/ . EiTibryol. exp. Morph. Vol. 44, pp. 227-241, 1978
Printed in Great Britain © Company of Biologists Limited 1978
227
Developmental analysis of the tumorous head
mutation in Drosophila melanogaster
By N. BOURNIAS-VARDIABASIS 1 AND M. BOWNES1
From the Department of Biology, University of Essex
SUMMARY
The extent and type of adult transformations in the tumorous head (tuh) mutation of
Drosophila melanogaster were studied. The observations indicate homeotic transformations,
duplications and deficiencies of eye antennal disc derivatives. Contrary to previous observations there is no transformation of eye to abdomen and the only homeotic transformations
identifiable are antenna to leg and rostralhaut to genitalia. Embryonic and post-embryonic
lethality was also examined. Specific anterior abnormalities were found in embryos leading
to early death. The amount of embryonic lethality was not affected by increased temperature
during oogenesis. When analysing the adult phenotype, however, the whole period of
oogenesis was sensitive to pulses of increased temperature; the pulses however did not have
to be restricted to any particular stage of oogenesis to be effective in increasing tuh penetrance.
The increase in the penetrance of the adult transformations is also exhibited when eggs are
moved from 25 °C to 29 °C from the 8th to the 12th hour of embryonic development.
Experiments indicate that the tuh-3 gene is active before the 8th hour of embryogenesis.
INTRODUCTION
Tumorous head (tuh) is a temperature-sensitive homeotic mutation affecting
the eye antennal disc of Drosophila melanogaster. The phenotype is determined
by the action of two genes tuh-1 (on the X chromosome) and tuh-3 (on the third
chromosome) (Gardner & Woolf, 1949). The adult phenotype includes homeotic
transformations of the rostralhaut and antennal regions, abnormal outgrowths
involving the facets and head cuticle and deficiencies and duplications of the
antenna and palpus. Extensive studies have been carried out on the maternal
effect and population genetics (Knowles, 1967; Gardner, 1970; Kuhn, 1970,1971,
1974). It has also been reported that tumorous head females have decreased
fertility and fecundity as compared to wild-type controls (Knowles, 1967;
Schneider and Schubiger, personal communication).
Temperature sensitivity in Drosophila melanogaster has proved to be an
invaluable tool in studying developmental processes (Suzuki, 1970). Many
homeotic mutations are temperature sensitive and exhibit a normal phenotype
at one temperature while the homeotic transformation is expressed at the
restrictive temperature (Ouweneel, 1976). Some homeotic mutants just show
1
Authors' address: University of Essex, Department of Biology, Colchester CO4 3SQ,
England.
228
N. BOURNIAS-VARDIABASIS AND M. BOWNES
a different degree of penetrance and expressivity with temperature. It has been
found that the temperature-sensitive periods of homeotic mutations are
restricted to specific stages of development except for tetraltera (tet) (Villee,
1942) and these stages might be indicative of the time of determination of the
imaginal disc cells (Ouweneel, 1976).
Tumorous head is an unusual mutant in that it is temperature sensitive during
oogenesis and embryogenesis. Increasing temperature increases the penetrance
while expressivity remains the same at 17 °C as at 29 °C (Bournias-Vardiabasis,
unpublished).
Postlethwait, Bryant & Schubiger (1972) described in detail the homeotic
effects of tumorous head. To understand this mutation however, it is essential
not only to identify the number and extent of adult abnormalities and the effect
increased temperature has on penetrance and expressivity but also to analyse
the type and extent of embryonic, larval and pupal lethality and other parameters of the phenotype.
MATERIALS AND METHODS
The tuh strain was obtained from the University of California, Irvine, and
has been kept at the University of Essex from January 1976 under selection. By
selection we mean that once flies have emerged in a bottle they were screened
for head abnormalities and only these were put in a new bottle to breed. The
penetrance is 50 % at 25 °C and 65 % at 29 °C. Our tuh stock is a tuh-1; tuh-3
strain with a third chromosome constitution of 3A/3A and with no Payne
inversion in the left arm of the third chromosome (Gardner, 1970; BourniasVardiabasis & Bownes, 1978). To ensure a standardized population, since the
penetrance varies from bottle to bottle, tuh females and tuh males were taken
from various bottles and pooled for use as parents for all the egg collections.
Morphological analysis of adults
The flies were raised on a cornmeal, yeast, agar and sucrose medium at either
25 or 29 °C. The heads were cut off and preserved in 70 % ethanol, mounted in
Gurr's water mounting medium and examined at x 160 magnification under
a Zeiss microscope. Expressivity was determined as the number of transformations, deficiencies and duplications per half head of tuh phenotype flies.
Analysis of eggs
Egg collections lasting 1 h were made on agar plates smeared with yeast
paste. The eggs were immediately dechorionated with 3 % sodium hypochlorite
for 3 min. They were then lined up on double sticky tape on a glass slide,
covered with Voltalef oil (Lehman and Voss) and kept in a moist chamber to
avoid desiccation. They were examined immediately after dechorionation under
tuh mutation in Drosophila
229
a Zeiss compound microscope and the majority were seen to be at stage 2
(nuclear multiplication) of development. Eggs were subsequently re-examined
at stages 5 and 6 (blastoderm), stage 8 (gastrulation) and stage 14 (immediately
prior to hatching) (Bownes, 1975).
In the first experiment eggs were raised at 25 and 29 °C and categorized as
follows: (1) Undifferentiated, eggs failing to develop beyond stage 2; (2)
abnormal, eggs developing past stage 2 but failing to hatch; (3) normal, eggs
which hatched 24 h after oviposition.
In experiment 2 tuh eggs raised at 25 °C and at 29 °C and categorized in terms
of the stage at which the particular abnormality was first noted. From the same
egg collection a description of the extent and location of the abnormalities found
was made along with the time of onset of the defect. Eggs were followed from
the time the abnormality was found until the time they would have been
expected to reach stage 14 if they had been normal.
Analysis of larvae and pupae
Eggs were collected for 2 h on agar plates covered with yeast and left on the
plate until they hatched into first instar larvae. Individual larvae were put into
small vials containing agar smeared with yeast paste, half were put at 25 °C
and half at 29 °C. They were examined under a Wild dissecting microscope
during second and third instar to detect any morphological abnormalities.
Oregon R eggs and larvae were used as controls.
Pupal lethality was also calculated and pharate adults were dissected out of
their pupal cases and examined for any morphological abnormalities.
Temperature sensitivity
Tumorous head eggs were collected over 1 h periods on agar plates smeared
with yeast. All pulses at 29 °C or shift up and shift down experiments to and
from 29 °C were corrected for the difference in developmental time between
oogenesis at 25 °C and at 29 °C (Suzuki, 1970). One hour at 25 °C being equal
to 45 min at 29 °C (personal communication, R. Arking). Time periods are
therefore always expressed in 25 °C equivalents.
(a) Oogenesis
Oogenesis takes approximately 72 h to complete at 25 °C (King, 1970), thus
the scheme seen on Table 3 was used in the first experiment to determine if
a particular 24 h period was more temperature sensitive than the other 2 days.
In experiment 2 the following scheme was used to determine if applying one
continuous pulse at the higher temperature at a specific time during oogenesis
would give the same increase in tuh penetrance as would three separate pulses
at the higher temperature which added up to the same time period as the one
continuous pulse. Thus initially an 8 h pulse was given during day 1 of oogenesis
to the first group of flies, an 8 h pulse on day 2 to the second group of flies
230
N. BOURNIAS-VARDIABASIS AND M. BOWNES
and an 8 h pulse to the third group of flies on day 3. Also a pulse totalling 8 h
at 29 °C spread over the 3 days was given to the fourth group of flies (2 h and
40 min of a 29 °C pulse for each of the 3 days). All eggs were raised at 25 °C.
The same scheme was used for the second series with a 16 h long pulse at 29 °C
and for the third series where a 24 h long pulse was applied. Control I refers to
tuh flies from the same population as those used for the several pulses on day
1, 2 or 3. Control II refers to tuh flies raised at 25 °C from the same population
as those used for the pulses on several days.
(b) Embryogenesis
In the first experiment the temperature-sensitive period was determined by
a series of temperature shifts up to 29 °C and shifts down to 25 °C every 24 h
during embryogenesis and post-embryonic development. Once the TSP was
established as being during the first 24 h of development the same scheme with
4 h shifts up and down was used to determine the TSP within the first 24 h of
development.
It has been previously reported (Gardner, 1970) that the maternal effect seen
in the F x progeny of the tuh females crossed to Oregon R males is also temperature sensitive during embryogenesis, the sensitive period being between 8 and
24 h of development, with any 4 h pulse at 29 °C giving an increase in the tuh
penetrance. In order to delimit the TSP of our stock and also to reinvestigate the
temperature sensitivity seen in the maternal effect, experiment 3 was carried
out. The following 4 h 29 °C pulses were given: 0-4, 4-8, 8-12, 12-16, 16-20
and 20-24 h post-oviposition for the following crosses: tuh females crossed to
tuh males; tuh males crossed to Oregon R females and Oregon R females crossed
to tuh males. Controls were raised at 25 and 29 °C.
In experiment 4, 1 h pulses were given between 8 and 12 h of embryogenesis
to determine if lh long pulses at the higher temperature were sufficient to increase
the penetrance. Finally in experiment 5 the following 4 h pulses were given to
clearly establish the most temperature sensitive period during embryogenesis:
4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10-14, 11-15 and 12-16 h after oviposition.
RESULTS
Adult abnormalities
Initially flies raised at 29 °C were examined separately frcm those raised at
25 °C. No significant difference was found between expressivity or areas involved
in the homeotic transformation between the two sets offlies,thus data was pooled
as to the type of abnormality. In fact, tumorous head flies which were raised
through several generations at 17 °C showed the same degree of expressivity as
those raised at 29 °C but a much lower penetrance of 15 % was found as compared to 80 % for 29 °C (Bournias-Vardiabasis, unpublished).
The range of abnormalities and areas of the head involved in the trans-
tuh mutation in Drosophila
231
allf
Fig. 1. (A) The normal head of Drosophila melanogaster. (B) Tuh eye where a strip
of head tissue is running across the middle of normal facets. Vibrissae-like bristles
out of large bracts with few smaller bristles near their base can also be seen. (C)
Tuh third antennal segment with the arista partially transformed to leg tissue. The
bristles seen on the transformed region are similar to those found on the second leg.
(D) Tuh palpus duplicated to twice its normal size, a, Antenna; alii, antennal segment III; ar, arista; e, eye; /, leg; o.g., outgrowth; p, palpus; r, rostralhaut; v,
vibrissae.
formation were generally similar to those obtained by Postlethwait et ah (1972).
However, no eye to adbomen transformations were observed even though our
stock was selected from that used by these authors. Eye abnormalities include
either small depressions on the facets or small patches of unidentifiable tissue,
which are also found in the head cuticle (vibrissae region and the occipital
bristle region) (Table 1) (Fig. 1B). Antennal transformations were always to leg
tissue and showed homology between the antennal segments and their counterpart leg segments. When there was suflficient differentiated leg tissue the pattern
of the bristles indicated that the transformation was to the second leg (Table 1)
(Fig. 1C). The rostralhaut region involved numerous genital transformations as
well as some large unidentifiable outgrowths (Table 1). The vibrissae bristles
were found often duplicated, missing or in the case where the eye was reduced the
vibrissae were found in an altered position, immediately beneath the remaining
eye facets. No homeotic transformations were found in the palpi, but they
were often duplicated, missing or reduced in size (Table 1) (Fig. ID).
232
N. BOURNIAS-VARDIABASIS AND M. BOWNES
Table 1. Adult abnormalities in tumorous head flies
Type of transformation
(%)
Antenna missing
Antenna duplication
Anenna to leg
Arista to leg
Total antennal transformations
3
3
1
3
10
Eye to head cuticle
Eye to unidentified
Eye missing
Eye reduced
Eye to palpus
Head cuticle to unidentified
Head vibrissae affected
Head cuticle to leg
Total eye and head transformations
Rostralhaut to anal and lateral plates
Rostralhaut to clasper teeth (males only)
Rostralhaut to unidentified
Rostralhaut to leg
Total rostralhaut transformations
15
2
1
12
1
10
13
1
55
10
2
8
2
22
Palpus missing
Palpus duplication
Total palpus transformations
11
2
13
Total number of transformations examined
Total number of half heads examined
Total number of transformations per half head
1810
1238
1 -46
Table 2. Viability of tumorous head eggs
Temperature
during oogenesis
and subsequent
embryogenesis
Total
number of
eggs laid
Eggs
undifferentiated
(%)
Eggs
abnormal
(%)
Eggs
hatched
(%)
25-25 °C
25-29 °C
29-29 °C
29-25 °C
2177
530
1697
1101
23
16
26
26
12
24
14
12
65
60
60
62
263
11
0
89
Control
OrR 25-25 c C
Embryonic lethality
The results of experiment 1, where tuh eggs were examined to determine the
degree of lethality, showed that tumorous head eggs cultured at 25 CC or at
29 °C showed a 61 % viability while Oregon R controls had an 89 % viability.
There was no difference in viability between tuh eggs raised at 29 °C from those
raised at 25 °C (Table 2).
tuh mutation in Drosophila
233
In experiment 2, where the stage at which an abnormality was first noted
was recorded, undifferentiated eggs were the most frequent class (20 %). It has
been established that tuh females have reduced fecundity and tuh males have
increased sterility compared to wild-type flies and this probably contributed to
the high percentage of undifferentiated eggs (Knowles, 1967). It was during
blastoderm formation and gastrulation that eggs most frequently developed
abnormalities (13 %). Defects initiated at later embryonic stages were rare (1 %).
In experiment 3 the embryos were classified according to the areas showing
abnormalities. Most abnormalities (75 %) were restricted to the anterior
one-quarter of the egg. Abnormalities which involved the anterior one-half of
the egg accounted for 10 % of the cases while totally abnormal embryos were
found in 10 % of the eggs studied. Embryos with posterior abnormalities were
found in only 4 % of the cases and only involved the posterior one-eighth of
the embryo.
Finally, a description of the abnormalities found and the time of onset of
the defects was made. When abnormalities were noted in the early stages of
egg development the extent of area involved varied from one-eighth to onequarter of the total egg. The embryo, in some cases, continued to develop to
stage 14 while the anterior remained undifferentiated with the columnar layer
of blastoderm cells around the surface of the embryo failing to form (Fig. 2 A).
When the onset of the abnormality was during the blastoderm stage and during
gastrulation, the anterior one-eighth to seven-eighths of the embryo was
granular while the rest developed normally (Fig. 2B). Occasionally the head is
developed but is pushed towards the posterior of the embryo. Mouthparts are
abnormal in such cases. Finally when the embryo becomes abnormal after the
first 8 h of development a variety of abnormalities were found. The extent of
the area involved varies from one-eighth to one-quarter of the egg. There are
embryos which appear normal at stage 10 (8 h post-oviposition) but fail to
develop any further. When only the anterior one-eighth is abnormal the
posterior of the embryo develops normally while the anterior develops abnormal
mouthparts (Fig. 2D). When the anterior one-quarter is abnormal the head
fails to involute and becomes arrested at stage 10 while the posterior develops
normally. Furthermore, when the anterior one-half is abnormal there is a complete disorganization of the organs present in that region or only gut tissue is
present while the posterior half is normal with segments and spiracles (Fig. 2C).
Stage-13 and stage-14 embryos which become abnormal usually involve malformed mouthparts which are pushed back or against the vitelline membrane
and thus the embryos are unable to hatch. In a few cases there are embryos
with only the gut misplaced or the head pushed to the side leaving an area of the
egg empty while the rest of the embryo is normal.
Fig. 3 is a diagrammatic representation of some of the abnormal developmental pathways tuh embryos follow.
234
N. BOURNIAS-VARDIABASIS AND M. BOWNES
ant.
synbl. -r~
g.m
post.
Fig. 2. (A) Abnormal tuh pre-blastoderm-stage embryo. The nuclei have failed to
migrate at the anterior. (B) Abnormal tuh blastoderm-stage embryo. A small
quantity of yolk is present but blastoderm cells have formed a complete layer
behind the yolk. (C) Stage-14 tuh embryo. The posterior contains all the normal
segments and spiracles but the anterior has only gut mass. (D) Abnormal tuh
embryo. Posterior is normal but the abnormal mouthparts in anterior make it unable
to hatch, ant., Anterior; bl., blastoderm; g.m., gut mass; m.p., mouthparts; post.,
posterior; synbl., syncitial blastoderm; t, trachae; y, yolk.
Larval and pupal lethality
Tuh larvae showed high mortality compared to Oregon R controls. While
Oregon R larvae had a 6-0 % post-embryonic lethality, tuh larvae showed an
average lethality of 31 %. There was no difference in lethality whether tuh larvae
were raised at 25 °C or at 29 °C. Of 374 first instar larvae raised at 29 °C, 69 %
pupated and 62 % hatched into adults. Of 129 first instar larvae raised at 25 °C
69 % pupated and 60 % hatched into adults.
The larvae died during late first instar and early second and showed no
obvious morphological abnormalities. Pupae that failed to eclose were dissected
out and 85 % showed the tuh phenotype while the expressivity was the same as
found with the tuh flies eclosed from the same population, with a high number
of pharate adults having reduced eyes and missing palpi.
tuh mutation in Drosophila
BL
CLF
BL
235
BL
Y
BL
Ga
P in
BL
Fig. 3. A diagrammatic representation of development of tuh eggs leading to
embryonic lethality, (a) Blastoderm nuclei migrate properly, blastoderm formation is normal, but stage-14 embryos have abnormal mouthparts or head
formation, (b) Nuclei are late in migration to the surface of the egg, a small
portion of yolk remains at the anterior pole. The nuclei finally migrate but the
embryo develops normally at the anterior. Stage-14 embryos do not hatch, (c)
The nuclei fail to migrate into the anterior regions of the embryo which then
develops with a gap in blastoderm thus the anterior is abnormal but the posterior
develops normally. BL, Blastoderm; CLF, cleavage furrow; M, micropyle; S,
segments; T, trachae; Y, yolk.
Table 3. Localization of the temperature-sensitive period during oogenesis
Temperature during embryogenesis
A
25 °C
29 °C
A
A
N
Temperature shift scheme
during oogenesis
Total flies
examined
(
1
Total flies
examined
Penetrance
(%)
654
1155
(31)
(63)
1215
976
Penetrance
(%)
(44)
(70)
542
(50)
683
(56)
650
(55)
840
(59)
789
(52)
548
(59)
689
(54)
498
(54)
29 °c
_i
25 °c 0 1 2 3
810
(62)
569
(68)
"I
29 °c
25 °c 0 1 2
511
(49)
762
(45)
25 °c control
29 °c control
29 °c
L_
25 °c 0 1 2
3
r~
29 °c
25 °c 0 1 2
3
29 °c
I
25 °c 0 1 2 ~3
1—
29 °c
25 °c 0 1 2 3
JZL
3
Refers to number of days pulse was at tha t temperature.
236
N. BOURNIAS-VARDIABASIS AND M. BOWNES
Table 4. Temperature-sensitive period during oogenesis
Temperature pulse scheme
6 h pulse at 29 °C
16 h pulse at 29 °C
24 h pulse at 29 °C
Developmental time
of oogenesis
Total flies Penetrance
examined
(%)
Day 1
Day 2
Day 3
Control I*
Days 1+2 + 3
Control II*
Day 1
Day 2
Day 3
Control I
Days 1+2 + 3
Control II
1657
948
1442
341
583
207
(37)
(41)
(37)
(29)
(35)
(26)
526
265
543
755
320
(45)
(42)
(42)
(28)
(51)
(40)
Day 1
Day 2
Day 3
Control I
Days 1+2 + 3
Control II
200
248
286
200
442
169
(63)
(62)
(63)
(46)
(64)
(47)
641
* Control I applies to day 1, 2, 3 and Control II to days 1+2 + 3.
Temperature sensitivity during oogenesis
In experiment 1, where 24 h long 29 °C pulses during the 3 days of oogenesis
were given, it was shown that there was no distinct 24 h period which increases
tuh penetrance more than any other period. Thus the entire period of oogenesis
seems to be temperature sensitive (Table 3).
In experiment 2, where an 8 h pulse at 29 °C during oogenesis was applied,
there was no statistically significant (P > 005) increase in penetrance. Pulses
of 16 and 24 h at 29 °C given on any day gave the same increase in penetrance
as did the cumulative 16 and 24 h pulses spread over the 3 days. Both increases
were statistically significant when compared to controls raised at 25 °C
(P < 0-05) (Table 4).
Temperature sensitivity during embryogenesis
The initial 24 h shifts up to 29 °C and shifts down to 25 °C place the TSP
within the first 24 h of embryonic development (Fig. 4a). In experiment 2,
where 4 h shifts up and shifts down were given during the first 24 h of development, the TSP was delimited to 8-12 h after oviposition (Fig. 4b).
The results of experiment 3, where 4 h pulses at 29 °C were given at 0, 4, 8,
12, 16, and 20 h after oviposition, to the progeny of different matings are
shown in Fig. 5 and clearly the most sensitive time in embryogenesis for tuh
237
tuh mutation in Drosophila
70 |(c)
»
1
60 V
50 -
40
1
1
1
5
6
1
1
I
7
8
I
I
9
I
I
1
1
1
1
1
1
1
10 11 12 13 14 15 16 17 18
Developmental time (h)
Fig. 4 (a) Temperature-sensitive period during embryogenesis. Localization of a 24 h
period, (b) Temperature-sensitive period during embryogenesis. Localization of
a 4h period, (c) Localization of the most temperature-sensitive 4 h period in
embryogenesis. • , Represents a shift up to 29 °C; • , represents a shift down to
25 °C; H , represents a 29 °C pulse.
males crossed to tuh females is the 8-12 h period. The results from the second
cross of tuh females mated to Oregon R males indicate that although the
maternal effect is temperature sensitive, 4 h pulses at 29 °C were not sufficient
to raise the penetrance substantially. As was expected the Oregon R females
mated to tuh males gave no tuh phenotype and increased temperature had no
effect (Fig. 5).
In experiment 4, it was found that 1 h pulses at 29 °C were insufficient to
increase the penetrance of tuh. A pulse from 8-9 h after oviposition gave
16
EMB
44
238
N. BOURNIAS-VARDIABASIS AND M. BOWNES
29 C 100
control
25 UC
control
50
40
30
20
10
OrR $ x
tuh
l
l
I
l
l
l
!
8
I
I
I I I
10
12
14
16
Developmental time (h)
I
I
I
18
I
I
20
22
I
I
24
Fig. 5. Temperature-sensitive period during embryogenesis. Comparison between
progeny of tuh females mated to tuh males (|——), and tuh females mated to
Oregon R males (I
1). Oregon R females mated to tuh males gave 0 % tuh
penetrance.
a 52 % penetrance, a 9-10 h pulse gave also a 52 %, a pulse at 10-11 h gave
57 % penetrance and an 11-12 h pulse gave 58 % penetrance while the 8-12 h
pulse at 29 °C gave a 72 % penetrance.
Finally the 4 h long pulses at 29 °C given at 4-8, 5-9, 6-10, 7-11, 8-12, 9-13.
10-14,11-15 and 12-16 h post-oviposition show clearly that the highest increase
in penetrance was found when the 29 °C pulse was applied from 8 to 12 h
during embryogenesis (Fig. 4c). The other pulses gave smaller increases in
penetrance.
DISCUSSION
Tumorous head has been examined for many years and its homeotic effect
along with its embryonic lethality have been already noted. The present study
confirms the extensive homeosis found in the adult head involving the eye.
antennal and rostralhaut regions but does not confirm the transformation to
abdominal tergites. It is essential to see precisely which homeotic transformations occur in the eye antennal disc. The antennal to leg transformations
are also observed in antennapedia and aristapedia and the rostralhaut to
genitalia is a genuine transformation. However, from our observations it is not
entirely certain whether the eye undergoes homeotic transformations. Many of
the structures which initially appeared to be homeotic transformations on closer
analysis seem to be head structures such as vibrissae, probably resulting from
intercalary regeneration (French, Bryant & Bryant, 1976) after the extensive
cell death in the imaginal discs observed in tumorous head eye antennal discs
tuh mutation in Drosophila
239
(Bournias-Vardtabasis, unpublished). Further evidence that the outgrowths are
head cuticle is that amongst the bristles a second abnormal palpus is sometimes
observed. Homeotic transformations are usually limited to a specific type of
change which is also segmentally restricted. The eye has been observed to transform to wing tissue (ophthalmoptera) but no other eye transformations are
known and it is thus likely that tumorous head is less of an exception than
previously thought when it was reported that the eye transforms to abdomen.
Tumorous head is unusual in that there are at least two transformations to
different segmental derivatives but this may be because during evolution the
eye antennal disc has arisen by fusion of head segments. One may see antenna
to leg or rostralhaut to genitalia transformation or both in one half head.
Embryonic lethality has been examined in detail. Defects in the anterior of the
egg were observed at blastoderm formation and at later embryonic stages.
Anterior larval structures were often missing or malformed and eventually led
to embryonic death. Since the eye antennal disc is in the anterior of the embryo
there is correlation between the areas of the embryo and the adult which were
affected by the tuh mutation. We have recently been able to separate the tuh-1
and tuh-3 genes from our tuh-1, tuh-3 stock and will now be able to analyse the
contributions of the two genes to the various aspects of the tuh-1, tuh-3
phenotype.
Tumorous head flies possess a temperature-sensitive period which spans all of
oogenesis. This was shown by the results of experiments where 16 and 24 h
long pulses at 29 °C given during 1 day or spread over 3 days gave the same
increase in penetrance. One possible explanation for this phenotype could be
that the tuh-] gene codes for or induces the synthesis of a protein, at the beginning of oogenesis, which is present throughout oogenesis. It is thus altered by
an increased temperature at any time during oogenesis. Much experimental
data points to the fact that the tuh-1 gene is responsible for the maternal effect
(Gardner, 1970). Siervogel (1972) proposed a model concerning the action of
the tuh-1 gene which produces a maternal substance which is deposited in the
egg and can be regulated by various modifiers. Much later in development the
gene product of tuh-3 interacts with the substances produced by tuh-1 and this
interaction results in the homeotic phenotype. His model may be applied to
our data on temperature sensitivity. The existence of the temperature sensitivity
during the entire time of oogenesis and the embryonic temperature-sensitive
period of 8-12 h suggest that perhaps tuh-3 gene is involved in producing
a protein (it does not necessarily mean that tuh-3 is the structural gene for that
protein) which interacts with the tuh-1 product. Either the protein itself or the
product of the interaction may be temperature sensitive. The results of analysing
temperature sensitivity during embryogenesis of various types of embryos
provide some interesting information on the time of action of the tuh-3 gene.
When the tuh-1 and tuh-3 genes are present in the heterozygous condition in
the zygote and come from the homozygous mother the embryos are slightly
16-2
240
N. BOURNIAS-VARDIABASIS AND M. BOWNES
temperature sensitive. However, when the tuh-3 gene is also provided by the
sperm and is present in the homozygous condition in the zygote the temperature sensitivity during embryogenesis is much greater. This suggests that the
paternal tuh-3 gene is active and its product interacts with products under the
control of the maternal tuh-1 gene before the 8th hour of development.
Since Ouweneel (1976) maintains that homeotic mutants with an early temperature-sensitive period are most likely to be involved with imaginal disc precursor cell determination, it seems most likely that tumorous head which has an
early temperature-sensitive period during embryogenesis, involves a cell
determination process. It is interesting to note that only two other homeotic
mutants exhibit early temperature-sensitive periods {{Ophthalmoptera (OptG)
Lederman-Klein, 1962; and ophthalmoptera (opht) Ouweneel, 1969). All other
homeotic mutants exhibit TSPs during the early third instar (for extensive
review see Ouweneel, 1976). The mutations with later temperature-sensitive
periods are probably involved in the maintenance of disc determination rather
than with the initial determination event.
N. B. V. wishes to thank her parents for theirfinancialsupport. This work was supported
in part by a Science Research Council grant to M. B.
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{Received 8 September 1977, revised 27 October 1977)