Copyright 0 1986 by the Genetics Society of America

THE GENETICS OF THE DORSAL-BICAUDAL-D REGION OF DROSOPHILA MELANOGASTER

RUTH STEWARD'.' AND CHRISTIANE NUSSLEIN-VOLHARD'

Freidrich Miescher Laboratorium der Max Planck Gesellschaft, Tubingen, Federal Republic of Germany, and Department of Biology, Princeton University, Princeton, New Jersey 08544

Manuscript received December 16, 1983 Revised copy accepted March 17, 1986

ABSTRACT

The chromosomal region 36C on 2 L contains two maternal-effect loci, dorsal (d l ) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respec- tively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mu- tagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(ZL)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of Kelch, two previously identified maternal- effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subre- gions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.

HE cytological interval 36C contains two maternal-effect genes, dorsal (dl) T and Bicaudal-D (Bic-D), which are involved in the establishment of em- bryonic polarity. The dorsal gene is essential to establishing dorsal-ventral po- larity during embryonic development. Homozygous mutant d l females produce embryos which, irrespective of the embryonic genotype, fail to develop the normal dorsal-ventral pattern. In the most severe alleles, the embryos com- pletely lack all ventrally and laterally derived elements, and the mature embryo consists of a tube of dorsal cuticle. Furthermore, mutants at the dl locus also exhibit a temperature-sensitive dominant phenotype, which depends on the

' Present address: Department of Biology, Princeton University, Princeton, New Jersey 08544. ' To whom correspondence should be addressed. a Present address: Max-Planck-Institut fur Entwicklungsbiologie, Spemannstrasse 35, D-7400 Tubingen, Fed-

eral Republic of Germany.

Genetics 113: 665-678 July, 1986.

666 R. STEWARD AND c . NUSSLEIN-VOLHARD

genetic background. Heterozygous mutant dl females at 29 O produce embryos which show weak dorsalization and fail to hatch (NUSSLEIN-VOLHARD 1979a).

Dominant mutations at the Bic-D locus affect anterior-posterior embryonic polarity. Heterozygous Bic-D females and, with a higher penetrance, homozy- gous Bic-D females produce bicaudal embryos with two posterior ends arranged in mirror-image symmetry. The penetrance of the Bic-D phenotype is cold- sensitive. At 18"C, homozygous mutant females lay up to 95% bicaudal em- bryos. In addition to the three dominant Bic-D alleles, R26, an X-ray-induced revertant of one of the dominant Bic-D alleles has been isolated that shows complete reversion of the dominant bicaudal phenotype but is a recessive sterile. Homozygous R26 females lay no eggs and their ovaries are full of partially developed egg chambers that contain 16 nurse cells and no oocyte. Moreover, the chorion of about 95% of eggs laid at 18" by all homozygous, hemizygous or trans-heterozygous Bic-D and R26 mutant females exhibit fused dorsal appendages. This phenotype is temperature-dependent, strictly recessive and does not depend on genetic background (MOHLER and WIESCHAUS 1985, 1986 and personal communication).

Both genes, dorsal and Bicaudal-D, were mapped to a small cytological in- terval (36C2-4; 36C11) on the left arm of the second chromosome (NUSSLEIN- VOLHARD 1979a; MOHLER and WIESCHAUS 1986). Since both genes play an important role in the establishment of embryonic polarity along the two major axes, the two genes and the region surrounding them warranted further anal- ysis; therefore, we sought to investigate how closely the two genes are linked, if there is any interaction between them and if other maternal-effect genes with related phenotypes map in the region.

In order to establish a detailed map of the region, we performed a muta- genesis screen using X rays as a mutagen to isolate DNA rearrangements in the region. In this screen, the dominant conditional dl-phenotype was used as a criterion for the identification of X-ray-induced alleles. Subsequently, we performed an EMS mutagenesis designed to isolate female-sterile, lethal and visible mutations in the dl-Bic-D region uncovered by one large deficiency, Df(2L)H20. These mutations were mapped into the different intervals defined by several deficiencies, allowing us to establish a genetic map of the region and also to identify genes which map into the same cytological interval as dl and Bic-D.

MATERIALS AND METHODS

The markers, aberrations and balancer chromosomes used are described in LINDSLEY

Cy0 = In(2L.R) 0, dp'"' Cy p r cn; homozygous Cy0 individuals die in larval stages. Cy0 DTS51? and Cy0 DTSIUU are CyO-derived chromosomes carrying a dominant

temperature-sensitive lethal mutation (FALKE and WRIGHT 1973). The temperature- sensitive period of DTS51? is restricted to embryogenesis. The temperature-sensitive period of DTSlUU starts after embryogenesis and lasts throughout the life cycle (C. NUSSLEIN-VOLHARD, unpublished results).

DTS9I is a dominant temperature-sensitive mutation on the second chromosome with an embryonic temperature-sensitive period (SUZUKI and PROCUNIER 1969; NUSSLEIN- VOLHARD, WIESCHAUS and KLUDIG 1984).

and GRELL (1968).

GENETICS OF THE DORSAL REGION

TABLE 1

Cytogenetic data on the 2L chromosomal aberrations

667

Deficiencies" Breakpointsb and inversions distal/proximal Reference

Df(2L)H20 36A6,7-A10,11; E3,4-F1,2 This report Of (2L)H68 36B1,2-C1,2; 37AI-Bl E. F. WIFSCHAUS (personal communication) Df(2L)MJB 36B3-B8; 36Dl-El J. MOHLER and E. F. WIFSCHAUS (personal

Df(2L)T317 Associated with 2;4 transloca- This report

Df(2L)TW119 Not visible WRIGHT, HODCETTS and SHERALD ( 1 976) Df(2L)TW137 36C2-C4; 37B9-Cl WRIGHT, HODGETTS and SHERALD (1 976) Df(2L)VA18 36C3,4-D1,2; 37CZ-D1,2 T. R. F. WRIGHT (personal communication) Df(2L)MHs5 36D1-El; 36F6,7-37A1,2 LINDSLEY and GRELL (1 968); WRIGHT,

Df(2L)TW330 Breakpoints not determined T. R. F. WRIGHT (personal communication) Df(2L)TW201 36EF-37A T. R. F. WRIGHT (personal communication) Df(2L)TW203 36E4-F1,2; 37B9-37C,1,2 WRIGHT et al. (1981) Df(2L)TW202 36F1,2-F5,6; 37B T. R. F. WRIGHT (personal communication) In(2L)dlH 36C; 37B13-C1,2 This report In(2L)dlT 21E1,2-F1; 36C This report

communication)

tion

HODGETTS and SHERALD (1 976)

a Deficiencies listed in order of distal breakpoint. ' Each breakpoint falls between the extremes of the intervals shown.

FsD is a dominant female-sterile mutation on the second chromosome. Heterozygous FsD females lay, with low frequency, abnormally shaped eggs that never develop (YAR- GER and KING 1971).

The deficiencies used in this work are listed in Table 1, and most of them were obtained from T. R. F. WRIGHT (WRIGHT, HODCETTS and SHERALD 1976; WRIGHT et al. 1981; LINDSLEY and GRELL 1968).

Fly food Flies were grown on standard medium, containing (per liter of water) 8 g of agar, 18 g of dried yeast, 10 g of soybean meal, 7 g of molasses, 80 g of malt extract, 80 g of cornmeal and 6.3 ml of propionic acid. Eggs were collected on apple- juice agar plates: 1 liter of apple juice, 3 liters of water, 95 g of agar, 100 g of sugar and 40 ml of 15% Nipagin in 95% ethanol.

X-ray mutagenesis: A total of 10,000 males carrying the lethal-free, previously iso- genized, second chromosome with the markers b pr cn sca were irradiated with 4,000 r and were crossed to Df(2R)vgD/Cy0 virgins, as described in Figure 1. Single F1 females carrying the mutagenized chromosome over a Df(2R)vgD second chromosome were set up for laying eggs in blocks at 29", as described by NUSSLEIN-VOLHARD (1977)

The plates containing the egglays were screened, under oil, for embryos which did not hatch and showed a d l dominant phenotype. If a putative mutant egglay was found, the corresponding female and the eggs she had laid were recovered from the block kept at 18" and were transferred to a vial. Her offspring were then further tested for complementation with d l alleles to confirm the mutation in d l . The mutant chromo- somes were further tested, both genetically and cytologically, for deletions, inversions or translocations. EMS mutagenesis: In order to reduce the number of lines that would have to be

established and tested for female sterility, we chose a relatively high concentration of EMS, 50 mM, to mutagenize 5000 males. The high dose of EMS resulted in low fertility in the treated males and, therefore, in a rather small number of F1 progeny.

A lethal-free second chromosome carrying the markers b pr cn wxt bw was isogenized, and 5000 homozygous males were treated with EMS essentially as described by LEWIS

668 R. STEWARD AND c. NUSSLEIN-VOLHARD

set up single females f o r egglays a t 29'

mature plates w i t h egglay 24h a t 2 9 '

screen egglays f o r mutants 1

f l i e s i n blocks to 18"

1 iso late mutant females

FIGURE 1.-Diagram of the X-ray mutant screen.

and BACHER (1968). The males were starved for 4 hr and were then transferred to bottles containing two Kleenex tissues saturated with 10 ml of 1% sucrose, 50 mM EMS solution. After 24 hr the males were mated (100 pairs per bottle) to 5000 b TftlCyO DTSlOO virgins at 18", as described in Figure 2. F1 virgin females and males were collected. Individual (b pr cn wxt bw)*/CyO DTSlOO virgins were mated with three b Df(2L)H20 pr cn sca/FsD males in a vial (Figure 2a). The offspring were exposed for 3 days to 29" so that the FP individuals carrying a Cy0 DTSlOO chromosome would die. Putative lethal mutations were recognized by the absence of orange-eyed progeny. If the (b pr cn wxt bw)*/b Df(2L)H20 pr cn sca flies survived, the flies from the vial were shaken into a tube of an egg-collecting block for analysis of female fertility. No sorting was required; as FsD females are sterile, all eggs produced must be from defi- ciency heterozygous females. Putative lethal or female-sterile mutations were recovered by crossing F2 (b pr cn wxt bw)*/FsD males to Df(2L)H2O/CyO virgins in order to establish a stock and to retest the mutation in the next generation.

Alternatively, the F1 (b pr cn wxt bw)*/CyO DTSlOO males were mated singly in vials to two b Df(2L)H20 pr cn sca/CyO DTS513 virgin females (Figure 2b). After 10 days at 18" the parents were removed, and the vials were transferred for 3 days to 29" so that the individuals carrying a Cy0 DTSlOO chromosome would die. Lethal mutations were recognized by the absence of straight-winged orange-eyed flies. From viable lines, the females carrying the mutagenized chromosome over the deficiency were sorted out and set up in blocks for analysis of female fertility. Putative female-sterile or lethal mutations were established as a stock from the (b fir cn wxt bw)*/CyO DTS513 males and females.

Test of the EMS dose: To test the effectiveness of the relatively high dose of EMS used in the mutagenesis experiment, we checked for the percentage of second chro- mosomes that obtained at least one lethal hit. One hundred two males carrying the mutagenized chromosome over Cy0 DTSlOO were mated singly with females of the genotype DTS91 b pr cn sca/CyO (NUSSLEIN-VOLHARD, WIESCHAUS and KLUDIG 1984). The vials were kept at 29" for 7 days (the parents were removed after 5 days) and

GENETICS OF THE DORSAL REGION

cy4 wss1oo

sl

FsD

18. for 10 days

&tar 5 days remove parents

29" for 2 days I check for /b pr cn wxt b w l */b Df/2f /HZ0 pr cn scu f l i e s (orange eyes)

i f absent, establish stock i f present, tes t females for f e r t i l i t y a t 25'

669

i f absent, establish stock i f present, tes t females f o r f e r t i l i t y a t 25O

FIGURE 2.-Diagrams of the EMS mutant screen.

were then shifted to room temperature. The only offspring, mutagenized chromosome over CyO, were shaken into a new vial and their offspring analyzed for lethals. Of the 102 males, 14 had no offspring, and 80 of the remaining 88 (91%) were lethal.

Determination of the order of the genes in the dl-Bic-D interval: In order to map dorsal and quail relative to each other, we took advantage of the fact that a recombi- nation event between the two female-sterile loci results in one fertile and one sterile chromosome. The markers present on the fertile chromosome will indicate the location

670 R. STEWARD AND c. NUSSLEIN-VOLHARD

el dp b dl pr cn sce sp que cn bw sp

Cy0 D T S l O O dd

D f L?LLTW/ l9 cn bw dd

cyu D T S / O O

7 CY0

el dp b dl pr cn sce sp w * qua cn bw sp

2 days egglay a t 25"

shake parents in to new bot t les

s h i f t bot t les t o 29" f o r 5 days

t r a n s f e r F 2 t o new bot t les

&move parents a f t e r 5 days

1 check f o r offspring

i f present, t e s t f o r genet ic markers and female f e r t i l i t y

FIGURE J.-Diagram of mapping dorsal relative to quail.

of the two genes relative to one another. The scheme for mapping dl and quail is shown in Figure 3. Trans-heterozygous a1 d p b dl ' cn sca sp/quawP20 bw sp virgins were mated to Df(ZL)TW119 cn bw/CyO DTSlOO males. After 2 days the parents were re- moved, and the bottles were shifted for 5 days to 29" so that all the surviving flies carried either of the parental chromosomes over Df(ZL)TWI19. The Ff progeny were transferred to new bottles and were tested for fertile females.

The same screen was used to map the lethal complementation group l(2L)HTI rel- ative to d l . In this test, one of the parental chromosomes, b dl , is sterile over the deficiency, and the other, b I(2L)HTI p r cn wxt bw, is lethal over the deficiency. From a recombination event between the two mutations, we would expect one fertile viable recombinant while the reciprocal product is lethal.

We used the same approach to map the Bic-D locus relative to dl . In this recombi- nation analysis, we used the recessive-sterile R26 revertant of the dominant B ~ c - D " ~ ~ allele. The parental chromosomes were b dl p r cn wxt bw and the other d p b R26. A recombination event between the two female-sterile mutations would result in one crossover chromosome carrying both female-sterile mutations, whereas the reciprocal crossover chromosome carries neither.

RESULTS

Isolation of chromosomal aberrations uncovering the dl locus: In this screen, designed to isolate rearrangements affecting the d l gene, we took ad- vantage of the temperature-sensitive dorsal-dominant phenotype. Depending o n genetic background, a t 29", dl/+ females lay eggs that develop the dorsal- dominant phenotype and fail to hatch. This phenotype is characterized by a n often severe reduction of the width of the ventral denticle bands, as well as a twisted appearance of the embryos, which lack all muscular movement. T h e dorsal-dominant phenotype is greatly enhanced by the deficiency Dj(2R)vgD,

GENETICS OF THE DORSAL REGION 67 1

and embryos from dl +/+ Df(2R)ugD females develop a uniformly strong dorsal- dominant phenotype and do not hatch. At 18", however, the same females lay eggs that develop normally (NUSSLEIN-VOLHARD 1979a). The temperature-sen- sitive period of this dominant phenotype lasts from a few hours before egglay to the blastoderm stage in the embryo (C. NUSSLEIN-VOLHARD, unpublished results). Females carrying the mutant chromosome will, therefore, begin to lay normal eggs within a few hours after being shifted to the permissive temper- ature. This makes it possible to identify a dl mutation in eggs laid by a het- erozygous mutant female at 29" and to isolate the mutant chromosome from progeny obtained from the same mutant female after a downshift to 18".

The 24- to 36-hr-old 29" egglays of 22,000 females heterozygous for an X- ray-treated second chromosome and Df(2R)ugD were scored for the dl dominant phenotype (see Figure 1). Eleven percent of the egglays contained nonfertilized eggs, or too few eggs to be scored. Among the remaining egglays, we detected and isolated a total of about 33 putative mutant females; these fell into two groups. In the first group of about 25 , the embryos within one egglay exhibited a range of phenotypes. Some embryos showed a dl dominant-like phenotype, whereas others stopped developing at an early stage, and a few others devel- oped normally. In the second group of eight egglays, all embryos showed the dl dominant phenotype uniformly. All the mutant chromosomes were isolated and retested in complementation tests over dl ' , a strong mutant allele of the dorsal gene (NUSSLEIN-VOLHARD 1979b). All the mutations in the first group, which did not show a uniform embryonic phenotype, proved not to affect the dl gene and were analyzed no further. Of the eight females in the second group, one died before laying any wild-type eggs at 18", and one was sterile at all temperatures, and thus the mutant chromosome could not be isolated. The other six mutants all proved to affect the dl gene. In a complementation test with Df(2L)TW119 (Table 1; Figure 4), three of the six mutants were lethal, whereas the other three were viable. In compleqentation tests with the other mutations in the region, the three hemizygous' lethal chromosomes proved to be deletions: Df(2L)H20, Df(2L)T317 (see Tab1.e 1) and Df(2L)T43. Segregational and cytological analysis of the deletions showed that Df(2L)T31 7 is associated with a 2;4 translocation and Df(2L)T43 with a 1;2 translocation (precise breakpoints not defined). That latter translocation caused male infer- tility and was subsequently lost.

The other three mutants were tested for their recombination frequency between b and pr , and two of them showed a severely reduced frequency, indicating that the dl mutation might be associated with an inversion. Cytolog- ical analysis of the mutant chromosomes confirmed this observation. Both chro- mosomes have a breakpoint in the 36C region; Zn(2L)dlH has a second break- point between 37B13-C1,2, and Zn(2L)dZT has a second breakpoint between 21E1,2-F1 (Table 1). The sixth mutant isolated in this screen showed no visible cytological aberration and in recombination tests behaves as a point mutation.

Isolation of EMS-induced lethal and female-sterile mutations in the dl- Bic-D region: The aims of this screen were (1) to identify new lethal and female-sterile complementation groups in the dZ-Bic-D region that in turn,

672 R. STEWARD AND c. NUSSLEIN-VOLHARD

* \ 1 + 1

'F

D f f2f )H20

D f f2f )H68

nfzk mia D f L?f ) 73 I ?

Dff2f ) 7 W / I 9

D f f2f ) 7 W l3?

Df fZf )NH s.5

D f f 2 L ) Y A I 8

D f f 2 1 ) 7 W 3 3 0

D f f21 I7 W 2 0 I

Dff2f ) 7 W 2 0 3

D l ( 2 L ) 7 W 2 0 2 FIGURE 4.-Complementation matrix of the genes in the 36A-36F region with the 2L deficien-

cies. *, Number of alleles isolated in the EMS screen; #, total number of alleles known; +, relative position of complementation groups not accurately known. Brackets indicate that the respective complementation groups have not been mapped relative to each other.

would help us to map the breakpoints of the deficiencies in the region; (2) to identify how many and what kind of genes were closely linked with d l and Bic- D; and (3) to isolate new alleles of the known genes in the region. The defi- ciency Df(ZL)H20, which we had isolated in the X-ray mutagenesis screen, deletes the cytological interval of 36C and additional bands flanking this in- terval; therefore, it seemed well suited as a chromosome to use in the screen for EMS-induced mutations. For the EMS mutagenesis (Figure 2), we treated males with the relatively high dose of 50 mM EMS to reduce the number of chromosomes that would have to be screened. The screen was designed for easy identification of lethal mutations in the F2 (pr cn homozygous flies have orange eyes) and also to facilitate the screening of the chromosomes that are nonlethal-bearing for female fertility. When the "agenized chromosomes

GENETICS OF THE DORSAL REGION 673

were isolated through the females, the vials containing flies without a lethal hit in the Df(2L)H20 region could be directly analyzed for female fertility, since females carrying the FsD chromosome are sterile (YARGER and KING 197 1). When the "agenized chromosome was isolated through the males, the females not carrying a lethal mutation in the deficiency region were sorted out and tested for female fertility.

We collected 765 virgin females (Figure 2a) and 950 males (Figure 2b) heterozygous for an EMS-treated second chromosome and established a line from each. Of the females, 14% did not produce offspring; of the males, 19% failed to reproduce. Thus, a total of 1428 chromosomes were tested for le- thality over Df(2L)H20. A total of 1225 chromosomes that were not lethal over Df(2L)H20 were further tested for female sterility; 594 came from cross (a) in Figure 2, and 631 from cross (b). We isolated 48 putative lethal muta- tions, which were then tested for complementation with the different deficien- cies (Table 1). This procedure allowed us to map the breakpoints of the defi- ciencies relative to each other. The mutants in the different deficiency intervals were then crossed inter se to assign them to complementation groups (see Figure 4). In the complementation tests we found that four mutations were only semilethal and could not be mapped properly; these were discarded. We also isolated 38 putative female-sterile mutations, 21 of which could not be confirmed upon retest. In complementation tests with previously identified female-sterile mutations in the region we found four alleles of dl, six quail (qua) (T. SCHUPBACH, personal communication) and one allele of kelch (kel) (T. SCHUPBACH, personal communication). Six female-sterile mutations which pro- duced predominantly small eggs, turned out to be caused by a dominant fe- male-sterile mutation elsewhere on the second chromosome and were not ana- lyzed further.

In all, we isolated 55 mutants mapping within Df(2L)H20, of which 41 are lethals, three are semilethals and show a visible phenotype, and 11 are female sterile. The 55 mutants could be assigned to 15 complementation groups, which averages to 3.6 hits per complementation group.

The deficiency intervals: Six of the complementation groups fall into the deficiency interval delimited distally by the distal breakpoint of Df(2L)H20 and proximally by the distal breakpoint of Df(2L)H68 and DF(2L)MI8 (Table 1). The three alleles of one complementation group, which we called pillow ( j l l ) , are semilethal over a deficiency and, as trans-heterozygotes, show a visible recessive phenotype, i.e., small-lobed eyes. One of the five lethal complemen- tation groups (one allele) has been shown to be allelic to the myosin heavy- chain gene (K. MOGAMI, personal communication). Mutants at this locus die at a late embryonic stage. Mutations in the other four complementation groups all lead to larval lethality. It is possible that one or more of these complemen- tation groups do not actually fall into Df(2L)H20 but that they are allelic to possible other lethal mutations induced on the deficiency chromosome during the X-ray mutagenesis. However, these would have to be localized on the left arm of the Df(2L)H20 chromosome, because we had replaced the right arm with a lethal-free right arm.

674 R. STEWARD AND c . NUSSLEIN-VOLHARD

In the next deficiency interval, delimited distally by the distal breakpoint of Df(2L)H68 and Df(2L)MI8 and proximally by Df(2L)T317 (Table l), we have identified two lethal complementation groups. Mutations in both genes cause lethality during the larval period. Trans-heterozygotes for mutations of a third complementation group 1 (2L)HT7 (seven alleles) rarely survive to adulthood, and these escapers show a “naked” phenotype, reduced bristles on the scutum and scutellum. In the next interval, delimited proximally by the distal break- point of Df(2L)TW119 and Df(2L)TW137 (Table l), we identified one larval lethal complementation group.

The next interval is bordered proximally by the distal breakpoint of several deficiencies: Df(2L)VAI8, Df(2L)MHs5 and DF(2L)TW330 (Table 1). This in- terval contains four complementation groups: qua, d l , Bic-D, and 1 (2L)HTI. Homozygous qua females are defective in oogenesis. The cytoplasmic contents of the nurse cells are not deposited into the oocyte, but the follicle cells produce a normal chorion around the small oocyte, resulting in uniformly small eggs, about half the normal size, which fail to develop (T. SCHUPBACH, personal communication). We isolated six alleles of qua in our screen. Of the four newly isolated d l alleles, one, d15, is a hypomorphic allele, showing a relatively weak phenotype similar to the d12 allele, whereas d16, dl’ and d l s show a strong dorsal phenotype indistinguishable from the previously known allele dl’ (NUSSLEIN-VOLHARD 1979b). We did not recover any dominant or recessive alleles of Bic-D. In the same interval, however, we did identify one additional lethal complementation group (five alleles) that has its lethal phase during the larval period.

l(2)Bld (LINDSLEY and GRELL 1968), of which we isolated seven alleles, falls into the next interval bordered proximally by the proximal breakpoint of Df(ZL)M18 and the distal breakpoint of Df(2L)201 and of Df(ZL)TW203 (Table 1). The next interval is delimited by the proximal breakpoint of Df(2L)TW119 (Table 1) and it contains the gene reduced ocelli (rdo) (LINDSLEY and GRELL 1968). We did not test for alleles of this mutant in our screen.

The last interval contains the locus ninal), essential for proper photoreceptor function. Mutants at this locus show decreased rhodopsin contents in all classes of photoreceptors, and concommitant defects in the electroretinogram (W. L . PAK, personal communication); we did not test for alleles of this gene. This last interval also contains one more female-sterile locus kelch (Kel) of which we isolated one allele. Homozygous mutant kel females are defective in oogenesis. The nurse cell cytoplasm fails to become incorporated into the oocyte. The follicle cells do not deposit egg membranes around the whole of the oocyte, resulting in small eggs that are “open” at the anterior pole (T. SCHUPBACH, personal communication). This interval is delimited by the proximal breakpoint of Df(2L)HZO and Df(2L)T317 and the distal breakpoint of Df(2L)TW202 (Fig- ure 4).

Order of complementation groups in the dorsal interval: We mapped quail relative to dorsal as indicated in the crosses outlined in Figure 3 and described in MATERIALS AND METHODS, taking advantage of the fact that, although each mutation over a deficiency results in a sterile phenotype, a recombination event

GENETICS OF THE DORSAL REGION 675

between the two mutations results in one chromosome that carries both mu- tations and is female’ sterile over a deficiency, and the reciprocal chromosome that should be wild type for both genes and, therefore, fertile. We set up 5000 F2 females to check for possible recombination events and found three fertile females of the genotype + + + + pr cn sca sp/Df(2L)Twl19, which indicated that dl maps distally to qua. Since we could not identify the reciprocal recom- bination event, the three recombinants represent half of the recombination events, indicating that quail maps 0.12 centimorgans (cM) proximal to dorsal [following STEVENS (1942), the 95% confidence limits were determined to be 0.26 and 0.044 cM].

The third gene mapping into this interval is the lethal complementation group l(2L)HTI. T o map this complementation group relative to dorsal, we used the same scheme as outlined in Figure 3 and in MATERIALS AND METHODS. A recombination event between dl and l(2L)HTl results in one fertile, viable chromosome and one lethal chromosome. In 1935 F2 females tested for re- combination events representing a total of nearly 4000 chromosomes exam- ined, we did not find any recombinant, indicating that l (2L)HTl maps closer than 0.025 cM to dorsal (the 95% confidence upper limit was determined to be 0.09 cM).

T o map the fourth gene in the interval, Bic-D, relative to d l we took advan- tage of the female-sterile revertant R26. In a first step we checked whether the recombination frequency between b and pr is affected by the R26 mutation and found that frequency to be normal. In the recombination experiment outlined in MATERIALS AND METHODS, we tested 25,000 putative recombinants for female fertility and found one fertile female which had the genotype d p b pr cn. This genotype, however, could have resulted from a different rare event; for example, from an unequal crossing over. Therefore, this result indicates that dl and the lesion in the R26 map closer than 0.004 cM (95% confidence limits: 0.022 and 0.0001) from each other and that, if it was a bona fide recombination event, R26 lies proximal to dorsal. Thus, the order of the genes in this interval from distal to proximal is probably dl , Bic-D, qua; but the position of l(2L)HTl is uncertain.

DISCUSSION

The goal of this work was to analyze the dl-Bic-D region genetically in order to know what kind of genes (lethal or female sterile) surround dorsal and Bicaudal-D, and how closely these genes map to one another. In our X-ray mutagenesis, we screened for the dl dominant phenotype and isolated six new mutants affecting dorsal, three of which are deletions, two are inversions, and one is a point mutation.

Among the 33 putative dl mutants which we first isolated in the X-ray mutagenesis screen, all the six mutations which showed the dorsal-dominant phenotype uniformly in all the embryos proved to be allelic to d l . This finding argues for dl being unique among the “dorsal group” genes (ANDERSON and NUSSLEIN-VOHLARD 1984) to show a fully penetrant, dominant, temperature- sensitive phenotype.

676 R . STEWARD AND c. NUSSLEIN-VOLHARD

In the EMS screen we isolated a total of 55 mutants, which we could assign to 15 complementation groups. The already existing and new deficiencies al- lowed us to group the known and newly identified genes into the different deficiency intervals (see Figure 4).

To make this analysis of the genes in the dorsal region meaningful, we must be reasonably certain that we identified virtually all the lethal and female- sterile complementation groups in the region. In our test of the effectiveness of the relatively high dose of 50 mM EMS used in the mutagenesis experiment, we found that 91% of the chromosomes had obtained at least one lethal hit. Assuming a Poisson distribution of lethal hits, we can calculate the number of hits induced per second chromosome using the formula, P(0) = e-m, whereby p(0) is the fraction of chromosomes without a lethal hit (0.09), and m is the number of hits per chromosome. The calculation yields an average of 2.4 hits per chromosome, for a total of 3427 lethal hits on the second chromosomes that we screened. Assuming equal mutability of all 1700 lethal loci on the second chromosome (NUSSLEIN-VOLHARD et al. 1984), we should have hit each lethal gene 2.0 times.

For two reasons we believe that we are close to saturation in our screen. First, our average allele frequency for lethal and female-sterile Complementa- tion groups is 3.6. On the basis of our calculation of the number of hits for each mutagenized second chromosome, we estimate 2.0 mutagenizing events per lethal gene. Our allele frequency in the 36C region of 3.6 hits per gene indicates that few, if any, of the lethal and female-sterile complementation groups were missed. Second, we did isolate at least one allele from five of the six previously known lethal and female-sterile genes in the region. However, we did not isolate an allele of the Bic-D locus. Dominant alleles of the Bic-D locus show an antimorphic phenotype (MOHLER and WIESCHAUS 1986), and it may be that a point mutation is not sufficient to create such a phenotype. Similarly, the R26 revertant of a dominant Bic-D allele, which is a recessive female sterile, might not be due to a lesion that would yield an abnormal phenotype in an otherwise wild-type background. Alternatively, the Bic-D gene might, for example, be tandemly repeated, and therefore, both genes would have to be altered in order to obtain a hypomorphic or amorphic mutation.

The four female-sterile loci mapping into Df(2L)H20 region represent a higher than average frequency. GANS, AUDIT and MASSON (1975) have esti- mated that there are a total of 150 female-sterile loci on the whole X-chro- mosome, which represent one-seventh of the lethal loci. Therefore, the rela- tively high number of four female-sterile loci and only 1 I lethal complemen- tation groups indicates some clustering, particularly for the three genes, d l , Bic-D and qua, which are very closely linked. However, all three genes com- plement each other and therefore seem to function independently. It is espe- cially noteworthy that two genes which are involved in establishing polarity are so closely linked, and it remains to be determined if they, and possibly also quail, are derived from a common ancestoral gene, are functionally related or are coordinately regulated.

Our genetic analysis not only proved to be successful in terms of its initial

GENETICS OF THE DORSAL REGION 677

goals but also the two inversions, I n (2L)dZH and I n (2L)dZT, proved to be essen- tial in the cloning and molecular analysis of the dorsa2 locus (STEWARD, MCNALLY and SCHEDL 1984).

We thank T . SCHUPBACH for communicating her results to us before publication and for her help with this paper, and T. R. F. WRIGHT for giving us the deficiencies in the dorsal region. We also thank ULRIKE SCHIER for technical assistance, and our colleagues KATHRYN ANDERSON, ERIC WIESCHAUS, GERD JURGENS, HELEN SALZ and PAUL SCHEDL for helpful discussions. R.S. has been supported by a grant from the National Institutes of Health.

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