MULTIPLE ALLELE APPROACH TO THE STUDY OF GENES IN DROSOPHILA MELANOGASTER THAT ARE INVOLVED

IN IMAGINAL DISC DEVELOPMENT*

ALLEN SHEARN, GRAFTON HERSPERGER, EVELYN HERSPERGER, ELLEN STEWARD PENTZ AND PAUL DENKER

Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

Manuscript received September 10, 1977 Revised copy received January 26, 1978

ABSTRACT

The phenotypes of five different lethal mutants of Drosophila nelanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. TO examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromo- some containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no sig- nificant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were com- pared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles a t these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect sig- nificant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold- sensitive mutants.

T H E mutational dissection of development proceeds by analyzing the con- sequences of mutations in genes whose functions are essential for normal

development. The application of this approach to imaginal disc development has recently been reviewed (SHEARN 1978). One strength of this approach is that such genes may be identified without knowledge of their direct gene products. However, a weakness of the approach is that without the ability to assay a gene’s

* Research supported by grant GB40737 from the National Science Foundation; Contribution 950 from the Depart- ment of Biology, Johns Hopkins University, Baltimore, Maryland 21218.

Genetics 89: 355-370 June, 1978.

356 A. SHEARN et al.

product directly, it is not possible to evaluate the extent to which a given muta- tion in that gene affects the activity of its product. This limitation makes it difficult to infer the function of the wild-type product of such a gene. There are at least two genetic solutions to this problem: One, the gene-dosage method, was described by MULLER (1932). The idea of that method is to define operationally the effect of a mutation on a gene’s activity by examining the effect on its pheno- type of adding one or two doses of the wild-type allele and of heterozygosity for a deletion of the gene. From such a study it may be inferred whether a mutation causes its effect by eliminating, reducing, altering, or increasing the wild-type gene function. A problem with this approach is that it requires considerable manipulation of aberrant chromosomes to analyze a single mutation.

The other approach, which is used in this study, may be called the multiple- allele method. This approach has been applied to several loci in Drosophila (reviewed by FRISTROM and YUND 1973). The idea of this method is to isolate a series of alleles of a reference mutation and then to compare the phenotypes of homozygotes of each allele to each other and to hybrids of different alleles. If the reference mutation causes elimination of a gene product, then no other allele should cause a more severe phenotype, although some may cause a less severe phenotype. If, on the other hand, the reference mutation merely reduces the activity of a gene product, then among its alleles some might eliminate the activity and thus cause a more severe phefiotype. Similarly, if the reference mutation alters the function of the wild-type gene product, then different alleles may be expected to express different phenotypic alterations. By limiting such studies to recessive mutants, it is assumed that mutations causing increased gene product levels would not be identified.

This study began with developmental analyses of five different lethal mutants, each of which has a third-chromosome mutation that causes the imaginal discs to express the small-disc phenotype (SHEARN et al. 1971, SHEARN and GAREN 1974). To identify alleles of these mutations, a chromosome was synthesized, by recombination, containing all five lethal mutations and three markers. Hybrids, having one quintuply lethal third chromosome and one third chromo- some derived from a mutagen-treated male, were tested for lethality. This iden- tified those chromosomes containing new mutations allelic to one of the five reference mutations. Each new mutation was then tested for complementation with each of the five reference mutations to identify in which of the five genes each is located. The mutants within each of the five groups were then compared to their respective reference mutants in terms of stage of lethality and imaginal disc morphology. Similar comparisons were made of hybrids of the different alleles at each locus. The results of this study indicate that for many loci (three of the five considered in this report) the analysis of a single mutant allele, even if it is a lethal, may not be adequate to infer the developmental function of those loci. A preliminary report of this work was presented at a symposium sponsored by the American Society of Zoologists (SHEARN 1977).

ALLELES O F SMALL DISC MUTATIONS 35 7

MATERIALS A N D METHODS

Stocks The five small-disc reference mutations analyzed in this study were among those third-

chromosome lethals described in a previous report (SHEARN et al. 1971). They were isolated in a stock marked with multiple wing hairs (mwh, 3-0.0) and ebony (e, 3-70.7) and are main- tained as balanced lethal stocks with TM3. Two of the mutations, [Z(3)052, 3-47.0 t 0.9 and 1(3)c21r, 3-68.0 * 0.51 were induced by ethyl methanesulfonate (EMS), and one [1(3)ZX-11, 3-81.7 & 0.61 was induced by the acridine compound, ICR-170. The other two [1(3)1602, 3-19.6 i: 1 .O and 1(3)1902, 3-30.9 i: 0.71 were induced by N-methyl-N’-nitro-N-nitrosoguani- dine (NG) in a stock that was also marked with red Malpighian tubules (red, 3-53.6). For a description of markers and balancers used, see LINDSLEY and GRELL (1968). All stocks and crosses were maintained in glass vials on a medium of cornmeal, molasses, yeast, and agar a t 20”, unless stated otherwise.

Measurements of discs To quantitatively compare the sizes of normal and mutant discs, we estimated their areas

to be the product of their lengths and widths, which were measured with an ocular micrometer. To examine whether this product is a good estimate of the area, photographs at the same mag- nification of 28 discs of various sizes were cut out and weighed. The relationship between the estimate of their areas by the product of their lengths and widths and by weighing their photo- graphs is shown in Figure 1 . Over a wide range, the relationship of these two estimates is linear. Since the discs are basically flat, especially during early development, it is assumed that there is also a linear relationship between the area of a disc and its mass. During the third larval instar, normal discs become folded; then their areas underestimate their mass.

Stage of lethality A mutant is considered to be a larval lethal if it does not form puparia; if it forms puparia

but does not metamorphose it is a prepupal lethal; if it metamorphoses but does not eclose, it is a pupal lethal.

Assays of developmental capacity b y disc injections Mutant discs cultured in normal adult females: Wing discs and eye-antenna-brain complexes

from late second-instar mutant larvae were measured and then injected into the abdomens of zero to two-day-old wild-type adult females (Canton-S). After seven days the discs were recov- ered, measured, and then re-injected into metamorphosing larvae to test for their ability to differentiate. The details of the injection technique have been described by URSPRUNG (1967). Normal discs from the marker stock mwh red e were used as controls.

Normal discs cultured in mutant laruae: Wing discs from early third-instar larvae of the marker stock, mwhrede, were measured and then injected into late second or early third- instar mutant larvae. After three to seven days, the diws were removed from their mutant hosts, measured, and then reinjected into mid-third-instar mwh red e larvae, which were allowed to metamorphose. Differentiated implants derived from such injections were cleared and mounted in Faure’s solution and examined with the light microscope.

Somatic recombination Somatic recombination was induced by y-irradiation (452 r/min) at several stages during

embryogenesis and larval life. The sizes and frequencies of Sb+ clones induced in heterozygotes with the genotype l(3)cZir e/bld Sb*s were compared to such clones induced in the controls that had the genotype e/bld Sb63. The mutant bald (bld, 3-48), described by GARCIA-BELLIDO and DAPENA (1974), causes the reduction of pigment in cuticular structures. The details of this analysis will appear elsewhere (PENTZ and SHEARN, manuscript in preparation). The sizes and frequencies of y ;mwh clones induced in heterozygotes with the genotype y / y ; mwh 1(3)1902/

358 A. SHEARN et al.

I I .025 .050 Area (mm21

FIGURE 1.-Relationship of two different estimates of the area of imaginal discs. The products of the lengths and widths of a mixture of imaginal discs fram normal larvae were used as one estimate of the area of such discs. Photographs of the same discs were weighed and used as a different estimate of their relative areas. A least-squares line fitted to the data is drawn through the points.

scJ4, y + were compared to such clones in the controls, which had the genotypes y / y ; mwh/ scJ4, y + . The details of this analysis will also appear elsewhere (TEITELBAUM and SHEARN, manuscript in preparation).

Synthesis of the quintuply lethal chromosome

The five lethal mutations combined in the quintuply lethal chromosome were chosen in part, because they map relatively far from each other. The first step in the synthesis of the quintuple lethal was t o make two double mutants, 1(3)1602 with 1(3)1902 and l(3)cZlr with Z(3)ZX-Il. The general scheme used for making such double mutants is otulined in Figure 2.

I

II

ALLELES O F SMALL DISC MUTATIONS

11 e + + + e ! 2 + 9 9 - ttftt I TM3*Sb+ e + Ser T M I , + + +

J 11 e + DTS GI +++ + e 1 2 I TM3,+ +Sbe Ser Q Q - =###

dd

d

1 , e + .+ + e!,+ + + ? e ? T M I , + + + TM3,Sb+ e+% DTS GI + + +

359

d

127"

? e ? ? e ? ? e ? + ? e ? +

R I e + + e 12 TMI ,+++ TM3,Sb+e+Ser #I==##='-'-

FIGURE 2.-General method of synthesizing double lethal mutants. In the first generation males of one mutant stock are mated to females of the other. It is not necessary at this step for the two stocks to have different balancers; different balancers are necessary, however, in the third generation. Female hybrid progeny of the correct genotype, recognizable by the e pheno- type, are then mated to males heterozygous for a dominant heat-sensitive (DTS) mutation and a balancer. Individual male progeny heterozygous for the DTS are then mated in a single vial to two females from each of the single mutant stocks at 27". The presence among the progeny of some individuals carrying each balancer insures that both genotypes of females participated in the cross. The absence of any e progeny indicates the presence in generation three of a recombinant male carrying both lethal mutations. DTS-2 was described by HOLDEN and SUZUKI (1973).

The double mutant l(3)cZIr l (3)IX-II was then combined with the remaining single mutant 1(3)052 to make a triple mutant. Finally this triple mutant was combined with the other double mutant. The quintuply lethal chromosome carries the markers mwuh, red and e and is balanced over TM3, which carries Sb, e, and Ser. Surprisingly enough, this stock is healthy and not difficult to maintain.

Mutagenesis and isolation of alleles

The new mutant alleles were all induced in chromosomes marked with red. To be sure that these chromosomes were free of lethal mutations prior to mutagenesis, the first three crosses out- lined in Figure 3 (labeled I, 11, and 111) were repeated regularly. Adult red males, 0-48 hours old, were treated with EMS according to the procedure of LEWIS and BACHER (1968), but the concentration was lowered to 12.5 mM. At this dosage, 48% of the third chromosomes recovered

360 A. SHEARN et al.

are expected to have at least one lethal mutation (RICE 19'73). These treated males were mated to females heterozygous for a dominant, heat-sensitive mutation and a balancer and cultured at a restrictive temperature. Male progeny of this cross were mated individually to females carrying the quintuply lethal chromosome. No phenotypically red progeny, at either 20" or 27", will appear among the progeny of those crosses involving males carrying an allele of any one of the five lethal mutations contained in the quintuple lethal. Red progeny at one tempera- ture, but not the other, would suggest the presence of a temperature-sensitive allele. The newly isolated alleles were maintained as balanced lethals over TM3. To identify the reference muta- tion to which each new mutation is allelic, males from each uew mutant stock were mated in separate tests to females of each of the five single-mutant reference stocks.

Complemeniation All pairwise combinations of the 24 alleles isolated in this study were tested for comple-

mentation. Within each of the five groups of alleles, this procedure was a test for intra-cistronic complementation.

RESULTS

All five of the reference mutants manifest the small-disc phenotype (SHEARN and GAREN 1974). Mutants in this group have imaginal discs that are smaller than normal and cannot differentiate into adult structures when injected into metamorphosing normal larvae. In each of these five mutants, all of the major discs (eye-antenna, leg, wing, haltere, and genital) are abnormal; however, the extent to which the discs are reduced in size in each mutant differs (Table 1).

TABLE 1

Autonomy of imaginal disc phenotype in five reference mutants

Parameter examined mwh red e 1(3)1602 Genotype

I(3)1902 1(3)052 1(3)c21r l ( 3 ) I X - i l

Disc size*,+ 1 7 9 t 7 2 2 1 (mm2 x 103) (4) (32)

Growthof youngdiscs, 81 t 24 3 t 1

(mm2 x 103) cultured in vivo+,$ (7) (10)

Subsequent differentiations + - In vivo culture of + embryonic cells

Recovery of normal clones + by somatic recombination

5 2 1 NR 5 O t 1 8 NR (15) (13)

- NA - NA

+

* Area of wing discs t standard deviation from late third instar larvae or latest stage attained

+The number of discs measured is in parentheses. $ Mean increase in area t standard deviation of discs dissected from second instar larvae and

+ = normal differentiation; - = implants recovered contained no characteristic adult -/+ = normal size and frequency of clones recovered only if induced late in development.

by mutant larvae.

cultured in adult females for seven days. NR = no implants ever recovered from hosts.

structures; NA = not applicable.

ALLELES O F SMALL DISC MUTATIONS 361

The maximum size of discs from 2(3)1602 and l(3)IX-11 is less than the size of discs dissected from late, second-instar, normal larvae (IO * 5 mm2 X ; the maximum size of discs from l(3)1902 and l(3jo52 is greater than that from late second-instar larvae, but less than that from young third-instar normal larvae (24 f 8 mmz X ; the maximum size of discs from l(3)c21r is similar to that from mid-third-instar normal larvae. In the case of this latter mutant, l(3)c21r7 but not the other four, we have found that the discs actually decrease in size in situ. They reach their maximum size in seven-day-old larvae (at 25”) and then degenerate. (PENTZ and SHEARN, manuscript in preparation).

Autonomy of imaginal disc defects For each of the mutants we wish to discover whether the observed imaginal

disc defects result from the loss of function of a gene, which normally acts in imaginal disc cells, or if those defects are the secondary effects of disc develop- ment in a defective larval environment. In other words, are these mutations “disc autonomous”? The fact that discs from none of the five mutants differentiate adult structures when injected into metamorphosing larvae is not a critical test of autonomy, for two reasons. First, small discs from young normal larvae are not competent to differentiate (BODENSTEIN 1939; GATEFF 1972, MINDEK 1972, SCHUBIGER 1974) ; second, even if the discs were not directly affected by these mutations, they might have already suffered irreversible damage by the time of dissection. Three kinds of experiments were performed to examine this issue: in vivo culture of discs from second-instar mutant larvae, in uiuo culture of cells from mutant embryos and somatic recombination in mutant heterozygotes.

In vivo culture of young discs: If any mutation were not disc autonomous, it should be possible to dissect discs from mutant larvae before some critical time, culture them in uivo to allow them to mature, then inject such cultured discs into metamorphosing larvae, and recover differentiated adult structures. Since all five of the mutants apparently develop normally until the end of the second instar, we tested discs from this stage. This is, in any case, the youngest stage from which we find it technically feasible to dissect discs. These experiments were performed with eye-antenna-brain complexes (data not shown) and wing discs from all five mutants (Table 1). Young normal discs (mwh red e ) , which served as the control, increase less in area when cultured in adult females for seven days than they do during the third larval instar in situ. Discs from two of the mutants [l(3)052 and l (3)IX-l1] were never recovered after seven days of culture. In one set of experiments, we found that these discs actually decreased in size for the first three days, after which they were no longer detectable. Discs from two others of the mutants [l(3)1602 and l(3)1902] grew slightly, but did not even reach the size of discs from early third-instar normal larvae. Even after five transfers through adult hosts, discs from l(3)1902 did not grow larger than they do in situ. Discs from 2(3)c21r, however, did grow substantially in adult hosts and continued to grow when transferred to fresh hosts. Nevertheless, neither l(3)c21r discs nor l(3)1602 and l(3)1902 discs were able to differentiate characteristic adult structures when injected into metamorphosing larval hosts.

362 A. SHEARN et al.

From these results, we tentatively conclude that all five mutations are disc autonomous.

In vivo culture of embryonic cells: While it is not technically feasible to dissect discs from mutant larvae prior to the end of the second instar, it is possible to culture cells in vivo from a mixture of mutant and normal embryos. Imaginal cells derived from the mutant embryos can be recognized by hair and pigment markers. This technique has previously been applied to one of the five reference mutants, l(3)052 (SHEARN and GAREN 1974). Structures derived from all of the imaginal discs were recovered from such mutant embryos at the same fre- quencies as from the control. Our results showed that, at an early stage in devel- opment, this mutation is not autonomous. The other four reference mutants have not yet been tested using this technique (Table 1 ) .

Somatic recombination: I t is not necessarily the case that a mutation which is disc autonomous is also cell autonomous. We have tested two of the reference mutations [l(3)c21r and l(3)1902] for cell autonomy by examining clones of homozygous mutant cells produced by somatic recombination in mutant het- erozygotes. Clones of l(3)c21r induced early in development, until the middle of the second instar, are significantly smaller than control clones; whereas, at later times, the frequency and size of mutant clones are indistinguishable from control values (PENTZ and SHEARN, manuscript in preparation). Since this mutation appears to be cell autonomous, at least during early stages of larval development, we conclude that the wild-type allele of l(3)c21r does directly act in imaginal disc cells. Clones of l(3)1902 contain morphologically abnormal structures, indicating that this mutation is also cell autonomous (Table 1). The other three reference mutations have not yet been tested for cell autonomy.

Evidence of larval defects

We examined all five mutants for larval defects. If mutant larvae were defec- tive by either of the two criteria we used, failure of puparium formation or fail- ure to support growth of normal discs, we would conclude that a defect occurred in some nonimaginal tissue. We assume that any defect limited to the imaginal discs would not cause larval defects. This assumption is based on the finding that neither the absence of discs (SHEARN et al. 1971) nor the presence of large discs (MARTIN, unpublished observation) causes larval defects.

Puparium formation: The puparium is formed by the tanning of the third- instar larval cuticle. We have isolated homozygous larvae from all five mutant stocks, and allowed them to continue development away from their heterozygous siblings. Larvae from three of the mutants [l(3)1602, 1(3)052 and 2(3)c21~] never form puparia (Table 2). By contrast, more than 50% of l(3)1902 and l(3)ZX-ll mutant larvae form puparia, although none of them metamorphose.

Growth of normal discs in mutant larvae: The ability of larvae to support the growth of normal discs (SHEARN and GAREN 1974) is a minimal criterion for evaluating the absence of a larval defect. Larvae from two of the mutants, [1(3)1602 and 1(3)1902], supported nearly as much growth as normal larvae (Table 2). Larvae from the three other mutants, l(3)052, l(3)c21r7 and

ALLELES O F SMALL DISC MUTATIONS

TABLE 2

Evidence of larval defects in five reference mutants

363

Genotype Parameter examined mwh red e 1(3)1602 1(3)1902 1(3)052 l(3)cZIr I(3)IX-I1

+ Puparium formation + Growth of injected 135 t 101 8 0 2 55 93 f 23 15 t 2 @ t 19t 9s 29 t 1% normal wing discs* (38) (17) (37) (7) (21 1 (32) (mmz x 103)

- - + -

~

* Mean increase in area +. standard deviation. The number of recovered discs is in parentheses. I. Significantly different from control according to t test ( t = 2.91; P < 0.01). $ Significantly different from control according to t test ( t = 4.33; P < 0.01). 0 Significantly different fro mcontrol according t o t test ( t = 4.76; P < 0.01).

l(3)ZX-ll did not support as much growth as normal larvae. In every case, normal discs, recovered after culture in mutant larvae, were reinjected into metamorphosing normal larvae to test for differentiation, In no case did such in vivo culture affect the ability of normal discs to differentiate subsequently.

Isolation of lethd alleles A total of 13,197 EMS-treated third chromosomes were tested for the presence

of mutations allelic to any of the five mutations in the quintuply lethal chromo- some, using the scheme indicated in cross V of Figure 3. Each of the 24 mutations recovered failed to complement the quintuple lethal at both 20" and 27O. These 24 were tested for complementation with each of the five reference mutations of the quintuple lethal (Table 3). The distribution of the alleles among the five genes is not significantly different from that expected if the probability of induc- ing a mutation in each of the five genes were identical (x2 = 1.83, P > 0.50).

The alleles were induced with an EMS dose expected to produce at least one lethal mutation in 6392 (48%) of the treated chromosomes (RICE 1973). Dividing this number by the average number of alleles isolated per locus (4.8) , we estimate that there could be 1332 genes on the third chromosome with a similar mutation frequency. The one gene: one salivary gland chromosome band hypothesis suggests that there should be a total of 2030 genes on the third chromo- some. According to this hypothesis, the five genes we have under study do not appear to have a mutation frequency greater than that of most third chromo- some genes.

Assuming an equal probability of mutation of all genes on the third chromo- some, we expected, on the basis of the Poisson distribution, that 70.8% of the lethal mutations would be recovered as single mutations. Therefore, 70.8% of the alleles (1 7/24) should have been free of additional lethal mutations. In fact, only 46% of the alleles (11/24) were free of additional mutations (Table 3). The probability of getting a difference this large by chance is less than 1% (G = 7.26; 1 d.f.) . This excess of multiple mutations could be accounted for if some genes on the chromosome have an exceptionally high frequency of muta-

A. SHEARN et al. 364

I

II

m

m

D T S G I + + + TM3, + + Sb e Ser red

DTS GI + 4- + red + + + x -d

T M 3 , + Sb e Ser

red + + 4- red + ++ TM3,+ Sb e Ser TM3,+ Sb e Ser

DTS GI + + + r e d H E M S 99- x *66

TM3,+ + Sb e Ser red

mwh 1602 1902 052 red +c21 e E-11 + * red + + + Y??? x ----ctttd

TM3,+ + + + +Sb + e + Ser T M 3 , + Sb e Ser FIGURE 3.-Outline of scheme for isolating lethal alleles. The first three generations lead to

the production of stocks homozygous for isogenic third chromosomes and therefore free of lethal mutations. Males from such stocks are treated with EMS and mated with females heterozygous for a dominant, heat-sensitive mutation and a balancer (Generation IV). Male progeny from this cross are mated individually to females heterozygous for the quintuple lethal and a balancer chromosome (generation V). The absence of phenotypically red adult progeny at either 20" or 27" or both temperatures indicates the presence of an allele of one of the five mutations or of a dominant temperature-sensitive mutation.

tion. If that number were relatively small, then mutations in some of them might have occurred more than once even in our small sample. To examine this possibility, we tested all 24 alleles for inter-cistronic complementation. Among the 13 multiple mutants, two pairs failed to complement. This indicates that both pairs are double mutants. Each has alleles of different reference mutations and alleles of some other gene with an exceptionally high frequency of muta- tion. If these two pairs are subtracted from the number of multiples, then the

ALLELES O F SMALL DISC MUTATIONS

TABLE 3

Summary of properties of alleles

365

Reference mutation Property 1(3)1602 1(3)1902 1(3)052 1(3)c21r l (3)ZX-lf

Number of alleles 4 7 3 5 5

Number with additional mutations when isoslated

1 4 0 4 4

- + + + Significant phenotypic variation - among alleles

Intracistronic complementation - - - - + observed number of multiples 9/24 is not significantly different from the expected number, assuming an equal probability of mutation in all genes (G 1 3.23; p > 0.05). Complementation and phenotype of lethal alleles

We compared each reference mutant to its alleles in terms of stage of lethality and imaginal disc morphology. In order to make such comparisons for the mul- tiple mutants, it was necessary to remove nonallelic mutations by recombination.

Alleles of 1 (3) 1602 and 1 (3) IX-11: Newly isolated alleles of two of the genes studied are phenotypically identical to their respective reference mutants, 1(3)1602 and 1(3)ZX-11. This suggests that these reference mutants are express- ing the null activity or amorphic phenotype. To test this hypothesis, mutants in both groups were tested in all pairwise combinations for partial intra-cistronic complementation. Each hybrid was analyzed in terms of stage of lethality and imaginal disc morphology. No complementation was observed in any of the hybrids; their phenotypes were identical to that of their respective mutant homozygotes .

Alleles of 1(3)c21r: Of the five alleles of 1(3)c21r isolated, four have a pheno- type indistinguishable from it, but one is quite different. This allele, called NC806, has normal appearing imaginal discs; it completes metamorphosis, but dies at the pharate adult stage. Examination of these pharate adults did not reveal any striking morphological defects. This allele is apparently hypomorphic or “leaky.” All hybrids of NC806 with other alleles of 1(3)c21r are phenotypically identical to NC806 homozygotes, and all hybrids not involving NC806 are identical to the other homozygotes; i.e., there is no complementation observed.

Alleles of 1(3)052: The phenotypes of 1(3)052 and its three alleles represent the widest range we have observed. At one extreme, allele MV1422 is lethal shortly after the first larval molt, when discs are not detectable by dissection even in normal larvae. At the other extreme, there is an allele which is lethal after metamorphosis; mutant pharate adults have no detectable imaginal disc defects, but do not eclose. These four mutants may be ranked according to the

366 A. SHEARN et al.

NU808, NHO06

NV931 M Z 1007, NW522

M Y 9 3 9 NY721 FIGURE Lt.-Complementation map of alleles of 2(3)1902. Overlapping bars indicate combina-

tions of alleles that do not complement.

seventy of their defects. When we tested by complementation, we found that the phenotype of each hybrid was intermediate between that of each allele when homozygous. This indicates that the three less severe alleles do not entirely elim- inate the activity of this gene’s product.

Alleles of l ( 3 ) 1902: Although all seven of the alleles of l(3)1902 allow pup- arium formation, their effects on imaginal discs range from ones that are sim- ilar to l(3)1902 to one that has normal appearing discs capable of normal differ- entiation. This group of alleles has significant differences from the other four groups studied because several pairs of alleles manifest intra-cistronic comple- mentation. In this case, a positive complementation result means that hybrid larvae have normal imaginal discs and they metamorphose to produce pharate adults. Since none of these hybrids eclose, this result is termed partial comple- mentation. A complementation map summarizing these results is presented in Figure 4.

Dominant cold-sensitive mutants: The protocol for identifying new mutations outlined in Figure 3 is, incidentally, a screen for dominant cold-sensitive muta- tions. We recovered two such mutations among the 13,179 chromosomes tested. This low frequency may account for the failure to find such mutations in an earlier study (ROSENBLUTH, EZELL and SUZUKI 1972). This interpretation is strengthened by our finding that the two dominant cold-sensitive mutations we identified are allelic (SHEARN and DENKER. manuscript in preparation).

DISCUSSION

The mutational dissection of development depends upon analyzing the con- sequencies of mutations in genes essential for normal development. A large num- ber of such genes have been ident!fied in Drosophila melanogaster by lethal mutations (SHEARN et al. 1971; STEWART, MURPHY and FRISTROM 1972; BRYANT and ZORNETZER 1973; RIPOLL and GARCIA-BELLIDO 1973; RUSSELL 1974; ARKING 1975; SIMPSON and SCHNEIDERMAN 1975). Among 134 third-chromo- some late-lethal mutations, 66 caused imaginal disc abnormalities ( SHEARN et al. 1971). However, more than 90% of the genetic loci identified by these lethal mutations were represented by a single allele. Since the products of these genes

ALLELES OF SMALL DISC MUTATIONS 367

could not be directly assayed, it was not possible to state whether the phenotypes observed resulted from the complete or partial loss of gene function. While the complete loss of function in most genes essential for development might lead to lethality, it does not follow that all lethal mutations result from the complete loss of gene function. This study is based on the assumption that, for any given gene, all mutations that cause a complete loss of function should have an identi- cal phenotype, and all other mutations in that gene should have a less severe phenotype. Our evidence, in keeping with this hypothesis, indicates that four of the five reference mutants analyzed [2(3)1602, 1(3)1902, Z(3)c2lr and Z(3)ZX-lll express null or amorphic phenotypes. Less severe alleles of two of these [Z(3)1902 and Z(3)c21r] have been recovered.

In an earlier study (SHEARN 1974) , no significant phenotypic differences were detected among ten pairs of noncorplementing, late-lethal mutants and their respective hybrids. Yet we detected, in the present study, such differences among alleles at three of five loci examined. We explain this apparent discrepancy, in part, by the fact that different methods of identifying mutants were used in the two studies. In the earlier study, lethal mutants that died late in development were recovered and then tested for complementation to see whether any pairs were allelic. In the present study, we first identified the alleles by their failure to complement and then examined them for their effect on development. Another explanation may well be that the probability of detecting phenotypic heterogene- ity among alleles at any locus depends upon the number of alleles examined. In the earlier study, each of the ten loci was represented by only two alleles, whereas in the present study each of the five loci was represented by four to eight alleles. Of the 29 mutants examined in the present study, 23 or 24 appeared to express a null activity phenotype. This fraction suggests that the majority of lethal alleles at other loci would express null activity phenotypes.

We examined the five reference mutants in this study for evidence of larval defects and autonomous expression in discs. According to these criteria, we infer that the primary defect in one mutant, 1(3)052, is not in imaginal tissue and that in another, Z(3)1902, the primary defect is in imaginal tissue. Analysis of alleles of these mutations provides evidence to support these inferences. The other three mutants, Z(3)1602, 2(3)c21r7 and Z(3)ZX-11, express larval defects in addition to autonomous imaginal disc defects. Thus, the disc defects observed in these three mutants in situ may not be entirely due to primary defects in imaginal tissue.

Since embryonic cells homozygous for 1(3)052 can differentiate normal imagi- nal structures after in vivo culture, and normal discs grow poorly in homozygous 2(3)052 larvae, we believe that the imaginal disc defects found in larvae of this mutant are the result of damage suffered by development in a defective larval environment. The finding of an allele of l(3)052 that is lethal early in larval life supports the idea that the gene product affected by this mutation is required by larval cells. Both complementation results and genetic mapping indicate that the phenotype of this extreme allele is due to a single mutation.

No larval defects are evident in larvae homozygous for Z(3)1902. The pheno- type of this mutant appears to result from the complete loss of a gene function,

368 A. SHEARN et al.

since none of its alleles have a more extreme phenotype. We infer that this gene might not be required to function in larval cells.

Our scheme for isolating alleles (Figure 3) was designed to identify tempera- ture-sensitive mutations. None of the 24 alleles recovered, however, are sensitive to either heat or cold. BAJLLIE, SUZUKI and TARASOFF (1968) estimated the fre- quency of temperature-sensitive mutants among EMS-induced, second chromo- some lethals to be 10.9%, and SHEARN (unpublished observation) estimates the frequency among third chromosome lethals to be 4.5 %. Thus the probability of not finding a single temperature-sensitive allele, by chance alone, among a sample of 24 mutations is greater than 10% (G = 2.7) or greater than 50% (G = 0.31) depending on which value of the observed frequency is used in the calculation. It is also possible that the products of the five loci studied here have a much lower than average probability of sustaining a temperature-sensitive alteration. For at least one of these five loci, it appears that the correct interpre- tation is that the sample size was too small. We have isolated 22 additional alleles of 1(3)1902, and two of them are heat sensitive (SHEARN, HERSPERGER and HERSPERGER 1978). Thus, two of 29, or 6.9%, of the alleles at this locus are temperature sensitive. It is perhaps significant that alleles of this locus also manifest intracistronic complementation, whereas alleles at the other four do not.

Two aspects of the multiple allele method employed in this study must be evaluated: (1) Is it useful to isolate and study more than one or two mutations in genes that are of developmental interest? (2) Is our method of isolating alleles worthwhile?

We have three principal reasons for believing that the multiple allele approach is useful. (1 ) It allows one to discover, for any given gene, the expression of the null phenotype; this knowledge is important in interpreting the role of such a gene in development. (2) Among the five small-disc reference mutants chosen for this study, we recovered lethal alleles of three of them with no detectable imaginal disc defects. This requires a reinterpretation of our earlier results that only 66 of 134 late lethal mutants caused imaginal disc abnormalities (SHEARN et al. 1971 ) . It is possible that additional alleles of the other 68 mutations would also cause such abnormalities. (3) One advantage of temperature-sensitive muta- tions is that their expressivity can be manipulated by the experimenter. In the absence of temperature-sensitive alleles, the availability of a series of alleles with varying expressivity can provide similar information.

There is no question that the protocol employed to isolate the 24 mutations in this study involved less labor than if they had been screened from among all lethal mutations. To identify these mutations by the latter method would have first required one additional generation for each of the 13,179 chromosomes tested. At the level of EMS dosage employed, 6392 of the third chromosomes recovered would have been expected to contain at least one lethal mutation. It would then have required 31,960 complementation tests, rather than 120 to identify which mutations were allelic to the five reference mutations. On the other hand the latter method would not have required synthesis of the quintuply lethal chromosome, and would have provided us with a large number of other

ALLELES O F SMALL DISC M U T A T I O N S 369

lethal mutations. On balance, however, since the amount of labor saved is sub- stantial and since unspecified lethal mutations are not an especially valuable commodity, we believe that the protocol for isolating alleles used in this study is worthwhile.

We thank PRESLEY MARTIN and WILLIAM SOFER for stimulating discussions during the course of this project.

LITERATURE CITED

ARKING, R., 1975

BAILLIE, D., D. T. SUZUKI and M. TARASOFF, 1968

Temperature-sensitive cell lethal mutants of Drosophila: Isolation and characterization. Genetics 80: 519-537.

Temperature-sensitive mutations in Dro- sophila melanogaster. 11. Frequency among second chromosome recessive lethals induced by ethyl methansulfonate. Can. J. Genet. Cytol. 10: 412-420.

Investigations on the problems of metamorphosis. V. Some factors deter- mining the facet number in the Drosophila mutant Bar. Genetics 24: 494-508.

Mosaic analysis of lethal mutations in Drosophila. Genetics 75: 623-637.

Genetic programming for development in Drosophila. CRC Critical Reviews in Biochemistry 1: 537-570.

Induction, detection and characterization of cell differentiation mutants in Drosophila. Molec. Gen. Genet. 12%: 117-130.

Developmental and histological studies of wild-type and mutant tissues of Drosophila melanogaster. Ph.D. Diss., University of California, Irvine.

Temperature-sensitive mutations in Drosophila melanogas- ter. XII. The genetic and developmental characteristics of dominant lethals on chromosome 3. Genetics 73: 445-458.

LEWIS, E. B. and F. BACHER, 1968 Method of feeding ethyl inethanesulfonate (EMS) to Dro-

BODENSTEIN, D., 1939

BRYANT, P. J. and M. ZORNETZER, 1973

FRISTROM, J. W. and M. A. YUND, 1973

GARCIA-BELLIDO, A. and J. DAPENA, 1973

GATEFP, E., 1972

HOLDEN, J. and D. T. SUZUKI, 1973

sophila males. Drosoph. Inform. Ser. 43: 193.

Carnegie Inst. Washington Publ. 627.

Entwicklungsmech. Org. 178: 353-356.

Int’l Cong. Gen. 1: 213-255. RICE, T. B., 1973

study of early determination in Drosophila melanogaster. Ph.D., Diss., Yale University.

RIPOLL, P. and A. GARCIA-BELLIDO, 1973 Nature New Biol. 241: 15-16.

ROSENBLUTH, R., D. EZELL and D. T. SUZUKI, 1972

LINDSLEY, D. L. and E. H. GRELL, 1968 Genetic variations in Drosophila melanogaster.

MINDER, G., 1972 Metamo’rphosis of imaginal discs of Drosophila melanogaster. Roux’ Arch.

MULLER, H. J., 1932 Further studies on the nature and causes of gene mutation. Proc. of 6th

Isolation and characterization of maternal effect mutants: An approach to the

Cell autonomous lethals in Drosophila melanogaster.

Temperature-sensitive mutations in Drosophila melanogaster. IX. Dominant cold-sensitive lethals on the autosomes. Genetics 70: 75-86.

RUSSELL, M. A., 1974 Pattern formation in the imaginal discs of a temperature-sensitive, cell

SCHUBIGER, G., 1974 Acquisition of differentiation competence in the imaginal leg discs of

lethal mutant of Drosophila melanogaster. Develop. Biol. 40 : 24-39.

Drosophila. Roux’ Arch. Entwicklungsmech. Org. 174: 303-312.

3 70 SHEARN, A., 1974

A. SHEARN et al. Complementation analysis of late lethal mutants of Drosophila melanogaster.

Genetics 77: 115-225. --, 1977 Mutational dissection of imaginal disc development in Drosophila melanogaster. Amer. Zool. 17: 585-594. __ , 1978 Mutational dissec- tion of imaginal disc development. In: The Genetics and Biology of Drosophila, Vol. 2b, edited by M. ASHBURNER and T. R. F. WRIGHT, Academic Press, New York and London (in press).

Genetic control of imaginal disc development in Drosophila. Proc. Nat. Acsd. Sci. U.S. 71: 1393-1397.

Genetic analysis of two allelic, tempera- ture-sensitive mutants of Drosophila melanogaster, both of which are zygotic and maternal- effect lethals. Genetics 89: 341-353.

SHEARN, A., T. RICE, A. GAREN and W. GEHRING, 1971 Imaginal disc abnormalities in lethal mutants of Drosophila. Proc. Nat. Acad. Sci. U.S. 48: 2594-2598.

SIMPSON, P. and H. A. SCHNEIDERMAN, 1975 Isolation of temperature-sensitive mutations blocking clone development in Drosophila melanogaster, and the effects of a temperature- sensitive cell lethal mutation on pattern formation in imaginal discs. Roux’ Arch. Entwick- lungsmech. Org. 178: 247-275.

The recovery and preliminary charac- terization of X-chromosome mutants affecting imaginal discs of Drosophila melanogaster. Develop. Biol. 27: 71-83.

URSPRUNG, H., 1967 In vivo culture of Drosophila imaginal discs. ~ p . 485-492. In: Methods in Developmental Biology. Edited by F. H. WILT and N. K. WESSELLS. Thomas Y. Crowell Co., New York.

Corresponding editor: B. H. JUDD

SHEARN, A., and A. GAREN, 1974

SHE~RN, A., G. HERSPERGER and E. HERSPERGER, 1978

STEWART, M., C. MURPHY and J. W. FRISTROM, 1972