/. Embryol exp. Morph. Vol. 34, 1, pp. 19-31, 1975
19
Printed in Great Britain
Analysis of gene expression
during development in the homeotic mutant
Contrabithorax of Drosophila melanogaster
By GINES MORATA 1
From the Institute) de Genetica y Antropologia, Centro de
Investigaciones Biologicas, Madrid, Spain
SUMMARY
Contrabithorax, a mutant of the bithorax system in Drosophila melanogaster produces
a partial homeotic transformation of mesothorax (wing) into metathorax (haltere). The
wing of a fly homozygous or heterozygous for the mutant is a mosaic of wing and haltere
structures. A genetic analysis of the mutant suggests that its phenotype is due to some form
of derepression in the wing of two other genes of the bithorax system {bithorax and postbithorax) which are not normally active there. This repression is not complete. The activity
of the two genes is below the normal level resulting in only a partial transformation of wing
into haltere.
Clones of marked cells were generated by X-rays and were found to include both transformed
(haltere) and untransformed (wing) territory; this was true even for those generated late in
development. Thus the final expression of a cell depends not on its immediate ancestry but
perhaps on the level of the products of the wild-type alleles of bithorax and postbithorax.
INTRODUCTION
Mutants belonging to a pseudoallelic series, the 'bithorax' system, produce
several kinds of homeotic transformations involving the mesothoracic, metathoracic and first abdominal segments of Drosophila melanogaster (Lewis, 1963,
1964). Amongst them, the mutant Contrabithorax (Cbx) leads to the differentiation in the mesothorax (wing) of structures typical of the metathorax (haltere).
At the phenotypic level the main characteristic of Cbx is that it presents
partial expression, that is, only a fraction of the cells in the wing segment show
mutant phenotype, the rest of them being wild type. Since the cells are genetically
identical and develop in the same developmental system, we are faced with
the problem of what mechanisms are responsible for this differential behaviour.
Moreover, assuming that the development of a haltere or a wing requires the
function of not one but several specific genes, the expression in the Cbx mesothoracic cells of the mutant (haltere) or wild-type (wing) phenotype requires the
co-ordinate induction and/or repression of specific sets of genes.
1
Author's address: MRC Laboratory of Molecular Biology, University Postgraduate
Medical School, Hills Road, Cambridge, U.K.
20
G. MORATA
One possible explanation is that phenotypic mosaicism might stem from a
determinative event somewhere in the development of the mutant disc which
produces separate lineages of cells which either differentiate the mutant or the
wild-type phenotype (see Postlethwait & Schneiderman, 1969, 1971).
Taking advantage of the previous genetic information about the bithorax
system (Lewis, 1963,1964,1967) and how it controls the development of various
segments (Morata & Garcia-Bellido, in preparation), I have made a genetic
and clonal analysis of the mutant Cbx in an attempt to understand the processes
that lead to the expression of this phenotypic diversity.
The main results suggest that: (1) the partial expression of the Cbx genotype
is due to partial expression of the bithorax system in the mutant wing; (2) those
cells which express the mutant phenotype are not related to each other: small
clones generated by X-irradiation late in development often include both transformed and untransformed cells.
MATERIALS AND METHODS
bithorax mutants
The following mutants were used in this work: bx3,pbx, Cbx, Ubx1 and Ubx130.
All map at 3-58-8. Several alleles are known of bx and Ubx but only one for
pbx and Cbx. The mutant Ubx1Z0 is associated with In (3LR) Ubx130 (see Lindsley
& Grell, 1968).
The translocation T(13)P115 was also used. It is an insertional translocation,
discovered by E. B. Lewis, carrying a fragment of the third chromosome
(including all bithorax genes) to the first chromosome proximal to forked. The
aneuploid segregant Df{3)P115f + is viable but sterile and shows the same phenotype as Ubxj +.
Cell marker mutants
The mutant multiple wing hairs (mwh) was used. It is located at 3-0-0 and
affects trichome differentiation producing several processes per cell instead of
one. It can be identified even in very small clones in both wing and haltere
(Garcia-Bellido & Merriam, 1971a; Morata & Garcia-Bellido, in preparation).
Irradiation
In mitotic recombination experiments, larvae were irradiated at 1000 R
(R = 2-58 x 10~4 C/kg) using an X-ray machine Phillips 151 Be at standard
working conditions of 150 kV at 15 mA with a 2 mm aluminium filter.
Larvae of all ages were irradiated and their ages at irradiation calculated
according to the time between irradiation and pupariation.
Hatched adults were treated with hot KOH 10 % for lOmin to digest soft
parts, washed in alcohol and mounted in Euparal for microscopical examination.
Gene expression in Cbx
21
RESULTS
(1) Some characteristics of the bithorax system
Almost all the information known today about the bithorax system comes
from the studies of Lewis (1963, 1964, 1967) and the reader is strongly advised
to consult his articles for a full description of the different mutants.
The bithorax system contains at least five loci, three of which produce recessive
mutations {bithorax, bithoraxoid and postbithorax) and two others (Ultrabithorax and Contrabithorax) produce dominant ones. (Another two mutants,
Contrabithoraxoid and Ultraabdominal, have been described by Lewis (1968)
but as yet little information about them is available.) Although the homeotic
transformations produced by these mutants affect both dorsal and ventral
structures, only dorsal transformations will be considered in this report.
The mutants in the bithorax (bx) locus produce a transformation of the anterior haltere into anterior wing, the mutant postbithorax (pbx) transforms the
posterior haltere into posterior wing. Thus flies homozygous for bithorax and
postbithorax have four wings and no halteres. The mutant bithoraxoid (bxd)
transforms the first abdominal segment into metathorax and it presents also
postbithorax transformation because of its effect on the wild allele of postbithorax located to its right (Lewis, 1963, 1964).
The mutants in the Ultrabithorax (Ubx) locus are homozygous lethal, showing
in heterozygous condition a slight enlargement of the halteres plus a variable
and small (1-6) number of wing-type bristles. All of them show mutant phenotype when in trans with any of the recessive mutants. It has been shown (Lewis,
1963; Morata & Garcia-Bellido, in preparation) by mosaic analysis that the
phenotype of Ubx homozygous clones is that of the triple combination for the
three recessive mutants, suggesting that mutants in Ubx locus produce an inactivation of the whole bithorax system. This interpretation is supported by the fact
that the heterozygous deficiency for the whole bithorax system, DfP115/ +
shows Ubx phenotype.
Some of the Ubx alleles, like Ubx130, produce very extreme mutant phenotypes in trans with bx, pbx and bxd which are not distinguishable from those
produced by the same mutants in trans with their deficiency. Thus bx3/Ubx1S0
has the same extreme phenotype as bx3/DfPU5, pbx/Ubx1™ has the same as
pbx\DfP115, etc.,. . . (Morata, 1973). This result suggests that the mutant
Ubx1™ produces a complete lack of function of the bithorax system equivalent
to the physical deficiency.
All of the mutants described so far affect only the haltere or the first abdominal
segment. Moreover the apparent lack of function of the bithorax system in
genetic combinations like Ubx1S0IDfPU5 (which is lethal in individuals but
viable in somatic spots) only affects the differentiation of the cells in the haltere
and in the first abdominal segment and does not affect the differentiation of cells
of any other segment including the wing (Morata, 1973). This fact suggests that
22
G. MORATA
B
\
Fig. 1. (A) Cbx fly showing complete transformation of wing into haltere. (B)
Example of incomplete transformation of Cbx. The mutant wing is a mixture of wing
and haltere structures.
the activity of the bithorax system is only required in the haltere and first abdominal segment.
In spite of this, there is a mutant of the series, Contrabithorax (Cbx), which
affects the wing partially transforming it into haltere. The phenotype of Cbx
can be explained (Lewis, 1963, 1964) by supposing that the mutant produces
a derepression in the wing of the wild-type alleles of bithorax and postbithorax.
As the normal function of these genes can be defined as preventing wing formation and directing the disc to develop as a haltere, their activity in the wing
would lead to haltere formation there, producing Cbx phenotype.
This view is consistent with Cbx being dominant (due to the activity of the
dominant alleles bx+ and pbx+) and also with some other characteristics of
the mutation (Lewis, 1963).
(2) Gene expression in Cbx flies
(i) The phenotype of Cbx. The normal wing and haltere have been described
by several authors (see, for example, Garcia-Bellido & Merriam (1971 a) for
the wing and Loosli (1959) andOuweneel & van der Meer (1973) for the haltere).
In those cases of extreme expression, homozygous Cbx flies produce an almost
complete transformation of wing into haltere (Fig. 1 A) but it is a rare event and
in the majority of flies this transformation is not complete, the wing blade being
Gene expression in Cbx
23
Fig. 2. (A) Mosaic wing showing wing (W) and haltere (H) trichomes. Notice the
presence in the haltere tissue of several small bristles typical of the haltere (arrows).
(B) Trichomes of intermediate size between wing and haltere in the mutant wing
(arrow). Compare with those of typical wing (above right) and of typical haltere
(below). (C) Small group of haltere trichomes in the anterior wing (arrow). (D)
Typical wing costa and triple row bristles surrounded by haltere trichomes.
a mosaic of wing and haltere differentiations (Fig. 1 B). Mature Cbx wing discs
also show characteristics of both wing and haltere discs.
In the majority of cases, each cell differentiates wing or haltere structures and
intermediates do not appear (Fig. 2 A). However, trichomes can be detected
which are an intermediate size between those of wing and haltere (Fig. 2B).
Those trichomes might be secreted by 'hybrid' wing-haltere cells.
Usually the mutant wing is composed of large patches of wing and haltere
tissue as those shown in Fig. 2 A but together with these, other small haltere
patches can frequently be detected (Fig. 2C). The minimum size of these small
patches is about 30 cells.
It is interesting to note that in the case of extreme expression, the only wing
structures seen are wing bristles that are completely surrounded by haltere
trichomes (Fig. 2D).
The expression of the mutant genotype is variable in the different regions of
the mutant wing. Fig. 3 shows the percentage of transformation in the major
24
G. MORATA
Fig. 3. Degree of transformation in different regions of the wing. Percentages were
calculated in a sample of 100 Cbx homozygous wings. NO, Notum; SC, scutellum;
CO, costa; TR, triple row; II, III, IV, V, veins.
TR
Fig. 4. Correspondence between different regions of wing and haltere. MS, Mesonotum; CO,costa (P, proximal; M, medial; D,distal); TR, triple row; MT, metanotum;
SCA, scabellum; PE, pedicellum; CA, capitellum.
Gene expression in Cbx
25
Table 1. Homeotic transformations of haltere into wing (i/-> W) and wing into
haltere {W->H) in different genotypes involving Cbx and other mutants of the
bithorax system
The symbol + indicates slight transformation and + + more complete transformation.
Phenotype
Genotype
H -> W
W -»H
Cbx/+
Cbxjbx^
Cbx/pbx
—
++
++
++
+
+
+
+
+
+
CbxI Ubx1
Cbx I Ubx1™
CbxjDfPUS
regions of the wing. In the posterior part (defined by the line between veins III
and IV) the expression is 100 % (a complete transformation). In the anterior
regions there is variable degree of transformation ranging from 90 % in C to
11 % in A'. The degree of transformation is weaker the more anterior are the
regions in the wing blade.
The fact that most of the mutant wings are mosaics of wing and haltere
structures allows us to make correlation between the type of haltere cells and
their location in the wing. This is expressed in Fig. 4 and shows a correspondence
between homologous regions along the proximo-distal axis so that the proximal
haltere regions (metanotum) appear in the proximal wing regions (notum) the
intermediate haltere regions (scabellum and pedicellum) appear in the intermediate wing regions (proximal and medial costa) and the distal haltere (capitellum) appear in distal wing (distal costa and distal wing blade). This type of
homologous correspondence has also been described for other homeotic mutants
(Gehring, 1966; Gloor & Kobel, 1966; Postlethwait & Schneiderman, 1971) and
also appears in the opposite transformations by bithorax and postbithorax
mutants (Ouweneel, 1973; Morata & Garcia-Bellido, in preparation).
(ii) Possible causes of the partial expression of Cbx. It has been observed by
Lewis (1963) that Ultrabithorax mutants in trans with Cbx produce a slight
bithorax and postbithorax phenotypes which is stronger than that produced by
the same Ubx mutants in trans with a wild-type chromosome. This observation
suggests that there is some hypofunction of bx+ and pbx+ genes of the Cbx
chromosome.
In order to confirm this hypothesis flies of different genetic constitutions were
synthesized and their phenotypes were analysed for homeotic transformation
both in wings and halteres. As shown in Table 1, flies of the genotypes Cbx/bx3
and Cbx/pbx have a Cbx phenotype as expected and also show no sign of transformation in the halteres. However, flies of the genotypes Cbx/Ubx1, Cbx/Ubx130
26
G. MORATA
Table 2. Number and size o/mwh clones found after X-irradiation
at different developmental ages
Age calculated in hours between irradiation and pupariation. W clones embracing
only wing trichomes. H clones embracing only haltere trichomes. W + H clones
embracing both. Average size of clones in parentheses.
Age at
irradiation
0-24
24^8
48-72
72-96
96-120
No. of
wings
No. of
W clones
No. of
H clones
50
104
44
180
146
48 (40)
40(190)
7 (68-6)
15(163)
5(680)
52 (4-3)
29 (28-7)
2 (400)
21 (127)
3 (400)
No. of
W + H clones
0
6 (52-6)
2 (850)
4(204)
6(630)
and Cbx/DfPlJS presented together with Cbx phenotype a slight but clear
bithorax and postbithorax phenotype in the halteres, confirming the earlier
observation of Lewis and inicating that there is some hypofunction of wild-type
alleles of bx and pbx when in cis with Cbx.
Thus, the evidence suggests that although Cbx flies have activity of bx+ and
pbx+ alleles in the mesothorax, this activity is below normal. Concentrations
of bx+ or pbx+ products that are near to threshold could explain the varied
effect on mesothoracic cells. Thus partial phenotypic expression could reflect
partial genetic activity.
(3) Clonal analysis
The clonal analysis was performed in the mutant wing disc to study the lineage
relationships of the wing and haltere structures, specially to investigate whether
there are independent cell lineages for each type of structure.
Larvae of the genetic constitution Cbx/mwh were irradiated at different ages
and wings of hatched adults were searched for mwh spots. Because mwh is
much easier to detect in the wing blade, only clones in this region were considered.
The clones found were classified into three groups: (1) forming exclusively
wing structures, (2) forming exclusively haltere structures, (3) forming both
wing and haltere structures.
The number and average cell numbers in clones of the three types which arose
after irradiation at different developmental ages are shown in Table 2. Clones
embracing wing and haltere structures were found in all ages except in flies
irradiated 0-24 h before pupariation (Fig. 5). This result suggests that there are
not separate cell lineages for wing and haltere during most of development.
The lack of this type of clone in the flies irradiated in the latest stage may be
due to the small size (Table 2) of the clones so that the probability of crossing
a border between wing and haltere is very low. As shown in Table 2 as the
clones are larger the proportion of clones embracing wing and haltere
increases.
27
Gene expression in Cbx
Fig. 5. mwh clones embracing wing and haltere trichomes.
Table 3. Proportion of wing and haltere cells in all mwh clones that were
found to embrace wing and haltere trichomes
Age calculated in hours between irradiation and pupariation. Except for the very
small clones in which the number of trichomes was counted directly, the size was
calculated according to the area occupied by each type of trichome with corrections for different cell densities.
Age at
irradiation (h)
Total no.
of cells
24-48
40
24
12
100
100
40
70
100
160
300
100
250
300
300
1000
1000
1000
200
48-72
72-96
96-120
No. of
wing cells
30
6
6
60
20
10
5
25
150
150
30
100
100
280
500
300
200
100
No. of
Ratio wing/
haltere cells haltere cells
10
18
6
40
80
30
65
75
10
150
70
150
200
20
500
700
800
100
3/1
1/3
1/1
3/2
1/4
1/3
1/13
1/3
15/1
1/1
3/7
2/3
1/2
14/1
1/1
3/7
1/4
1/1
28
G. MORATA
The continuity of all of the clones embracing wing and haltere argues against
the possibility of two independent events of mitotic recombination since the
probability of conjunction of two independent clones to form a continuous
pattern must be very low.
The supposed influence of division rate on homeotic expression has been
emphasized several times (Ouweneel, 1969; see Postlethwait & Schneiderman,
1971, for discussion on this point). The results presented here argue against
this hypothesis since the sizes of clones producing haltere or wing are more or
less similar, suggesting that division rate does not influence the final differentiation. It also has been shown that the normal haltere and wing had very similar if
not equal division rate (Morata & Garcia-Bellido, in preparation). Furthermore,
the analysis of clones differentiating wing and haltere (Table 3) shows that the
proportion of wing and haltere tissue in those clones is very variable, suggesting
that the division rate of the cells within a given clone does not affect the
differentiation.
DISCUSSION
As shown previously (p. 30) in Cbx flies the homeotic transformation of wing
into haltere is likely due to the derepression in the mesothorax of the bithorax
and postbithorax genes which are those responsible for the anterior and posterior
transformation respectively. However, the genetic analysis shows that despite
their derepression in the mesothorax, the activity of bx+ and pbx+ when in cis
with Cbx is below the level found in wild-type flies. This hypofunction may
explain the partial expression of Cbx genotype; that is, some cells might have
enough bx+ or pbx+ product to differentiate mutant haltere phenotype while
others would not and so produce normal wing cells.
In this context it is interesting to note that in many aspects the developmental
pathways leading to wing and haltere formation are similar. Both discs grow
during the same larval stages, have similar division rate and have an apparently
homologous process of compartmentalization (Morata & Garcia-Bellido, in
preparation; Garcia-Bellido, Ripoll & Morata, 1973). These results suggest that
only the presence of bx+ and pbx+ gene products is enough to direct development into haltere while their absence produces wing structures.
Moreover, it has been inferred from mosaic analysis (Morata & GarciaBellido,in preparation) that during most of the developmental period of the haltere
disc the cells require the function of bithorax and postbithorax genes continually
so that the removal of the wild-type alleles is expressed directly by a change in
the state of determination of the affected cells. Only when the change is induced
after 24 h before puparium formation (that is, in the late development of the
disc) does the removal of the wild-type alleles fail to affect the final differentiation
of the cells. This inability of the cells to respond to a change in the genetic constitution after a given moment has been called 'perdurance' by Garcia-Bellido &
Merriam (1971 b). These results are in line with those obtained with other genes
Gene expression in Cbx
29
like hairy and Hairy wing (Hw) (Garcia-Bellido & Merriam, 19716) engrailed
(Garcia-Bellido & Santamaria, 1972) and spineless-aristapedia (Postlethwait &
Girton, 1974) and suggest that this continuous requirement is a general feature
of the homeotic genes. One can speculate that all show perdurance in the late
stages because of the persistence of the normal gene products during the remaining few divisions, rather than because of the attainment of an irreversible state
of determination. This interpretation is supported by the observation of GarciaBellido (unpublished) that the perdurance of bithorax is lost after in vivo culture.
The fact that there is such a continuous dependence by the cells on the state of
activity of their homeotic genes may help us to understand the development of the
Cbx wing. Due to the hypofunction of bx+ and pbx+, the mutant cells may have
an unstable state of determination that could change from one cell generation to
another depending on the level of activity in each cell of bx+ andpbx + products.
What actually decides the final fate of the cells is the level of these products in
the last divisions.
This instability of the state of determination is reflected by the clonal analysis
since clones embracing wing and haltere structures were found in all except the
last stage examined. The fact that these clones were not found in this last stage
may be due either to the small size of the clones at this period (about four
cells/clone, with little probability of crossing a border between haltere and wing)
or to the perdurance of bx+ and pbx+ products in the last two-three divisions.
If this latter possibility were true then the cells would be finally committed to
differentiate wing or haltere at a very late stage of development, on average
two-three divisions before the disc stops growing. In any case it is clear that
during most of the development of Cbx wing disc the cells remain undetermined
to produce wing or haltere.
In some respects these results are similar to those of Postlethwait & Schneiderman (1969, 1971) in their experiments with Antpn. They were unable to find
clones crossing the border between leg and antenna structures after 72 h of
development, that is the third larval period. As they point out recently (Postlethwait & Schneiderman, 1974) the mutation Antpn produces an unstable state
of determination in the mutant antennal disc.
Despite this lack of stable determination in the Cbx wing disc some regions
show a consistently higher degree of expressivity than others (see Fig. 4). The
reasons that may lead to this region-specific expressivity are not fully understood,
they may reflect different synthetic rates of bx+ and pbx+ products or different
regional requirements.
Together with these regional differences some wing structures seem to be more
easily transformed than others. In the cases of extreme expression it is often found
(see Fig. 2D) that the only wing structures that can be observed are the bristles
whereas all of the surrounding trichomes are typical of haltere. This differential
behaviour of bristles versus trichomes in wing-haltere transformation has also
30
G. MORATA
been observed in experiments (Morata & Garcia-Bellido, in preparation) in
which bxs/bxs clones (marked with y mwhjv) were induced in normal (bxz/ + )
halteres. Clones were detected which presented wing bristles surrounded by
marked (and therefore belonging to the same clone) haltere trichomes.
The causes of this difference between bristles and trichomes are not clear.
The bristles appear consistently in the margin and it has been shown (Santamaria
& Garcia-Bellido, in preparation) that bristle differentiation in the wing margin
is dependent on interactions between cells in the dorsal and ventral surface. It
is conceivable that those specific inductive stimuli demand a longer period of
bithorax gene activity in the affected cells (it is known that presumptive bristle
cells perform several extra divisions whereas presumptive epidermal cells do not
(Lawrence, 1966), and this may uncover the hypofunction of the controlling
genes in some cells. Other cells outside the margin do not receive the stimuli and
remain haltere trichomes.
Another characteristic of the Cbx transformation is the proximo-distal
correspondence between wing and haltere regions (see Fig. 4). This type of
correspondence has been observed previously in other homeotic mutants
(Gehring, 1966; Gloor & Kobel, 1966; Postlethwait & Schneiderman, 1969,
1971) as well as in bithorax mutants (Ouweneel, 1973; Morata & GarciaBellido, in preparation) and has been discussed in detail by Postlethwait &
Schneiderman (1971). This correspondence probably indicates identical positional information (Wolpert, 1969) along at least the proximo-distal axis in
the haltere and wing discs.
This identity in the positional information in homologous regions of wing
and haltere probably produces the same developmental properties, whether the
cells will differentiate haltere or wing depending exclusively on the activity of
inactivity of the bithorax genes.
I thank Drs A. Garcia-Bellido, P. Santamaria and P. Ripoll for fruitful discussions. I am
most grateful to Dr P. A. Lawrence for his comments and help during the preparation of
the manuscript.
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{Received 15 November 1974, revised 10 March 1975)
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