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.