Development 113, 373-384 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

373

Mutations in a newly identified Drosophila melanogaster gene, mago nashi,

disrupt germ cell formation and result in the formation of mirror-image

symmetrical double abdomen embryos

ROBERT E. BOSWELL, MARY E. PROUT and JESSICA C. STEICHEN

Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347, USA

Summary

The mago nashi (mago) locus is a newly identified strictmaternal effect, grandchildless-like, gene in Drosophilamelanogaster. In homozygous mutant mago femalesreared at 17 °C, mago+ function is reduced, the inviableembryos lack abdominal segments and 84-98 % of theembryos die. In contrast, at 25°C, some mago allelesproduce a novel gene product capable of inducing theformation of symmetrical double abdomen embryos.Reciprocal temperature-shift experiments indicate thatthe temperature-sensitive period is during oogeneticstages 7-14. Furthermore, embryos collected frommago1 homozygous females contain no apparent func-

tional posterior determinants in the posterior pole. Inviable Fx progeny from mago mutant females, regardlessof genotype and temperature, polar granules arereduced or absent and germ cells fail to form (thegrandchildless-like phentoype). Thus, we propose thatthe mago+ product is a component of the posteriordeterminative system, required during oogenesis, bothfor germ cell determination and delineation of thelongitudinal axis of the embryo.

Key words: Drosophila, maternal effect, anteroposteriorpolarity.

Introduction

With the possible exception of mammalian eggs,pattern formation in the early embryo is initiated bymaternally encoded cytoplasmic factors (determinants)asymmetrically distributed in the egg. By providingpositional information, cytoplasmic determinants re-strict early embryonic cells to particular developmentalpathways necessary for the establishment of the majorbody axes of the organism and, in Drosophila andXenopus, cytoplasmic factors localized within the germplasm have been implicated in the determination of thegerm cell lineage (Wilson, 1925; Davidson, 1986;Wilkins, 1986). Presumably, these determinants specifycell fates by directly or indirectly regulating theexpression of genes within cells that inherit them. Thus,early spatial differences in the pattern of cellulardifferentiation can reflect: (1) localization of cytoplas-mic determinants during oogenesis (Wilson, 1925;Boswell and Mahowald, 1985a; Davidson, 1986); (2)reorganization of the cytoplasmic constituents afterfertilization resulting in local activation and/or localiz-ation of these determinants (Davidson, 1986; Klingler etal. 1988; Sprenger et al. 1989; Strecker et al. 1989); and(3) a graded distribution of these factors (Davidson,1986; Driever and Niisslein-Volhard, 1988a,6; Driever,et al. 1990). In any case, it is the underlying spatial

organization of these determinants within the egg thatwill ultimately direct early embryonic events. Thus,how the spatial organization of the egg is establishedand how this organization becomes reflected in the finalpattern of the embryo remains a fundamental problemin developmental biology.

In Drosophila, the posterior pole plasm has beendemonstrated to be essential both for germ celldetermination and delineation of the anteroposterioraxis of the insect embryo (Illmensee and Mahowald,1974, 1976; Frohnhofer et al. 1986). Results ofconstriction and cytoplasm removal and/or transferindicate that two morphogenetic centers, one at eachpole of the embryo, interact to establish the longitudi-nal body pattern (Schubiger et al. 1977; Schubiger andNewman, 1982; Frohnhofer et al. 1986; Sugiyama andOkada, 1990). Removal of cytoplasm from the anteriorpole results in the reduction or complete loss of anteriorstructures and a replacement of these structures byasegmental tail (telson) structures. Moreover, whenposterior pole cytoplasm is transplanted to the anteriorpole of an embryo from which anterior cytoplasm hasbeen removed double abdomen embryos are formed. Incontrast, removal of cytoplasm from the posterior tipresults in a loss of abdominal segments, but is notaccompanied by the acquisition of anterior structures atthis tip or a reduction in telson structures. Thus,

374 R. E. Boswell and others

cytoplasm removal and/or transfer experiments indi-cate that localized anterior and posterior factors exhibitlong-range organizing abilities with respect to longitudi-nal axis formation in the embryo. Furthermore, thesefactors have the properties expected for morphogens inthat they exhibit inductive properties when trans-planted to ectopic sites within the embryo.

Genetic analysis in Drosophila melanogaster hasdenned more than 20 strict maternal effect genes thatare required for anteroposterior axis formation (Akam,1987; Nusslein-Volhard et al. 1987; Ingham, 1988;Manseau and Schiipbach, 1989a). These genes aredivided into categories based on the regions of thenormal embryonic pattern elements altered in embryosfrom mutant mothers, and can be phenocopied bycytoplasm removal and/or transfer experiments. Forexample, anterior group genes such as bicoid (bed) arerequired for the formation of head and thoracicsegments (Frohnhofer and Nusslein-Volhard, 1986).Thus, embryos from homozygous bcdm mothers lackhead and thoracic segments, which are replaced bytelson structures. Genes within the posterior group(nanos and pumilio) are necessary for segmentation ofthe embryonic abdomen (Lehmann and Nusslein-Volhard, 1987; Nusslein-Volhard et al. 1987) and asubset (cappuccino, oskar, staufen, spire, tudor, vasaand valois) are required both for abdominal segmen-tation and pole cell formation (Boswell and Mahowald,19856; Lehmann and Nusslein-Volhard, 1986; Schiip-bach and Wieschaus, 1986; Manseau and Schiipbach,19896). Mutations in the spatial coordinates genes(bicaudal, Bicaudal-C and Bicaudal-D) result in thereplacement of head and thoracic segments by telsonand abdominal segments in reverse polarity to thenormal posterior structures (Bull, 1966; Nusslein-Volhard, 1977; Mohler and Wieschaus, 1986). A classthat has not been phenocopied is the terminal groupgenes (e.g., torso and trunk) in which the terminalstructures (asegmental head and telson) are deleted(Schiipbach and Wieschaus, 1986; Nusslein-Volhard etal. 1987). Except for the terminal group genes, theobserved pattern defects are similar to those obtainedin experimental manipulations in which the anteriorand posterior organizing centers are disrupted (Frohn-hofer et al. 1986; Nusslein-Volhard et al. 1987).Therefore, the genes within the posterior and anteriorgroups are currently thought to be required for theestablishment and/or function of their respectiveorganizing centers.

In a continued effort to identify and characterizegenes essential for pattern formation and germ celldetermination in Drosophila melanogaster, we haveisolated maternal effect mutations that disrupt theseprocesses. Here we describe the genetic and develop-mental analysis of a previously unidentified strictmaternal effect locus, mago nashi (mago). We provideevidence that mutations of the locus disrupt anoogenetic process required for normal functioning ofthe germ plasm and that these same mutations result inthe production of a novel gene function capable ofaltering the fate of cells along the entire anteroposterior

axis of the embryo. The phenotypic analysis of inviableembryos from mutant mago mothers indicates that themago locus is unique among maternal effect genesrequired for the establishment of the anteroposterioraxis, both in the variety and types of segmentationpattern abnormalities that it produces when mutated.

Materials and methods

Strains, culturing and isolation of mutantsDrosophila melanogaster strains were cultured on standardcornmeal, agar, molasses and yeast medium in 180ml plasticbottles or 8 dram vials. Except for the mago nashi (mago)alleles and where otherwise indicated, all mutations andbalancers are described in Lindsley and Grell (1968). Theoriginal mago mutation, mago1, was A'-ethyl-A'-nitrosourea(ENU)-induced in a en bw sp background, as described byLee (1987). mago1 was identified in a genetic screen in whichfemales homozygous for the mutagenized en bw sp chromo-some produced sterile F! progeny. The mating scheme usedwas similar to that described below for the isolation of mago2

and mago3. Approximately 2600 second chromosomes werescreened and we obtained one allele of mago and two allelesof tud (tud was used to monitor the efficacy of the screen). Thename mago nashi means without grandchildren, in theJapanese language.

The mating scheme used to identify mago2 and mago3 isdescribed below. 100 males of the genotype en bw sp/cn bw spor Sco en bw sp balanced by In(2LR)SM6a, al2 Cy dpM prcn2P sp2 (SM6a) were fed 25 mM ethyl methanesulfonate(EMS) in a 1 % sucrose solution for 24 h (Lewis and Bacher,1968). In new bottles containing standard medium, 10mutagenized males of the genotype en bw sp (or Sco en bwsp)/SM6a were crossed to 20 virgin females of the genotypenub b Sco It stw3/SM6a . The males were discarded after fourdays. F] en bw sp (or Sco en bw sp)/SM6a males werecollected and crossed individually to virgin cv-2 mago1 bwsp/SM6a females in 8 dram vials. F2 en bw sp (or Sco en bwsp)/SM6a males were collected and later used to constructstocks. F2 females of the genotype en bw sp (or Sco en bwsp)/cv-2 mago1 bw sp were selected and mated to siblingmales for 4 days, after which all adults were discarded. The Fjprogeny of en bw sp (or Sco en bw sp)/cv-2 mago1 bw spfemales were transferred to new vials and, after 14 days, thevials were inspected for the presence of eggs. When no eggswere found, the reproductive organs of the males and femaleswere inspected by light microscopy. In this manner, weidentified mago alleles fully penetrant for the sterilityphenotype.

To obtain a precise localization of the locus, we determinedits cytological position by testing the ability of variouschromosomal deletions to complement mago1. We initiallylocalized the locus to the polytene chromosome interval57B5;58Bl-2, deleted by Df(2R)D17 (O'Donnell etal. 1989).Of the available chromosomal deletions within this polytenechromosome interval, mago fails to complement Df(2R)PC18(57B16;57C6), Df(2R)F36 (57B17-20;57C6), and Df(2R)PL3(57B20;57Dl,2). but complements Df(2R)CC2 (57C2;57C5)and Df(2R)MPl (57C2;57D1,2) (O'Donnell et al. 1989);therefore, the mago locus must reside in the polytenechromosome interval 57B20;57C2. This localization wasfurther confirmed by the isolation of recombinants betweencrossveinless on chromosome 2 (cv-2) and mago1, and Punch(Pu) and mago1. The cv-2 and Pu loci map at positions 96.2and 97, respectively. Thus, both recombination and deletion

mago nashi and anteroposterior patterning 375

mapping place the locus in the interval 57B20;57C2, betweenthe cv-2 and Pu loci.

Furthermore, for each mago allele, meiotic recombinationwas used to remove accessory mutations that might lie outsideof the cv-2 to Pu interval. For each allele a number ofrecombinant lines were tested. In this manner, it was possibleto exclude the possibility that the observed phenotypes weredue to accessory mutations outside of the cv-2 to Pu interval.

Egg collections, analysis of cuticle, and cytoplasmictransfer experimentsEggs were collected for egg counts and cuticle observation on35x10 mm plates containing a molasses agar medium (Bos-well and Mahowald, 1985b). At 25 °C, eggs were collected for12 h and the embryos were aged 2 days. At 17°C, eggs werecollected for 12 h and embryos were aged for 6 days. Thefrequency of egg hatching was determined, and the inviableembryos were prepared for cuticle observation using themethod of Van der Meer (1977). Embryos were viewed byphase-contrast microscopy.

For cytoplasmic transfer experiments, eggs were collectedat 25°C during 30min intervals and, at 17°C, eggs werecollected during 1—1.5 h intervals. Embryos were prepared forcytoplasmic transfer as described by Lehmann and Niisslein-Volhard (1986). All recipient embryos were early cleavagestage embryos and donor embryos were of the stages specifiedin the text. Injected embryos were developed two days at25 °C and 4 days at 17 °C.

Ultrastructural studies of embryos and oocytesFor electron microscopy (EM) and thick-section analysis,embryos were collected on agar plates as above. Collectionswere made at 2-4 h intervals at 25 °C and 6-16 h intervals at17°C. Embryos were dechorionated in 3% sodium hypo-chlorite for 1.5 min, washed 3 times with PBS (130 raM NaCl,20 mM KPi pH7.4) and then fixed in a 1:1 mixture of heptane:25 % glutaraldehyde for 1-2 min Embryos were then fixed for1.5 h in 4% glutaraldehyde, 2% formaldehyde, 2.5%DMSO, lmM CaCl2, 0.1M sodium cacodylate, and 1%Acrolein. After hand dissection from the vitelline mem-branes, the embryos were rinsed 4 times for 5 min each in0.1M sodium cacodylate and postfixed in 1% OsO4 in 0.1Msodium cacodylate for 1.5 h at room temperature. They werethen rinsed 3 times for 5 min each in distilled water, 2 times,10 min each in 0.1M sodium acetate and stained with 0.5%uranyl acetate in 0.1M sodium acetate overnight at 0°C. Theembryos were rinsed twice in distilled water for 5 min eachand dehydrated in a series of solutions of increasing acetoneconcentrations, 10 min each followed by three 10 min washesin 100% acetone. Acetone was substituted with epon (1:1acetone:epon/Araldite for 5h, followed by 100% epon/Araldite overnight. The embryos were then embedded in100% epon/Araldite between two glass slides and polym-erized overnight at 60°C. Embryos were cut out and mountedonto epon blocks for sectioning.

Longitudinal thick (0.5 mm) and thin (70 nm) sections ofeach embryo were taken. Both ends of the thin-sectionedembryos were inspected on a Phillips CM10 transmission EMfor the presence of polar granules.

Determination of the temperature-sensitive periodsThe temperature-sensitive periods for both mago1 and mago2

were determined using females heterozygous for the mutantallele and the chromosomal deletion Df(2R)F36 (O'Donnell,et al. 1989). Egg collections were done on molasses agarplates. Females heterozygous for mago1 or mago2 andDf(2R)F36 were raised at 25°C or 17°C and then shifted to

17°C and 25°C, respectively. Beginning 24 h before the shift,eggs were collected at 12 h intervals from mago1/Df(2R)F36females, or 8h intervals from mago2 / Df{2R)F36 females.Eggs were collected continuously at these intervals for 8 daysto ensure that the last eggs collected had completed oogenesisat the post-shift temperature. The eggs were aged for 2 days at25°C or 6 days at 17°C, and then scored for the percentage ofeggs that hatched. The temperature-sensitive periods werethen determined based on the effect of the temperature shifton the viability of the embryos. To determine whether theembryonic lethality and the phenotype of the inviableembryos exhibit the same temperature-critical periods,inviable embryos from mago1 / Df(2R)F36 females werecollected and prepared for cuticle observation, as describedabove.

Results

The three mago alleles described here are strictmaternal effect mutations identified solely on the basisof the production of sterile F[ progeny by hemi- andhomozygous mothers (grandchildless-like phenotype).In grandchildless-like mago alleles, no apparent tem-perature dependence of the sterility phenotype isobserved. As with many other loci required for germcell formation, mutations at the mago nashi locus alsocause embryonic lethality. Both the frequency ofembryonic death and the phenotype of the inviableembryos are allele-specific and temperature-depen-dent. The partially permissive temperature is 25 °C andthe non-permissive temperatures are below 18° andabove 26°C. Here we present: (1) an analysis of thecuticular pattern defects observed in embryos producedby mago mothers maintained at 17° or at 25°C; (2) anultrastructural analysis of the germ plasm of oocytesand embryos derived from mago females; (3) anexamination of the temperature-sensitive periods ofmago1 and mago2; and (4) cytoplasmic transplantationexperiments to test for the presence of functionalposterior determinants in embryos collected fromhomozygous mago1 females reared at 17 °C.

Pattern abnormalities in embryos from mago mothersInviable embryos collected from homozygous, hemizy-gous or heteroallelic mago mothers reared at 17 °Cresemble embryos collected from mothers mutant forposterior group genes, such as oskar (osk) and tudor(tud) (Boswell and Mahowald, 19856; Lehmann andNiisslein-Volhard, 1986). In the most extreme cases, theembryos lack all ventral cuticular pattern elements ofthe abdomen (Fig. IB). Approximately 84-98% of theembryos die whether the mother is homozygous,hemizygous or heteroallelic for mago1 or mago2 (seeTable 1). The extreme phenotype, the complete lack ofventral abdominal cuticular pattern elements, isexhibited by approximately 90-96% of the inviableembryos (see Table 2). The remaining embryos exhibitrelated abdominal cuticular pattern defects that in-clude: (1) a single broad abdominal denticle beltconsisting of a mirror-image duplication of similarabdominal-like hairs, or (2) embryos with two or moreabdominal segments. Embryos with two or more ab-

376 R. E. Boswell and others

B

A2 A3 A4 A5 <»

Fig. 1. The cuticular phenotype of embryos derived from mago mothers reared at 17 °C. (A) Cuticular preparation of awild-type embryo viewed by phase-contrast microscopy. The dorsal surface is on top and the ventral surface is on thebottom. From anterior to posterior the structures are the antennal sense organ (AntSO), maxillary sense organ (MxSO),mouth hooks (mh), the thoracic segments (Tl, T2 and T3), ventral pits (vp), Keilin's organ (ko), the abdominal segments(A1-A8), anal pad (ap), telson (Te), Filzkorper (fk) and posterior spiracles (ps). (B) An embryo derived from amagol/magol female reared at 17°C. The embryo lacks abdominal segments (A1-A8), and is representative of an embryowith severe abdominal segmentation defects. Telson structures are present, but the posterior spiracles are abnormal and thenormally extended Filzkorper form circular structures.

Table 1. Inviability of embryos derived from mutant mago mothersGenotypeof mother

mago1

mago'

mago'~Df(TR)F36*

mago1

mago'

mago'~Df(TR)F36

mago'mago2

mago'mago

Temp.

17°C

17 °C

25 °C

25 °C

17 °C

25 °C

*For a description of

Total no.embryos

991

1626

1084

3056

1989

3538

Inviableembryos

9 8 % ± 1

97% ±3

65% ±4

24% ±5

95% ±3

46% ±2

Genotypeof mother

mago2

mago2

mago2

Df(2RjF36

mago2

mago2

mago2

Df(2RjF36

the chromosomal deletion Df(2R)F36

Temp

17 °C

17 °C

25 °C

25 °C

see O'

Total no.embryos

434

3757

815

3129

Donnell et al.

Inviableembryos

94 % ±2

84% ±2

6 1 % ±4

26% ±3

(1989).

Genotypeof mother

mago'mago3

mago2

mago3

mago'mago3

mago2

mago3

Temp.

17 °C

17 °C

25 °C

25 °C

Total no.embryos

1528

1285

5773

3429

Inviableembryos

92%±1

85%±1

53% ±3

44% ±3

dominal segments always have an apparently normalfirst abdominal setal belt (Al). Therefore, at 17°C, asmall fraction (<10%; see Table 2) of the inviableembryos derived from homozygous and hemizygousmago1 or mago2 females exhibit some normal abdomi-nal cuticular structures. Moreover, mago1 and mago2

display a similar, but incomplete, embryonic lethalitywhen homozygous or hemizygous females are main-tained at 17°C. Thus, at this temperature, mago1 andmago2 are most likely hypomorphic (reduced function)mutations. Furthermore, the extreme abdominal cu-ticular defects displayed by inviable embryos derivedfrom mago females reared at 17°C are identical to thephenotype observed in embryos derived from femaleshomozygous or hemizygous for apparent amorphic(null) mutations in other genes of the maternal effect

posterior group (Niisslein-Volhard et al. 1987; Manseauand Schiipbach, 1989a).

However, unlike genes of the maternal effectposterior group some mutations of the mago locusappear to result in the production of altered geneproducts at 25 °C. As shown in Table 1, at 25 °C,approximately 60-65 % of the embryos from mago1 andmago2 homozygous females die. In contrast, at thissame temperature, only 26 % and 24 % (see Table 1) ofthe embryos from hemizygous mago2 and mago1

females die, respectively. In all cases, the surviving F :progeny are phenotypically normal, but fail to formgerm cells. Moreover, the frequency of embryoniclethality observed in embryos from +/magol (17/713 or2 % embryonic lethality) and +/mago2 (76/736 or 10 %embryonic lethality) females is similar to that observed

mago nashi and anteroposterior patterning 377

Table 2. The phenotypes observed in embryos derived from mutant mago nashi mothers reared at 17°C and25° C

Genotype of mothermago1/mago

mago1/Df(2R)F36

mago2/mago'

mago'/Df(2R)F36

mago1/mago'

mago11mago

mago'/mago

17°CAbdominal defects8

Severe abdominal defects'1

Total no. embryos

25 °CNormalHead defectsLabrumLabrum/abdominalHead/abdominal defectsHead+telsonc

Double abdomenAbdominal defects'1

Added posterior structurese

Otherf

Total no. embryos

11119

1119

1435

108

(8%)(92%)130

(7%)(12%)

-(9%)(2%)(3%)(67%)--

160

20204

2410

511

46

(9%)(91 %)224

(28%)(11%)

-(6%)(1%)(1%)(53%)

-87

10(4%)262 (96%)

272

5(3%)

--

4(2%)--

190 (95%)--

199

9174

1626

11

2111

(5%)(95%)183

(11%)(1%)(4%)(7%)--(1%)(76%)--

148

8(4%)218(96%)

226

118(8%)13 (6%)

--

6 (3%)-

9(4%)185 (80%)

--

231

16384

1122

22132226

(4%)(96%)400

(5%)(10%)----(10%)(61%)(10%)(3%)215

9118

6

2601812

(7%)(93%)127

(2%)

-----(88%)(6%)(4%)296

a Embryos that display one or more abdominal setal belts.b Embryos completely lacking normal abdominal cuticular pattern elements.cHead+telson embryos are embryos that are missing head structures and these structures are replaced by posterior structures, but the

thoracic and abdominal segments are normal.dThese embryos completely lack abdominal segmentation or exhibit more than one normal abdominal denticle belt.eEmbryos of this class display a normal first abdominal denticle belt, a lawn of denticles throughout the remaining abdominal region,

supernumerary posterior-spiracle-like structures and Filzkorper, and sometimes a normal eighth abdominal denticle belt.These embryos exhibit a variety of both head and abdominal defects.

in control embryos from a wild-type strain, Oregon R(data not shown). Together, these results indicate thatmago1 and mago2 are recessive antimorphic maternaleffect mutations at 25 °C.

Also, unlike genes of the posterior group, and unlikethe results at 17°C, the inviable embryos from magofemales reared at 25 °C exhibit a continuum of cuticularphenotypes that most closely resemble the cuticularpattern defects observed in embryos collected fromhomozygous or hemizygous bicaudal females (Niisslein-Volhard, 1977). The range of cuticular pattern abnor-malities observed is illustrated in Fig. 2. Embryos thatdo not fall within the above continuum display theventral cuticular pattern defects observed in embryoscollected from mago1 and mago2 homozygous andhemizygous females reared at 17°C (not illustrated butidentical to the embryo in Fig. IB). The mago allelescan be distinguished, one from another, by thefrequency with which they produce inviable embryosthat fall into any of the phenotypic classes just described(see Table 2). For example, mago1 homozygous fe-males produced the highest frequency of doubleabdomen embryos, approximately 3%. In contrast,mago2 homozygous females were not observed toproduce double abdomen embryos. Instead, the ma-jority of embryos from mago2 homozygous females lackabdominal segments, but have normal head, thoracicand telson structures (see Table 2). Interestingly,approximately 46% of the embryos derived frommago11 mago mothers (reared at 25 °C) die, and ofthese approximately 4% exhibit the double abdomenphenotype. Furthermore, although hemizygous mago2

females produce double abdomen embryos at a low

frequency (~1 %), approximately 11 % of the embryoshave labrum-specific defects or labrum and abdominaldefects. The labrum is the preoral lobe of the head andis the most anterior portion of the blastoderm fate mapof the cuticle of the larval head (Jiirgens et al. 1986).The labrum defects observed in embryos produced bymago2 mothers are rarely, if ever, seen in embryos frommago1 females. The labrum defect represents the leastsevere alteration of the cuticular pattern in embryos inwhich head structures are deleted and replaced byabdominal segments. The most severe transformationof this kind that we observe is the double abdomenphenotype observed among the progeny of mago1

females. Thus, mago1 is more capable of leading to areplacement of head segments by segments of aposterior origin than is mago2.

The third allele, mago3, is not viable when homo-zygous or hemizygous. We have examined the cuticularstructures of presumed homozygous, inviable, mago3

embryos and observed no apparent cuticular defects.When embryos were collected from mago1 /mago3 ormago2/mago3 females reared at 17°C, approximately 92and 85 % (Table 1) of the embryos die, respectively.The inviable embryos lack the ventral cuticular patternelements of the abdomen, but exhibit normal head,thoracic and telson structures (Table 2). Approximately53 and 44 % of the embryos from mago /mago3 andmago2/mago3 females reared at 25°C die, respectively.At 25 °C, mago1 /mago3 females produce double abdo-men embryos (~10% of the inviable embryos), and arange of cuticular phenotypes typically observed inembryos derived from mago1/mago mothers (Table 2).However, a novel phenotypic class is also observed that

378 R. E. Bos weII and others

r"T"'

, * f < .

Fig. 2. The range of cuticularphenotypes observed inembryos derived from magofemales reared at 25 °C. Theventral cuticular patternsillustrated here represent thecontinuum from wild-type tosymmetrical double abdomenphenotypes, observed inembryos derived from mago1

and mago2 homozygous andhemizygous mothers. Theventral cuticular patterns ofembryos that best illustrate thisgraded replacement of headstructures by posteriorstructures are depicted: (A) anembryo with an apparentlywild-type ventral cuticularpattern; (B) an embryo lackingmost head structures, but withnormal thoracic segments; (C)an embryo in which head,thoracic and telson structuresare present, but Filzkorpermaterial is observed in the headas well (the Filzkorper materialin the head is indicated by anarrow); and (D) a symmetricaldouble abdomen embryo.

consists of embryos lacking the abdominal segments,but having supernumerary posterior spiracle-like andFilzkorper structures anterior to the normal posteriorstructures (this class is —10% of the inviable embryos;see Fig. 3 and Table 2). mago2/mago3 females produceembryos that exhibit supernumerary posterior struc-tures at a similar frequency, 6%. Embryos collectedfrom mago1 /mago3 and mago2/magoi females can bedistinguished, one from the other, because embryosfrom mago2/mago3 mothers do not display the doubleabdomen phenotype. Supernumerary posteriorspiracles and Filzkorper have not been observed inembryos derived from mothers mutant in maternaleffect genes of the posterior group or the spatialcoordinates class, and thus, this represents a novelphenotypic class.

Ultrastructure of the germ plasmAs stated earlier, all surviving ¥i progeny from magohomozygous, hemizygous or heteroallelic mothers aresterile, and exhibit no temperature-dependent variationof the sterility phenotype. Among mutations within theposterior group genes (cappuccino, oskar, staufen,spire, tudor, vasa and valois) that result in the completeabsence of pole cells, it has been observed that the polargranules, germ plasm-specific electron-dense cytoplas-mic structures, are reduced or absent (Boswell andMahowald, 1985b; Lehmann and Niisslein-Volhard,1986; Schupbach and Wieschaus, 1986; Manseau andSchiipbach, 1989ft). In mutations at one of these loci,tud, the ability of embryos to form germ cells has beenshown to be allele-specific and to correlate with the

total amount of assembled polar granule materialobserved in the posterior pole plasm, i.e., as theamount of polar granule-like material is reduced thereis a concomitant reduction in the ability to form germcells (Boswell and Mahowald, 1985ft). Thus, ultrastruc-tural analysis of the germ plasm has become a reliableassay for the integrity of the germ plasm. Therefore, todetermine whether there were germ plasm defects, weexamined the posterior pole plasm of embryos frommago1 and mago2 homozygous or hemizygous femalesat the ultrastructural level. In an analysis of 72 embryosderived from mutant mago females reared at 17° and25 °C, none were observed to have normal polar granulemorphology. In some embryos (16/72), infrequent,reduced polar granule-like material was observed in theposterior pole plasm (illustrated in Fig. 4). Thismaterial was less dense than that observed in wild-typeposterior pole plasm, was associated with polysome-likestructures, and frequently remained attached to mito-chondria. Thus, these less dense polar granule-likestructures exhibit the typical features of polar granules,and may represent polar granule remnants. However,in the majority of embryos (56/72) no polar granule-likematerial was evident in the posterior pole, nor did weobserve ectopic polar granules.

Rather than a specific defect in the determinativeprocess, mutations in maternal genes required for germcell formation and segmentation of the embryonicabdomen may instead cause: (1) a delay in themigration of cleavage-stage nuclei into the posteriorpole, as observed in grandchildless of D. subobscura(Mahowald et al. 1979) and gs(l)N26 (Niki and Okada,

mago nashi and anteroposterior patterning 379

Fig. 3. The novel cuticularphenotype observed in inviableembryos produced byheteroallelic mago3 mothers,reared at 25 °C. The embryoillustrated here has normalhead, thoracic and firstabdominal cuticular patternelements. Dorsal is at the top,ventral is at the bottom,anterior is to the left andposterior is to the right. Theremaining abdominal regionexhibits a lawn of denticlesand an apparently normaleighth abdominal denticle belt.The homologous structures areindicated by arrows of thesame type; the arrows indicatethe presence of supernumeraryFilzkorper and posteriorspiracle-like structures anteriorto the normal Filzkorper andposterior spiracles. In the toppanel, a region of the dorsalsurface has been magnified; inthe bottom panel, a region ofthe ventral surface has beenmagnified.

mm*-m*\^ h.

i f

- , * • •

^r v ̂ mM^?•n

* • *

^ ' J « - v , * . . ^ : . - : " * l v , v•^*-^4'*:.

1981); and (2) incomplete cellularization at the blasto-derm stage, observed in valois (Schiipbach and Wie-schaus, 1986), when females are reared at 22°C. It was,therefore, possible that the defects associated withmago mutations result from a disruption of nuclearmigration to the posterior pole or a failure incellularization. Thus, to examine this possibility,embryos from mutant mago females were prepared forobservation both by light and scanning electronmicroscopy. Neither delayed nuclear migration norfailed cellularization were ever observed (not illus-trated). Therefore, in mago mutants, neither the failureto form germ cells, nor the aberrant segmentation of theembryonic abdomen can be accounted for by delayedmigration of cleavage-stage nuclei to the posterior tip or

Fig. 4. Transmission electron micrographs of the posteriorpole plasm of syncytial blastoderm embryos. Electronmicrographs of the posterior pole plasm of embryos fromen bw sp (A) and hemizygous mago2 females (B). Thegerm plasm of the embryo derived from a hemizygousmago female, illustrated in Fig. 4B, contains polargranule-like material (arrows) associated withmitochondria. These polar granule-like structures are,however, less dense than those observed in en bw spembryos (the genetic background in which mago alleleswere induced). In embryos from mothers hemizygous,homozygous or heteroallelic for various mago allelesnormal polar granule morphology is never observed and inmost embryos (52/72) polar granule-like material is absent.Mitochondria (M), ribosomes (R) and vesicles (V). Thebar represents 0.5

380 R. E. Boswell and others

a failure to properly cellularize at the cellular blasto-derm stage.

Determination of the temperature-sensitive periods ofmago1 and mago2

Mutations of the mago locus are strict maternal effectmutations indicating that mago+ product is requiredduring oogenesis and/or before the onset of zygoticgenome transcription. It has been observed that thetemperature-sensitive period (TSP) of strict maternaleffect mutations is limited to oogenesis, or occursduring early embryogenesis. Two independently iso-lated mago alleles were found to be cold-sensitivemutations, and thus, could be used to measure thetemperature-sensitive period of these mutants. Sincemago2 is not viable in the homozygous or hemizygousstate, it was not possible to determine whether thisallele is a temperature-conditional mutation. Tempera-ture-sensitive mutations of the Notch and shibire loci(Poodry etal. 1973; Shellenbarger and Mohler, 1978), inD. melanogaster, have been found to express distinctTSPs for lethality and morphological defects. Mutationsof mago exhibit both embryonic lethality and segmen-tation pattern abnormalities; thus, it was of interest toexamine whether the two defects displayed the same orindependent TSPs. Therefore, we used two differentcriteria to measure the TSP of mago mutants: (1) theembryonic lethality of progeny derived from hemizy-gous mago1 or mago2 females; and (2) the frequencyand kind of cuticular pattern defects observed in thedeveloping embryos laid by hemizygous mago females.Fig. 5 shows the results of reciprocal temperature-shiftexperiments as measured by the inviability of embryosderived from females maintained at the non-permissivetemperature (17°C) and then shifted to the permissivetemperature (25°) at defined intervals or vice versa (seeMethods and Materials for details). Although theresults for mago1 are not described in Fig. 5, the TSPsof mago1 and mago2 are indistinguishable, by eithercriterion used. The TSPs for both mutations begin atapproximately stage 7 and end at approximately stage14 of oogenesis. The data clearly indicate that the TSPof mago and mago1 is during oogenesis, is limited tooogenetic stages 7 through 14, and that the lethality andmorphological defects of mago mutations display thesame TSP.

The posterior pole of embryos from mutant mago1

females contains no functional posterior determinantsIn Drosophila, cytoplasmic transfer experiments haveserved as a powerful tool to examine the spatialdistribution and activity of particular maternal geneproducts (Anderson, 1987; Niisslein-Volhard et al.1987; Manseau and Schiipbach, 1989a). Thus, weemployed cytoplasmic transfer experiments to deter-mine: (1) whether the abdominal segmentation defectsobserved in embryos collected from homozygous magofemales reared at 17°C could be alleviated by cytoplasmcollected from wild-type embryos; and (2) if embryoscollected from homozygous mago females reared at

e so.

192

•64

-144 -96-48 -32

Developmental Time (h)

•48

-16 tegg deposition

• 16 1 7 X25°C

Fig. 5. The temperature-sensitive period for embryoniclethality of mago2. Each measurement represents theinviability of embryos collected from hemizygous mago2

mothers, during an 8h interval (see ExperimentalProcedures for details). Each time point represents theanalysis of the viability of 200 to 1000 embryos. The topabscissa represents the oogenetic stages, as defined by King(King, 1970). The bottom abscissa depicts thedevelopmental times, at 17°C (top) and 25°C (bottom),and the arrow indicates egg deposition. Thus, the negativetimes represent time during oogenesis prior to oviposition.The 17° and 25 °C time scales have been normalizedaccording to Suzuki (1970) and the oogenetic stages weredetermined as described by David and Merle (1968).

17 °C contained a functional posterior morphogeneticcenter.

Wild-type donor cytoplasm can alleviate the abdomi-nal cuticular defects observed in embryos derived fromhomozygous mago1 females reared at 17 °C (seeTable 3). Approximately 7 % (18/265) of the uninjectedembryos collected from homozygous mago1 femalesreared at 17°C exhibit 5=3 abdominal segments.Therefore, the frequency with which injected embryosformed 5=3 abdominal segments was used as an assay todetermine the presence of functional posterior determi-nants. Wild-type donor cytoplasm must come from 0 to10 % egg length (EL; 0 % represents the posterior and100% the anterior pole of the embryo) and is mosteffective when transplanted into the presumptiveabdominal region (40-50 % EL; data not shown) of therecipient embryo. Once the donor embryos havereached the cellular blastoderm stage (~2.2-2.8h) thedonor cytoplasm is no longer able to correct theobserved cuticular defects (Table 4). Thus, the site ofaction (40-50% EL) of the cytoplasmic componentsrequired for correcting the abdominal segmentationdefects observed in mago is at a distance from thesource of these components (0-10%) and, althoughthe TSP for mago1 is during stages 7-14 of oogenesis,the cytoplasmic components capable of alleviating theabdominal cuticular defects can act as late as ~ 1.5 h in

mago nashi and anteroposterior patterning 381

Table 3. Alleviation of abdominal segmentation defects in embryos collected from homozygous mago7 femalesreared at 17°C

Maternal genotype ofcleavage stage donor

wild-typewild-typewild-typewild-typewild-typemago (17°C)osk

0-10 % EL mago cytoplasmto osk recipient embryo

Controlsmago injected with oilmago uninjectedosk uninjectedosk injected with 0-10% ELwild-type cytoplasm

* Of the embryos with S3 abdominal segments

Position indonor(%

egg length)

0102050

1000-100-10

29 of 34 had 6 to {

Developed(«)

50383753422824

26

702657850

i abdominal segments.

Abdominal segments formedin recipien

2==

16(32%)32(84%)35(95%)53 (100%)41 (98%)26(93%)23(96%)26(100%)

64 (91%)247(93%)78(100%)30 (60%)

t embryos

S3

34(68%)*6(16%)2(5%)0(0%)1 (2%)2(7%)1(4%)0(0%)

6(9%)18(7%)0(0%)

20 (40%)

Table 4. The ability of wild-type posterior polecytoplasm from various stages of embryogenesis to

alleviate abdominal defects in embryos collected fromhomozygous mago7 females reared at 17°C

Abdominal segments formedin embryos collected

from homozygous magofemales reared at 17°C

Developmentalstage of donor

Developed() 2s=

Early cleavageSyncytial blastodermCellular blastodermUninjected control

502829

297

16(32%)20(71%)27(93%)

276 (93%)

34(68%)8(29%)2(7%)

21 (7%)

embryogenesis. Furthermore, 0-10% EL cytoplasmcollected from embryos derived from homozygousmago1 females reared at 17°C does not alleviate theabdominal segmentation defects observed in embryosderived from mago1 homozygous females reared at17°C, nor does it correct abdominal defects observed inembryos collected from homozygous osk166 females(see Table 3). Taken together, these results suggest thatfunctional posterior determinants are not present in theposterior pole of embryos derived from homozygousmago1 females reared at 17°C. Therefore, these resultsprovide direct evidence that mutations of the magolocus disrupt the ability of the posterior organizingcenter to function. Moreover, these results are consist-ent with results obtained when wild-type or mutantcytoplasm is transferred to embryos collected fromfemales mutant in other posterior group maternal effectgenes (Lehmann and Niisslein-Volhard, 1986, 1987).

Discussion

In this report, we have described the phenotypic

characteristics associated with mutations of a newlyidentified maternal effect locus, mago nashi. Theexperimental results presented provide compellingevidence that the wild-type gene product of the magonashi locus is vital for the establishment of longitudinalaxial polarity in the Drosophila embryo. Specifically,we provide evidence suggesting that: (1) mago*function is required during a discrete period ofoogenesis for segmentation of the embryonic abdomen;(2) at the partially permissive temperature mutations ofthe locus result in the production of a novel genefunction capable of altering the fates of cells along theentire anteroposterior axis; and (3) mago* function isrequired for germ cell determination. Moreover, magonashi is novel among maternal effect genes requiredboth for segmentation of the embryonic abdomen andgerm cell determination in two respects. First, mu-tations of the mago nashi locus can result in a deletionof anterior embryonic pattern elements and a replace-ment of the deleted elements by a mirror-imageduplication of posterior abdominal and tail structures.Second, mothers heteroallelic for particular magoalleles produce embryos lacking pattern elements of theabdomen and these pattern elements are replaced bysupernumerary tail structures. Thus, as discussedbelow, we have identified a gene that produces a crucialcomponent of the posterior organizing center.

The cuticular pattern abnormalities associated withmutations of mago suggest that the mago* product isessential in an oogenetic process necessary for segmen-tation of the embryonic abdomen. When femaleshomozygous, heteroallelic or hemizygous for eithermago1 or mago2 are reared at 17°C approximately 90 %of their progeny die during embryogenesis, and themajority (~93%) of the inviable embryos lack normalcuticular pattern elements of the embryonic abdomen.This aberrant cuticular pattern is indistinguishable from

380 R. E. Boswell and others

a failure to properly cellularize at the cellular blasto-derm stage.

Determination of the temperature-sensitive periods ofmago1 and mago2

Mutations of the mago locus are strict maternal effectmutations indicating that mago+ product is requiredduring oogenesis and/or before the onset of zygoticgenome transcription. It has been observed that thetemperature-sensitive period (TSP) of strict maternaleffect mutations is limited to oogenesis, or occursduring early embryogenesis. Two independently iso-lated mago alleles were found to be cold-sensitivemutations, and thus, could be used to measure thetemperature-sensitive period of these mutants. Sincemago2 is not viable in the homozygous or hemizygousstate, it was not possible to determine whether thisallele is a temperature-conditional mutation. Tempera-ture-sensitive mutations of the Notch and shibire loci(Poodry etal. 1973; Shellenbarger and Mohler, 1978), inD. melanogaster, have been found to express distinctTSPs for lethality and morphological defects. Mutationsof mago exhibit both embryonic lethality and segmen-tation pattern abnormalities; thus, it was of interest toexamine whether the two defects displayed the same orindependent TSPs. Therefore, we used two differentcriteria to measure the TSP of mago mutants: (1) theembryonic lethality of progeny derived from hemizy-gous mago1 or mago2 females; and (2) the frequencyand kind of cuticular pattern defects observed in thedeveloping embryos laid by hemizygous mago females.Fig. 5 shows the results of reciprocal temperature-shiftexperiments as measured by the inviability of embryosderived from females maintained at the non-permissivetemperature (17°C) and then shifted to the permissivetemperature (25°) at defined intervals or vice versa (seeMethods and Materials for details). Although theresults for mago1 are not described in Fig. 5, the TSPsof mago1 and mago2 are indistinguishable, by eithercriterion used. The TSPs for both mutations begin atapproximately stage 7 and end at approximately stage14 of oogenesis. The data clearly indicate that the TSPof mago and mago2 is during oogenesis, is limited tooogenetic stages 7 through 14, and that the lethality andmorphological defects of mago mutations display thesame TSP.

The posterior pole of embryos from mutant mago1

females contains no functional posterior determinantsIn Drosophila, cytoplasmic transfer experiments haveserved as a powerful tool to examine the spatialdistribution and activity of particular maternal geneproducts (Anderson, 1987; Niisslein-Volhard et al.1987; Manseau and Schiipbach, 1989a). Thus, weemployed cytoplasmic transfer experiments to deter-mine: (1) whether the abdominal segmentation defectsobserved in embryos collected from homozygous magofemales reared at 17°C could be alleviated by cytoplasmcollected from wild-type embryos; and (2) if embryoscollected from homozygous mago females reared at

-144 -96 -48-48 -32 -16

Developmental Time (h)tegg deposition

• 16 17X25°C

Fig. 5. The temperature-sensitive period for embryoniclethality of mago2. Each measurement represents theinviability of embryos collected from hemizygous mago2

mothers, during an 8h interval (see ExperimentalProcedures for details). Each time point represents theanalysis of the viability of 200 to 1000 embryos. The topabscissa represents the oogenetic stages, as denned by King(King, 1970). The bottom abscissa depicts thedevelopmental times, at 17°C (top) and 25°C (bottom),and the arrow indicates egg deposition. Thus, the negativetimes represent time during oogenesis prior to oviposition.The 17° and 25 °C time scales have been normalizedaccording to Suzuki (1970) and the oogenetic stages weredetermined as described by David and Merle (1968).

17 °C contained a functional posterior morphogeneticcenter.

Wild-type donor cytoplasm can alleviate the abdomi-nal cuticular defects observed in embryos derived fromhomozygous mago1 females reared at 17 °C (seeTable 3). Approximately 7 % (18/265) of the uninjectedembryos collected from homozygous mago1 femalesreared at 17°C exhibit 5=3 abdominal segments.Therefore, the frequency with which injected embryosformed 5=3 abdominal segments was used as an assay todetermine the presence of functional posterior determi-nants. Wild-type donor cytoplasm must come from 0 to10 % egg length (EL; 0 % represents the posterior and100% the anterior pole of the embryo) and is mosteffective when transplanted into the presumptiveabdominal region (40-50 % EL; data not shown) of therecipient embryo. Once the donor embryos havereached the cellular blastoderm stage (~2.2-2.8h) thedonor cytoplasm is no longer able to correct theobserved cuticular defects (Table 4). Thus, the site ofaction (40-50% EL) of the cytoplasmic componentsrequired for correcting the abdominal segmentationdefects observed in mago is at a distance from thesource of these components (0-10%) and, althoughthe TSP for mago1 is during stages 7-14 of oogenesis,the cytoplasmic components capable of alleviating theabdominal cuticular defects can act as late as ~ 1.5 h in

mago nashi and anteroposterior patterning 381

Table 3. Alleviation of abdominal segmentation defects in embryos collected from homozygous mago7 femalesreared at 17°C

Maternal genotype ofcleavage stage donor

wild-typewild-typewild-typewild-typewild-typemago (17°C)osk

0-10 % EL mago cytoplasmto osk recipient embryo

Controlsmago injected with oilmago uninjectedosk uninjectedosk injected with 0-10% ELwild-type cytoplasm

* Of the embryos with S3 abdominal segments

Position indonor(%

egg length)

0102050

1000-100-10

29 of 34 had 6 to {

Developed(«)

50383753422824

26

702657850

i abdominal segments.

Abdominal segments formedin recipien

2==

16(32%)32(84%)35(95%)53(100%)41 (98%)26(93%)23(96%)26(100%)

64 (91%)247(93%)78(100%)30 (60%)

t embryos

S3

34(68%)*6(16%)2(5%)0(0%)1 (2%)2(7%)1(4%)0(0%)

6(9%)18(7%)0(0%)

20 (40%)

Table 4. The ability of wild-type posterior polecytoplasm from various stages of embryogenesis to

alleviate abdominal defects in embryos collected fromhomozygous mago7 females reared at 17°C

Abdominal segments formedin embryos collected

from homozygous magofemales reared at 17°C

Developmentalstage of donor

Developed2s=

Early cleavageSyncytial blastodermCellular blastodermUninjected control

502829

297

16(32%)20(71%)27(93%)

276 (93%)

34(68%)8(29%)2(7%)

21 (7%)

embryogenesis. Furthermore, 0-10% EL cytoplasmcollected from embryos derived from homozygousmago1 females reared at 17°C does not alleviate theabdominal segmentation defects observed in embryosderived from mago1 homozygous females reared at17°C, nor does it correct abdominal defects observed inembryos collected from homozygous osk166 females(see Table 3). Taken together, these results suggest thatfunctional posterior determinants are not present in theposterior pole of embryos derived from homozygousmago1 females reared at 17°C. Therefore, these resultsprovide direct evidence that mutations of the magolocus disrupt the ability of the posterior organizingcenter to function. Moreover, these results are consist-ent with results obtained when wild-type or mutantcytoplasm is transferred to embryos collected fromfemales mutant in other posterior group maternal effectgenes (Lehmann and Niisslein-Volhard, 1986, 1987).

Discussion

In this report, we have described the phenotypic

characteristics associated with mutations of a newlyidentified maternal effect locus, mago nashi. Theexperimental results presented provide compellingevidence that the wild-type gene product of the magonashi locus is vital for the establishment of longitudinalaxial polarity in the Drosophila embryo. Specifically,we provide evidence suggesting that: (1) mago*function is required during a discrete period ofoogenesis for segmentation of the embryonic abdomen;(2) at the partially permissive temperature mutations ofthe locus result in the production of a novel genefunction capable of altering the fates of cells along theentire anteroposterior axis; and (3) mago* function isrequired for germ cell determination. Moreover, magonashi is novel among maternal effect genes requiredboth for segmentation of the embryonic abdomen andgerm cell determination in two respects. First, mu-tations of the mago nashi locus can result in a deletionof anterior embryonic pattern elements and a replace-ment of the deleted elements by a mirror-imageduplication of posterior abdominal and tail structures.Second, mothers heteroallelic for particular magoalleles produce embryos lacking pattern elements of theabdomen and these pattern elements are replaced bysupernumerary tail structures. Thus, as discussedbelow, we have identified a gene that produces a crucialcomponent of the posterior organizing center.

The cuticular pattern abnormalities associated withmutations of mago suggest that the mago* product isessential in an oogenetic process necessary for segmen-tation of the embryonic abdomen. When femaleshomozygous, heteroallelic or hemizygous for eithermago1 or mago2 are reared at 17°C approximately 90 %of their progeny die during embryogenesis, and themajority (~93%) of the inviable embryos lack normalcuticular pattern elements of the embryonic abdomen.This aberrant cuticular pattern is indistinguishable from

382 R. E. Boswell and others

the phenotype observed in embryos derived fromfemales homozygous or hemizygous for apparentamorphic (null) alleles of posterior group maternaleffect genes, such as osk (Lehmann and Nusslein-Volhard, 1986). Furthermore, embryos collected frommago1 homozygous females contain no apparent func-tional posterior determinants in the posterior pole.However, the incomplete penetrance of the embryoniclethality and the small percentage (<10%) of inviableembryos with more than two abdominal segmentssuggest that mago1 and mago2 are not amorphic alleles,but hypomorphic (reduced function) alleles. Thus, inthe absence of a completely functional mago+ product(17°C) abdominal segmentation rarely occurs.

In contrast, at 25°C, there is_an increased viability ofthe embryos derived from mago1 or mago2 mothers, butembryos from females homozygous for these allelesexhibit a higher incidence of embryonic lethality thando embryos from mothers that are hemizygous foreither allele. Although further dosage studies arenecessary to determine the exact nature of the magomutations, the genetic results described here clearlyindicate that: (1) 25°C is the more permissive tempera-ture; and (2) at 25 °C mago mutations result in theacquisition of a novel gene function rather thaneliminating or reducing the function of the gene. Thus,at 25 °C, mago mutations behave as recessive antimor-phic alleles (m/m>m/Df>m/+ = + / + , where m rep-resents the mutant allele and the severity of thephenotype is depicted in decreasing order; cf Muller,1932). Moreover, at 25°C, mago1 homozygous orhemizygous females produce inviable embryos thatdisplay a range of phenotypes representing a continuumfrom symmetrical double abdomens at one extreme tomorphologically wild-type embryos at the other. At25 °C, homozygous mago2 females produce no doubleabdomen embryos and hemizygous mago2 femalesproduce them infrequently (1 % of the inviable em-bryos are double abdomen embryos). In addition,mago2 hemizygous females produce embryos lackinglabrum structures. If the continuum from doubleabdomen to wild-type embryos represents the gradedeffect of a gene product capable of inducing thereplacement of head and thoracic structures by pos-terior abdominal and asegmented tail structures, thenthe labrum defects observed in embryos derived fromhemizygous mago2 females may be considered to be theleast extreme expression of this replacement. The mostextreme expression of this defect would then be thedouble abdomen embryos derived from mago1

mothers. Most of the inviable embryos lack abdominalsegmentation and, thus, represent a phenotype thatdoes not lie within the continuum described above.

Formation of a functional posterior organizing centerin the Drosophila embryo requires the products of atleast nine genes (Niisslein-Volhard etal. 1987; Manseauand Schiipbach, 1989a). Together, these genes areinvolved in the synthesis, assembly and proper distri-bution of the components of the posterior organizingcenter. Although amorphic alleles of mago are notavailable, it is clear that at 17°C mutations of mago

disrupt two of the functions attributed to the posteriororganizing center, germ cell formation and segmen-tation of the embryonic abdomen. Therefore, wepropose that mago+ function is required for the properassembly or distribution of the components of theposterior organizing center. The isolation and charac-terization of amorphic alleles of mago should allow usto determine whether mago+ function is specificallyrequired, during oogenesis, for the formation of afunctional posterior organizing center. Furthermore,epistasis studies and a determination of the distributionof maternal and zygotic gap gene products in embryosderived from mutant mago females should provideinsights into the functional relationships between magoand other genes required for delineation of theanteroposterior axis.

As demonstrated by the phenotypes of the inviableembryos at 25°C (the more permissive temperature),the products of mago1 and mago2 are capable ofchanging the fate of cells along the anteroposterior axisof the embryo, although the mago2 product is not asefficient as the mago1 product at causing the replace-ment of anterior structures with posterior structures.Dominant mutations of the Bicaudal-D (Bic-D) locusproduce double abdomen embryos by affecting thelocalization of both anterior and posterior determi-nants. Both the anterior and posterior pole cytoplasmof embryos collected from Bic-D1 /Bic-D2 motherscontain functional posterior determinants (Lehmannand Niisslein-Volhard, 1986). In addition, bed RNA isunstable and bed protein (the anterior morphogen) isabsent (Driever and Niisslein-Volhard, 1988a,b; Whar-ton and Struhl, 1989). Taken together, these resultssuggest that Bic-D mutations result in the mislocaliza-tion of posterior determinants to the anterior pole and adestabilization of the anterior determinants in theanterior pole; thus, forming an active posterior organiz-ing center at the anterior pole of the embryo. However,the germ plasm of embryos collected from Bic-D1 /Bic-D2 females is localized normally and the embryos formpole cells only at the posterior end. Thus, although thespecific mechanism by which mago mutations inducedouble abdomen embryos is not known, it is reasonableto propose that mutations at the mago locus may resulteither in the mislocalization of the posterior organizingcenter in the anterior pole or stabilization of posteriordeterminants in the anterior pole. With both mago1 andmago2, the majority of embryos collected from homo-zygous or hemizygous females reared at 25 °C lacknormal segmentation of the embryonic abdomen. Theexistence of embryos that lack abdominal segmentationmay be due to the lability of the mago gene productwhen the mago locus has been mutated, although wehave no direct evidence in support of this idea.Nevertheless, this scheme does explain the range ofphenotypes observed among mago embryos and isconsistent with results from studies in a variety ofinsects suggesting that double abdomen embryos can beinduced by experimental interference of normal devel-opment (Sander, 1976; Frohnhofer et al. 1986; Kalthoffand Elbetieha, 1986) or by mutation (Bull, 1966;

mago nashi and anteroposterior patterning 383

Niisslein-Volhard, 1977; Mohler and Wieschaus, 1985;Suter et al. 1989; Wharton and Struhl, 1989)].

In some heteroallelic combinations (mago1 /mago3

and mago2/mago3) approximately 7 % of the inviableembryos exhibit a novel phenotype in which they lackabdominal segments, but display supernumerary pos-terior spiracles and Filzkorper structures anterior to thenormal set of posterior structures. The inability ofmago3 homozygotes or hemizygotes to survive, and thefailure of mago3 to complement loci within thechromosomal interval containing the mago locussuggest that mago3 is a chromosomal rearrangement,although it is not cytologically visible. However,chromosomal deletions that completely remove boththe mago locus and loci flanking mago produce adifferent range of phenotypes than do mago1/mago3

and mago2/mago3 females. Therefore, if mago3 is achromosomal deletion, it probably does not remove theentire locus. Whether mago3 is a small chromosomaldeletion or a small inversion, it is possible that themago3 product is modified such that components of theposterior organizing center become improperly concen-trated within the posterior end of the embryo. Thus, inthese heteroallelic combinations posterior determinantsare active at an ectopic site within the embryo,resulting in the production of posterior tip structures ina region that normally exhibits abdominal patternelements.

The mago locus provides a unique opportunity tostudy the mechanisms involved in germ cell determi-nation and delineation of the anteroposterior axis.First, it can be considered to be a posterior groupmaternal effect gene required both for germ celldetermination and segmentation of the embryonicabdomen. This was demonstrated both by the lack ofabdominal segments in inviable embryos derived frommutant mothers reared at 17 °C and the absence of germcells in embryos derived from such mothers. Second, atthe partially permissive temperature of 25°C magomutations produce viable embryos that lack germ cellsand normal polar granules, and the inviable embryoscan exhibit mirror-image duplications of posteriorabdominal and telson structures. Previously, the forma-tion of double abdomen embryos has only beenobserved in embryos derived from mothers mutant inthe spatial coordinate genes, bicaudal, Bicaudal-C andBicaudal-D (Bull, 1966; Nusslein-Volhard, 1977;Mohler and Wieschaus, 1985). Disruption of germ cellformation, however, is never observed in embryos frommothers mutant for spatial coordinates genes. Thisindicates that mago nashi exhibits features of both theposterior group and spatial coordinate maternal effectgenes. Thus, the mago locus is unique in that embryosderived from mutant mothers lacking a functional germplasm can form posterior abdominal segments and formposterior abdominal structures in place of anteriorpattern elements of the head and thorax. Therefore,molecular and further genetic analysis of the magolocus and its interactions with other genes involved inestablishing anteroposterior polarity should provide

new insights into the mechanisms underlying earlypattern formation.

We thank Phillip Newmark and Drs Susan Dutcher,George Golumbeski and David Prescott for critical reading ofthe manuscript. We are grateful to Virginia Fonte fortechnical assistance with transmission electron microscopy.This work was aided by grant no. NP-52138 from theAmerican Cancer Society. R.E.B. is also a recipient of anaward from the PEW Scholars program.

References

AKAM, M. (1987). The molecular basis for metameric pattern inthe Drosophila embryo. Development 101, 1-22.

ANDERSON, K. V. (1987). Dorsal-ventral embryonic pattern genesin Drosophila. Trends Genet. 3, 91—97.

BOSWELL, R. E. AND MAHOWALD, A. P. (1985a). Cytoplasmicdeterminants in embryogenesis. In Comprehensive InsectPhysiology, Biochemistry and Pharmacology, vol. 1 (ed. G. A.Kerkut and L. I. Gilbert), pp. 387-405. Oxford: PergamonPress.

BOSWELL, R. E. AND MAHOWALD, A. P. (19856). tudor, a generequired for assembly of the germ plasm in Drosophilamelanogaster. Cell 43, 97-104.

BULL, A. L. (1966). Bicaudal, a genetic factor which affects thepolarity of the embryo in Drosophila melanogaster. J. exp. Zool.161, 221-242.

DAVID, J. AND MERLE, J. (1968). A reevaluation of the duration ofegg chamber stages in oogenesis of Drosophila melanogaster.Drosophila Info. Serv. 43, 122.

DAVIDSON, E. H. (1986). Gene Activity in Early Development.Academic Press: New York.

DRIEVER, W. AND NUSSLEIN-VOLHARD, C. (1988a). A gradient ofbicoid protein in Drosophila embryos. Cell 54, 83-93.

DRIEVER, W. AND NOSSLEIN-VOLHARD, C. (1988b). The bicoidprotein determines position in the Drosophila embryo in aconcentration-dependent manner. Cell 54, 95-104.

DRIEVER, W., SIEGEL, V. AND NCSSLEIN-VOLHARD, C. (1990).

Autonomous determination of anterior structures in the earlyDrosophila embryo by bicoid morphogen. Development 109,811-820.

FROHNHOFER, H. G., LEHMANN, R. AND NUSSLEIN-VOLHARD, C.

(1986). Manipulating the anteroposterior pattern of theDrosophila embryo. J. Embryol. exp. Morph. 97 Supplement,169-179.

FROHNHOFER, H. G. AND NUSSLEIN-VOLHARD, C. (1986). The

organization of anterior pattern in the Drosophila embryo bythe maternal gene bicoid. Nature 324, 120-125.

ILLMENSEE, K. AND MAHOWALD, A. P. (1974). Transplantation ofposterior polar plasm in Drosophila. Induction of germ cells atthe anterior pole of the egg. Proc. natn. Acad. Sci. U.S.A. 71,1016-1020.

ILLMENSEE, K. AND MAHOWALD, A. P. (1976). The autonomousfunction of germ plasm in a somatic region of the Drosophilaegg. Expl Cell Res. 97, 127-140.

INGHAM, P. W. (1988). The molecular genetics of embryonicpattern formation in Drosophila. Nature 335, 25-34.

JORGENS, G., LEHMANN, R., SCHARDIN, M. AND NOSSLEIN-VOLHARD, C. (1986). Segmental organisation of the head in theembryo of Drosophila melanogaster. A blastoderm fate map ofthe cuticle structures of the larval head. Roux's Arch, devl Biol.195, 359-377.

KALTHOFF, K. AND ELBETIEHA, A. (1986). Transplantation oflocalized anterior determinants in Chironomus eggs bymicroinjection. J. Embryol. exp. Morph. 97 Supplement,181-196.

KING, R. C. (1970). Ovarian Development in Drosophilamelanogaster. Academic Press: New York.

KLINGLER, M., ERDEXYI, M., SZABAD, J. AND NUSSLEIN-VOLHARD,

C. (1988). Function of torso in determining the terminal anlagenof the Drosophila embryo. Nature 335, 275-277.

384 R. E. Boswell and others

LEE, C. S., CURTIS, D., MCCARRON, M., LOVE, C , GRAY, M.,BENDER, W. AND CHOVNICK, A. (1987). Mutations affecting theexpression of the rosy locus in Drosophila melanogaster.Genetics 116, 55-66.

LEHMANN, R. AND NUSSLEIN-VOLHARD, C. (1986). Abdominalsegmentation, pole cell formation, and embryonic polarityrequire the localized activity of oskar, a maternal gene inDrosophila. Cell 47, 141-152.

LEHMANN, R. AND NUSSLEIN-VOLHARD, C. (1987). Involvement ofthe pumilio gene in the transport of an abdominal signal in theDrosophila embryo. Nature 329, 167-170.

LEWIS, E. B. AND BACHER, F. (1968). Method of feeding ethylmethane sulfonate (EMS) to Drosophila males. Drosophila Info.Serv. 43, 193.

LINDSLEY, D. L. AND GRELL, E. H. (1968). Genetic variations ofDrosophila melanogaster. Carnegie Institution of Washington:No. 627.

MAHOWALD, A. P., CAULTON, J. H. AND GEHRING, W. J. (1979).Ultrastructural studies of oocytes and embryos derived fromfemale flies carrying the grandchildless mutation in Drosophilasubobscura. Devi Biol. 69, 118-132.

MANSEAU, L. J. AND SCHUPBACH, T. (1989a). The egg came first,of course! Anterior-posterior pattern formation in Drosophilaembryogenesis and oogenesis. Trend Genet. 5, 400-405.

MANSEAU, L. J. AND SCHUPBACH, T. (19896). cappuccino and spire:two unique maternal-effect loci required for both theanteroposterior and dorsoventral patterns of the Drosophilaembryo. Genes Dev. 3, 1437-1452.

MOHLER, J. AND WIESCHAUS, E. F. (1985). Bicaudal mutations ofDrosophila melanogaster: alteration of blastoderm cell fate. ColdSpring Harb. Symp. quant. Biol. 50, 105-111.

MOHLER, J. AND WIESCHAUS, E. F. (1986). Dominant maternal-effect mutations of Drosophila melanogaster causing theproduction of double-abdomen embryos. Genetics 112, 803-822.

MULLER, H. J. (1932). Further studies on the nature and causes ofgene mutations. Proc. 6th Inter. Congr. Genet. 1, 213-255.

NIKI, Y. AND OKADA, M. (1981). Isolation and characterization ofgrandchildless-hke mutants in Drosophila melanogaster. WilhelmRoux'Archiv. devl Biol. 1981, 1-10.

NUSSLEIN-VOLHARD, C. (1977). Genetic analysis of patternformation in the Drosophila melanogaster embryo.Characterization of the maternal effect mutation bicaudal.Wilhelm Roux Arch, devl Biol. 190, 1-10.

NUSSLEIN-VOLHARD, C , FROHNHOFER, H. G. AND LEHMANN, R.(1987). Determination of anteroposterior polarity in Drosophila.Science 238, 1675-1681.

O'DONNELL, J., BOSWELL, R., REYNOLDS, T. AND MACKAY, W.(1989). A cytogenetic analysis of the Punch-tudor region ofchromosome 2R in Drosophila melanogaster. Genetics 121,273-280.

POODRY, C. A., HALL, L. AND SUZUKI, D. T. (1973).Developmental properties of shibire: a pleiotropic mutationaffecting larval and adult locomotion and development. DevlBiol. 32, 373-386.

SANDER, K. (1976). Specification of the basic body pattern inInsect embryogenesis. Adv. Insect Physiol. 12, 125-238.

SCHUBIGER, G . , MOSELEY, R. C. AND WOOD, W. J. (1977).Interaction of different egg parts in determination of variousbody regions in Drosophila melanogaster. Proc. natn Acad. Sci.U.S.A. 74, 2050-2053.

SCHUBIGER, G. AND NEWMAN, J. S. M. (1982). Determination inDrosophila embryos. Am. Zool. 22, 47-55.

SCHUPBACH, T. AND WIESCHAUS, E. (1986). Maternal-effectmutations altering the anterior-posterior pattern of theDrosophila embryo. Roux's Arch. Devl Biol. 195, 302-317.

SHELLENBARGER, D. L. AND MOHLER, J. D. (1978). Temperature-sensitive periods and autonomy of pleiotropic effects of /(7)7Vsl,a conditional Notch lethal in Drosophila. Devl Biol. 62,432-446.

SPRENGER, F., STEVENS, L. M. AND NOSSLEIN-VOLHARD, C. (1989).The Drosophila gene torso encodes a putative receptor tyrosinekinase. Nature 338, 478-483.

STRECKER, T. R., HALSELL, S. R., FISHER, W. W. AND LIPSHITZ,H. D. (1989). Reciprocal effects of hyper- and hypoactivitymutations in the Drosophila pattern gene torso. Science 243,1062-1066.

SUGIYAMA, S. AND OKADA, M. (1990). Cytoplasmic factorsdetermining anteroposterior polarity in Drosophila embryos.Rouxs Arch, devl Biol. 198, 402-410.

SUTER, B., ROMBERG, L. M. AND STEWARD, R. (1989). Bicaudal-D,a Drosophila gene involved in developmental asymmetry:localized transcript accumulation in ovaries and sequencesimilarity to myosin heavy chain tail domains. Genes Dev. 3,1957-1968.

SUZUKI, D. T. (1970). Temperature-sensitive mutations inDrosophila melanogaster. Science 170, 695-706.

VAN DER MEER, J. M. (1977). Optical clean and permanent wholemount preparation for phase-contrast microscopy of cuticularstructures of insect larvae. Drosophila Info. Serv. 52, 160.

WHARTON, R. P. AND STRUHL, G. (1989). Structure of theDrosophila BicaudalD protein and its role in localizing theposterior determinant nanos. Cell 59, 881-892.

WILKINS, A. S. (1986). Genetic Analysis of Animal Development.Wiley-Interscience Publication: New York.

WILSON, E. B. (1925). The Cell in Development and Heredity.Macmillan Co.: New York.

{Accepted 31 May 1991)