The Ketel^D Dominant-Negative Mutations Identify Maternal Function of the Drosophila Importin-ß Gene Required for Cleavage Nuclei Formation

Corresponding author: János Szabad, Faculty of General Medicine, Department of Biology, University of Szeged, H-6720 Szeged, Somogyi B. u. 4, Hungary., szabad{at}comser.szote.u-szeged.hu (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Ketel^D dominant female-sterile mutations and their ketel^r revertant alleles identify the Ketel gene, which encodes the importin-ß (karyopherin-ß) homologue of Drosophila melanogaster. Embryogenesis does not commence in the Ketel^D eggs deposited by the Ketel^D/+ females due to failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, cytoplasm of the Ketel^D eggs does not inhibit nuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel⁺ transgenes slightly reduce effects of the Ketel^D mutations. The paternally derived Ketel^D alleles act as recessive zygotic lethal mutations: the Ketel^D/- hemizygotes, like the ketel^r/ketel^r and the ketel^r/- zygotes, perish during second larval instar. The Ketel maternal dowry supports their short life. The Ketel^D-related defects originate most likely following association of the Ketel^D-encoded mutant molecules with a maternally provided partner. As in the Ketel^D eggs, embryogenesis does not commence in eggs of germline chimeras with ketel^r/- germline cells and normal soma, underlining the dominant-negative nature of the Ketel^D mutations. The ketel^r homozygous clones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in most larval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.

THE commencement of embryogenesis is one of many intriguing biological phenomena and raises questions about the origin and function of factors required for the initiation of a new life. What are those factors? Where and how are they made? What are their functions? It has long been known that most of the factors that are required during early embryogenesis are deposited into the egg cytoplasm during oogenesis and are maternally provided. The importance of maternal contribution is emphasized by the fact that there is very little, if any, zygotic gene expression during the initial cleavage divisions in Drosophila.

Genetic dissection and the use of female-sterile mutations have been an efficient approach to identifying genes coding for the maternally provided factors required during Drosophila embryogenesis (WIESCHAUS 1996 ). During genetic dissection of maternal effects in Drosophila melanogaster, we isolated 75 dominant female-sterile mutations including the four Ketel^D alleles (ERDELYI and SZABAD 1989 ; SZABAD et al. 1989 ; ERDELYI et al. 1997 ). Since embryogenesis is terminated at the very beginning in the so-called Ketel^D eggs deposited by the Ketel^D/+ females, we assumed that the normal Ketel gene product is maternally provided and performs essential functions during the commencement of embryogenesis. As described in the accompanying article (LIPPAI et al. 2000 ), we cloned the Ketel gene and learned that it encodes the Drosophila homologue of importin-ß, a protein believed to be essential for import of some types of nuclear proteins.

Biochemical studies have clarified a number of nuclear import and export processes and several of the components (for recent reviews see MATTAJ and ENGLMEIER 1998 ; WEIS 1998 ; WOZNIAK et al. 1998 ; GORLICH and KUTAY 1999 ). However, the developmental role of proteins involved in nuclear transport and genetic complexity of the process have been poorly studied. To understand Ketel gene requirement during development and reveal unknown aspects of nuclear protein import, we analyzed the mutant phenotypes associated with the Ketel^D gain-of-function and the ketel^r loss-of-function alleles, constructed different types of genetic mosaics, and studied the expression pattern of a Ketel promoter-operated lacZ reporter gene. The present article describes that function of Drosophila importin-ß is required—in association with a maternally provided component—for the formation of cleavage nuclei. We propose that several of the dominant female-sterile (Fs) mutations identify maternal function of essential zygotic genes and the functions become manifested following association of the Fs-encoded mutant gene products with a maternally provided partner "designed" for the initial stages of embryogenesis.

We also report that clones homozygous for ketel^null alleles are viable in the female germline, the follicle epithelium, and wing and tergite cells referring to the existence of nuclear protein import mechanisms that compensate lost Ketel gene functions. With few exceptions the Drosophila importin-ß encoding the Ketel gene is expressed in mitotically active cells and is not expressed in those larval and adult cells that are mitotically inactive.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Ketel alleles:
The four Ketel^D alleles were isolated in a screen for dominant female-sterile mutations. The 27 recessive ketel^r alleles were generated through second mutagenesis of the Ketel^D mutations (SZABAD et al. 1989 ; ERDELYI et al. 1997 ). The ketel^r/ketel^r homozygotes and the ketel^r/- hemizygotes were produced by crossing y/y; ketel^r/y⁺CyO or y/y; ketel^rX32/y⁺CyO females with y/Y; ketel^r/y⁺CyO males. [The ketel^rX32 allele is a small deficiency that removes the Ketel and a few neighboring loci (ERDELYI et al. 1997 ).] The y⁺CyO balancer chromosome carries a y⁺ transgene (TIMMONS et al. 1993 ). Head skeleton and ventral setae of the descending y/y (or y/Y); ketel^r/ketel^r (or ketel^r/-) zygotes are yellow and thus the homo- and the hemizygous larvae can be separated from the heterozygous nonyellow (y⁺CyO) sibling larvae in which the chitin structures are dark. The Ketel^D/- hemizygotes were produced by crossing y/y; ketel^rX32/y⁺CyO females with y/Y; Ketel^D/y⁺CyO males. For explanation of the genetic symbols throughout this article see LINDSLEY and ZIMM 1992 . The Drosophila cultures were kept at 25°.

The Ketel^D mutant phenotypes:
The Ketel^D dominant phenotype was established by cytological analysis of Ketel^D eggs. Squashes were prepared from eggs newly deposited by Ketel^D/+ females. The egg squashes were stained by Feulgen and Giemsa, a procedure appropriate for the staining of chromosomes, centrosomes, and the spindle apparatus (PURO and NOKKALA 1977 ; PURO 1991 ).

Cytoplasm injections:
Two types of cytoplasm injections were carried out:

1. A sample of 40 pl Ketel^D1 egg cytoplasm (0.4% total egg volume) was injected on one side into the presumptive head region at 70% egg length and 70% egg diameter of wild-type embryos. The antennal and the maxillary sense organs, characteristic landmark structures, derive from the chosen blastoderm region (JURGENS et al. 1986 ). The donor and the recipient embryos were <30 min old. In another set of cytoplasm injections the wild-type embryos received 300 pl Ketel^D1 egg cytoplasm. Cuticles of the developing embryos and larvae were analyzed (JURGENS et al. 1986 ; WIESCHAUS and NUSSLEIN-VOLHARD 1986 ). As a control, cytoplasm samples of <30-min-old wild-type embryos were injected.

2. A small sample (20 µg/ml) of the red fluorescent classic nuclear localization signal-phycoerythrin (cNLS-PE; CSERPAN and UDVARDY 1995 ) was first injected into Ketel^D1 eggs. A sample of 200 pl cNLS-PE containing Ketel^D1 cytoplasm was subsequently injected into wild-type cleavage embryos. In the control the cNLS-PE substrate solution was first injected into newly deposited wild-type eggs and the cNLS-PE containing wild-type egg cytoplasm was subsequently injected into wild-type cleavage embryos. Import of the cNLS-PE substrate into the cleavage nuclei was followed in a Zeiss (Thornwood, NY) LSM410 confocal microscope. The injections were done at 20°.

The Ketel^D/+/+ and the Ketel^D/+/+/+ females:
We constructed Ketel^D/+/+ and Ketel^D/+/+/+ females which, in addition to the Ketel^D allele, carried two and three normal Ketel gene (+) copies. Egg and progeny production of the females was monitored. The extra Ketel gene copies (+) were introduced through the Tp(2;Y)G chromosome or through one of the Ketel⁺ (K⁺) transgenes described in the accompanying article (LIPPAI et al. 2000 ).

The Tp(2;Y)G chromosome: For production of XXTp(2;Y)G; Ketel ^D/+ females, XXTp(2;Y)G; Df(2L)Sd68, pr/CyO, pr females were mated with XY; Ketel^D/CyO, pr males. [In the Tp(2;Y)G chromosome the 36B5-C1 to 40F segment of the second chromosome—including the Ketel and the purple (pr) loci—was transposed onto a Y chromosome. The Df(2L)Sd68 deficiency removes both the Ketel and the pr loci (LINDSLEY and ZIMM 1992 ).] The XXTp(2;Y)G; Ketel^D/CyO, pr females were mated with XTp(2;Y)G; pr/pr males. As determined in a cross between XXTp(2;Y)G; Df(2L)Sd68, pr/CyO, pr females and XY; pr/pr males, 39% of the progeny females are XXTp(2;Y)G.

The Ketel⁺ (K⁺) transgenes: To study the effects of the K⁺ transgenes, we generated females that were heterozygous for one of the Ketel^D alleles and carried one or two additional Ketel gene copies in one of the three different types of K⁺ transgenes. The K⁺ transgenes were labeled with the mini-white marker gene that ensured, on white background, light yellow and orange eyes in one and two copies, respectively. One group of the K⁺; Ketel^D/+ and the K⁺/K⁺; Ketel^D/+ females was mated with wild type (+/+), the other group with K⁺/Y; +/+ males (see Table 1).

Germline chimeras:
We constructed germline chimeras through the transplantation of pole cells, ancestors of the germline (ILLMENSEE 1973 ). In the chimeras functionally normal soma surrounded the germline cells that were hemizygous for ketel^rX13, a ketel^r null allele. (In ketel^rX13 9/10 of the 3' end of the open reading frame was deleted.) In practice, pole cells of the embryos from a cross between ketel^rX13/Bc Gla females and ketel^rX32/CyO males were transplanted into host embryos that derived from wild-type (+/+) females and Fs(1)KI237/Y males. Fs(1)K1237 (= ovo^D1) is a dominant female-sterile mutation that disrupts function of the germline cells without affecting the soma (BUSSON et al. 1983 ; KOMITOPOULOU et al. 1983 ). The eclosing K1237/+ females were mated with lt bw/lt bw males. [The ketel^rX13 and ketel^rX32-carrying chromosomes are labeled with the lt and the bw recessive marker mutations (LINDSLEY and ZIMM 1992 ; ERDELYI et al. 1997 ).]

Follicle cell mosaics:
Follicle cell clones homozygous for ketel^rX13 were generated through mitotic recombination. In practice, ketel^rX13 pr/Bc Gla adult females were mated with Fs(2)Ugra/Bc Gla males. [Three independent ketel^rX13 pr lines were recovered following meiotic recombination to remove possible second-site lethal mutations that were induced during (i) the EMS treatment to induce the Ketel^D1 mutation and (ii) the X-ray treatment when the ketel^rX13 revertant allele was generated (SZABAD et al. 1989 ; ERDELYI et al. 1997 ). The three lines gave identical results in the clonal analysis experiments and hence pooled data are presented in Table 3 and Table 4. Fs(2) Ugra (= Ugra) is a dominant female-sterile mutation that disrupts follicle cell function without affecting the germline cells (SZABAD et al. 1989 , SZABAD et al. 1991 ).] Early third instar larvae were X-irradiated for the induction of mitotic recombination by 1500 R of X rays (150 kV; 0.5 mM Al filter, 1000 R/min). The eclosing ketel^rX13/Ugra and the control lt bw/Ugra adult females were tested for offspring production. Whether the ketel^rX13/ketel^rX13 homozygous follicle cells can support egg development was decided by comparing the frequencies of follicle cell mosaicism in the ketel^rX13/Ugra and the lt bw/Ugra control females (SZABAD et al. 1989 ).

Wing and tergite mosaics:
Clones homozygous for the ketel^rX13 allele were induced through mitotic recombination in f^36a/Y; ketel^rX13/f⁺ ck pwn larvae. The f⁺ symbol stands for a forked⁺ transgene in the 30B cytological region. It compensates effects of the f^36a mutation (P. MARTÍN and A. GARCÍA-BELLIDO, personal communication). The f^36a, ck, and pwn symbols represent cell marker mutations that allow recognition of the different types of clones. Mitotic recombination was induced in young third instar larvae 72–80 hr after egg deposition (1500 R, 150 kV, 0.5 mm Al filter, 1000 R/min). Following mitotic recombination, the majority of the forked (f) clones are homozygous for ketel^rX13. The ck pwn twin clones served as reference in analysis of the f clones. Wings and abdomens of the f^36a/Y; ketel^rX13/f⁺ ck pwn males were mounted and analyzed for clones in a compound microscope. Types, frequencies, and clone sizes were recorded.

The Ketel-lacZ reporter gene:
We constructed a reporter gene in which a 1378-bp upstream segment of the Ketel gene between positions -1336 and +42 was combined with the Escherichia coli lacZ gene. (See the corresponding sequence under the accession no. AJ002729 in the EMBL nucleotide sequence database.) The 1378-bp fragment contains the entire Ketel promoter with the transcription start site (LIPPAI et al. 2000 ). The 1378-bp segment was cloned into the pP(CaSpeR-AUG-gal) P-element transformation vector (THUMMEL et al. 1988 ). Four germline transformant lines of the reporter gene were generated using the CaSpeR vector with the mini-white marker gene. Two of the transgenes were inserted into the X and two into the 3rd chromosome. Flies homozygous for any of the transgenes were viable and fertile. ß-Galactosidase activities of transgene homozygotes were studied in adult, embryonic, and larval stages of development according to standard procedures.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Ketel^D mutant phenotype suggests NE-related function of the normal Ketel gene product:
The four Ketel^D dominant female-sterile mutations of D. melanogaster emerged following EMS mutagenesis (SZABAD et al. 1989 ). The Ketel^D/+ females and the males are fully viable and male fertility is normal. However, the Ketel^D/+ females are sterile. Development of the egg primordia is normal in all the Ketel^D/+ females and the meiotic divisions are indistinguishable from wild type (J. PURO, unpublished data). The Ketel^D/+ females deposit normal numbers of normal-looking so-called Ketel^D eggs. All the Ketel^D eggs are normally fertilized as revealed by the presence of sperm tail in the egg cytoplasm. As in wild type, the nuclei are well contoured in the newly deposited Ketel^D eggs, suggesting compact nuclei (Fig 1A and Fig B). The four haploid nuclei, products of the meiotic divisions, appear compact in the unfertilized Ketel^D eggs deposited by virgin Ketel^D/+ females. The bundle of microtubules (MTs) between the two inner haploid nuclei is most likely the central spindle pole body that persists following the second meiotic division, a phenomenon never seen in wild-type eggs, whether fertilized or not (Fig 1B; PURO 1991 ; RIPARBELLI and CALLAINI 1996 ). Severe Ketel^D-related defects appear 6–7 min after fertilization, during the commencement of embryogenesis when the female and the male pronuclei become juxtaposed (Fig 1, C–E). In wild type, the daughter centrosomes separate and move around the perimeter of the male pronucleus to the opposite pole and organize the first mitotic spindle (Fig 1D and Fig F; FOE et al. 1993 ; CALLAINI and RIPARBELLI 1996 ). Two nuclei form shortly in wild-type eggs and cleavage divisions commence and follow with a cycle of 8–10 min (Fig 1H; FOE et al. 1993 ). In 6- to 7-min-old fertilized Ketel^D eggs the female and the male pronuclei are poorly contoured and suggest nuclear envelope (NE) defects (Fig 1C and Fig E). As a rule, disorganized masses of MTs form instead of the gonomeric spindle. The MT mass appears as a prominent sperm aster and persists for several minutes (Fig 1E and Fig G). The centrosome replicates; however, the daughter centrosomes cannot separate in absence of intact NE (Fig 1G). The chromosomes fail to segregate in absence of the gonomeric spindle and disintegrate within minutes (Fig 1G and Fig I). The centrosomes may replicate two to three times but instead of separation they organize rudimentary asters of MTs along with a general decay of the egg cytoplasm. (Behavior of the polar body nuclei was not different in wild-type and in Ketel^D eggs and served as a reference for timing the initial events of embryogenesis.) The Ketel^D mutant phenotype is identical for the four Ketel^D alleles.

View larger version (130K): In this window In a new window Download PPT slide   Figure 1. The first embryonic division cycle in Feulgen-Giemsa-stained egg squashes of wild type (A, D, F, and H) and KetelD1/+ females (B, C, E, G, and I). The left and right panels correspond to roughly identical stages as inferred from appearance of the polar body nuclei. Thin arrows point to centrosomes. (A) Female and male pronuclei come to lie side by side following fertilization. The centrosome is attached to the NE of the male pronucleus. The three polar body nuclei are shown on the right. The well-defined nuclei imply the presence of intact NE. (D) In wild type one of the daughter centrosomes migrates to the opposite pole along the male pronuclear envelope; the other remains attached to the sperm tail (arrows). (Three polar body nuclei are shown on the right.) (F) The first mitotic spindle (that is gonomeric in Drosophila) in wild type. The maternal (right) and paternal (left) sets of chromosomes are separated. (Chromosomes of the polar body nuclei are shown on the right.) (H) Two zygotic nuclei form in wild type following completion of the first cleavage division. The centrosomes have already replicated and are about to become separated. (B) Four well-contoured meiotic products in an egg of a virgin KetelD1/+ female. The dark structure most likely represents the central spindle pole body, a remainder of the meiotic divisions. (C) The pronuclear stage in a fertilized KetelD1/+-derived egg. The fuzzy nuclei suggest decomposing NE. (Only one polar body nucleus is shown on the right.) The sperm-tail-associated sperm aster, which disassembles in wild type, persists. (E) NE of the pronuclei seems to be missing. The sperm aster persists. (G) Centrosome migration does not take place in the KetelD1/+-derived eggs and bipolar spindles fail to form. The chromosomes embedded in the microtubular aggregate disintegrate. Enlarged polar body chromosomes are shown in insert on the right. Arrowheads point to sister chromatids and refer to chromosome replication during the pronuclear stage. (I) NE and nuclei never form in the KetelD1/+-derived eggs. While the chromatin (thick arrows) disintegrates the centrosomes pass through two to three rounds of replication but fail to separate and are surrounded by asters of microtubules. Bars, 10 µm.

The Ketel^D egg cytoplasm prevents cleavage nuclei formation:
Since the Ketel^D mutations have been known to be gain-of-function type (SZABAD et al. 1989 ), the above-described defects are most likely brought about by Ketel^D-encoded mutant gene products. To elaborate the above possibility we carried out egg cytoplasm injections:

1. First a sample of 40 pl Ketel^D egg cytoplasm was injected into one side of the presumptive head region of each of 59 wild-type cleavage embryos. (In the control, where wild-type cytoplasm was injected into wild-type embryos, only 5 of the 72 larvae showed minor head defects.) The corresponding head structures were invariably missing at the site of injection (Fig 2). The anterior structures were entirely missing following the injection of 300 pl Ketel^D egg cytoplasm per wild-type embryo (44 embryos).

   
   

   View larger version (95K): In this window In a new window Download PPT slide   Figure 2. Phase-contrast photomicrographs of the head of first instar larvae as seen in cuticle preparations. About 40 pl wild-type (A) or KetelD egg cytoplasm (B) was injected into the presumptive head region of wild-type cleavage embryos. Blastoderm cells that originate from the injected region give rise to the antennal (AntSO) and the maxillary sense organs (MxSO; JURGENS et al. 1986 ). Note the absence of sense organs on the side injected with KetelD egg cytoplasm (B). Bar, 50 µm.

2. In the second set of experiments, a small sample of cNLS-PE solution was first injected into newly deposited Ketel^D1 and (as control) into wild-type eggs. A sample of the cNLS-PE containing egg cytoplasm was subsequently injected into wild-type cleavage embryos and the fate of the red fluorescent cNLS-PE substrate was followed in a laser scanning microscope. Whether the cNLS-PE was introduced in wild-type or in Ketel^D egg cytoplasm, the cNLS-PE substrate readily entered the cleavage nuclei, implying that the Ketel^D-encoded molecules do not prevent nuclear import of the cNLS-PE substrate (Fig 3). In the control the cNLS-PE substrate was essentially homogeneously distributed during mitosis in the egg cytoplasm (Fig 3B). The cNLS-PE substrate highlighted the nuclei—which doubled in numbers—upon onset of the next interphase (Fig 3C). Following Ketel^D1 egg cytoplasm injections some nuclei appeared normal; however, many small nuclei appeared (Fig 3D). Both the small and the normal-sized nuclei entered mitosis (with some delay as compared to control); however, nuclei did not form at the end of mitosis as indicated by the homogeneously distributed cNLS-PE substrate in the egg cytoplasm (Fig 3F).

   
   

   View larger version (123K): In this window In a new window Download PPT slide   Figure 3. Import of the cNLS-PE red fluorescent substrate into nuclei of wild-type cleavage Drosophila embryos. The cNLS-PE substrate was coinjected with wild-type (A–C) or with KetelD1 (D–F) egg cytoplasm at positions shown by the arrows. Import of the cNLS-PE molecules into the nuclei was followed in a laser scanner microscope. A and D, B and E, and C and F photographs were taken 15, 22, and 29 min following cytoplasm injections, respectively. Bar, 100 µm.

The Ketel^D alleles are very strong antimorph mutations:
To find out whether the Ketel^D-encoded products participate in the same process as the wild-type counterpart (i.e., the Ketel^D alleles are antimorph mutations; MULLER 1932 ) or whether they disrupt a process in which the normal Ketel gene products are not involved (i.e., the Ketel^D alleles are neomorphs), we constructed females that, in addition to a Ketel^D allele, carried two or three normal Ketel gene copies and studied whether dominant female sterility can be overcome in the Ketel^D/+/+ and in the Ketel^D/+/+/+ females. Two systems were analyzed:

1. In the XXTp(2;Y)G; Ketel^D/CyO females the Tp(2;Y)G chromosome carried a normal Ketel gene as part of the second chromosome transposed onto a Y chromosome. The females were mated with XTp(2;Y)G; +/CyO males. About 1% of the eggs of the 446 XXTp(2;Y)G; Ketel^D2/CyO females turned brown, indicating the progression of embryogenesis to the stage of embryonic cuticle formation. [An analysis of the cuticles of the deceased embryos revealed gross cell death apparently without any preference to the different body regions (J. SZABAD, unpublished results).] In addition, 56 offspring descended from the total of 466 XXTp(2;Y)G; Ketel^D2/CyO females during the 3-wk test period. [The rate of offspring production was 5.6 x 10^-3 offspring/(female x day)]. Of the 56 offspring, 13 females carried the Ketel^D2 allele and were sterile. Analysis of the Ketel^D2/+/+ females showed the strong antimorph nature of Ketel^D2 and implies involvement of the Ketel^D2-encoded and the normal Ketel gene products in the same process. The Tp(2;Y)G chromosome did not reduce sterility imposed by the other three Ketel^D alleles: every egg remained white and not a single offspring descended from the 1196, 1341, and 945 XXTp(2;Y)G; Ketel^D/CyO females that carried the Ketel^D1, the Ketel^D3, and the Ketel^D4 alleles, respectively.

2. In the second set of experiments we made use of three types of seven Ketel⁺ (K⁺) transgenes linked to the X and to the third chromosomes (LIPPAI et al. 2000 ). Since effects of the transgenes were very similar, results related to only one of the X-linked K⁺ transgenes are presented. The transgene carries a 22-kb genomic fragment including the promoter and the structural parts of the Ketel gene. Flies homozygous for the transgene are fully viable and fertile. We constructed both K⁺/X; Ketel^D/+ and K⁺/K⁺; Ketel^D/+ females in which the wild-type:Ketel^D ratios were 2:1 and 3:1, respectively. One group of the females was mated with wild-type (X/Y; +/+), the other group with K⁺/Y; +/+ males (Table 1). As in the case of the Tp(2;Y)G chromosome, a slight reduction of female sterility was apparent in the K⁺/X; Ketel^D2/+ females: cuticle developed in 1% of the eggs and a few offspring descended (Table 1). Apparently offspring production of the K⁺/X; Ketel^D2/+ females significantly increased when they were mated with K⁺/Y; +/+ males showing contribution of paternal rescue of the mutant phenotype. Female sterility was further reduced in the K⁺/K⁺; Ketel^D2/+ females, especially when they were mated with K⁺/Y; +/+ males (Table 1).

As expected, one copy of the K⁺ transgene did not overcome female sterility brought about by the other three Ketel^D alleles, whether or not the K⁺/X; Ketel^D/+ females were mated with wild-type or with K⁺/Y; +/+ males (Table 1). The K⁺/K⁺; Ketel^D/+ females were also sterile when mated with wild-type males (Table 1). However, when mated with K⁺/Y; +/+ males, the K⁺/K⁺; Ketel^D1/+ and the K⁺/K⁺; Ketel^D3/+ females yielded a few offspring (Table 1). About 50% of the progeny females carried the Ketel^D allele. Results of the transgene experiments confirmed (i) the highly toxic nature of the Ketel^D-encoded gene products and (ii) that three of the Ketel^D alleles are very strong antimorph mutations. Furthermore, the paternal rescue of embryonic lethality shows expression and function of the zygotic Ketel gene during embryogenesis.

The ketel^r alleles reveal the zygotic requirement of the Ketel gene:
We generated, through second mutagenesis of the Ketel^D alleles, 27 loss-of-function ketel^r alleles. Twenty-five of the 27 ketel^r alleles are recessive zygotic lethal mutations. The most severe ketel^r/ketel^r homozygous and the ketel^r/- hemizygous combinations, including ketel^rX13/-, bring about death during the second larval instar. [The - symbol stands for ketel^rX32, a small deficiency that removes the Ketel and a few adjacent loci (ERDELYI et al. 1997 ).] The mutant larvae become sluggish and decease within a day without any apparent morphological defect. Since second larval instar death is the most severe defect associated with the ketel^r alleles, it most likely represents the complete loss-of-function mutant phenotype. The zygotic lethal nature of the ketel^r alleles clearly shows the zygotic requirement of the Ketel gene.

When paternally derived the Ketel^D mutations behave as the zygotic lethal ketel^r alleles, the Ketel^D/ketel^r and the Ketel^D/- larvae also perish during second larval instar and cannot be distinguished from the ketel^r/- ones.

Germline chimeras without a functional Ketel gene revealed important features of Ketel gene function:
To decide whether the short life of the ketel^r/- hemizygotes is made possible by the normal Ketel gene products provided by the +/- mothers, we constructed germline chimeras with normal soma and ketel^r/- female germline cells that lacked a functional Ketel gene (Table 2). The chimeras deposited normal-looking eggs. Although the eggs were fertilized, embryogenesis did not commence inside them due to the lack of cleavage nuclei formation following fertilization, and the defects were indistinguishable from those described for the Ketel^D eggs. Analysis of the germline chimeras revealed three features of Ketel gene requirement: (1) Function of the Ketel gene is not required in the female germline since the ketel^r/- cells are viable and are sources of normal-looking eggs; (2) since embryogenesis does not commence in eggs of the above chimeras, development of the ketel^r/- larvae to the second larval instar must be supported by the Ketel maternal dowry present in the egg cytoplasm [maternal support of embryogenesis is a rather general phenomenon in Drosophila (for a recent review see SZABAD 1998 ).], and (3) the identical phenotype seen in eggs of the above germline chimeras and in the Ketel^D eggs shows that the Ketel^D-encoded mutant gene products impede function of the normal Ketel gene products; i.e., the Ketel^D alleles are dominant-negative mutations.

To further clarify the function of the Ketel^D alleles, we constructed germline chimeras with normal soma and Ketel^D1/- germline cells (Table 2). As in the ketel^r/- germline chimeras, the Ketel^D1/- germline cells allowed proliferation of the female germline cells and were sources of normal-looking eggs. However, embryogenesis did not commence in their eggs due to the lack of cleavage nuclei formation.

Function of the Ketel gene is not required in the follicle cells:
The normal-looking eggs that derive from mosaic egg primordia with ketel^r/- germline cells and normal follicle cells may develop because (i) function of the Ketel gene is not required in the germline or (ii) the follicle cells compensate Ketel gene function absent in the germline. To distinguish between the above possibilities, we generated mosaic egg primordia in which some or all the enveloping follicle cells lacked Ketel gene function and the germline cells were normal. To produce the latter type of mosaic egg primordia, we X-irradiated ketel^rX13/Ugra larvae for the generation, through mitotic recombination, of ketel^rX13/ketel^rX13 follicle cell clones. Egg and progeny production of the eclosing females were monitored. Results of the experiment can be summarized as follows (Table 3): (1) Similar frequencies of the ketel^rX13/Ugra and the +/Ugra control females were mosaic and (2) the two types of mosaic females produced eggs with similar rates. However, the larvae hatched from eggs of the ketel^rX13/Ugra females with a reduced rate as compared to the control females (Table 3).

The ketel^null homozygous clones are fully viable on the wings and the tergites:
To further characterize the requirement of the Ketel gene in cell types of the soma, we analyzed clones of ketel^rX13 homozygous wing and tergite cells. The ketel^rX13 homozygous clones were induced through mitotic recombination in f^36a/Y; ketel^rX13/f⁺ ck pwn young third instar larvae. Most of the forked homozygous clones were homozygous for ketel^rX13. As summarized in Table 4, frequencies and sizes of the different types of clones were similar in the ketel^rX13 and in the control experiments in both the wings and the tergites. Several of the f clones included as many as 70–100 cells. More than seven rounds of cell divisions are required following mitotic recombination to reach clones of that size. Features of the f clones revealed that Ketel gene function is not required for life and function of the wing imaginal disk and the abdominal histoblast cells.

Expression pattern of the Ketel gene as revealed by a reporter gene:
To study the expression pattern of the Ketel gene, we generated four transgenes in which the Ketel promoter regulated expression of a lacZ reporter gene and analyzed ß-gal activities in embryos as well as in different larval and adult tissues. The reporter gene is expressed in the ovaries and its expression pattern in the ovaries and during embryogenesis is identical with that detected by RNA in situ hybridizations and by the anti-Ketel antibody. [For details on Ketel gene expression during oogenesis and embryogenesis see LIPPAI et al. 2000 .] The reporter gene is also expressed in the testes and sperm pump. However, the reporter gene is not expressed in the bulk of the adult tissues. (Some ß-gal activities appeared in the central nervous system. The blood-producing organ could not be included in the reporter gene analysis due to its intrinsic ß-gal expression.) In third instar larvae the reporter gene is intensively expressed in all the imaginal discs, in the larval gonads, in the imaginal ring cells of salivary glands, and in the ring gland (Fig 4). There was no ß-gal activity present in most larval cells, including the salivary glands, fat body, larval epidermis, larval musculature, and Malpighian tubules (Fig 4).

View larger version (81K): In this window In a new window Download PPT slide   Figure 4. Expression pattern of a Ketel promoter-regulated lacZ reporter gene in different tissues of late third instar larvae. Blue staining corresponds to expression of the Ketel gene as revealed through activities of ß-galactosidase molecules. The reporter gene is expressed in all the imaginal discs (id), in the larval ovaries (lo) and testes (lt), in imaginal rings of the salivary glands (ir), and in the ring gland (rg). The reporter gene is not expressed in the larval epidermis and the overlying muscles (lem), the fat body (fb), the salivary glands (sg), the central nervous system (cns), the gut (gt), and theMalpighian tubules (mt). (The slight staining in different sections of the digestive tract is due to endogenous ß-galactosidase activities.) Bar, 1 mm on A–C and 100 µm on D–H.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Ketel^D-encoded importin-ß molecules prevent cleavage nuclei formation:
Embryogenesis fails to commence in the Ketel^D eggs deposited by the Ketel^D/+ females: the Ketel^D-encoded mutant gene products prevent formation of the first zygotic nuclei. Instead of cleavage nuclei formation MT bundles and asters persist. As results of cytoplasm injections revealed, the defects are brought about by "toxic" components in the Ketel^D egg cytoplasm that "poison" wild-type cleavage embryos through the blocking of cleavage nuclei formation following cleavage mitoses.

In higher eukaryotes the nuclei disassemble upon the initiation of mitosis. The process is under the control of p34^cdc2, the cyclin-B-dependent mitotic kinase (GANT and WILSON 1997 ; MARSHALL and WILSON 1997 ; COLLAS 1998 ). Phosphorylation of the lamins, the nucleoporins, and the integral nuclear membrane proteins are important events of NE disassembly. Some of the disassembled nuclear membrane complexes disperse in the cytoplasm; others are stored in vesicles (MARSHALL and WILSON 1997 ). In Drosophila NE disassembly is complete only opposite the spindle poles. The nuclear pore complexes (NPCs) and the nuclear lamina dissociate from the rest of the nuclear membrane and so-called spindle envelopes form (FOE et al. 1993 ).

Formation of nuclei, as studied mostly in Xenopus egg extracts, begins with reassembly of the NE, a process proposed to occur in the following major steps (SUTOVSKY et al. 1998 ; ZHANG and CLARKE 2000 ). The process begins during telophase when Ran-GDP associates with chromatin (ZHANG et al. 1999 ). [Ran is a Ras-related G protein without membrane anchoring site (for a recent review see AZUMA and DASSO 2000 ).] The chromatin-associated Ran-GDP promotes (i) the binding of membrane vesicles to chromatin; (ii) the recruitment of RCC1 (regulator of chromatin condensation), a chromatin-associated nuclear protein that is the only known guanine nucleotide exchange factor for Ran; and (iii) the association of nucleoporins (GOLDBERG et al. 1997 ; GANT et al. 1998 ). RCC1 generates Ran-GTP from Ran-GDP, and Ran-GTP causes fusion of the vesicles and formation of a double nuclear membrane (GANT et al. 1998 ; ZHANG and CLARKE 2000 ). Formation of the NE with NPCs establishes conditions for resumed nuclear protein import and the formation of functional nuclei. The process proceeds in vitro where NE forms from egg cytoplasm extract components over the demembranated sperm chromatin (BURKE and GERACE 1986 ) in a process similar to NE assembly around the sperm chromatin during male pronucleus formation following fertilization (SUTOVSKY et al. 1998 ). As described recently by ZHANG and CLARKE 2000 , functional NEs form over Sepharose beads loaded with Ran-GDP in Xenopus egg extract in the absence of DNA or chromatin.

The intrinsic GTPase activity or Ran is activated by RanGAPs, the RanGTPase activating proteins. The RanGAPs are cytoplasmic and hence Ran-GTP is nuclear and Ran-GDP is cytoplasmic. Ran carries out its GTPase cycle to supply energy for the nucleocytoplasmic transport of macromolecules. During this cycle it shuttles between the nucleus and cytoplasm (MELCHIOR and GERACE 1998 ; AZUMA and DASSO 2000 ).

Two possible mechanisms seem feasible to explain the Ketel^D-related defects, i.e., the failure of cleavage nuclei formation following mitosis:

1. Since the Ketel gene encodes the Drosophila homologue of importin-ß, a component of nuclear protein import (LIPPAI et al. 2000 ), the Ketel^D-encoded protein molecules may simply block the import of molecules required for the formation of cleavage nuclei following cleavage mitoses. This possibility is supported by two observations: (i) Ketel^D egg cytoplasm and ovary extracts of Ketel^D/+ females (LIPPAI et al. 2000 ) do not disrupt already established NEs and (ii) the formation of miniature nuclei besides the normal-sized ones (Fig 3D). The cNLS-PE experiments do not rule out the above possibility. The cNLS-PE molecules may be imported into the nuclei through an alternative pathway (MATTAJ and ENGLMEIER 1998 ; PEMBERTON et al. 1998 ; GORLICH and KUTAY 1999 ). The possible existence of alternative nuclear protein import pathway(s) is supported by two findings described in this article: (a) Cells in which the Ketel gene is expressed function normally without Ketel gene product and (b) the Ketel gene is not expressed in most of the terminally differentiated and nondividing cell types.

2. Importin-ß (the Ketel protein) has been known to interact with importin-, Ran, and a number of nucleoporins during nuclear protein import, and the regions of interactions have been determined (KUTAY et al. 1997 ; WOZNIAK et al. 1998 ). In the case of human importin-ß, as in other members of the importin-ß, the Ran binding domain resides at the N-terminal region. Truncated importin-ß molecules lack the N-terminal sections and thus cannot bind Ran, can associate with NPCs, and have a dominant-negative effect on nuclear protein import as determined in the digitonin-permeabilized HeLa assay system (KUTAY et al. 1997 ); the NPC binding domain slightly overlaps the Ran binding domain and resides toward central regions of the protein. The importin- binding domain is located toward the C terminus.

Abnormal interactions between the Ketel^D-encoded molecules and components of the nuclear transport process may lead to the failure of cleavage nuclei formation. In fact, dominant-negative mutations in both Ran and RCC1 have been known to disrupt NE formation. The RanT24N (substitution of Thr at position 24 by Asn) dominant-negative mutant allele of Ran encodes a protein that is defective in nucleotide binding and profoundly disrupts NE assembly in the Xenopus laevis egg extract system (DASSO et al. 1994 ; ZHANG and CLARKE 2000 ). Similarly to RanT24N, the nonhydrolyzable guanosine 5'-triphosphate analogues have also been known to inhibit NE assembly through disrupted function of Ran, which has been known to play a key role in NE integrity and exit from mitosis (DEMETER et al. 1995 ; MACAULAY and FORBES 1996 ). The pim1-d1ts mutant allele of RCC1 of Schizosaccharomyces pombe prohibits the reestablishment of the NE following mitosis (KORNBLUT et al. 1994 ). Mutations in a number of nucleoporins have also been reported to lead to nuclear fragmentation in yeast (reviewed in CORBETT and SILVER 1997 ).

The disturbing effects on NE organization of the Ketel^D-encoded molecules is also supported by persistence of the MT bundles and asters seen in the Ketel^D eggs. We interpret the persistence of the MT bundles and asters by leakage of Ran-GTP from the nuclei through the inappropriately assembled NE. Ran-GTP has recently been reported to stabilize microtubule asters and promote spindle assembly (for reviews see KAHANA and CLEVELAND 1999 ; NISHIMOTO 1999 ; AZUMA and DASSO 2000 ). Evidently several types of molecules participate in NE disassembly and reassembly upon the onset and toward the end of mitosis and the Ketel^D mutations should help in understanding the role of importin-ß in the process.

The Ketel^D alleles are dominant-negative mutations:
Four observations show the gain-of-function nature of the Ketel^D mutations and that mutant gene products bring about the Ketel^D-related defects: (1) The Ketel^D egg cytoplasm is toxic: when injected into wild-type embryos it prevents formation of cleavage nuclei; (2) following the induction of +/+ clones (through mitotic recombination) in the germline of Ketel^D/+ females, the +/+ clones appear with reduced frequencies and with several days delay due to perdurance of the Ketel^D-encoded mutant gene products in the +/+ cells (SZABAD et al. 1989 ); (3) the gain-of-function features of the Ketel^D alleles can be eliminated during second mutagenesis and loss-of-function ketel^r recessive alleles are generated (SZABAD et al. 1989 ; ERDELYI et al. 1997 ); and (4) the deleterious effects of three of the four Ketel^D alleles can be reduced by the addition of normal (+) Ketel gene copies: a few offspring derive from the Ketel^D2/+/+/+ females. Interestingly, mating of the Ketel^D2/+/+/+ females with males that provided two wild-type Ketel genes significantly increased the rate of offspring production (as compared to the wild-type males that provided only one Ketel gene). In fact, the Ketel^D1/+/+/+ and the Ketel^D3/+/+/+ females yielded a few offspring only when their male partners provided two normal Ketel gene copies during fertilization. The paternal rescue effect on Ketel^D-associated maternal-effect lethality clearly shows expression of the Ketel gene during embryogenesis.

Analysis of the Ketel^D/+/+/+ females revealed the strong antimorph nature of the Ketel^D mutations and imply involvement of the normal and the Ketel^D-encoded mutant gene products in the same process (MULLER 1932 ). Since products of the antimorph mutations impede action of the normal gene products they are also called dominant-negative mutations. The gain-of-function and loss-of-function mutant phenotypes are expected to be identical in the case of strong antimorph mutations. [The Tomaj^D mutations, which identify the Tubulin67C gene of Drosophila, represent a typical example (MATHE et al. 1998 ).] Although the Ketel^D alleles are very strong dominant-negative mutations, the gain-of-function and loss-of-function mutant phenotypes are not identical: failure of the commencement of embryogenesis in the Ketel^D eggs vs. death during second larval instar of the ketel^r homo- and hemizygotes. When, however, the Ketel gene product is removed from the eggs (in eggs of the germline chimeras with normal soma and ketel^r/- hemizygous germline cells) the gain-of-function and the loss-of-function Ketel mutant phenotypes become identical: they lack cleavage nuclei formation. Obviously, the normal Ketel gene products, as part of the maternal dowry provided by the +/- mothers, support life of the ketel^r/- hemizygotes up to second larval instar when they perish.

The Ketel^D-related defects are brought about following interaction of the Ketel^D-encoded molecules with maternally provided partner(s):
In understanding the mode of Ketel^D action, the following facts should be considered:

1. The Ketel maternal dowry and the "toxic" nature of the Ketel^D egg cytoplasm imply deposition by the Ketel^D/+ mothers of both the normal and the Ketel^D-encoded mutant gene products into the egg cytoplasm.

2. Lethality of the ketel^r homo- and hemizygotes implies expression of the normal Ketel and the Ketel^D alleles during development. Yet the Ketel^D-encoded mutant gene products exert their deleterious effects only on cleavage embryos.

3. When paternally derived, the Ketel^D alleles behave as the ketel^r loss-of-function alleles: they do not disturb but also cannot support cell functions.

We interpret the deleterious effects of the Ketel^D mutations on cleavage embryos by interaction of the Ketel^D-encoded molecules with a maternally provided partner present in the egg cytoplasm but absent from the somatic cells. The complexes not only inhibit cleavage nuclei formation but also impede activities of the complexes composed from normal Ketel molecule(s) and the maternally provided partner. During cellular stages of development, when the maternally provided partner is absent, the Ketel^D-encoded gene products may well be present but cannot exert their deleterious activities in the absence of an appropriate partner and consequently the cells survive and function normally. Thus the Ketel^D mutations identify maternal function of a zygotically essential gene and the identified function, if manifested, following interaction with a maternally provided partner. The maternally provided component that interacts with the Ketel and the Ketel^D-encoded molecules remains to be identified.

Requirement and expression patterns of the Ketel gene indicate parallel nuclear protein import pathways:
We assumed that Ketel gene function was required in every cell for the import of cNLS-containing nuclear proteins. Surprisingly, cells in the female germline, follicle epithelium, and wing and abdominal histoblasts are viable and function normally without the Ketel gene. The following possible explanations may be considered to account for viability of the above cells:

1. Perdurance of the normal Ketel gene products in clones of cells without a functional Ketel gene is not likely the reason for survival of the cells for the following reasons. In female germline chimeras the pole-cell-derived normal Ketel gene products are expected to decay and/or dilute between pole cell formation and egg production. Even if they survived it is unlikely that they can support the immense biochemical activities associated with egg cytoplasm production. It is also rather improbable that perdurance of the normal Ketel gene products supports life of the wing disc cells over more than seven rounds of cell divisions following the induction of mitotic recombination.

2. Nonautonomy of the lack of Ketel gene function (i.e., the supply with normal Ketel gene products of the mutant cells by the normal cells) is also unlikely since the 94-kD Ketel protein lacks signal sequences required for excretion and uptake from and into cells.

3. Most likely parallel nuclear import pathways substitute lost Ketel gene function and proteins normally imported through the importin-ß route are imported into the nucleus through another system. The human ribosomal protein L23a, for example, can be imported through at least four routes (JAKEL and GORLICH 1998 ). Similarly, transportin, a member of the importin-ß superfamily, recognizes and assists import of rather different types of proteins into the nucleus (reviewed in GORLICH and KUTAY 1999 ). However, function of the Ketel gene must be essential in some cell type(s) since the ketel^rX13/- hemizygotes die during second larval instar. "Focus," i.e., the cell type in which function of the Ketel gene is essential, remains to be elucidated.

4. It is also possible that the Ketel gene is not expressed in the studied cell types. To clarify this possibility, we constructed a reporter gene in which the Ketel promoter regulated expression of the lacZ reporter gene. In adult females ß-gal activities appeared in the ovaries, including the female germline and the follicle cells. Expression patterns of the reporter and the Ketel genes—as determined by in situ hybridizations and Western blot analysis—were in harmony (see also the accompanying article by LIPPAI et al. 2000 ). In harmony with the genetic data, the egg cytoplasm was loaded with ß-gal and there was ß-gal activity present in virtually every cell of the cleavage and cellular embryos. However, except for minor ß-gal activities in the brain, no other cells in the adult females possessed ß-gal activities. In late third instar larvae ß-gal activity appeared in imaginal discs, larval gonads, imaginal rings of the salivary glands, and in the ring gland. However, the other larval tissues did not possess ß-gal activity. It thus appears that the Ketel gene is expressed, with exception of the ring gland cells, in certain types of the mitotically active cells. (The neuroblast cells in the central nervous system and the histoblast cells, for example, do not possess ß-gal activity.) The role of the Ketel protein in cell cycle progression remains to be elaborated. Interestingly, the Drosophila importin-ß coding (Ketel) gene is not expressed in the mitotically quiescent cells. This observation raises questions about import of cNLS-containing nuclear proteins in, e.g., the larval cells.

	ACKNOWLEDGMENTS

We thank Révész Kati and Kissné Ani for excellent technical help and our colleagues Drs. I. Belecz, E. Máthé, J. Mihály, Z. László, and A. Udvardy for stimulating discussions. We are grateful to Drs. A. Garcia-Bellido for the f⁺/ck pwn system and A. Shearn for the y⁺CyO chromosome. We express our gratitude to Dr. David Glover, who organized support through the Préadhésion pour les Dix Pays d'Europe Centrale et Orientale No. CEC ERB CIPD CT 94 0049 EC Cell Cycle Network program. Support for the "Ketel project" came from the following additional sources: OTKA 922, OTKA T5537, OTKA T32540 from the Hungarian National Science Foundation, FKFP grant 1348/1997 from the Hungarian Education and Science Foundation and the Poland and Hungary: Action for the Restructuring of the Economy-ACCORD Program No. H-9112-0528.

Manuscript received January 27, 2000; Accepted for publication September 6, 2000.

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SUT