Copyright 0 1982 by the Genetics Society of America

STUDIES ON THE SEX-SPECIFIC LETHALS OF DROSOPHILA MELANOGASTER. IV. GYNANDROMORPH ANALYSIS OF THREE

MALE-SPECIFIC LETHALS, mle, m ~ l - 2 ~ ~ AND mle(3)132

T. UENOYAMA, S. UCHIDA, A. FUKUNAGA AND K. OISHI"

Department of Biology, Faculty of Science, Kobe University, Nada, Kobe 657, japan

Manuscript received December 3, 1981 Revised copy accepted May 21, 1982

ABSTRACT

Mutants at three male-specific lethal loci of Drosophila melanogaster (mle, m ~ l - 2 ~ ' and mle(31132) were examined by gynandromorph analysis. In all cases only a very few gynandromorphs with small X/O patches appeared. Most of these small X/O patches were in the abdomen, and the structures in these X / O regions were reduced in size. These results indicate that the primary effects of these mutants are not on any particular organs or tissues, but rather on individual cells. mle and msl-2 have been shown by BELOTE and LUCCHESI (1980a) to be defective in dosage compensation in X/Y males. We suggest that this dosage-compensation defect results in the expression of Minute-like phe- notypes in X/O cells, and hence results in the death of X/O males and flies with large X/O tissue areas.

HE analysis of sex-specific lethals in Drosophila melanogaster that are T lethal specifically to chromosomal females or males may be expected to provide clues to the understanding of the two processes, dosage compensation and sex determination, both of which are known to be based on the X:A balance. Sex-specific lethals should represent the loci that are normally ex- pressed in only one sex, but whose products are essential for that sex and/or deleterious for the other. The recent finding that several male-specific lethal loci and a female-specific lethal locus all show a profound effect on the rate of transcription of X-linked genes (BELOTE and LUCCHESI 1980a; LUCCHESI and SKRIPSKY 1981) emphasizes the importance of the studies on these lethals. An earlier suggestion by CLINE (1978) that the sex-specific lethal effects of da (daughterless, 2-41.5) and various alleles (both male-specific and female-specific) of Sxl (Sex-lethal, 1-19.2) are caused by their involvement in the process of dosage compensation has thus been supported.

Gynandromorph analyses of sex-specific lethals have been performed by CLINE (1976, 1979) for da and SxlM#' (a dominant male-specific lethal). The survival of da gynandromorphs was very low and the X/X tissues occupied only a small fraction. Furthermore, the differentiation of X/X tissues was very poor and abnormal. The Sxl'#' /+//SxIM#l/O gynandromorphs also appeared at a very much reduced frequency and the X/O tissues were a small fraction compared to the control. These X/O tissues in gynandromorphs differentiated as females in sexually dimorphic structures indicating a role of Sxl in primary sex determination. It was concluded that there were no single local lethal foci for da and SxlM#'.

In our previous paper (TANAKA, FUKUNAGA and OISHI 1976), we reported that To whom correspondence should be addressed

Genetics 102 223-231 October, 1982.

224 T. UENOYAMA ET A L .

males homozygous for mle (maleless, 2-55.2), a nonmaternal-effect male-specific lethal, can often differentiate head and thoracic structures (first legs with the sex-comb) as well as genitalia (male) in dead pharate adults but rarely abdominal tergites and sternites. It thus appeared important to determine whether there is a distinct focus of primary action in mle. We here present results of gynandro- morph analyses using three nonmaternal-effect autosomal male-specific lethals and discuss our results in the light of the recent finding noted above.

MATERIALS AND METHODS

Ten chromosomes each of which conferred male-specific, nonmaternal-effect lethality when homozygous were examined (Table 1). These were grouped into five genes by complementation tests (data not shown). Three chromosomes each carrying a different male-specific lethal were chosen, and their effects in gynandromorphs were examined. Gynandromorphs heterozygous for each male-specific lethal served as internal controls.

Two kinds of crosses were made to generate gynandromorphs. Cross 1 involved X, y w spl/X, In(1)w"'"; Balancer/male-specific lethal females and X, y w spl/B"Y; Bolancer/mole-specific lethal males. In Cross 2, In(l)wWc was introduced through the male parents: y w spl/y w spl; Balancer/ male-specific lethal X In(l)wuC/BSY; BaJancer/male-specific lethal. Progeny flies were examined at -4 X 20 magnification by using the y character as a marker for X/O in cuticle, hairs and bristles, and the w character in compound eyes. In the present experiments, gynandromorphs appeared at lower and In(l)w"'/B"Y males at higher frequencies in contrast to the generally high and low, respectively, frequencies in original stocks. The reason for this is not clear, but perhaps reflects the particular genetic background or stabilization of the In(l)wUc chromosome. For examples of the mechanism of gynandromorph production and the reasoning behind gynandromorph analysis, see HALL, GELBAKT and KANKEL (1976) and ZALOKAR, EKK and SANTAMARIA (1980). For characteristics of mutants and chromosomes refer to LINDSLEY and GKELL (1968).

Imaginal disc transplantation experiments were performed as previously described (TANAKA, FUKUNAGA and OISHI 1976).

Flies were reared at 24 k 1' on a standard yeast-sucrose-agar medium with propionic acid (3.5 ml/liter of medium) as a mold inhibitor.

RESULTS

Autosomal, nonmaternal-effect male-specific lethals: Nine independently de- rived second chromosomes, each of which was homozygous male-specific lethal, were examined (Table 1). None of these chromosomes exhibited maternal effects; that is, homozygous males died regardless of the maternal genotype. Complementation tests revealed that these comprise four groups: (1) 350, K25, 79K, 23, and mlets; (2) msl-2; (3) msl-2 and 27; (4) C52. Since the alleles 350, K25 and 79K were recovered from the same natural population at Katsunuma it is likely that they are repeats of the same mutant. 350 (= mle) and mletS have been characterized previously (FUKUNAGA, TANAKA and OISHI 1975; TANAKA, FUKUN- AGA and OISHI 1976; BELOTE and LUCCHESI 1980a, b). Thus there are at least three alleles of independent origin at the mle locus: mle, mleL3, and mlet5. Homozygous 13 females showed an apterous (ap, 2-55.2) phenotype and died within a day after eclosion. Three up alleles (apXa, ap5? and ap4) were each test-crossed with 13. In all combinations, both females and males eclosed and showed the ap phenotype. These results indicate either that the 13 chromosome carries two independently induced close mutations or that it has a small deletion that uncovers both mutant loci.

Group 2 has two mutant allelesi msl-1 and msl-lb (BELOTE and LUCCHESI 1980b). The two alleles in group 3 have different phenotypes: homozygous 27 males are slow in development and die as small third instar larvae whereas

SEX-SPECIFIC LETHALS OF DROSOPHILA 225

TABLE 1

Chromosomes with male-specific, nonmaternal-effect lethal actions examined ~~ ~ .~

Chromo- Lethal

350 1 (Katsunuma, Japan)

some gene Origin Discoverer Reference

OSHIMA (1971) FUKUNAGA, TAN- AKA and OISHI (1975)

TANAKA, FUKUN-

mle 2-55.2 Spontaneous in a natural population

K25 79K J 13

mlek

msl-1

msl-2

27

C52‘

132

m 1 e ~ z 5

m1e13

mle” 2-56.8

msl-1 2-53.3

msl-2 2-9.0

m ~ J - 2 ~ ~

(41A-43A)

(36F7-37B8)

multiple

mle(3)232 3- 25.8

EMS induced in an Oregon-R chromo- some

EMS induced in a cn bw chromosome

EMS induced in a cn bw chromosome

EMS induced in a cn bw chromosome

EMS induced in an Oregon-R chromo- some

Spontaneous in a lab- oratory population (Mishima, Japan)

natural population (Katsunuma, Japan)

Spontaneous in a

AGA and OISHI (1976)

WATANABE (1978) WATANABE (1979) SUGIMOTO and

OISHI (1980)

BELOTE (1977)

BELOTE (1977)

BELOTE (1977)

BELOTE and Luc- CHESI (1980a, b)

BELOTE and Luc- CHESI (1980a, b)

BELOTE and Luc- CHESI (1980a, b)

OISHI (1976)

WATANABE (1976)

WATANABE (1979) UCHIDA, UENO- YAMA and OISHI (1981)

_ _ _ _ _ ~ ~

Probably represent the same allele recovered repeatedly from the same population. Slightly leaky in male-killing action.

homozygous msI-2 males develop up to the late third instar or prepupal stage (BELOTE and LUCCHESI 1980b). The last group contains only one chromosome, C52. Its lethal action is slightly leaky. We were not able to map the lethal gene to a single locus. Instead, it appeared that the lethality was caused by at least two loci both of which had to be homozygous. Furthermore, homozygous C52 females are completely sterile and have grossly underdeveloped ovaries; this phenotype also appears to be under polygenic control.

There is at present only one male-specific lethal on the third chromosome, mIe(3)132 (UCHIDA, UENOYAMA and OISHI 1981). Some additional information is now available. X/Y; mIe[3)132/mIe[3)132 males live as larvae for more than 10 days but never pupariate. The 10-day-old larvae are about the size of normal mid-third instar larvae, but the imaginal discs are disproportionately small. Most discs are recognizable only by their positions and not by their character- istic shapes, Nevertheless, when transplanted onto mid-third instar Oregon-R male larvae and induced to metamorphose, some discs show nearly complete (wing, leg) or partial (eye-antennal) differentiation. All four host males that eclosed from larvae that were implanted with discs from the wing-disc position had upon dissection wing and notum structures with hairs and bristles com- pletely differentiated. Two of four host males that eclosed after implantation of discs from the leg-haltere position had differentiated structures; in both cases

226 T. UENOYAMA ET AL.

the tissue was leg. Sex combs and other bristles were recognizable but the claws were missing. Three of six hosts that eclosed after implantation with eye- antennal discs (barely recognizable by their characteristic shape in the donor) contained differentiated structures. The antennal segments were complete but compound eyes were not recognizable; no ommatidia were observed. In all these cases the differentiated structures appeared normal in size. This may suggest that the imaginal disc cells are able to divide and grow when trans- planted into the normal mid-third instar hosts.

Altogether, it is apparent that the number of male-specific, nonmaternal- effect lethals on the second and third chromosomes is not very large since a limited number of loci on the second chromosome has been recovered repeat- edly in separate experiments (see also BELOTE and LUCCHESI 1980b).

Gynandromorph analysis af mle, msl-ZZ7, and mle(3)132: Three alleles each representing a different locus were chosen to be examined by gynandromorph analysis. All of these alleles are known to be lethal only to single-X individuals regardless of the phenotypic sex (FUKUNAGA, TANAKA and OISHI 1975; UENOY- AMA, unpublished; UCHIDA, UENOYAMA and OISHI 1981; BELOTE and LUCCHESI 1980b).

Table 2 shows the results for mle. In Cross 1, in which the unstable ring-X chromosome, Ir1(2)w"~, was introduced maternally and lost in the progeny to produce gynandromorphs, no X/Y or X/O; mle/mle progeny flies appeared and oniy 2 gynandromorphs were obtained. The percentages of progeny of various genotypes relative to y w spl/y w spl are shown in parentheses. If X/O cells of putative gynandromorphs had been killed in homozygous mle and those indi- viduals had survived as X/ln(2)wuc, there should have been proportionally more y w spl/In(l)wUC females in mle/mle than in the mle/SMZ control; the converse was observed. Moreover, few of the y w spl/In(l)w"C flies had gross structural deficiencies that might be caused by extensive cell death. Consequently, the very few homozygous mle gynandromorphs (3% of the expected number) that survived do not appear to be an underestimate.

Among the total of seven mle/mle gynandromorphs (Table 2), five had a small y (presumably X/O) patch in the abdomen, one had a single j r ( X / O )

TABLE 2

Results of two gynandromorph-producing crosses for mle

Crass 1 Cross 2 - -_ -____.

Genotype CY Non-Cy CY Nun-Cy

- Y w SPVY w SPl 4522 (100) 1841 (100) - y w spl/B"Y 4150 0 6019 0

In (I) w U r / ~ " Y 501 0 - - y w spl/y w spl/B"Y 0 1 1 0 y w spl/In(l)w""/B"Y 7 2 1 0 Y w SPl/O 302 0 362 0 Gynandromorph 177 (4) 2 (0.1) 168 5

y w spl/In(l)w"C 2094 (46) 768 (42) 2655 941

Cross 1, Y w sPl/ln(l)w"L'; mWSM1, Cy x y w spl/B'Y; mle/SMl, Cy. Cross 2, y w spl/y w spl; mle/SMl, Cy x In(l)wvC/B"Y; mle/SMZ, Cy. The percentages of progeny of various genotypes relative to y w spl/y w spl are shown in parentheses.

SEX-SPECIFIC LETHALS OF DROSOPHILA 227

thoracic bristle, and the last had a large area of X/O tissue over the head, thorax and abdomen. In all of the first five mosaics above the y (X/O) bristles themselves were very small and the hemitergites were reduced in size (Figure 1). The mosaic with a single y bristle might have been a somatic mutation. The last mosaic individual was abnormally small in size.

Table 3 shows the results for m~J-2'~. They are very similar to those for mle in all respects except that 4 X/Y; m ~ 1 - 2 ~ ~ / m s l - 2 ~ ~ flies survived in Cross 1, and two mosaic individuals in which the y structures were female in phenotype occurred in the control. The latter probably represent 3X-2X mosaics resulting from the loss of In(1)wuC in 3X individuals that were produced by X chromosome nondisjunction.

The only gynandromorph homozygous for m ~ l - 2 ~ ~ obtained here had six abdominal hemitergites and four hemisternites with y (X/O) phenotype. Again the hemitergite and sternite structures as well as the bristles themselves were very much reduced in size.

Table 4 shows the results for mIe(3)132. Two out of three mosaics obtained had X/O tissues in the abdomen (Figure 2). Again both the X/O structures and bristle size were very much reduced. The third fly had two y bristles on the thorax. Although not recorded exactly, we noted that more than 10 y w spl/ In(l)wuc; mfe(3)132/mIe(3)132 flies out of 750 had part of their abdominal structures (mostly tergites) deleted. Only a few (less than 5 among 1888) y w spl/In(Z)wuc; mle(3)132/TM3 control flies were found with similar defects. These apparent nonmosaics may also have been gynandromorphs in which the male tissue failed to produce cuticle.

FIGURE 1.-Examples of mosaic structures in mle/mle gynandromorphs. X/O areiis iire shown by dotted line. vp. Vaginal plate: ap. anal plate: 31-71. third to seventh tergite: 4s-7s. fourth to seventh sternite. Arrow indicates spermatheca which remained in preparation. A and R are the same individual. Note reduced hemitergite and smaller bristles in X/O areas.

228 T. UENOYAMA ET AL.

TABLE 3 Results of two gynandromorph-producing crosses for msl-2"

Cross 1 Cross 2

Genotype CY Non-Cy CY Non-Cy

3957 (100) 1764 (100) - - Y w SPJ/Y w SPJ y w spl/B"Y 3759 4 1425 0

In(l)w"'/B"Y 95 0 y w spl/y w spl/B"Y 0 0 0 1 y w spl/ln(l)w"C/BsY 7 10 0 0 Y w SPV0 213 0 89 0

Gynandromorph 145 (4) l(O.1) 52 0

y w spI/In(1)wUC 2072 (52) 813 (46) 912 360 - -

3X-ZX mosaic 2 0 0 0

Crosses 1 and 2 are the same as in Table 2 except that 1nsJ-2~~ is used in place of mle.

TABLE 4 Results of gynandromorph-producing cross for mle (3)132: y w spl/In(l)wVC;

mle(3)132/TM3, Sb Ser X y w spl/B"Y; mle(3)132/TM3, Sb Ser

Genotype Sb Ser Non-Sb Ser

Y w SPVY w SPl 3576 (100) 1413 (100)

y w spl/ln(l)wuc 1888 (53) 750 (53) y w spJ/B"Y 3293 0

ln(l)w"'/ B'Y 159 0 y w spJ/y w spJ/B"Y 0 1 y w spl/In(l)w"C/BsY 13 3 Y w SPl/O 169 0 Gynandromorph 161 (5) 3 (0.2)

DISCUSSION

The present results show that only a very few gynandromorphs homozygous for any one of the three autosomal male-specific lethals examined survive to adulthood, and that in those rare cases the X/O cuticular regions are primarily seen in the abdomen, are small in size, and the structures which they encompass such as bristles are themselves smaller in size. It would thus appear that none of these lethals have a well defined focus of primary actions (either larval, adult external or adult internal). Ordinarily these results would indicate that the male-specific lethals act as cell lethals. We did not, however, observe deletions or duplications, which would be expected if these were cell lethal, in adult cuticular structures except perhaps some in the abdomen.

Our results also show that at least certain imaginal discs of mie(3)132/ mle(3)132 males can grow and differentiate into adult structures when trans- planted into Oregon-R male larvae. Similarly major imaginal discs (eye-anten- nal, leg, wing, haltere, and genital) of mJe/mle larvae were shown to differentiate when transplanted into Oregon-R hosts (TANAKA, FUKUNAGA and OISHI 1976). Furthermore, adult head and thoracic structures were often observed to be completely differentiated whereas abdominal structures were rarely so in the dead mle/mJe pharate adults (TANAKA, FUKUNAGA and OISHI 1976). These observations and those mentioned above seem to contradict and need to be explained.

In all the studies concerned with autosomal male-specific lethals (TANAKA,

SEX-SPECIFIC LETHALS OF DROSOPHILA 229

FIGURE 2.-Examples of mosaic structures in mle(3)132/mle(3)132 gynandromorphs. X/O areas are shown by dotted line.

FUKUNACA and OISHI 1976 BELOTE and LUCCHESI 1980b; UCHIDA, UENOYAMA and OISHI 1981; and present results), it has been noted that homozygous male larvae are slow in development and have late lethal phases. These results may suggest the involvement of these lethals in the slow rate of cell multiplication. This is consistent with the observations of BELOTE and LUCCHESI (1980a). In male larvae homozygous for autosomal male-specific lethals, mle", msl-I, msl- Ib and msl-2, significant and specific reduction (to ca. 60% of the control values) were observed in the enzyme activities of X-linked genes. They also showed for mleb male larvae that the polytenic X chromosome was thinner (although its DNA content appeared normal) and that the rate of X chromosome transcription was reduced to ca. 65%.

Incomplete dosage compensation in these males would result in effective aneuploidy, which by itself might explain the lethality. We might also draw the analogy to the actions of Minute loci. There exist seven or more Minute+ loci on the X chromosome. Mutations that impair the function of these loci, known as Minute (M), are lethal in homozygous and hemizygous conditions, cause slow development as well as morphological abnormalities such as smaller bristles, and are dosage sensitive (LINDSLEY and GRELL 1968). MORATA and RIPOLL (1975) have shown by somatic crossing over experiments that M/M+ cells divide slowly and that M/M+ clones in an M+/M+ background are found only when they are induced relatively late in development and consist of a few cells when examined in the wing disc derivatives. Apparently M/M+ clones induced early in development are outgrown by rapidly dividing M+/M+ cells. This cell com-

230 T. UENOYAMA ET A L .

petition occurs, however, only mildly when M/M' clones are induced in abdominal histoblasts, perhaps reflecting the distinctive behavior of these cells in multiplication pattern (MADHAVAN and SCHNEIDERMAN 1977). We therefore assume that incomplete dosage compensation in larval tissues is primarily responsible for the death of males homozygous for these male-specific lethals, with one of the overt effects being the expression of M. We expect that X/Y; male-specific lethal/maJe-specific lethal cells have lowered amounts of various M + products and are strongly M in phenotype with slow cell division rate and slow development. In fact BELOTE and LUCCHESI (1980b) observed that homo- zygous mletS males that appeared under the permissive condition had thin bristles and rough eyes, a phenotype similar to that of M.

Gynandromorphs that have only a few cells XO are expected to survive; these cells may be recovered as adult structures if they happen to occupy abdominal histoblasts, but in major imaginal discs they would be outgrown by surrounding X/X cells and would not appear in adults. Gynandromorphs that have large numbers of X/O cells die, but transplanted imaginal discs from third- instar mJe and mle(31232 males survive and produce normal-looking adult structures. We suggest that when these imaginal cells have the opportunity to extend their development, as they do in the transplantation experiments, no matter how slowly they divide, they may produce normal adult tissues; however, if they are in a gynandromorph, then differentiation is precocious for the X/O cells and they produce reduced structures. Here we note similarities to l(2)ts- 2226 reported by SIMPSON and SCHNEIDERMAN (1976). These authors showed that pulses of restrictive temperature given during the third larval instar pro- duced no delay in subsequent development hut resulted in small-sized flies, whereas pulses during earlier stages caused prolonged development but normal- sized adults. Furthermore, they observed that homozygous mutant clones on the tergites had shorter and stunted bristles.

We thank T. K. WATANABE, J. C. LUCCHESI and P. OSTER for kindly supplying us with fly stocks.

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