The segment polarity gene costal-2 in Drosophila

DEVELOPMENTAL

BIOLOGY

122, 186-200 (1987)

The Segment Polarity Gene costd-2 in Drosophila I. The Organization

of Both Primary and Secondary Fields May Be Affected YVES

Laboratoire

de G&&tique

Embryonic

GRAU AND PAT SIMPSON

Molkulaire des Eucaryotes du CNRS, Unit6 184 de Biologic MolSculaire et de Ginnie Ghe’tique de Chimie Biologique, Facultg de Midecine, 11 Tue Humann, 67085 Strasbourg Chdex, France Received November

24, 1986; accepted in revised form

February

de 1‘INSERM,

Institut

10, 1987

A series of loss of function alleles at the costal- locus is described. Embryos mutant for lethal alleles that are derived from a mutant female germ line display polarity defects on the larval segments. A posterior part of the segmental denticle belt is missing and in its place is a mirror-image duplication of the anterior part including the segment boundary. Maternally rescued embryos are lethal but have normal morphology. Hypomorphic alleles escape to adults that display pattern duplications on the wings and halteres. Dominant gain of function alleles at the Costa&l locus are also described and data are presented that argue that these are neomorphic and act in trans to impair functioning of costal-2. Some wild-type isoalleles of costal- are particularly sensitive to interference from Costal-l mutations and different combinations of these alleles with Costal-l can lead to embryos in which the primary embryonic field is disrupted (bicaudal phenotype) and adults with pattern duplications on the anterior compartment of most body segments. 0 1987 Academic

Press, Inc.

In this paper we describe a series of alleles at the costal- locus and show that this gene falls into the segment polarity mutant class. In 1976, Whittle described an unusual type of wing duplication in flies simultaneously heterozygous for the mutations Costal-l and costal-2. Here we show that leaky alleles of costal- display wing duplications and that these are part of a larger syndrome of polarity defects that affect the embryonic and the imaginal segment. We also describe a series of alleles at the Costal-l locus, most of which are semiclominant and also cause wing duplications. They represent a gain of function and are thought to act in bans to impair functioning of the costal- gene. Such impaired functioning of the costal- locus causes abnormal patterning of the entire embryo on the one hand and pattern duplications within each segment on the other. We conclude that at least some aspects of positional signaling at these two stages may be the same.

INTRODUCTION

The question of how positional information and polarity are acquired in the early embryo has long been of interest to developmental biologists. A genetic approach to this problem, used in the study of Drosophila development, has proved rewarding. Recent studies have shown that the initial spatial cues are placed in the egg before fertilization, through the action of maternally acting genes (Niisslein-Volharcl, 1979; Anderson et al., 1985a, 1985b; Schupbach and Wieschaus, 1986; Mohler and Wieschaus, 1986). Mutation of maternal effect genes can disrupt the entire body of the embryo. Subsequent development along the anterior-posterior axis leads to the subdivision of the embryo into repeated metameric units. A series of mutants called the segment polarity mutants has been described that disrupts the pattern within each segment (Niisslein-Volhard and Wieschaus, 1980). Generally, a part of the segment is deleted and is replaced by a mirror-image duplication of a remaining part. Since different genes are involved in the generation of patterns at these different stages, it is possible that the specification of position within the entire embryonic field relies upon a mechanism different from that used to specify position within each segment. On the other hand, many arguments have been formulated in favor of the universality of positional signaling both at different stages in the life cycle of an individual and between different species (Wolpert, 1969, 1971; French et ah, 1976). 0012-1601X37 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND

METHODS

Stocks, Origin of Mutants costal- (cos-2) alleles. costal-2w1 (cos-.z~I) was kindly supplied by R. Whittle (1976). Other alleles (see Table 1) were induced in mutagenesis screens following standard procedures using ethyl methanesulfonate (Lewis and Bather, 1968) or X-rays at a dose of 4000 rads (100 kV, 10 mA given for 5 min, 1.5-mm aluminum filter, Philips MG102 constant potential X-ray system, beryllium window). They were isolated in an experiment designed 186

GRAU

AND SIMPSON

Segment Polarity

187

Mutant

TABLE 1 costal- ALLELES Viability Allele designation

Chromosome mutagenized

Cytology of chromosome

Mutagen

cn bw sp cn bw sp cn tn.0 sp tYK cn bw sp cn bw sp cn bw sp

X-ray X-ray EMS EMS EMS EMS EMS

Normal Normal N.D. N.D. N.D. N.D. N.D.

cn bw sp bprcnbw Df(2L)fn2 pr cn b el’ rds pr cn dl’ cn sea

EMS Unknown Unknown Unknown Unknown

N.D. N.D. N.D. N.D. N.D.

Nofe. N.D., not determined. a COY-2s’ was isolated by R. Whittle (1976). *These alleles were found to exist on the various

laboratory

stocks listed and were recombined

to detect lethal mutations by their failure to survive when transheterozygous with cos-2” Cos-1’. These mutations were later found to be allelic to cos-.z~I. We initially named the locus Enhancer of Epaulette. cos-zK5was also recovered in this screen. Crosses involving these mutant alleles were performed using the original mutagenized chromosome, except for cos-26 for which the following recombinant was used: pr pk cos-26 en bw sp. cos-2”‘, cos-2y cos-2y and cos-2v4 were found to exist on the various laboratory stocks listed in Table 1. The last three were separated by recombination from these stocks to give the pr cos-2” cn, cos-2” en, and cos-2v4 en sea chromosomes that were used in this study. Costa&l (Cos-1) alleles. The following alleles of Costal1: cos-1 u”, Cos-lA’, Cos-lx, and Cos-l’were gifts from R. Whittle (1976), M. Ashburner, M. C. Mariol, and Ch. Niisslein-Volhard, respectively. Cos-lA1 was induced simultaneously with In(2L)Epa (In(2L)2611-2;35DI-2) and subsequently separated from the inversion. Other alleles (see Table 4) were induced in mutagenesis screens following standard procedures using ethyl methanesulfonate or X-rays at a dose of 4000 rads. Crosses involving these mutant alleles were performed using the original mutagenized chromosome (see Table 4), except for Cos1” and Cos-1’ which were separated from costal-2”(cos2”‘) to give b pr Cos-1” and Cos-1s chromosomes. We initially named the locus Epaulette, allelism to cos-lwz was discovered later. Revertants were isolated by mutagenizing b pr cos-2” en cos-1” bw, b pr en Cos-lA’, or b pr cos2”* cn cos-1s I?Wchromosomes. The complete genotypes of the cos-2 COS-1 recombinants used are: cos-2” en Cos-l’, cos-2v3cn Cos-l’,

Homozygote

Hemizygote

Lethal Lethal Lethal Lethal Lethal Lethal Semiviable, wing duplications Viable Viable Viable Viable Viable

Lethal Lethal Lethal Lethal Lethal Lethal Lethal

out, see Materials

Viable Viable Viable Viable Viable

and Methods.

cos-2” en Cos-l’, b pr cos-2’l en Cos-1’ bw, and cos-2” en cos-1”

bw.

Df(2R)pk7Xk, Df(2R)pk7X”, and Df(2R)STl were obtained from D. Gubb and M. Ashburner. A T(2;3)P32/TMl stock was obtained from E. Lewis. The large Dp(2;3)P32 with cytology as described in Lindsley and Grell (1968) was isolated from this stock. A small deficiency, Df(2R)CA58, that is not as described in Lindsley and Grell was also isolated. The cytology of this was determined by M. Ashburner. A description of marker mutations and balancer chromosomes can be found in Lindsley and Grell (1968). Viabilities of mutants were calculated relative to the balancer classes. Flies were raised on a soybean flour, cornmeal, molasses, yeast extract, malt extract agar medium. All crosses were reared at 25°C. Observation of embryos. From crosses between heterozygotes balanced over CyO, all lethal embryos are assumed to be mutant, as CyO homozygotes hatch as larvae. The calculations of viability of cos-2 embryos given in Table 7 were obtained as (number of lethal embryos)/(25% of the total number of eggs laid) X 100 (n > 200 eggs for each case). The occurrence of aneuploid crossover products was not taken into account. Late embryos were mounted in Hoyer’s medium (van der Meer, 1977) and examined with phase-contrast optics. Imaginal cuticle preparations. Flies were cooked in 10% KOH, mounted in Euparal, and examined under brightfield. Production of germ line clones. Clones homozygous for ~0.9-2 were produced in the germ cells of 72- to 96-hr-old

188

DEVELOPMENTAL BIOLOGY

larvae heterozygous for Fs(2)D and cos-2, obtained by crossing cos-2/CyO females to Fs(2)D/W males at 18°C. The larvae were irradiated with 1000 rads (100 kV, 3 mA for 3 min 20 set) and the resulting females were crossed to cos-2/CyO, Df(2R)pkTsk, cos-2-/CyO, or Oregon R males. They were placed in batches of five in vials containing agar medium and a drop of live yeast. When the females laid eggs these were allowed to develop for 48 hr after which unhatched embryos or dead larvae were mounted for examination of the cuticle. Live larvae were transferred to vials containing standard medium and left to develop. Those that developed to the adult stage were scored for the presence of Cy and in the case of cos-d a few were tested in further crosses for the presence of

VOLUME 122, 1987

izygotes (73%)dying as embryos, followed by cos-@(3%); lethality for the other alleles being mainly larval. The morphology of the cuticle of the lethal embryos appears normal. cos-9 appears to be closest to the amorphic condition since Df(2R)pk78k/Df(2R)ST1 transheterozygotes also survive to the end of embryogenesis and show normal cuticular morphology. cos-27is a hypomorphic allele that is hemizygous lethal but homozygous viable. Alone of the lethal alleles, cos-P is semiviable over cos-27.cos2’/c0s-2~ and ~os-$/~os-2~flies display local pattern duplications on the anterior margin of the wings and halteres similar to those described by Whittle (1976) for Cos-l/cos-2 transheterozygotes. They are sick and sterile. After a recombination experiment in which a freshly cos-2. synthesized chromosome bearing cos-.@was generated, a few homozygotes survived to late pupal stages: these RESULTS flies too displayed pattern duplications, sometimes on Genetic Characterization of costalmany body segments. After establishment of this chromosome in a balanced stock it became entirely embryonic We have mapped cos-2 to position 57 on the right arm and early larval lethal. Both the lethality of the nonof chromosome II, 0.25 units distal to prickle and 0.08 viable alleles and the phenotype of cos-~/cos-27 and cosunits proximal to cinnabar. The gene order in this in2’/c0s-2~ flies are covered by Dp(2;3)P32, cos-2+. The alterval has been determined to be prickle spiny legs pawn cos-2 cinnabar. Both a lethal allele, cos-9, and a semi- leles can therefore be ordered according to the severity viable allele, COS-~~, were mapped independently to this of the mutant effect: cos-d > cos-d > cos-2w* = cos-21 interval. The locus has been mapped cytologically to the = cos-5 > cos-9 2 cos-2? Five other alleles, the Vseries, are described. Apart three to four band interval between 43B3-5 and 43C3 by means of the deletions shown in Fig. 1. The locus is cov- from cos-2v5that was EMS-induced they were found to be present in various laboratory stocks. These alleles ered by Dp(2;3)P32 (41A; 44CD inserted at 89). Neither of the two X-ray-induced alleles were cytologically ab- appear to be wild type in that homozygotes or hemizygotes are viable and morphologically normal and also normal. Twelve alleles are described, see Table 1. Six alleles they fully complement the lethal alleles. However, they fail to complement the lethal alleles, cos-2 deletions, or are homozygous and hemizygous lethal and are lethal in all transheterozygous combinations. Lethality occurs one another in flies that are simultaneously heterozygous for Cos-1 mutants (see Table 2). Cos-1 maps to position in the late embryonic stages and the first larval instar. cos-29is the strongest allele, with the majority of hem- 61 on chromosome 2 and mutant alleles are semidominant (the heterozygous bear wing duplications; see “Genetic Characterization of Costa&l Alleles”). They are believed to act in trans to impair the functioning of cos-2. cos2 h The nature of the synergism between Cos-1 and cos-2 is discussed under “Cos-1 Mutations Act in Tram to Impair I t Df CA50 (43A3;43F6) Functioning of cos-2.” The Valleles of cos-2are uniquely Df pk 78s sensitive to interference from Cos-1 mutants. The nature ci (4261.7;43F5.8)‘---: of the Valleles is not clear. We have interpreted them to be wild-type isoalleles. cos-2” was also mapped Df pk 78k (42E3;43C3) H meiotically very close to cn. This was done by crossing b pr cos-2” en bw/+ females to b p cos-2v1cn Cos-l2 bw/ w--i Df ST1 (4383.5;43E1.8) CQOmales and scoring the progeny for the presence of wing duplications. (Selection for Cos-l2 was relaxed prior to the experiment so that the dominant phenotype was not expressed, see section on Cos-1 alleles.) Among the recombinant chromosomes, 100/103 that carried cn bore pattern duplications whereas only 101434 en+ flies carried duplications (total number of progeny from the FIG. 1. Chromosomal rearrangements used to localize cos-2. cross was 3095).

GRAU AND SIMPSON

Segment

cos-~‘~Cos-1/cos-2~ flies are poorly viable and bear wing and haltere duplications of the same nature as cos-@6/ cos-z7 and cos-~~/cos-2~ homozygotes. The V alleles can be arranged in a continued allelic series according to the penetrance of the wing duplications: cos-2” 2 cosgvh = cos-2”’ 3 cos-2v’ > cos-2” (see Table 2). The effect of dosage of cos-2”’ was measured in the presence of Cosl’, see Table 3. The last four genotypes in Table 3 were all obtained in one cross in order to minimize variation in genetic background, dominant expression of Cos-1’ being variable depending on background. These results indicated that with respect to the synergism with Cos1, cos-2” is hypomorphic. Genetic Characterization

TABLE2

COMPLEMENTATION PATTERNSOFTHE

V

SERIESOFcos-2ALLELES

IN THE PRESENCE OF Cos-1’ Percentage viability relative to Cy sibs

cos-13/t cos-2v’

CwlS/

t

cos-P co,s-18/cos-2v’ cos-dV2 cos- 13/cos-2vA cos-P cos-1~/cos-2?” cos-2v~ Cos-lvCoB-Lv~ cos-2v~ cos-l~/cos-Pvs I < co.s-2v’ cos-1J/cos-2”2 cos-2v’ cos-2v'

cos-13/cos-LvJ co.;-l~/cos-3L'~ ,. I

cos-2L” cos-1~/cos-2v5 cos-2” cos-2” cos-Pvs cos-2” cos-2v’

Cos-ls/Df(2R)CA58 Cos-Is/Df(2R)CA58 Cos-13/Df(2R)CA58 Cos-13/Df(2R)CA58 cos-13/cos-2’ i

co.9P2 cos-ls/cos- -2) cos-2v3 cos-1s/cos-2” cos-P cos-P

cos-lg/cos-Ys co.91 8/cos-2”

Note.

T/CyO Methods.

Crosses or

were

189

Mutant TABLE3

EFFECTOFDOSAGEOFcos-2" IN THE PRESENCEOFCos-1’ Penetrance wing Dosage cos-2”’

Genotype

duplications (%:)

n

124 DpP3d/+ 4. cos-2” O,s-l’/t; 5. cos-2”’ Cos- I’/+;

COS-P"'/COS-P/+

Ki/+ DpPW+

cos-!P/ + COS-P/+/+

0 34 0

158 213 205

of Costa&l Alleles

Seven mutant alleles at this locus were initially studied, see Table 4. Five of these are dominant and when heterozygous cause local pattern duplications of wings and halteres. Two alleles are recessive but also cause a dominant expression of the same phenotype when transheterozygous with lethal alleles or deletions of the cos-2 gene. Mutational events at the cos-1 locus are not rare. Cos-lA1, Cos-lR, Cos-lg, and Cos-lwl were recovered fortuitously in other laboratories by virtue of the dom-

Genotype

Polarity

95 44 6 12 26 2 1 26 17 7

10 Lethal Lethal Lethal Lethal 3 5 6 Lethal Lethal P cos-2 Cos-l/CyO

Percentage flies with wing duplications 3 26 100 83 17 100 100 100 79 100 100 -

n 115 223 33 36 30 27 25 30 57 17 31 -

100 100 100

21 18 29

X 8 Ore R, Df(2R)CA58,

cos-d/Q/O. For complete genotypes see Materials n, number of flies of genotypes shown in column 1.

cos-

and

inant phenotype. From a screen of 6000 EMS-treated chromosomes, and in the presence of a viable wild-type isoallele of the Vseries of cos-2 mutants, the two alleles Cos-1” and Cos-1’ were isolated. From an additional screen in which 5000 EMS-treated chromosomes were tested for lethality over Cos-l’, Cos-l’was recovered. All alleles are homozygous lethal and lethal in all bans combinations except for Cos-lA1 which is homozygous viable and viable over the other alleles. None of the irradiation-induced alleles of Cos-1 are associated with cytological aberrations of salivary gland chromosomes. Independent meiotic mapping experiments for Cos-lA’, Cos-12, and Cos-1’ place the locus between cn and vg at 61 on chromosome 2R. Penetrance and expressivity of the dominant phenotype are variable in the balanced stocks but the flies can be selected to give high (80-95%) or low (3-11%) penetrance. Penetrance and expressivity are consistently higher when the mutation is inherited from the female rather than from the male parent. In order to recover revertants of Cos-1 mutations it was necessary to find conditions in which the penetrance of the phenotype was 100%. This was done in the following manner. When Cos-1 mutations are placed in bans with deletions of the cos-2 locus nearly all flies display the wing duplication phenotype. This occurs whether or not the lines have been previously selected. Penetrance for Cos-1” and Cos1’ is 100% whereas it is about 80% for Cos-lA1. Furthermore, if the Cos-1 chromosomes also carry COS-~~,a viable wild-type isoallele of cos-2, then cos-2”’ Cos-l/cos2- flies are generally lethal and escapers invariably bear extensive pattern duplications (cos-2v*/cos-~ flies are wild type). Under such conditions of complete penetrance, revertants were selected by mutagenizing flies carrying cos-2”’ Cos-1”. cos-2”’ Cos-l’, and cos-2” Cos-lA1 chromosomes and crossing them to Df (2R)CA58, cos-2-. Twenty-four revertants were isolated, see Table 4. The frequency of reversion in these experiments was l/5200

190

DEVELOPMENTAL BIOLOGY

VOLUME 122, 1987

TABLE 4 Costal-1 ALLELES

Allele designation Dominant COS-lA’a

Chromosome mutagenized

Mutagen

Cytology chromosome

Viability homozygote

Viability over Cos-1 dominants

Viability over Co.91 revertants

b cn

7-w

Normal

Viable

Semiviable

Viable

cos- 11

b pr cos 2” cn bw

EMS

N.D.

Lethal

Lethal

Viable

cos-IS

b pr cos 2” en bw

EMS

N.D.

Lethal

Lethal

Viable

cos-1””

Oregon 6

EMS

N.D.

Lethal

Lethal

Viable

COS-P”

cn bu! sp

EMS

N.D.

Lethal

Lethal

Viable

cn bw

EMS

N.D.

Lethal

Lethal

Viable

cn bw sp

EMS

N.D.

Lethal

Lethal

Viable

cos-I2

X-ray X-ray X-ray

Normal Normal Normal

Lethal Viable Lethal

Viable Viable Viable

cos-IS

X-ray X-ray

Normal Normal

Viable Lethal

Viable Viable

cos- 1 s

X-ray

Normal

Lethal

Viable

cos-I3 cos-I8 cos-I8 co.9IS cos-l3 cos-l8 cos-lS cm-lS

EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS EMS

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. Normal

Viable Viable Viable Lethal Lethal Lethal Lethal Lethal Lethal Lethal Viable Viable Viable Lethal Viable Lethal Lethal Viable

Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable

Viable Viable Lethal over R + 5 andR+6 Viable Lethal over R + 3 and R+6 Lethal over R + 3 and R+5 N.D. N.D. N.D. Viable Viable Viable Viable Viable Viable Viable N.D. N.D. N.D. Viable N.D. Viable Viable Viable

Recessive cos-lw’” COS-P

Revertants cos-PR+ cos-lSR+* COS-13R+S

cos-lS cos-lS

cos-IS

Cos-lSR+G

cos-lS

cos-lS cos-lS cos-lS cos-lS cos- 1 s cos- 1 J cos-P cos-13 Cos-lA’

X-ray

Note. N.D. = not determined. ’ Cos-lA’ was isolated by M. Ashburner,

Cos-1' by M.C. Mariol,

Cos-l9 by Ch. Niisslein-Volhard,

for X-irradiated chromosomes and l/2000 for chromosomes treated with EMS. Some revertant chromosomes are homozygous viable and without a mutant phenotype whereas others are lethal. All revertants, however, are viable over the seven cos-1 alleles described above. Crosses generating all

Viability over DfCA58,cos-2m

Semiviable, wings Semiviable, wings Semiviable, wings Semiviable, wings Semiviable, wings

duplicated

Semiviable, wings Semiviable, wings

duplicated

duplicated duplicated duplicated duplicated

duplicated

Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable Viable

and Cos-Iw’ by R. Whittle

(1976).

possible trans combinations of lethal revertant chromosomes (and some viable ones) were performed. Except for cos-lJRc3, COS-I~~+~, and COS-~‘~~~, the revertants are viable over one another and the flies are morphologically normal. Cos-lsR+‘, Cos-lsRi5, and Cos-1sR’6 were among the X-ray-induced revertants and they form a lethal

GRAU

AND SIMPSON

complementation group. Homozygotes and transheterozygotes of these three are lethal as embryos and have a common phenotype (normal cuticle but U-shaped embryos) that is quite distinct from that of Cos-1 homozygous embryos (see section on embryonic phenotypes). Presumably these three revertant chromosomes have small deletions that remove a nearby lethal gene. The cytology of the salivary gland chromosomes is, however, normal. All revertant chromosomes are viable over Df(ZRjCA58, cos-2- and the flies are morphologically normal. In the course of these experiments, nine putative revertant chromosomes were recovered that proved to be due to a second site suppressor mutation. In these cases the suppressor mutation was separable by recombination from the original Cos-1 allele which then recovered the dominant phenotype. The suppressor mutations suppressed the dominant phenotype but did not suppress the lethality of the original chromosome nor its lethality analover other Cos-1 alleles. Extensive recombination yses were also carried out with COS-lJRi3, Cos-lJRi5, Cos1‘RR+‘,Cos-lJRi”‘, and COS-I’~+~~‘. Females of genotype b pr cos-2”‘cn Cos-lRibw/f were crossed to b pr cos-2”cn bw and Df(ZR)CA58, cos-2- males. Homozygosity for cos2I.I or heterozygosity for Df(ZR)CA58 cause Cos-1 to express the wing duplication phenotype with a high penetrance. In no case was it possible to recover the dominant phenotype of the original Cos-1 mutation (n = a3000 chromosomes tested for each revertant). We conclude from these experiments that reversion of Cos-1 leads to viable, fertile, and morphologically normal flies. Presumably a number of revertants will be null alleles, so Cos-1 is a gene that is not required for viability, fertility, or normal morphology, It therefore follows that the dominant phenotype and the recessive lethality of Cos-1 mutants cannot be attributed to a loss of function of the Cos-1 gene itself but that rather the mutant alleles are neomorphic (Muller, 1932), the phenotype resulting from impaired functioning of another gene(s) or gene product(s). This conclusion is consistent with the observation that the dominant alleles retain an unchanged mutant phenotype when in trans with the revertant, presumed null alleles (Table 5). Cos-1 Mutations Act in Trans to Impair Functioning of cos-2. The pattern duplications seen in flies heterozygous for dominant Cos-1 alleles are indistinguishable from those seen in flies homozygous for leaky mutant alleles of cos2. This phenotype is not a common one, no other mutants in Drosophila have been described that give pattern duplications of the same nature. Unlike Cos-1, cos-2 mutations are recessive and represent a loss of function. In

Segment Polam’ty

191

Mutant

TABLE 5 EFFECT OF Cos-1 REVERTANTS ON THE DOMINANT PHENOTYPE OF Cos-IA’ Cos-lA’/Cos-lR+

Revertant R+l R+2 R+S R+4 Rf5 RfEI R+E.$

Percentage flies with wing duplication

Percentage viability relative to bd’sibs

Cos-lA’/bu~D Percentage flies with wing duplication

Number of flies from cross

95 101 98 92 so 95 88

15 12 12 12 8 10 18

212 184 279 309 154 227 301

10 8 11 15 9 16 10

Note. Crosses were P Cos-lA’/CyO tYL0I).

X d b pr cos-2” cn COS-~‘+~ bwu,/

this section we present arguments that the dominant phenotype seen in flies heterozygous for the neomorphic Cos-1 mutants is actually the result of a reduced level of functioning of the cos-2 locus. If this were the case, then lowering the number of wild-type copies of cos-2+ should lead to a more extreme dominant phenotype of Cos-1 heterozygotes, whereas the addition of extra copies should decrease it. The data in Table 6 show that, indeed, haploidy for cos-2 enhances whereas triploidy for cos-2 suppresses the phenotype of Cos-1 heterozygotes. The results given in Table 6 were obtained immediately after a prior selection for the wing duplication phenotype in the balanced Cos-1 stocks for three generations. The results are presented for Cos-lA’, Cos-l”, and Cos-1’ but pertain to all other Cos-1 mutant alleles including the two recessive alleles Cos-lwl and Cos-1’ which present the phenotype when in trans with cos-2 deletions. Two other cos-2 deletions, Df(2R)pkrXk and Df(ZR)STl, have the same effect (results not shown). Cos-l/cos-2 transheterozygotes also present wing duplications as was first observed by Whittle (1976). The data in Table 7 show that for a given Cos-1 allele the frequency of duplications is correlated with the strength of the mutant cos-2 allele. Six cos-2 alleles have been ordered by virtue of the percentage of homozygotes or hemizygotes dying as embryos. Both Cos-1’ and Cos-1’ present a high frequency of duplications when in trans with strong alleles such as cos-2s or cos-d and little or no duplications when transheterozygous with leaky alleles such as cos-9 and cos-2’. The data in Table 7 were collected without prior selection for Cos-1” or cos-1’. cos-2 Has Both a Maternal

and a Zygotic Act&L

Wild-type embryos of Drosophila are characterized externally by the differentiation of three thoracic and

192

DEVELOPMENTALBIOLOGY

TABLE 6 EFFECTOFDOSAGEOF COS-2+ ON THE DOMINANT PHENOTYPE OF Cos-I Percentage

Percentage

viability relative to Cv or Me sibs

flies with wing duplications

Number of flies”

106 40 21 52

0 70 100 100

364

610 254 472

2

98

2 2

105 91

30 23 28

383 291 305

3 3 3 3

81 94 96 83

0 0 0

306 279 142 119

cos-2-/CyO,

Ore R

Dosage Genotype +/DfCA58 Cos-lA’/DfCA58 Cos-1~/DfCA58 Cos-ls/DfcA58 Cos-IA’/+ CO.9P/t

Co.9P/t t/t;

Cos-F/t;

Cos-P/t; Cos-I’/+;

DpP32 DpP32 DpP32 DpP32

cm-z+

1 1 1

1

Note. Crosses were 9 Cos-l/CyO or Dy(z$)P32, cos-Z+/TMl.

4

or Ore R X d Df(2RKA58,

‘Total number of flies from cross.

eight abdominal segments. Each segment bears a belt of denticles in the anterior part; these differ in morphology among the different segments, see Fig. 2A. On the abdominal segments 1 to ‘7, the denticles are small anteriorly and increase in size toward the posterior. Embryos homozygous for strong cos-2 alleles that are derived from heterozygous parents are lethal but have an essentially normal segmental pattern of cuticular denticle belts. Close inspection reveals occasional perturbations in the polarity of ventral denticles especially along the midline but these defects are minor. However, cos-2 also acts maternally and the strongest mutant phenotype is seen when both the female parent and the zygote are mutant. Flies of this type were obtained by

VOLUME122. 1987

the production of homozygous germ line clones. cos-2/ Fs(2)D females at 18”C, that had been irradiated between 72 and 96 hr after oviposition, were crossed to cos-2/CyO or cos-2-/CyO males and scored for the production of eggs, see Table 8. No clones were recovered for cos-.P, this is an X-ray-induced allele that perhaps carries a small nonvisible rearrangement affecting another gene(s). For all other alleles studied, there was a clear bimodal distribution of phenotypes. About half of the embryos in each case showed normal segmentation but displayed an abnormal deletion-duplication phenotype within each segment. These are presumed cos-2 homozygotes. The remaining embryos have a much more normal, though not completely wild-type, phenotype. These are presumed cos-2/CyO zygotes and in fact some of these survive to adults and are invariably Cy (a few of these were tested in subsequent crosses and proved to be cos-2/CyO). The imagoes are morphologically normal. Some cos-?/Fs(2)D females were crossed to Oregon R males and embryos developing from this cross do not display deletion-duplication patterns and many of them develop to adults. Therefore, for the majority of alleles studied, one dose of cos-2+ in the zygote can rescue some, but not all, embryos derived from cos-2 females. It appears that in this experiment the majority of recombination events took place proximal to both Fs(2)D and cos-2 (Schupbach and Wieschaus, 1986). Four clones resulting in entirely wild-type progeny were obtained, however (two for cos-9 and two for cos-9). These are presumed to result from recombination between Fs(2)D and cos-2 and are not shown in Table 8. One allele, COS-~~*,behaved somewhat differently from the others. All embryos were lethal and although half of the embryos displayed the deletion-duplication phenotype within each segment, embryos of both classes

TABLE 7 EMBRYONIC

LETHALITY

OF DIFFERENT cos-2 ALLELES AND FREQUENCY OF PATTERN ON WINGS OF COS-I/COS-2 TRANSHETEROZYGOTES

In trans with Cos-l3 Percentage Allele

cos-2$ cos-2p cos-2w' cos-25 cos-2@ co.92'

Homozygote 32 16 12 13 100” 7

dying as embryos Hemizygote

Percentage duplicated wings

73 32 24

18 20 14

Note. For calculations of embryonic lethality see Materials and Methods. a This chromosome is thought to carry an extraneous lethal. b n, total number of flies of genotype Cos l/cos 2.

100 100 80 74 21 0

DUPLICATIONS

In trans with Cos-1’

nb 87 31 52 42 48 25

Percentage duplicated wings 100 100 92 92 80 0

nb 34 28 38 29 59 63

FK. 2. (A) A wild-type embryo (Oregon R). Note the three thoracic and eight abdominal segmental denticle belts. (B) A homozygous cos-2 embryo from a female germ line mutant for cos-25. Note the absence of thoracic denticle belts and the abnormal shape and reduced width of the abdominal belts. The denticles are all small and the segment boundaries are duplicated. (C) A presumed cos-SW’ heterozggote from a homozygous cos-2” female germ line. Segmentation is abnormal. (D) A presumed cos-2 WI homozygote also from a homozygous cos-Zw’ female germ line. Only a few posterior segments have differentiated cuticle and the denticle belts show the deletion duplication phenotype. (E) A presumed cos-2 heterozygote from a mutant cos-P female germ line. All segmental denticle belts are seen but they are not entirely wild type. (F) A 03~1~ homozygous embryo (from a cross between heterozygous parents). The embryo is virtually identical to that seen in (B). The duplicated segment boundaries can be seen. (G) An assymetrical bicaudal embryo derived from a cross between cos-2”’ Cos-Y/CyO females and Oregon R males. (H) A symmetrical bicaudal embryo derived from a cross between cos-2 v’ Cos-1’/Cy0 females and cos-?/CyO males. The denticle belts also show abnormal polarity. 193

194

DEVELOPMENTAL BIOLOGY

VOLUME 122, 1987

TABLE 8 GERM LINE CLONES PRODUCED BY IRRADIATION-INDUCED MITOTIC RECOMBINATION

Somatic genotype of irradiated female

Genotype of male

No. of females tested

No. of females laying morphologically normal eggs

No. of fertilized eggs

No. of lethal embryos with pattern duplications

Embryos

without

No. dying as embryos or Ll larvae

pattern

duplications

No. dying as pupae

No. Cy imagoes

COS-~‘/FS(~)D COS-~~/FS(~)D cos-2’/Fs(2)D cos-2’/Fs(2)D

cos-2J/CyO Ore-R cos-2s/cyo cos-2~/cyo

445 265 315 450

9 5 7 0

87 48 33

43 0 14

12 12 10

7 5 3

25 31 6 -

cos-2’/Fs(2)D

cos-2- or cos-2J/CyO cos-2J/CyO

365

6

35

17

13

1

4

170

2

21

21”

0

0

0

cos-Bw’/Fs(2)D

Note. For complete genotypes see Materials a See text.

and Methods.

also showed abnormal segmentation. The maternal effect of cos-2w1 thus appears to be stronger than that of the other alleles tested, because of the mutant phenotype, on the one hand, and because one dose of cos-2+ in the zygote is unable to effect a significant rescue. Since for most alleles tested the presence of one dose of cos-2' in the zygote can partially rescue embryos derived from a mutant germ line, we looked to see whether 2 doses of cos-2’ in the female parent would permit survival of cos-2 homozygotes. cos-P cn bw sp/CyO; Dp(2;3)P32, cos-2’ en+/+ females were crossed to cos-@ cn bw sp/CyO or cos-,@cn bw sp/CyO males. No Cy’ en flies were recovered. Embryonic

Phenotypes

Mutant embryos derived from a mutant germ line. The deletion-duplication phenotype seen on the cos-2 zygotes is shown in Fig. 2B. The phenotype is very similar to that of patched (Niisslein-Volhard and Wieschaus, 1980). The posterior part of the abdominal segmental belts is missing and in its place there is a mirror-image duplication of the anterior part. This generally includes the segment boundary which is therefore duplicated. The thoracic denticle belts are absent. This may be due to deletion and duplication of a naked part of the segment or to extreme ventral reduction, see next paragraph. The extent of the deleted area and the size of the duplication varies between alleles but is remarkably constant among individuals of any one allele. For strong alleles such as cos-22 a rather small part of the denticle belt is absent and the duplication involves quite a large area. For cosd embryos the entire belt is composed on average of seven to eight rows of denticles (Fig. 3D) whereas embryos mutant for cos-@, an intermediate allele, display on average five to six rows of denticles per belt (Fig. 3E). For weak alleles such as cos-27 the deletion involves

virtually the entire denticle belt, only one or two of the anterior-most rows of denticles remaining (Fig. 3F); most cos-P embryos bear no duplication. In all cases the embryos are shorter than wild type so presumably part of the naked region of the segmental cuticle is also deleted. In summary it appears that the part of the pattern that is deleted varies from allele to allele and that for all alleles, except perhaps the hypomorphic COS-~~,the deletions are accompanied by a duplication of a remaining part. Apart from the deletion-duplication phenotype an overall effect on the width of the denticle belts is apparent. The first abdominal belt is extremely narrow, often the rows are composed of only three to four dentitles. The belts become progressively wider the more posterior the segment and often the last two or three segments bear belts of the correct width, see Fig. 2B. Furthermore, a few embryos from each cross (between 3 and 10% depending on the allele) fail to secrete any cuticle at the anterior pole, they are thus open at the anterior end and lack thoraces and sometimes even the anterior-most abdominal segments. This phenotype is even more marked for cos-zwl embryos which were far more abnormal than embryos mutant for the other alleles. Although it is possible that the cos-2w1chromosome carries an unrelated mutation, these embryos are strongly reminiscent of Cos-1 homozygotes, see section on the effects of Cos-1 mutations. In addition to the deletion-duplication phenotype that was present within each segment (half of the embryos), all embryos developed fewer than the normal number of segments. Some embryos developed fewer but larger segments and the entire space within the egg was occupied by the embryo (Fig. 2C). Furthermore, these sometimes showed mirror-image duplication patterns between segments. The majority of embryos, however, show very little cuticular differentiation, often only a few posterior segmental

GRAU

AND SIMPSON

Segment

Polarity

Mutant

195

FIG. 3. The denticle belt of the second abdominal segment, or of abdominal segments 2 and 3, in animals of different genotypes. (A) A wildtype embryo. (B) A cos-I’homozygote showing deletion-duplication phenotype. (C) A cos-$ heterozygote from a homozygous cos-$ female germ line. Note the abnormal size and orientation of the rows of denticles in some positions, (D-F) Mutant cos-2 embryos derived from a mutant female germ line showing the large duplications due to the strong cos-P allele (D), the smaller duplications caused by an intermediate allele, ~0.~9, (E), and the small remaining row of anterior denticles in the case of the hypomorphic cos Z7 allele (F).

denticle belts are seen (Fig. 2D). Therefore, the anteroposterior pattern of the entire embryo can be disrupted in ~0.7-2mutant embryos. Heteroxygous embryos derived from a mutant germ line. The phenotype of the cuticle of cos-2 heterozygotes that develop from a homozygous mutant germ line is not entirely wild type. No clear deletion-duplication phenotypes are observed. Instead small local perturbations in the size and orientation of denticles are ob-

served. The small anterior denticles are often of a large size characteristic of more posterior ones. In contrast small denticles are often found in a more posterior position. Occasionally all the denticles point anteriorly and sometimes only some of the rows do. These phenotypes are much more marked in the case of the strong cos-9 allele (Fig. 3C). The effects of Cos-1 mutations. Consistent with the hypothesis that Cos-1 mutations affect the expression of

196

DEVELOPMENTALBIOLOGY

the cos-2 locus, very similar defects are observed in Cos1 homozygotes which die as embryos. In contrast to cos2 embryos which show a constant phenotype for any one Cos-1 allele, embryos derived from crosses involving mutant alleles are variable in phenotype. Examples of crosses with Cos-1 mutants are shown in Table 9. Eighteen percent of Cos-1 homozygotes show the same intrasegmental deletion-duplication phenotype typical of cos2 mutants (Figs. 2F and 3B), and ‘75% show a disturbed denticle polarity phenotype that resembles that of the paternally rescued cos-2 embryos (see Fig. 2E). In addition to these intrasegmental defects a number of embryos fail to differentiate anterior parts. Defects of the head, as visualized by the mouthparts which are abnormally formed or are missing, are the most frequent, followed by absence of the cuticle of the thorax and then the anterior abdominal segments. No abnormalities of posterior parts are observed without all parts anterior to them being affected. The defects seem, therefore, to begin at the anterior pole and spread further and further posteriorly. A similar range of phenotypes is seen when Cos-l/CyO females are crossed to lethal alleles of cos-2 or cos-,Zdeletions (results not shown). The Valleles of cos-2 are particularly sensitive to interference from Cos-1 mutations and this is illustrated for crosses involving chromosomes carrying both cos-2” and a Cos-1 mutation (crosses 2 and 3, Table 9). (A similar range of phenotypes was seen for crosses with cos-zvZ Cos-l’, cos-2” cos-l’, and cos-Zv4 Cos-1’ chromosomes; results not shown.) They result in embryos with defects similar to those from cross 1, but when cos-2” is inherited from the female parent the abnormalities are far more extensive. In addition a new phenotype is observed, that of bicaudal embryos. In such embryos the anterior half of the body is deleted and is replaced by a posterior part with reversed symmetry. Bicaudal embryos may be completely symmetrical, that is with the same number of segments in the original and in the duplicated abdomen, or assymetrical, bearing fewer segments in the duplicate (Figs. 2G and 2H). Such embryos may also show the intrasegmental abnormalities. Bicaudal embryos are only observed when both Cos-1 and cos-2’l are inherited from the female parent (crosses 2 and 3). This is consistent with the known maternal effect of cos-2. A slight dominant maternal effect of cos-2”’ Cos-1 heterozygotes is also observed (crosses 4 and 5). cos-2”’ Cos-11 + females when crossed to cos-~~‘/cos-2~~ or Df(2R)pkrak, cos 2-/CyO males also show the same range of defects described above (results not shown). Since it is thought that the embryonic abnormalities associated with Cos1 mutations are due to reduced or altered expression of the cos-z locus, then additional cos-2+ alleles should suppress or at least reduce the frequency of defects. This is the case as shown in crosses 6 and ‘7 of Table 9.

VOLUME122,1987

GRAU AND SIMPSON

Phenotypes: Only the Anterior Compartment Is Afleeted

Imaginal

Flies heterozygous for Cos-1 or homozygous for leaky develop to adults that display wing and haltere duplications. An intermediate state between the weak hypomorphic phenotype on the one hand and the early lethality seen for the lethal alleles on the other can be obtained with some combinations of Cos-1 and cos-2 alleles. Here we describe the pattern defects of cosZvl Cos-l’/Df(ZR)CA58, cos-2- individuals. Such animals die at various stages of development and the most excos-2 mutants

Segment

Polarity

Mutant

197

treme imaginal phenotypes are observed in those flies that metamorphose but fail to hatch from the pupal case. These animals display pattern duplications on all segments of the body (Fig. 4). All parts of the fly body, with the possible exception of the clypeolabrum, can be affected. It is a general rule that the mutants carry duplications of pattern elements of only the anterior compartment. Representative anterior (A) and posterior (P) structures of the proboscis, head, wings, and legs were scored for frequency of duplications and are presented in Table 10. P structures are very rarely affected. Extra bristles are formed in the anal plates of both sexes, extra

FIG. 4. Imaginal pattern duplications seen for flies of the genotype cos-PL” Cos-lz/Df(2R)CA58, COSX. (a) Proboscis with additional pseudotracheae. (b) Head showing duplicated aristae. (c) Extra sex combs on the foreleg of a male fly. (d) Duplicated wing containing the pattern elements: costa, triple row of bristles, and double row of bristles. (e) Duplicated haltere. (f) Duplicated tergite: large anteriorly pointing bristles replace the smaller posteriorly oriented bristles in the anterior part of the segment.

198

DEVELOPMENTAL BIOLOGY TABLE

FREQUENCY WITH WHICH

VARIOUS

Proboscis A

CUTICULAR

STRUCTURES

Head A

P

VOLUME 122, 1987 10

ARE DUPLICATED

IN FLIES OF THE GENOTYPE

A

Pseud

Ml

M2

n

Ar

FRN

Pa

Zb

30

0

0

30

42

47

2

0

12 60

SC 100

P scl 63

BH2

Cos-Iz/Df(2R)CA58,

Midleg

Foreleg P

cos-2”’

sell 0

Wing P

A

COS-$-

A

n

Ab

Spb

scl

sell

n

60

35

78

32

0

60

Co 96

TR 100

P DR 80

PR 0

Al 0

12 50

Note. Figures are given in percentages. Pseud, pseudotrachea; Ml, medial bristle 1; M2, medial bristle 2; Ar, arista; FRN, frontal bristles; Pa, palpus; Zb, zahnborsten; SC, sex comb, scl, sensilla campaniformia on femur; BH, bristle on hairy island on coxa; sell, group of 11 sensilla campaniformia on coxa; Ab, apical bristle; Spb, spur bristles; Co, costa; TR, triple row of bristles; DR, double row of bristles; PR, posterior row of hairs; Al, alula; A, anterior compartment; P, posterior compartment. In the proboscis, additional pseudotrachea are added onto the lateral side and thus in the anterior compartment (Struhl, 1977). -

clasper teeth were observed in male genitalia, and extra vaginal teeth were seen in female genitalia. These pattern duplications do not represent a replacement of one part of the pattern by another, since only very small parts of the pattern, if any, are absent. They are formed almost exclusively of the addition of extra pattern elements. The duplications seen in the abdominal tergites are different from those seen in the imaginal disc derivatives in that they are not composed of additional pattern elements but clearly replace missing parts of the original pattern. In the wild-type fly, only the anterior part of each tergite bears bristles (this is believed to be the anterior compartment, see Madhavan and Madhavan, 1980), the posterior part is naked. The posterior-most bristles are larger than the rest and reside on a band of dark pigment. The abdomens of cos-2’l Cos-12/ Df(2R)CA58 flies have a very constant phenotype, the smaller anterior bristles are replaced by larger bristles, similar to those seen posteriorly, which point anteriorly (see Fig. 4f). Additionally the dark pigment is also seen anteriorly. This phenotype, a deletion of a part of the pattern and its replacement by another part with reversed polarity, is formally more similar to the phenotypes seen in COS-2mutant embryos. The duplicate develops as a function of positicm within the segment. As the duplications occur only in the anterior compartment of each segment we asked the question whether they develop as a function of position within the segment or whether they occur only in cells bearing the label “anterior” versus those bearing the label “posterior.” This was done by constructing Costal1 engrailed flies. engrailed (en) partially transforms the cells of the posterior compartment into anterior. If the duplications attributable to cos-2 affect anterior cells, then a second duplication might be expected to occur in the transformed posterior. This was found not to be the case, Cos-lJA en’ flies carry duplications only in the original anterior compartment and not in the transformed posterior. We therefore conclude that the duplications develop as a position response.

DISCUSSION

Cos-1 mutations act in bans on cos-2. We have shown that the majority of Cos-1 mutant alleles are semidominant and cause the formation of wing duplications in the heterozygotes. Even the two recessive alleles express the dominant phenotype when in bans with deletions of cos-2. The dominant alleles represent a gain of function since they can be reverted and then lose the phenotype. The revertants fail to cause wing duplications even in the presence of cos-2 deletions. Although we have not attempted to revert the recessive alleles (due to lack of complete penetrance of the phenotype) it is likely that they too are a gain of function mutants since the revertants, many of which are presumably null alleles, are without any apparent effect on the fly. Cos-li is therefore not required for viability, fertility, or normal morphology. One possibility is that the gene is redundant. This hypothesis however makes it difficult to understand the nature of the mutant alleles. These must be neomorphs (Muller, 1932). Neomorphic mutations are rather rare in Drosophila and are mostly caused by unique events. Cos-1 mutations are not rare, however, and can in fact be obtained fairly readily in standard mutagenesis experiments. Another possibility is that the mutations result in the constitutive or inappropriate expression of a gene that is not normally expressed in the ectoderm of the fly. Alternatively they all result in an altered product of a nonvital function. The unusual imaginal phenotype of flies heterozygous for Cos-1 mutations is very similar to that seen in flies homozygous for leaky mutants of cos-2. Furthermore, the abnormalities seen in the lethal Cos-1 homozygous embryos are also similar to those of cos-2 mutant embryos derived from a mutant germ line. The cos-2+ function is vital since mutations of this gene are all recessive, cause a loss of function, and most result in early lethality. These considerations, together with the observation that haploidy for cos-2 enhances whereas triploidy suppresses the dominant phenotype of Cos-1 mutants, lead us to conclude that Cos-1 mutants affect the expression of cos-2. The fact that Cos-l/cos-2 transheterozygotes

GRAU

AND SIMPSON

also bear the same wing duplication phenotype, and that penetrance of this is correlated with the strength of the cos-z mutant allele, is consistent with this hypothesis. Under this view the mutant phenotypes of Cos-1 are attributable either to a loss of cos-2+ function or to an altered cos-2 function. cos-2 is a segment polarity gene. Seven loci detected in systematic screens for zygotic lethals were classified as belonging to the segment polarity class (Ntisslein-Volhard and Wieschaus, 1980; Wieschaus et al, 1984). These are hedgehog, patched, gooseberry, cubitus interruptus”, armadillo, fused, and wingless. To this list can be now added cos-2, mutants of which would not be detected in systematic screens due to the lack of mutant phenotype of the lethal homozygous embryos derived from heterozygous parents. Of the segment polarity mutants, fused and armadillo, like cos-2, have a maternal as well as a zygotic effect (Counce, 1958; Wieschaus and Noell, 1986). In the case of cos-2 both the maternal and the zygotic action are vital: two doses of cos-2+ in the female parent are not sufficient to rescue homozygous embryos and one dose in the zygote does not completely rescue embryos derived from a homozygous germ line. The phenotype of cos-2 mutant embryos derived from a mutant germ line most closely resembles that of patched. The mutant embryos have a posterior portion of the denticle belt deleted and a duplication of the anterior part including the segment boundary. The deletion appears to be more posteriorly situated within the segment in the case of strong alleles and more anteriorly situated for weak alleles. Since it is not possible to make germ line clones homozygous for cos-2- deletions, we do not know if any of these phenotypes represent the amorphic condition. It is clear, however, that they do represent a loss of function and thus the major effect of a loss of function of cos-2 is to disrupt the polarity within each segment. These effects could be caused by cell death and subsequent regeneration. Cell death has been detected, for example, in fused embryos (Martinez-Arias, 1985). However, it appears that the pattern duplications are cell autonomous for both armadillo and fused and that the phenotype does not necessarily arise through a regenerative response to cell loss (Gergen and Wieschaus, 1986). A mosaic analysis and molecular studies should provide a clearer view of the role of cos-2 in segment polarity. The deletion-duplication phenotypes seen on embryos mutant for the segment polarity genes may be considered to be due to a disruption of secondary embryonic fields within each segment. The insect segment has long been considered to possess the properties expected of an embryonic field (see Lawrence, 1981, for recent review). Grafting experiments have revealed the existence of an A-P gradient that provides positional information and is repeated in each segment (Locke, 1959). This gradient

Segment Polarity

Mutawt

199

determines polarity and since it is reiterated an abrupt change in scalar value occurs at every segment boundary. This is revealed by grafts of the intersegmental boundary which are associated with changes in polarity (Piepho, 1955). However, segments are subdivided into clonally restricted anterior and posterior compartments (Garcia-Bellido et ah, 1973, 1976; Steiner, 1976; Struhl, 1977; Morata and Lawrence, 1978) and a polarity change may occur at each anterior-posterior compartment boundary (Simpson and Grau, 1987). The segmental defects of gooseberry, hedgehog, and cubitus interruptus dominant are restricted to the anterior compartment (Martinez-Arias and Ingham, 1985). We have shown here, through a study of the imaginal pattern duplications, that the effects of cos-2 are similarly restricted to the anterior compartment. Furthermore, observation of Cos-1 en’ wings reveals that this is the result of a localized defect in an anterior position in the segment and is not related to the anterior versus posterior labels of the cells. The effect of cos-2 on the imaginal discs is not the result of gene action at a later stage of development, since we have shown elsewhere that cos-2+ is not required after 3 hr of development (Simpson and Grau, 1987). The imaginal pattern duplications probably arise as the consequence of a polarity defect on the embryonic segment. In the embryo the imaginal discs are formed from a group of anterior and posterior cells that are initially continuous with the larval epithelium (Szabad et al., 1979). At this stage, therefore, cell patterning events are common to both larval and imaginal cells. One other segment polarity mutant, wingless, also causes imaginal pattern duplications and appears similarly to be active only during embryogenesis (Babu, 1977). Unlike the other segment polarity mutants, cos-2 also appears to affect anteroposterior coordinates of primary embryonic fields. For all alleles we observe a gradient in the width of abdominal denticle belts, anterior ones being very small and posterior ones larger. The most striking effects on the primary embryonic field are seen with the allele cos-Zwl which has a stronger maternal effect than the other alleles and results in embryos with fewer segments. It is possible that this could be due to the presence of another mutation on this chromosome arm. However, there is a striking similarity between cos-2w1 embryos and embryos resulting from crosses involving the neomorphic Cos-1 mutations. Cos-I mutants display patterns characteristic of both strong and weak cos-2 alleles. It is therefore likely that at least in part Cos-1 actually reduces the level of functioning of cos-2. Thus Cos-1 homozygotes show the same range of phenotypes seen for the germ line clones of all cos-2 alleles. However, when Cos-1 and the Vseries of wild-type variant cos-2 alleles are combined and as a result of both the maternal and the zygotic effects, more extreme phenotypes, like those of COS-~~‘,are observed. In some cases

200

DEVELOPMENTAL BIOLOGY

a change in the entire embryonic fate map may occur as in the case of bicaudal embryos. An extra dose of cos2’ in the female parent causes a sharp decrease in the frequency of such embryos, consistent with the notion that Cos-1 is affecting the expression of cos-2. It is likely that these more extreme embryonic patterns are not merely the result of a reduced cos-2 function but also result from an altered activity of the cos-2 gene. If it is true that cos-1 mutations are acting exclusively on the ~0.~2 gene and that the phenotype of cos-zw’ is due exclusively to this mutant allele, then these results provide evidence that a single gene can play a role in cell patterning both of the entire embryo and the individual segment. This then lends some support to the idea that secondary fields rely on a mechanism for positional signaling similar to that used in primary embryonic organization. It has been generally assumed that the basic mechanism whereby cells know their position in a developing tissue will be the same at different stages in development and within different tissues, and indeed may be the same in widely differing animals (Wolpert, 1969,197l). Recent models show, for example, how spatial information in the imaginal discs could be a direct consequence of that formed in the embryo (Meinhardt, 1983) and how a two-dimensional system of polar coordinates could specify pattern in many systems (French et al., 1976). The excellent technical assistance of Cathie Carteret and Claudine Ackerman was greatly appreciated. We thank Robert Whittle, David Gubb, Michael Ashburner, Christane Niisslein-Volhard and MarieChristine Mario1 for generously providing mutant stocks, Geoffrey Richards and Michael Ashburner for help with the cytology, Marc Bourouis and Peter Lawrence for comments on the manuscript, and Pierre Chambon for providing excellent working conditions. This work was supported in part by grants from the INSERM (PRC 134025) and the DGRST (8131086). Y.G. was supported by the Fondation pour la Recherche Medicale. REFERENCES ANDERSON, K. V., BOKLA, L., and NUSSLEIN-VOLHARD, C. (1985a). Establishment of the dorsal-ventral polarity in the Drosophila embryo: The induction of polarity by the Toll gene product. Cell 42,791-798. ANDERSON, K. V., JURGENS, G., and NUSSLEIN-VOLHARD, C. (198513). Establishment of dorsal-ventral polarity in the Drosonhilu embryo: Genetic studies on the role of the Toll gene product. Cell 42,779-789. BABU, P. (1977). Early development subdivisions of the wing disc in Drosophila.

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VOLUME 122, 1987

giant, and unpaired mutations in mosaic embryos. Wilhelm Roux’s Arch. Dev. Biol. 195, 49-62. LAWRENCE, P. A., (1981). The cellular basis of segmentation in insects. Cell 26, 3-10. LEWIS, E., and BACHER, G. (1968). Method of feeding ethyl methane sulfonate (EMS) to Drosophila males, Drosophila Inj Serv. 43,193. LINDSLEY, D. L., and GRELL, E. H. (1968). Genetic variations of Drosophila melanogaster. Carnegie Inst. Washington Publ. 627. LOCKE, M. (1959). The cuticular pattern in an insect, Rhodnins prolixw. J. Exp. Zool. 36, 459-477. MADHAVAN, M. M., and MADHAVAN, K. (1980). Morphogenesis of the epidermis of the adult abdomen of Drosophila. J. Embryo1 Exp. Morphol. 60, l-31. MARTINEZ-ARIAS, A. (1985). The development offused embryos of Drosophila melanogaster. J Embryol. Exp. Morphol. 87, 99-114. MARTINEZ-ARIAS, A., and INGHAM, P. W. (1985). The origin of pattern duplications in segment polarity mutants of Drosophila melanogaster J. Embryol. Exp. Morph01 87, 129-135. MEINHARDT, H. (1983). Cell determination boundaries as organizing regions for secondary embryonic fields. Dew. Biol. 96,375385. MOHLER, J., and WIESCHAUS, E. (1986). Dominant maternal effect mutations of Drosophila melunogaster causing the production of double abdomen embryos. Genetics 112,803-822. MORATA, G., and LAWRENCE, P. A. (1978). Anterior and posterior compartments in the head of Drosophila. Nature (London) 274,473-474. MIJLLER, H. J. (1932). Further studies on the nature and causes of gene mutations. Proc. Int. Cong. Genet. 6th, 213-255. N~SSLEIN-VOLHARD, C. (1979). Maternal effect mutations that alter the spatial coordinates of the embryo of Drosophila melanogaster. In “Determinants of Spatial Organization” (S. Subtelny and I. R. Konigsberg, Eds.), Academic Press, New York. N~SSLEIN-VOLHARD, C., and WIESCHAUS, E. (1980). Mutations affecting segment number and polarity in Drosophila. Nature (London) 278, 795-801. PIEPHO, H. (1955). Uber die polare orientierung der Bilge und Schuppen dem schmetterlingsrumpf. Biol Zbl. 74,467-474. SCHUPBACH, T., and WIESCHAUS, E. (1986). Germ line autonomy of maternal effect mutations altering the embryonic body pattern of Drosophila Dev. Biol. 113, 443-448. SIMPSON, P., and GRAU, Y. (1987). The segment polarity mutant costal2 in Drosophila II. The origin of imaginal pattern duplications. Dev. armadillo,fused,

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