on the functions of both the oocyte and the posterior follicular cells (reviewed by LASKO 1995 ), we examined whether the focus of the Rab11 mutations is in the germline or in the somatic follicular cells. Rab11 homozygous germline clones were induced by the ovoD-FRT method (CHOU and PERRIMON 1996 ) and analyzed by osk mRNA in situ hybridization. In homozygous germline clones of the medium-strong Rab11^j2D1 allele we could recover mislocalized osk mRNA phenotypes that were similar to those of Rab11 heterozygotes. In the ovoD-free egg chambers we observed the three types of osk localization defects that we had earlier detected in viable Rab11 heteroallelic combinations (Table 3). Thus, the Rab11 gene has a germline autonomous effect on osk mRNA localization. However, eggs from homozygous Rab11^j2D1germline clones were frequently collapsed and never developed into larvae, indicating that the Rab11 gene has pleiotropic functions during egg development. In the case of the strong mutations, Rab11^EP3017 and Rab11^E(To)11, we never detected egg chambers older than stage 4, when the ovoD dominant selective marker mutation blocks egg chamber development. The absence of the homozygous egg chambers older than stage 4 indicates that Rab11 gene function is necessary for early germline development, too.

Rab11 is necessary for the regulation of the oocyte cytoskeleton:
Mislocalization of osk mRNA to the center is observed in several mutants such as Notch, PKA, and laminin A (RUOHOLA et al. 1991 ; LANE and KALDERON 1994 ; GONZALEZ-REYES et al. 1995 ; ROTH et al. 1995 ; DENG and RUOHOLA-BAKER 2000 ). These mutations affect the follicle cell-to-oocyte signaling, required for triggering reorganization of the oocyte microtubule cytoskeleton. These mutations result in a mutant, symmetric microtubule cytoskeleton in the oocyte resulting in osk mRNA in the oocyte center and bcd mRNA at both the anterior and posterior poles. The symmetric oocyte microtubule cytoskeleton frequently coincides with a failure of the oocyte nucleus to move to the anterior dorsal corner of the oocyte (GONZALEZ-REYES et al. 1995 ; ROTH et al. 1995 ). In symmetric mutant egg chambers, Kinesin:ß-galactosidase, a plus-end-directed microtubule motor fusion protein, localizes to the center of the oocyte (CLARK et al. 1994 ; GONZALEZ-REYES et al. 1995 ; ROTH et al. 1995 ). In the Rab11^93Bi/Rab11^EP3017 mutant combination we also observed a centrally localized Kinesin:ß-galactosidase accumulation (Fig 4). However, unlike Notch, PKA, and Laminin A mutations, Rab11 mutations never resulted in a symmetric microtubule cytoskeleton as we showed by direct visualization of the oocyte microtubules (MICKLEM et al. 1997 ). Tau:green fluorescent protein (GFP), a fusion between a microtubule-binding protein and GFP, never accumulated at the posterior pole in the nine-stage Rab11^j2D1/Rab11^93Bi oocytes (Fig 4). The absence of a focus of the posterior Tau:GFP fluorescence at stage 9 indicates that posterior MTOC disassembled normally. In Rab11 mutant oocytes, bcd mRNA consistently localizes solely to the anterior pole as in the wild type, and the oocyte nucleus was always found at the wild-type position (Fig 5A). Furthermore, grk mRNA, which localizes in the close vicinity of the oocyte nucleus throughout oocyte development, showed wild-type localization (Fig 5B). These results indicate that the follicle cell-to-oocyte signal, which triggers the rearrangement of the oocyte cytoskeleton, is not affected in Rab11 mutants.

View larger version (121K): In this window In a new window Download PPT slide   Figure 4. Micrographs of ß-galactosidase-stained wild-type (A) and Rab1193Bi/Rab11EP3017 (B) egg chambers. Micrographs of Tau:GFP expressing wild-type (C) and Rab11j2D1/Rab1193Bi (D) living oocytes.

View larger version (66K): In this window In a new window Download PPT slide   Figure 5. Micrographs of bicoid (A) and gurken (B) mRNA in situ hybridization of stage 9 Rab11j2D1/Rab1193Bi egg chambers. Micrographs of lacZ expression of slbo1 enhancer trap line in a stage 9 Rab11EP3017/Rab1193Bi mutant egg chamber (C).

Signaling from the oocyte to the follicle cells is also intact in Rab11 mutants. In wild-type egg chambers, the oocyte sends a signal to the posterior follicle cells. This gurken-mediated signal is responsible for posterior polar follicle development. In grk mutants, due to the lack of the germline-to-soma signal, the posteriorly located follicle cells adopt the default anterior polar follicle cell fate and express the anterior-specific enhancer trap marker slbo¹ (MONTELL et al. 1992 ; GONZALEZ-REYES et al. 1995 ). On the contrary, in the Rab11^EP3017/Rab11^93Bi and Rab11^E(To)11/Rab11^93Bi egg chambers only the normal anterior slbo expression was detected at stage 9, demonstrating that gurken-mediated signaling was functional and that Rab11 is not required for gurken signaling (Fig 5C).

However, Tau:GFP visualization of the Rab11 mutant oocytes revealed a significant difference from the wild-type microtubule orientation (Fig 4). Instead of the wild-type anterior-to-posterior gradient, we observed mainly cortical Tau:GFP accumulation in the stage 9 mutant oocytes.

Taken together, the Rab11 mutant phenotypes are different from those of mutants that interfere with the mutual signaling between the oocyte and the follicle cells. The effect of Rab11 mutations on microtubule orientation and osk localization is rather reminiscent of that of par1 (SHULMAN et al. 2000 ; TOMANCAK et al. 2000 ). Par1 is a recently described posteriorly localized kinase, which has a proposed role in a novel step of the anteroposterior polarization of the oocyte that is necessary for osk but not for bcd localization (SHULMAN et al. 2000 ).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Here we report a successful application of an interaction type of genetic screen, designed to study the polar plasm assembly in the Drosophila oocyte. Much of our understanding about polar plasm assembly and function stems from studies of MEL mutations of posterior group genes. MEL mutations were identified in screens for homozygous viable mutations when the homozygous phenotypes were directly analyzed. However, detailed genetic analyses revealed that most of the MEL mutations are specific alleles of pleiotropic genes, with functions in other developmental processes, too. It is likely that there exists a group of pleiotropic genes involved in polar plasm assembly that has not been identified in the direct screens. Several experimental strategies are available to analyze the maternal effects of pleiotropic genes and some of them have been used in systematic mutant isolation experiments for genes involved in posterior body patterning. Screening lethal P-element insertions by mosaic technique, Ruden and co-workers (RUDEN et al. 2000 ) found a lethal P insertion that causes a mislocalization of osk mRNA in homozygous clones. Wilson and co-workers published a suppressor screen of a gain-of-function allele of osk (WILSON et al. 1996 ). Maternal effect alleles of the lethal polycomb group genes were found to suppress nanos mutations (PELEGRI and LEHMANN 1994 ).

Instead of isolating suppressors of gain-of-function or hypomorphic alleles, we carried out an enhancer screen. We developed and used osk and TmII mutant genetic backgrounds and thus identified 14 enhancer mutations of a weak oskar-like posterior phenotype. Following EMS mutagenesis, 0.4% of the third chromosomes carried E(To) mutations while, according to the control experiment, each chromosome bore one recessive lethal mutation on average. Complementation analysis of the E(To) mutations revealed that most of the genes were identified by a single allele, indicating that our screen is far from the saturation level, and more than 13 E(To) mutable genes in principle can be identified by our TmII-osk-sensitized system. Since a given sensitive genetic background may preferably identify only a special subset of the pleiotropic posterior group genes, we estimate that the number of genes that have roles in polar plasm function is even higher than identifiable in a single sensitized background.

Three of the 13 genes we identified by E(To) mutations are the known posterior group genes: osk, mago nashi, and vasa. Since the sensitive genetic background contained the osk mutation, identification of a new osk allele was due to a simple noncomplementation. However, the mago-nashi and the vasa alleles were isolated on the basis of dominant genetic interactions. Identification of the vasa gene, which encodes a polar plasm component physically interacting with osk protein, clearly validates our screen and proves that the genetic background we used was selective for posterior group genes (HAY et al. 1988A , HAY et al. 1988B ; BREITWIESER et al. 1996 ). Most of the E(To) mutations (11/14) were recessive lethal. These genes would not have been identified in a direct MEL screen.

We have isolated two lethal alleles of the Drosophila homologue of the Rab11 gene by their dominant E(To) phenotypes. Rab11 is a subfamily of the large rab/Ypt gene family of small monomeric GTPases, which are essential components of vesicular traffic. Rab/Ypt members are involved in targeting of transport vesicles to different subcellular compartments (NUOFFER and BALCH 1994 ). More than 40 mammalian Rab family members have been published so far, which play roles in virtually all membrane trafficking pathways (NUOFFER and BALCH 1994 ). The Rab11 subfamily members Rab11a, Rab11b, and Rab25 have been reported as regulators of endocytic membrane recycling of both polarized and nonpolarized cells of various organisms (ULLRICH et al. 1996 ; REN et al. 1998 ; CASANOVA et al. 1999 ; COX et al. 2000 ; WANG et al. 2000 ). Beyond its function in endocytosis, Rab11 is also involved in exocytosis. For instance, Golgi to plasma membrane transport of the vesicular stomatitis virus G protein can be blocked by a gain-of-function allele of the Rab11 gene (CHEN et al. 1998 ). In the neuroendocrine cell line, PC12, Rab11 localizes to both constitutive and secretory vesicles (URBE et al. 1993 ). Finally, ora3, a Torpedo marmorata Rab11 homologue, is associated with cholinergic synaptic vesicles, suggesting a role of Rab11 in specialized recycling membranes (VOLKNANDT et al. 1993 ). Hence, the Rab11 family itself has been reported to function in rather different cell biological events.

Whatever the role of the Drosophila Rab11 gene in the osk localization pathway, it must be indirect and act in the reorientation of the oocyte microtubule network, as seen by the Tau:GFP microtubule visualization and by mislocalization of the Kinesin:ß-galactosidase fusion protein to the center in the Rab11 mutants. Central mislocalization of the Kinesin:ß-galactosidase protein has been observed in mutants that impair any step of the reciprocal signaling events between the oocyte and the posterior follicle cells. Given that the best-characterized role of the Rab11 proteins is the targeting of recycling endosomes or trans-Golgi vesicles to the plasma membrane, it seemed to be plausible that Rab11 mutants would exert their E(To) phenotypes by blocking signaling events between the oocyte and the follicular cells. However, two types of evidence suggest that both the oocyte-to-follicle cells and the follicle cells-to-oocyte signals are functional in the Rab 11 mutants. First, absence of expression of an enhancer trap in follicle cells at the posterior cap indicates that the posterior polar follicle cell fate is properly adopted. Second, a focus of microtubules at the posterior pole was never observed by Tau:GFP labeling, indicating that posterior MTOC disassembles. Consistently, mislocalization of the bcd mRNA to the posterior pole was never observed, indicating again that the back signaling from the posterior polar follicle cells is received, and the MTOC and the minus ends of the microtubules disappear from the posterior pole. However, working with hypomorphic allele combinations, we cannot exclude the possibility that mislocalization of bcd mRNA was not detected because of its relative insensitivity to the microtubule reorientation. The analysis of the hypomorphic phenotypes also supports this interpretation. We observed and showed an intermediate osk mislocalization phenotype in Rab11 mutants, when osk mRNA was detected in the center and simultaneously at the normal posterior position in the same oocyte. This indicates that in such mutant oocytes the MTOC does indeed disappear and the minus ends of the microtubules are replaced by plus ends at the posterior. Rab11 phenotypes are reminiscent of that of par1. In par1 mutant oocytes, the posterior MTOC also disappears but central osk mRNA localization is observed. We therefore conclude that even though the posterior MTOC normally disassembles, the reorientation of the oocyte microtubule network is incomplete in Rab11 mutants. We propose that instead of having reverse polarity when the microtubules nucleated predominantly from the anterior, in Rab11 mutants only a subset of microtubules, which are nucleated over the entire cortex of the oocyte, is intact (THEURKAUF and HAZELRIGG 1998 ) driving Kinesin:ß-galactosidase motor protein and osk mRNA to the center of the oocyte. In Rab11 mutants, osk mRNA mislocalization phenotype is not fully penetrant and transient, and by stage 10–11 egg chambers exhibit wild-type osk mRNA localization. We suggest that the Rab11-dependent localization pathway for osk RNA itself is redundant and the recovery observed in later stages is due to an alternative, Rab11-independent osk localization mechanism when cytoplasmic streaming, which begins at stage 10, directs the osk mRNA to the posterior pole (GLOTZER et al. 1997 ). Our results indicate that posterior MTOC breakdown may not be sufficient for reorientation of the microtubule network during stages 6–8 of the Drosophila oocyte; rather, the reorientation process depends on other factors too, like internal membrane functions. The precise mechanism by which Rab11 contributes to the microtubule reorientation is still unclear. A similar phenotype, characterized by osk mislocalization to the center of the oocyte, was observed in Ter94 mutant ovaries (RUDEN et al. 2000 ). Ter94 encodes an AAA type ATPase that is also responsible for internal membrane trafficking, namely, for homotypic fusion of endoplasmic reticulum vesicles. Rab11 and Ter94 phenotypes reveal that the internal membranous structures and the cytoskeleton of the Drosophila oocyte have a functional connection to conduct cytoplasmic mRNA localization.

	ACKNOWLEDGMENTS

We thank A. Ephrussi, D. St Johnston, I. Clark, and L. Sipos for providing cDNA clones and fly strains. We are grateful for the critical reading of the manuscript by Z. Györgypál and G. Tick. The work was supported by grant RG-356/97 from the Human Frontier Science Program (HFSP) and grant F019869 from the Hungarian National Science Foundation (OTKA). The research was largely supported by OTKA, providing a postdoctoral fellowship to M.E. This publication was supported by the Dr. Rollin D. Hotchkiss Foundation.

Manuscript received December 23, 2000; Accepted for publication April 30, 2001.

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				R. Sinka, F. Jankovics, K. Somogyi, T. Szlanka, T. Lukacsovich, and M. Erdelyi poirot, a new regulatory gene of Drosophila oskar acts at the level of the short Oskar protein isoform Development, March 9, 2003; 129(14): 3469 - 3478. [Abstract] [Full Text] [PDF]

				G. Dollar, E. Struckhoff, J. Michaud, and R. S. Cohen Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation Development, March 3, 2003; 129(2): 517 - 526. [Abstract] [Full Text] [PDF]

				J. Pellettieri and G. Seydoux Anterior-Posterior Polarity in C. elegans and Drosophila--PARallels and Differences Science, December 6, 2002; 298(5600): 1946 - 1950. [Abstract] [Full Text] [PDF]

				I. M. Palacios and D. S. Johnston Kinesin light chain-independent function of the Kinesin heavy chain in cytoplasmic streaming and posterior localisation in the Drosophila oocyte Development, January 12, 2002; 129(23): 5473 - 5485. [Abstract] [Full Text] [PDF]