J. Biosci., Vol. 12, No. 1, March 1987, pp. 1–11. © Printed in India.

Wingless mutation in Drosophila melanogaster

S. G. BHAT and P. BABU* Molecular Biology Unit, Tata Institute of Fundamental Research, Bombay 400 005, India MS received 13 June 1986; revised 6 December 1986 Abstract. A temperature sensitive lethal allele of the wingless locus of Drosophila melano- gaster together with previously studied lethal and viable alleles in this locus, has been used to study some properties of this locus. These studies show the existence of two lethal phases for the wingless lesion; one during embryogenesis and another during pupation. By growing embryos with temperature sensitive wingless lesion at the permissive temperature and letting the larvae develop at non-permissive temperature, a large-scale cell death and subsequent regeneration were seen to occur in the mutant wing discs. This cell death followed by re- generation alters the normal developmental potential of the wing disc. Disc transplantation experiments show that these discs are incapable of differentiating into wing blade structures.

Keywords. Drosophila; wingless; temperature-sensitive; disc transplantation.

Introduction

The fruitfly Drosophila melanogaster was chosen as the organism of choice by T. H. Morgan and his coworkers for genetic studies in the early part of this century. These studies have led to the discovery of several major aspects of genetics. Drosophila has again recently become a favoured organism for the study of the genetic basis of development. In order to understand the genetic programming of development, we could classify genes of Drosophila into those with a controlling role and those with a subsidiary function. The likely candidates for controlling genes are the homeotic loci. Homeotic mutations alter the developmental fate of a group of cells; in the mutant fly, instead of building a structure or organ normally produced by these cells, these cells now go on to build a structure or organ found elsewhere in the fly. Thus homeo- tic genes can be thought to control the fate of a battery of genes which have to act in concert to bring about the development of a specific organ or structure of the fly. Several homeotic genes have been identified in D. melanogaster. Mutations in the Bithorax-complex, one such homeotic gene complex, lead to replacement of one body segment or subsegment by another body segment or subsegment (Lewis, 1978) A particularly striking example of mutant combination in this gene complex trans- forms the two halteres into nearly perfect wings; the fly, instead of having two wings, ends up with 4 perfectly formed wings. The mutation wingless (wg1) isolated by Sharma and co-workers (Sharma, 1973; Sharma and Chopra, 1976) appears to be a candidate for a homeotic mutation. In flies homozygous for wingless, the wings are frequently. missing and are replaced by duplicated notum structures. A similar transformation occurs in the metathorax, leading to the frequent disappearance of the halteres. In the originally isolated wg1 mutation, only a fraction of the wings and halteres were transformed. This pene- trance of the mutation is dependent on the genetic background as well as the tempe-

*To whom all correspondence should be addressed. 1 2 Bhat and Babu rature. Earlier, we (Babu, 1977) used the temperature sensitivity of this mutation to show that the penetrance of the phenotype was dependent on the temperature during embryonic development alone. We also isolated new alleles in the wingless locus on the basis that the newly induced mutations fail to complement the wingless pheno- type; these new alleles so isolated were all recessive lethal mutations in the wingless locus (Babu, 1977). Lethal alleles of wingless were independently isolated by Nusslein- Volhard and Wieschaus (1980; also see Nusslein-Volhard et al., 1984) based on an entirely different criterion. In their search for mutations which cause defects in the segmentation pattern, they isolated lethal alleles of wg and showed that in embryos homozygous for these mutations, a defined fraction of each segment was deleted and replaced by mirror image duplication of the reminder. These studies with lethal alleles of wg showed that the pattern malformation and lethality occurred during embryogenesis. More recently, a temperature sensitive lethal allele of wg has been isolated (Nusslein-Volhard et al., 1984). By using this allele we show here that the wingless gene product is needed both in embryonic and larval stages. With embryos which have escaped lethality by completing embryonic development at the permi- ssive temperature, the larval development when subjected to the non-permissive temperature leads to early pupal lethality. The wing discs in these larvae suffer extensive cell death followed by regenerative growth. These discs are morphologi- cally abnormal. We show by disc transplantation experiments that these discs have lost their ability to develop wing-blade structures.

Materials and methods

Mutants in the wingless locus used are listed in table 1. Other mutants used are described by Lindsley and Grell (1968).

Table 1. Alleles of wingless used.

Observation of larval cuticle of wingless larvae Egg collected from the cross were maintained at 25°C for 36 h (or 70 h at 18°C). The unhatched embryos were fixed and cleared as described by Van der Meer (1977) and observed under phase- contrast microscope.

Embryonic temperature shift experiments

The embryos were washed and soaked in Voltalev oil (Lehman's, Hemburg). All the

Wingless mutation in Drosophila melanogaster 3 embryos at the blastoderm stage were set aside. Just at the onset of gastrulation, they were 'staged' and shifted to the required temperature. The growth and observations until 'staging' were done at approximately the same temperature as egg collection. Shift up experiments consisted of shifting the gastrulating embryos from 18°C to 25°C whereas in shift-down experiments, gastrulating embryos maintained at 25°C were shifted down to 18°C. All the unhatched embryos at the end of the experiments were confirmed to be of wingless genotype on the basis of larval cuticular phenotype, by mounting the embryos as described earlier.

Determination of post-embryonic temperature sensitivity

Eggs were collected from the cross

and maintained at 18°C for 40 h, to ensure that embryonic development is completed at permissive temperature. The larve were then shifted to 25°C. When identification by the absence of pigmentation of the malpighian tubules (due to the homozygosity of cn bw lesions) becomes possible, these wgts/wg1–l larvae were segregated and allowed to develop at 25°C.

Observation of cell death in imaginal discs

Degenerating cells were localised in the whole disc preparations using the method of Spreij (1971). Wing discs were dissected in Drosophila Ringer medium from second and third instar larvae and prepupae. The discs were placed in Acridine Orange (2×l0-6M in Drosophila Ringer) and photographed within 5 min using Zeiss Fluorescence Microscope. Cell death patterns were obtained from wing discs from the following: (i) wgts/wg1–1 larvae (second and third instar), and prepupae, shifted up from 18°C to 25°C just at the time of hatching. ts 1–1 (ii) wg /wg third instar larvae and prepupae, shifted up from 18°C to 25°C at early third instar stage. (iii) wgts/wg1–1 larvae entirely grown at 16°C.

(iv) larvae (control) grown at 25°C.

(v) wg1/wg1 larvae grown at 25°C.

Anatomy of discs

Discs were fixed in acetic acid-alcohol-formaldehyde=2:4:1 for 2h with two changes and embedded in Durcupan ACM (Fluka) araldite. Sections (2–3) µ thick) were cut using LKB Pyramitome and stained with 0·1% toulidine blue+0·1 % methylene blue in Borax solution. 4 Bhat and Babu

Disc transplants

Discs from larvae were dissected out as described earlier, in sterile Drosophila Ringer and were injected into young third instar, y wch larvae. The host larvae were successively washed with 30% (v/v) ethanol, 1% (w/v) trichloroacetic acid followed by 3–4 changes of distilled water and anaesthetised with ether prior to injection. These larvae after injection were grown at 18° or 25°C in various experiments. After the adults emerged, the metamorphosed mass was dissected out from the abdomen, washed in alcohol and mounted in Struhl's mounting medium (Struhl, 1981).

Results

Preliminary observations on the temperature sensitive lethality of the wgts allele either as homozygote or in trans with the lethal alleles wg1–1 or wgl –6 indicated that there are two separate temperature sensitive lethal phases, one embryonic and another late larval/pupal. Further, for the embryonic development, temperatures upto 20°C were found to be permissive whereas 16°–18°C was found to be the permissive temperature for larval development. Even when grown at 16°C during the entire life cycle, only a small proportion of wgts/wg1–1 (or wgts/wg1–6) adult flies emerge from pupae; large majority of such adults are unable to eclose. These wgts/ wg1–1 (and wgts/wg1–6) flies grown at permissive temperatures have normal wings, halteres and eyes unlike wg1/wg1 flies. However, a large proportion of them have defective legs. We list below our observations on the effect of wg lesion on embryogenesis and on the development of imaginal discs during larval stage.

Larval cuticle and germband extension wgts/wgts and wgts/wg1–2 embryos, when grown at non permissive temperature show impaired segmentation pattern similar to that of wg1 homozygotes (Nusselien Volhard and Wieschaus, 1980). One such cuticle is shown in figure 1A. When embryos were collected at 18°C and shifted to 25°C soon after gastrulation, partial cuticular defective phenotype, as shown in figure 1B were often obtained. It was also seen that in wgts/wgts or wgts/wg1–2 embryos grown at 25°C the germ band did not extend properly, when observed in oil. The extension of the germband, which is part of the gastrulation process is probably the earliest developmental event of embryogenesis where an abnormality is noticeable in wingless lethal embryos.

Embryonic temperature sensitive period

The results of shift up and shift down experiments on the embryos carrying the temperature sensitive allele are shown in figure 2. It is clear from this figure that the temperature sensitive period for embryonic lethality lies approximately 2h after gastrulation. This corresponds to a stage when the normal extension of germband is believed to be completed. Wingless mutation in Drosophila melanogaster 5

Figure 1. Typical examples of cuticular patterns of wgts/wg1-2 embryos. The embryos were grown (A) entirely at the non-permissive temperature (25°C) and (B) grown at the permissive temperature (16°C) until just before the onset of gastrulation and shifted to the non-permissive temperature since that stage. Partial cuticular mutant pattern can be seen in (B).

Figure 2. Estimated embryonic lethality (per cent) of wgts/wg1-1 embryos. The embryos were shifted up from the permissive (16°C) to the non-permissive (25°C) temperature at various times after onset of gastrulation (●) and shifted down from the non-permissive to the permissive temperature after onset of gastrulation (Ο). The percentages are estimated by multiplying the proportion of dead embryos (confirmed for wingless cuticular phenotype) by 4 from all eggs laid in the cross:

Development time at 16°C is divided by 2 to normalise it to a developmental rate at 25°C.

6 Βhat and Babu

Post-embryonic temperature sensitivity wgts/wgts and wgts/wc1 larvae when shifted to 25°C after embryogenesis appear to grow normally. However these larvae on pupation turn brown and die. Thus there is a second temperature sensitive lethal period in the development of wgts homozygotes (or trans heterozygotes with other lethal alleles studied) some time during larval or prepupal stage. No attempt was made to determine the precise developmental stage in the larval period which is temperature sensitive. When these larvae are grown at 25°C, the discs are abnormally small in size and lack all the folds of a wild type disc. In the latter half of third instar, i.e. towards the end of larval phase, the wing disc starts regenerating rapidly. We describe below our observations on the imaginal discs from mutant larvae grown at non-permissive temperature.

Cell death in imaginal discs

When stained for cell death, the mutant wgts/wg1–1 wing discs grown at non- permissive temperature exhibited extensive cell degeneration. The earliest observ- able cell death pattern emerged at late second instar larva (figure 3A,B). A band of degenerated cells can be localised in the central region of the wing disc, roughly perpendicular to the long axis of the disc. Figure 3C show cell death patterns at a somewhat later stage of development; the corresponding phase-contrast picture (figure 3D) indicates a complete lack of folds as compared to the wgts/wg1–1 disc grown at 16°C (figure 4D) or wild type disc (figure not shown). In the disc grown at the permissive temperature, cell death appears to be confined to the proximal regions. Even if the wgts/wg1–1 larvae were shifted to the non-permissive temperature as late as early third instar, considerable cell degene- ration appears in the disc by late third instar stage (figure 4A,B). Comparing the distribution of dead cells in this disc with a wgts /wg1 disc grown entirely at 16°C (figure 4C,D) it can be seen that cell death in the non-permissive temperature is spread over the entire disc, whereas cell death of wgts/wg1 disc at the permissive temperature (figure 4C) or in the wild type control disc (figure not shown) is confined to a small area. By the pupal stage, the mutant wing disc grown under non-permi- ssive temperatures shows very little cell degeneration; however the disc is abnormal in shape indicative of massive cell regeneration. Sharma and Shekaran (1983) have studied the cell death pattern of the mutant wg1 wing discs. The cell death pattern of wgts /wg1–1 discs grown at the non-permissive temperature during larval stage are somewhat similar to the pattern of cell death obtained for wg1.

Types of cells in the mutant wing disc

There is a population of cells referred to as adepithilial cells found in imaginal discs (for detailed description see Crassley 1978). These are believed to be the progenitors of certain muscles in the adult fly. These adepithilial cells are cytologically recognisable in the imaginal discs. Figure 5A shows 2 µ thick horizontal section of the wgts/wgl–l wing disc from a prepupa which was shifted to the non-permissive temperature (25°C) at the first instar stage. Comparing this section with the section

Wingless mutation in Drosophila melanogaster 7

Figure 3. Whole wing disc preparations from wgts/wg1–1 larvae showing cell death pattern. The eggs were collected and allowed to hatch at 18°C; subsequent development took place at 25°C. Acridine Orange was used as the vital dye to reveal dead cells and photographed with blue filter. A. Wing disc from second instar larvae (about 35 h at 25°C). B. Same disc in phase contrast. C. Wing disc from early third instar larva (about 55 h at 25°C). D. Same disc in phase contrast from control disc (i.e. wgts/wgl–l disc from larva grown at the permissive temperature) shown in figure 5B, it can be seen that adepithilial cells are more extensive and form a continuous layer when growth occurs at the non-permissive temperature whereas adepithilial cells are seen only in the proximal regions in the disc grown at the permissive temperature. This is an identifiable difference in the cell type distribution in the mutant wing discs grown in permissive or non-permissive temperature.

Differentiation potential of the mutant wing disc wgts/wgl–1 larvae were shifted to the non-permissive temperature (25°C) at early second larval instar stage and discs dissected out at late third larval instar stage.

8 Bhat and Babu

Figure 4. A. Whole wing disc preparation stained for cell death (as in figure 3). The figure shows extensive cell death in discs from late third instar larvae (of genotype wgts/wg1–1) shifted from the permissive to the non-permissive temperature at early third instar. B. Same disc in phase contrast. C. Wing disc of the same age and genotype grown entirely at permissive temperature. D. Same disc in phase contrast.

These discs were implanted in y wch larvae grown at 25°C (11 successful implants) or 18°C (3 successful implants). In all these cases the implants show only notal structures after metamorphosis. No wing blade structures are seen. Typical cases are shown in figure 6(A,B)· These studies show that irrespective of the temperature at which host larvae and pupae develop, the discs from donor larvae grown at the non- permissive temperature seem to have lost their potential to differentiate into wing blade structures. In contrast, wgts/wg1 discs from larvae grown entirely at the permissive temperature (16°C) differentiate wing blade structures even when host development takes place at 25°C. Seven such successful implants were observed and a typical case is shown in figure 6C. Figure 6D is that of a transplanted (mutant) wing disc from wg1 larvae, which also has only notal structures as expected. Wingless mutation in Drosophila melanogaster 9

Figure 5. A. Horizontal section through a wgts/wg1–1 wing disc from prepupa. The prepupa, was shifted to non-permissive temperature at first instar stage. B. Horizontal section through a wing disc of same genotype and same developmental stage, but grown at permissive temperature.

Discussion

The wingless locus, which was uncovered more than a decade ago by Sharma (1973), has proved to be an interesting developmental locus of Drosophila. The original temperature sensitive viable allele has an embryonic temperature sensitive period (Babu, 1977). The lethal alleles, isolated since, have defects in embryogenesis itself again demonstrating the need for wg+ gene product in embryogenesis (Nusslein- Volhard and Wieschaus, 1980). In this study using a temperature sensitive lethal allele, we find that wg+ gene product is needed during embryogenesis. But our studies also show that the role of wg+ product is not limited to the embryonic stage alone. Embryos carrying the temperature-sensitive lethal wg lesion and grown at permissive temperature during embryogenesis, when subjected to non-permissive temperature during larval development have abnormal wing discs. These discs have extensive cell death followed by regenerative growth; but these regenerated are morphologically and cytologically abnormal. They also have impaired differentia- tion potential. Thus wg+ gene product appears to be needed for normal development of the wing discs and presumably other discs as well. Garcia Bellido (1975) had suggested a classification of homeotic genes into two classes. One class, referred to as selector genes, are needed to select and maintain a particular developmental pathway. Such genes are also expected to be cell autono- mous. Mutations such as bithorax presumably belong to this category. Another class of genes, referred to as activator genes, whose products are needed to activate, once and for all times, a given state of development through a particular selector gene. These activator genes are likely to be cell non-autonomous. Unlike most known homeotic mutations, wingless mutation is cell non-autonomous (Morata and Lawrence, 1977) and hence-is a candidate for activator mutation. It is however, disc autonomous (Bhat, 1985; Babu and Bhat, 1986). Our observation that wingless gene product is needed during both embryonic and larval stages argues against the

10 Bhat and Babu

Figure 6. A and B. Implants after metamorphosis of wing discs from wgts/wg1–1 larvae shifted to non-permissive temperature at early third instar stage and discs dissected out at late third instar stage and injected into host larvae. A. Host larval and pupal development taking place at the non-permissive temperature. B. Host larval and pupal development taking place at the permissive temperature. C. Corresponds to donor embryonic and larval development taking place at the permissive, with host larval and pupal development taking place at the non-permissive temperature. D. Transplant of a (mutant) wg1 wing disc for comparison. Note that only (C) contains identifiable wing blade structures. Wingless mutation inDrosophilamelanogaster 11

possibility of wingless being an activator-like locus. Studies of the function of wingless product at the molecular level will presumably settle this question.

Acknowledgement

We thank Peter Lawrence for comments on an earlier version of this manuscript. Gary Struhl's keen interest in the embryological studies is gratefully acknowledged. We are grateful to Janni Nusslein-Volhard and Eric Wieschaus for providing the wgts allele used in this study. We thank the reviewers of this paper for help in improving upon our earlier draft. This work was initiated by one of us (S.G.B.) during a vsit to M.R.C. Laboratory of Molecular Biology at Cambridge; this visit was supported by the British Council.

References

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