isolation and characterization of dominant female sterile ... · isolation and characterization of...

Copyright 0 1989 by the Genetics Society of America

Isolation and Characterization of Dominant Female Sterile Mutations of Drosophila melanogaster. I. Mutations on the Third Chromosome

Miklos Erdhlyi* and Janos Szabad*+ *Institute of Genetics, Biological Research Center of the Hungarian Academy ofsciences, Szeged, Hungary,t and Howard Hughes

Medical Institute, Department of Biology, University of Utah, Salt Lake City, Utah 84112

Manuscript received August 12, 1988 Accepted for publication January 30, 1989

ABSTRACT Fifty-one dominant female sterile (Fs ) mutations linked to the third chromosome of Drosophila

melanogaster are described. EMS induced Fs mutations arise with the frequency of one Fs per about 2500 recessive lethals. Complementation analysis of the revertants showed that these Fs mutations represent 27-34 loci, about 60% of the third chromosome units mutable to dominant female sterility by EMS. The Fs mutations were mapped on the basis of mitotic recombination induced in the female (in 16 cases also in the male) germ-line. Behavior of the revertants and the Fs+ germ-line clones demonstrate the gain-of-function nature of the Fs alleles. With two exceptions, the Fs(3) mutations are germ-line dependent. Novel phenotypes appeared in most of the Fs mutations. With eight exceptions, the Fs(3) mutations are fully penetrant, in some cases with variable expressivity. One of the Fs(3) mutations is a non-ovary-dependent egg retention mutation, two others alter egg shape, and 27 bring about arrest in development at about the time of fertilization. In 21 of the Fs(3 ) mutations embryos develop to the larval stage of differentiation; this group includes 5 new alleles of Toll and 4 of easter.

S EVERAL systematic studies have been carried out to isolate and characterize recessive female sterile

vs) mutations of Drosophila melanogaster. Analysis of these mutations facilitated the genetic dissection of oogenesis, maternal-effects and embryonic pattern formation (for reviews see KONRAD et al. 1985; NUS- SLEIN-VOLHARD, FROHNHOFER and LEHMANN 1987; INCHAM 1988). Descriptions of several dominant female sterile (Fs ) mutants provided major contribu- tions to a basic understanding of the role of maternal- effects in the establishment of the dorso-ventral and the anterior-posterior embryonic polarities. These mutants are dominant alleles of dorsal (NUSSLEIN- VOLHARD 1979; NUSSLEIN-VOLHARD et al. 1980), Toll (ANDERSON, JURGENS and NUSSLEIN-VOLHARD 1985; ANDERSON, BOKLA and NUSSLEIN-VOLHARD 1985), easter (ANDERSON and NUSSLEIN-VOLHARD 1986), bi- caudal (MOHLER and WIESCHAUS 1986) and torso (KLINGLER et al. 1988).

A potentially attractive feature of the Fs mutants is that they allow convenient molecular cloning of the identified genes by isolation of dysgenic revertants first and subsequent cloning, as has been shown for Toll (HASHIMOTO, HUDSON and ANDERSON 1988) and for five of the mutations described in this paper (J. SZABAD and M. ERD~LYI, unpublished data). It is also possible that a few of the dominant female sterile mutations may identify important genes that escape screens designed to isolate recessive female sterile mutations.

Genetics 122: 1 1 1-127 (May, 1989)

The mutations Fs(2)D (YARGER and KING 1971), Fs(l)K1237 (also known as ovoD’; KOMITOPOULOU et al. 1983; BUSSON et al. 1983), and Fs(2)l (SZABAD, ERD~LYI and SZIDONYA 1987) proved to be useful tools in the analysis of germ-line functions of fs and zygotic lethal mutations by the so-called dominant female sterile technique (WIESCHAUS 1980; PERRIMON and GANS 1983). These Fs mutations are strictly germ- line dependent, and are agametic or allow formation of only a few rudimentary eggs. Mitotic recombinations were induced in females trans-heterozygous for the studiedfs (or zygotic lethal) and one of the above three Fs mutations and development of the egg primordia composed of fs homozygous and Fs-free germ- line cells and>/+ soma was analyzed. These studies demonstrated that the genetic requirements in the germ-line cells include most of the genome and also that mutations that interfere with sex determination have no effect on the germ-line (GARCIA-BELLIDO and ROBBINS 1983; PERRIMON and GANS 1983; PERRIMON, ENGSTROM and MAHOWALD 1984a, b, 1985a, b; Nus-

BACH and WIESCHAUS 1986; WIESCHAUS and NOELL 1986; TAUBERT and SZABAD 1987; SZABAD, REUTER and SCHRODER 1988; SCHUPBACH 1982). However, the fs and the zygotic lethals linked to the 3rd chromosome were excluded from these studies due to lack of proper Fs(3 ) mutations.

With the above perspectives in mind we isolated 51 Fs(3) mutations. These mutations represent 27-34

SLEIN-VOLHARD, KLUDING and JURGENs 1985; SCHUP-

112 M. Erdclyi and J. Szabad

loci, as deduced from complementation of the Fs revertants, about 60% of the units on the third chromosome that can be mutated to dominant female sterility by EMS. Unexpectedly, none of the 51 Fs(3 ) mutations appears to be an ideal tool for the dominant female sterile technique. Only Fs(3)Avar is agametic. However, it is a non-ovary-dependent egg retention mutation. One of the two egg-shape mutations (Fs(3)Apc) is follicle dependent while the other (Fs(3)VenceZZin) is not fully penetrant. Females carrying any of the remaining 48 F s ( 3 ) mutations deposit normal-looking eggs. Development is arrested at different stages of embryogenesis in these eggs. The Fs(3)-associated phenotypes are novel in the majority of these mutations.

This paper provides genetic and developmental characterization of 51 Fs(3 ) mutations, including the isolation, mapping, reversion, complementation analysis, phenotypes, and the effects of these mutations on female germ-line cells.

MATERIALS AND METHODS

Mutagenesis: All the described Fs(3 ) mutations were induced by EMS (ethyl methanesulfonate) on one of three isogenic chromosomes: wild-type (Canton-S), red e or mwh e. (For a description of the marker mutations, the multiply marked rucuca, the T M 3 and CxD balancer chromosomes, see LINDSLEY and GRELL 1968). Mutagenesis was achieved by feeding adult males with 25 mM EMS solution for 8 hr (LEWIS and BACHER 1968). Populations of the EMS-treated males were mated with CxDITM3, Sb Ser females (Figure 1). T o avoid isolation of clusters of Fs(3 ) mutations, the males were discarded 3-5 days following matings. Single mwh e/ T M 3 , Sb Ser males (each representing one mutagenized chromosome) were mated with Sb/TM3, Ser females. Only the mutagenized chromosome-carrying offspring survive in the subsequent generation. The eclosing offspring were transferred into fresh vials and tested for sterility. Vials in which no larvae developed were saved; the lack of larvae was due to one of two reasons. (1) A few of the vials did not contain females with the mutagenized chromosome. They all represented Y ; 3 translocations; the crosses between the parental males and attached-X females yielded fertile females. These lines were not further investigated. (2) When both males and females were present in the vials, the mwh e /TM3, Ser males were mated with CxDITM3, Sb Ser females for retesting dominant female sterility, and when it was confirmed, a stock was established in which F s ( 3 ) , mwh e / TM3, Sb Ser males are mated with T ( I ; 3)OR60/TM3, Sb Ser females. (The T ( I ; 3 ) O R 6 0 translocation is male lethal; LINDSLEY and GRELL 1968.) All the above experiments were carried out at 25“.

The efficiency of EMS-treatment, as characterized by the frequency of induction of lethal mutations, was determined in the following way. A few of the mwh e/CxD males (siblings of the mwh e /TM3, Sb Ser males) were individually mated with CxDITM3 females and the second generation was subsequently screened for the presence of the mwh e homozygous class to determine if the mutagenized chromosome carried lethal mutations.

Reversion of the Fs(3) mutations: For induction of revertants of the F s ( 3 ) mutations, populations of Fs(3 ) /TM3 adult males were either X-irradiated (4000 R; 150 kV, 0.5

C x D

EMS mutagenorls

ff mwh e*

TM3,SbSer mwh e

I 4

Sb 0

mwh e*

TM3,Ser TM3,SbSer

I mwh e*

Sb Screen tor

tortlllty

TM3,SbSer mwh e*

T(1:3)0R60 TM3,Ser 0 u

1 e“ sb + ;/3) TM3.SbSer

I

straln TM3,Ser +

TM3,SbSer I

FIGURE 1 .-Scheme for the isolation of Fs(3 ) mutations. The (*) symbol stands for an EMS-treated chromosome. Male parents are shown on the right side of the three crosses. t = lethal combination.

mm AI filter, 1000 R/min) or fed with 25 mM EMS solution for 8 hr. The males were subsequently mated with CxD/ T M 3 females and discarded 3-5 days following mutagenesis. The Fs(3 ) /TM3 and the Fs(3) lCxD progeny females were mated with CxD/TM3 males and screened for fertility in lots of 25. Only females representing Fs(3 ) revertant chromosomes give rise to offspring. The x-ray and the EMS-induced revertants are labeled as RX or RE. Females of a lot were individually tested when reversion of a F s ( 3 ) mutations was noticed. The revertants were used in complementation tests to test for allelism with known recessive maternal-effect lethal (mel) mutations and within the Fs(3 ) mutations. Those me1 mutations were used that bring about “dorsalized” embryonic phenotype: easter, nudel, pipe, pe l l e , snake, spatzle, Toll and tube (ANDERSON and NUSSLEIN-VOLHARD 1986) or lead to the formation of giant nuclei (FREEMAN, NUSSLEIN- VOLHARD and GLOVER 1986).

Mapping the Fs(3) mutations: Because the Fs-carrying females are sterile and there is no meiotic recombination in males, methods that make use of induced mitotic recombination were applied for localization of the Fs(3 ) mutations. First, mitotic recombination was induced (by 1500 R of x- rays) in Fs(3)lrucuca adult females (Figure 2). (The rucuca chromosome carries 8 recessive marker mutations including e’.) The females were 3-5 days old when irradiated and had been mated with rucuca males for stimulation of ovulations (6 SZABAD and FAJSZI 1982). Lots of 10 females plus 10- 15 males were transferred into vials and inspected for the production of offspring. Females were kept for a test period of 21 days necessary to recover most of the mosaics. Lots with mosaic females were kept for several more days. Also, females surviving for 21 days were counted in order to characterize the “viability” of the Fs(3)-carrying females.

Fs Mutations of Drosophila 113

Daughter cells

FIGURE 2.-Mitotic recombination between the Fs mutations and the centromere leads to the formation of an Fs(3)-free daughter cell (*). The symbols a, b, c and d stand for recessive marker mutations. Their wild-type alleles (located on the Fs-carrying chromosomes delineated by thick lines) are not shown. Only the wild- type (+) allele of the Fs(3) mutation is indicated. For details see text.

Mitotic recombination can lead to the formation of Fs- free oogonial stem line cells (Figure 2). When the Fs mutation interferes with functions of the germ-line, the Fs-free cells may continue development. Their genotype can be inferred in a test cross with rucuca males. Wild-type and mutant alleles of the marker mutations carried on the arm of chromosome three which does not contain the induced Fs mutation should appear at a 1:l ratio. Wild-type alleles of those marker mutations located on the same arm as Fs behave differently. While wild-type alleles of the marker mutations located between Fs and the centromere will appear, only mutant alleles of the marker mutations distally located from Fs are expected to be identified among the offspring. Hence, an interval, delineated by two marker mutations, can be ascertained for the location of an Fs mutation. Similar methods have been described by BUSSON et al. (1983) and SZABAD, ERD~LYI and SZIDONYA (1 987). It should be noticed that transmission of the Fs allele to the offspring is not expected when it interferes with germ-line functions.

In the case of nongerm-line-dependent Fs(3) mutations, the nonmutant soma may allow formation of eggs from which progeny can develop. In this case, mutant and wild- type alleles of all the marker mutations are expected to appear in a 1 : 1 ratio in the offspring, half of which will carry the Fs allele.

The relative frequencies of induction of Fs(3)+ clones was determined by analysis offs(1)KIO germ-line, y, mwh and Sb+ clones on the abdomens of yfs(l)KlO/+ +; mwh Sb63b/ + + females. These females were irradiated with the same parameters of x-rays as used for the induction of the Fs(3)+ clones. Two groups of females were analyzed; the first group was irradiated as early third instar larvae and the second as 5-day-old adults.fs( 1)KlO (=KlO) is a germ-line-dependent, egg-shape mutation (WIESCHAUS, MARSH and GEHRING 1978; WIESCHAUS and SZABAD 1979).

When the above procedure proved to be inadequate to generate a sufficient number of recombinants, Fs(3)/rucuca old larvae and/or young pupae were irradiated by 1500 R of x-rays. The testes are filled with mostly primary sperma-

tocytes at these stages (BODENSTEIN 1950; LINDSLEY and TOKUYASU 1980). Mitotic recombination can lead to the formation of recombinant chromosomes as shown in Figure 2. The eclosing males were mass mated with rucuca females. The recombinant female progeny were isolated and screened for sterility/fertility. WIE~CHAUS and JURCENS, as described in a paper by ANDERSON, JURGENS and NUSSLEIN- VOLHARD (1985), mapped two Toll dominant alleles using the same procedure.

Eight of the Fs(3) mutations proved to be incompletely penetrant in trans-heterozygous condition with rucuca. The Fs(3)/rucuca females were mated with rucuca males in these cases and the recombinant female progeny were screened for the presence of the Fs mutant phenotype. Standard mapping procedures were applied subsequently.

Germ-line and ovarian chimeras: Germ-line chimeras were constructed by transplantations of pole cells (LEHMANN and NUSSLEIN-VOLHARD 1986) to determine whether the mutant phenotype is brought about by altered functions of the germ line and/or the soma: rucuca females were mated with Fs(3)/TM3 males, and pole cells of the resulting embryos were implanted into host embryos that originated from the cross between wild-type (Ore-R) females and Fs(l)Kl237/Y males. Fs(l)Kl237 (=K1237) is an agametic, strictly germ-line-dependent dominant female sterile mutation of Drosophila melanogaster (KOMITOPOULOU et al. 1983; BUSSON et al. 1983). The +/K1237 females were mated with rucuca males. When an Fs(3) mutation proved to be nongerm-line-dependent, pole cells of y v f mal embryos were implanted into embryos that derived from rucuca females and Fs(3)/TM3 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 histochemical procedure described by JANNING (1 972).

Two types of ovarian chimeras were constructed by transplantation of larval ovaries (EPHRUSSI and BEADLE 1936) to determine whether the focus of the Fs(3) mutation is located in the ovaries or other parts of the soma. First, ovaries of Fs(3)/rucuca larvae were implanted into +/K1237 host larvae; these animals were screened for the Fs(3) phenotype after metamorphosis. Second, ovaries of y v f mal larvae were implanted into Fs(3)/rucuca larval hosts which were screened for production of y v f mal offspring after metamorphosis and mating toy v f mal males.

Analysis of the mutant phenotype: Three experimental procedures were applied. ( 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. (2) Embryos of Fs(3)/+ females that did not develop to the larval stage of differentiation were analyzed according to HANDKE-KOCIOK and LIEBRICH (1986) and WIESCHAUS and NUSSLEIN-VOLHARD (1 986): eggs were collected from Fs(3)/rucuca females for 4 hr (25"), dechorionated in NaOCl solution, washed, rinsed in heptane (to ren- der the vitelline membrane permeable), fixed in a mixture of methanokacetic acid 3: 1 for 30 min, stained in g/ml DAPI (diamino phenyl indole) 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 " (WIESCHAUS and NUSSLEIN-VOLHARD 1986). The only structures remaining after this preparation procedure are those made of larval cuticle.

114 M. Erdklyi and J. Szabad

Table 1 Characteristics of the fully penetrant dominant female sterile mutations

Total lots with N(i ) clonesb Frequency Offspring per lot

Total Females induction' of clone Total Fs' and Fs

Fs(3)" N N o N I N P N3 N I clones screened (%) 1 2 2 3 spring in progeny Off- alleles

Ape IOd

Avar'" Baksa'" Bercel"l'" Bereel'"

Botond I"' Bojla"

Damasa"" Damasa

ean*m 1022

can I z0 ea n2,m FarkasYd Gerec I" Hodos'" HorkaiZh Huban/'" HubaR"b Huba"' Hubs"' J u t a ~ ' ~ Kavar""' Kavar2" Kauar2le Keled " K e ~ e " ~ Kun 2ob

Laborc"' PalatfYb Pilts12f Teve14"

Toll I"' Toll lXa

Tomaj3" Tomaj'"''

Tonuz'" Tomajl"d

Zerind' "M Varas"

Zerind Zombor'"

~ 0 1 1 "

42 21 14.6 5.0 1.2 0.2 38 33 4.7 0.3

21 21 0.0 32 30

46 35 9.6 1.3 0.1 49 48 1.0 49 40 8.1 0.8 54 46 7.4 0.6 71 41 22.5 6.2 1.1 0.2 39 30 7.9 1.0 16 11 4.1 0.8 0.1 45 37 7.2 0.7 0.1 43 40 2.9 0.1 25 21 3.7 0.3 42 41 1.0 58 44 12.2 1.7 0.2 41 35 5.5 0.4 33 19 10.5 2.9 0.5 35 29 5.5 0.5 39 33 5.5 0.5

40 30 1.0 30 25 4.6 0.4

48 45 2.9 0.1 34 28 5.4 0.5

41 27 11.3 2.4 0.3 34 32 1.9 0.1

32 20 9.4 2.2 0.3 67 44 18.5 3.9 0.5 50 47 2.9 0.1 20 6 7.2 4.3 1.7 0.5 56 48 7.4 0.1

47 19 17.2 7.8 2.4 0.5 56 44 10.6 1.3 0.1

29 26 2.8 0.2 43 36 6.4 0.6 85 68 15.2 1.7 0.1 18 16 1.9 0.1 43 35 7.2 0.7 0.1 33 24 7.6 1.2 0.1 36 31 4.6 0.4

121 115 5.8 0.1 56 44 10.6 1.3 0.1 45 35 8.8 1.1 0.1

~

29 5

0 2

13 I

10 9

39 10 6 9 3 4

16 1

18 9

6 6 5 1 3 6 2

17 15 28

3 24

14 8

43 3 8

19 2

10 9

5 6

14 11

413 348 305 174 356

470 477

52 1 670 381

444 135

378 248 359 40 1 379 284 323

284 372

352 445 327 309 395 292 640 465 184 214 516 455 279 41 1 823 174

310 395

1137 336

535 400

7.0 0 2 19 1.4 0 1 4 0.7 0 0 2

3.7 0 0 0 4 1 6

0.2 0 0 1 2.1 7 1 1 1.7 5.8

2 2 4 15 6 9

2.6 7 0 2 4.4 2.0 6 1

3 0 2 1

0.8 2 1 0 1.6 3 0 0 0.3 0 1 0 4.0 4 2 8 1.6 6.3

3 2 1 7 6 1

1.9 1 2 3 1.6 4 1 1 1.8 0.3

3 0 2 0 0 1

0.7 1 0 2 1.8 2 1 3 0.7 1 0 1 4.3 5 1 8 5.1 5 3 4 4.4 12 4 7 0.7 3 0 0

13.0 5 1 8 3.7 2.7 6 0 6

3 2 3

9.5 10 6 12 1.1 1 1 1 2.0 4 1 2 2.3 14 1 2 1.2 1 1 0 2.3 3 2 3 3.2 6 0 3 1.5 1 0 4 0.5 5 1 0 2.6 6 2 4 2.8 3 3 4

183 76 14

33 0

7 14

149 64

33 28 13 4 3

85 2

22 13

15 10 17 3

19 1 1

36 5

66 42

3 74 32 30 72

21 8

24 3

42 16 25

7 31 52

23 12

32

4 I 1

0 0 0

8 0

5 0

3 2 7

22 12 24

13 14

1

4 0

2 0

0 0 0

0 0 29 10

5 2 3 4 6 3 3 1

2

1 0 3

6 7

0

1 0

6 6 0

10 14

6

1 0 0

17 2

6

17 0 2

23 19 3 0 2 0 6 0

2 0

3 4 9 7

0

I 1 1

21 14 0

0

settlements ( K R I S T ~ , MAKK and SZEGFU 1973, 1974). The Fs(3) mutations were named after Hungarian clans that vanished by the beginning of the 14th century but their names survived in the names of

The total number of clones divided by the number of females. Estimated on the basis of a Poisson distribution: N ( i ) = NP( i ) , where P ( i ) = u'e-"/i! and considering that e-" = N,/N.

Average f SD of females in a lot of 10 who survived 21 days following irradiations. * and ** = significantly different from the control (9.6 ? 0.7, for 20 mwh e/rucuca females) at P = 0.05 and 0.01, respectively.

cnl = centromeron, ND = not determined, NGLD = nongerm-line dependent.

RESULTS

Recovery of Fs(3) mutations: Following EMS treatment, 0.1 % (54/50,128, i .e . , 1 in 928) of the chromosomes carried a F s ( 3 ) mutation. Fifty-one of the 54 F s ( 3 ) mutations could be established as stocks. Symbols of the F s ( 3 ) mutations are listed in the first columns of Tabies 1 and 2. Only the abbreviated forms (three letters and the superscript in case of allelism) are used throughout the paper. All mutations isolated in a given experiment are assigned super- scripts which differ only in the final digit (number or letter). With the exception of Ven and Kar (that were induced on a red e chromosome), and Tom409' and Kav (induced on a Canton-S chromosome), all the F s ( 3 )

mutations are located on chromosomes that carry the recessive marker mutations mwh and e .

Forty of the 51 Fs(3 ) mutations have no effect on the viability of the carrier females: virtually the same proportion of the mutant-bearing or control mwh e / wmca females lived for 21 days (Table 1). Slight but significant reduction of the viability was noticed in 4 cases. Three of these F s ( 3 ) mutations, P i l , Ber8'Ia and Lev are late hatching due to Minute effects. Kev and Berg" are associated with dominant bristle phenotypes (Figure 3). Except for the 11 F s ( 3 ) mutations that reduce viability, none of the F s ( 3 ) mutations reduce male fertility.

Efficiency of mutagenesis: Of the 120 mwh e chromosomes assayed for lethality, 6 were homozygous


J k ~ k reconlhination Gernl-line cllilneras Revertants induced b y

Days u n t i l Progeny the No. of first Recon,- Mq) reconlhi- Females

Chimeras

h h p

11-111 None 9.8 f 0.5 74 I 4%c;l.l> 10 012938 013730 11-Ill IO- I 4 9.2 2 0.5 18 Q"':l." 2 01900 31. 15. 20 9.6 f 0.5 5453 44 0.8 ru-11 1 23 5 2 3/%4

position Fs' egg Surviv;lld Tot;11 binant % position nants screened Fs(3) Control EMS X-ray

ND 8.3 f 1.2** 24 2 5 31375 11143 ru-11 11-16 7.7 f ] . X * * 115 I5 I 1.500 31. 12-18 9.7 2 0.5 53.56 64 1.2 11-Ill 3 19 2 5 I/I339 3 L 14-20 9.6 2 0.7 21 7 4 114225

3L 13-18 9.3 f 1.2 21 2 3 71367.5 3R 13-17 9.8 f 0.3 4384 50 1.1 sr-e 2 25 7 1 1/1361 1/2310 3n 12-17 8.4 f 1.2** 2750 2 5 0.9 cu-e 3 14 1 I - 112709

ru-h 11-16 9.6 f 0.7 2/2400

sr-e 16-21 9.8 f 0.5 211 366 0/1236 sr-ca 14-19 8.8 & 1.3 511 I55 ru-sr 16-22 9.9 f 0.3 32 7 2 01675 h-th 8.5 f 1.2* 35 8 2 11873 ru-h 10-14 6.9 f 2.3** 24 I 4 3/2075 3R 12-17 9.2 f 0.9 58 8 I I l/925 h-th 12-17 8.6 f 1.2* 44 7 4 61697 11-th 12-17 9.2 2 1.0 42 3 7 41x97 ru-h 12-17 9.5 f 0.6 27 4 5 11925 th-sr 14-19 9.5 -c 0.8 79 18 12 2I683 3R 19-24 8.8 f 1.2* 4276 46 1.1 sr-e 3 33 4 5 1/220 ru-h 21-27 9.3 -C 1.1 37 5 9 21751 3 I. 12-1 9 9.6 f 0.6 112156 ND 12, 18 9.0 f 0.9* 211 985 cu-sr 10-14 9.6f 0.5 8 I I 112223 1/1200 h-th 13-39 9.1 f 1.1 2389 23 1.0 3L I 1 I6 3 2 3/39I 211297 e-ca 11-16 9.5 f 0.7 I 1 1 2 318.54 31. 14-19 9.3 f 0.7 64 6 8 214.525 ru-h None 9.2 f 1. I 10 1 3 If1340 sr-e 14-20 3.8 -C 1.4** 2132 2 0.1 3R 2 h-th 20-28 9.2 f 1.1 31 4 3 21896 512404 e-ca None 9.7 f 0.5 43 2 3 111 102 e-ca 13-1 8 9.6 f 0.9 312 19 sr-e 12-1 8 9.6 f 0.6 31 2 4 211530 h-th 14-2 1 9.7 f 0.5 3662 32 0.9 ru-cm I 34 8 6 11450 ND 17, 21 9.6 f 0.7 1623 21 1.3 3L 4 I 1848 ND 14-20 9.2 2 0.9 36 3 7 3/605 3R 17-22 9.4 f 0.9 4290 46 1.1 sr-ca 3 39 3 8 1/1511 ru-h 24-30 9.3 f 0.8 36 7 5 515350 3R 15-20 9.4 -C 0.9 394 I 48 1.2 st-cu 10 15 2 1 31638 e-ca 16-22 9.6 f 0.6 31 I 023 h-th 22-28 8.9 f 1.2 65 2 4 2129.5

FIGURE 3."Scutellar bristles of females that are wild-type (A) or carry one of the 5 Fs(3) mutations Pi1 (B), Ber""" (C). Leu (D), Keu (E) or Rer" (F). T h e Minute bristle phenotype and female sterility were sepa- rable in the case of Pi1 and Leu and inseparable for Eer""". The dominant bristle phenotypes seen in K e v and Ber'" females were inseparable from the dominant female sterility. Revertants of Keu and Eer'" do not show the dominant bristle phenotype, indicating that the two dominant phenotypes are consequences of common mutational lesions (bar = 100 pm).

viable. I t was estimated (by a Poisson distribution, distributed among the EMS-treated chromosomes) assuming that the lethal mutations were randomly that each EMS-treated chromosome carried on the

116 M. Erdilyi and J. Szabad

TABLE 2

Characteristics of the incompletely dominant female sterile mutations

Revertants induced by Male recombination Germ-line chimeras

Fertile Progeny fenlales Map No. of re-

Chimeras

Fs(3y (% total) position combinants EMS X-ray Total Recombinant % posltlon combinants screened Fs(S) Control Map No. of re- Females

Ka~tal'"~' 100 cu-ca 60 51149 21749 2246 45 2.0 cu-sr 4 14 3 1 Levente'f 10 80.7 58 7299 126 1.7 cu-ca 19 12 4 2 Pudur'"' 23 ru-cu 4 51194 112709 3344 57 1.7 st-CU 12 18 3 5 Rosd '" 14 cu-sr 3 411375 5035 50 1.0 cu-sr 5 20 2 4 Tollzb 23 89.5 80 21820 37 8 8 Tollzfh 65 89 10 111875 7158 77 1.1 sr-ca 8 Vence~lin4"22 81 28.1 54 11675 11688 ZoltaZf" 26 cu-sr 7

a See footnote a in Table 1.

average 2.7 lethal mutations. Thus the relative frequency of induction of Fs(3) mutations is 112524 zygotic lethals.

Phenotype of the Fs(3) mutations: Aua, an egg retention mutation: Of the 5 1 Fs(3 ) mutations only Aua is agametic. Although ovaries of the AualTM3 females are filled with mature eggs, these are not deposited. Aua is incompletely penetrant in a few genetic combinations, e.g., about 2% of the AualCxD and the Aual rucuca females deposit a few eggs. Larvae hatch from some of these eggs and develop to adulthood.

Egg-shape mutations: Anterior egg coverings are incompletely formed in Apc females. This often leads to leakage of the egg cytoplasm, followed by degenera- tion of several of the egg primordia, formation of flaccid eggs with rudimentary chorionic appendages. The Apc phenotype is brought about by altered functions of the anterior follicle cells (SZABAD and HOFF-

Eggs originating from Ven-carrying females are usually rudimentary and flaccid. The dorsal appendages are frequently missing, and short when present. Very often only a thickening remains from the chorionic appendages on the dorsal egg surface at a slightly more posterior position than seen in wild-type (Figure 4). Similar eggs are deposited by females homozygous for either of the fs mutations gurken or torpedo and the mutant phenotype is the result of a ventral shift in the dorsoventral egg polarity (SCHUPBACH 1987).

Early arrest in embryonic development: Embry- onic development is arrested before the blastoderm stage in 27 of the 51 Fs(3) mutations. Meiotic defects, as indicated by the lack of formation of the polar bodies, were observed only in the Z~?r'~~-derived eggs. Females that carry any of the 6 mutations Bak, Bot, Pud, Dami6', Dam2'* and Tom40y' deposit eggs that are fertilized, the meiotic divisions are completed, however virtually no further development takes place. Five nuclei can be identified in the DAPI-stained eggs

MANN 1989). I;ICURI'. 4.-1);1rL-field pllotographs of eggs from wild-type (A)

Ind Venderived females. The appendages seen on the dorsal ante- .ior surface of the wild-type egg form a knob of appendage material )n the Venderived eggs (arrow), an indication of ventrali7ation of he follicle cell epithelium during oogenesis (SCHUPRACH 1987).

n a typical alignment: nuclei of the three polar bodies, .he oocyte and that of the sperm (Figure 6A). Embry- ,genesis may occasionally proceed up to the blasto- ierm stage in a few of the Bot-, Pud- or Dam l6'-derived 'ggs (Figure 5). In a small fraction of the Tom4"'- lerived eggs, cleavage nuclei appear in the posterior .hird of the egg cortex besides the vitellophage nuclei .hat accumulate in the egg center along the antero- Iosterior egg axis.

The sperm pronuclei divide a few times in eggs of Ibn-, Var-, Kun-, Boj-, Lab-, Zer4i04 and Hub"'-derived emales while the oocyte pronuclei remain still. The zight sperm-derived nuclei line up along the antero- Iosterior egg axis in the Boj-derived eggs (Figure 6). 3ne to three large nuclei develop in the Lab-derived


e E

Nearly normal

z -Q 'Ventralized'

0 Some

L

0 - e a

Blastoderm

Cleavage def.

Pronuclear def.

Fertilized eggs

Meiotic defects

I

m I s c E

E

W

I

c

.- o 2 m c E - I - m

-0 2.5 L y yq..

x E, b ' E =

m 2 -

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e? C 0

I = 2 Z m E

m o U C a = . E m ~ W O

- 0

m I c v)

a _m d 3 3 g . E ; g " p d , D P P L L

p 5 f ; - m * .e 0

- , 0 _ m = = S z . E . ~ g g ~ m o n m m

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C I. ; g s a g . : ; : . : ; ; ; g;;; W

0 m - 5 S z I g g : P : : E o 5

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a

I a l i : L P

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0 L

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- 0 c a L = 0

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z z z e e e

0 0 0 : : : e

f Q W - 4 w o o z - 5 P Z Z B - 4

31: " 0 .;;&g$B$ u - : : g G E D ~ ~ E + Q Q Q Q . . Q L S . + - $ a b s - 8 ~ ~ ~ = z = = a c ~ a a - c - $ % ~ $ = ' , ~ 6 g % ~ $ $ Q O ~ Q c ~ ~ r ~ ~ ~ ~ ~ ~ o ~ ~ s ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ a ~ ~ ~ ~ s ~ ~ ~ ~ ~ ~ ~ ~

FIGURE Fi.--Schem;~tic illustratioll of the I.'s(3)-derivcd enlbryonic nwtant phenotypcs. 'fhe horizontal lines represent the stages up to which development may take place in the Fs(3)-derived eggs. The figures above the lines show the percentage of embryos that possess typical mut:lnt phenotypes. Ihtted and dashed lines represent occasional escapers and cases where different mutant phenotypes appear with frequencies > I 0%. respectively. The symbols are a s follows: a, the sperm pronuclei do not divide and b, do divide following fertilization; c, clr;~\;~gc. divisions do not take place; tl, fenlale and male-derived pronuclei divide a few times without fusion. Note: only few larval cuticle s t r ~ ~ c t ~ ~ r e s fornl i n the Tol/"'b-derived eggs, anlong which only structures that derive from ventral regions of the blastoderm fate map can be idcntifietl.

FIGURE 6.-Fluorescent nlicro- graphs of Fs(3)-derived embryos following DAPI staining in the c;lse of Pud (A), Var (R). Lab (C) , Roj ( D ) , J u f (E) and Kar (F).

eggs (Figure 6). They probably derive from multipli- cation of the sperm pronuclei without segregation of the chromosomes because nuclei of the oocyte and the polar bodies are present in these eggs. Cleavage divisions may proceed to the blastoderm stage in a few of the Kun-derived eggs.

In the eggs deriving from Ger, Hor, Tom3", Tomihd and Hub"'" females the female and male pronuclei approach each other and almost fuse; however, they

do not divide. Expressivity of the mutant phenotype for Tom i6d and Hor is not 100% and 20-30% of the embryos develop to the blastoderm stage.

In eggs of Far, Zom, Hod and Hub*/'' females the female and the male pronuclei divide a few times without fusion. Linear arrays of four haploid-like nuclei appear in the egg cortex in a small fraction of the H~b'/~'-derived eggs. These figures are reminiscent of the alignment of the oocyte and polar body nuclei

118 M. Erdglyi and J. Szabad

seen in wild-type eggs shortly after fertilization. Cleavage abnormalities were noticed in eggs de-

rived from Hub''*-, Jut-, Kar- and Lev-carrying females. Only three cleavage divisions take place in the Hub"'-derived eggs. In eggs of Jut females several groups of four small nuclei develop in the egg cortex reminiscent of the oocyte and polar body nuclei ar- rangement (Figure 6). Cleavage divisions proceed up to the syncytial blastoderm stage in about 20% of the Kar-derived embryos. However, relatively large areas are devoid of nuclei (Figure 6). Development of virtually all the Lev-derived embryos is terminated at the syncytial blastoderm stage.

Larval cuticle producers: In 21 of the F s ( 3 ) mutations 5 to 100% of the embryos developed to the larval stage of differentiation. Only chitin granules are seen in the BerX/la and Berya-derived embryos. In embryos that derived from Kel or Pil-carrying mothers, poorly formed cuticle fragments develop in about 50% of the embryos. These cuticle fragments represent mostly the posterior regions of the fate map: fragments of the filzkorper and ventral cuticle belts appear most commonly (4. LOHS-SCHARDIN, CREMER and NUSSLEIN-VOLHARD, 1979; JURGENS et al. 1986; JURGENS 1987; Figure 7).

About 20% of the embryos that derive from Tev females show the ventralized phenotype described for Toll and easterD mutations along with strong head lesions (ANDERSON, JURGENS and NUSSLEIN-VOLHARD 1985; ANDERSON and NUSSLEIN-VOLHARD 1986). In about 50% of the Kev-derived embryos the most ex- treme anterior and posterior structures develop (Fig- ure 7). Severe head lesions develop in embryos derived from Pal, Ros or Kav2'g females (Figures 5 and 7). Some posterior structures can be identified in about 5% of the Kav'" and KavZ"-derived embryos (Figure 7).

Embryos deriving from females carrying any of the new Toll alleles (TollZb, Toll", Toll'Ob, Toll'" and Tollzoi) or the new easterD alleles (eaD4Io2, eaD5OZ2, eaD1za and eaDzoa) possess typical ventralized phenotype described earlier (ANDERSON, JURGENS and NUSSLEIN- VOLHARD 1985; ANDERSON and NUSSLEIN-VOLHARD 1986). Chitin granules form in about 30% of the Tol13"-derived embryos. Almost normal-looking larvae develop in about 70% of the eggs deposited by Zol- carrying females. However, the dorsal arms of the cephalo-pharyngeal head skeleton bend up and out- ward.

Cases where larval cuticle structures developed in about 50% of the embryos are particularly interesting. It appears that in the case of Ros, Bergs and Kel, those embryos develop larval cuticle structures that inherit the + allele from the Fs(3) /+ mother, while those that inherit the F s ( 3 ) allele terminate development prior to formation of larval cuticle: in the cross where y/y;

F s ( 3 ) / y + , TM3 females were mated with y/Y; +/+ males, cuticle of all developing embryos was y+. This shows that they inherited the + (in fact the y+, TM3) and not the F s ( 3 ) homolog from the mother. Fs(3)+ mosaicism: Fs(3)lrucuca adult females were

X-irradiated in order to induce Fs(3)+ clones through mitotic recombination. Analysis of the Fs(3 )+ clones should provide information about germ-line depend- ence of the Fs(3 ) mutations and facilitate mapping of these mutations.

The frequency of mosaicism was less than 1% in 9 cases, fell between 1 and 3% for 20 of the F s ( 3 ) mutations and exceeded 7% only in two cases (Table 1). These frequencies were unexpectedly low.

For an estimation of the expected frequency of mosaicism, we irradiated y KIO/+ +; mwh Sb63b/+ + early third instar larvae for the induction of different types of clones through mitotic recombination. Of the 100 females, 46 deposited K I O eggs. It was estimated, based on a Poisson distribution, that these females carried 6 1 KIO homozygous germ-line clones. There were 31 3 y, 156 mwh and 140 Sb+ clones identified on the abdomens of the above females. When adult y K10/+ +; mwh Sb6j6/+ + females were irradiated, 73% of them deposited KIO eggs. It was estimated that 100 of these females carried about 13 1 KIO homozygous germ-line clones. The former figures were used to calculate the expected frequencies of Fs(3)+ mosaicism. Knowing that y and K10 are closely linked (WIESCHAUS, MARSH and GEHRING 1978) and assuming that mitotic recombinations take place with similar frequencies during larval and adult life on the X and the third chromosomes, the expected frequencies of Fs(3)+ mosaicism were about 48 and 44% for F s ( 3 ) mutations located in the vicinity of rnwh and Sb, respectively. As mentioned earlier, the observed frequencies were far lower than expected (Table 1).

A further unusual feature of the Fs(3)+ mosaicism was the very long time lapse between induction and detection of the mosaicism. It took 9-1 5 days to recover the first K l O eggs following irradiations of adult females (see also Figure 5 of WIESCHAUS and SZABAD 1979). In 27 of the Fs(3 ) mutations the first eggs from which progeny developed were deposited 12-22 days following irradiations, and in 5 cases this time period was as long as 19-30 days. No or minor delay was recorded for only 8 of the Fs(3) mutations (Table 1). The former observations made it necessary to test the irradiated females for 221 days (often to the end of their life) in order to recover the majority of the mosaics.

Most of the F s ( 3 ) mutations studied resulted in unusual patterns of production of progeny. When an Fs(3)+ oogonial stem line cell is created in an adult female, it may directly follow the pathway of egg differentiation and be the source of a single egg or it

Fs Mutations of Drosophila

may remain an undifferentiated stem line cell from which several eggs derive during her subsequent life (NOTHICER et al. 1978; WIESCHAUS and SZARAD 1979). T h u s a lot of 10 females with one Fs(3)+ germ- line clone is expected to yield a single offspring about half of the time, and several (23) offspring in the other 50% of the cases. Only one-fourth of the lots with two clones should produce two offspring each and three-fourths should produce 2 3 . Altogether,

119

FIGURE i.-lhrk-ficlcl micro- graphs ofcn1l)ryos th; l t derivrd from w i l d - t v l x (A ) and Fs(jr)-c;~rrving tnorllers Rerx"" (B). K m ( C ) , Kav'" (D). K t 1 (E), Ros (F). Rtr'" ( G ) , Pi1 (H). Pal ( I ) , Toll" (J) ;wcl ea"5"y2 (K) .

more than 50% of the lots are expected to give rise to 2 3 offspring each. These expectations are valid only when the Fs mutations interfere with functions of the germ-line cells (WIESCHAUS and SZARAD 1979; SZARAD and HOFFMANN 1989).

The offspring production pattern fit expectations for only four of the F s ( 3 ) mutations (Ber'", Kel , Tar and Pal; Table 1). Although the low frequency of mosaicism hindered analysis of the offspring-produc-

120 M . Erdklyi and J. Szabad

tion pattern for several of the Fs(3 ) mutations, in general many more of the lots yielded one or two offspring than expected, at the expense of those with 2 3 progeny (e .g . , Bot, Tom409', eaD4102, eaD12a; Table 1). In the cases of Apc and Ava however, none of the lots yielded a single offspring and in fact 1 3 were recovered from most of the lots which produced offspring. Both Apc and Ava proved to be nongerm-line dependent, as was shown by analysis of the germ-line chimeras. In summary, the observed frequency of Fs(3)+ clones was less than expected, clones appeared with several days of delay, and fewer offspring derive from the Fs(3)+ clones than expected.

Chimeras: The low frequency of Fs(3)+ mosaicism, the unusual offspring production pattern as well as the observation that the Fs(3) alleles were transmitted to the offspring of the irradiated females (as will be presented) suggested that the dominant female sterility may not merely be the consequence of disturbed germ-line functions. T o evaluate this possibility, Fs(3)/rucuca germ-line chimeras were constructed for 33 of the Fs(3 ) mutations by transplantation of pole cells (Table 1). With the exceptions of Apc and Ava, which are not germ-line dependent, presence of the Fs(3) mutation in germ-line cells was a sufficient condition for production of dominant female sterility. However, for several of the Fs(3 ) mutations studied, the development of embryos that derived from chimeras that carried Fs(3)/rucuca germ-line cells was different from that seen for embryos that derived from the Fs(3)lrucuca females (J. SZABAD, unpublished data). This observation indicates that contri- bution of both the germ-line and the soma may be essential for the production of the dominant maternal- effect phenotype in these cases.

Apc and Ava are nongerm-line dependent. Normal eggs and progeny derive from the Apclrucuca germ- line cells when surrounded by normal soma (SZABAD and HOFFMANN 1989).

Analysis of chimeras with Ava/rucuca germ-line cells revealed that the egg retention phenotype is not germ- line dependent: the Ava germ-line cells are the source of offspring when surrounded by normal soma. On the other hand, no offspring derived from the normal germ-line cells when implanted into Avalrucuca hosts, indicating that the focus of the mutant phenotype is located in the Ava soma (Table 3).

Analysis of ovarian chimeras showed that the focus of the Ava mutant phenotype is not located in the ovarian soma. The two chimeras with Avalrucuca ovaries deposited several eggs from which progeny developed. The five Avalrucuca chimeras that carried normal (in reality y v f mal) ovaries did not deposit eggs, although the implanted ovaries were attached to the oviduct of the Avalrucuca hosts. We propose that Ava interferes with the egg deposition step of the

TABLE 3

Germ-line chimeras of Fs(3)Avar"

A

Genotype of embryos Types of chimeras

Donor Host males Avalrucuca +'/rucuca Host fe-

Avalrucuca

+b/rucuca or * +"/K1237 18 3 2

B

Genotype of embryos Host females

Aualrucuca +'/lrucuca

Donor Host Total Chimeras Total Chimeras

Avalrucuca

+b/rucuca y u f m a l - or 39' 13 18 7

wild-type (Ore-R). TM3, Sb Ser.

'Six of the 39 Avalrucuca females deposited eggs. Two of then) yielded y v f mal offspring. The other 11 y v f mal//Ava/rucuca chimeras were identified on the basis of the mal homozygous, nonstaining egg primordia in their ovarioles.

female reproductive pathway (CJ: SZABAD and FAJSZI 1982).

Mapping the Fs(3) mutations: Eight of the 5 1 Fs(3) mutations are incompletely penetrant in trans-heterozygous condition with the rucuca chromosome. Ten to 100% of such Fs(3)/rucuca females gave rise to a variable number of offspring. Several of their progeny females were recovered and screened for the presence of the Fs(3) mutation. Standard mapping procedures were then used to localize these mutations on the meiotic map (Table 2).

Mapping of Apc and Ava, the soma-dependent Fs(3 ) mutations, was based on the recombinant progeny recovered from the chimeras. For example, 34 h Apc th+, 29 h+ Apc+ th, 13 h Apc+ th+, and 5 h+ Apc th recombinants were recovered from the 14 Apclrucuca germ-line chimeras, dividing the h-th distance at the ratio of 63: 18. Using the map positions of h and th as reference points (26.5 and 43.2 cM, respectively), the Apc locus was mapped at 39.5 cM. Similarly, the 14 h Ava th+, 13 h+ A m + th, 9 h Ava+ th+, and 10 h+ Ava th recombinant progeny females, recovered from the chimeras with Ava/rucuca germ-line cells or ovaries, divided the h-th distance in a ratio of 27: 19; thus the site of the Ava locus is 36.3 cM.

Mapping of most of the germ-line dependent Fs(3 ) mutations was based on analysis of progeny recovered from Fs(3)+ clones (see MATERIALS AND METHODS). Surprisingly, wild-type alleles of all the marker mutations appeared, although with very different frequencies, among the progeny of all the 42 Fs(3 ) mutations studied. For instance, while mutant and wild-type alleles of the marker mutations located on the right


th st cu FIGURE 8.--Schematic illustra- Fd3) Progeny 'i' tion of mapping six Fs(3 ) mutations.

Fs(3)lrucuca adult females were x- irradiated for the induction of mitotic recombination, and formation of Fs(3)-free germ-line clones (see

15 14 14 24 29 30 * 31 also Figure 2). Genotypes of the 16 17 17 7 2 1 0 progeny, recovered from the Fs(3)-

free germ-line clones following a test 54 cross with rucuca males, served to 3 l map the 2743) mutations. The num-

2 2 3 5 8 the marker mutations as identified in a a 7 5 2 the progeny are shown above and

under the lines associated with each

i U:l 7 i Zombor18e All 5 1 52 * 47 25 26 31

2 1 6 28 27 22

Zerind'" All

All 67 18

H O ~ O S ~ ~ ~ Fertile females 29

48 43 44 46 52 37 42 41 39 33 20 16 10 11 12 18

O * 9 13 19 18 17 ber of mutant and wild-type alleles of

Sterile females 1 9

All

K e ~ e l ' ~ Fertile females

Sterile females

33 26 27 22 23 26 33

30 40 39 44 43 38 36 10 6 4 4 3 0

7 4 6 6 7 3

1 1 6

2 4 5 5 5 4

o * 2 1 5

All 16 14 16 17 20 23 20

10 22 20 19 16 13 18

UeledQb Fertile females 4 5 6 5 4 3 2 3

6 6 6 6 3 4 2

0 0 0 O * : , 3' Sterile females

All 27 35 35

16 6 13

27 22 24 24 35 40 313 38 27

Toll3c Fertile females :5 5 6 11 11 19

28 34 18

15 10 10 2 6 7 5 6

3 5

11 12 14 13 14

* Sterile females :o

arm appeared at roughly the 1 : 1 ratio in the case of Zom, the ru/ru+, h/h+ and th/th+ alleles were recovered at the ratios of 51/2, 52/1 and 47/6, respectively (Figure 8). These data indicate that Zom is located between the marker mutations h and th. Dis- tribution of the mutant/wild-type pattern of the marker mutations was similar for the following 11 Fs(3) mutations: Bak, ZerI5', Ton, Kun, Kav'", Kav2",

, and Var. It is a common feature of these mutations that the Fs(3) allele is not (or very seldom) transmitted to the offspring (Table 1 )*

In 19 of the Fs(3) mutations mutant and wild-type alleles of all the marker mutations appeared with rather similar frequencies, rendering the mapping procedure unapplicable (Figure 8). It was a common feature of these mutations that the Fs as well as the Fs+ alleles were recovered among the offspring, although with rather different frequencies (Table l). Location of these Fs(3) mutations was established on the basis of the phenotype of the fertile and sterile offspring females. Figure 8 shows 4 typical examples of these 19 Fs(3) mutations.

For 12 additional Fs(3) mutations, only 3-8 offspring could be recovered from the Fs(3)lrucuca females following irradiations (Table 1). Reversion and the subsequent complementation analyses demonstrated that four of these mutations are allelic to other F s ( 3 ) mutations (Zer4'04, Tom3", Berg/'" and Kav2'g)

T ~ , eaD5022, eaD12a eaD4102

Fs(3) mutation. Since wild-type alleles of the marker mutations distally located from the Fs(3 ) mutation are not expected to appear in the offspring, the most likely site for Zom is the h-th chromosome interval. For cases in which mutant and wild-type alleles of the marker mutations appeared with rather similar frequencies, the phenotypes of the fertile and the sterile females were clues in es- tablishing the possible sites of the F s ( 3 ) mutations. The top thick line shows the standard genetic map with sites of the marker mutations present in the rucuca chromosome.

and three proved to be allelic to Toll or easter (Toll I O b ,

T011'~" and eaD20n). For 16 of the Fs(3) mutations, mitotic recombina-

tion was also induced in spermatogonial cells of Fs(3) / rucuca males and the recovered recombinant offspring were tested for the presence of the Fs(3) alleles. These 2743) mutations included several that could not be mapped on the basis of female recombination as well as five of the incompletely penetrant Fs(3) mutations.

In the control, where mwh e/rucuca males were irradiated, 1.5% (175/11,663) of the progeny flies were recombinants. In the Fs(3)lrucuca experiments 1-2% of the offspring were recombinant (Table 1). Thus, all of the 51 Fs(3) mutations could be mapped to intervals on the chromosome using one or more of three different mapping procedures (Figure 9).

Reversion of the Fs(3) mutations: Reversions of the Fs(3) mutations were induced to determine possible allelism with (1) already known recessive me1 mutations and (2) allelism among the Fs(3) alleles. Analysis of the revertants may also reveal the "loss-of- function" phenotypes. The revertants were induced in a second mutagenesis of the Fs(3)-carrying chromosomes, either by EMS or x-rays. The revertants appeared as fertile F1 females with rather different frequencies: while 3 of the 21 9 EMS-treated TolllOb chromosomes carried revertants, this frequency was only 1/4225 for Bot (Table 1). On the average 1 of about 350 chromosomes carried one F s ( ~ ) ~ mutation

122 M . Erdklyi and J. Szabad

ru h th st cu sr e' ca

1 u- 1 1 1

I

Ven

Var Bak

Hod Pal

Bot Lab

U Hub'vu HubsMD Hub'" Hub'B'

Ava mi Gel Zom Tev Kev Apc

Far

irrespective whether the revertants were induced by 25 mM EMS or 4000 R of x-rays. However, many more than 350 chromosomes were screened for a number of Fs(3) mutations without any indication of their reversion; e.g. none of the 2938 EMS and the 3730 x-ray treated Apc chromosomes carried a revertant. "Revertants" of the incompletely penetrant mutations emerged with rather high frequencies. Those revertants which retained a partial dominant phenotype were omitted from further studies. No revertants could be recovered for Apc, Ava, Far, Lev, Pi1 or 201. These mutations as well as Bot, whose only revertant is a partial one, were thus excluded from the complementation tests.

Almost all of the F~(3)~-bearing chromosomes behave as zygotic lethals in homozygous condition. Le- thality may be an intrinsic feature of at least some of the revertants or, more likely, is brought about by second site lethal mutations. A number of F s ( ~ ) ~ mutations are partial revertants.

Complementation analyses: The complementation analyses included 44 Fs(3) mutations and their 113 revertants. A first test determined whether the revertant-carrying chromosomes were viable over the original Fs(3) mutations from which they were derived. With only two exceptions, the F~(3) /Fs(3)~ combinations are lethal. One of the exceptions is KevRX2. The Kev/KevRX2 females are viable and fertile, most likely because KevRX2 is a dominant suppressor mutation of Kev. The other exception is Tev. All seven revertants of Tev are viable over the original Tev chromosome. The phenotype of the embryos produced by the Tev/TevR females are identical with those seen in case of the Tev/+-mothers.

For 31 of the Fs(3) mutations, two to seven revertants were recovered (Table 1). Individuals, trans- heterozygous for revertants of the same Fs(3) mutation did not usually develop either because the revertant homozygous condition is not viable and/or

Jut Lev Hor

Ton

Kun

f I I I I I I

I I

I !

FIGURE 9.-Localization of 5 1 F s ( 3 ) mutations on the meiotic nlap. The top thick line shows the standard genetic map with sites of the marker mutations present in the rucuca chromosome. The Fs(3) loci are located within intervals delineated by two neighboring dashed lines identified by the marker mutations. The F s ( 3 ) mutations in the framed boxes are allelic.

due to the second site lethal mutations present on the maternal Fs(3) chromosome. Exceptions were observed for Ber'l''" (Figure 1 l), Kev and Tev and included fertile as well as recessive female sterile combinations.

T o determine whether some of the Fs(3) mutations are dominant alleles of already known recessive me1 mutations, one revertant (all in the case of allelism) of those Fs(3) mutations that allow formation of at least some larval cuticle were combined with the "dorsaliz- ing" me1 mutations easter, nudel, pelle, pipe, snake, spatzle, TollgQRE (a revertant allele of Toll; ANDERSON, JURGENS and NUSSLEIN-VOLHARD 1985) and tube (AN- DERSON and NUSSLEIN-VOLHARD 1986). All 10 revertants of the 4 eaD alleles are viable over easter. The easter/eaDR females are sterile: their offspring possess typical "dorsalized" phenotype.

The 9 revertants of 5 Fs(3) mutations resulted in recessive female sterility in trans-heterozygous condition with TollgQRE. Typical dorsalized embryos developed in the eggs of these females, indicating that 5 of the newly induced 51 Fs(3) mutations are alleles of Toll. Results of the complementation analyses between the deficiencies Df(3R)roaob and Df(3R)TI 5BRXP and revertants of the new Toll alleles as well as their revertants are summarized in Figure 10. Females that carry TollzbRx2 and either of the me1 mutations spatrle or pelle give rise to dorsalized embryos, most likely because is a deficiency that deletes the Toll as well as the nearby spatzle and pelle loci. One revertant of Toll'Ob (RE3) shows recessive female sterility over the me1 mutation easter and the offspring embryos are dorsalized.

Large nuclei develop in the Lab-derived embryos, as described for the me1 mutation gnu (FREEMAN, NUSSLEIN-VOLHARD and GLOVER 1986). Females trans-heterozygous for gnu and Lab'7cR"' are fertile, indicating that gnu and Lab are nonallelic.

Initially, one revertant of each of the 44 Fs(3)


P

8 % 2

Fs(3) $ 2 9 e

and = s Q F s ( ~ ) ~ 6 8 g g g

!!a)* &

26

3c D t t D D F 2 b Rx2

D t t F F F 2bRX' D t D V V V

D t D F F F 20iRE' D D D V V V 20i D D D F F F 78aRx3 D t D F F F 78aRX7 D D V V V V 18a D D D F F D lobRE3 D D D F F F 70bRE2 D D D F F F 70bRf7 E E V V V V 106 D t t F F F 3CRX7 D t N C V V V

FIGURE 10.-Complementation between 5 new Toll alleles (2b , 3c , IOb, Z8a and 2Oi) and their revertants with a formerly described Toll revertant (YQRE) and two deficiencies that eliminate the Toll locus (Df(?R)roff0' and Df13R)TZ5BRXP (6 ANDERSON, JURGENS and NUSSLEIN-VOLHARD 1985) and with the me1 mutations spatzle, pelle and easter.

mutations was chosen for the complementation analysis among the F s ( ~ ) ~ mutations, and a panel of 924 crosses was set up. All revertants were included for the F s ( 3 ) mutations that map to common chromosome intervals and bring about similar mutant phenotypes.

The females trans-heterozygous for revertant alleles of the different F s ( 3 ) mutations were viable and fertile. However, in 32 cases the revertant trans-heterozygous conditions were not viable or resulted in female sterility. These cases are illustrated as framed boxes in Figure 11. All 7 revertant alleles of Dam21m are lethal over , and hence allelism was inferred. Similarly, all 3 RE alleles of

are lethal over the 3 RE alleles of indicating allelism of these F s ( 3 ) mutations. The RE alleles of Tom4'", Tom'" and Tom16d are viable in trans- heterozygous condition, however the females are sterile. One revertant of Berg" is lethal over all the four revertant alleles of Ber*'I", however the other allele (BerYaRE1) is viable and fertile (Figure 11). Because these two F s ( 3 ) mutations map to the same chromosome interval and result in very similar phenotypes, they were considered to be allelic.

The complementation pattern of the 5 RE alleles of the 3 Kav mutations is rather complex: lethal, fertile as well as recessive female sterile combinations were recovered (Figure 1 1). Females trans-heterozygous for Kav 18cRb2 and Kav211R"1 give rise to embryos with severe head defects. Embryonic development is ar-

or D~~ 16bRE2

zer 4104

rested prior to the blastoderm stage in the other 3 recessive female sterile combinations.

Complementation of the Toll revertants gave rise to lethal, female sterile and fertile combinations (Fig- ure 1 1). While most of the female sterile combinations resulted in formation of dorsalized embryos, no cuticle developed in the eggs of the ~~ll20iRR"l females.

DISCUSSION

This paper describes genetic and developmental characterization of 51 newly induced Fs(3) mutations. Following EMS treatments, one in about 1000 chromosomes carried an F s ( 3 ) mutation. Zygotic lethal mutations were 2500 times more frequent than the F s ( 3 ) mutations.

Reversion of 44 of the F s ( 3 ) mutations could be induced although with rather variable frequencies: one in 73 to 4225 of the EMS or x-ray treated Fs(3)- carrying chromosomes showed reversion of the Fs phenotype. Complementation analysis of the revertants demonstrated that the 44 of the Fs(3) mutations represent 27 units on the third chromosome mutable to dominant female sterility by EMS: 5 new alleles of TollD, 4 of easterD and Huba, 3 of Kavar and Tomaj, 2 of Bercel, Damasa and Zerind were recovered; in ad- dition, 19 units were represented by single F s ( 3 ) mutations. A rough estimation of the numbez- of the third chromosome units which are mutable by EMS to dominant female sterility can be provided if we allow a simplifying assumption: these units mutate with similar frequencies following EMS treatment. A Poisson distribution shows than that there are about 50 such units on the third chromosome of Drosophila melanogaster. Phenotypes and map positions of the 7 Fs(3) mutations not included in the complementation sug- gest that they probably represent additional Fs-mutable units. Thus it seems likely that with the 51 Fs(3) mutations we identified about 60% of the genes on the third chromosome that can be mutated to dominant female sterility.

Except the 5 new TollD and the four easterD alleles, the F s ( 3 ) mutations appear to identify novel genes and functions. Unexpectedly, none of the 51 Fs(3) mutations seem to be beneficial as a tool in the dominant female sterile technique as are the strictly germ- line dependent Fs mutations Fs(Z)D, Fs(I)K1237 and F s ( 2 ) 1 , that are agametic or allow formation of a few abnormal eggs only (WIESCHAUS 1980; PERRIMON and GANS 1983; SZABAD, REUTER and SCHR~DER 1988). Although the Fs(3)Avar-carrying females are agametic, Avar is a nonovary-dependent egg retention mutation. Of the two egg shape mutations Fs(3)Apc is follicle dependent (SZABAD and HOFFMANN 1989) while Fs(3)Vencellin is incompletely penetrant. Eggs produced by the Vencellin-carrying females are "ven-

124 M. Erddyi and J. Szabad

FIGURE 1 I.-Results of the complementation analyses between revertants of 44 Fs(3) mutations. The symbols are as follow. j- = lethal combination. S = female sterile conlbi- nation. D = female sterile combination where the females give rise to dorsalized embryos. F = fertile combination. In the major panel most of the combinations were fertile and for simplicity, the F synlbols were omit-

t, but B e r y & x t / e a n ~ z ~ ~ ~ l - ~ = E’. b = ted. Notes: a = BerY”RE’/eaD’ZaHb-”2 =

~~b 17~Rfi~l/Dam16bREl-2 = t but ~~b I7rREI / D a m 2 ~ m ~ f i : ~ = F, c = Tev4eHb.’l-

~ / ~ ~ n r u r e n x l = t. but TevKx“7/ea- usmmxt = F. d = females with Toll’(’bRE3 over any revertants of the 4 ea” alleles are sterile and produce dorsalized embryos. e = PalRE’/ Kauzl@Ef = j-, but palR~1/KaUZ16HEi = pal/+. f = Lab17~REl/pUdJ~21H“i-3 =

j - 9

but Lab’7‘R“1/PudM21RE4-7 = F. Inserts show results concerning complementation of the Tofl. Be7 and kav complementation groups.

tralized” in a manner similar to those deposited by the females homozygous for the mutations gurken or torpedo. Such observations emphasize the importance of close interactions between somatic and germ-line com- ponents in the developing egg primordia (SCHUPBACH 1987).

Females that carry any of the other Fs(3 ) mutations deposit normal-looking eggs. In 27 cases no or very little development takes place following fertilization. Several of these F s ( 3 ) mutations seem to interfere with fertilization (Zerind’jd), fusion, and division of the pronuclei. In eggs derived from Jutas or Huba8/ 4Q-carrying females, groups of four small nuclei appear among the cleavage nuclei in an array reminiscent of the polar bodies and the oocyte nuclei seen in the wild-type eggs shortly after fertilization. The large nuclei seen in Laborc-derived eggs are similar to that described for giant nuclei (gnu), a recessive me1 mutation that uncouples nuclear divisions from many cy- toplasmic events of mitoses in the Drosophila embryos (FREEMAN, NUSSLEIN-VOLHARD and GLOVER, 1986). However, Laborc and gnu are not allelic.

In 2 1 of the Fs(3 ) mutations development can proceed up to the larval stage of differentiation. One of these, Fs(3)Tevel, interferes with formation of the

dorsoventral embryonic polarity. Tevel is not a dominant allele of any of the known mel mutations that give rise to “dorsalized” embryos. Fs(3 ) mutations in the Palat, Rosd, Kavar and Keve loci interfere with formation of the anterio-posterior embryonic polarity. These Fs(3) mutations may represent important, pre- viously unknown gene functions involved in establishment of the segmented body plan of the Drosophila embryos (NUSSLEIN-VOLHARD and WIESCHAUS 1980; AKAM 1987; NUSSLEIN-VOLHARD, FROHNHOFER and LEHMANN 1987; INGHAM 1988).

Although the nature of the mutations that lead to the F s ( 3 ) phenotypes is not fully understood, observations indicate that several of them are “gain-of- function” types of mutations. This is best demonstrated by the observation that 94% of them could be reverted through second mutagenesis that inactivated or removed the Fs alleles, as was described for the TollD and torsoD alleles (ANDERSON, JURGENS and NUS- SLEIN-VOLHARD 1985; KLINGLER et al. 1988).

The “gain-of-function” nature of the Fs(3) mutations is also shown by the unusual behavior of the Fs(3)+ clones induced in stem line cells of the F s ( 3 ) / + adult females. The Fs(3)+ clones were 10-100 times less frequent than expected, appeared with a delay of


about 5-15 days when compared to the control, and very few offspring derived from the Fs(3)+ clones. These observations are consistent with the hypothesis that perdurance of the mutant gene products present in the Fs(3)/+ germ-line cells at the time of clone induction leads to a delayed expression of the nonmutant phenotype (GARCIA-BELLIDO and ROBBINS 1983; PERRIMON 1984). Whether the mutant phenotypes are brought about by production of mutant and/ or ectopic expression of the normal gene products will have to be evaluated through studies of the indi- vidual Fs(3) mutations. It appears that constitutive expression of normal gene products is the source of dominant female sterility in at least a few of the Fs(3) mutations. In the case of Berg”, Keled or Rosd, development of the +/+ embryos can proceed up to the larval stage of differentiation, implying that the mutant embryonic phenotype is brought about by maternal expression of the Fs(3) alleles. On the other hand, the Fs(3)/+ embryos receiving the Fs(3) allele from the mothers terminate development prior to formation of the larval cuticle, probably as a consequence of expression of the Fs(3) alleles in the young embryos. However, the +/Fs(3 ) embryos that receive the Fs(3) allele from the father develop to adulthood, indicating maternal-zygotic interactions in production of the mutant phenotypes. Similar types of maternal- zygotic interactions have been described by ROBBINS (1983), SIMPSON (1983) and TRICOIRE (1 988).

It is not clear, however, whether other F s ( 3 ) mutations are anti-, neo- and/or hypermorphs. Future studies should include analysis of the mutant phenotypes in Fs(3)/+/+ females as well as in those who represent the “loss-of-function’’ phenotypes, using revertants and newly isolated alleles or already available deletions. It has not been possible to test most Fs(3 ) mutations for “loss-of-function” phenotypes because, in some cases, the revertant homozygous condition is lethal (as was described for several of the Toll” revertant alleles; ANDERSON, JURCENS and NUSSLEIN- VOLHARD 1985) and in others second site lethal mutations present on the original Fs(3) allele-carrying chromosomes prevent the necessary tests of revertant- bearing chromosomes. Future determination of the Fs(3)/+/+ and the “loss-of-function’’ phenotypes may be possible following more precise localization of the F s ( 3 ) mutations.

Mapping of the Fs(3) mutations was based primarily on analysis of Fs(3)+ germ-line clones induced in adult females. Progeny may develop from the Fs-free daughter cells. In such progeny, wild-type alleles of the marker-mutations located distal from the Fs(3) mutations are not expected to be present; thus every F s ( 3 ) mutation should be localized between two of the marker mutations. Unexpectedly, high frequencies of wild-type alleles of these marker mutations appeared

for most of the Fs(3 ) mutations. This might be due to (1) double mitotic recombinations and/or (2) reversions of the Fs(3) mutations. I t is known that the frequency of double exchanges is 10-20 times higher than expected from the frequency of induced single mitotic exchanges (GARCIA-BELLIW 1972). Reversion (often partial) of the 2743) mutations usually takes place with a rather high frequency (BUSSON et al. 1983). Whether the high reversion frequencies reflect physically large targets or “inherent instability” (as suggested for the Toll gene by ANDERSON, JURCENS and NDSSLEIN-VOLHARD 1985) is not clear. Analysis of Fs(3 ) revertants that originate due to deletions, inversions or translocations or analysis of P-element- induced revertants by in situ hybridization should provide more precise localization of the F s ( 3 ) mutations. The latter types of mutations should also allow molecular cloning of the genes identified by the Fs(3) mutations and analysis of new aspects of oogenesis, maternal-effects and embryonic pattern formation through detailed analysis of single Fs(3 ) mutations.

We are grateful for the excellent technical help of ANDREA PAPP- MosONYl. We also greatly appreciate the help and discussions of several of our colleagues during the course of the ‘Fs (3 ) Project”: J ~ N O S CSIRIK, GYULA HOFFMANN, HENRIK GYURKOVICS, CHRIS- TIANE N~~SSLEIN-VOLHARD, who also provided several mutant strains, and GUNTER REUTER. We would like to thank JACK LEVY for proofreading the manuscript. We also thank T. I . HAZELRICC of the Howard Hughes Medical Institute for providing the resources of her laboratory. The research of this paper was largely supported by the grant T t 83/1987 from the Hungarian Academy of Sciences to J. S.

LITERATURE CITED

AKAM, M., 1987 The molecular basis for metameric pattern in the Drosophila embryo. Development 101: 1-22.

ANDERSON, K. A., and C. NUSSLEIN-VOLHARD, 1986 Dorsal-group genes of Drosophila, pp. 177-194 in Gametogenmis 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 Drosoph- ila embryo: the induction of polarity by the Toll gene product. Cell 42: 791-798.

ANDERSON, K. A., G. J ~ C E N S and C. NUSSLEIN-VOLHARD, 1985 Establishment of dorsal-ventral polarity in the Drosoph- ila 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 M. DEMEREC. John Wiley & Sons, New York.

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

EPHRUSSI, B., and G. W. BEADLE, 1936 A technique of transplantation for Drosophila. Am. Nat. 70: 218-225.

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

GARCIA-BELLIw, A., 1972 Some parameters of mitotic recombi- 457-466.

126 M. Erdclyi and J. Szabad

nation in Drosophila melanogaster. Mol. Gen. Genet. 115: 54- 72.

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.

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 T h e To11 gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 52:

INGHAM, P. W., 1988 T h e molecular genetics of embryonic pattern formation in Drosophila. Nature 335: 25-33.

JANNINC, W., 1972 Aldehyde oxidase as a cell marker for internal organs in Drosophila melanogaster. Naturwissenschaften 5 9 516-517.

JURGENS, G., 1987 Segmental organisation 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 organisation of the head in the embryo of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 195 359-377.

KLINCLER, 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. GANS, 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.

KRISTO, G., F. MAKKand L. SZEGFU, 1973 Data concerningnames of “early” Hungarian settlements (in Hungarian). Acta Hist. Univ. Szegediensis 44: 1-96.

KRISTO, G., F. MAKK and L. SZEGFU, 1974 Data concerning names of “early” Hungarian settlements (in Hungarian). Acta Hist. Univ. Szegediensis 48: 1-55.

LEHMANN, R., and C. NUSSLEIN-VOLHARD, 1986 Abdominal segmentation, 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. Serv. 43: 193.

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

LINDSLEY, D. L., and K. T . TOKYASU, 1980 Spermatogenesis, pp. 226-294 in The Genetics and Biology of Drosophila, Vol. 2d, edited by M. ASHBURNER a n d 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:

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

269-279.

239-255.

the spatial coordinates of the embryo of Drosophila melanogaster. Symp. SOC. Dev. Biol. 37: 185-2 1 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: 474-476.

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: 145-1 54.

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. GANS, 1983 Clonal analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female sterile mutation Fs(l)K12?7. Dev. Biol. 100 365-373.

PERRIMON, N., L. ENCSTROM and A. P. MAHOWALD 1984a T h e 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 pupa 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 .

ROBBINS, L. G., 1983 Maternal-zygotic lethal interactions in Dro- sophila melanogaster: zeste-white region single-cistron mutations. Genetics 103: 633-648.

SCHUPBACH, T. , 1982 Autosomal mutations that interfere with sex determination in somatic cells of Drosophila have no direct effect on the germline. Dev. Biol. 8 9 117-127.

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.

SIMPSON, P., 1983 Maternal-zygotic gene interactions during formation of the dorsoventral pattern in Drosophila embryos. Genetics 105: 615-632.

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

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

SZABAD, J., M. ERDELYI and J. SZIDONYA, 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 T h e effects of two mutations-connected with chromatin functions-on female germ line cells of Drosophila. Mol. Gen. Genet. 211: 56- 62.

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

TRICOIRE, H., 1988 Dominant maternal interactions with Dro- sophila segmentation genes. Wilhelm Roux’s Arch. Dev. Biol. 197: 115-123.


WIESCHAUS, E., 1980 A combined genetic and mosaic approach to the study of oogenesis in Drosophila, pp. 85-94 in Deuelop- ment and Neurobiology of Drosophila, edited by 0. SIDDIQI, P. BABU, L. M. HALL and J. C. HALL. Plenum Press, New York.

WIESCHAUS, E., and C. 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.

WIESCHAUS, E., and J. SZABAD, 1979 The development and function of the female germ line in Drosophila: a cell lineage study. Dev. Biol. 68: 29-46.

WIESCHAUS, E., and E. NOELL, 1986 Specificity of embryonic

lethal mutations in Drosophila analyzed in germ line clones. Wilhelm Roux’s Arch. Dev. Biol. 195: 63-73.

WIESCHAUS, E., J. L. MARSH and W. J. GEHRING, 1978 Fs(1 )KlO: a germ-line dependent female sterile mutation causing abnormal chorion morphology in Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 184: 75-82.

YARGER, R. J., and R. C. KING, 1971 The phenogenetics of a temperature sensitive, autosomal dominant, female sterile gene in Drosophila melanogaster. Dev. Biol. 2 4 166-177.

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