The Drosophila embargoed Gene Is Required for Larval Progression and Encodes the Functional Homolog of Schizosaccharomyces Crm1

Corresponding author: Simon Collier, University of Manchester School of Biological Sciences, 2.205 Stopford Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom., simon.collier{at}man.ac.uk (E-mail)

Communicating editor: T. SCHÜPBACH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

The CRM1 (Exportin 1) protein is a receptor for leucine-rich nuclear export signal sequences. We have molecularly characterized the Drosophila melanogaster embargoed (emb) gene and find that it encodes a product with 49 and 71% sequence identity to the fission yeast Schizosaccharomyces pombe and human CRM1 proteins, respectively. We show that expression of the emb cDNA is sufficient to suppress the growth phenotype of both conditional-lethal and null S. pombe crm1^- mutant strains, suggesting that emb encodes the functional homologue of the S. pombe Crm1 protein. Through mutagenesis screens we have recovered a series of recessive lethal emb mutations. There is a substantial maternal contribution of emb mRNA and animals hemizygous for our emb alleles can develop to second instar larvae but persist at this stage and consistently fail to undergo the molt to the third instar stage. We see a nuclear accumulation of endogenous actin in the intestinal epithelial cells of the emb mutant larvae, consistent with a role for the emb gene product in nuclear export of actin protein.

THE division of eukaryotic cells into nuclear and cytoplasmic compartments has required the development of mechanisms that actively and selectively transport macromolecules through pores in the nuclear membrane. The nuclear trafficking of proteins is mediated by a group of related receptors called karyopherins that bind a peptide signal on the protein cargo and carry it through the nuclear pore (OHNO et al. 1998 ). This article describes the molecular and genetic characterization of a Drosophila homolog of the export karyopherin CRM1 (chromosome region maintenance 1), also called Exportin I. Studies have shown that nuclear CRM1 binds cooperatively to a leucine-rich nuclear export signal (NES) on its target protein and to the small GTPase Ran in its active GTP-bound form (FORNEROD et al. 1997B ). The CRM1 protein then carries its protein cargo through the nuclear pore to the cytoplasm where it is released upon the hydrolysis of RanGTP to RanGDP.

It was originally believed that one of the primary roles of the CRM1 protein is to maintain chromosome integrity, because the first cold-sensitive fission yeast (Schizosaccharomyces pombe) crm1 mutants showed atypical chromatin structure when cultured at a restrictive temperature (ADACHI and YANAGIDA 1989 ). S. pombe crm1 alleles have since been recovered from a screen for caffeine resistance (KUMADA et al. 1996 ) and have also been shown to confer resistance to the antibiotics spaurosine and brefeldin A (TURI et al. 1994 ). However, both the chromatin and the multidrug-resistance phenotypes are suppressed by a mutation in pap1, which encodes an AP-1-like transcription factor, suggesting that they are a result of deregulated pap1 activity (TODA et al. 1992 ). It is now known that Pap1 is a substrate for CRM1-mediated nuclear export and that its nuclear accumulation in crm1 mutants mimics the normal translocation of Pap1 to the nucleus under oxidative stress, where it regulates gene expression including the activation of drug-resistance genes (TOONE et al. 1998 ). The primary role of S. pombe Crm1 in nuclear export is supported by the nuclear accumulation of the Dsk1 kinase in cold-sensitive S. pombe crm1 mutant cells at a restrictive temperature (FUKUDA et al. 1997 ).

A further source of S. pombe crm1 alleles was a screen for mutations that confer resistance to leptomycin B, a Streptomyces-derived antifungal antibiotic (NISHI et al. 1994 ). Treatment of S. pombe crm1⁺ cells with leptomycin B produces phenotypes, such as disorganized chromatin, that resemble those of crm1 mutants. It is now known that leptomycin B inhibits Crm1 activity directly by disrupting its interaction with the NES and RanGTP (FORNEROD et al. 1997B ). This property of leptomycin B has allowed its use as a tool to study the effects of blocking CRM1-mediated nuclear export in both vertebrate (OSSAREH-NAZARI et al. 1997 ; YANG et al. 1998 ) and invertebrate (ABU-SHAR et al. 1999 ; BERTHELSEN et al. 1999 ) cell culture systems.

Much of our understanding of the mechanisms of nuclear export has come from genetic studies in yeast and from Xenopus oocytes and mammalian cell culture systems (OHNO et al. 1998 ). In contrast, the study of nuclear trafficking in Drosophila is still in its infancy (DAVIS 1997 ). In this article we present a molecular and genetic characterization of the Drosophila embargoed (emb) gene and show that it encodes the functional homologue of S. pombe Crm1. We describe a series of lethal emb alleles recovered from mutagenesis screens and present evidence that they are associated with a defect in nuclear export.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Molecular characterization of the emb gene:
Restriction fragments from the genomic phage clone W5-p9 (NEUMANN-SILBERBERG and SCHUEPBACH 1993 ) were used to screen 5 x 10⁵ clones of an Oregon-R 12–24-hr embryonic plasmid cDNA library by the standard protocol (BROWN and KAFATOS 1988 ). The longest emb cDNA recovered (emb2-4) was subcloned into Bluescript SK+ (Stratagene Ltd., Cambridge, UK) and exonuclease III deletion constructs from 5' to 3' of the sense strand were sequenced from the M13-20 primer (ABI Prism system). Sequence data were assembled into a contig using the GCG (Genetics Computer Group, Inc.) GELASSEMBLE program. The emb2-4 sequence was confirmed by sequencing the opposite strand from antisense oligonucleotide primers. The same primers were used to characterize subclones of fragments from the genomic phage clone W5-p9 to establish the emb intron/exon structure. The best open reading frame within the emb2-4 cDNA sequence was identified by the positional base preference method in the standard Staden programs for nucleotide interpretation. The encoded polypeptide was compared to protein database entries using the blastp program (ALTSCHUL et al. 1990 ) and aligned to human and S. pombe Crm1 amino acid sequences using the ClustalW program.

Whole mount in situ hybridization:
Digoxigenin-labeled riboprobes corresponding to the sense and antisense strands of the 3' untranslated region of our full-length emb cDNA were synthesized from the T7 and T3 promoters, respectively, of the emb cDNA subclone 2-4XN. The 2-4XN construct consists of a 700-bp fragment of the cDNA clone emb2-4, extending from the XhoI site immediately 3' of the emb coding sequence (see Fig 1B) to the downstream pNB40 vector NotI site, ligated into the XhoI/NotI polylinker sites of the Bluescript plasmid vector. In situ hybridization of the emb riboprobes to 0–24-hr Oregon-R embryos was undertaken essentially as described by TAUTZ and PFEIFFLE 1989 except that prehybridization and hybridization steps were carried out at 56° rather than 45° because of the greater stability of RNA/RNA duplexes in comparison to RNA/DNA hybrids.

View larger version (101K): In this window In a new window Download PPT slide   Figure 1. (A) Alignment by homology of the predicted amino acid sequence of the emb gene product with the S. pombe and human CRM1 proteins by the ClustalW program. Identical amino acid residues are boxed together. (B) Molecular map of the emb locus at cytological location 29C1.2. Transcripts are indicated by boxes, the shaded portions of which represent the extent of coding sequence. The location of the P-element insertion sites of the emb mutants l(2)k16715 and l(2)k06303 are indicated by shaded arrowheads. The region deleted by Df(2L)N22-14 is represented by a box, the unshaded portion of which indicates uncertainty about the distal breakpoint of the deletion. Restriction sites are B, BamHI; E, EcoRI; H, HindIII; and X, XhoI. The lowercase "e" is a polymorphic EcoRI site present on cn bw sp and derivative chromosomes. GenBank accession numbers are AF179360 for the emb cDNA and AF179361 for the emb genomic sequence.

Construction of emb expression constructs:
The pRE PCD1emb and pREPCD41emb constructs were made in two steps. First, a restriction fragment extending from the internal emb2-4 cDNA BamHI site (see Fig 1B) to a downstream pBluescript vector NotI site was ligated within the pREPCD1/41 polylinker to make a primary construct. Second, the product of a PCR amplification of the emb2-4 cDNA, using a primer (GTCA GGATCC ATG GCG ACA ATG TTG ACA TC) that introduces a BamHI restriction site immediately 5' of the putative emb start codon and a primer downstream of the internal emb BamHI site, was BamHI digested and ligated to the primary construct. The correct orientation and sequence of the cloned PCR fragment was confirmed by restriction mapping and DNA sequencing. The pREP42emb construct was also made in two steps. First, a PCR primer (CAAA CAT ATG GCG ACA ATG TTG ACA) was used to introduce a NsiI recognition site overlapping the putative emb start codon in an amplification reaction with a second primer downstream of the internal emb BamHI site. The PCR product was digested with NsiI and BamHI and ligated within the pREP42 polylinker to make a primary construct. The fidelity of the cloned PCR product was checked by DNA sequencing. Second, the remainder of the coding region plus the nmt1 polyadenylation site was added by ligating the BamHI/SstI insert from the pREPCD1emb construct (see above) to the primary construct.

Suppression of fission S. pombe crm1 mutants by emb expression:
Standard procedures for S. pombe genetics were followed (MORENO et al. 1991 ). The S. pombe strains used in this study are shown in Table 1. For suppression of the conditional lethal mutants, crm1-809 and crm1-1R strains were transformed with the plasmids pREPCD1emb and pREPCD41emb using the lithium method (ITO et al. 1983 ) and grown on plates containing 1.6% agar. For suppression of the null crm1 mutation, diploid strains heterozygous for the crm1⁺ gene (TP45, Table 1) were transformed with multicopy plasmids containing emb (pREP42-emb) or crm1⁺ (pKK1, KUMADA et al. 1996 ). Leu⁺ transformants were allowed to sporulate on minimal plates in the absence of thiamine and remaining unsporulated diploids were killed by treatment with 0.5% glusulase. Free spores were spread in minimal plates supplemented with adenine. Leu⁺ Ura⁺ Ade^- colonies (crm1::LEU2-containing plasmids, TP45-1 and TP45-2, Table 1) were picked and analyzed.

Isolation and characterization of embargoed mutants:
Flies were cultured at 25° on yeasted cornmeal agar unless stated otherwise. To screen for mutations lethal with T(Y;2)fy⁴, X-ray- or EMS-mutagenized b cn males were crossed to SM6a/Gla females. Single b cn/SM6a male progeny were crossed to C(1)M4; T(Y;2)fy⁴/CyO females and single b cn/SM6a female progeny crossed to T(Y;2)fy⁴/CyO males. F₂ progeny were screened for inviability of the T(Y;2)fy⁴/b cn class. The lethal phase of hemizygous emb mutants was determined by collecting larval progeny of Df(2L)N22-14/Gla Bc males and emb/Gla Bc females and identifying the furthest developed Bc⁺ animals. To identify the emb¹ lesion, homozygous emb¹ second instar larvae were selected from the progeny of emb¹/Gla Bc parents on the basis of a Bc⁺ phenotype. Genomic DNA was prepared from the homozygous larvae and the emb locus PCR amplified using gene-specific oligonucleotide primers. PCR products were directly sequenced from emb antisense primers (see above). The P-element insertion sites of l(2)k16715 and l(2)k06303 were identified by plasmid rescue of the P{lacW} element and flanking sequences using standard procedures (HAMILTON et al. 1991 ). Informative restriction enzyme digests were PstI for l(2)k16715 and BglII and BamHI for l(2)k06303. Rescued plasmids were sequenced from an oligonucleotide primer complementary to the P-element terminal repeat (CGACGGGACCACCTTATG).

Nuclear and actin staining of Drosophila gut epithelia:
Homozygous y w; emb second instar larvae were selected from the progeny of y w; emb/CyO [y⁺] parents on the basis of mouth hook pigmentation. The emb mutant larvae and control homozygous y w second instar larvae were dissected and fixed in PBS with 4% paraformaldehyde. Fixed larvae were incubated with an actin antibody (Sigma A2066, raised in rabbit) at 1:200 dilution and secondary goat anti-rabbit (FITC, Jackson Immunoresearch, West Grove, PA) at 1:200 dilution following standard methods for imaginal disc staining (WHITE 1998 ), but including 20 µg/ml propidium iodide in the final phosphate buffered TWEEN wash to stain nuclei. Intestinal epithelia were dissected from the stained larvae and then mounted. Images were captured from a Leica confocal microscope using the TCS-NT program and were processed in Adobe Photoshop 4.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Molecular characterization of the emb gene:
In the course of screens for expressed sequences in the Drosophila chromosomal region 29C we isolated a class of cDNAs encoding a polypeptide with strong homology to the S. pombe Crm1 protein (ADACHI and YANAGIDA 1989 ). We originally called this gene Crm1 (COLLIER and GUBB 1997 ), but have now renamed it embargoed (emb) to reflect the nuclear export defects of the mutants (see below). We have recovered emb cDNA clones from 12–24-hr embryo and imaginal disc plasmid cDNA libraries (BROWN and KAFATOS 1988 ) and a 0–24-hr pupal cDNA library. The longest of the emb cDNA clones is 4.2 kb and contains an open reading frame of 1063 amino acids that is 49 and 71% identical to the S. pombe (ADACHI and YANAGIDA 1989 ) and human (FORNEROD et al. 1997A ) CRM1 protein sequences, respectively (Fig 1A). The emb cDNA maps to a genomic region of just 4.5 kb (Fig 1B), with the coding sequence split by five small introns. The emb transcription unit lies immediately proximal on the chromosome to the tissue polarity gene fuzzy (fy; COLLIER and GUBB 1997 ), and the two genes are divergently transcribed with a little over 600 bp of genomic sequence between the 5' ends of our longest respective cDNAs. [A recent publication (FASKIN et al. 2000 ) describes an embargoed cDNA with an extra 131 nucleotides of 5' sequence of which 86 nucleotides form an additional untranslated exon. This reduces the distance between the emb and fy transcripts to just 132 bp.]

emb gene expression:
Our longest emb cDNA hybridizes to two bands of ~4.2 and 3.5 kb on a Northern blot of Drosophila mRNA, the larger band having a severalfold higher intensity in all developmental stages tested (data not shown). Our emb cDNAs fall into two classes: a single pupal cDNA is polyadenylated just over 100 nucleotides 3' of the termination codon and the other four cDNA clones, from embryonic and pupal libraries, are polyadenylated 660 nucleotides further downstream (Fig 1B). We conclude that the two size classes of emb mRNA result from differential polyadenylation and that both transcripts encode the same peptide sequence.

We have hybridized a riboprobe corresponding to an antisense strand of the 3' untranslated region of our full-length emb cDNA to mixed-stage wild-type (Oregon-R) Drosophila embryos (Fig 2). We find ubiquitous expression of the emb transcript at all stages of embryonic development (Fig 2, A–D), as befits a gene that encodes a central component of the nuclear trafficking system. This includes a substantial maternal contribution of emb mRNA that is evident in very early embryos (Fig 2A). However, from the cellular blastoderm stage (Fig 2B) onward the levels of emb expression vary across the developing embryo. Specific tissues in which emb expression is relatively high are the brain, hind gut, and posterior spiracles shortly before dorsal closure (Fig 2C) and the ventral nerve cord, midgut, and somatic musculature shortly after dorsal closure (Fig 2D). In each case emb expression seems to increase when the tissue is well developed, suggesting that emb is required for the function or maintenance of these tissues rather than for their formation.

View larger version (100K): In this window In a new window Download PPT slide   Figure 2. emb expression in wild-type (Oregon-R) embryos. Anterior is left and dorsal up unless stated otherwise. (A) Lateral view of Stage 1 embryo. (B) Lateral view of Stage 5 embryo. (C) Dorsal view of Stage 13 embryo. br, brain; hg, hindgut; ps, posterior spiracles. (D) Ventral view of Stage 15 embryo. mg, midgut; sm, somatic musculature; vnc, ventral nerve cord. Photographs were taken at x25 magnification.

Suppression of S. pombe conditional-lethal crm1 mutations by emb expression:
The high sequence homology between the emb gene product and the S. pombe Crm1 protein prompted us to ask if the function of these proteins has also been conserved. We initially addressed this question by testing if the expression of an emb cDNA in conditional-lethal S. pombe crm1 mutants could suppress the growth phenotype seen at restrictive conditions. The emb coding region was ligated immediately downstream of the thiamine-repressible nmt1 promoter of the pREPCD1 expression vector and the attenuated nmt1 promoter of pREPCD41 (MAUNDRELL 1993 ). The pREPCD1emb and pREPCD41emb constructs were introduced into a cold-sensitive (crm1-809; ADACHI and YANAGIDA 1989 ) and a temperature-sensitive (crm1-1R; KUMADA et al. 1996 ) S. pombe crm1 mutant. The expression of the emb cDNA gave analogous results for both strains (Fig 3A and Fig B). At the permissive temperature and in the presence of the thiamine repressor, the emb cDNA did not affect growth (Fig 3, Ai and Bi), but in the absence of thiamine, expression of the pREPCD1emb construct inhibited growth, although pREPCD41emb did not (Fig 3, Aii and Bii). We conclude that the levels of the emb gene product produced by expression from the wild-type nmt1 promoter are toxic to the yeast cell. There are precedents for this observation since it is known that high level expression of the human Crm1 protein in S. pombe prevents growth (KUDO et al. 1997 ) and that overexpression of the endogenous CRM1 protein in the budding yeast Saccharomyces cerevisiae prevents sporulation (TODA et al. 1992 ). The toxicity of high levels of emb expression in S. pombe may result from a dominant-negative interaction, as it produces elongated cells that resemble cold-sensitive crm1 mutant cells incubated at the restrictive temperature (ADACHI and YANAGIDA 1989 ). In the presence of thiamine, the pREPCD1emb and pREPCD41emb constructs did not permit growth of the crm1-809 and crm1-1R mutants at the restrictive temperature (Fig 3, Aiii and Biii). In the absence of thiamine, the pREPCD1 emb construct also failed to support growth, presumably due to the toxicity of high levels of the emb gene product (Fig 3, Aiv and Biv), but the pREPCD41emb construct restored growth close to the level achieved by the endogenous crm1+ gene (Fig 3, Aiv and Biv). From these data we conclude that an appropriate level of emb gene expression can suppress the growth phenotype of both cold- and temperature-sensitive S. pombe crm1 mutants.

View larger version (104K): In this window In a new window Download PPT slide   Figure 3. Suppression of the growth phenotype of conditional-lethal S. pombe crm1 alleles by expression of the emb cDNA. (a) Results of emb expression in the cold-sensitive crm1-809 allele. (b) Results of emb expression in the temperature-sensitive crm1-1R allele. Expression constructs present in the crm1 mutant strains are indicated on the template above. Incubation conditions are indicated adjacent to each image. Thi, thiamine.

Suppression of an S. pombe crm1 null mutation by emb expression:
The suppression of conditional-lethal S. pombe crm1 mutant growth phenotypes by the expression of the emb cDNA suggests that the emb gene encodes the functional homologue of S. pombe Crm1. However, it remained possible that the Drosophila protein can only substitute for one activity of the yeast protein that is sensitive to the introduction of conditional-lethal mutations. We therefore attempted to suppress a null crm1 mutant that had been made by inserting the S. cereviseae LEU2 gene at the crm1 locus (ADACHI and YANAGIDA 1989 ), by emb expression. The emb cDNA sequence was ligated downstream of the thiamine repressible promoter of the ura4⁺ pREP42 vector and was used to transform S. pombe diploid heterozygous crm1 mutants (crm1::LEU2/+,TP45, Table 1), as haploid crm1 null mutants are inviable (ADACHI and YANAGIDA 1989 ). As a positive control the same stain was transformed with a multicopy plasmid carrying the crm1+ gene (pKK1; KUMADA et al. 1996 ). Diploid transformants were allowed to sporulate and free spores were germinated on minimal plates supplemented with adenine. In the presence of thiamine Leu⁺ Ura⁺ Ade^- (crm1::LEU2) haploid cells were obtained from the diploid containing pKK1, but not from those containing pREP42emb (Fig 4I). However, in the absence of thiamine the diploids containing pREP42emb also produced Leu⁺ Ura⁺ Ade^- haploid cells (Fig 4ii). This result implies that the expression of the emb gene is sufficient to suppress the growth phenotype of a crm1 null mutant and that the emb gene product is functionally homologous to the S. pombe Crm1 protein. This, in turn, suggests that the mechanism of CRM1-mediated nuclear export has been highly conserved through evolution and means that experiments in Drosophila can complement the ongoing studies in S. pombe and vertebrate cell culture systems.

View larger version (68K): In this window In a new window Download PPT slide   Figure 4. Suppression of the growth phenotype of a S. pombe crm1 null mutant by expression of emb. Independent crm1-deleted haploids were streaked on minimal plates (supplemented with adenine) and incubated at 30° for 3 days. Expression constructs present are indicated on the template above. Incubation conditions are indicated adjacent to each image. Thi, thiamine.

Identification of emb mutants:
The chromosome translocation T(Y;2)fy⁴ breaks in the cytological region 29B4-C2 and was recovered from an X-ray screen for new fy alleles (COLLIER and GUBB 1997 ). Flies heterozygous for T(Y;2)fy⁴ and a fy null allele have the typical cold-sensitive amorphic fy phenotype. Although the T(Y;2)fy⁴ break places heterochromatin adjacent to the fy locus, the associated fy phenotype does not appear to be subject to position effect variegation as it is not ameliorated by introducing additional Y chromosome material (data not shown). Unlike other amorphic fy alleles, however, T(Y;2)fy⁴ is lethal over the deficiency Df(2L)N22-14 (29C1.2;30C8.9). To investigate the source of this lethality we performed F₂ lethal X-ray and EMS mutagenic screens. Four lethal alleles (l(2)fy^4-1, l(2)fy^4-2, l(2)fy^4-3, and l(2)fy^4-5) were recovered from 2496 chromosomes in EMS screens and a single lethal allele (l(2)fy^4-4) from 2076 in an X-ray screen, all five alleles belonging to a single complementation group (Table 2). The T(Y;2)fy⁴ break must be close to the fy locus as it is associated with an amorphic, nonvariegating fy phenotype, but cannot map significantly distal to fy as it is also lethal with Df(2L)N22-14 (see Fig 1B). Therefore, the best candidate for the l(2)fy⁴ lethality was the next essential gene proximal to fy and most probably emb. To test this, we PCR-amplified and sequenced the emb locus from genomic DNA prepared from l(2)fy^4-1 (emb¹) homozygous second instar larvae and found a G-to-A transition at the 5' donor site of intron 4. The splice site mutation was not found on the b cn progenitor chromosome or in other emb alleles from the same EMS screen. Since intron 4 is 66 nucleotides in length with no in-frame termination codons, the emb¹ mutant transcript is expected to encode a protein with an additional 22 residues inserted at amino acid position 872. The high degree of sequence conservation along the entire length of the S. pombe, Drosophila, and human CRM1 proteins (Fig 1A) suggests that this insertion will seriously compromise the function of the emb product and, indeed, the lethal phenotype of emb^1/ hemizygotes is indistinguishable from that of a putative emb null [l(2)k06303, see Table 2 and below].

Our emb alleles fail to complement the Berkeley Drosophila Genome Project P-element insertion lines l(2)k06303 and l(2)k16715, which had both been mapped to 29C3-C4 (TOROK et al. 1993 ; SPRADLING et al. 1995 ). By plasmid rescue of the P{lacW} element and flanking sequences we have found that the l(2)k06303 insert is within exon 4 and the l(2)k16715 insert is ~400 bp upstream of the emb transcription start site (Fig 1B). The lethal phenotypes of l(2)k16715 and l(2)k06303 hemizygotes are indistinguishable (Table 2). The fact that the l(2)k16715 insertion site is within the 5' untranslated exon of the emb gene that has recently been described (FASKIN et al. 2000 ) explains why its associated phenotype is as severe as l(2)k06303, which we believe to be an emb null allele. However, despite the l(2)k16715 insertion being within 200 bp of the fy transcription start site, it does not significantly compromise fy expression, as flies heterozygous for l(2)k16715 and an amorphic fy allele have no detectable tissue polarity phenotype when cultured at the restrictive temperature of 18° (COLLIER and GUBB 1997 ; data not shown).

The similar lethal phase of the emb alleles suggests either that they are all genuine amorphs, perhaps reflecting the vulnerability of the highly conserved CRM1 protein to introduction of null mutations. Alternatively, the emb alleles are of differing strengths, but a high demand for the emb gene function prior to the second larval molt means that even hypomorphic emb alleles fail to support ecdysis (see below).

The lethal stage of emb mutants:
We identified the lethal stage of hemizygous emb flies by collecting larval progeny from emb/Gla Bc females crossed to Df(2L)N22-14/Gla Bc males (see Table 2). For all emb alleles, emb hemizygous Bc⁺ second instar larvae were abundant, but no hemizygous third instar larvae were seen. This was not due simply to developmental delay, as isolated second instar emb hemizygotes can survive for up to 7 days at 25° and still do not undergo the L2/L3 molt. This absolute block of ecdysis might mean that some component of the ecdysone signaling pathway is dependent upon emb-mediated nuclear export. Such consistent arrest in larval development is reminiscent of hypomorphic mutations of the stranded at second (sas) gene that encodes a cell surface receptor protein containing fibronectin type III class repeats (SCHONBAUM et al. 1992 ). In common with sas mutants, second instar emb mutant larvae are usually smaller than wild type and can display unusually convoluted tracheae. It is possible that sas activity is dependent upon nuclear export or that the sas receptor is a component of a signaling pathway required for emb expression.

The CRM1 protein is a fundamental component of the nuclear export machinery that we would expect to play a vital role in the development of the embryo to larval stages. Therefore, the survival of animals carrying emb null alleles to second instar larvae is probably dependent upon the maternally contributed emb mRNA present in early embryogenesis (Fig 2A), particularly as there is no closely related protein encoded by the Drosophila genome that might readily substitute for emb function.

Nuclear accumulation of actin in gut cells of emb mutant larvae:
To confirm the role of the emb gene product in nuclear export we looked for an abnormal subcellular distribution of actin in our emb mutants. Due to its low molecular weight (~42 kD), actin is believed to migrate freely through nuclear pores, but becomes localized exclusively in the cytoplasm due to CRM1-dependent nuclear export (WADA et al. 1998 ). Our analysis of the six characterized Drosophila actin proteins (actin 5C, 42A, 57B, 79B, 87C, and 88F; FLYBASE 1999 ) reveals that they are all identical in sequence to mammalian -actin at the sites of the two internal NES sequences, suggesting that they are also substrates for Crm1-mediated nuclear export. We stained intestinal epithelia of emb and y w mutant second instar larvae with an actin antibody and with propidium iodide to highlight nuclei (Fig 5). In wild-type epithelial cells actin is exclusively cytoplasmic (Fig 5A), but in ~50% of cells from emb¹/emb² mutant larvae actin staining is both nuclear and cytoplasmic (Fig 5C). Similar nuclear accumulation of actin occurs when rat tissue culture cells are incubated with the CRM1 inhibitor Leptomycin B (WADA et al. 1998 ), suggesting that our observations reflect a deficiency in nuclear export caused by the emb mutation. Surprisingly, ~50% of cells in emb¹ heterozygotes also show some nuclear localization of actin (Fig 5B), although this is apparently benign, as heterozygous emb¹ animals are viable and appear phenotypically wild type. This dominant emb¹ phenotype may result from nuclear NES sequences being in excess of the reduced number of export receptors or, alternatively, from the emb¹ gene product actively blocking export perhaps by binding to the NES sequences, but failing to mediate export. From these observations we conclude that the emb gene product is required to actively export endogenous actin from the nucleus in an analogous fashion to CRM1 in mammalian tissue culture cells. This result not only confirms the role of the emb gene product in nuclear export, but could potentially provide an in vivo assay for emb-mediated export activity in Drosophila.

View larger version (49K): In this window In a new window Download PPT slide   Figure 5. Actin distribution in intestinal epithelial cells of wild-type (WT) and emb mutant second instar larvae. Antibody staining of actin is green and propidium iodide staining of nuclei is red; nuclear actin appears yellow. (A) y w, (B) emb1/+ heteroygotes, (C) emb1/emb2 mutant.

	FOOTNOTES

¹ Present address: School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
² Present address: Department of Biology, University of Pennsylvania, Philadelphia, PA 19104–6018.

	ACKNOWLEDGMENTS

Many thanks to David Hughes for supplying the pREP vectors and to Martin Baron and Marian Wilkin for helping S.C. to complete this work in Manchester. Thanks also to John Roote, David Gubb, Rachel Drysdale, Anne Beyer, and Haeryun Lee for useful discussion. This work was funded by a Medical Research Council program grant to Michael Ashburner and David Gubb, a National Institutes of Health grant to Paul Adler, a Hong Kong Croucher Foundation Scholarship to Edwin Chan, and an Imperial Council Research Fund grant to Takashi Toda. Confocal microscopy was carried out at the Cambridge University Multi-imaging Centre and was supported by a Wellcome Trust grant.

Manuscript received October 18, 1999; Accepted for publication April 21, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

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