Download Wingless mutation in Drosophila melanogaster

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 melanogaster 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 regeneration 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 homeotic 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 transforms 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 penetrance of the mutation is dependent on the genetic background as well as the tempe*To whom all correspondence should be addressed.
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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 phenotype; these new alleles so isolated were all recessive lethal mutations in the wingless
locus (Babu, 1977). Lethal alleles of wingless were independently isolated by NussleinVolhard 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 permissive 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 morphologically 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 phasecontrast microscope.
Embryonic temperature shift experiments
The embryos were washed and soaked in Voltalev oil (Lehman's, Hemburg). All the
Wingless mutation in Drosophila melanogaster
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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.
(ii) wgts/wg1–1 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.
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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.
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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.
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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 nonpermissive temperature exhibited extensive cell degeneration. The earliest observable 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 degeneration 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-permissive 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
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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.
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Bhat and Babu
Figure 4. A. Whole wing disc preparation
shows extensive cell death in discs from
shifted from the permissive to the
instar. B. Same disc in phase contrast.
grown entirely at permissive temperature.
stained for cell death (as in figure 3). The figure
late third instar larvae (of genotype wgts/wg1–1)
non-permissive temperature at early third
C. Wing disc of the same age and genotype
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 nonpermissive 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
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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 (NussleinVolhard 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 differentiation 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 autonomous. 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
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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.
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