/ . Embryol. exp. Morph. Vol. 53, pp. 225-235, 1979
Printed in Great Britain © Company of Biologists Limited 1979
225
The time of action of three mutations affecting
Drosophila eye morphogenesis
By ROBERT RANSOM 1
From the lnstitut fur Biologie III Universitdt Freiburg,
West Germany
SUMMARY
Histology and clonal analysis are used to look at the time of action of the mutant Drosophila genes eyeless2, eyeless dominant and sine oculis. The findings suggest that eyeless
dominant has its effect during the first two larval instars, whilst eyeless2 and sine oculis act
during the third larval instar.
INTRODUCTION
The three genes studied here, so, sine oculis; eyB, eyeless dominant and ey2,
eyeless2 all affect the development of the Drosophila eye and head. Histological
observation by Fristrom (1969) has already suggested that cell death occurs in
the ey2 late third larval instar. How do eyB and so compare with this? A combination of histology and clonal analysis have been used to investigate the
time of action of the three genes.
Genetically, two chromosomal regions are involved, so is a recessive point
mutation on the right arm of the second chromosome mapping at position 56-7.
ey2 is likewise a recessive mutation on the fourth chromosome (4-2-0). eyB
is dominant, and has been identified cytologically as a reversed repeat of about
11 bands (Bridges, 1935) in the same region as ey2.
MATERIALS AND METHODS
For staged histological studies, eggs of appropriate genotypes were laid for
3 h periods, and cephalic complexes of the resulting larvae were dissected out
and fixed at 12 h intervals from 72 to 132 h. A minimum of three head discs
of each genotype was studied at each age. All rearing was done at 25 °C.
Imaginal discs or adult heads fixed and embedded in Araldite (Hofbauer &
Campos-Ortega, 1976) were used for observation by light microscope. Discs
or heads (1-2 /tm) were sectioned on an LKB ultramicrotome and were stained
with 1 % toluidine blue. Pycnotic cells are stained an intense blue by this
method.
1
Author's present address: Department of Biology, The Open University, Walton Hall,
Milton Keynes, MK7 6AA, U.K.
226
ROBERT RANSOM
Fig. 1. For legend see opposite.
Drosophila eye mutations
227
For cell lineage analysis, the mutant genotypes were marked with yellow
white forked36'1 (y, 1-0-0; w, 1-1 -5;/ 3 6 a , 1-56-7), y vt;/36a females being mated to
males of the appropriate eyeless genotype. For the ey2 and so series, the females
were also homozygous for ey2 and so respectively. The female offspring of the
crosses, genotypically y wfSGa/ + + + on the first chromosome had clones of
homozygous y jv/ 36a tissue after X-ray induced mitotic recombination. Details
of all these genotypes may be found in Lindsley & Grell (1968).
Eggs and larvae were irradiated with 800R X-rays (125 kv, 15 mA 0-7 mm
Al nitration, dose of 400R/min). This relatively low dose was used firstly to
minimize eye scarring due to cell death, especially in the third larval instar, and
also to reduce lethality in X-ray-sensitive eyB larvae. Female flies emerging were
scored for white clones in the eyes under a binocular microscope, and were then
decapitated, the heads being either mounted on slides in DPX after a brief
sojourn in 95% alcohol to dissolve away red eye pigment or else used for
histology. The mounted heads were scored for y /3Gft bristles. Irradiations were
performed on eggs laid over a 24 h period, with mean ages at irradiation of 12
up to 108 h in 12 h steps. To check for abnormalities in general growth rates of
flies carrying mutant eye genes, y w3Ga clones were scored on the legs of flies
of the four genotypes studied, after mounting on slides in DPX. Descriptions of
the phenotypes of the mutants are given in an abbreviated form in Lindsley &
Grell (1968) and further descriptions of ey2 in Medvedev (1935), El Shatoury
(1963) eyB in Arking, Putnam & Schubiger (1975); so in Milani (1946), Hofbauer
& Campos-Ortega (1976). Fig. 1 and Tables 1 and 2 show the most important
details of the expression of the mutations.
RESULTS
1. Histological observations
Previous studies of eyeless2 imaginal discs (Fristrom, 1969) indicated that
cell death is instrumental in producing this phenotype in the third larval instar.
Similarly, Hofbauer & Campos-Ortega (1976) observed cell death in sine oculis
imaginal discs. Neither of these sources gives detailed information on either
FIGURE 1
Scanning EM picture of heads of the flies of the genotypes studied here.
(a) eyn*. Bristle groups are: IOC, interocellar; OC, ocellar; F, frontal; OB, orbital;
VB, vibrissae; VT, vertical; PO, postorbital; PV, postvertical. E represents the
compound eye with palp P. The normal perimeter of the wild-type eye is marked a.
No front orbital bristles (region FO) are present in ey2.
(b) eyv. E represents eye region here replaced by cuticle.
(c) so. Note E, eye on pronounced bulge from head. No ocelli are present. A
cuticular 'bristle plate' occupies part of the eye region on the side of the head
lacking an eye.
228
ROBERT RANSOM
Table 1. Eye phenotypes of the mutants so, ey2 and eyD
Size as percentage
of wild type±s.d.
2
52 ± 12%
ey°
46-5 ± 27 %
ey
so
2±6%
Morphology
Rounded, sometimes with cuticular ingrowths or
palps. Ommatidial disruption minimal
Occasionally completely eyeless. Eye field positioned
in dorsal region of area corresponding to wild-type
eye, and often truncated ventrally. Some ommatidial
disruption with some receptor cells missing
Eyefield is sometimes in form of an 'eyestalk' protruding from the head with an irregular arrangement
of ommatidia. Heavy ommatidial disruption with
many receptor cells missing
Table 2. Head phenotypes of the mutants so, ey2 and
Size as
percentage
of wild type
+ s.d.
ey
2
80 ± 8 %
ey°
87±8%
so
78 ± 9 %
Morphology
Lacks front orbital bristles,
all others normal (some
difference in vibrissal
number)
Changes in most bristle group
numbers. Vibrissal number
may be double wild type
Most dorsal and posterior
bristles resemble wild type.
Abnormalities restricted to
orbitals, front orbitals,
vibrissae and post orbitals
Other features
Homozygous lethal: nonemergent pupae are pharate.
8 % of ey° flies possess
antennal duplications.
(Arking et al. 1975) Also
duplications of male sex
combs
Temperature sensitive.
Lethality increases with
temperature. Optimum eye
size at 25 °C (Milani, 1946,
Ransom, 1979)
proportion of discs studied which showed cell death, or the pattern and time of
death.
In the present study, Araldite embedding of osmium-fixed cephalic complexes
was performed, with dissection of the head discs themselves only at the stage
of orientation of the material in blocks containing liquid Araldite prior to
polymerization.
Fig. 2 shows the average number of dead or dying cells found per disc at
each developmental stage studied for each genotype. 'Cell death' has been
scored by the presence of dark-blue-staining pycnotic granules, found after
Drosophila eye mutations
229
90-
^ 6 0 -
30-
84
108
132
Age at fixation (h)
Fig. 2. Numbers of dead cells seen in histological sections of discs of the different
genotypes. Each point (12 h increments from 72 to 132 h) is an average of data from
at least three serially sectioned discs. + = wild type; O = so; • = ey2; • = eyD.
staining the sectioned material with toluidine blue. Although the number of
such granules differed greatly from disc to disc, neither wild type nor ey° discs
showed any localized patches of death. An interesting feature of the pronounced cell death in both ey2 and so is that it seems to start in the form of a
distinct band at about 84 h larval development and later appears generally over
the whole disc. No clear pattern of cell death differences could be established
between these two genotypes, although so cell death appeared more intense
over a shorter time span (see Fig. 2). In contrast to ey2, the absence of cell
death in eyB demonstrates a clear difference in the development of the two
genotypes.
2. Clonal analysis
Previous use of clonal analysis has given information on cell loss during
larval growth, for example Santamaria & Garcia-Bellido (1975) showed cell
loss to occur in the third larval instar in the wing mutant Beadex of Jolios. As
a complement to the histological methods described above, a similar approach
has been used here. It is difficult to draw firm conclusions on the time and extent
of cell loss using clonal analysis because of the variability between stocks. For
this reason, the results presented here should be used only as indicators of
mutant action, and not as reliable data for speculation on the nature of the
aberrant process itself.
230
ROBERT RANSOM
1001—
(a)
•2
10 -
36
12
60
84
age at irradiation (hrs)
10
(b)
0-1
12
36
60
84
108
Age at irradiation (h)
Fig. 3. (a) Size and (Jb) frequency of clones in the eyes of the mutations studied and
in wild-type controls. Limits of confidence are 95 % for frequencies and 68 % for
sizes.
Drosophila eye mutations
10 r-
231
(a)
12
36
60
Age at irradiation (h)
84
108
12
36
60
Age at irradiation (h)
84
108
a 0-1
001
Fig. 4. (a) Size and (b) frequency of y / 36a cuticle clones in the mutants studied.
Symbols and limits of confidence as in Fig. 3.
Figs. 3 and 4 illustrate clone sizes and frequencies in the eye and on the head
cuticle for the three genotypes and for wild-type controls. Because later eye
clones were too small to be reliably scored under the dissecting microscope,
individual heads were fixed and embedded in Araldite and sections were then
examined for pigment cell clones.
In order to standardize clone frequencies, mutant clone frequencies have
been multiplied by 100/n, where n is the average percentage of wild-type ommatidia possessed by each mutant genotype.
Eye clones induced before 72 h
In the pre-third instar ey2 flies, clones showed about the same frequency and
size as wild type. No clones in sine oculis were quantitatively scored before the
232
ROBERT RANSOM
third larval instar because the low number of so flies with eyes made this
impracticable. Clones in ey° flies showed a drop in frequency when induced
from about 36 h after egg laying onwards, although their average size over the
same period was larger than normal. In spite of this, the ranges of eyD eye
clone sizes, particularly at 36-72 h of development, were very great.
The shapes of clones in ey2 and eyP flies induced before the third larval
instar seem to obey the characteristic anterior-posterior, longitudinal pattern
first observed in the wild-type eye by Becker (1957). No 'fragmented' clones
were seen, either using microscopic examination of clone borders, or by general
observations of whole clones.
Eye clones induced in the third larval instar
Here, ey® showed similar frequency and size values to wild-type clones,
whilst both ey2 and so showed abnormalities. Apart from the 84 h clone frequency, all third-instar values were reduced in ey2. Sine oculis frequencies were
higher than wild type at 84 and 108 h, but were significantly lower at 96 h. This
is in agreement with the sharp peak of cell death seen in discs fixed at 108 h.
The sizes of clones in ey2 were lower at all times up to 108 h. Clones in so were
smaller at 84 and 96 h. The clone frequency reductions seen in third-instar ey2
and so clones are interesting because, even if a large amount of cell death occurs
it should be impossible for late induced clones to be less frequent than early
induced clones. Several possible explanations are that cells at the 'cell death'
period are sensitized in some way and are killed by the X-ray treatment itself
or that cells become briefly insensitive to the crossing-over-generating properties
of the X-rays.
Head clones
The head cuticle clones showed the following major features. Clones in
eyD, as with the eye, were bigger than wild-type, in this case throughout development. The frequencies of ey° head clones were about the same as wild type.
Clones in ey2 were similar in size to wild type, but showed a pronounced drop
in frequency during the third larval instar: clones in so heads were larger than
wild type at 48 h, and particularly at 96 h. However, the most obvious feature
of the so head clonal analysis was that clones overall were much more frequent
than clones of all other genotypes studied.
In order to look at regional differences in clone frequency, the head was
arbitrarily split into three bristle regions; upper anterior (frontals, front orbitals,
orbitals, ocelli, interocelli), lower anterior (vibrissae), and posterior (verticals,
postverticals, postorbitals, occipitals). Flies were irradiated early (0-48 h),
mid (48-72 h) and late (72-120 h). The proportion of clones within these groups
was then calculated for mutant strains and for wild type as shown in Fig. 5.
If the regional differences are omitted, the clonal analysis of the head cuticle
indicates third instar cell loss or lower cell division rates in ey2 only. The larger
233
Drosophila eye mutations
Early (0-48 h)
Mid (48-72 h)
Late (72-120 h)
UA
^~
'0-280 / \
<6-480J P
0-240\ /
/ 0-355 /
\
f 0-324
<p-290
\0-354\
/
0-354 ey°
Fig. 5. Regional distribution of head epidermis clones in the three regions LA
(lower anterior); UA (upper anterior); P (posterior). The figures shown are proportion of all clones obtained by irradiation at the given age in the given region.
For emphasis, arrows point from lower than expected proportions to greater than
expected proportions when regions differ from wild-type values by more than 20%.
number of so clones throughout development was observed with clones on other
parts of the body and probably represents some general effect of the genotype.
The regional analysis gives a fuller picture. The number of lower anterior
cuticle clones produced in eyB flies before 72 h is very low, whilst the variation
of both eyz and so clone frequencies in particular regions is significant only
when induction was later than 48 h. With so a reduction in vibrissal clone
frequencies was noted: this agrees with the observation that the most normal
so bristles are those placed dorsally and posteriorly (see phenotypic analysis
section). There is a large reduction in ey% upper anterior clone frequency at
72-120 h clone initiation, and a smaller general anterior reduction at 48-72 h.
234
ROBERT RANSOM
30
10
12
36
60
84
108
Age at irradiation (h)
30
i-
(b)
10
0-1
005
12
36
60
84
108
Age at irradiation (h)
Fig. 6. (a) Size and (b) frequency of yf3&!l clones on the legs of the mutants studied
and in wild-type controls. Symbols and limits of confidence as in Fig. 3.
Leg clones
Figs. 6a and b show clone size and frequency respectively for clones on the
legs of flies of wild type, eyB, ey% and so genotypes. Although there is a slightly
higher frequency of so clones throughout development, echoing the results of
the head clone analysis, no significant variations in clone size and frequency
were seen apart from this single anomaly.
CONCLUSIONS
It is concluded that the ey® mutant effect is active early in larval development
and that ey2 and so mutant activity occurs in the third larval instar. The evidence
for ey2 and so is based both on histological analysis and clonal analysis, whilst
the eyB effect has only been observed by using clonal analysis. The negative
Drosophila eye mutations
235
evidence that cell death does not occur in third in star ey® head discs should also be
taken into account. All three mutant genotypes seem to affect the development
of particular regions of the head capsule.
The localization of clonal abnormalities to head and eye clones, demonstrated
by the comparison of Fig. 6 with Figs 3 and 4, suggests that there are no general
growth abnormalities in flies of the mutant eye genotypes. The higher frequency
of sine oculis clones found on the cuticle might suggest that so is associated with a
gene which increases mitotic recombination.
The above observations are in agreement with the previous work of Fristrom
(1969), who showed third-instar cell death in eyeless2. Arking et al. (1975) have
suggested that the primary effect of the eyeless Dominant mutation is to cause
a reduction in the number of neuroblasts at an early stage of development. The
low frequency of clones induced in the first and second larval instars in the head
disc is compatible with this hypothesis.
I would like to thank Jose A. Campos-Ortega, Sigried Krien, Alois Hofbauer, Gerd
Jurgens, and the other inhabitants of room 567 who helped me during my stay in Frieburg.
Financial support was given by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 46) to Professor Campos-Ortega, and during part of the period during
which this work was carried out I was a recipient of a European Molecular Biology Organisation Fellowship.
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(Received 14 August 1978, revised 9 May 1978)
ARKING,