Translational recruitment of maternal mRNAs is an essential process in early metazoan development. To identify genes required for this regulatory pathway, we have examined a collection of Drosophila female-sterile mutants for defects in translation of maternal mRNAs. This strategy has revealed that maternal-effect mutations in the cortex and grauzone genes impair translational activation and cytoplasmic polyadenylation of bicoid and Toll mRNAs. Cortex embryos contain a bicoid mRNA indistinguishable in amount, localization, and structure from that in wild-type embryos. However, the bicoid mRNA in cortex embryos contains a shorter than normal polyadenosine (poly(A)) tail. Injection of polyadenylated bicoid mRNA into cortex embryos allows translation, demonstrating that insufficient polyadenylation prevents endogenous bicoid mRNA translation. In contrast, nanos mRNA, which is activated by a poly(A)-independent mechanism, is translated in cortex embryos, indicating that the block in maternal mRNA activation is specific to a class of mRNAs. Cortex embryos are fertilized, but arrest at the onset of embryogenesis. Characterization of grauzone mutations indicates that the phenotype of these embryos is similar to cortex. These results identify a fundamental pathway that serves a vital role in the initiation of development.

The initiation of early development requires the presence of preexisting molecules in the maternal cytoplasm (Davidson, 1986; Evans et al., 1992; St Johnston and Nüsslein-Volhard, 1992). Posttranscriptional regulation of these maternal factors is essential to many crucial processes such as oocyte maturation, fertilization and the initial divisions of the zygote. One class of maternal molecules is dormant mRNAs that are translationally silent following synthesis, but are activated during early developmental events where they participate in oogenesis and embryogenesis. For example, translational recruitment of the c-mos proto-oncogene is required for oocyte maturation and completion of meiosis (Gebauer et al., 1994; Sheets et al., 1995). A variety of mechanisms can modulate maternal mRNA translation including RNA masking, cytoplasmic elongation of the poly(A) tail, and localization (Standart, 1992; Wickens, 1992; Gavis and Lehmann, 1994).

In Drosophila, embryonic asymmetry is established by maternal factors deposited in the early oocyte. These maternal components constitute four patterning systems: anterior, posterior, dorsoventral, and terminal (St Johnston and Nüsslein-Volhard, 1992). In each of these systems, at least one pivotal maternal mRNA is translationally regulated: bicoid (anterior), nanos (posterior), Toll (dorsoventral), and torso (terminal) (Driever and Nüsslein-Volhard, 1988; Gavis and Lehmann, 1994; Gay and Keith, 1992; Casanova and Struhl, 1989). Three of these mRNAs, bicoid, Toll and torso, are cytoplasmically polyadenylated concomitant with translational recruitment. For bicoid mRNA, elongation of the poly(A) tail has been shown directly to be necessary for translation (Sallés et al., 1994). These observations demonstrate that mRNA translational activation by cytoplasmic polyadenylation is a crucial prerequiste for establishing the Drosophila body pattern.

In this report, we have examined a set of Drosophila femalesterile mutations for defects in dormant mRNA activation. This analysis has identified two genes, cortex (cort) and grauzone (grau), that are required for both translation and cytoplasmic polyadenylation of one class of maternal mRNAs. Mutations in these genes also disrupt the onset of embryogenesis, thereby linking mRNA activation with the initiation of development.

Drosophila strains

Fly strains were generously provided as follows: (1) EMS-induced second chromosome maternal-effect mutations were from T. Schüpbach (Princeton University). Mutant chromosomes contained cn, bw, and were balanced by CyO513 (Schüpbach and Wieschaus, 1989). From this collection, we obtained two alleles of cortex, cortQW55 and cortRH65, and three alleles of grauzone, grauQQ36, grauRG1 and grauRM61. (2) Two deficiencies that delete Toll, Df(3R)Tl 9QRX, and Df(3R)ro XB3, were from C. Hashimoto (Yale University), and balanced by TM1. The Toll gene is physically absent in Df(3R)Tl 9QRX / Df(3R)ro XB3 flies (Anderson et al., 1985). (3) A strain carrying the dominant female-sterile gene, P[ovoD1], on the second chromosome was from J. Duffy and N. Perrimon (Harvard University). (4) The Df(2L)chiffon 64/Cyo strain was from J. Tower (UCLA). Balancer chromosomes and marker mutations are described by Lindsley and Zimm (1992). The wild-type strains were either Canton-S or homozygous for mutations in the yellow and white genes.

Whole-mount antibody stains

Embryos (0–3 hour) were dechorionated, permeabilized, fixed, devitellinized and stained (Kania et al., 1990). Fixed embryos were rehydrated in phosphate-buffered saline (PBS) and blocked with 2% normal goat serum (Vector) for 30 minutes at room temperature. Embryos were incubated with either monoclonal anti-bicoid antibodies (line 733.3) or rabbit anti-nanos antibodies (gift from E. Gavis and C. Wang) in a solution containing PBS, 1% BSA, and 0.1% Tween 80 overnight at 4°C. After 12 washes in PBS, 1% BSA, 0.1% Tween 80, preabsorbed secondary antibodies conjugated to biotin (Vector) were added in the same solution and incubated for 2 hours at room temperature. Following 12 additional washes in PBS, 01% Tween 80, antibody/antigen complexes were detected by the HRP Vectastain Elite kit (Vector) (Kania et al., 1990). Embryos were dehydrated in 100% ethanol, cleared with Histoclear (National Diagnostics), mounted in Permount (Fisher), and visualized with a Zeiss microscope using Nomarski optics. The following second chromosome mutations did not interfere with bicoid protein expression: bientotSD06, earlyQP71, fruhHM21, mat(2)early QA26, mat(2)early QD68, mat(2)early QM47, mat(2)early RL4, mat(2)syn PL63, mat(2)early RU28, remnantsHG24, prestoPL10. A description of these genes can be found in Schüpbach and Wieschaus (1989) and Lindsley and Zimm (1992).

Prior to use, monoclonal anti-bicoid antibodies were concentrated by ammonium sulfate precipitation (Harlow and Lane, 1988). Antibicoid and anti-nanos antibodies were preabsorbed against either aged (>4 hour) embryos or 0to 3-hour embryos, respectively.

In situ hybridization

Drosophila embryos 0to 3-hours old were prepared, and hybridized with a digoxigenin-labeled antisense bicoid probe according to the method of Klingler and Gergen (1993). The bicoid RNA probe (Tsai and Gergen, 1994) was a generous gift from C. Tsai.

Poly(A) tests and assessment of bicoid mRNA structure

For poly(A) tests (PAT) of bicoid, Toll, and torso, oocyte RNA was isolated from 2-week old wild-type, cortQW55/cortRH65, and grauQQ36/grauRG1 females as described for mouse oocytes (Huarte, 1987). Embryos were collected at half hour intervals, aged appropriately, and total RNA was extracted as above. Total RNA from both oocytes and embryos was treated with RNAase free DNAase (Promega) in 1× Superscript buffer (Gibco-BRL) for 15 minutes at 37°C. RNAs were phenol extracted, ethanol precipitated, and resuspended in 20 μl DEPC-treated H2O. 1 μl of RNA from each sample was subjected to PAT cDNA synthesis with oligo(dT) followed by PCR amplification with a message specific primer and Primer T (Sallés and Strickland, 1995). PCR products were resolved in 6% denaturing polyacrylamide gels. Since the amount of RNA for each time point was not determined, quantitative estimates cannot be made. Message specific primers were as follows:

bicoid, 5′CATTTGCGCATTCTTTGACC3′;

Toll, 5′GTATCAACTGTAATCTCACGCCCA3′;

torso, 5′CCAGAAAGGCTGAAACAACTGCAAG3′.

To assess the poly(A) tail lengths of mRNAs in early oocytes (stage 12 or before), ovaries from 3-day old wild-type and cortQW55/cortRH65 females were fractionated by the method of Theurkauf (1992). Total RNA was isolated and subjected to PAT cDNA synthesis as described in Sallés and Strickland (1995).

To examine bicoid mRNA structure, 0to 0.5-hour cDNAs from wild-type, cortQW55/cortRH65 and grauQQ36/grauRG1 embryos (prepared as above) were PCR amplified with two bicoid specific primer pairs as follows:

No. 1 sense, 5′CGAAGCAGTGGATCGCAA3′;

No. 1 antisense, 5′GCTGGAAGTCAAAGTGATGGT3′; No. 2 sense, 5′AATCGGATCAGCACAAGGAC3′;

No. 2 antisense, 5′CCCGAGTAGAGTAGTTCTTATATATTTTCGTAATTAAAAATACAAAGC3′.

A 37 cycle PCR amplification was performed for both pairs at: 45 minutes at 93°C, 1 hour at 60°C, 3 hour at 72°C for pair no. 1 (4.5 hours at 72°C for pair no. 2), with a final extension of 7 hours at 72°C. PCR products generated with primer pairs no. 1 and no. 2 correspond to nucleotides 4-1058 and 618-2455 of the 2455 nucleotide bicoid cDNA, respectively (Berleth et al., 1988).

Injection of bicoid mRNA into cortex embryos

Capped wild-type bicoid transcripts labeled with [α-P32]UTP were synthesized from NotI-linearized pBCDwt (Sallés et al., 1994) using a T3 message machine kit (Ambion) (specific activity 2.5×104 cpm/μg). After purification, transcripts were resuspended in filtered H2O at 250 ng/μl. 10 μg of resuspended bicoid mRNA was in vitro polyadenylated with 3 units of poly(A) polymerase (Gibco-BRL) and 300 μM ATP for 30 minutes at 37°C. Polyadenylated mRNAs were purified by one round of isopropanol precipitation followed by an ethanol precipitation and resuspended in filtered H2O at 250 ng/μl. To confirm integrity and polyadenylation of the transcripts, mRNAs were resolved in formaldehyde-agarose gels and transferred to Duralon membranes (Stratagene). The extent of polyadenylation was estimated from DNA size markers resolved in parallel. Polyadenylated and nonpolyadenylated bicoid mRNAs were injected into 0.5to 1.0-hour old cortQW55/cortRH65 embryos. Embryos were aged an hour, collected, and fixed in 3.7% formaldehyde, PBS for 15 minutes. Vitelline membranes were removed by rolling fixed embryos between a coverslip and a frosted glass slide, and stored at −20°C in methanol (Theurkauf et al., 1992). Translation of injected bicoid mRNAs was assessed by staining with an anti-bicoid antibody in situ (see above). For these whole-mount experiments, trace numbers of uninjected 1.5to 4.0-hour old wild-type embryos were added to all assay wells to serve as internal standards for production of bicoid protein.

Western blot analysis

Extracts from 0to 3-hour old wild-type, cortQW55/cortRH65, and Df(3R)Tl 9QRX/Df(3R)ro XB3 embryos were prepared (Driever and Nüsslein-Volhard, 1988). Embryos were dechorionated in chilled 50% bleach, washed in 10 mM Tris (pH 7.4), 300 mM NaCl, 0.1% Triton X-100, and allowed to settle to determine volume (20-40 μl). After freezing with liquid nitrogen, embryos were homogenized with 3 volumes of 2× sample buffer, 6 M urea. Extracts were incubated for 5 minutes at 95°C, clarified by low-speed centrifugation at 4°C, and stored at −80°C. 1 μl of each extract was resolved by SDS-PAGE in a 7% polyacrylamide gel and electroblotted to nitrocellulose. The efficiency of electroblotting was assessed by the transfer of prestained molecular mass markers (BioRad). The blot was blocked in trisbuffered saline (TBS), 5% non-dry fat milk (Carnation) and incubated with polyclonal anti-Toll antibodies (a gift from C. Hashimoto) diluted in TBS, 0.1% Tween 20 for 1 hour at room temperature. After one 10-minute and 3 five-minute washes in TBS, 0.1% Tween 20, the blot was probed with goat anti-rabbit antibodies conjugated to HRP (Life Sciences) for 1 hour at room temperature. Antibody/antigen complexes were detected by ECL according to the manufacturer’s protocol (Amersham). Total protein on the blot was visualized by incubation with 5% India ink in PBS and by staining extracts resolved in parallel with Coomassie blue (Harlow and Lane, 1988).

Generation of cortex germ-line mosaics

To test for a germ-line requirement of cortex, 320 virgin cortRH65cn bw/SM6a females were mated with 60 yw/Y ; P[ovoD1]/CyO males and the F1 larvae were irradiated with 1000 rads using a cesium-137 source. After eclosion, approximately 1400 virgin cortRH65cn bw/P[ovoD1] females were isolated, and mated with cn bw males. Embryos were collected on apple juice agar plates for 3 hours, aged an additional hour, and transferred to microscope slides where they were placed under halocarbon oil. Embryonic morphology was assessed with a Nikon microscope. Overnight collections were also performed upon irradiated cortex/P[ovoD1] females yielding 203 embryos arresting with a cortex phenotype and 100 undergoing vacuolar degeneration.

Cellular analysis

0to 2-hour wild-type and cortQW55/cortRH65 embryos were collected, fixed and examined according to the method of Theurkauf (1992). To examine the microtubules, embryos were incubated with a monoclonal anti-α-tubulin antibody (DMA 1, Sigma) in PBS, 0.05% Triton X-100 (PBST), washed in PBST, and then with secondary antibodies conjugated to fluorescein (Boehringer Mannheim) in PBST. Secondary antibodies were preabsorbed against wild-type embryos overnight at 4°C. To visualize sperm tails, cortex embryos were incubated 1:1 with a monoclonal anti-sperm tail antibody (Karr, 1991) in PBST and then with secondary antibodies as above. Chromosomes were visualized by staining embryos with either propidium iodide (1 μg/ml) or incubating with a monoclonal anti-histone antibody (ChemiCon) and then with fluorescein-conjugated secondary antibodies. Propidium iodide and the monoclonal anti-α-tubulin antibody were used to double stain for microtubules and chromosomes. Embryos were resuspended in a mounting medium of 1× PBS, 90% glycerol, and 1mg/ml p-phenylene diamine, and visualized with a BioRad MRC 600 laser scanning confocal attachment and a Nikon Diaphot inverted microscope.

Temperature-shift experiments

Embryos from cortQW55/Df(2L)chiffon 64 females reared at 18°C were collected up to 6 hours, aged an hour and dechorionated. Embryos were devitellinized and fixed by agitation in a heptane/methanol solution and stored in methanol at −20°C. To examine if cortex is required during early embryogenesis, embryos from cortQW55/Df(2L)chiffon 64 females were collected at half-hour intervals at 18°C and shifted to 29°C on prewarmed apple juice agar plates. Transferring these collections to 29°C was accomplished in approx. 2-3 minutes. After incubation at 29°C, the cortQW55/Df(2L)chiffon 64 embryos ranged in age from 1.5 hours to 3.5 hours and were processed as described above. To test when cortex is required during oogenesis, cortQW55/Df(2L)chiffon 64 females were shifted to 29°C and embryos were collected at 3-hour intervals over the next 53 hours. Each collection of embryos was aged an additional hour and processed as described above. Analogous temperature shift experiments were also performed with cortQW55/Cyo and Df(2L)chiffon 64/Cyo embryos and females to serve as controls.

Mitotic cycling in all embryos was assessed by examining the number of nuclei. Embryos were rehydrated in PBST, and incubated with 4,6-diamino-2-phenylindole (DAPI) for a half-hour at room temperature. After 5 washes in PBST embryos were mounted in 1× PBS, 90% glycerol, and 1mg/ml p-phenylene diamine, and scored by fluorescence microscopy.

The cortex and grauzone genes are necessary for bicoid protein expression

Mutations in genes required for translational activation of maternal mRNAs should have profound effects on early embryonic development by impairing production of an entire class of proteins. Therefore, mutations affecting this activation pathway might produce a maternal-effect lethal phenotype by disrupting early developmental processes such as completion of meiosis, oocyte maturation, or the initial cleavage divisions of the zygote. To identify genes that participate in this process, we examined a collection of female-sterile mutations for defects in dormant mRNA activation.

Since bicoid mRNA is translationally activated during early embryogenesis (Driever and Nüsslein-Volhard, 1988), expression of bicoid protein can serve as a marker for maternal mRNA activation. We therefore tested embryos from second chromosome maternal-effect mutants that arrest early in embryogenesis (Class I and II, Schüpbach and Wieschaus, 1989) for production of bicoid protein by immunostaining. Bicoid protein is normally produced during the first hour of embryogenesis from an anteriorly localized mRNA, generating an anterior-to-posterior concentration gradient that guides head and thorax formation (Fig. 1A,C; Frohnhöfer and Nüsslein-Volhard, 1986; Driever and Nüsslein-Volhard, 1988). Mutations in eleven different female-sterile loci did not impair formation of the bicoid protein gradient, with at least 90% of embryos producing a strong bicoid expression pattern (mutant loci listed in Materials and Methods).

Fig. 1.

Bicoid protein expression is reduced in cortex and grauzone embryos. Collections of 0to 3-hour old wild-type (A,C), cortQW55/cortRH65 (B) and grauQQ36/grauRM61 (D) embryos were stained for production of bicoid protein with a monoclonal antibicoid antibody. The wild-type embryos (A and C) were positive controls of independent immunostainings of cortex and grauzone, respectively.

Fig. 1.

Bicoid protein expression is reduced in cortex and grauzone embryos. Collections of 0to 3-hour old wild-type (A,C), cortQW55/cortRH65 (B) and grauQQ36/grauRM61 (D) embryos were stained for production of bicoid protein with a monoclonal antibicoid antibody. The wild-type embryos (A and C) were positive controls of independent immunostainings of cortex and grauzone, respectively.

Mutant embryos from homozygous cortQW55/cortQW55 females, however, exhibited a profound reduction in bicoid protein expression. In approximately 90% of these embryos, bicoid protein was undetectable, with the remaining 10% expressing the protein at levels slightly above background. An identical reduction in bicoid protein was observed with females trans-heterozygous for two independently isolated cortex alleles, cortQW55/cortRH65, indicating that this biochemical phenotype is due to mutation of a single genetic locus (Fig. 1A,B). Mutant embryos from grauQQ36/grauRM61 females also exhibited a reduction in bicoid protein expression when compared to wild-type (Fig. 1C,D). Although significantly lower than wild-type, grauzone embryos appear to express slightly more bicoid protein than cortex. We therefore focused our analysis on the cortex locus.

Mutations in cortex impair bicoid mRNA translation by disrupting cytoplasmic polyadenylation

The reduction of bicoid protein in cortex embryos could be due to defects in transcription, mRNA processing, message localization, or translation. To address these possibilities, bicoid mRNA was visualized by in situ hybridization with an antisense probe. By this assay, both wild-type and cortex embryos contained similar amounts of bicoid mRNA that was localized to the anterior pole (Fig. 2A, B). These observations indicate that bicoid transcription and localization are not grossly affected by mutations in cortex. To assay for alterations in message processing and splicing, cDNAs were prepared from both wild-type and cortex embryos, and the entire bicoid message examined by RT-PCR using two specific primer pairs. No migration differences were detected between the PCR products generated from wild-type and cortex cDNAs (Fig. 2C). Restriction enzyme digestion confirmed that the amplified DNAs were bicoid specific (data not shown). Taken together, these data indicate that cortex embryos contain properly localized and processed bicoid mRNA, and imply that this mutation impairs translational activation.

Fig. 2.

Mutations in cortex do not interfere with bicoid mRNA expression, localization, or processing. Spatial localization of bicoid mRNA in wild-type (A) and cortQW55/cortRH65 (B) embryos was detected with an antisense probe by in situ hybridization. A schematic diagram of bicoid mRNA is presented with darkened lines representing the 5′ and 3′ UTRs (C). The approximate size of bicoid PCR products amplified with primer sets no. 1 and no. 2 are indicated. PCR amplification of wild-type and cortQW55/cortRH65 cDNAs with bicoid specific primer pairs no. 1 and no. 2 is shown. The expected size of the bicoid PCR products is 1054 nucleotides and 1837 bases, using primer sets no. 1 and no. 2, respectively. The PCR products were resolved in a 1.5% agarose gel along with indicated DNA markers.

Fig. 2.

Mutations in cortex do not interfere with bicoid mRNA expression, localization, or processing. Spatial localization of bicoid mRNA in wild-type (A) and cortQW55/cortRH65 (B) embryos was detected with an antisense probe by in situ hybridization. A schematic diagram of bicoid mRNA is presented with darkened lines representing the 5′ and 3′ UTRs (C). The approximate size of bicoid PCR products amplified with primer sets no. 1 and no. 2 are indicated. PCR amplification of wild-type and cortQW55/cortRH65 cDNAs with bicoid specific primer pairs no. 1 and no. 2 is shown. The expected size of the bicoid PCR products is 1054 nucleotides and 1837 bases, using primer sets no. 1 and no. 2, respectively. The PCR products were resolved in a 1.5% agarose gel along with indicated DNA markers.

Cytoplasmic polyadenylation regulates the translation of maternal mRNAs in mouse, Xenopus and Drosophila (Huarte et al., 1989; Simon et al., 1992; Sheets et al., 1994; Wharton and Struhl, 1991; Sallés et al., 1994). In these systems, dormant maternal mRNAs are synthesized during oogenesis and contain short poly(A) tails. During either oocyte meiotic maturation or early embryogenesis, these mRNAs are further polyadenylated, resulting in translational activation. In the fly, elongation of the poly(A) tail is necessary for bicoid mRNA translational activation (Sallés et al., 1994). To examine if bicoid mRNA polyadenylation was altered in the mutant embryos, wild-type and cortex RNAs collected from whole ovaries and from embryos aged at half-hour intervals were subjected to a PCR poly(A) test (PAT) analysis (Sallés and Strickland, 1995). In wild-type embryos during the first 1.5 hours of development, bicoid mRNA acquires a poly(A) tail length extending to approx. 140 nucleotides concomitant with translational activation (Fig. 3A). In cortex embryos, bicoid mRNA is maximally elongated only to approx. 80 nucleotides by 1.5 hours of embryo development (Fig. 3B). These results indicate that cortex is required for the proper cytoplasmic polyadenylation of bicoid mRNA. In grauzone, bicoid mRNA is also present, and processed normally (data not shown), but its cytoplasmic polyadenylation is impaired (Fig. 3C). These results suggest that mutations in grauzone, like cortex, disrupt bicoid mRNA translational activation and cytoplasmic polyadenylation.

Fig. 3.

Disruption of bicoid mRNA cytoplasmic polyadenylation impairs translational activation in cortex embryos. RNAs from wild-type, cortQW55/cortRH65 and grauQQ36/grauRG1 ovaries and embryos aged at half-hour intervals were isolated and subjected to PAT cDNA synthesis (Sallés and Strickland, 1995). The poly(A) status of bicoid mRNA in wild-type (A), cortex (B) and grauzone (C) embryos was assessed by amplification with a message specific primer and Primer T. By 0.5-1.0 hours, bicoid mRNA was elongated to a maximum length of 140 nucleotides in wild-type and 80 nucleotides in cortex. The size of bicoid amplification products without a poly(A) tail was 286 nucleotides (256 bases of bicoid cDNA plus 30 nucleotides of Primer T). Tail lengths were estimated from DNA size standards resolved in parallel. Cortex embryos were injected with bicoid mRNA at 250 ng/μl containing either no poly(A) tail (D) or a poly(A) tail of 180 As (E). After 1 hour of aging, injected embryos were dechorionated, fixed, devitellinized and assayed for production of bicoid protein with a monoclonal antibody. For both D and E, collections of uninjected 1.5to 4.0-hour old wild-type embryos were added to the assay wells to serve as an internal standard for bicoid protein. Wild-type embryos were distinguished from injected cortex embryos using morphological criteria. Mutant embryos injected with bicoid mRNA containing no poly(A) tail expressed bicoid protein (C), but at levels significantly lower than uninjected 1.5to 4.0-hour old wild-type embryos (data not shown). When this bicoid mRNA was polyadenylated with 180 As in vitro and injected into cortex embryos at the same concentration, the injected embryos expressed bicoid protein (E) at levels comparable to the wild-type embryos included in the assay well (data not shown).

Fig. 3.

Disruption of bicoid mRNA cytoplasmic polyadenylation impairs translational activation in cortex embryos. RNAs from wild-type, cortQW55/cortRH65 and grauQQ36/grauRG1 ovaries and embryos aged at half-hour intervals were isolated and subjected to PAT cDNA synthesis (Sallés and Strickland, 1995). The poly(A) status of bicoid mRNA in wild-type (A), cortex (B) and grauzone (C) embryos was assessed by amplification with a message specific primer and Primer T. By 0.5-1.0 hours, bicoid mRNA was elongated to a maximum length of 140 nucleotides in wild-type and 80 nucleotides in cortex. The size of bicoid amplification products without a poly(A) tail was 286 nucleotides (256 bases of bicoid cDNA plus 30 nucleotides of Primer T). Tail lengths were estimated from DNA size standards resolved in parallel. Cortex embryos were injected with bicoid mRNA at 250 ng/μl containing either no poly(A) tail (D) or a poly(A) tail of 180 As (E). After 1 hour of aging, injected embryos were dechorionated, fixed, devitellinized and assayed for production of bicoid protein with a monoclonal antibody. For both D and E, collections of uninjected 1.5to 4.0-hour old wild-type embryos were added to the assay wells to serve as an internal standard for bicoid protein. Wild-type embryos were distinguished from injected cortex embryos using morphological criteria. Mutant embryos injected with bicoid mRNA containing no poly(A) tail expressed bicoid protein (C), but at levels significantly lower than uninjected 1.5to 4.0-hour old wild-type embryos (data not shown). When this bicoid mRNA was polyadenylated with 180 As in vitro and injected into cortex embryos at the same concentration, the injected embryos expressed bicoid protein (E) at levels comparable to the wild-type embryos included in the assay well (data not shown).

Although the reduction in poly(A) tail length in cortex embryos appears modest, a truncated bicoid RNA with 50 As is not translated, whereas the same RNA with 175 As is translated (Sallés et al., 1994). Taken together, these results suggest that defective translational activation of bicoid mRNA in cortex embryos might be due to impaired cytoplasmic polyadenylation. To test this prediction, we injected cortex embryos with bicoid mRNA that contained either no poly(A) or a poly(A) tail of approx. 180 As. Expression of these transcripts was assessed by staining injected cortex embryos with anti-bicoid antibodies and comparing protein production with that in wild-type uninjected embryos.

Translation of bicoid mRNA without a poly(A) tail was severely reduced in injected cortex embryos (Fig. 3D). If the lack of translation of injected poly(A)-minus bicoid mRNA in cortex embryo was due to disruption of polyadenylation, then providing a poly(A) tail should bypass the block. Indeed, polyadenylated bicoid mRNA was effectively translated when injected into cortex embryos (Fig. 3E). Since providing a polyadenylated bicoid mRNA circumvents the translational block, these results indicate that the failure to translate endogenous bicoid mRNA in cortex embryos is due an insufficiently long poly(A) tail, suggesting that this mutation disrupts the polyadenylation machinery.

Cytoplasmic polyadenylation and translation of maternal patterning mRNAs is perturbed in cortex embryos

Embryos that contain no maternal bicoid protein proceed through the syncytial blastoderm divisions and develop extensively, but do not form head and thorax structures (Frohnhöfer and Nüsslein-Volhard, 1986). Since cortex embryos do not develop to the cellular blastoderm stage (Schüpbach and Wieschaus, 1989), their phenotype cannot be explained solely by an inability to translate bicoid mRNA. However, cortex might be required for cytoplasmic polyadenylation and translation of a class of mRNAs, and the absence of these maternal proteins could produce the mutant phenotype. To investigate this possibility, we examined the translational activation of Toll mRNA by resolving total protein from wild-type, cortexand Toll embryo extracts and by probing with anti-Toll antibodies. A Toll-specific band of approximately 145×103Mr was detected in the wild-type but not in the Toll extract (Fig. 4A; Hashimoto et al., 1991). Toll protein expression in the cortex extract was reduced as compared to wild-type. All three extracts contained comparable levels of total protein as determined by Coomassie blue staining of embryo extracts resolved in parallel (Fig. 4B) and India ink staining of the blot (data not shown). Since Toll mRNA is cytoplasmically polyadenylated concomitant with protein production (Sallés et al., 1994), we tested if the reduction of Toll protein in cortex embryos was also accompanied by a disruption of polyadenylation. Within 1.5 hours of embryonic development, wild-type embryos elongate Toll mRNA to a length of approx. 250 As, whereas cortex embryos lengthen this message only to approx. 150 nucleotides (Fig. 4C,D). These data on Toll mRNA and protein, in conjunction with those on bicoid, demonstrate that cortex is required for both proper cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs.

Fig. 4.

Poly(A) tail elongation and translational activation of Toll mRNAs is perturbed in cortex embryos. Translational recruitment of Toll mRNA was assessed in wild-type, cortex and Toll 0to 3-hour old embryo extracts (A). Protein extracts from each genotype were prepared (see Materials and methods) and resolved by SDS-PAGE. After electroblotting to nitrocellulose, Toll protein was detected by probing the membrane with polyclonal anti-Toll antibodies and visualized using ECL (Amersham). A prominent Toll-specific band was detected in wild-type but not in the Toll extracts and migrated as a species similar to those reported elsewhwere (Hashimoto et al., 1991). Toll protein expression in the cortex extract appeared reduced as compared to wild-type extracts. Identical extracts were also resolved in parallel with (A) and were stained with Coomassie blue (B). PAT analysis of Toll mRNA was performed upon wild-type (C) and cortQW55/cortRH65 (D) ovaries and embryos aged at half-hour intervals. By 0.5-1.0 hours of embryogenesis, Toll mRNA was elongated to a maximum length of 250 bases in wild-type and 150 nucleotides in cortex. The size of Toll amplification products without poly(A) tails was 335 nucleotides (305 nt. of Toll cDNA plus 30 bases of Primer T).

Fig. 4.

Poly(A) tail elongation and translational activation of Toll mRNAs is perturbed in cortex embryos. Translational recruitment of Toll mRNA was assessed in wild-type, cortex and Toll 0to 3-hour old embryo extracts (A). Protein extracts from each genotype were prepared (see Materials and methods) and resolved by SDS-PAGE. After electroblotting to nitrocellulose, Toll protein was detected by probing the membrane with polyclonal anti-Toll antibodies and visualized using ECL (Amersham). A prominent Toll-specific band was detected in wild-type but not in the Toll extracts and migrated as a species similar to those reported elsewhwere (Hashimoto et al., 1991). Toll protein expression in the cortex extract appeared reduced as compared to wild-type extracts. Identical extracts were also resolved in parallel with (A) and were stained with Coomassie blue (B). PAT analysis of Toll mRNA was performed upon wild-type (C) and cortQW55/cortRH65 (D) ovaries and embryos aged at half-hour intervals. By 0.5-1.0 hours of embryogenesis, Toll mRNA was elongated to a maximum length of 250 bases in wild-type and 150 nucleotides in cortex. The size of Toll amplification products without poly(A) tails was 335 nucleotides (305 nt. of Toll cDNA plus 30 bases of Primer T).

To determine if perturbations in the poly(A) status of maternal mRNAs are a general feature of cortex embryos, we examined the poly(A) tail of torso mRNA which is also cytoplasmically polyadenylated during translational recruitment (Sallés et al., 1994). By the first half hour of development, wild-type embryos lengthen torso mRNA to approx. 120 nucleotides, and it is then apparently deadenylated by 3 hours after egg deposition (Fig. 5A). Cortex embryos also show a difference in poly(A) status with torso mRNA, which is deadenylated earlier than in wild-type embryos, between 0.5 and 1.0 hour after egg deposition (Fig. 5B). Combined, these results show that the regulation of poly(A) status of bicoid, Toll and torso mRNAs is perturbed in cortex embryos, demonstrating that this gene affects cytoplasmic polyadenylation.

Fig. 5.

The poly(A) profile of the torso mRNA is altered in cortex embryos. The poly(A) status of torso mRNA was assessed in wildtype (A) and cortQW55/cortRH65 (B) ovaries and embryos using PCR PAT analysis. By 0.0–0.5 hours, wild-type and cortex embryos lengthened torso mRNA to a similar length. In 2.0– to 2.5-hour old wild-type embryos, an apparent deadenylation of torso mRNA was observed. This removal of torso poly(A) began precociously in cortex embryos at 0.5–1.0 hours. The size of torso amplification products without poly(A) tails was 279 nucleotides (249 nt of torso cDNA plus 30 bases of Primer T). Tail lengths were estimated from DNA molecular size markers resolved in parallel.

Fig. 5.

The poly(A) profile of the torso mRNA is altered in cortex embryos. The poly(A) status of torso mRNA was assessed in wildtype (A) and cortQW55/cortRH65 (B) ovaries and embryos using PCR PAT analysis. By 0.0–0.5 hours, wild-type and cortex embryos lengthened torso mRNA to a similar length. In 2.0– to 2.5-hour old wild-type embryos, an apparent deadenylation of torso mRNA was observed. This removal of torso poly(A) began precociously in cortex embryos at 0.5–1.0 hours. The size of torso amplification products without poly(A) tails was 279 nucleotides (249 nt of torso cDNA plus 30 bases of Primer T). Tail lengths were estimated from DNA molecular size markers resolved in parallel.

Mutations in cortex do not block poly(A)independent translational activation of nanos mRNA

Although mutations in cortex and grauzone disrupt expression of bicoid protein, this phenotype could reflect a generalized defect in translation. One translationally regulated mRNA, nanos, is localized to the posterior pole of the embryo (Gavis and Lehmann, 1994; Wang et al., 1994), and is not cytoplasmically polyadenylated concomitant with protein production (Sallés et al., 1994). Therefore, translational activation of this localized mRNA occurs by a polyadenylation-independent pathway, and generates a posterior-anterior protein concentration gradient (Fig. 6A,C; Barker et al., 1992). To determine if cortex and grauzone embryos translate this mRNA, mutant embryos were stained with a polyclonal anti-nanos antibody. Cortex and grauzone embryos produce nanos protein in a pattern indistinguishable from wild-type (Fig. 6B,D). These results indicate that these genes do not affect translational recruitment of all mRNAs, and suggest that the translational defect in the mutant embryos is specific to mRNAs that are cytoplasmically polyadenylated.

Fig. 6.

Cortex and grauzone embryos translate nanos mRNA. Nanos protein expression was assessed in wild-type (A,C), in cortQW55/cortQW55 (B), and in grauQQ36/grauRM61 (D) embryos by whole-mount staining with a polyclonal antinanos antibody. Nanos protein expression appeared indistinguishable between wild-type, cortex and grauzone embryos. Identical results were obtained with cortex trans-heterozygous embryos.

Fig. 6.

Cortex and grauzone embryos translate nanos mRNA. Nanos protein expression was assessed in wild-type (A,C), in cortQW55/cortQW55 (B), and in grauQQ36/grauRM61 (D) embryos by whole-mount staining with a polyclonal antinanos antibody. Nanos protein expression appeared indistinguishable between wild-type, cortex and grauzone embryos. Identical results were obtained with cortex trans-heterozygous embryos.

The cortex gene is required for initiation of development

Since mRNA activation is required for critical processes in development and is impaired by mutations in cortex, we further examined the cortex phenotype by determining the number of nuclei and the microtubule organization in mutant embryos. The microtubules and chromosomes were visualized with confocal microscopy after staining with anti-α-tubulin and anti-histone antibodies, respectively. There was a complete absence of microtubule-containing spindles throughout the central regions of mutant embryos (data not shown). This finding indicates that cortex embryos do not initiate the mitotic divisions, and suggests that meiosis may be affected. In early wild-type embryos, completion of meiosis is heralded by formation of one or two microtubule-associated polar bodies (Fig. 7A,B). In no instances were pole bodies detected in cortex embryos. However, single and double spindles were observed at equal frequency in the anterodorsal portion of the embryos (Fig. 7C), the region where female meiosis occurs. Chromosomes were present on these spindles with some at the metaphase plate and others in an anaphase configuration (Fig. 7D). The presence of one or two spindles located anterodorsally in the embryos and the absence of polar bodies indicate that mutations in cortex prevent the completion of meiosis. The differing stages of meiotic arrest may be due to residual cortex activity, or alternatively, a null mutation at the cortex locus might produce a pleiotropic phenotype. Examination of grauzone embryos reveals a block in meiosis similar to that observed in cortex embryos (data not shown). Therefore, both cortex and grauzone are required for completion of oocyte meiosis.

Fig. 7.

Mutations in cortex disrupt meiosis and alter microtubule reorganization. Wild-type and cortQW55/cortRH65 embryos were double stained for microtubules (A and C, respectively) and for chromosomes (B, D, respectively) and visualized by confocal microscopy. Oocyte activation and fertilization is normally accompanied by polar body formation and the disassembly of a dense array of cytoplasmic microtubules that fills the mature oocyte. As a result, very early embryos do not contain free cytoplasmic microtubules (A), but contain one or two microtubule-associated polar bodies (B). In cortex embryos, by contrast, polar bodies are not formed and chromosomes are found on one or two spindles (C,D). In addition, these mutant embryos are filled with cytoplasmic microtubules (compare C and A).

Fig. 7.

Mutations in cortex disrupt meiosis and alter microtubule reorganization. Wild-type and cortQW55/cortRH65 embryos were double stained for microtubules (A and C, respectively) and for chromosomes (B, D, respectively) and visualized by confocal microscopy. Oocyte activation and fertilization is normally accompanied by polar body formation and the disassembly of a dense array of cytoplasmic microtubules that fills the mature oocyte. As a result, very early embryos do not contain free cytoplasmic microtubules (A), but contain one or two microtubule-associated polar bodies (B). In cortex embryos, by contrast, polar bodies are not formed and chromosomes are found on one or two spindles (C,D). In addition, these mutant embryos are filled with cytoplasmic microtubules (compare C and A).

Unfertilized eggs have previously been shown to complete meiosis (Doane, 1960). Nevertheless, to determine if a defect in oocyte fertilization could be a component of the cortex phenotype, we stained mutant embryos with a monoclonal antisperm tail antibody (Karr, 1991). Sperm tails were present in cortex embryos, indicating that fertilization is unimpaired (data not shown).

At the end of Drosophila oogenesis, wild-type stage 14 oocytes contain a microtubule network that is composed of short, randomly-oriented fibers (Theurkauf et al., 1992). After oocyte activation, this microtubule cytoskeleton rapidly disassembles (Fig. 7A). In cortex embryos, the microtubule network does not disassemble, and resembles a stage 14 oocyte (Fig. 7C). These results, along with an arrest in meiosis, indicate that mutations in cortex disrupt events surrounding the onset of embryogenesis.

Cortex functions in the germ-line at multiple stages of development

Although translational impairment in the mutant embryos appears specific to an entire class of mRNAs, this phenotype could be an indirect developmental defect. If the cortex gene product serves a direct role, then the gene should be required in germ cells. To examine this possibility, we generated flies deficient in cortex only in germ cells, using mitotic recombination (Wieschaus, 1980; Perrimon and Gans, 1983) with a dominant P[ovoD1] mutation present on the second chromosome (Chou et al., 1993). Irradiated females that carry one P[ovoD1] and one cortex second chromosome can only produce oocytes if the P[ovoD1] mutation has been lost by mitotic recombination, thereby generating germ-line clones that are homozygous for cortex. Collections of 1to 4-hour old embryos from irradiated P[ovoD1]/cortex females were analyzed by light microscopy. All 50 of the embryos did not develop, with 49 containing rounded anterior poles and cytoplasmic clearings surrounding the entire periphery. These morphological features are distinctive of the cortex mutant phenotype (Schüpbach and Wieschaus, 1989; M. Lieberfarb, unpublished observations). The remaining embryo arrested as presyncytial blastoderm. Non-irradiated control P[ovoD1]/ cortex females did not produce any embryos. These results indicate that cortex is required in the germ-line, and suggest that mutation of the cortex gene product in germ cells impairs polyadenylation and mRNA translation.

To investigate when in development cortex is required, we took advantage of a deficiency, Df(2L)chiffon 64 (Tower et al., 1993), that behaves as a temperature-sensitive allele of cortex (M. Lieberfarb, unpublished observations). At 18°C, 80% of Df(2L)chiffon 64/cortQW55 embryos develop normally (Table 1, row 1), whereas at 29°C no embryos complete development. Therefore, Df(2L)chiffon 64/cortQW55 embryos or females were shifted to the non-permissive temperature at various times and embryonic development assessed by staining DNA with DAPI.

Table 1.

Determination of the temporal requirement for cortex during oogenesis and embryogenesis using a temperaturesensitive allele

Determination of the temporal requirement for cortex during oogenesis and embryogenesis using a temperaturesensitive allele
Determination of the temporal requirement for cortex during oogenesis and embryogenesis using a temperaturesensitive allele

Development decreased from 80% to 42% when embryos were transferred to the restrictive temperature (Table 1, row 2), suggesting a requirement for cortex during early embryogenesis. Development was further decreased to approx. 4% when females were maintained at 29°C for 3 hours, indicating that cortex is required late in oogenesis. After 36 hours at the restrictive temperature, development decreased further from approx. 4% to 0.7%, suggesting that cortex may also be necessary during mid-oogenesis.

cortex perturbs the poly(A) status of torso mRNA during mid-oogenesis

Previous studies in mouse have shown that maternal mRNAs regulated by cytoplasmic polyadenylation are synthesized with a long poly(A) tail that is shortened following exit from the growing oocyte nucleus (Huarte et al., 1992). Regulation of maternal mRNA poly(A) tail length thus begins before during early oogenesis. Cortex could therefore act during this initial modification of the poly(A) tail. We examined the poly(A) status of bicoid, Toll, and torso mRNAs in cortex oocytes during mid-oogenesis. RNAs from wild-type and cortex oocytes before stage 13 were isolated and subjected to PCR PAT analysis. The poly(A) tails lengths of bicoid and Toll mRNA in cortex oocytes were 60 and 90 nucleotides, respectively, and appeared identical to wild-type (data not shown). However, torso mRNA contained a poly(A) tail of approx. 30 nucleotides in cortex oocytes, in contrast to a length in wildtype oocytes of approx. 70 nucleotides (Fig. 8). This reduction in torso mRNA poly(A) tail length was also detected in early grauzone oocytes (data not shown). These results show that the cortex and grauzone gene products modulate poly(A) tail lengths during mid-oogenesis well before later events in oogenesis, such as activation of the oocyte and completion of meiosis.

Fig. 8.

The poly(A) status of torso is altered in cortex oocytes before meiotic maturation. Wild-type and cortex RNAs from oocytes that have not entered meiosis (stage 12 or before) were prepared and subjected to PAT cDNA synthesis. Torso mRNA contained a poly(A) tail of approx. 30 nucleotides in cortex oocytes. In contrast, the poly(A) tail length of torso mRNA in wild-type oocytes was approx. 70 nucleotides. Tail lengths were estimated from DNA molecular size markers resolved in parallel.

Fig. 8.

The poly(A) status of torso is altered in cortex oocytes before meiotic maturation. Wild-type and cortex RNAs from oocytes that have not entered meiosis (stage 12 or before) were prepared and subjected to PAT cDNA synthesis. Torso mRNA contained a poly(A) tail of approx. 30 nucleotides in cortex oocytes. In contrast, the poly(A) tail length of torso mRNA in wild-type oocytes was approx. 70 nucleotides. Tail lengths were estimated from DNA molecular size markers resolved in parallel.

cortex and grauzone: potential regulators of cytoplasmic polyadenylation

We have identified two genes that are indispensable for maternal mRNA activation and the initiation of development. For all phenotypes examined, grauzone and cortex embryos are comparable, strongly suggesting that their gene products participate in the same developmental pathway. For bicoid mRNA, we have shown that the lack of translation in cortex embryos is due to insufficient elongation of its poly(A) tail during early embryogenesis. These results implicate cortex and grauzone in regulation of the poly(A) status of maternal mRNAs.

The cytoplasmic regulation of poly(A) tail length is determined by an equilibrium between two competing reactions, polyadenosine addition and removal (Wickens, 1992). The poly(A) tail of a mRNA could be inappropriately short either by decreasing the rate of poly(A) addition or increasing the rate of removal. One possible explanation for our results is that the cortex gene encodes a component of the polyadenylation apparatus, and that catalysis of poly(A) elongation is reduced. Examples of such components include poly(A) polymerase, CPSF, and CPEB (Bienroth et al., 1991; Paris et al., 1991; Murthy and Manley, 1992; Whale and Keller, 1992; Bilger et al., 1994; Hake and Richter, 1994).

An alternate possibility is that cortex encodes a regulator of the polyadenylation machinery. This hypothesis is attractive for several reasons. (1) Mutations in cortex influence the extent of polyadenylation in bicoid, Toll and torso mRNAs in the embryo, but these perturbations are not identical, and differ in timing and extent of the alteration. (2) cortex mutations disturb the poly(A) status of torso mRNA in the oocyte, consistent with the regulation of polyadenylation during oogenesis (Huarte et al., 1992). (3) In addition to a reduction in polyadenylation, we have observed mRNAs whose poly(A) tail in cortex embryos is longer than in wild-type (data not shown). Taken together, our observations suggest that disruption of the cortex and grauzone genes produces a perturbation in the regulation of polyadenylation.

Mutations in the cortex and grauzone genes produce multiple biological phenotypes

Mutations in both cortex and grauzone not only affect mRNA translation, but also produce defects in microtubule regulation and in meiotic maturation. The precise reason why mutation in a single genetic locus produces these diverse biological phenotypes is not known. Since cortex is required during the latest stages of oogenesis, one interpretation is that the primary defect is in oocyte activation. Defective activation might prevent the completion of meiosis, alter microtubule regulation, and interfere with translational recruitment of maternal mRNAs during embryogenesis. Two pieces of evidence make this interpretation unlikely. (1) Accumulation of nanos protein in the embryo occurs after oocyte activation (Wang et al., 1994). Since both cortex and grauzone embryos translate nanos mRNA (Fig. 6), these mutations do not completely block oocyte activation. (2) Mutations in cortex alter the poly(A) status of torso mRNA before oocyte activation. The contribution of this disturbance to the cortex phenotype is unclear. Nevertheless, an effect on polyadenylation this early indicates that cortex is not exclusively a mutant in oocyte activation.

Alternatively, polyadenylation and translational recruitment of maternal mRNAs could be required for oocyte activation and maturation. There is direct evidence for this model since cytoplasmic polyadenylation and translation of c-mos mRNA are required for mouse and Xenopus oocyte maturation (Gebauer et al., 1994; Sheets et al., 1995). The defects in meiotic maturation and in translational activation observed in cortex and grauzone embryos are consistent with this paradigm. Moreover, recent experiments in Drosophila have shown that translational regulation of maternal mRNAs is not limited to early embryogenesis, but is also vital during oogenesis to establish embryonic asymmetry: specification of the posterior patterning system requires translational regulation of oskar mRNA during mid-oogenesis (Kim-Ha et al., 1995). When oskar mRNA is precociously translated, defects along the anteroposterior axis ensue. These findings underscore the importance of translational control of maternal mRNAs during oogenesis to development.

The diverse biological phenotypes in cortex and grauzone embryos document their vital role in development. These genes thus provide a genetic means to unravel the interrelationships of polyadenylation-dependent mRNA activation, microtubule reorganization, and meiosis, three critical processes that participate in the initiation of development.

We are very grateful to the following for Drosophila stocks, reagents and/or insightful discussions and comments on the manuscript: Kathryn Anderson, Bin Chen, Joseph Duffy, JoAnne Engebrecht, Elizabeth Gavis, Carl Hashimoto, Ruth Lehmann, Terry OrrWeaver, Jennifer Perrone, Fernando Sallés, Jennifer Schisa, Trudi Schüpbach, Allan Spradling, John Tower, Chicheng Tsai, Arturo Verrottti, Charlotte Wang, James Wells, Robin Wharton, and members of our laboratories. This work has been supported by grants from NIH to S. S. (GM51584) and to W.T. (GM50898), an American Cancer Society Faculty Research Award to J. P. G. (FRA-428), and a Hoffmann-La Roche predoctoral fellowship from the Institute of Cell and Developmental Biology to M. E. L.

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