Requirements for zygotic gene activity during gastrulation in Drosophila melanogaster

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Requirements for Zygotic Gene Activity during Gastrulation in Drosophila meknogaster SUSAN Department

B. ZUSMAN AND ERIC F. WIESCHAUS

of Biology, Princeton

University,

Princeton,

New Jersey OS.544

Received December 4, 1.98.&;accepted in revised form May 6, 1985 (tsg) loci interfere with early morphogenetic moveMutations at thefolded gastrulation (fog) and twisted gastdation ments in Drosophila melanogaster. fog embryos do not form a normal posterior midgut and although their germbands do elongate, they do not extend dorsally. As a result, when normal embryos have fully extended germbands, the germbands in mutant embryos
INTRODUCTION

Recent mutagenesis experiments have identified a number of zygotically active loci in Drosophila required for normal gastrulation. Among the earliest acting are two X-linked recessive embryonic lethals called folded gastrulaticm (fog) and twisted gastrulaticm (tsg), both of which show obvious effects on germband extension. These mutations were isolated following an EMS (ethyl methane sulfonate) mutagenesis and were mapped to bands 2OA.S2OB and llAl-llA7, respectively, using overlapping deletions (Wieschaus et al., 1984). The experiments described below were designed to test: (1) whether the fog and tsg genes are only required in the zygotic genome during embryogenesis; (2) whether the dosage of wild-type genes in the mother has any effect on gastrulation; and (3) whether the fog and tsg mutations affect only particular regions of the embryo. Morphological and genetic techniques were used to examine the zygotic and maternal expression of the two loci. In addition, fog and tsg adult and larval gynandromorphs were produced using the loss of an unstable ring X chromosome during early cleavage.

In the embryos of multi~cellular organisms, early morphogenetic movements play an important role in the proper formation of normal pattern. Most often such movements depend on localized changes in cell shape and adhesive properties, as well as on directed growth and cell proliferation. Though these processes have been well described in a variety of organisms (Trinkaus, 1969; Spooner et al., 1973; Saunder, 1976) the specific mechanisms involved and the genetic regulation of these events are not well understood. Embryonic development in Drosophila melanogaster appears to be a good experimental system to approach this problem. Extensive and well-characterized morphogenetic movements occur throughout embryogenesis and are extremely reproducible from one individual to the next. They are particularly notable during gastrulation when they can be followed in living embryos using relatively simple optical techniques. The initial stages of Drosophila gastrulatio’n last about 40 min at room temperature, and occur .primarily through cell shape changes and cell movements with little cell proliferation (Sonneblick, 1950; Bownes, 1975; Fullilove and Jacobson, 1976; Hartenstein and Campos-Ortega, 1985; Technau and Campos-Ortega, 1985).

MATERIALS

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METHODS

Mutant strains. Unless otherwise indicated, the fog allele used in these experiments was fwti and the tsg allele 359

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used was tsgB8.Both muta.tions produce embryonic phenotypes identical to those observed in embryos hemizygous for small deficiencies of the respective regions. Mutant chromosomes carrying the marker mutations yellow, white, forked, or shavenbaby, alone or in various combinations, were kept over the balancer chromosome FM7 (Merriam and Duffy, 1972). In addition, males with fog and y+Ymal+ (Schalet and Lefevre, 1976) or tsg and Dp(l$)~““~ (Craymer and Roy, 1980), balanced over CyO, were kept in stocks with C(l)DXfemales. For a detailed description of most of the marker mutations, see Lindsley and Grell (1968).

Observation of living embryos, cuticle preparation, and ,whole mounts. Eggs were collected from females for 1530 min at 25°C on agar plates coated with yeast paste. Eggs were submerged in Voltalef 3s oil to make the egg coverings transparent, and embryonic development was observed under a dissecting microscope. Time-lapse videotapes of single embryos were made using a Panasonic NV-8050 recorder and WV-1800 high-resolution video camera. Photographs of living stages were made with a Zeiss photomicroscope II, using embryos from a mutant stock homozygous for the maternal effect mutation klarsicht. klarsicht has no effect on embryonic development but increases the contrast between cells and yolk thus making the eggs more suitable for photographic purposes (Nusslein-Volhard, unpublished). Cuticle preparations of mutant embryos were made following the procedures of van der Meer (1977). To make whole mounts of mutant and wild-type embryos at early developmental stages, the ernbryos were dechorionated in sodium hypochlorite, fixed using the method described by Zalokar and Erk (1977), dehydrated, and mounted in cedarwood oil. Germline clones. Mitotic recombination was induced by y irradiation (1500 r) in the germline of heterozygous fog or tsg larvae, 48-66 hr old. The dominant female sterile mutation OvoD’ (=KlZ37) (Jimenez and CamposOrtega, 1982; Garcia-Bellido and Robbins, 1983; Perrimon et al., 1984), was used to identify mosaic females. Clone-containing females were transferred to plastic cups over agar plates supplemented with yeast for egg collection. The resulting eggs were then submerged in Voltalef 3s oil and embryonic development was examined. To ensure that normal egg production was not due to a strong perdurance of the wild-type gene product made before mitotic recombination, clones containing

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females were tested for egg production for 2 weeks. Production of adult mosaics. Approximately 300 fog gynandromorphs were collected from a cross in which females carrying the unstable ring X chromosome, In(l)wVc (for a description of this chromosome, see Hinton, 1955; Hall et al., 1976) were mated to y f fog males carrying a Y-linked duplication covering the mutation (y’Ymal+). fog control gynandromorphs were obtained by mating In(l)w”“/FM7 females with y w f/y+Ymal+ males. In the tsg experiment, both mutant and control mosaics were obtained by crossing In(l)w”“/FM7 females with y tsg/Y; Dp(l,Z)~““~/Cy0 males. Control mosaics were derived from In(l)w”“/y tsg; Dp(l$)~“~“/+ flies, and could be distinguished from the tsg mosaics which were CyO and lacked the duplication. In addition, approximately 200 fog gynandromorphs and 200 tsg gynandromorphs were collected from crosses in which y f fog/FM7 or y tsg/FM7 females were mated to In(l)w’lc males. Control gynandromorphs for these experiments were obtained from the following cross: y w f/FM7

X In(l)w”“/

Y

All adult gynandromorphs were prepared for microscopic examination using the method described in Szabad (1978). The genotype of 20 landmark structures was recorded; the left and right landmark structures were scored separately. From this data, the maleness average was determined as the number of times a structure is mutant divided by the number of times it was scored. Significant differences between experimental and control values were detected using x2 contingency tables (P

G 0.05). Production of larval mosaics.fw, tsg, and control larval gynandromorphs were produced from crosses in which males carrying the unstable ring X chromosome, In(l)wDc, were mated to sub fog/FM7, y sub tsg/FM7, and y sub/FM7 females. shavenbaby (sub), an X-linked recessive mutation which makes larval denticles shorter and removes dorsal hairs (Wieschaus et al., 1984; Gergen and Wieschaus, 1985) was used to mark mutant patches on larval gynandromorphs. Embryos from the gynandromorph-producing crosses were fixed and mounted using the method described in Gergen and Wieschaus (1985). The genotype of denticle bands and dorsal cuticle of mosaic larvae and embryos was scored under a compound microscope and schematic drawings were prepared of each mosaic; the left and right sides of the larvae and embryos were scored sep-

FIG. 1. Photographs of living normal, tsg, fog, and tsg fog embryos at room temperature during the process of germband elongation. (a-c) Normal gastrula at developmental stages approximately 10, 20, and 40 min after the onset of gastrulation, respectively. Arrows indicate the position of the posterior midgut invagination and the posterior dorsal fold. (d-f) fbg gastrula approximately 5, 10, and 25 min after the onset of gastrulation, respectively. (g-j) kg gastrula approximately 10, 30, 50, and 90 min after the onset of gastrulation, respectively. (k) fog tsg gastrula approximately 25 min after the onset of gastrulation. (1) Heterozygousfbg gastrula from a mother with homozygous fog germ cells approximately 25 min after the onset of gastrulation.

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arately. From this data, maleness average scores for individual larval segments were calculated and the lethal foci of the gastrulation mutations were mapped. RESULTS

Early Abnormalities in fog and tsg Embryos In wild-type Drosophila embryos, gastrulation begins with ventral furrow formation. This invagination brings the future mesoderm into the interior of the embryo and results in a clearly visible “germ band” on the embryo’s ventral side (Figs. la-c). About 5 min after this furrow first forms, the cells underlying the pole cells at the posterior end of the embryo become columnar and their nuclei migrate basally (Scriba, 1964). These cell shape changes result in the formation of a cup-shaped posterior midgut. During subsequent gastrulation, the germband expands, pushing the posterior midgut onto the dorsal side of the embryo. One hour after the invagination of the ventral furrow, the germband has extended to about 2; times its initial length and the posterior midgut has come to lie one-third the way from the anterior end of the embryo (Fig. 2a). Embryos hemizygous for folded gastrulation (=fog) initiate ventral furrow formation normally but do not form a posterior midgut (Figs. Id-f). The pole cells and underlying blastoderm cells are shifted slightly to the dorsal side of the embryo but move no farther anteriorly. As the germband increases in length, it folds into the interior of the embryo rather than moving onto the dorsal side (Figs. le, f; 2b). These ventral folds persist until germband shortening. These embryos then continue through subsequent stages of development but never revert to normal morphology. At final differentiation, the cuticle of fog embryos has two large ventral holes at the anterior and posterior end. The posterior hole corresponds in position to the anal opening and anal plates. The anterior hole is more variable in extent. Embryos hemizygous for tsg become unambiguously distinguishable from their heterozygous siblings when their posterior midgut has moved onto the dorsal side of the embryo. At this point in normal development, the cells on the dorsal side are thrown into folds, then shifted laterally to make way for the extending germband. In tsg embryos, the dorsal cells remain fixed on the dorsal side and are pushed into increasingly deep folds by the pressure of the advancing posterior midgut (Figs. lg-i). This results in a temporary blockage of germband extension. Subsequently, the posterior midgut buckles under the dorsal cells and the folds on the dorsal side are released (Fig. lj). At the end of gastrulation, tsg embryos have an abnormally positioned posterior midgut, abnormally thickened dorsal cells, and an abnormally deep cephalic furrow (Fig. 2~). tsg embryos continue through

b

FIG. 2. Whole mount preparations of a normal,.&, and tsg gastrula under Nomarski optics, (a) Normal gastrula with a fully extended germband. (b) An embryo hemizygous for fog at a similar developmental stage. (c) An embryo hemizygous for tag at a similar developmental stage. The embryos were mounted as described under Materials and Methods, Anterior is left; ventral side is down.

subsequent stages of development, but their morphology remains abnormal. At the completion of embryonic development, tag cuticles show head defects and condensed, retracted posterior spiracles. In order to see if the defects caused by the fog and tsg mutations affect independent processes of germband extension, the two mutations were recombined onto the same chromosome. Both fog traits, such as folds in the germband and an abnormal posterior midgut invagination, and tsg traits, such as the abnormally deep dorsal folds and cephalic furrow, can be seen in these embryos (Fig. lk). The result suggests that fog and tsg act on different pathways and is consistent with our subsequent conclusion that the primary effects of the two mutations can be localized to different regions of the embryo.

ZUSMAN AND WIESCHAUS

Zygotic and Maternal

Activity

The folded gastrulatio?z and twisted gastrulation mutations identify genes on. the X chromosome whose activity is required in zygotic nuclei after fertilization. When mutant chromosolmes are marked with cuticle markers such as yellow or shavenbaby, the phenotypes are only observed in embryos hemizygous or homozygous for the mutations and not in their heterozygous siblings. Mutant embryos show the same phenotypes regardless of whether they are derived from heterozygous mothers or totally wild-type attached-X mothers with two normal copies of each gene. Although these results indicate that wild type fog and tsg gene function is needed in the embryo for normal development, they do not exclude the possibility that the genes are expressed in the mother’s germ cells during oogenesis. To study the eflect of total elimination of wildfrom maternal germ cells, germline type gene product clones were produced in females heterozygous for each mutation and the dominant female sterile mutation OvoD’ (Jimenez and Campos-Ortega, 1982). Females with germ cell clones homozygous for fog or tsg produce eggs as often as the control mosaics (Table 1). Eggs fertilized by nonmutant sperm develop to adults and mosaic females continue to produce progeny for at least 2 weeks following eclosion. The latter result implies that the ability of homozygous germ cells to produce eggs and viable offspring is not due to the perclurance of wildtype gene product made prior to clonal induction (Perrimon et al., 1984). fog and tsg genes, therefore, do not need to be expressed in the mother’s germ cells during oogenesis; their activity is only needed in the zygote for complete embryonic development and survival. Mutant embryos derived from germline clones show the same phenotypes observed earlier in embryos from heterozygous mothers. No effect of germline homozygosity is observed in the development of tsg/+ embryos. TABLE 1 EGG PRODUCTION FROM.~~QAND tsg GERMLINE CLONES

Genotype of irradiated flies y (4’f/ovoD’ y tsg B8/ovoD’ y tsg Ng/cbJoD’ y fog 4A/0voD’ y lll)l14/ovoD’

No. flies examined 434 351 260 356 265

No. flies which produce eggs

No. of flies which continued to produce eggs for at least 2 weeks

14 11 9 12 9

13 10 9 10 7

” Mosaic females which produce more than 10 daughters were regarded as having homozygous germline clones.

No. of flies with homozygous

fog or tsg germ cells”

10 9 11 8 and no sons

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Heterozygous embryos derived from fog germline clones, however, do not gastrulate in the typical wild-type pattern. They often show deep ventrolateral folds (Fig. ll), reminiscent of those observed in hemizygous fog/ Y embryos. Such embryos did form an apparently normal cupshaped posterior midgut and underwent a full germbancl extension onto the dorsal side of the embryo. Subsequent development in these embryos appeared normal, and in spite of the initial folds, the embryos hatched and formed fertile adults. Although we do not understand the nature of the altered pattern or its relationship to the hemizygous phenotype, two further observations suggest that it is indeed caused by the lack of normal fw gene product in germ cells. The deepened lateral folds were never observed in heterozygotes derived from normal mothers (200 embryos examined), but were produced in embryos derived from germ cells homozygous for a second fog allele (l(l)ll4, Schalet and Lefevre, 1973). The latter result argues that the effect is not due to other extraneous X-linked mutations on the original fog chromosome. Collectively, these results indicate that the fog gene may normally be transcribed during oogenesis, even though the maternal gene product is not required for normal germline development, and even though its presence is irrelevant for the ultimate survival of a genetically wildtype embryo. Gynandrommph

Analysis

In order to determine if the fog and tsg mutations are cell viable (Bryant and Zornetzer, 1973; Rip011 and Garcia-Bellido, 1973) gynanclromorphs with mutant and wild-type cells were produced using the unstable ringX chromosome In(l)wvc (Catchesicle and Lea, 1945; Hotta and Benzer, 1972). For each mutant, two different crosses were used. Since the frequency of ring-X loss varies in different stocks, a y w f control cross was run simultaneously with each experiment using ring-X individuals from the same bottles used in the experimental crosses. The frequency of mosaic production and the distribution of male tissue in these control mosaics were used to estimate the starting situation in the experimental crosses. Both fog and tsg mutations allow adult mosaics to survive. Patches of mutant tissue were normal in appearance and could be found anywhere on the adult cuticle. Up to 88% of the adult cuticle in surviving fog gynanclromorphs could be mutant and up to 97% of the adult cuticle in surviving tsg gynanclromorphs could be mutant. These observations indicate that the mutations are cell viable and the wild-type genes are not needed during subsequent imaginal disc development. In each experiment, mosaics for fog or tsg survive less well than corresponding control mosaics. When crosses yielding similar control maleness averages are com-

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pared, the fog mosaics survive less well than the tsg mosaics (24% compared to 79% survival in experiment I and 45% compared to 93% survival in experiment II). Since the maleness average in the control is not affected by the presence of a lethal mutation on the rod chromosome, it provides an estimate for the probability that any given blastoderm cell in a mosaic is mutant. The lethality of fog mosaics is higher than the frequency with which any single cell is mutant. This implies that the viability of fog mosaics depends on the genotype of more than one cell, as would occur if the animal dies when any cell in a hypotheticalfog “lethal focus” is mutant. In classical gynandromorph terminology (Hotta and Benzer, 1972, 1973), the fog lethal focus behaves in a “domineering” fashion. If the observed lethality of fog mosaics does in fact result from those cases where the lethal focus is totally or partly mutant, the fraction of mosaics which die can be used to estimate the frequency of mosaicism within the lethal focus and thus, the size of the region where fog’ activity is required (Table 2). The measured mosaic frequencies (62,64% ) are roughly equal to those obtained for the entire thorax and abdomen on one side of the animal (67, 64%), an area of about 300 blastoderm cells (Fig. 3). To determine whether fog gene products are required only in particular regions of the embryo, we measured the frequency with which various adult structures are mutant. The maleness average values are nonuniform and position dependent, indicating that mosaics with mutant clones in certain areas of the blastoderm are more likely to survive than others. The maleness average values decrease in the anterior to posterior direction; there is no significant change in the dorsal to ventral direction. The genitalia in fog gynandromorphs are mutant less often than any other scored structure and are mutant significantly less often than control genitalia (Table 2). Although the total number of mosaics scored (505 individuals, 1010 sides) is not large enough to show that the genitalia are significantly closer to the focus than tergites 4 through 7 or sternites 3 through 6, the tendency of the maleness average to decrease for structures more and more posterior indicates that the primary site of fw’ action is most probably at the posterior end of the embryo (Fig. 3b), in the region which gives rise to posterior midgut or proctodeum. In both experiments, the lethality of the tsg mosaics is lower than the control maleness average. This indicates that tsg+ function can be supplied by a number of different cells in the embryo, such that there is no single point on the blastoderm surface which must be wild type for embryonic survival. This might occur if tsg+ product is nonautonomous or could diffuse from wild type to mutant cells. If all regions of the embryo can supply wildtype product equally, one would predict that all struc-

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tures in surviving mosaics would be mutant with the same frequency. In both tsg experiments, however, the humerus was scored as mutant less often than other adult structures and was mutant significantly less often than in the control (Table 3). Although the 525 adult mosaics scored (1050 sides) are not sufficient to show that the humerus is mutant significantly less often than tergites 2 through 7, there is a significant drop in maleness average from the head, legs, and genital structures to the humerus (see tsg cross 3, P < 0.05). Consistent with this result, all surviving mosaics have at least some tag+ cells in their dorsal structures (humerus, scutellum, tergites l-4), whereas in 16 cases, all ventral structures (legs, sternites, genitalia) are mutant. These results indicate that the cells on the dorsal side of the embryo are the most effective source of wild-type tsg product. This might occur if these cells are closest to the area of the embryo where the gene product is actually needed. Alternatively, it is possible that tsg+ activity is restricted to a particular region on the dorsal side of the embryo, such as the amnion serosa cells (Fig. 3~). This lethal focus might be “submissive,” such that any wild-type cells within the area are sufficient for survival. Under this hypothesis, the only mosaic individuals which die are those whose foci are entirely mutant. To account for the high survival of tsg mosaics, the frequency of mosaicism within the tsg focus would have to be about 4448%,equal to that of the abdomen on one side and corresponding to an area on the blastoderm of about 200 cells.

Determination of the Lethal Foci Using Larval Mosaics The interpretation of the adult mosaic data is complicated by uncertainties about the embryonic origins of the various adult structures scored. Moreover, since the mosaics which die cannot be examined, conclusions are necessarily indirect. Therefore, a second set of fog and tsg mosaics were produced in which the genotype of the epidermal cells could be identified at the end of embryogenesis. To do this, we used the X-linked marker shavenbaby which causes shortened ventral denticles and removes dorsal hairs (Wieschaus et al., 1984; Gergen and Wieschaus, 1985). Mosaics were obtained by mating In(l)wVc males to females heterozygous for subfog or svb tsg (see Materials and Methods). The resultant embryos were classified as normal, typically mutant, or otherwise defective, and their genotype with respect to svb was recorded. The “normal” class included embryos which had hatched themselves, unhatched embryos which were apparently normal, and embryos with minor defects in the head and segmentation pattern. Defects of this kind are often produced in crosses involving the ring-X; in our exper-

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TABLE 2 CALCULATED MALENESS AVERAGE SCORESFOR LANDMARKS IN CONTROL AND~O~ ADULT GYNANDROMORPHS Control -

Nonmosaics” Mosaics Mosaic survival’ Lethality” Mean maleness average Mosaicism of lethal

focusd

Landmarks Ocellar bristle Probosis Humerus Scutellum Leg 1 Leg 2 Leg 3 Tergite 1 Tergite 2 Tergite 3 Tergite 4 Tergite 5 Tergite 6 Tergite 7 Sternite 2 Sternite 3 Sternite 4 Sternite 5 Sternite 6 Genitalia

cross 1

fog Cross 1

Control

cross 2

fog Cross 2

IYL(l)uY” x y Y Fm 7

In (I) l(JUC:: Y fw Y Fm7

vfw :: In(l)uPc Fm7 y+ Ymal+

228 232 (-1 C-1

830 199 0.24 0.76

502 324 C-1 C-1

1044 306 0.45 0.55

0.45

28

0.23

0.15

C-J

0.62

(-)

0.64

Maleness average score

Maleness average score

Maleness average score

Maleness average score

0.46 0.44 0.49 0.48 0.48 0.45 0.46 0.47 0.45 0.45 0.44 0.45 0.44 0.43 0.47 0.44 0.43 0.43 0.41 0.41

0.46 0.44 0.46 0.40* 0.38* 0.36* 0.34* 0.30* 0.29* 0.26* 0.23* 0.19* 0.18* 0.13* 0.25* 0.25* 0.22* 0.20* 0.17* 0.07*

0.19 0.23 0.24 0.25 0.25 0.24 0.23 0.25 0.24 0.25 0.25 0.26 0.25 0.25 0.20 0.20 0.21 0.22 0.21 0.21

0.21 0.15* 0.18* 0.1s* 0.19* 0.17* 0.17* 0.17* 0.16* 0.16* 0.14* 0.14* 0.14* 0.11* 0.12* 0.12* 0.11* 0.10* 0.09* 0.09*

’ Nonmosaics include only Zn(l)w”“/y w (fog) flies. * Survival = [(No. fog mosaics)/(No. control mosaics)] X [(No. control nonmosaics)/(No. fog nonmosaics)]. ‘Lethality associated with fog in mosaics; l-survival rate of fog mosaics. d The frequency with which lethal foci are mosaic in a given experiment was calculated on the assumption that for domineering foci, the fraction of gynandromorphs which die is equal to the frequency with which the lethal focus is either totally male or mosaic (i.e., L = f (8) + f(m)). Also, the maleness average in the experimental animal at the blastoderm stage should be equal to both the final values observed in the control (X,) and to the frequency of totally mutant foci plus one-half the frequency of mosaic foci (i.e., X, =Ad) + if(m)) since mosaic foci should on the average be one-half mutant and one-half wild-type cells. *jio,9 maleness average scores significantly less than corresponding control values. Significance was measured using x2 contingency tables (P < 0.05). Since right and left halves of each mosaic were scored independently, the sample size for each landmark is double the number of mosaics obtained. For example in the scutellum of cross 1, the number of mutantfog scutella is 159 and the x2 value is 5.4.

iments they occurred in both mosaic and nonmosaic individuals and with equal frequency in the experimental and control cross. The svb fog mosaics of “normal” morphology were recovered at 42% the frequency with which svb mosaics were found among fog’ c’ontrols. The similarity of this survival frequency with those obtained in the adult experiment suggests that most, if not all of the fog-related deaths in mosaics can be attributed to abnormalities

during embryogenesis. The mosaics of “normal” morphology also show the same nonrandom distribution of mutant patches observed in the adult mosaics. The maleness averages decrease in the anterior-posterior direction, with the lowest values in the eighth abdominal segment (5% ventral, 6% dorsal) and anal tuft (6%)(Table 4). Among the unhatched individuals derived from the fog cross, we found 61 abnormal embryos which were

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FIG. 3. The lethal foci offog and tsgplaced on a blastoderm fate map. (a) The fate map was constructed by placing various embryonic, larval, and adult structures on a blastoderm split opened such that dorsal was closest to the middle of the two halves. The blastoderm consists of 6000 cells and was drawn such that each half was approximately 100 cells long (anterior to posterior) and 80 cells wide (dorsal to ventral). Embryonic and larval structures were positioned on the map based on the results of morphological studies (Sonneblick, 1950; Bownes, 1975; Fullilove and Jacobson, 1976; Turner and Mahowald, 1977; Hartenstein et aL, 1985; Hartenstein and Campos-Ortega, 1985; Zusman and Weischaus, unpublished observation), ablation studies (Lohs-Schardin et al, 1979; Underwood et ah, 1980), and injection studies (Technau and CamposOrtega, 1985). The tergites and sternites scored in adult gynandromorphs were positioned on the map using the known location of their

ZUSMAN AND WIESCHAUS

clearly mosaic for sub. Only 12 of these showed the typical fog cuticle phenotype. The remainder had more heterogeneous defects, generally intermediate between fog and wild type. Most had almost normal heads and smaller holes or scars at their posterior ends (Fig. 4a). We interpret these intermlediate patterns as due to mosaicism in the lethal focus. In contrast to the distribution of sub patches observed in mosaics of normal morphology, the abnormal mosaics were five times more likely to have sub denticles in their posterior ventral belts than in their anterior ones (61~% compared to 12%, respectively, in 54 measured individuals.) Based on the frequency of ring-X loss observed in the control cross, the fog experiment should have produced about 224 mosaics. Since only 95 of these developed normally, the expected number of dead mosaics is about twice the number (61) we actually obtained. The fbg cross also produced, however, 95 additional embryos with more severe heterogeneous defects. The extremely abnormal patterns of the ventral denticle belts in those animals often made the identification of sub patches unreliable (Fig. 4~). Although they were classified as sub+, it is possible that many of them were mosaic, particularly given that embryos with such abnormalities were not detected at significant frequencies in the control cross. Regardless of whether this is the case, our examination of unhatched embryos does indicate that most of the mosaics which die due to fog do not show a typical fog phenotype, at least with respect to the Enal cuticle pattern. Consistent with the observation made on adult gynandromorphs, the yield of morphologically normal tsg mosaics was only slightly less than the value obtained from the nonmutant controls (Table 4). These tsg mosaics showed the same dorsal to ventral bias observed in adult tissue with respect to location of the mutant patches. All had at least some sub+ tsg+ cells in their dorsal side between T2 and A6. Some of the mosaics were totally mutant except for small wild type patches corresponding to about one-fifth of the dsorsal surface.

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The predicted number of tsg mosaics which die is too small to allow a detailed analysis of the distributions of sub and sub’ patches. At least one phenotypically tsg mosaic, however, was unambiguously scored as having a completely sub dorsal cuticle and some sub+ ventral denticles. The existence of dead phenotypically tsg mosaics suggests that, like fw, the lethality associated with tsg adult mosaics is due to abnormalities during embryogenesis. DISCUSSION

The existence of both maternal and zygotic mutations affecting Drosophila gastrulation suggests that the process is under the control of both the maternal and zygotic genomes. The known maternal effect mutations can be grouped into two classes. A larger and rather heterogeneous class seems to affect gastrulation secondarily, with a primary effect on the earlier cellularization, cleavage, nuclear migration, or ploidy (Rice and Garen, 1975; Zalokar et al., 1975). The smaller class is potentially more relevant for studies of gastrulation in that the mutations allow normal cellular blastoderm formation but seem to alter the positional coordinates supplied to the individual cells at that stage (Nusslein-Volhard, 1979). Such mutants alter not only the final differentiative fates of blastoderm cells but also their behavior during gastrulation. Although the nature of the positional information in the Drosophila egg is not understood, the gastrulation effects of such mutants indicate that the altered positional information has already been “read” by the onset of gastrulation and that this information provides the direct or indirect basis for the localized cell shape changes which characterize the beginning stages of gastrulation. A priori one might expect zygotic gene function to be required both to detect positional information and to respond to it in terms of localized differences in cell behavior. Although our experiments clearly indicate the

precursors (= histoblasts) in the larval hypoderm (Szabad et al., 1979). The other adult structures were located by triangulation from the tergites and sternites using the distance measured between adjacent tergites (6 sturts) to convert any given sturt value to the actual distance on the embryonic fate map. The :probosis and humerus have been shifted one-half segment anteriorly, to give them a position on the fate map consistent with their assumed segmental origin (Struhl, 1981). (b) The limits of the fog lethal focus are indicated by a series of circles drawn around the various adult and larval structures (Ts, T?, &, G, TU) shown to be closest to the focus. The radii of the circles are equal to the frequency with which each structure was male in surviving mosaics and presumably represent the number of times the mosaic boundary falls between the structure and the nearest point on the lethal focus (see Results and Discussion for the “domineering” model). This restricts the focus to an area including the PR, PMG, and ventral posterior germband (stippled area) (see Results). (c) Squares indicate adult structures closest to the tsg lethal focus (see Results). Under certain conditions, the limits of the tsg focus can be defined by the frequencies with which a mosaic boundary cuts between an adult structure and the farthest point of the lethal focus (see Results and Discussion for the “submissive” model). In our experiments, however, such frequencies were too large to allow accurate gynandromorph mapping (a36 sturts). Since morphological studies have shown the amnion serosa cells to be abnormal in tsg embryos while the dorsal hypoderm differentiates normally (Zusman and Wieschaus, unpublished results), amnion serosa precursor cells have been stippled to indicate the most likely region needing tsg+ activity. Embryonic and larval structures: P, pole cells; PMG, posterior midgut; TU, tuft; PR, proctodeum; MS, mesoderm; AMG, anterior midgut; St, stomodeum; AS, amnion serosa; Mx, maxillary segment; Md, mandibular segment; L, labial segment; dotted line, cephalic furrow. Adult structures: Oc, ocellar bristles; IHu, humerus; Pr, probosis; SCT, scutellar bristles, L,-> Lt, Legs l-> 3; T,-> T7, tergites l-> 7; S,-> S,, sternites 2%> 6; G, genitalia.

368

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VOL~JME 111. 1985 3

CALCULATEDMALENESSAVERAGESCORESFORLANDMARKSINCONTROLAND tsg ADULTGYNANDROMORPHS Control

Nonmosaics” Mosaics Survival * Lethality” Male meaness average Mosaicism of lethal focusd

Control

cross 1

In(l)w”” Y

x yw Fm7

tsg Cross 1 In(l)uY Y

XYQ Fm7

228 232 (-) (-) 0.45 C-1

272 219 0.79 0.21 0.44 0.48

Maleness average score

Maleness average score

0.46 0.44 0.49 0.48 0.48 0.45 0.46 0.47 0.45 0.45 0.44 0.45 0.44 0.43 0.47 0.44 0.43 0.43 0.41 0.41

0.46 0.46 0.40* 0.50 0.48 0.47 0.49 0.44 0.43 0.42 0.43 0.43 0.42 0.42 0.44 0.44 0.43 0.43 0.40 0.37

cross 3

-.y tsg Dp(1,2)vGSb x In(1)uY Fm7 Y’ CYO Control 801 332 C-1 (-) 0.29 (-)

kg 793 306 0.93 0.07 0.26 0.44

Maleness average score Landmarks Ocellar bristle Probosis Humerus Scotellum Leg 1 Leg 2 Leg 3 Tergite 1 Tergite 2 Tergite 3 Tergite 4 Tergite 5 Tergite 6 Tergite 7 Sternite 2 Sternite 3 Sternite 4 Sternite 5 Sternite 6 Genitalia

Control 0.30 0.32 0.32 0.34 0.31 0.31 0.31 0.29 0.31 0.30 0.29 0.29 0.28 0.27 0.28 0.30 0.28 0.27 0.27 0.25

txl 0.33 0.29 0.22* 0.29* 0.29 0.29 0.28 0.25 0.26* 0.26 0.24* 0.25 0.23* 0.23 0.25 0.24* 0.23* 0.23 0.24 0.24

a Nonmosaics include In(l)w”“/y w (tsg) and y tsg/In(l)w”; Dp(1,2)dSb/+, or y tsg/In(l)uY; CyO/+ flies. * Survival = [(No. tsg mosaics)/(No. control mosaics)] X [(No. control nonmosaics)/(No. tsg nonmosaics)]. ‘Lethality associated with kg in mosaics; l-survival rate of kg mosaics. ‘The frequency with which lethal foci are mosaic in a given experiment was calculated on the assumption that for submissive foci, the fraction of gynandromorphs which die is equal to the frequency with which the lethal focus is totally male (i.e., L = f(a)). Also, the maleness average in the experimental animals at the blastoderm stage should be equal to both the final values observed in the control (X,) and to the frequency of totally mutant foci plus one-half the frequency of mosaic foci (i.e., X, = .f(s) + if(m)) since mosaic foci should be on the average one-half mutant and one-half wild type. * tsg maleness average scores which are significantly less than the corresponding control values. Significance was measured using x’contingency tables (P < 0.05). Since right and left halves of each mosaic were scored independently, the sample size for each landmark is double the number of mosaics obtained. For example in cross 1, the total number of mutant tsg humera is 175 and the x2 value is 7.2.

zygotic nature of the requirement for wild-type fog and products, they do not allow us to distinguish whether the genes are involved in the initial determination or in the immediate differentiation which follows. The phenotypes of the mutations do indicate the importance of zygotic transcription for the normal course of gastrulation. Although the abnormalities of both mutations are most visible at completion of germband extension, they can be traced back to earlier stages. Hemizygous embryos mutant for fog and tsg can be unambiguously t.sg

identified by 10 and 20 min, respectively, after the onset of gastrulation. Our results indicate that the amorphic phenotype depends on the embryo’s own genotype and occurs only in genetically mutant individuals. In addition, it appears to be independent of the number of wildtype genes in the mother. These results argue that the only relevant expression of both fog and tsg occurs after fertilization and has reached sufficient levels to affect cellular behavior by early gastrulation. The most striking feature of the two mutations, how-

ZUSMAN

AND

WIESCHAUS

Localized TABLE

CALCULATED

MALENESS

AVERAGE

In(l)uY Y

Gene

369

Activity

4

IN CONTROL fog, AND

SCORES FOR LANDMARKS

Control

Zygotic

tsg LARVAL GYNANDROMORPHS OF NORMAL MORPHOLOGY

cross

fog Cross

:: y svb Fm7

In(l) ida’ x svb fog Fm7 Y

tsg Cross In(l) Y

:: Y svb tw Fm7

Total progeny Total mosaicsa Normal mosaics* Survival” Lethalityd Abnormal mosaics observed Total abnormal’

977 102 102 (1.0) (0.0) 0 4

2148 95 0.42 0.58 61 156

1008 (105) 101 0.96 0.04 3 12

Landmarks

Maleness average score

Maleness average score

Maleness average score

T, ventral T, dorsal Tz ventral T2 dorsal T3 ventral T3 dorsal A, ventral A, dorsal AZ ventral A2 dorsal A3 ventral A3 dorsal A, ventral A4 dorsal A5 ventral A5 dorsal A6 ventral AC dorsal A7 ventral A7 dorsal A8 ventral A8 dorsal Tuft

0.40 0.47 0.38 0.37 0.40 0.35 0.38 0.36 0.39 0.37 0.37 0.35 0.34 0.33 0.36 0.34 0.35 0.31 0.31 0.29 0.34 0.29 0.42

0.27* 0.43 0.25* 0.27* 0.24* 0.23* 0.23* 0.22* 0.19* 0.18* 0.17* 0.15* 0.16* 0.14* 0.11* 0.10* 0.09* 0.10* 0.07* 0.09* 0.05* 0.06* 0.06*

0.39 0.35* 0.36 0.25* 0.35 0.26* 0.35 0.26* 0.33 0.25* 0.32 0.27 0.31 0.27 0.31 0.27 0.31 0.27 0.30 0.25 0.32 0.23 ND

Mean maleness average

0.36

0.17

0.30

(224)

Abbreviations: Ti-T3, thoracic segments l->3; Al-As, abdominal segments l->8; ND, not determined. ’ Values in parenthesis are estimates based on the frequency of ring loss obtained in the control (41.8%)and the fact that in each cross, only one-quarter of the total progeny are heterozygous for the ring. *In each case, between 18 and 20% of “normal” mosaics had head defects associated with a dominant lethality of In(f)w”“. ’ Survival = number of “normal” mosaics/total number of mosaics expected based on control values. ‘Lethality associated with the mutants in mosaics; l-survival rate of mutant. e Excludes hemizygous embryos totally svb in genotype, as well as those which were poorly differentiated. *.fij,o and ts,y maleness average scores which are significantly less than corresponding control values. Significance was measured using x2 contingency tables.

ever, is their apparent specificity for gastrulation. fog and tsg mosaics survive to adult stages with up to 90% of their surfaces mutant, indicating that the wild-type gene products are probabl:y not required for later morphogenetic events occurring in individual imaginal discs. Given the high correlation in mosaic animals between the genotype of internal and external adult structures (Kankel and Hall, 1976), the survival of such mosaics also indicates that the respective gene products are probably not required for the development of most internal organs. The distribution of mutant patches in-

dicates that fog’ gene function is required at the posterior pole of the embryo and although the interpretation is more ambiguous, tsg+ gene function is probably needed only on the dorsal side of the embryo. One very rigorous test for specificity is provided by the behavior of germline clones homozygous for each mutant. Many randomly chosen lethal mutations cause cell death in homozygous female germ cells or result in the formation of eggs which are unable to support embryonic development (Garcia-Bellido and Robbins, 1983; Perrimon et al., 1984). This observation correlates well

370

DEVELOPMENTAL BIOLOGY

VOLUME 111. 1985

c a

FIG. 4. Abnormal cuticle phenotypes of nonhatching progeny from the cross used to produce larval and embryonicfog gynandromorphs (see text). The embryo in (a) is a sub/sub+ mosaic with the atypical fog phenotype. This embryo has an almost normal head and a posterior hole (large arrow) associated with svb denticles (small arrow) in the sixth and seventh denticle bands (the eighth denticle band is not storable). The mosaic embryo in (b) shows a typicalfog phenotype with two ventral holes (large arrows) at opposite ends of its cuticle. The mosaic border in this embryo runs through ventral denticles A,-> As (ventral denticles T,-> T3 and A,-> As are not storable). svb denticles can be seen in this photograph in denticle bands Aa and A3 (small arrows). Abnormalities other than those associated with the typical& phenotype, such as randomly sized, scattered holes (large arrow) and abnormal patches of denticles can be seen in embryo (c). Although it was scored as svb+, we cannot exclude the possibility that it contains some regions of sub morphology.

with the high synthetic activity of such cells during oogenesis. Loci which have no effect on germline clones are those with relatively specific phenotypes with respect to embryonic pattern (Ultrabithorax, Kerridge and Dura, 1982; Antennapedia, Sex combs reduced, engrailed, Lawrence et al., 1983; Kruppel, Wieschaus et al., 1984). Although we have observed a slight effect of germ line homozygosity for fog on the gastrulation of heterozygous progeny, the transcription occurring during oogenesis is clearly not necessary for embryonic survival. The only source of fog and tsg products normally relevant for survival is the embryonic genome. When that source is eliminated, the embryo dies and shows characteristic

fog and tsg abnormalities regardless of the genotype of the mother. What remains to be done for both mutants is an exact and detailed description of the initial cellular abnormalities which result in the phenotypic defects at gastrulation. The mosaic data presented in this paper are extremely useful in that they define the regions of the embryo to be subject to the most detailed morphological analysis. We are grateful to Dr. Trudi Schupbach, Dr. Peter Gergen, Dr. Douglas Coulter, and Dr. Saul Zackson for their helpful comments on the manuscript, and Karin O’Hara for her assistance in preparation of this manuscript for publication, This work was supported by NIH

ZUSMAN AND WIESCHAUS Grant HD15587 and a Basil O’Connor March of Dimes Research Grant 5-389 to Dr. Eric Wieschaus and by an NSF Graduate Fellowship to Susan Zusman.

Localized

Zygotic

Gene Activity

371

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