The mode of action of “discless” mutations in Drosophila melanogaster

DEVELOPMENTAL

93, 240-256 (1982)

BIOLOGY

The Mode of Action of “Discless” Mutations in Drosophila melanogas ter JANOS SZABAD' AND PETER J. BRYANT’ Develqpmental

Biology

Center and Department

of Developmental

Received March

and Cell Biology,

University

of California,

Irvine,

Califwnia

92717

15, 1982; accepted in revised form May 3, 1982

The development of five different “discless” lethal mutants of Drosophila melanogaster was studied by using whole mounts and histological procedures as well as by radiation-induced mitotic recombination. Rudimentary imaginal discs were found in both newly hatched first-instar and late-third-instar larvae homozygous for each of the mutations. Rather than interfering with the establishment of imaginal discs, the mutations were found to interfere with cell proliferation not only in the imaginal discs but also in the larval central nervous system, the imaginal rings of the salivary gland and foregut, and the gonads. Production of blood cells by the lymph gland was severely reduced in all of the mutants. Autonomous effects on the growth of homozygous clones in a heterozygous background were demonstrated for four of the five mutations. Although some of the mutations had, in general, a more extreme effect on cell proliferation than did others, the effects on different organ systems were not clearly correlated. The mutations also caused a slight reduction in the final cell size in the nonproliferating larval epidermis and histoblasts, but only the most extreme mutation studied caused a reduction in cell number in these tissues. It is concluded that these mutations are not imaginal disc specific, but that they interfere with functions required for cell proliferation in general. Several possible explanations for their lack of effects on embryonic cell proliferation are discussed. INTRODUCTION

There are two major developmental pathways established during the embryogenesis of the higher dipterans, one for the larval and one for the imaginal tissues. Cells of the larval tissues do not divide during larval life but rather grow in size and develop large polytene nuclei. On the other hand the presumptive imaginal cells remain diploid and proliferate in the imaginal discs and other localized cell groups during larval and early pupal life. The growing imaginal cells presumably depend on the larval tissues for a supply of essential nutrients and hormones, but the larval cells seem to be totally independent of the imaginal tissues as shown by the normal larval development of “discless” mutants (Shearn et al., 1971; Shearn and Garen, 1974). The way in which the larval and imaginal tissues are segregated from one another in the embryo is not clear. However, there are indications that the epidermis of the larva and of the presumptive adult share common precursor cells during early embryogenesis (Szabad et al., 1979) and the two lineages are probably distinct by the time the imaginal discs are histologically recognizable in the newly hatched larva (Auerbach, 1936). The segregation between larval and presumptive i Present address: Institute of Genetics, Biological Post Office Box 521, H-6701 Szeged, Hungary. * To whom correspondence should be addressed. 0012-1606/82/090240-17$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

Research Center,

240

adult cell lineages is presumably under the control of zygotic genes, and we have begun a study to try to identify these genetic functions. The discless lethal mutations are candidates for defects in these functions and are the subject of the present report. The discless phenotype could in principle be produced by mutations in hypothetical genes which are involved in the segregation of the two pathways, so that larval cells but no imaginal cells would be produced. However, other defects could also bring about the discless phenotype. For example, presumptive imaginal cells might fail to proliferate either as a result of a disc-autonomous defect or because of a defective larval milieu. Alternatively, presumptive imaginal cells might be formed but later degenerate. Several discless mutants have already been isolated (Shearn et al., 1971; Shearn and Garen, 1974; Kiss et al., 1976a) and Shearn and Garen (1974) have estimated the number of loci which can mutate to produce either discless or “small disc” phenotypes to be about 1000, or 20% of the genome. In the present study we have investigated five of the discless mutants with the goal of determining in each case whether all or only some of the imaginal cells are affected, whether imaginal cells form during embryogenesis, and whether they proliferate. The results indicate that these five mutations act by inhibiting proliferation of imaginal cells, rather than by interfering with the establishment of the developmental pathway leading to imaginal structures.

SZABADAND MATERIALS

AND

BRYANT

METHODS

Mutant stocks. The following recessive late larval or early pupal lethal discless (dl) mutations were studied: l(l)dl-1 (l-35) (Kiss et al., 1976b), Z(3)eZO(3-25.9 + 3.2), 1(3)g30 (3-50.3 + 0.5) l(3)ky (3-52.1 t 1.6), 1(3)12m 137 (3-106). In the original description of the discless mutations (Shearn and Garen, 1974), e20 and g30 were listed as Al and A3, respectively. The third chromosome mutants are from the collection of Dr. Allen Shearn, who also provided their unpublished map positions. The third chromosomes carried, in addition to the discless lethals, the following recessive marker mutations: mwh (all but ml.%“), red (e20 and m137), e (all stocks). They were kept over a y+, TM3 Sb Ser balancer chromosome and the first chromosomes in these stocks were marked with y. The chromosome carrying dl-1 was marked with y, w, and sn’, and kept over the balancer chromosome Binsn. For detailed descriptions of the marker mutations and the balancer chromosomes, see Lindsley and Grell (1968). Larvae homozygous for each dl mutation were collected from the balanced stock; they all had yellow mouth parts and ventral setae (due to the presence of y and absence of y’), and in addition white (in case of dl-1) or red (for e20 and m137) malpighian tubules. The cultures were raised on standard Drosophila cornmeal-molasses medium supplemented with live yeast, and all the experiments were carried out at 25°C. Histology. Histological analyses were performed in order to search for disc tissue in homo- or hemizygous dl larvae. Third instars were fixed by injecting Kahle’s fixative into the larvae after which they were cut open and processed for histological analysis according to the method of Madhavan and Schneiderman (1977). Fourmicrometer serial sections (both transverse and longitudinal) in paraffin of at least 10 larvae from each dl mutant were cu? stained with Feulgen and Fast Green, and analyzed in -base contrast (250-1000~). Newly hatched first-instar .~rvae were also sectioned in order to determine whether i, Taginal discs had formed during embryogenesis; in these cases homo- or hemizygous dl larvae were identified by their y mouth parts. Such larvae should represent the conditions established by the 12th hr of embryogenesis because no cell proliferation takes place between this time and the time of hatching of the larva at 22-24 hr after fertilization (Madhavan and Schneiderman, 1977). Young first instars, at least three of each dl mutant, and a few third-instar larvae were fixed by injecting 4% gluteraldehyde, and embedded in plastic. Serial 2-pm sections (both transverse and longitudinal) were cut (Campbell, 1981), stained with Azure II-Methylene blue, and analyzed in phase contrast (625-1560X). Whole mounts and cell counts. A search for rudimen-

“Discless” Mutations

241

tary imaginal discs in whole mounts of late-third-instar larvae was carried out using fluorescence labeling of nuclei and immunofluorescence labeling of cells of the disc epithelium. dl larvae were injected with fixative (2% formaldehyde in phosphate buffer, pH = 6.9-7.1) and cut open along the dorsal midline. The internal organs were then removed without destroying the more anterior regions. The larval epidermal preparations were washed, incubated in 1O-4 M Hoechst-33258 (dissolved in 0.7% NaCl solution) for a few minutes, and then incubated with the D.A.lB6 antibody specific for diploid epithelia (Brower et al., 1980) followed by treatment with fluorescein-conjugated rabbit anti-mouse IgG (Miles). The preparations were mounted in 0.7% NaCl solution and inspected under a Zeiss fluorescence microscope. The larval epidermis, as a representative of the larval tissues, was fixed by injecting Kahle’s fixative into larvae ready to pupariate (spiracles everted); the larvae were then cut open along the dorsal midline. After removal of the internal organs the epidermis was stained with Feulgen, dehydrated, and mounted in Euparal. The number of larval epidermal cells and histoblasts was determined in the first four abdominal segments, which contain roughly equal numbers of cells. The number of larval epidermal cells per segment was estimated by comparing the number of nuclei in three to five areas (3400-9600 wrn’, free of histoblasts) per segment to the total area of the segment which was determined by planimetry of camera lucida drawings. The surface area of the cuticle of late-third-instar larvae was also measured by planimetry of camera lucida drawings of such larvae prepared by the method of Szabad (1978). The imaginal rings of the salivary glands were studied in whole mounts under phase contrast, and those of the foregut were studied on sections of third-instar larvae. The formation centers of the adult optic lobes were analyzed on sections of the central nervous system (CNS) of third-instar larvae (Gateff, 1978; White and Kankel, 1978). The number of cells in the larval CNS was determined using a hemacytometer after cell dissociation in 0.35 M citric acid (Martin, 1982). The lymph glands were analyzed on sections of third-instar larvae, and the concentration of blood cells was determined as follows. A larva was opened in a drop of immersion oil, and the volume of the resulting hemolymph sphere was calculated after measuring its diameter. The hemolymph sphere was then brought into contact with the microscope slide by means of a tungsten needle, whereupon a layer of hemolymph formed. After removal of the excess oil, the number of blood cells was counted in phase contrast with no coverslip. Gonads. Gonads were dissected from late-third-instar larvae and their diameters were measured. The function

242

DEVELOPMENTAL BIOLOGY

of the gonads was tested by transplanting them into host larvae. Ovaries were transplanted into larvae homozygous for the marker mutations fs(l)KlO (Wieschaus et al., 1978) and w. The w mutation results in white malpighian tubules thus allowing for selection of homozygous fs(l)KlO host larvae from a balanced stock. The fs(l)KlO homozygous females produce abnormally shaped eggs, while eggs derived from the implanted dl ovaries, if they are produced, should have normal shape. The ovaries become attached to the oviducts after pupariation, and when the implanted ovary as well as at least one of the host ovaries becomes attached the female lays two types of eggs. Eggs can mature in the implanted ovary whether or not it becomes attached to the oviducts. Eggs were collected from host females for at least 7 days, after which the females were dissected open and their abdomens were inspected for functioning ovaries. Testes of dl larvae were implanted into w hemizygous larvae (except in the case of dl-1, where the dl chromosome carried w, and the hosts were w+). Males hemizygous for w have colorless testes sheaths in contrast to the yellow sheath of the w+ males. Each host adult male was mated with 8-10 females and the progeny were screened to determine whether any of them could have been derived from sperm from the dl testis. Host males were dissected and the abdomens inspected for testes and the color of the testis sheath. The affects of dl mutaEflects on cell proliferation. tions on cell proliferation were studied either in squashes of the larval CNS following colchicine incubation (Gatti et al., 1974) or in clones homozygous for dl mutations. dl homozygous clones were generated by induction of mitotic recombination using gamma rays (13?Cs, 0.622 MeV, 1170 rpm). In one experiment (Fig. la) dl-1 homozygous clones were induced by X-irradiation (150 kV, 0.5 mm Al filter, 1000 rpm). dl homozygous clones were identified by flanking cell marker mutations as shown in Fig. 1. A full description of most of the marker mutations may be found in Lindsley and Grell (1968), but forJ@ see Garcia-Bellido and Dapena (1974). Bsb is a dominant marker mutation which removes most of the macrochaetes and shortens the microchaetes (Baker, 1980). Flies eclosing from the irradiated larvae were prepared for microscopic analysis according to Szabad (1978) and screened under the compound microscope. The type, size, and location of the clones were recorded. Clones consisting of a single bristle were omitted from calculations of clone size since many of these are thought to represent clones suffering mitotic inhibition caused by the irradiation (Haynie and Bryant, 1977). However, this procedure could not be applied with Ki+ clones, for reasons discussed later. Clone sizes are expressed in terms of clone size class, which is the number

VOLUME 93, 1982 FIRST y I

a. b.

, y,

AND

sn3dl-1 I I

Y C.

CHROMOSOMES

0

,

,

0 f360

,

1

mwh I I

1

s,n3d,l-1

o

rnwhTmo Y

THIRD

I 1 mwh

f 0

mwh ,I

0

I

, e20 r

1 1

I

flr3

Y

d.

mwh t 1

)

930

k43 f

I

I Ki

Y e.

0

I

0

‘

ml37 1 I Pr Bsb

FIG. 1. Location of five dl and eight cell marker mutations on the meiotic map, showing the genotypes used in the mitotic recombination experiments. ml37 and Bsb both map to the tip of the right arm of the third chromosome. (a) For production of y sn’dl-l//fs6” twin spots. (b) For production of mwh sn’ dl-l//f twin spots. (c) For production of mwh e%V/flr? twin spots, with y clones as internal control. (d) For production of g.30 Ki’ or k&’ Ki+ clones, with y and mwh clones as internal control. (e) For production of ml.37 Pr’ Bsb+ clones, with y clones as internal control.

of cell divisions needed to reach the observed size, starting from the single progenitor cell. Growth of wild-type implants. We also tested whether dl mutant larvae could support the growth of wild-type imaginal discs. Wing discs of freshly ecdysed wild-type third-instar larvae were removed and implanted into early- to mid-third-instar dl larvae. The implants were removed after a 3-day culture period and their size was measured by preparing camera lucida drawings and determining the surface area of the implants from these drawings. Some of the recovered implants were dissociated in 0.35 M citric acid and the number of cells was determined using a hemacytometer (Martin, 1982). RESIJLTS

Growth of dl larvae. The term “discless” mutation (dl) indicates that no imaginal discs can be identified in third-instar larvae upon dissection under the dissecting microscope (Shearn et al., 1971). In a preliminary screen of 11 dl mutations, 5 fulfilled this criterion; no discs could be detected in the homozygous mutant larvae after dissection. During the course of our studies we found no evidence for any variation in expressivity, or for incomplete penetrance of any of these mutations. dl homozygous larvae showed good viability and most of them developed to the third larval instar. Except in the case of dl-1, the majority of the dl larvae pupariated (Table l), but there was a 2- to S-day delay, as compared to dl heterozygous sibs, in the time of pupariation. In most cases the puparia had a normal appearance. The

SZABADAND BRYANT

“Dixless”

Mutations

243

pupal molt was complete over the abdomen and the pupal cuticle could usually be pulled out as a bag-like structure from the pupal case with the debris of the histolyzing larval tissue inside. In no case could we identify metamorphosed adult cuticle over the head, thorax, or abdomen. dl-1 larvae reached and terminated development at early- to mid-third-instar and never pupariated. They stopped feeding shortly before their heterozygous sibs pupariated. dl larvae (except those homozygous for m137) were generally smaller than their heterozygous sibs as indicated by the surface areas of late-third-instar larvae (Table 1). The reduced surface area of the larvae could be attributed partly to the presence of fewer epidermal cells in dl-1 larvae, but segments of eZ0, ~30, k43, and ml37 larvae contained numbers of larval epidermal cells which were not significantly different from those of controls (P > 0.05, t test). The larval epidermal nuclei were smaller than those of controls, their volumes amounting to about 30% (g30), 40% (dl-1, m137), or 60% (e20, K43) that of the controls (Table 1). This was associated with reduced larval size in all of the mutants except m1.37, where the larvae grew to normal size in spite of the reduced nuclear size of the larval epidermal cells (Table 1). Imaginal discs. Primordia of almost all of the major imaginal discs could be identified in sections of newly hatched first-instar larvae homozygous for each dl mutation. The one exception was dl-1 in which we were unable to find any wing or haltere discs. The discs were located in the positions described by Auerbach (1936) and Madhavan and Schneiderman (1977) for wild-type larvae, and the morphology of newly hatched dl larvae was not noticeably different from that of wild type. Leg disc primordia were seen as thickenings of the larval epidermis slightly posterior to the Keilin’s organs. Their size and shape were similar to those of wild type (Fig. 2), Small groups of cells corresponding to all of the major imaginal discs could also be identified in late-third-instar larvae homozygous for each of the dl mutations. They were slightly larger than those seen in the first instar, except in the case of dl-1 where there had been no apparent growth. The leg disc primordia were still very close to the larval epidermis, about 16-20 pm behind the Keilin’s organs (Fig. 3), and apparently attached to the neural processes which connect the larval epidermis with the ventral ganglion. In none of the disc primordia of dl larvae did we observe a lumen, and there was no indication of cell death at the stages studied. As with the first-instar larvae, we were unable to identify wing or haltere discs in dl-1. Rudimentary imaginal discs were also identified in third-instar larval epidermal preparations by exam-

244

DEVELOPMENTAL

BIOLOGY

ining the DNA-specific fluorescence after incubation with Hoechst 33258, and by examining the binding pattern for the diploid epithelium-specific monoclonal antibody D.A.lB6 (Brower et al., 1980). All of the leg discs were identified as small groups of cells with small, presumably diploid nuclei showing Hoechst fluorescence (Fig. 4a). The number of cells in each disc was (except in the case of m137) in the range reported by Madhavan and Schneiderman (1977) for discs of newly hatched wild-type larvae. The dl cells also bound the D.A.lB6 antibody (Fig. 4b) indicating that at least the rudimentary leg discs express an antigen which is characteristic of diploid epithelial cells. Antibody-binding capacity was strong for ml37, moderate for e.20, 9.30, and k&3, and very weak for dl-1. Of the dl mutations studied the late third instars of ml37 larvae contained the largest disc primordia, some of them containing as many as 200 cells, while the leg discs of dl-1 larvae each contained about 12 cells. A wild-type wing disc at this stage contains about 40,000 cells (Martin, 1982). Histoblasts. Histoblasts, which are progenitor cells of the adult abdomen, were present in all of the dl mutants. The histoblast nests were surrounded by larval epidermal cells in the abdominal segments at the locations described earlier for wild type (Szabad et al., 1979). The histoblast nests of dl larvae contained numbers of cells very similar to those of the heterozygous sibs, with the exception of dl-1 larvae where only 57% of the histoblast cells were present (Table 1). Histoblast nuclei

FIG. 2. Transverse 2-pm section of a newly eclosed OreRlarva at the level of the Keilin’s organs of the mesothoracic segment. E, epidermis; K, Keilin’s organ; L, leg disc; M, muscle.

VOL~JME 93, 1982

FIG. 3. Transverse 2-pm section of a third-instar c&l larva in the metathoracic segment. E, epidermis; L, leg disc; M, muscle.

were smaller than those of controls for all of the dl mutants, but they reached a final size which bore a fairly constant relationship to the final size of larval epidermal nuclei (Table 1). Imaginal rings. The diploid cells of the imaginal ring of the salivary glands were missing in the dl-1 larvae as studied on whole mounts in phase contrast (Fig. 5). There were a few imaginal cells at the expected positions in g30 and k.&’ larvae, while this imaginal ring seemed to be unaffected in late-third-instar larvae homozygous for e20 or m137. These results were confirmed when the fluorescence patterns of Hoechst 33258-stained preparations were analyzed, except that two to four apparently diploid nuclei were seen in each pair of dl1 salivary glands. Except for dl-1, the salivary glands of all of the dl mutants showed binding of the diploid epithelium-specific antibody in the region of the imaginal ring. Binding approached control levels in ezo and ml37 salivary glands, but was weaker in g30 and k43. The number of cells in the imaginal ring of the foregut of late-third-instar larvae was normal in m137, reduced in e20 and g30, and we were unable to detect any such cells in dl-1 and k&?, using sectioned material (Table 2).

SZABAD AND BRYANT

“Discless”

245

Mututions

FIG. 4. Fluorescence of a rudimentary mesothoracic leg disc of a late-third-instar rrc197 homozygous Hoechst-33258 (a) and with the fluorescein-labeled diploid epithelium-specific antibody D.A.lBG (b).

Lymph glands. The size of the lymph glands, which are known to produce blood cells in Drosophila (Srdic and Reinhardt, 1980), was reduced in dl-1 and e20 but

FIG. 5. Imaginal ring of the salivary cells; D, diploid cells; S, salivary duct.

gland of (a) wild-type

larva after

staining

the nuclei with

these structures were fairly normal in g30, k43, and ml37 larvae (Table 2). Even though the lymph glands appeared normal in size they may not have functioned

and (b) l(l)dl-1

homozygous

larva. Whole mounts, phase contrast,

P, polytene

246

DEVELOPMENTAL

EFFECTS

OF dl MUTATIONS

Imaginal

dl mutation Control

dl-1 e20

cl30 k.&’ ml37

* Significantly

different

ON IMAGINAL

BIOLOGY

RINGS,

VOLUME

TABLE 2 LYMPH GLANDS,

93. 1982

AND ON THE NUMBER

OF BLOOD CELLS

ring of

Salivary gland

Foregut

Size of lymph glands

Normal Missing Normal Reduced Reduced Normal

Normal Missing Reduced Reduced Missing Normal

Normal Reduced Reduced Normal Normal Normal

from controls;

Cells in 1 ml hemolymph (X104) mean f SD (n = 8) 191 5.6 31.5 30.7 34.2 7.0

Number of blood cells as percentage of control

i: 58 + 3.7* +- 22.1* +- l&5* + 9.7* +- 4.0*

100 3 17 16 18

4

P < 0.01.

properly, since the number of cells in the hemolymph was drastically reduced in all of the mutants (Table 2). The number of blood cells was 16-B% that of heterozygous sibs for e20, g30, and k&f? and only 3-4% in dl-1 and m137. This feature of the dl mutations was especially striking for m137, because larvae of this genotype grew to full size, contained at least the normal amount of hemolymph, and had seemingly normal lymph glands, but we were unable to find any blood cells in about half of the 6- to 7-day-old mutant larvae. Gonads. Gonads of late-third-instar dl larvae were in every case smaller than those of the heterozygous sibs (P < 0.05, t test), their volumes amounting to 3% (g30, m137), to almost 70% that of the control (e20, k&3; Table 3). The ovaries were always smaller than the testes thus allowing sexing of the dl larvae in the functional tests described below. In order to test gonad function, the larval dl gonads were implanted into larval hosts which were then allowed to metamorphose. When ovaries of the late-third-instar dl larvae were transplanted into

w homozygous larval hosts, all of the resulting adult females laid only KlO eggs; when dissected, none of them carried a third, even rudimentary, ovary in their abdomens (Table 3). Instead, a residual brownish body was recovered, indicating that ovaries of dl larvae degenerate and die because the dl mutations interfere with ovarian development. In the control experiment each of the 11 host females carried an extra ovary in its abdomen, with several mature oocytes, derived from a dl heterozygous donor larva. Four of these control females laid two types of eggs: those with the KlO phenotype originating from the host; those with the nonKlO phenotype, from the donor ovary. There was no indication that the transplanted testis survived in the case of dl-1,930, or m137, which are the dl mutations in which the testes remain very small (Table 3). However, when e20 and k43 larval testes were transplanted into w hemizygous host larvae, patches of yellow testis sheath differentiated in the host males (in 4 and 3 cases out of 10 and 8 host males, respectively;

fs(l)KlO

TABLE EFFECTS

3

OF dl MUTATIONS

ON THE GONADS

Testes Ovaries

dl mutation

Diameter” (4

Control

85 k 11

e.20 k&’ ml37

Development in wild-type host

n

12

3.2

Yes

10

8 6 5 5

2.0 0.2 2.2 0.3

No No No No

10

-d

dl-1 NO

n

Volume* (pm3 X 105)

72 32 75 40

+ lO* f 8** f 10 k 6**

a For fully grown larvae, mean * Calculated as sphere. ’ Calculated as oblate spheroid. d No female larva available. * Significantly different from ** Significantly different from

-

-

and SD. Average controls, controls,

diameter

ratio

0.05 > P > 0.01. P < 0.01.

= 1.32.

4 8 11

Larger diameter” 6.4 280 135 235 93

+ f i +

22 17** 15** 13**

n 10 6 9 5

165 -+ 17**

11

91 * 14**

5

Volume” (gm3 X 105) 87.1 9.8 51.5 3.2 17.8 3.0

Development in wild-type host Yes No Partial No Testis sheath No

n 6 13 4 11

10 10

We estimated the number of cells in the larval CNS of dl mutants after dissociation in citric acid, and we also screened for mitotic figures in orcein-stained squashes of the CNS. With the exception of late-thirdinstar larvae homozygous for the mutation m13?‘, all of the dl larval CNSs contained fewer cells than did the dl heterozygous controls (Table 4). The low cell numbers are presumably related to the fact that very few if any of the cells of the larval CNS divide in the dl larvae (Table 4). Instead, abnormal chromatin condensation was observed in a large fraction of the nuclei of the dl CNS. The majority of dl-1 CNS nuclei contained three to four heavily stained nucleoli and densely stained chromatin granules were often observed. The diameter of the nuclei was 2.5-5 wrn both for the control and the dl larvae. No chromosomes were seen in squashes of 29 dl-1 larval CNS. There were a few mitotic figures in the CNS of young e.20homozygous larvae, but in the case of late-third instars only eight mitotic figures (in two of the 5 squashes) were seen, a result which is similar to one obtained by B. Baker (personal communication). The number and morphology of the chromosomes were normal in the case of e.20. Seven mitotic figures were observed in 20 CNS squashes of 930 homozygous larvae and in many of the chro-

Table 3). In 1 case an uncoiled e20 adult testis was recovered with spermatogonial cells and sperm inside. In the control a third testis with a yellow sheath was recovered in each of the 6 host males (Table 3). These testes were derived from dl heterozygous donor larvae. One of them was attached to the ejaculatory duct and yielded sperm as shown by the results of a test cross. The fact that gonads from the dl mutants usually failed to function even when transplanted into wildtype hosts might be interpreted as showing a detrimental effect of the mutant larval environment on gonad development in the larval stage, before transplantation. However, in view of the cell-autonomous effects of these mutations on cell proliferation in imaginal discs and histoblasts (see later) we consider it more likely that the gonad abnormalities are also a result of cell-autonomous effects on proliferation. Central nervous system. The formation centers of the optic lobes were in general much smaller and contained fewer cells in dl larvae as compared with the control larval brains, but a few large neuroblasts could still be identified. We found no evidence for cell death in sections of the CNS of either newly hatched or late-thirdinstar larvae. Nuclear sizes in the CNS appeared to be normal. TABLE THENUMBEROFCELLSANDMITOTICFIGURES(MEAN

247

“LXsc1es.s” Mututifnls

SZABADAND BRYANT

4

ANDSD)INTHECENTRALNER~OUSSYSTEMSOF~~LARVAE? Days after oviposition

dl mutation

Feature

4

n

2.7 i- 0.1 127 f 54 0.47

2 6

Cells (X104) +SD Mitotic figures tSD % cells dividing

1.7 k 0.2 0** 0

2 11

eL0

Cells (X10”) *SD Mitotic figures *SD % cells dividing

1.9 F 0.2** 25.3 c 7.0** 0.13

4 6

s30

Cells (X104) &SD Mitotic figures &SD YC cells dividing

2.3 -c 0.3 0.6 1 l.O** 0.003

3 10

kW

Cells (X10’) &SD Mitotic figures *SD R cells dividing

1.8 + 0.2* 0** 0

3 8

ml27

Cells (X104) +SD Mitotic figures *SD % cells dividing

-

Control

Cells (X104) *SD Mitotic figures +-SD % cells dividing

dl-1

” Data concerning mutant larvae on Days 5 and 7-8 were compared to controls of the mutants. * Significantly different from controls; 0.05 > P > 0.01. ** Significantly different from controls; P < 0.01.

16.0 k 1.3 600 k 240 0.38 1.0 * 0** 0

0.3*

2.2 * 0.7 0.3 t 0.7** 0.001

17,

7-8

n

5

7 7 2 11

1.3 t 0** 0

0.2**

5 7

3.1 -+ 0.4** 1.6 f 2.6** 0.005

3 5

1.6 rf- 0.2** 0.5 f 0.6** 0.003

3 4

2 10

1.8 1?I 0.3 0** 0

2 7

4.4 ri- 0.3 35.6 +- 27.9** 0.08

2 7

on Days 4 and 5, respectively,

15 230

k 2.3 f loo* 0.15

due to the delay in pupariation

3 4

248

DEVELOPMENTALBIOLOGY

mosomes the sister chromatids were closely attached along the proximal heterochromatin. Altogether three mitotic figures were detected in 19 CNS squashes from kJ+3homozygous larvae. The chromosomes were unusually short and granulated. In three nuclei one or two chromosomes were visible while the rest of the chromatin was highly condensed and granular. There were no abnormalities detected in the nuclei or mitotic figures of ml.37 homozygous larvae, and in mid-third-instar larvae 0.08%, while in late-third instars 0.15% of the nuclei were arrested in mitosis. It may be relevant to note that ml37 homozygous larvae contain very little fat body up to the end of the third larval instar, but then it increases to almost the normal level in many of the larvae. Ability of dl larvae to suppvrt imaginal disc growth. The function of the larval milieu of the dl mutants was tested by culturing wild-type wing discs in dl larvae for 3 days. The implanted discs grew in every case although to differing extents in different dl hosts (Table 5), and the typical folding pattern developed. The implanted disc evaginated in some of the larvae of all of the dl mutations studied. The growth of the implanted discs was due to an increase in the number of cells and not due to enlargement or flattening, since the recovered and dissociated implants contained many more cells as compared to those which were dissociated without culture (Table 5). Clonal analysis of dl mutations. In this study we analyzed the ability of dl homozygous cells to proliferate and metamorphose in both imaginal discs and histoblasts. dl homozygous cells were generated in heterozygous larvae by mitotic recombination induced by either gamma or X rays. E(l)dl-1. When y sn’ dl-l/fS6” larvae were irradiated (Fig. la) no y sn3//fj6” twin and very few y sn’ single TABLE 5 THE

SIZE AND THE NUMBER OF CELLS (MEAN f SD) IN WILD-TYPE WING DISCS AFTER 3 DAYS CULTURE IN dl HOST LARVAE

Host Control dl-1

e20 Q30 k4s ml37 Noncultured wing disc

Surface area of implant after culture” (pm’ X 10”) 7.9 + 3.5 + 6.3 f 20.4 k 9.1 f 4.6 k

3.1* 0.6’ 1.4* 3.5* 3.1* 1.2*

2.0 * 0.4

n

Number of cells

3 3 5 3 5 3

9.7 f 0.7* 14.9 f 3.0* 25.7 20.8 f 1.6*

6

3.0 f 0.1

a Evaginated discs were not included. *P < 0.01 as compared with noncultured wing discs (t test).

VOLUME93, 1982

spots were found on the head and thorax of the adults while in the controls without dl-1 most of the clones were either y sn3//f36” twin or y sn’ singles (Table 6a). Many fs6a single clones developed in the experimental series but the total frequency of f”“” clones (including those in twin spots) was not significantly different from that observed in the control (P > 0.05, x2 test). Presumseries ably, the f36a single spots in the experimental originated from twin spots in which the y sn’ dl-1 partner did not develop. This result, therefore, shows that dl-1 homozygosity interferes with the ability of disc cells to grow and form storable regions of the head and thorax. In contrast to the head and thorax, about 20% of the expected number of twin spots (based on the control frequencies) developed on the adult abdomen. The y sn’ dl-1 clones were significantly smaller than the f36atwins (P < 0.01, t test) and in 92% of the cases consisted of only one or two bristles (Table 6a). In the above experiment the clones could be scored only if they included bristle cells. In order to examine the effect of dl-1 on nonbristle cells, we induced mwh sn3 dl-l//f twin spots (Fig. lb) and screened wing blades for clones. The results of this experiment, where the genotype of each cell could be determined, are shown in Fig. 6 and Table 6b. In the control series about half the clones were mwh sn'//f twin spots and more than 90% of the singles were mwh. When dl-1 was included in the genotype and clones were induced at the beginning of the third larval instar (70-72 hr after egg laying) very few m,wh sn’ dl-l//f twin but many f single spots were recovered. In the seven twin spots five of the mwh sn’ dl-1 clones consisted of only one or two cells while the f twins were much larger. This result suggests that indeed most of the dl-1 homozygous clones either do not develop or remain very small in the developing wing blade. However, when clones were induced around the middle of the third instar (94-98 hr after egg laying) mwh sn'//f twin and mwh single clones developed with very similar frequencies in both the control and the experimental series, and there was no significant difference between the size distributions of mwh clones in the control and the experimental series (P > 0.05, x2 test; Table 6b). Several of the mwh sn’ dl-1 clones 12 consisted of eight cells implying that dl-1 homozygous wing disc cells can divide as many as three times after induction of mitotic recombination and are still able to 5 metamorphose. dl-1 homozygous clones larger than 5 eight cells apparently are unable to differentiate, and perhaps even die. We found no abnormal clones adja1 2 cent to the f clones when the mwh twin would have been homozygous for dl-1 suggesting that the cells of 5 such clones are actually lost. l(3)eZO. Mitotic recombination was induced in y/ +;mwh e20/f lr3 larvae. Since e20 was in the cis position

“Discless” 1 .

khtatim

249

with the cell marker mutation mwh, mitotic recombination between e20 and centromere could result in the formation of mwh e20 homozygous cells (Fig. le), but recombination between e20 and mwh could also provide clones homozygous for mwh but not for e20. However, only proximal recombination results in fk” homozygosity (Fig. IC), so that mwh clones can be assumed to be homozygous for e20 when they are in twin with fir” (Fig. 7). The characteristics of such clones on the wing are shown in Table 7a. The frequency of mwh clones was not significantly different from the control in any of the series (P > 0.05, x2 test) suggesting that most if not all of the mwh. e.20 homozygous clones develop and differentiate on the adult wing. Unfortunately the site of recombination could not be determined for the majority of the clones in the 70- to 74- and 11% to 122-hr series because fLrS is an incompletely expressed cell marker; it is usually not expressed in wing clones smaller than four cells per clone. Also none of the 150 fir” clones we observed included more than 16 cells while several mwh clones with more than 100 cells were identified. In the experimental series there was a tendency for single mwh clones to be larger than *mwh clones in twin spots, suggesting that e20 might retard the growth of clones which are homozygous for the mutation. However, the effect was seen only at the earliest stage of irradiation and the level of statistical significance was not great. With irradiation at later stages the wbwh e20 clones grew as large as the control clones, indicating that e20 homozygous cells can go through six or perhaps even more rounds of cell division before they differentiate. Clones of homozygous e20 cells on the abdomen were indistinguishable in frequency and size from the controls (P > 0.05, x2 and t test, respectively; Table 7b), showing that e20 does not interfere with cell viability in abdominal clones. e20 homozygosity was not accompanied by abnormal trichome or bristle morphology on either the wing blade or the abdomen. 1(3)g30. g30 maps to the proximal part of the right arm of the third chromosome (A. Shearn, personal communication) so Ki, which is nearer to the centromere than 930 (Fig. Id); (Garcia-Bellido, 1972), was used as a marker mutation to identify 930 homozygous clones. After mitotic recombination in the proximal heteroand Ki+ g3O/Ki+ g30 sister homozychromatin Ki/Ki gous cells are formed. The progeny of the former produce short bent bristles as do Ki heterozygous cells, while descendants of the g3O/g30 cells produce normal bristles (Fig. 8). Ki+ clones formed with much lower frequency than y clones which were introduced as an internal control (Table 8). This can be understood because Ki is close to the centromere and mitotic recombination would be expected to occur only rarely prox-

DEVELOPMENTALBIOLOGY

250

TABLE

VOL~JME 93, 1982

6b

CLONALANALYSISOFdl-1: DISTRIBUTIONOF mwh ANDf CLONESAMONGSIZE CLASSESAFTERIRRADIATION OFLARVAE WITH 1.5 krad OFGAMMA RAYS” Age at irradiation (hr AEL) 70-74

Genotype

Number of wings scored

Number of clones

6

61

Control

dl-I

Clones in size classes

47

21

Phenotype of clones

Control

2

148

183

mwh/mwh.

6

7

8

Total

1 2

2 3

2 2

10 5

3 9

7 8

4 1

32

mwh f mwh f

5

2

0

0

1 0

3 1

5 0

9 1

2 0

0 0

1 0

4 0

0 0

1 1

1 2

0 2

0

0

mwh

2 0

3 0

0

1

0

2

2

1

11

f

0

1

4

9

12

3

29

mwh f

6 8

14 24

32 25

15 12

2 0

0 0

0 0

0 0

69

mwh

13 2 3 8

33 2 19 9

18 2 31 30

8 0 25 21

1

0

0

0

0 2 11

0 0 0

0 0 1

0 0 0

80

11 0

12 0

38 2

30 1

6 2

0 0

0 1

0 0

97 6

Twin

Twin

Twin

mwh

f

mwh

f yx

5

Single

Single a Genotypes: control, y sn’/mwh+,

4

3 2

f 6

3

mu?h f

Single dl-r

2

Twin

Single 94-98

1

1

1

27

2 7

73 6

dl-1, y sn’ d&l/m wh’, yL mwh/mwh

imal to this marker (Becker, 1974). The frequency of and experimental series (P > 0.05, x2 test) in the frebetween the con- quency of Ki’ clones in the head and thorax, but there trol and the experimental series when heads and tho- were slightly fewer abdominal Ki+ clones in the experimental series than in the control. The size of the k&3 races were scored, indicating that 930 homozygosity does not interfere with survival and metamorphosis of abdominal clones was significantly smaller than that 930 homozygous cells. The Ki/Ki’ system does not, how- of the Kit control clones (P < 0.01, t test), indicating ever, make it possible to determine how large the g30/ that the dl mutation k.43 does not permit full development of the clones over the abdomen. k.43 homozygosity 930 clones could have grown because (1) only bristles were genetically marked and they represent only a did not result in abnormal bristle or trichome phenosmall fraction of the marked cells; (2) even in the con- types. 1(3)12m-137. ml37 is located at the tip of the right trols, Ki’ clones were always significantly smaller on the head and thorax than the y clones (P < 0.05, t test) which were produced by X-chromosome mitotic recombination as an internal control. They were also smaller than y sd, f96a(Table 6), and Pr’ Bsb+ (Table 10) clones. On the abdomen, Ki+ clones grew as large as those of other genotypes (y, fYGa,Pr+ Bsb’) so that we were able to study clone size as well as frequency. Slightly, but not significantly (P > 0.05, x2 test) fewer 930 clones were formed on the abdomens, and their size was significantly smaller than those in the control (P < 0.05, t test). Therefore, the dl mutation 930 does have a slight effect on the development of homozygous clones. 930 homozygosity did not result in abnormal bristle or trichome phenotypes. 1(3)k43. k43 is located slightly distal on the third chromosome to Ki and 930 so that the genetic system used for mitotic recombination (Fig. Id) was similar to that used for 930. The characteristics of k&‘clones are shown FIG. 6. mwh sn’//f twin spot on the wing blade in Table 9. There was no difference between the control Kit clones did not differ significantly

SZABADAND BRYANT

FIG. ‘7. mwh//fl?

“Discless”

251

Mutati0n.s

twin spots on the wing blade (a) and on the abdomen (b).

arm of the third chromosome, close to the dominant marker mutation Bsb (Baker, 1980). Pr (in cis configuration with Bsb, Fig. le), another dominant cell marker mutation, was also used to identify ~1137 homozygous clones. Pr maps close to Bsb, so that most of

the mitotic recombination takes place proximal to Pr, yielding Pr’ Bsb’ clones. These are homozygous for ~137, and such clones should develop normal bristles unless ~137 interferes with clone development. Bristles should be either missing or very much reduced in size

TABLE 7a CLONALANALYSISOF 1(3)e&O.DISTRIBUTIONOF mloh ANLI~? Wrx: CLONESAMONGSIZE CLASSES" Number Age at irradiation (hr AEL) 70-74

Genotype Control e20

94-98

11x-122

” Clones or 1.0 krad ” Except * 0.05 >

Number of wings scored 29

of clones Size classes in It+

muth/

Jr” 1

mwh

clones

Jr”

25

0

1

2

3

4

5

6

7

8

9

10

Twin Single

0 0

0 0

0 0

0 0

0 1

0 3

1 5

0 3

0 10

0 3

2 0 4861

0

0

0 0

0 2

Clone size and SD 36 138

t 98

32

4

21

0

Twin Single

0 0

0 0

1 0

1 0

Control

6

50

53

12

Twin Single

1 5

2 10

10 13

21 16

13 3

2 3

1 2

0 1

0 0

0 0

9.7 k 7.0 2

e20

6

59

59

10

Twin Single

0 10

3 14

12 7

21 12

20 9

7 7

0 0

0 0

0 0

0 0

7.6 -+ 5.4 8.4 -t 13.1

Control

8

11

493

5

Twin Single

5 369

5 114

1 10

0 0

0 0

0 0

0 0

0 0

0 0

0 0

1.6 + 0.7 1.3 i- 0.7

e20

8

3

508

5

Twin Single

1 323

0 164

2 21

0 0

0 0

0 0

0 0

0 0

0 0

0 0

2.7 2 1.4 t

were induced in y/+; mwh jr” as control (188- to 122-hr series) of gamma rags. two large clones in size class 10. P > 0.01 as compared with singles.

and in y/+;

ncwh e30/jr”

individuals

8.5 2 4.9* 30.5 k 16.9” 6.7 6.9

1.5 0.7

bg 1.5 krad (70- to 74- and 94- to 98-hr series)

252

DEVELOPMENTAL BIOLOGY

VOLUME 93. 1982

TABLE 7b CLONAL ANALYSIS OF 1(3)e20: CHARACTERISTICS OF ABDOMINAL CLONES’ Clone size (bristles/clone f SD)b Clones Age at irradiation (hr AEL) 70-74

Dose of irradiation (krad) 1.5

Twin Genotype

Abdomens analyzed

Control e.20

15 15

Single

mwh/

Jfr”

mwh

J%”

g

mwh

n

22 30

13 14

4 5

63 65

4.6 k 1.6 4.4 k 2.2

15 23

fr”

4.4 + 1.4 3.8 + 2.1

?l

mwh

n

14 15

4.3 ? 1.6 3.8 * 1.5

8 9

Jw

3.5 + 2.1 4.3 + 0.6

n

2/

2 3

3.9 2 2.2 3.5 ? 1.7

’ Clones were induced by 1.5 krad of gamma rays. For genotype see Table 7a. *Clones with one bristle/clone were omitted. Number of bristles in mwh clones was estimated by counting those bristles which were surrounded by mwh trichomes, since mroh does not affect bristles.&’ affects both bristles and trichomes (Garcia-Bellido, 19’73;Garcia-Bellido and Dapena, 1974).

when produced by Pr Bsb heterozygous cells (Fig. 9). The characteristics of ml37 homozygous clones are summarized in Table 10. They were detected at a lower frequency over the head and the thorax, as compared to controls, and they were significantly smaller than the Pr’ Bsb’ clones observed in the controls (P < 0.05, t test). The lower frequency of ml37 clones may, in fact, be simply a reflection of their smaller size since in this experiment only bristles were scored, and smaller clones are more likely to be devoid of bristles than are larger clones. ml37 clones developed with almost the expected frequency over the abdomen and their size did not differ from that of the controls, indicating that the mutation ml37 does not interfere with development of abdominal clones. Homozygosity for m137, as with the other four dl mutations, did not alter the morphology of cuticular structures.

FIG. 8. Ki’

DISCUSSION

Although the five mutants we have studied were originally isolated as discless mutants, histological analysis of both newly hatched and late-third-instar homozygous larvae revealed the presence of rudimentary imaginal discs at both of these stages in all five mutants. The rudimentary discs were also demonstrated by analysis of fluorescence-labeled nuclei in whole mounts of the larval integument; groups of lo-200 diploid nuclei identified the rudimentary imaginal discs in the thoracic segments of old larvae, their positions being close to the larval epidermis and characteristic of the original positions of disc primordia (Auerbach, 1936; Madhavan and Schneiderman, 19’77) rather than the positions of mature discs. The cells of these rudimentary discs express a diploid epithelium-specific antigen as shown by

clones on the head (a) and abdomen (b).

‘Discless”

SZABAD AND BRYANT TABLE

253

Mutations

8

gso/Ki LARVAE WERE IRRADIATED WITH 1.5 krad OF GAMMA RAYS

CLONAL ANALYSIS OF 1(3)@0. y/+;

Abdomen

Head and thorax

Flies Genotype scored

Kit clones

y clones

Kz+ clones Age at irradiation (hr AEL)

Number of clones

Clone size”

Flies scored

Number of clones

Clone size”

Flies scored

Number of clones

1/ clones Clone size”

R

Flies scored

Number of clones

Clone size”

12

46-50

Control Q30

35 42

5 2

1.8 ? 1.0 1.0 * 0.0

15 -

14 -

17.4 + 2.3 -

35 42

29 32

4.1 f 1.9 20 2.7 + 1.08 15

15 -

50

3.9 + 1.7 -

39

94-98

Control #JO

56 65

27 33

1.0 + 0.2 1.1 ? 0.2

15 15

56 95

1.6 f 0.9 1.8 + 1.2

56 65

25 14

3.7 k 1.7 2.1 + 0.4*

15 15

42 71

3.8 f 1.4 2.9 + 0.9

20 33

13 6

a Averaee and standard deviation. In the abdomen clones with only one bristle were omitted. * Signif;cantly different from controls; 0.05 I P > 0.01.

affect imaginal discs, but they seem to block proliferation in all of the larval organs that normally contain diploid, dividing cells. Table 11 summarizes the effects of these mutations on several organ systems. The formation centers in the larval brain are rudimentary and the larval central nervous system contains far fewer cells and mitotic figures than wild type; the imaginal rings of the salivary gland and foregut are often reduced or missing; the lymph glands are sometimes reduced and always produce very few blood cells; the larval gonads remain rudimentary and cannot function even in a wild-type host (with the exception of testicular cells in some cases). The abdominal histoblasts appear to be normal in most of these mutants but these cells do not proliferate even in the wild type until after pupariation (Roseland and Schneiderman, 1979). Thus, the mutations seem to interfere with functions required for cell proliferation in general. The effects are most severe on imaginal discs, the CNS and gonads where extensive cell proliferation occurs during larval life, and relatively minor in organs whose cells do not proliferate. Since all five dl mutations affect proliferating cell populations in most of the organs where proliferation occurs, although to different extents which are not clearly

their binding of the monoclonal antibody, D.A.lB6 (Brower et al., 1980). Six more putative dl mutants have been studied in whole mounts and all of them show rudimentary imaginal discs. Consequently, none of the 11 dl mutants we have examined is strictly discless. There seems to be only a quantitative difference between discless and small disc mutations, the former representing stronger alleles. In fact different mutations of one locus can result in either the discless phenotype or the small disc phenotype (Shearn and Garen, 1974). Failure of the disc primordia to grow during the larval period might in some cases be due to cell death which exactly balances proliferation, but we found no evidence for cell death in our histological studies. Therefore, we suggest that these dl mutations are in genes whose functions are necessary for cell proliferation. This conclusion is suggested by the observation that none or very few mitotic figures were observed in squashes of the larval central nervous systems, and that abnormal chromatin condensation was seen in a significant fraction of the nuclei in all of these mutants except m137, which will be discussed later. The five dl mutations we studied in detail do not just TABLE

CLONALANALYSISOF~(~) k&L g/+; k.@/Ki

9

LARVAEWEREIRRADIATEDWITHL~ krad OF GAMMARAYS

Head and thorax Ki’ clones Age at irradiation (hr AEL)

Genotype

Flies Number scored of clones

Abdomen 2/ clones

Clone size”

Flies Number scored of clones

Ki’ clones Clone size”

Number Flies scored of clones

2/ clones

Clone size”

n

Flies scored

Number of clones

Clone size”

n

70-74

Control w

64 27

10 5

1.2 + 0.4 3.0 k 2.4

20 13

24 38

3.4 +- 2.1 2.4 + 1.8

64 27

32 8

3.4 k 1.1 2.0 + o.o*

21 3

20 13

58 44

3.1 + 1.2 3.5 i- 1.7

24 29

94-98

Control k49

56 17

27 9

1.0 * 0.2 1.1 5 0.3

15 10

56 40

1.6 + 0.9 1.7 + 1.4

56 17

25 7

3.7 + 1.7 2.0 + o.o*

13 2

15 10

42 33

3.8 + 1.4 3.4 ? 1.3

20 22

118-122

Control k&

52 84

20 24

1.2 + 0.5 1.0 ? 0.0

12 8

95 38

1.3 2 0.6 2.Ok1.4

52 84

14 20

3.0 ? 1.4 2.3 + 0.5

4 8

12 8

34 35

2.5 k 0.9 2.8 k 0.8

12 17

a Average and standard deviation. In the abdomen clones with only one bristle were omitted. * Significantly different from controls; 0.05 > P > 0.01.

254

DEVELOPMENTAL

BIOLOGY

VOLUME

FIG. 9. Pr+ Bsb+ clones on the thorax

correlated, we conclude that many of the same genes function in cell proliferation in different organs during larval life. None of the dl mutations seems to interfere with chromosome replication as indicated by the presence of polytene nuclei in larval cells which are known to be in permanent interphase, having only G and S phases of the cell cycle (Pearson, 1974a,b; Nagl, 1978). However, the dZ mutations all appear to have some effect on cell growth, since both histoblast and larval epidermal cell nuclei fail to achieve their normal final volumes in all of the dl mutations. The larval environment provides nutrients for proliferating cells, and it has been shown that an abnormal larval milieu can bring about the small disc phenotype (Shearn and Garen, 1974; Pentz and Shearn, 1979).

CLONAL

ANALYSIS

OF l(SJm137:

y/+;

m137/Pr

93, 1982

(a) and abdomen (b).

However, the dl mutations seem to exert most of their effects directly on developing cell populations, rather than acting indirectly by rendering the larval environment unable to support growth. This is shown by the fact that homozygous dl larvae are able to support apparently normal growth and cell division in implanted wild-type imaginal discs, as was also shown for two other dl mutants by Shearn and Garen (1974). Furthermore, clonal analysis revealed that most of these mutations can inhibit the growth of at least some types of somatic clones of homozygous cells, showing that the effects of the mutants are expressed in a cell-autonomous fashion. The mutant ml.37 may be an exception in that it appears also to be defective in the larval environment it provides for disc growth. The fat body lobes are very

TABLE 10 Bsb LARVAE WERE IRRADIATED

WITH 1.5 krad

Pr+ Bsb+ clones

RAYS

Abdomen

Head and thorax

Age at irradiation (hr AEL)

OF GAMMA

Pr’

y clones

Bsb’ clones

y clones

Genotype

Flies scored

Number of clones

Clone size”

Flies scored

Number of clones

Clone size”

Flies scored

Number of clones

Clone size”

n

Flies scored

Number of clones

Clone size”

12

V-74

Control rnlS7

23 25

32 23

2.3 + 2.0 1.4 ? 0.8

23 25

67 54

2.2 + 1.8 2.2 f 1.2

23 25

57 63

4.1 + 2.0 3.7 T 1.9

42 50

23 25

65 73

3.4 k 1.9 3.7 f 1.6

35 45

94-98

Control mu7

13 25

51 49

1.5 t 1.1 1.2 c 0.6

12 25

43 46

1.2 + 0.6 1.9 + 0.9

13 25

29 37

3.5 t 1.5 4.3 2 1.9

17 25

12 25

33 61

3.3 2 1.4 2.9 * 1.0

22 37

’ Average

and standard

deviation.

In the abdomen clones with only one bristle

were omitted

SZABAD AND BRYANT TABLE 11 SUMMARY OF THE EFFECTS OF dl MUTATIONS Mutant Effect on Pupariation Pupariation time Larval size Larval cell number Larval cell size Histoblast cell number Histoblast cell size Imaginal ring salivary gland Imaginal ring foregut Lymph gland size Blood cell number Ovary size Ovary function Testis size Testis function CNS cell number CNS mitotic figure frequency Clone frequency, thorax Clone size, thorax Clone frequency, abdomen Clone size, abdomen Note. 0, no effect; +, moderate plicable or not determinable.

dl-I

e20

NO

kW

ml37

+ -

0 + + 0 +

0 ++ + 0 ++

0 + + 0 +

0 ++

0 + + ++

++ ++ ++ ++ ++ ++

0 + 0 + + + + ++ + + ++ ++ 0 0

0 + ++ ++ ++ ++ ++ ++ 0 -

0 + + ++ + + ++ ++ 0

++ ++

0 0

+

+ +

0 + 0 0 + 0 + 0 0 0 ++ ++ ++ ++ ++ ++ + + + 0 0

++ ++ + + + ++ ++ + ++ -

+ +

+

effect; ++, strong effect; -, not ap-

small (and possibly do not function properly) in ml37 homozygous larvae at early third instar and only very few of the cells in the CNS are mitotically active. However, at the end of the third instar the amount of fat body increases to almost the normal level and a significant fraction of the CNS cells start to divide at this stage. Such larvae can also provide growth conditions for implanted wild-type imaginal discs and presumably also for their own imaginal discs. However, since they pupariate shortly after developing the normal fat body lobes, the discs never grow beyond the rudimentary stage. If the mutations we have studied interfere with the proliferation of diploid cells in general, we need to account for the fact that the nuclear and cell divisions in the early embryo appear to proceed normally in the homozygotes or hemizygotes. In four of the dl mutations (eZO,g30, k43, and m137), the larval epidermis develops with a normal number of cells, suggesting that the nine nuclear, four syncytial blastoderm, and at least two postblastoderm divisions (Szabad et al., 1979) had occurred in the epidermis. Furthermore the larval central nervous systems contain neurons, and larval behavior appears normal, which suggests that neuroblasts were formed and executed their programs of cell division normally. We suggest that these early divisions are supported in the dl embryo by maternal dl+ gene products,

“Lkcless”

Mutation.5

255

which either compensate for the dysfunctional dl gene products of the embryo or which support development of the embryo before the zygotic dl or dl+ genes are activated. Such maternal support appears to be limited to the early embryo where the total volume of cytoplasm is not increasing. It appears to become ineffective shortly after the establishment of imaginal discs and other proliferating diploid cell populations, either because the maternal gene products are rapidly diluted out or because they have a finite lifetime. An alternative to the maternal-support idea, which cannot at present be excluded, is that separate sets of genes control proliferation during embryonic and larval life, and that we have been studying only the latter set. A direct demonstration of the suggested maternal effect would have to include a study of the development of homozygous dl/dl embryos derived from similarly homozygous mothers. This is, of course, difficult to arrange with a nonconditional lethal and in the cases we have studied, the problem cannot be overcome by transplantation of homozygous ovaries into wild-type hosts since the ovaries of dl larvae are also affected by the dl mutations. Perhaps a homozygous dl/dl germ line could develop in a normal host after pole cell transplantation or induction of mitotic recombination in a dl heterozygous germ line. Our clonal analysis supports the idea that the dl+ gene products can support growth and development long after the dl+ gene is removed from the cell. dl/dl homozygous cells could undergo several rounds of cell division after origination from a dl/dl+ heterozygous cell, showing that the dl+ gene product has a relatively long “perdurance” (Garcia-Bellido and Merriam, 1971). Similar observations were made for other cell lethal mutations (Rip011 and Garcia-Bellido, 1973). However, we cannot exclude the possibility that in some cases the dl/dl clone develops nonautonomously, its growth being supported by dl+ gene product from the surrounding heterozygous cells, The dl-1 mutation seems to interfere with the development of larval structures more than is the case with the other dl lethals, since the larval epidermis of dl-1 hemizygous larvae contains fewer cells than are found in wild type. Whether this is due to a reduced number of nuclear and/or cell divisions during embryogenesis, or to later degeneration, is not clear. However, the effect seems to be related to a more severe interference with cell proliferation than is the case with the other mutants, since dl-1 homozygous cells can divide very few times in clones after the induction of mitotic recombination. The dl-1 phenotype is fully cell autonomous, as shown in gynandromorphs where the female (dl-Udl-I+ heterozygous part) is normal, with imaginal discs, in contrast to the male (dl-1 hemizygous part)

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which lacks imaginal discs (Szabad and Kiss, unpublished). In gynandromorphs the female larval epidermis forms puparium but the male epidermis fails to do so, giving rise to so-called larval-pupal gynandromorphs (Kiss et al., 1976b). The dl-1 mutation seems to be the most extreme of those we studied, showing a more complete block to clone development as well as probable effects on embryogenesis. It is conceivable that even more extreme mutants could be found in which cell division was blocked earlier in embryogenesis. The fact that such mutations are missing from the present collection does not necessarily show that all such mutants will be rescued from embryonic effects by maternal gene products, since the present collection was selected to include only those mutants with good larval viability. We are grateful to Dr. Juliana Szabad-Juhasz for histological work, Dr. A. Shearn for providing stocks of discless lethals, Dr. D. Brower for a supply of D.A.lBG antibody, and Dr. Bruce Baker for the Bsb stock. Dr. Kaushal Kumar kindly provided data on the clonal analysis of al-1 wing y sn3//jY6” twin spots. This investigation was supported by Grant HD 06082 from the National Institues of Health. It was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt International Cancer Fellowship awarded by the International Union Against Cancer to Dr. Janos Szabad. REFERENCES AUERBACH,C. (1936). The development of the legs, wings, and halteres in wild type and some mutant strains of Drosophila melanogaster. Trans. Roy. Sot. Edinburgh

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CAMPBELL,R. D. (1981). A method for making ribbons of semithin plastic sections. Stain Tech. 56, 247-249. GARCIA-BELLIDO,A. (1972). Some parameters of mitotic recombination Mol. Gen. Genet. 115, 54-72. GARCIA-BELLIDO,A. (1973). The corrected number of aduit epidermic cells of the tergites. L?ros.In&r. Seru. 50, 99. GARCIA-BELLIDO,A. and DAPENA, J. (1974). Induction, detection and characterization of cell differentiation mutants in Drosophila Mol. Gen. Genet. 128, 117-130. GARCIA-BELLIDO,A., and MERRIAM, J. R. (1971). Genetic analysis of cell heredity in imaginal discs of Drosophila melanogaster. Proc. Nat. Acad Sci. USA 68, 2222-2226.

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VOLUME93, 1982

GATTI, M., TANZARELLA,C., and OLIVIERI, G. (1974). Analysis of the chromosome aberrations induced by X-rays in somatic cells of Drosophila melanogaster. Genetics 77, 701-719. HAYNIE, J. L., and BRYANT, P. J. (1977). The effects of X-rays on the proliferation dynamics of cells in the imaginal wing disc of Drosophila melanogaster. Wilhelm Roux’s Arch. 183, 85-100. KISS, J., BENCZE, G., FODOR,A., SZABAD, J., and FRISTROM,J. W. (1976a). Pupal-larval mosaics in Drosophila melanogaster. Nature (London) 262, 136-138. KISS, J., BENCZE,G., FEKETE, E., FODOR,A., GAUSZ, J., MAROY, P., SZABAD,J., and SZIDONYA,J. (1976b). Isolation andcharacterization of X-linked lethal mutations affecting differentiation of imaginal discs in Drosophila melanogaster. .I Them: Appl. Genet. 48217-226. LINDSLEY, D. L., and GRELL, E. H. (1968). Genetic variations of Drrr sophila melanogaster. Carnegie Inst. Wash. Publ. No. 6%. MADHAVAN, M. M., and SCHNEIDERMAN,H. A. (1977). Histological analysis of the dynamics of growth of imaginal disc and histoblast nests during the larval development of Drosophila melanogaster. Wilhelm Roux’s Arch. 83, 269-305.

MARTIN, P. (1982). A technique for the determination of cell number in imaginal discs. Submitted for publication. NAGL, W. (1978). “Endopolyploidy and Polyteny in Differentiation and Evolution,” pp. l-283. North-Holland, Amsterdam. PEARSON,M. J. (1974a). Polyteny and the functional significance of the polytene cell cycle. J. Cell Sci. 15, 457-479. PEARSON,M. J. (1974b). The abdominal epidermis of Calliphora cry throcephala (Diptera). I. Polyteny and growth in the larval cells. J. Cell Sci. 16, 113-131. PENTZ, E. S., and SHEARN, A. (1979). Analysis of the autonomy of imaginal disc defects in a small disc mutant of Drosophila melanogaster. Dev. Biol. 70, 149-170. RIPOLL, P., and GARCIA-BELLIDO,A. (1973). Cell autonomous lethals in Drosophila melanogaster. Nature New Biol 241, 15-16. ROSELAND,C., and SCHNEIDERMAN,H. A. (1979). Regulation and metamorphosis of the abdominal histoblasts of Drosophila melanogaster. Wilhelm Roux’s Arch. 186, 235-265. SHEARN,A., and GAREN, A. (1974). Genetic control of imaginal disc development in Drosophila Proc. Nat. Acad. Sci. USA 71,1393-1397. SHEARN,A., RICE, T., GAREN, A., and GEHRING,W. (1971). Imaginal disc abnormalities in lethal mutants of Drosophila. Proc. Nat. Acad. Sci. USA 68,2594-2598. SRDIC,Z., and REINHARDT, C. (1980). Histolysis initiated by “lymph gland” cells of Drosophila. Science 207, 1375-1377. SZABAD,J. (1978). Quick preparation of Drosophila for microscopic analysis. L?ros.Inf Serv. 53, 215. SZABAD,J., SCH~~PBACH, T., and WIESCHAUS,E. (1979). Cell lineage and development in the larval epidermis of Drosophila melanogaster. Dew. Biol. 73, 256-271. WHITE, K., and KANKEL, D. R. (1978). Patterns of cell division and cell movement in the formation of the imaginal nervous system in Drosophila melanogaster. Dev. Biol. 65, 296-321. WIESCHAUS,E., MARSH, J. L., and GEHRING, W. (1978). fs(l)KlO, a germ-line dependent female sterile mutation causing abnormal chorion morphology in Drosophila melanogaster. Wilhelm Roux’s Arch. 184, 75-82.