Isolation and characterization of dominant female sterile mutations of Drosophila melanogaster. II....

Copyright zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA0 1989 by the Genetics Society zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof America

Isolation and Characterization of Dominant Female Sterile Mutations of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBADrosophila melanoguster. 11. Mutations on the Second Chromosome

Janos Szabad,*,? Miklos Erd&lyi,*, Gyula Hoffmann,* Janos Szidonya* and Theodore R. F. Wright*

*Institute zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Genetics, Biological Research Center of the Hungarian Academy $Sciences, H-6701, P.O. Box 521, Sze ed, Hungary, ?Howard Hughes Medical Institute, Department of Biology, The University of Utah, Salt Lake City, Utah 84112 and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAf Department of

Biology, University of Virginia, Charlottemille, Virginia 22901 Manuscript received January zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA18, 1989

Accepted for publication April 14, I989

ABSTRACT Twenty-four, second chromosome, dominant female sterile zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(Fs) mutations in Drosophila are de-

scribed. Fs(2) were isolated at a frequency of approximately l per 1000 EMS-treated chromosomes screened. In comparison the isolation of frequency for second chromosome zygotic recessive lethal mutations was approximately 550 per 1000. Complementation analysis of the Fs(2) revertants showed that the 24 Fs(2) mutatations identify 13-15 loci, calculated to be about 65-75% of the second chromosome genes EMS mutable to dominant female sterility. Two of the Fs(2) mutations are useful tools for the dominant female sterile technique: Fs(2)I for induction and detection of germ-line clones and Fs(2)Ugra for follicle cell clones. Several of the Fs(2) mutations bring about novel mutant phenotypes. Seven of them alter egg shape, whereas the others arrest development primarily at two stages: around fertilization by five Fs(2) and during cleavage divisions [by Fs(2) in three loci]. The remaining that allow development to the larval stage of differentiation include four new dorsal alleles and one dominant torso allele. Analysis of germ-line chimeras revealed that with two exceptions all the Fs(2) mutations are germ-line dependent. The Fs(2) mutations were mapped mainly on the basis of mitotic recombination induced in the female germ-line cells of adult females. That most of the Fs(2) may be gain-of-function mutations is indicated by the unusual behavior of the Fs+ germ-line clones and also by the fact that 90% of the could be induced to revert.

M UCH of our understanding about oogenesis and maternal control of embryonic pattern forma-

tion in Drosophila melanogaster stems from studies of female sterile mutations of which only a few are dominant (for reviews see KONRAD et al. 1985; AKAM 1987; NUSSLEIN-VOLHARD, FROHNOFER and LEH- MANN 1987; INCHAM 1988). A few dominant female sterile (Fs) mutations are gain-of-function alleles of the recessive maternal-effect lethal (mel) mutations dorsal, Toll, easter and torso, and their analysis, along with the analysis of the BicaudalC and BicaudalD mutations, led to a basic understanding of the role of maternal control in the establishment of the dorso- ventral and the anterior-posterior embroyonic polar- ities (NUSSLEIN-VOLHARD 1979; NUSSLEIN-VOLHARD et a l . 1980;ANDERSON, BOKLA AND NUSSLEIN-VOL- HARD 1985;ANDERSON, JURGENS and NUSSLEIN-VOL- HARD 1985; ANDERSON and NUSSLEIN-VOLHARD 1986; MOHLER and WISCHAUS 1986; KLINGLER et a l . 1988). In an experiment in which about 60% of the genes EMS-mutable to dominant female sterility on the third chromosome were identified, six new loci were identified that play a role in the establishment of the anterior-posterior embryonic polarity along with and one new locus that affects dorso-ventral

Generics 122 823-835 (August, 1989)

polarity (ERD~LYI and SZABAD 1989). Although these mutations have not been the subject of thorough analysis yet, they are expected to reveal further important features of the control of embryonic pattern formation. Similarly, a few of the Fs(2) mutations identify as yet unknown genes that upon analysis may reveal new aspects of genetic control of oogenesis and embryogenesis. Several of the dominant female sterile mutations identify important genes that escape screens designed to isolate recessive female sterile mutations. A further attractive feature of the Fs mutants is that the isolation of dysgenic revertants will facilitate the subsequent cloning of the gene a manner similar to that for Toll (HASHIMOTO, HUDSON and ANDERSON 1988).

Three of the Fs mutations, Fs(2)D, Fs(I)K1237 (also known as zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAovoD1) and Fs(2)l are strictly germ-line dependent and are essentially agametic (YARGER and KING 197 1; KOMITOPOULOU et al. 1983; BUSSON et al. 1983; SZABAD, ERD~LYI and SZIDONYA 1987). They have been useful tools in analysis of gene expression in female germ-line cells by facilitating the identification of germ-line clones homozygous for recessive mutations: the so-called dominant female sterile technique (WIESCHAUS 1980; PERRIMON and GANS 1983).

824 J. Szabad zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet al.

In this procedure, mitotic recombination is induced in females trans-heterozygous for either a recessive female sterile zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(fs) or zygotic lethal zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA( 1 ) mutation and one of the above zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs mutations. Subsequently, development of the induced mosaic egg primordia is fol- lowed in germ-line cells that are Fs-free but homozy- gousfs or 1 and that are surrounded by phenotypically normal soma. These studies have implicated the genetic requirement for most of the genome for the normal development of female germ-line cells zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(GAR- CIA-BELLIDO and ROBBINS 1983; PERRIMON and GANS 1983; PERRIMON, ENGSTROM and MAHOWALD 1984a,b, 1985a,b; NUSSLEIN-VOLHARD, KLUDING and

JURGENS 1985; SCHUPBACH and WIESCHAUS 1986; WIESCHAUS and NOELL 1986; TAUBERT and SZABAD 1987; SZABAD, REUTER and SCHRODER 1988). Some of the newly isolated Fs(2) mutations may be useful tools for the dominant female sterile technique and thus facilitate the analysis of expression of genes located on the second chromosome.

With the above perspectives in mind we isolated zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA24 Fs(2) mutations. The complementation analyses of their revertants indicate that they represent 13-15 loci, which have been calculated to be 65-75% of the genes on the second chromosome ethylmethane-sul- fonate (EMS)-mutable to dominant female sterility. One of them, Fs(2)1, is a good tool for the identification of germ-line clones. Another, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs(2)Ugra is an agametic, follicle cell dependent mutation, which should be a useful tool in the study of gene expression in follicle cells. Six of the 24 Fs(2) mutations alter egg shape, and one leads to formation of weak egg shell. Fifteen of the 24 Fs(2) mutations allow deposition of normal shaped eggs. Embryogenesis is arrested shortly after its initiation in eight cases. In seven of the Fs(2) mutations, embryogenesis can proceed up to the larval stage of differentiation. Five of the Fs(2) mutations are allelic to previously described maternal effect mutations, i e , four are dorsal alleles and one is a torso allele. In addition, one appears to be a dominant Bicaudal allele and yet another one also interferes with the establishment of anterior-posterior embryonic polarity. Several of the Fs(2) mutations produce novel mutant phenotypes.

In this paper we describe the genetic and developmental characterization of 24 new Fs(2) mutations, including isolation, brief mutant phenotyps, mapping, the effects on female germ-line cells, reversion and complementation analyses.

MATERIALS AND METHODS

Mutagenesis: The Fs(2) mutations were induced by EMS or in the case of dlD6, dlD7 and Etre by a combined treatment by EMS and 4000 R of gamma-rays (WRIGHT et al. 1982) on the following isogenic chromosomes: wild-type (Ore-R, Canton-S or the second chromosome from the strain red e),

pr or It bw. (For a description of the marker mutations, the CyO, SM5 and Zn(2LR)Pm chromosomes as well as the T( 1;2)Bld and T( 1;2)0R64 translocations see LINDSLEY and GRELL 1968.) For the matagenesis, adult males were fed with a 25 mM EMS solution for 8 hr (LEWIS and BACHER 1968). Populations of the EMS-treated males were mated with females carring the SM5 balancer chromosome.To avoid isolation of clusters of Fs(2) mutations, the males were discarded 3-5 days following matings. Single SM5 carrying males, each representing one mutagenized second chromosome, were mated with CyO/Zn(2LR)Pm females. Since the SM5/CyO and the SM5/Zn(2LR)Pm combinations are lethal, the only surviving progeny will carry the mutagenized chromosome over the Cy0 or the Zn(2LR)Pm balancer chromosomes. These flies were transferred into fresh vials and tested for fertility. The Cy0 carrying males were isolated from vials in which larvae did not develop. These males were mated with Bc Gla/Cy Roi females and their progeny used to confirm dominant female sterility. The Cy Roi progeny males were subsequently used to establish strains in which T( 1;2)Bld/T( 1;2)0R64 females are mated with Fs(2)/ T( 1;2)Bld males (YARGER and KING 197 1). (The T( 1;2)0R64 translocation is male lethal; LINDSLEY and GRELL 1968.)

Analysis of the mutant phenotype: Three experimental procedures were used. (1) When the Fs-carrying females deposited abnormal eggs, these were washed in a detergent solution, fixed in acetic acid:glycerol (1: 1) mixture for 1 hr at 60", mounted in Hoyer's medium and cleared at 60" for 1 day (WIESCHAUS and NUSSLEIN-VOLHARD 1986). (2) Em- bryos of Fs(2)/+ females that did not develop to the larval stage of differentiation were analyzed according to a procedure described by HANDKE-KOCIOK and LIEBRICH (1 986) and WIESCHAUS and NUSSLEIN-VOLHARD (1986). Eggs were collected from Fs(2)/+ females for zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA4 hr (25"), dechorionated in NaOCl solution, washed, rinsed in heptane (to render the vitelline membrane permeable), fixed in a mixture of methano1:acetic acid (3:l) for 30 min, stained in a g/ml diaminophenylindole (DAPI) solution for labeling the nuclei and analyzed in a fluorescent microscope. (3) When the embryos developed to the larval stage of differentiation eggs were collected, dechorionated, washed, mounted in Hoyer's:lactic acid 1: 1 medium and cleared for 1 day at 60" (WISCHAUS and NUSSLEIN-VOLHARD 1986). The only structures remaining after this preparation procedure are those made of larval cuticle. The larval cuticle preparations were analyzed according to LOHS-SCHARDIN, CREMER and NUS- SLEIN-VOLHARD (1979), JURGENS et al. (1986) and JURCENS (1987).

Mapping of the Fs(2) mutations: Because the Fs(2) mutations do not give rise to offspring and there is no recombination in the Drosophila males, methods that make use of induced mitotic recombination were employed for localization of the Fs(2) mutations. Mitotic recombination was induced in 3-5-day-old Fs(P)/al dp b pr c p x sp or Fs(2)lal nub It stw sca sp females by 1500 R of X-rays. The females had been mated with a1 dp b pr c p x sp or a1 nub It stw sca sp males prior to irradiation to stimulate ovulation (6 SZABAD and FAJSZI 1982). Lots of 10 females (5 in the case of 4 of the Fs(2) mutations) and 10-1 5 males were placed in vials and inspected for the production of offspring. To identify most of the mosaics the vials were screened for 2 1 days and those with offspring were kept for several more days. Fe- males surviving for 21 days were counted in order to doc- ument the longevity of the Fs(2)-carrying females.

Mitotic recombination can lead to the formation of Fs- free cells following irradiation of the adult females (Figure 1). If Fs-free oogonial stem line cells continue development, their genotype can be inferred following test crosses. Wild-

Fs Mutations of Drosophila zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA825 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAMitotlc zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA-‘e recombination

Daughter cells

FIGURE 1 .-Mitotic recombination between the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs mutation and the centromere leads to the formation of an Fs-free daughter cell zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(*). The symbols zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa, b, c and d stand for recessive marker mutations. Their wild-type alleles, located on the Fs-carring chromosome delineated by thick line, are not shown. Wild-type allele (+) of the Fs mutations is shown. A, Mitotic recombination between the centromere and b. B, Mitotic recombination between Fs and b. For details see text.

type and mutant alleles of the marker mutations located on the chromosome arm opposite to that of the Fs mutation should appear in the progeny at a 1: 1 ratio. On the other hand, while wild-type alleles of the marker mutations located between the Fs mutations and the centromere may appear in the test cross progeny, no wild-type alleles of those markers located distal to the Fs mutation are expected to be recovered among the offspring of the irradiated Fs- carrying females (Figure 1). Therefore, an interval delineated by two marker mutations can be determined for every Fs mutation (see also BUSSON et al. 1983; SZABAD, ERD~LYI and SZIDONYA 1987; ERDLLYI and SZABAD 1989). Trans- mission of the Fs allele to the offspring of the irradiated females is not expected if the Fs mutation represents a gene whose function is required for germ-line development.

When the Fs phenotype is the consequence of altered somatic functions in the female, the Fs-free somatic clones may allow the production of Fs-bearing and Fs-free eggs which on fertilization can develop into adult progeny. In this case, however, wild-type and mutant alleles of all the marker mutations are expected to appear in the 1 : 1 ratio in the offspring of the irradiated females and, in addition, half the offspring should carry the Fs allele (SZABAD AND HOFF-

In the case of Fs(P)Ugra, Ugrallt bw individuals were X- irradiated with 1000 R at various stages during development: at the blastoderm stage (2.5-3.5 hr after egg laying, hAEL), at the end of the second/third instar boundary (70- 74 hAEL), 1 day after pupariation (142-146 hAEL) and also 5-day-old adults. The eclosing females were mated with It bw males and individually tested for egg production for at least 24 days.

For torsoD402’, mitotic recombinations were induced in spermatogonial cells of Fs(2)al dp b c p x sp old larvae and/ or young pupae by irradiation with 1500 R of X-rays. The testes are filled mostly with primary spermatocytes at these stages (BODENSTEIN 1950; LINDSLEY and TOKUYASU 1980). The eclosing males were test-crossed with a1 dp b p r c p x sp females. The recombinant female progeny were isolated and screened for fertility.

Germ-line and ovarian chimeras: Germ-line chimeras were constructed by transplantations of pole cells (LEHMANN and NUSSLEIN-VOLHARD 1986). Pole cells of embryos produced by al dp b pr c p x sp or cn bw sp females mated to

MANN 1989).

Fs(P)/Cy Roi males were transplanted into embryos originat- ing from wild-type (Ore-R) females mated to zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs( IKI.237 males. Fs(IK1237 (=K1237) is an agametic, strictly germ- line dependent dominant female sterile mutation (BUSSON et al. 1983; KOMITOPOULOU et al. 1983). The +/K1237 females were mated with a1 dp b pr c p x sp or cn bw sp males. When an Fs(2) mutation proved to be non-germ-line dependent, pole cells of y v f mal embryos were implanted into embryos that derived from a1 dp b pr c px sp or cn bw sp females and Fs(Z)/Cy Roi males. The host females were mated withy v f mal males. The mal homozygous cells lack aldehyde oxidase activity and do not stain in the histochem- ical procedure described by JANNING (1972).

Ovarian chimeras were constructed to determine whether the foci of the Fs(2) mutations were located in the ovaries or in other parts of the soma (EPHRUSSI and BEADLE 1936). Ovaries of Fs(Z)/al dp zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA6 pr c p x sp or Fs(P)/cn bw sp larvae were transplanted into +/K1237 host larvae. The donor larvae originated from crosses between al dp b pr c $x sp or cn 6w sp females with Fs(P)/Bc Gla males. Bc is a dominant larval marker mutation that allowed identification of the Fs(2)-carrying larvae (GRELL 1969).

Reversion: For induction of revertants of the Fs(2) mutations, populations of Fs(P)/Cy Roi males were irradiated by 4000 R of X-rays (150 kV; 0.5 mm AI filter, 1000 R/ min) or fed with 25 mM EMS solution for 8 hr. The males were subsequently mated with Bc Gla/Cy Roi females and discarded 3-5 days after matings. The Fs(P)/Cy Roi progeny females were mated with Bc Gla/Cy Roi males in lots of 25. Only females carrying Fs(2) revertants (or suppressors) will give rise to offspring. Females in a lot were individually tested when evidence of a reversion of an Fs(2) mutation was noticed. The revertants were used in inter se complementation assays and also in tests for allelism with known recessive maternal-effect lethal (mel) mutations that change egg morphology or alter the embryonic pattern, i e . , gurken, torpedo, Bicaudal, dorsal, exuperantia, stauffen, torso, trunk, tudor, vasa and valois (SCHUPBACH 1987; MOHLER and WIFS-

and WIESCHAUS 1986). CHAUS 1986; NUSSLEIN-VOLHARD et al. 1980; SCHUPBACH

RESULTS

Recovery of the Fs(2) mutations: The 24 Fs(2) mutations were derived from three sources. Etre, Hont, dlD6 and dlD7 were isolated in T. R. F. WRIGHT’S laboratory, Billa was kindly provided by G. REUTER and all the others were recovered in screens in Szeged. Most of the described Fs(2) mutations were induced by EMS in lethal-free chromosomes: wild types of different origin or those labeled with recessive marker mutations (Table 1). In Szeged, 0.1% (20/18,723 i.e., 1 in 936) of the chromosomes carried an Fs(2) mutation. Except for one, all these mutations could be established as stocks and are kept as described by YARGER and KING (1 97 1): females trans-heterozygous for the reciprocal translocations T( 1;2)Bld and T( Z;2)OR64 are mated with Fs(2)/T( Z;2)Bld males. (The T(1;2)0A64 translocation is male lethal; LIN- DSLEY and GRELL 1968). Symbols of the Fs(2) mutations are listed in Table 1. Although Himca and Tekele, Bursa and Hont, Ketel and Told were recovered in the same mutagenesis experiment, they do not represent

826 J. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBASzabad et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAal.

clusters since the mutant phenotypes are different and they map to different chromosome intervals.

Out of a sample of 984 EMS-treated chromosomes marked with It and bw that were assayed for lethal mutations, 549 were homozygous viable. It was estimated, using a Poisson distribution assuming that the lethal mutations are randomly distributed among the EMS-treated chromosomes, that each mutagenized chromosome carried on the average 0.6 lethal mutation. Thus the relative frequency of Fs(2) mutation induction zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAus. zygotic lethal induction is 1:546.

Nine of the Fs(2) mutations significantly reduce the longevity of the carrier females (Table 1). These mutations also bring about a slight reduction in male fertility.

Phenotype zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs(2) mutations: Fxcept for Vaja, all the Fs(2) mutations are fully penetrant in all of the 7 genetic backgrounds so far tested. Of the 24 mutations, Ugra is the only agametic mutation. Develop- ment of the egg primordia proceeds normally up to about stage 10 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(6 KING 1970) with the normal number of follicle cells surrounding the oocyte and its 15 sister nurse cells. The follicle cells migrate over the oocyte, and vitellogenin uptake commences. Subse- quently the nurse cell nuclei become pycnotic with heavily condensed chromatin, and the egg primordia degenerate (Figure 2).

Egg shape mutations: Seven of the Fs(2) mutations alter egg shape. The Fs(2)Z-carrying females deposit very few, mostly flaccid eggs that never bear dorsal chorionic appendages (SZABAD, ERD~LYI and SZI- DONYA 1987). Females that carry any of the Bursa alleles deposit eggs slightly shorter than normal with short, wide dorsal appendages. These dorsal appendages are often fused at the base of their origin, which is shifted to a position more posterior than the wild- type position (Figure 3). While embryogenesis very seldom starts in Bursa’, Bursa’ and Barsa3-derived eggs, embryos develop to the larval stage of differentiation in several of the Bursa4-derived zygotes. This reflects the slight variability of the mutant phenotype of Bursa4. These embryos often appear normal with minor defects in the cephalo-pharyngeal skeleton. Eggs deposited by the Billa-carrying females appear very much like those produced by the more severe Bursa alleles. Eggs of the Dorog-carrying females are normal in size, but the dorsal appendages are strongly reduced. Eggs of the Etre females are normal in size with two normal sized but very thin appendages. The chorion cell-imprint pattern is very weak most likely because the chorion of the Etre-derived eggs is much thinner than in wild type.

Early arrest in development: Mutations to be discussed in this section allow deposition of apparently normal eggs. However, development is arrested at different stages in these eggs. Meiotic defects, as vis-

ualized by the lack or abnormal formation of polar body and oocyte nuclei in the DAPI-stained eggs, are apparent in eggs of the Hont, Ketel’ and Kete13 females. About 50% of the Ketel’lCy Roi-derived eggs appear unfertilized, but cleavage divisions can proceed as far as the syncytial blastoderm stage in the other 50%. We also observed that expressivity of the Ketel’ mutant phenotype varies by changing the genetic back- ground. For example, while typical Ketel’ phenotypes develop and no adult offspring are produced when females carry chromosomes from the strains Bc Gla/ Cy Roi, rucuca, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa1 nub It stw sca sp,3-60% of the females in Canton-S, Oregon-R or a1 dp b pr zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAc px sp backgrounds will give rise to a few adult offspring. The Himca- carrying females deposit eggs that are fertilized as shown by the presence of five haploid nuclei. How- ever, no further development takes place in these

eggs- Abnormalities develop during cleavage divisions in

the eggs derived from Kompolt, Tarhos or Tekele-carrying females. In the eggs of Kompolt females 20-28 nuclei are found in the egg cytoplasm (Figure 4). In eggs of Tarhos females cleavage nuclei are absent in the posterior 10% of the egg cortex. In eggs of Tekele females the cleavage nuclei are not uniformly distributed.

Larval cuticle producers: Seven of the 24 Fs(2) mutations allow development to proceed to the larval stage of cuticle differentiation. In virtually 100% of the eggs derived from any of the four new dorsalD alleles, dlD4, dlD5, dlD6 and dlD7, mutant phenotypes are found with range from embryos with disarranged head skeleton to those with the “tail up” phenotype (cf. NUSSLEIN-VOLHARD 1979; NUSSLEIN-VOLHARD et al. 1980). F ~ ( 2 ) t o r s o ~ ~ ~ ~ ’ is a dominant allele of the recessive maternal-effect mutation torso (SCHUPBACH and WIESCHAUS 1986). Embryonic development proceeds normally to the blastoderm stage in the torso- D402’-derived eggs. At 18” only the most posterior larval structures develop. At higher temperatures, posterior abdominal segments and also the anterior head segments may develop in a fraction of the embryos (KLINGLER et al. 1988). Severe head defects are found in about 15% of the Told-derived eggs: the anterior segments are often missing and are very distorted when present. Embryos deriving from Vaja- carrying females exhibit a wide range of head defects ranging from almost wild-type with reduced mouth parts to those lacking the entire head. Vaja is incompletely penetrant and the degree of expression of the mutant phenotype depends on the genetic back- ground.

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828 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAJ. Szabad zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet al.

I : IGL .RE :3.-l)ark-field microgr;~phs of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAq g s laid by wild-tyx! zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(A) females and those that carried one of the following zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs(2) mutations Rilla (B). Barsa' (C), Dorog (D), Barsa' zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(E) or Barsa' (F and G). (A wild-type egg is about zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA500 nm long.)

to map the Fs(2) mutations and determine whether they alter the function of the germ-line or the soma.

The Fs(2)+ mosaicism showed three unexpected features. (1) For 16 of the 23 Fs(2) mutations studied, the frequency of mosaicism was much lower than the expected 17-32%. Only four of the Fs(2) mutations approached the expected values (Table 1 and APPEN-

DIX). (2) Eggs from which progeny developed were usually deposited 9- 14 days following clone induction in those F s ( 2 ) mutations producing clones at the frequency of >lo%, as was described in the case of the recessive egg shape marker mutation@( I ) K 1 0 (WIES- CHAUS and SZABAD 1979). For those Fs(2) mutations producing clones at less than a 10% frequency, a 2- 10-day delay in the deposition of progeny-producing eggs was noted. (3) Unusual progeny production pattern was observed for the majority of the Fs(2) mu-

FIGURE 2.-Micrographsof DAPI- ,tained (A and B) and semi-thin ,ections (C and D) of wild-type (A md C) and Fs(2)Ugra/+ (B and D) :gg primordia (bar = 50 nm).

tations. An Fs(2)+ oogonial stem line cell may directly follow the pathway of egg differentiation and be the source of a single egg or it may remain an undiffer- entiated stem cell giving rise to several eggs during the subsequent life of the female (NOTHIGER et al. 1978; WIESCHAUS and SZARAD 1979). Thus, a group of females with only one Fs(2)+ germ-line clone is to yield a single offspring in about half of the cases and several offspring in the other half. Of those groups of females with two clones, one-quarter should produce two progeny each, and theoretically three-quarters are expected to yield several, 23 offspring each. These considerations hold for the germ-line dependent Fs(2) mutations (WIFSCHAUS and SZABAD 1979; SZABAD and HOFFMANN 1989). The above offspring production pattern was observed only for Fs(2)1, Kete12 dlD4 and Told. All the other Fs(2) mutations produced many more single or two offspring progeny than expected (Table 1).

Germ-line chimeras: The low frequency of Fs(2)+ mosaicism, the unusual offspring production pattern as well as the occasional transmission of the Fs(2) allele to the offspring, as described in the Mapping section, raise the question of whether the dominant female sterility is the consequence of disturbed germ- line functions. For the evaluation of this question, germ-line chimeras were constructed in which Fs(2)/ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa1 d p b pr c p x sp or Fs(2)lcn b sp germ-line cells were surrounded by phenotypically normal soma. Except for 2 of the 20 Fs(2) mutations tested, the presence of only the mutant germ-line cells was sufficient for the dominant female sterility (Table 1). However, while larval cuticle developed in virtually 100% of the eggs derived from dlD4, dlD5 or Told heterozygous

Fs zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAMutations zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Drosophila 829

TABLE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA2

F'IGLXE 4.--Slicrographs of I ~ ~ l l ' l - s t a i ~ ~ ~ d rggs of \v i ld- type zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(;\) females and those produced by females that carried one of the following zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs(2) mutations Kompolt zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(R), Tarhos (C) or Tekele (D).

females, this ratio was only about 50% when the eggs derived from chimeras with mutant germ-line cells and normal soma ~.SZABAD, unpublished results). This observation indicates that in these mutations the germ-line and the soma are both involved in the generation of the mutant embryonic phenotype.

Ugra and Etre are not germ-line dependent. Of the nine chimeras recovered six carried Ugralal d p b pr c px sp germ-line cells, as was deduced from the phenotype of their progeny (Table 2A). Similarly, normal- looking eggs, from which progeny developed, derived from the Etrelcn bw sp germ-line cells when surrounded by normal soma (Table 2A). Consistent with the above results, no normal (y zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAv zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAf zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAmally v f mal) progeny were produced by any host Fs(2) /+ female even though four Ugra/+ and six Etre/+ host females were subsequently determined to carry chimeric ovaries. Homozygous y v f mal nonstaining germ-line cells were identified when their ovarioles were stained for aldehyde oxidase (Table 2B).

That the Ugra and Etre mutant phenotypes depend on the genotype of the ovarian soma was shown by analysis of ovarian chimeras. Typical Fs(2) mutant phenotypes developed when or Ugra/+ or Etre/+ larval ovaries were implanted into the +/KZ237 agametic hosts (3 and 5 ovarian chimeras, respectively). On the other hand, Ugra or Etre host females provided excellent milieu for the development of the implanted y v f mal ovaries (2 and 3 chimeras, respectively). Normal eggs derived from the implanted y v f mal ovaries and gave rise to y v f mal offspring.

X-irradiation of Ugrallt bw zygotes at various stages in development gave rise to several Ugrallt bw females with Ugra-free clones which permitted them to deposit eggs. The frequency of egg producing females was 3.8, 10.1 and 12.8% when blastoderm-stage embryos, late second/young third instar larvae or l-day- old pupae were irradiated and was as low as 1.3% following irradiation of adult females (Table 3). Features of mosaicism indicate a clonal origin of the eggs. For

Germ-line chimeras of Ugra and Etre A

Parents of embryos Host

Types of chimeras

Donor Host females' +'/Fs(2) +'/Cy Roi

+a/+. +h/+h

X + X 51 6 3 UgralCy Roi K1237/Y

EtrelCy Roi K I237/Y

B Parents of embryos Host females'

+a fFs(2) +=/Cy Roi

Donor Host Total Chimera Total Chimera zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA+"I+" + a / v

+d/Y UgralCy Roi

+"/+" +C/+C

+d/Y EtrelCy Roi

x + X 20 4 30 6

X + X 15 6 17 9

a = a1 d p b p r c p x sp; b = wild-type (Ore-R); c = cn bw sp d = y v f m a l ; e, these females were test-crossed with a1 dp b pr c px sp males in the case of Ugra and cn bw sp males for Etre; f, host females were mated with y v f m a l males.

example, fewer eggs were deposited when irradiation took place during more advanced stages, and longer screening time was required to identify egg-producing females. This was as long as 18 days when adult females were irradiated, and it took 24 days to determine that 5 females were producing a few normal eggs (Table 3). While approximately 95% of the eggs were apparently normal following irradiation of blastoderm-stage embryos or larvae with approximately 70% hatching, only about 50% of the eggs appeared normal following irradiation of adult females and only one in about 5 hatched (Table 3). The abnormal egg class included mostly flaccid eggs often with dissimilar dorsal appendages shorter than normal (cf: SZABAD and HOFFMANN 1989).

Mapping: Of the 24 Fs(2) mutations only Vaja is incompletely penetrant. About 30% of the Vajalal nub It stw sca sp females gave rise to a few offspring. Recombinant offspring females were isolated, following a test cross with a1 nub It stw sca males, and tested for the presence of the Vaja allele. Analysis of the recombinants located Vaja at 2-15.1 (1 3 recombinants between a1 and Vaja and 36 between Vaja and nub).

In the case of Ugra and Etre the chimeras with Fs(2)- carrying germ-line cells and normal soma gave rise to several offspring that were recombinants, which were tested for the presence of the Fs(2) allele. There were 3 d p Ugra b+, 4 dp+ Ugra+ b, 13 d p Ugra+ b+ and 19 dp+ Ugra b recombinants recovered that divided the dp-b interval at a 7/32 ratio. Relative to the site of the

830 J. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBASzabad zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet al. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBATABLE 3

Characteristics of Fs(Z)Ugra+ mosaicism

Females Days till Hatched Abnormal

Stage irradiated Screened Mosaics zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA% (average zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAir SD) clone/dav of clones' (% total) (% total) Eggs per female" EgpSl detection eggs eggs

Control zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA51 1 4 0.8 3.0 f 2.4 0.14 - 22.1 41.7 Blastoderm 1209 46 3.8 29.1 f 56.0 2.01 3.8 69.2 4.7 Young third inst. 1059 107 10.1 5.8 f 9.4 0.22 5.7 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA66.0 9.6 One-day old pupae 171 22 12.8 2.7 f 1.8 0.13 7.9 32.0 24.7 Adult 374 5 1.3 2.6 f 1.5 0.1 1 17.7 17.2 46.2

Individuals were irradiated (1000 R of X-rays) during different stages of development and screened for production of eggs following

' Throughout the test period of 24-28 days. eclosion.

Average from eclosion of the (irradiation in the case of the adult) females. For recovery of 95% of the mosaics, females had to be screened for 8, 12, 13 and 24 days in the case of the stages from the blastoderms on.

d p (2-1 3.0) and the b loci (2-48.5), Ugra was located at 2-19.4. Similarly, the 19 cn Etre bw+, 17 cn+ Etr+ bw, 43 cn Etre+ bw+ and 43 cn+ Etre bw recombinants placed Etre at 2-7 1.4.

Twenty-two of the Fs(2) mutations were mapped on the basis of the pnenotype of the progeny recovered following irradiation of the Fs(2) heterozygous females. As expected, mutant and wild-type alleles of the marker mutations located on one of the chromosome arms appeared at a roughly 1: 1 ratio placing the Fs(2) mutation on the other arm. On the other hand, mutant and wild-type alleles of the marker mutations located on the same chromosome arm as the Fs(2) mutation appeared with different frequencies (Table 4). For example, while there were 26 c and 46 c+, 29 p x and px+ 43 progeny flies recovered among the offspring of the irradiated Kompolt/al d p b pr c p x sp females, in the offspring 72 d p and 0 dp+, 66 b and 6 b+ were found, locating Kompolt in the dp- b chromosome interval (Table 4). Similarly, while 167 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa1 and only 5 al+ alleles were recovered among the offspring of the irradiated Fs(P)l/al bp pr c p x sf females, 133 d p and 39 dp+ alleles were found, locating Fs(2)l in the al-dp region (Table 4). In fact, no al+ were expected to appear. However, (1) double mitotic recombinations, which are more frequent than expected on the basis of the singles (GARCIA-BELLIDO 1972), (2) reversion of the Fs(2) alleles and/or (3) Fs- free mosaicism within germ-line components of the egg primordia (as will be discussed in a subsequent paragraph) may be responsible for the appearance of wild-type alleles of the marker mutations located distal to the Fs(2) mutations.

In the case of e.g., Kete12, mutant and wild-type alleles of all the marker mutations were found with rather similar frequencies among the progeny of the irradiated females providing no clue for the localization of Kete12 (Table 4). The reocombinant females were then recovered and analyzed for the presence of the Fs allele. Based on these studies Ketel' was localized in the interval delineated by the nub and sca marker mutations (Table 4).

In a number of Fs(2) mutations very few offspring could be recovered. These mutations were located on the basis of allelism with other Fs(2) mutations or known recessive female sterile stations (as described in a subsequent section).

In the case of torsoD4o2' mitotic recombinations were induced in spermatogonial cells of nub It stw sca sp old larvae and/or young pupae. Among 10,959 offspring, 44 recombinants were recovered and tested for the presence of the Fs(2) allele. Analysis of these recombinants placed torsoD4"' in the stw-sca interval where the torso locus is located (ScHupbach and WIESCHAUS 1986). The different mapping procedures allowed the localization of all the 24 Fs(2) mutations (Table 1).

Reversion of the Fs(2) mutations: Reversion of the Fs(2) mutations took place with rather different frequencies following either EMS or X-ray treatment of the Fs(2)-carrying chromosomes. While one in approximately 300 of the Tarhos or Vaja-carrying chromosomes reverted following EMS mutagenesis, only 1 in 1280 Bursa' or one in 6900 Ugra-carrying chromosomes were reverted (Table 1). Analysis of the revertants in outcrosses with recessive maternal-effect mutations and with revertants of other Fs(2) mutations revealed that several of them are partial revertants that retained some dominant female sterility. The partial revertants were especially frequent in the case of Told, Dorog and Kompolt. Revertants of 22 of the 24 Fs(2) mutations were isolated. The two exceptions include Billa and Tekele (Table 1).

Complementation analyses: Twenty-one of the 24 Fs(2) mutations were included in the complementation analyses. (Billa and Tekele were excluded due to lack of revertants and also Told because its revertants are partials.)

The combinations between any Fs(2) mutation and its revertants were lethal. Whether lethality is an inherent feature of the F~(2) /Fs(2)~~" combinations or is the consequence of second site lethal mutations present on the Fs(2)-carrying maternal chromosomes

Fs zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAMutations of Drosophila zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA83 1

TABLE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA4

Mapping the Fs(2) mutations

F@., Marker mutations and the number of phenotypically mutant and wild-type offspring

mutatlon Offspring analyzed zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAal/al+ dpjdp+ bjb+ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAPdP.’ c/c+ pxjpx’ spjsp’ of Fs(2)

Location

Fs(2) I Total 16715 133139 136136 139133 86/86 85/87 85/87 al-dp Barsa4 Total 4110 3912 3912 3318 12/29 13/28 13/28 al-dp Kompolt Total 7210 7210 6616 44/28 26/46 29/43 29/43 dp-b

aL/aL+ nub/nub zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA+ Lt/lt’ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAsku/sku’ sca jsca’ sfi/sfi+

Teh le Total 3410 3311 22/12 12/22 11/23 9/25 al-nub Ketel‘ Total 20122 261 16 22/20 28/14 29/13 24/18 ?

Fertile females 614 91 1 1010 812 713 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA515 nub-sca Sterile females 519 211 2 0114 011 4 1/13 717 nub-sca

The number of phenotypically mutant and wild-type offspring produced by Fs(2)lal dp b pr c p x sp or Fs(2)lal nub It shu sca sp adult females which were X-irradiated and test- crossed with appropriate males.

has not been determined. Similarly, the revertant homozygous combinations are not viable either due to their lethal features or, more likely, because of homozygosity of second site lethal mutations. Fur- thermore, different revertants of the same Fs(2) mutation are lethal in the trans-heterozygous condition.

Revertants of different Fs(2) mutations usually com- plemented. Not only were the trans-heterozygotes viable, but these females were fertile. The exceptions identified three complementation groups with 2 3 alleles each. Females trans-heterozygous for revertants of any of the four Fs(2) mutations dlD4, dlD5, dlD6 and dlD7 are sterile and typical “dorsalized” embryos develop in their eggs ($. NUSSLEIN-VOLHARD et al. 1980). Revertants of Barsa’, Bursa2, Bursa3 and Bursa4 are not viable when heterozygous in trans with each other thus establishing the Bursa complementation group. Revertants of Ketel’ are not viable over the only revertant of Kete12, but Ketel’lKetel’ revertant heterozygotes exhibit recessive female sterility iden- tifying the Ketel complementation group. The 21 Fs(2) mutations could thus be classified into 13 com- plentation groups.

We also tested whether a few of the Fs(2) mutations are dominant alleles of previously described recessive female sterile mutations. These experiments showed that four of them are dominant alleles of dorsal. Revertants of dlD4, dlD5, dlD6 and dlD7 result in recessive female sterility in trans-heterozygous condition with dorsal and their descendent embryos were “dorsalized.” Females trans-heterozygous for any revertant of torsoD402’ and torso are sterile. Embryos lacking the most anterior and posterior structures develop in their eggs a phenotype characteristic for torso (SCHUPBACH and WIESCHAUS 1986) indicating that torso4”’ is a dominant allele of torso (KLINGLER et al. 1988). Re- vertants of Vaja are female sterile in truns-heterozygotes with the incompletely penetrant dominant female sterile mutation B i ~ a u d a l ~ ” . ~ ~ (MOHLER and WIESCHAUS 1986). Typical bicaudal embryos develop

in about 50% of their eggs indicating that Vaja is most likely a new Bicaudal dominant allele. Revertants of the Fs(2) mutations Bursa and Dorog, which produce “ventralized” chorion cell imprint patterns similar to that described for the recessive female sterile mutations gurken and torpedo (SCHUPBACH 1987), fully com- plement gurken. We did not identify dominant alleles of the recessive female sterile mutations torpedo, exuperantia, stauffen, trunk, tudor, vasa, and valois.

DISCUSSION

Following EMS mutagenesis, only about 0.1% of the chromosomes carried Fs(2) mutations while 55% of the chromosomes carried zygotic lethals. Comple- mentation analysis of the revertants showed that 21 of the Fs(2) mutations represent 14 genes. Bursa and dorsalD were represented by four alleles each, Ketel by three alleles and the other 10 Fs(2) mutations appeared in single copies. If one assumes equal mutabil- ity of these genes, it can be calculated, on the basis of a Poisson distribution, that there are 20 genes on the second chromosome of Drosophila that can be mutated to dominant female sterility by EMS. The 24 Fs(2) mutations we isolated mutated 13-15 of these 20 genes or 65-75%.

Of the 24 Fs(2) mutations only Fs(2)1 is suitable for induction and detection of germ-line clones by the dominant female sterile technique (MACDONALD and STRUHL 1986; IRISH and GELBART 1987; SZABAD, REUTER and SCHRODER 1988). Fs(2)I-carrying females deposit only a few rudimentary eggs, the mutation is strictly germ-line dependent and high frequencies of germ-line clones can be recovered (SZA- BAD, ERD~LYI and SZIDoNYA 1987). zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Fs(2)Ugra renders females agametic. As was shown by analysis of germ-line and ovarian chimeras, the mutant phenotype depends on the genotype of somatic components of the ovaries. Analysis of Ugra+ clones indicate that the mutation interferes with func-

832 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAJ. Szabad zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAal.

tion of the follicle cells. Of the irradiated Ugra/+ females only about 1.3% deposited eggs. Half of these eggs were flaccid and only about 20% hatched. Fur- thermore, several of these eggs developed dissimilar dorsal appendages. (It is unfortunate that the Ugra+ clones cannot be identified on the chorion due to lack of appropriate marker mutation.) It has been known that the dorsal appendages are products of the follicle cells that cover the anterior dorsal surface of the oocyte (KING 1970; MARGARITIS 1985; SZABAD and HOFFFMANN 1989). Several of the follicle dependent recessive female sterile mutations are associated with flaccid eggs (WIESCHAUS, AUDIT and MASSON 198 1; PERRIMON and GANS 1983).

An additional feature of the Ugra’ clones induced in adult females is the delay in the deposition of eggs until 18-24 days following irradiation. It is not clear whether this long time lapse is the consequence of (1) perdurace of the mutant gene product (as suggested for several of the germ-line dependent dominant female sterile mutations, GARCIA-BELLIDO and ROBBINS 1983; PERRIMON 1984; ERD~LYI and SZABAD 1989), (2) partial nonautonomy of the Ugra mutant phenotype and/or (3) unknown characteristics of prefollicle cell proliferation. It is possible that contribution of a single or a few Ugra+ follicle cells, which participate in establishment of the follicle cell envelope of a newly established egg primordium, is insufficient to rescue the mutant phenotype. However, proliferation of Ugra+ prefollicle stem cells provide a sufficiently large number of nonmutant cells, that when surround the 16 germ-line derived cells allow partial or complete rescue of the Ugra phenotype.

In contrast to the irradiation of adults, the Ugra+ clones induced during early stages of development appeared with rather high (4-10%) frequencies. The majority of these eggs were normal and hatched. Since precursors of the follicle cells proliferate throughout the larval life (POULSON 1950; KING 1970), the Ugra+ clone be comprised of large numbers of cells and thus establishment of follicle envelopes from these cells is likely. The above considerations render Ugra an appropriate tool for analysis of gene expression in follicle cells, an extension of the dominant female sterile technique to the follicle cell components of the ovary. For example, mitotic recombinations could be induced in preferably late second/early third instar larvae truns-heterozygous for Ugra and an zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAfs or a zygotic lethal mutation. Egg production by the eclosing females should reveal whether the gene represented by the fs or the zygotic lethal mutation is required for function of the follicle cells.

Females that carry Fs(2)1, Billa, Dorog or any of the four Bursa alleles deposit eggs with “ventralized” chorion imprint pattern as described for t he4 mutations gurken and torpedo and also for Fs(3)Vencellin (SCHUP-

BACH zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1987; ERD~LYI and SZABAD 1989). Although the chorion is the product of the follicle cells, the mutant phenotype is the consequence of altered gene expression in the germ-line cells of the above Fs(2) mutations. These observations, along with those reported previously, underline the importance of cell commu- nication between the oocyte, its 15 sister nurse cells and the enveloping follicle cells (WIESCHAUS, MARSH and GEHRING 1978; WIESCHAUS, AUDIT and MASON 198 1; PERRIMON and GANS 1983; S C H ~ B A C H 1987; ERD~LYI and SZABAD 1989; GALANOPOULOS et al.

So far very little is known concerning the genetic control of the initial steps of embryogenesis in Dro- sophila, including fertilization, completion of the first and second meiotic divisions and initiation of cleavage divisions (FREEMAN, NUSSLEIN-VOLHARD and GLOVER 1986). Analysis of the 8 Fs(2) mutations reported in this paper, along with the Fs(3) mutations described by ERD~LYI and SZABAD (1989) should provide a means for the genetic dissection of early embryogenesis.

Of the 7 Fs(2) mutations that allow development to the larval stage of differentiation, the four newly isolated dorsalD alleles interfere with formation of the dorso-ventral embryonic polarity (NUSSLEIN-VOL- HARD 1979; NUSSLEIN-VOLHARD et al. 1980; for reviews see AKAM 1987; NUSSLEIN-VOLHARD, FROHN- HOFER and LEHMANN 1987; INGHAM 1988) the other three disturb formation of the anterior-posterior embryonic polarity. Several aspects of torsoD4o2’ have already been described (KLINGLER et al. 1988). Char- acteristics of Vuja are reminiscent of the incompletely penetrant dominant female sterile mutation BicaudalD (MOHLER and WIESCHAUS 1986) while Told, similar to most of the F5 mutations, has not been subjected to a thorough analysis yet.

Most of the Fs(2) mutations appear to be dominant gain-of-function type mutations. This is indicated by the abnormal behavior of the Fs+ clones induced in the Fs(Z)/adult females: (1) the frequency of the Fs+ clones was unexpectedly low, (2) the clones appeared only after several days of delay and (3) were frequently very small. This behavior of the Fs+ clones can be explained by the perdurance of the mutant gene products as was proposed for Fs( 1 )K237 and several of the Fs(3) mutations (GARCIA-BELLIDO and DEL PRADO 1979; GARCIA-BELLIDO and ROBBINS 1983; PERRIMON 1984; ERD~LYI and SZABAD 1989).

The gain-of-function nature of the Fs(2) mutations is best shown by the induction of revertants for 90% of them, albeit, with rather different frequencies. A few of the Fs(2) mutations that we failed to revert may represent germ-line haplo-insufficient loci as was reported for the BicaudalC mutation as well as for additional loci (MOHLER and WIESCHAUS 1986; GAR-

1989).

Fs zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAMutations zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Drosophila 833

CIA-BELLIDO and DEL PRADO 1979). The different, frequently rather high, reversion frequencies may re- flect different target sizes and/or “inherent instability” of the zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAFs(2) mutations as proposed for the Toll alleles (ANDERSON, JURCENS and NUSSLEIN-VOLHARD 1985). Whether the “gain-of-function” feature of the Fs(2) mutations reflects a hypermorphic, antimorphic or neomorphic nature or perhaps even ectopic gene expression remains to be determined upon detailed analysis of the individual mutations.

Such analyses will require more precise localization of the mutations. Mapping of the Fs(2) mutations was mostly based on analysis of progeny descending from the Fs+ clones induced by mitotic recombination in the Fs(2) /+ females. This procedure allowed mapping of the Fs(2) mutations into intervals delineated by two recessive marker mutations. The mapping procedure provided two unexpected features. (1) Wild-type alleles of the marker mutations located distal to the Fs mutations appeared in the offspring of the irradiated females. (2) Although the Fs(2) mutations are germ- line dependent (except Ugra and Etre), the Fs alleles were occasionally transmitted to the offspring (as was also reported for the Fs(3) mutations; ERD~LYI and SZABAD 1989). The first observation could partially be the consequence of double mitotic exchanges and/ or reversion of the Fs(2) mutations. Double mitotic recombinations have been known to occur with frequencies 10-20 times higher than expected on the basis of the single exchanges (GARCIA-BELLIDO 1972). The second observation can be best explained by Fs+ mosaicism among germ-line constituents of the devel- oping egg primordia. An Fs+ cell may originate during one of the four mitotic divisions needed to generate the oocyte and its 15 sister nurse cells (6 KING 1970; MAHOWALD and KAMBYSELLIS 1980). Mosaic egg primordia that are composed of at least a few Fs+ nurse cells while the genotype of the oocyte is Fs/+ may be the source of offspring. In such cases the progeny will carry the Fs mutant allele and, in addition, wild-type alleles of all the marker mutations.

More precise localizations of the Fs(2) mutations should include analysis of X-ray-induced revertants. Several of these revertants are expected to be associated with chromosome rearrangements whose break points are likely to define the site of the gene represented by the Fs mutation. Furthermore, the X-ray- induced revertants should allow for a determination of the loss-of-function phenotypes as well. In situ hy- bridizations of the P element-induced dysgenic revertants could provide another way to localize the Fs mutations. Furthermore, these types of revertants should also allow cloning of the genes identified by the Fs mutations as has been described for the Toll gene (HASHIMOTO, HUDSON and ANDERSON 1988). The forthcoming analyses of the Fs mutations may

reveal important new aspects of oogenesis, maternal control of embryogenesis as well as gene regulation.

We are grateful for the excellent technical help of ANDREA PAPP- MOSONYI. We also greatly appreciate the help and discussions of several of colleagues during the course of the “Fs project”: J ~ N O S

CSIRIK, Henrik Gyurkovics, Martin Klingler, Christiane Niisslein- Volhard and GUNTER REUTER. We also thank TRUDI S C H ~ B A C H

for providing mutant strains and TULLE I. HAZELRICC of the Howard Hughes Medical Institute for providing the resources of her laboratory. The research of this paper was largely supported by a grant, T t 83/1987, from the Hungarian Academy of Sciences to J. Sz.

LITERATURE CITED

AKAM, M., 1987 The molecular basis for metameric pattern in the Drosophila embryo. Development zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA101: 1-22.

ANDERSON, K. A., and C. NUSSLEIN-VOLHARD, 1986 Dorsal-group genes of Drosophila, pp. 177-194 in: Gametogenesis and the Early Embryo, edited by J. G. GALL. Alan R. Liss, New York.

ANDERSON, K. A., L. BOKLA and C. NUSSLEIN-VOLHARD, 1985 Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product. Cell 42: 791-798.

ANDERSON, K. h., G. JURGENS and C. NUSSLEIN-VOLHARD, 1985 Establishment of dorsal-ventral polarity in the Drosophila embryo: genetic studies on the role of the Toll gene product. Cell 42: 779-789.

BODENSTEIN, D., 1950 The postembryonic development of Dro- sophila, pp. 275-367 in Biology of Drosophila, edited by zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAM. DEMEREC. Wiley, Now York.

BUSSON, D., M. GANS, K. KoMITowuLou and M. MASSON, 1983 Genetic analysis of three dominant female sterile mutations located on the X chromosome of Drosophila melanogaster. Genetics 105: 309-325.

EPHRUSSI, B., and G. W. BEADLE, 1936 A technique of transplan- tation for Drosophila. Am. Nat. 7 0 218-225.

ERD~LYI, M., and J. SZABAD, 1989 Isolation and characterization of dominant female sterile mutations of Drosophila melanogaster. I. Mutations on the third chromosome. Genetics 122: 111-127.

FREEMAN, M., C. NUSSLEIN-VOLHARD and D. M. GLOVER, 1986 The dissociation of nuclear and centrosomal division in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAgnu, a mutation causing giant nuclei in Drosophila. Cell 46: 457-466.

GALANOPOULOS, V. K., W. Orr, J. Szabad and F. C. KAFATOS, 1989 Genetic analysis of chorion formation in Drosophila melanogaster. 1. The effects of one somatic specific and seven germ-line specific mutations. Dev. Genet. 1 0 87-97.

GARCIA-BELLIDO, A., 1972 Some parameters of mitotic recombination in Drosophila melanogaster. Mol. Gen. Genet. 115: 54- 72.

GARCIA-BELLIDO, A., and M. DEL PRADO, 1979 Genetic analysis of maternal information in Drosophila. Nature 478: 347-348.

GARCIA-BELLIDO, A., and L. G. ROBBINS, 1983 Viability of female germ line cells homozygous for zygotic lethals in Drosophila melanogaster. Genetics 103: 235-247.

GRELL, E .H., 1969 Bc: Black cells. Drosophila Inform. Serv. 4 4 46.

HANDKE-KOCIOK, M., and W. LIEBRICH, 1986 A simple method for staining chromosomes in whole embryos of Drosophila. Drosophila Inform. Serv. 63: 142.

HASHIMOTO, C., K. L. HUDSON and K. V. ANDERSON, 1988 The Toll gene of Drosophila, required for dorsal-ventral embryonic

834 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAJ. Szabad zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet al.

polarity, appears to encode a transmembrane protein. Cell zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA52:

INGHAM, P. W., zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1988 The molecular genetics of embryonic pattern formation in Drosophila. Nature 335 25-33.

IRISH, V. E., and W. M. GELBART, 1987 The decapentaplegic gene is required for dorsal-ventral patterning in the Drosophila embryo. Genes Dev. 1: 868-879.

JANNING, W., 1972 Aldehyde oxidase as a cell marker for internal organs in Drosophila melanogaster. Naturwissenschaften 5 9

JURGENS, G., 1987 Segmental organization of the tail region in the embryo of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 196 141-157.

JURGENS, G., R. LEHMANN, M. SCHARDIN and C. NUSSLEIN-VOL- HARD, 1986 Segmental organization of the head in the embryo of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 195: 359-377.

KING, R. C., 1970 Ovarian development in Drosophila melanoqaster, pp. 1-227. Academic Press, New York.

KLINGER, M., M. ERD~LYI, J. SZABAD and C. NUSSLEIN-VOLHARD 1988 The role of torso in determining the terminal anlagen of the Drosophila embryo. Nature 335: 275-277.

KOMITOPOULOU, K., M. CANS, L. M. MARGARITIS, F. C. KAFATOS and M. MASSON, 1983 Isolation and characterization of sex- linked female-sterile mutants in Drosophila melanogaster with special attention to eggshell mutants. Genetics 105: 897-920.

KONRAD, K. D., L. ENGSTROM, N. PERRIMON and A. P. MAHOWALD, 1985 Genetic analysis of oogenesis and the role of maternal gene expression in early development, pp. 577-617 in Devel- opmental Biology, Vol. 1 , edited by L.W. BROWDER. Plenum, New York.

KRIST~, G., F. MAKK and L. SZEGFU, 1973 Data concerning names of “early” Hungarian settlements (in Hungarian). Acta Hist. Univ. Szeged. 44: 1-96.

K R I S T ~ G., F.MAKK and L. SZEGFU, 1974 Data concerning names of “early” Hungarian settlements (in Hungarian). Acta Hist. Univ. Szeged. 48: 1-55.

LEHMANN, R., and C. NUSSLEIN-VOLHARD, 1986 Abdominal seg- mentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila. Cell 47: 141-152.

LEWIS, E. B., and F. BACHER, 1968 Method for feeding ethyl methane sulphonate (E.M.S.) to Drosophila males. Drosophila Inform. Sew. 43: 193.

LINDSLEY, D. L., and E. H. GRELL, 1968 Genetic Variations zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627.

LINDSLEY, D. L., and K. T. TOKUYASU, 1980 Spermatogenesis, pp. 226-294 in The Genetics and Biology zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Drosophila, Vol. 2d, edited by M. ASHBURNER and T. R. F. WRIGHT. Academic Press, New York.

LOHS-SCHARDIN, M., C. CREMER and C. NUSSLEIN-VOLHARD, 1979 A fate map of the larval epidermis of Drosophila melanogaster: localized cuticle defects following irradiation of the blastoderm with an ultraviolet laser microbeam. Dev. Biol. 73:

MACDONALD, P. M., and G. STRUHL, 1986 A molecular gradient in early Drosophila embryos specifying the body pattern. Na- ture 324 537-545.

MAHOWALD, A. P., and M. P. KAMBYSELLIS, 1980 Oogenesis, pp. 141-225 in The Genetics and Biology of Drosophila, Vol. 2d, edited by M. ASHBURNER and T . R. F. WRIGHT. Academic Press, New York.

MARGARITIS, L. H., 1985 Structure and physiology of eggshell, pp. 153-230 in Comprehensive Insect Biochemistry, Physiology and Pharmacology, Vol. 1 , edited by L.I. GILBERT and G. A. KER- KUT. Pergamon Press, Oxford.

MOHLER, J., and E. F. WIESCHAUS, 1986 Dominant maternal- effect mutations of Drosophila melangaster causing the produc-

269-279.

516-517.

239-255.

tion of double-abdomen embryos. Genetics 112: 803-822. NOTHIGER, R., T. SCHUPBACH, J. SZABAD and E. WIESCHAUS,

1978 Stem cells and tissue homeostasis in insect development, pp. 71-85 in Stem Cells and Tissue Homeostasis (British Society for Cell Biology), Cambridge University Press. Cambridge.

NUSSLEIN-VOLHARD, C., 1979 Maternal effect mutations that alter the spatial coordinates of the embryo of Drosophila melanogaster. Symp. SOC. Dev. Biol. 37: 185-21 1 .

NUSSLEIN-VOLHARD, C., M. LOHS-SCHARDIN, K. SANDER and C. CREMER, 1980 A dorso-ventral shift of embryonic primordia in a new maternal-effect mutant of Drosophila. Nature 283:

NUSSLEIN-VOLHARD, C., H. KLUDING and G. JURGENS,

1985 Genes affecting the segmental subdivision of the Dro- sophila embryo. Cold Spring Harbor Symp. Quant. Biol. 50:

NUSSLEIN-VOLHARD, C., H. G. FROHNHOFER and R. LEHMANN, 1987 Determination of anteroposterior polarity in Drosoph- ila. Science 238: 1675-1681.

PERRIMON, N., 1984 Clonal analysis of dominant female sterile, germline-dependent mutations in Drosophila melanogaster. Ge- netics 108: 927-939.

PERRIMON, N., and M. CANS, 1983 Clonal analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female sterile mutation Fs(I)K1237. Dev. Biol. 100 365-373.

PERRIMON, N., L. ENGSTROM and A. P. MAHOWALD, 1984a The effects of zygotic lethal mutations on female germ line functions in Drosophila. Dev. Biol. 105 404-414.

PERRIMON, N., L. ENCSTROM and A. P. MAHOWALD, 1984b Developmental genetics of the 2E-F region of the Drosophila X chromosome: a region rich in “developmentally important” genes. Genetics 108: 559-572.

PERRIMON, N., L. ENGSTROM, and A. P. MAHOWALD 1985a A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster. Dev. Biol. 114: 480-491.

PERRIMON, N., L. ENGSTROM and A. P. MAHOWALD, 1985b Developmental genetics of the 2C-D region of the Drosophila X chromosome. Genetics 11 1: 23-4 1 .

POULSON, D. F., 1950 Histogenesis, organogenesis and differentiation in the embryos of Drosophila melanogaster Meigen, pp. 168-274 in Biology of Drosophila, edited by M. DEMAREC. Wiley, New York.

SCHUPBACH, T., 1987 Germ line and soma cooperate during oogenesis to establish the dorsoventral pattern of egg shell and embryo in Drosophila melanogaster. Cell 4 9 699-707.

SCHUPBACH, T., and E. WIESCHAUS, 1986 Maternal-effect mutations altering anterior-posterior pattern of the Drosophila embryo. Wilhelm Roux’s Arch. Dev. Biol. 195 302-317.

SZABAD, J., and C. FAJSZI, 1982 Control of female reproduction in Drosphila: genetic dissection using gynandromorphs. Ge- netics 100 61-78.

SZABAD, J., and G. HOFFMANN, 1989 Analysis of follicle cell functions in Drosophila: the Fs(3)Apc mutation and the development of chorionic appendages. Dev. Biol. 131: 1-10.

SZABAD, J., M. ERD~LYI and J. SZIWNYA, 1987 Characterization of Fs(2)1, a germ line dependent dominant female sterile mutation of Drosophila. Acta Biol. Hung. 38: 257-266.

SZABAD, J., G. REUTER and M. B. SCHRODER 1988 The effects of two mutations-connected with chromatin functions-on female germ line cells of Drosphila. Mol. Gen. Genet: 211: 56- 62.

TAUBERT, H., and J. SZABAD, 1987 Genetic control of cell proliferation in germ line cells of Drosophila: mosaic analysis of five discless mutations. Mol. Gen. Genet. 209 545-55 1.

WIESCHAUS, E., 1980 A combined genetic and mosaic approach to the study of oogenesis in Drosophila, pp. 85-94 in Develop-

474-476.

145-154.

Fs zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAMutations zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAof Drosophila 835 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAment and Neurobiology of Drosophila, edited by zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA0. SIDDIQI, P. BABU, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAL. M. HALL and J. C. HALL. Plenum Press, New York.

WIESCHAUS, E., and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAC. NUSSLEIN-VOLHARD, 1986 Looking at embryos, pp. 199-227 in Drosophila: A Practical Approach, edited by D. B. ROBERTS. I.R.L. Press, Washington D.C.

WIFSCHAUS, E., and J. SZABAD, 1979 The development and function of the female germ line in Drosophila: a cell lineage study. Dev. Biol. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA68: 29-46.

WIFSCHAUS, E., and E. NOELL, 1986 Specificity of embryonic lethal mutations in Drosophila analyzed in germ line clones. Wilhelm Roux's Arch. 195 63-73.

WIESCHAUS, E., J. L. MARSH and W. J. GEHRING 1978 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAf s ( l )K lO : a germ-line dependent female sterile mutation causing abnormal chorion morphology in Drosophila melanogaster. Wilhelm Roux's Arch. Dev. Biol. 184: 75-82.

WIESCHAUS, E., C. AUDIT and M. MASSON, 1981 A clonal analysis of the roles of somatic cells and germline during oogenesis in Drosophila. Dev. Biol. 8 8 92-103.

WRIGHT, T. R. F., B. C. BLACK, C. P. BISHOP, J. L. MARSH, E. S. PENTZ, R. STEWARD and E. Y. WRIGHT, 1982 The genetics of dopa decarboxylase in Drosophila melanogaster. V. Ddc and l(2)amd alleles: isolation, characterization and intragenic complementation. Mol. Gen. Genet 188 18-26.

YARGER, R. J., and R. C. KING, 1971 The phenogenetics of a temperature sensitive, autosomal dominant, female sterile gene

in Drosophila melanogaster. Dev. Biol. 24: 166-1 77. Communicating editor: W. M. GELBART

APPENDIX

The estimated frequency of Fs(2)+ mosaicism was studied as follows. In order to inducefs(1)KIO germ-line y, mwh and Sb+ abdominal reference clones, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAyfs( I)KIO/++; mwh Sb63b/++ early third instar larvae and adult females were X-irradiated with pameters identical to those used to induce the Fs(2)+ clones. Following irradiation of larvae, 46% of the females deposited K10 eggs. One of these females carried on the average 0.61 KIO germ-line clones, as estimated on the basis of a Poisson distribution. These females carried at the same time 3.13 y, 1.56 mwh and 1.40 Sb+ clones per abdomen. When adult females were irradiated, 73% of the females deposited KIO eggs indicating the 1.31 K l O clones/female. Furthermore, it has been known that mitotic recombinations take place with similar frequencies on the left and right arms of the third and second chromosomes. For example, while on the average 0.49 mwh and 0.42 Sb+ clones developed on one abdomen following X-irradiation by 500 R, there were 0.39 0.29 stw and 0.60 sCs2" clones detected on the same abdomens [GARCIA-BELLIDO (1972), Table 71. As may be estimated based on the above figures, the frequency of Fs(2)+ germ-line clones should be about 17, 22 and 32% for the Fs(2) mutations located in the vicinity of the marker mutations stw or d 2 , repectively.

Isolation and characterization of dominant female sterile mutations of Drosophila melanogaster. II....

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