The Wilhelmine E. Key
1992 Invitational Lecture
Phenotypic Analysis of the Dopa
decarboxylase Gene Cluster Mutants in
Drosophila melanogaster
T. R. F. Wright
Theodore R.F. Wright is Professor Emeritus at the University of Virginia, Charlottesville, VA, where he conducted research on molecular genetics of Drosophila
melanogaster since 1965. Dr. Wright received his Ph.D
in genetics from Yale University in 1959 after undergraduate study at Princeton and masters research at
Wesleyan University. He has enjoyed continuous grant
support from National Science Foundation and National Institutes of Health from 1960 through 1993. He has
authored numerous scientific publications and served
as co-editor of Advances in Genetics and Associate Editor of Developmental Genetics.
From the University of Virginia, Department of Biology,
Charlottesville, VA 22903-2477. This lecture was delivered on March 11, 1992 at the 33rd Annual Drosophila
Research Conference, held at the Wyndham Franklin
Plaza in Philadelphia. It is the 25th in the series of Key
lectures established by the American Genetic Association through funds bequeathed to the association by
Dr. Wilhelmine E. Key for support of lectures in genetics. 1 would like to acknowledge the efforts of the numerous individuals who over 25 years have contributed in one way or another to the results summarized
in this article. They include graduate students Allen F.
Sherald (deceased), Marianne Dudick Huntley, Clifton
P. Bishop, John Kullman, Mark E. Freeman, Jie Shen,
Gerald R. Hankins, Dean G. Stathakis, Martha Corjay,
and Nathaniel J. Pearlson; postdoctoral fellows and research associates Eileen Y. Wright, John C. Sparrow,
Allen F. Sherald (deceased), Glen C. Bewley (deceased), J. Lawrence Marsh, Ruth Steward, Bruce C.
Black, Wilson E. Mclvor, Ellen S. Pentz, and Theodore
Homyk; and technicians Charlotte A. Dillard, Barbara
J. Robertson, Mary D. Morton (deceased) Mary Helen
Graziano, Maria Angeles Mena (Yebenes), Janina L.
Kaars, Kay S. Greisen, Rita Tickel, Lee E. Hendrickson,
Anne D. Tomsett, Lee T. Stinchfield, Karen A. Ussher,
Robert L Youngken, Patricia 0. Cecil, Karen Wade, Lee
Litvinas, Pamela Neff, and Robert E. McMichael III.
Progress on this work was dependent on the collaboration of Ross G. Hodgetts, Wolfgang Beermann, Jay
Hirsh, Denise Gilbert, J. Lawrence Marsh, and E. McCrady. Barry Ganetzky, John Brittnacher, Dan L. Lindsley, J. Lawrence Marsh, Pierre Gay, Didier Contamine, and Trudi Schupbach contributed valuable
Drosophila stocks. This research was supported primarily by NIH Research Grant GM19242 to T.R.F.W. Additional support came from NIH Research Grants
GM31217 and GM332217 to B.C.B., NIH Training Grants
GM01450 to A.F.S. and G.C.B. and HDO7192 to B.C.B.,
E.S.P., and C.P.B., NSF Grant GU 1531 to J.C.S., and
Swiss National Science Foundation Postdoctoral Fellowship to R.S.
Journal of Heredity 1996:87:175-190; 0022-1503/96/$5.00
Mutations in a majority of the 18 loci of the Dopa decarboxylase (Ddc) gene cluster
effect similar morphological defects of the cuticle and/or catecholamine-related abnormalities. Mutations in 14 loci affect cuticle formation, cuticle sclerotization, or
cuticle melanization, with mutations in 11 of these same loci (including Ddc and
amd) producing melanotic psueudotumors, symptomatic, perhaps, of abnormal catecholamine metabolism. Mutations in seven of the genes perturb catecholamine
pool levels during prepupal and pupal development, all of which also form melanotic pseudotumors, suggesting several of these genes may encode proteins involved in catecholamine metabolism. Thus, the Ddc gene cluster represents in
higher eukaryotes an unusual example of a large cluster of functionally related
genes involved in a common physiological process.
The importance of catecholamines as neurotransmitters or neuromodulators in all
higher eukaryotes is well documented
(Cooper et al. 1991; Nagatsu 1973). In insects, catecholamine metabolism not only
plays a fundamental role in neurophysiology (Brown and Nestler 1985; Buchner
1991; Pichon and Manaranche 1985; Ristifo
and White 1990), but also in the formation,
sclerotization, and melanization of the
exoskeletal cuticle (Hopkins and Kramer
1992; Wright 1987a). A key catecholamine
in both of these fundamental biological
processes is dopamine (DA), because this
compound is not only a neurotransmitter,
but also plays a central role in the pigmentation of the cuticle and in the production of sclerotization precursors involved in the cross-linking of proteins and
chitin in the cuticle (Figure 1). The biosynthesis of DA occurs in both dopaminergic neurons and epidermis by the same
two steps in the pathway illustrated in Figure 1. In the first step, tyrosine 3-hydroxylase (TH) (E.C.I.14.16.2) along with a
pterdine cofactor catalyzes the hydroxylation of L-O-tyrosine (TYR) to produce
3,4-dihydroxy-L-phenylalanine (DOPA). The
decarboxylation of DOPA to form DA is the
second step and is catalyzed by the enzyme DOPA decarboxylase (DDC) (E.C.
4.1.1.28) and its cofactor pyridoxal phosphate.
Because of DDC's central role in DA biosynthesis and its apparent regulation by
the molting hormone ecdysone during de-
velopment in insects (Shaaya and Sekeris
1965), Ross B. Hodgetts and I independently began to search for the DDC structural gene in Drosophila melanogaster. In
an effort to recover mutations in DDC, my
laboratory conducted screens designed to
isolate mutations based on their sensitivity to the DDC inhibitor a-methyl DOPA
(aMD) (Sherald and Wright 1974; Sparrow
and Wright 1974; Wright et al. 1976a). Although seven mutant alleles exhibiting
dominant hypersensitivity to aMD were
isolated, DDC activity in these mutants
was normal, and, therefore, it was not possible to conclude that they were mutations in the DDC structural gene (Sparrow
and Wright 1974; Wright et al. 1976a). All
seven of these mutant alleles were recessive lethal and were mapped to 2-53.9, a
locus found in the vicinity of 37C on the
left arm of chromosome 2. Because all the
mutations in this locus were hypersensitive to aMD, it was designated as the alpha
methyl dopa hypersensitive (amd) gene.
Significantly, using segmental aneuploids
constructed by crossing a series of overlapping translocations (Lindsley et al.
1972), Hodgetts (1975) located the only
DDC dosage sensitive region in the genome to 36EF to 38D, a region that includes amd at 37C. In order to delineate
further the DDC dosage sensitive region
and provide a means of recovering putative lethal mutations in the structural gene
for DDC, a series of overlapping deletions
was isolated (Figure 2) (Wright et al.
175
0
NH 2
0
11
C-CH 2 -CH 2
o-p-o
II
i
'H
'
Diphenol Oxidase*
HO
'N-y8-alanyl dopamine
0 Tyroslne-O-PO 4
N-y9-alanyl dopamine quinone
I
\
/3-alanyl dopamine
synthetase
tf(3-70.7)
/9-alanyI dopamine
hydrolase
Alkaline Phosphatose
Aph ( 3 - 4 7 . 3 )
1
H C-CH 2 -CH 2
N
NH 2
CH2-C-COOH
0"
NH 2
Sclerotin
/>(2-48.5)
5(1-43.0)
CH2-COOH
CH 2 -NH 2
Tyrosine
I
Tyrosine Hydroxylase
pie ( 3 - 1 8 . 8 )
NH 2
/9-olonine
NH 2
CH 2 -C-COOH
DopaH
decarboxylase
HO
HO
•Uracll
Ddc\ 2-53.9)
Dopa
Diphenol Oxidase*
HO.
II
CH 2 -C-H
'
II
HO
Dopamine
H
Dopamine
N-acetyltransferase
Dot (2-107.3)
N-acetyldopomine
Diphenol Oxidase*
Diphenol Oxidase* Jl_
lndole-5,6-quinone
N-acetyl dopamine quinone
NH,
1
'
CH--C-COOH
c
1
H
Melanin
Diphenol Oxidase:
Melanin
Sclerotin
; A 3 -A?,r-J(2-53.1+);
Figure 1. Proposed pathway for catecholamlne metabolism In Drosophila. The asterisks Identify reactions Involving dlphenol oxidase activity. Mutations affecting dlphenol
oxidase activities are listed at the bottom of the figure. Numbers In brackets after gene symbols specify chromosome and locus. References for gene assignment! to reactions
In the pathway are as follows: Aph, Harper and Armstrong (1974); pie, Neckameyer and White (1993); Ddc, Wright et al. (1976a); Dat, Huntley (1978); / and e, Black (1988); b,
s, and dy, Black BC, Hankins GR, and Wright TRF, unpublished data; Dox-A2, Pentz et al. (1986); Dox-3, Rlzki et al. (1985); and qs and lyr-1, Pentz et al. (1990). Dox-1 (Deng
and Rlzkl 1989) also aftects dlphenol oxidase.
1976b). Lethal mutations in one of the
complementation groups isolated over
one of these overlapping, DDC dosage sensitive deficiencies reduced DDC activity
when heterozygous over the CyO balancer
(Wright et al. 1976a), and it was subsequently established that these mutations
were in the DDC structural gene (Wright
1977, 1978; Wright et al. 1981, 1982). That
Ddc and amd were separate genes was
clearly demonstrated by the fact that Ddc
mutations are not aMD hypersensitive,
that all recessive lethal Ddc mutations
complement all recessive lethal amd mutations, and that two pairs of Ddc and amd
mutant alleles were mapped genetically to
be 0.002 cM apart (Wright et al. 1976a,
1 7 6 The Journal of Heredity 199687(3)
1981). However, while the amd locus does
not encode the structural gene for DDC, its
hypersensitivity to levels of dietary aMD
and its effect on the integrity of the embryonic cuticle (see below) strongly argue
for its involvement in catecholamine metabolism and cuticle development. Therefore, these initial studies not only recovered recessive lethal mutations in the DDC
structural gene, they also suggested the
existence of a cluster of functionally related genes involved in catecholamine metabolism. Subsequently, the smallest DDC
dosage sensitive deletion in the region,
Df(2L)TW130, an 8-12 band deficiency
from 37B10;37Dl, has arbitrarily served to
define the cluster of functionally related
genes often referred to as the "Ddc gene
cluster."
To date, mutations have been assigned
to 18 loci in the Ddc cluster. That many, if
not most, of these genes are functionally
related is suggested by the fact that mutations in 14 of the genes affect the formation, sclerotization, and/or melanization of cuticle, catecholamine pool levels
are perturbed by mutations in eight loci,
melanotic pseudotumors are found in mutants of 11 of the genes, and the normal
activity of 13 of the genes is required for
complete female fertility. It is the purpose
of this article to document the phenotypes produced by mutations in all the
loci of the Ddc gene cluster except hk.
Dfmj)TW130
. Df(2L)NST
.Df(2L)VA17
1
Df(2L)TW203 .
Df(2UTE37B7
. Df(2UTE37C1
Dff2LJ0D15.
.Df{2LJVA12
Df(2UhkUC1 .
.Df(2UVA19
Oft2L)hk18_
.Df(2UVA13
DfQUVA 18 .
. Df(2L)Sd57
Dff2UhkUC2 .
. Df(2JJSd77
Df(2L)VA21 .
. Df(2USd37
DISTAL
-100 kb
-80 kb
1
-60 kb
1
hk Be BcBaB2Bb Dox B1
-20 kb
1
Bd
Okb
—I—
+ 20 kb
1
amd C2 Ddc Cc Cb Cd Ca Ca Cg
+ 40 kb
1
+ 60 kb
+ 80 kb
PROXIMAL
brat TW1
Figure 2. Location of deficiency breakpoints within the Ddc gene duster. Deficiency breakpoints located within the Ddc gene duster are shown In relation to the DNA
coordinate system used In this region. A solid horizontal line Illustrates the extent of a deletion. A dashed horizontal line, bracketed by solid vertical lines, Indicates the
region of mapping uncertainty for the breakpoint. Distal and proximal are relattve to the centromere. The zero coordinate corresponds to the Hpal restriction endonuclease
site near the 3' end of the Ddc gene, and the coordinates are positive In the direction of the centromere and negative In the direction of the telomere. The diagram Is drawn
to scale. Gene symbols are abbreviated (see Figure 3 legend).
Genetic and Molecular Organization of
the Ddc Cluster
Approximately 340 mutations have been
isolated In the 8-12 band Df(2L)TW130 region (37B9-Cl;Dl-2), which defines the Ddc
cluster. They have been assigned to 18
genes: 16 vital genes, including Ddc and
amd, one female sterile gene, fs(2)TWl,
and one recessive visible gene, hook, mutations in which produce hooked bristles
(Stathakis et al. 1995; Wright 1987a; Wright
et al. 1976b, 1981).
The Ddc gene was initially cloned by
Hirsh and Davidson (1981), and subsequently chromosome walks cloned ~160
kb of contiguous DNA in the Df(2L)TW130
region (Gilbert and Hirsh 1981; Hankins
1991; Stathakis et al. 1995; Steward et al.
1984). The breakpoints of 17 overlapping
deficiencies have been physically located
in the cloned DNA (11 depicted in Figure
2), making it possible to assign all 18
genes to 10 segments of DNA (Black et al.
1987; Gilbert et al. 1984; Hankins 1991;
Kullman 1989; Pearlson 1991; Pentz and
Wright 1986; Stathakis et al. 1995). By
means of Northern blot analysis of mRNA
and the isolation of cDNAs, 21 transcription units have been definitively identified
in this region, and there are undoubtly
several more based on reverse Northern
blot surveys (Pentz ES, unpublished data).
No mutations have been recovered in
three of the 21 transcription units, TU37B1
(Bh), TU37B2, and TU37C2 (Cs), although
cDNAs were isolated for all three of them
(Freeman 1989; Spencer et al. 1986a,b; Sta- ters: a proximal subcluster with eight
thakis et al. 1995).
genes, amd, Ddc, l(2)37Cc, l(2)37Cb,
Restriction fragment length polymor- l(2)37Cd, l(2)37Ca, l(2)37Ce, and l(2)37Cg,
phism (RFLP) analysis of 245 of the 340
plus one unmutated transcription unit,
mutant strains in the region identified 15
TU37C2(Cs), in - 2 3 kb of DNA and a distal
RFLPs in 12 of the 18 genes, permitting the subcluster with six of the genes, hk,
provisional assignment of hk, l(2)37Be, l(2)37Be, l(2)37Bc, l(2)37Ba, l(2)37Bb, and
l(2)37Bc, l(2)37Bb, Dox-A2, l(2)37Bd, amd, Dox-A2, and two unmutated transcription
Ddc, l(2)37Cc, l(2)37Cb, l(2)37Cd, and brat units, TU37B2 and TU37B1 (Bh), in 27.4 kb
to definitive transcription units within the
of DNA. Approximately 53 kb of DNA sep10 segments of DNA defined by the defi- arate the two subclusters. Four "scattered
ciency breakpoints (Black et al. 1987; Free- genes," l(2)37Bg l(2)37Bd, brat, and
man 1989; Hankins 1991; Kullman 1989; fs(2)TWl, are not included in the two subPearlson 1991; Pentz and Wright 1986).
clusters. Both l(2)37Bd and l(2)37Bg are
Subsequently, the rescue of lethality of lelocated between the two subclusters. Inthal/Df(2L)TW130 hemizygotes by trans- terestingly, because of the large size deformed fragments of DNA delineated pre- duced for l(2)37Bd (-24 kb), >44% of this
cisely the extent of the DNA required for
intervening region is occupied by this
the activity of 15 of the 16 vital genes. This gene (Pearlson 1991; Stathakis et al. 1995).
was effected by the germline transforma- The last two scattered genes, brat and
tion of >26 different DNA fragments along fs(2)TWl, are clustered together -27 kb
with the recovery of >75 transformed
proximal to the proximal subcluster withstrains (Freeman 1989; Hankins 1991; Kull- in a region of no more than 15 kb.
man 1989; Marsh et al. 1985; Schlonick et
The density of clustering in the two subal. 1983; Stathakis et al. 1995; Wang and
clusters is emphasized by the fact that
Marsh, 1995). No attempt was made to
75.2% of the DNA is transcribed in the
transform DNA fragments for hk and
proximal (Ddc) subcluster and 82.1% in
fs(2)TWl, and the rescue of l(2)37Bd with the distal (Dox) subcluster. Furthermore,
its 20-kb intron was not effected (Pearlson
there are two pairs of overlapping genes
1991).
in the proximal subcluster and one pair in
In Figures 2 and 3, summarizing the ge- the distal subcluster. TU37C2(Cs) and Ddc
netic and molecular organization of Ddc share an 88-bp overlap at the 3' end of
both genes (Eveleth and Marsh 1987;
cluster, it is quite apparent that the genes
Spencer et al. 1986a,b), and the noncoding
are not evenly distributed in the 160 kb of
3' end of l(2)37Cb has a 260-bp minimum
cloned DNA, but that 14 of the 18 genes
are segregated into two distinct subclus- overlap with the noncoding 5' end of
Wright • Ddc Gene Ouster Mutants 1 7 7
Be
hk
Lk V
i
Ba
Be
k*\
TU37B2 Bb
Dox-A2
rti
ni*;
TU37B1
r
Distai Subcluster 27.4 kb (over 82.1% transcribed)
brat TW1
DISTAL |—
-80 kb
PROXIMAL
I
-80 kb
+40 kb
+60 kb
Proximal Subcluster 23.0 kb (75.2% transcribed)
amd
TU37C2
Ddc
Cc
Cb
Cd
Ca
Ce Cg
1.0 kb
Figure 3. Molecular organization of subclusters within the Ddc gene cluster. The deduced location, structure, and transcriptlonal direction of genes within the distal and
proximal subclusters are shown. Exons are represented by rectangles and introns by lines. Genes that have their exons colored medium gray Indicate structure known by
DNA sequencing, genes with light gray exons Indicate position deduced by restriction enzyme mapping. The direction of transcription Is Indicated by the bent arrow. See
Figure 2 for a description of the DNA coordinate system. The diagram Is drawn to scale, with the 1.0-kb scale corresponding to the enlarged subcluster regions. The 1(2)37
prefix has been omitted from the following g«ne symbols In the figure. Be, Be, Bo, Bb, Bg. Ba\ Cc, Cb, Cd, Ca, Ce, and Cg. TWl = fs(2)TWl
l(2)37Cc (Kullman 1989). In the distal subcluster, the 42-bp overlap in the untranslated 3' ends of l(2)37Bb and TU37B2 includes putative poly-A addition signals for
both genes (Freeman 1989). The functional significance of these overlaps, if any, is
unknown.
The Ddc gene cluster appears evolutionarily conserved, because subcluster integrity is maintained in related Drosophila
species. In D. virilis and D. psuedoobscura,
although the proximal and distal subclusters are separated, the subclusters themselves are intact and are still located on
the same chromosome (Wright TRF, unpublished data). Therefore, the maintenance of subcluster integrity appears
functionally important enough to conserve.
Mutations Affecting Enzymes in
Catecholamlne Metabolism
Three genes contained within the Ddc
gene cluster encode enzymes involved in
biogenic monoamine metabolism. Two of
these enzymes, DDC and DOX-A2, participate in catecholamine biosynthesis (Pentz
et al. 1986; Wright et al. 1982).
Dopa decarboxylase (Ddc). Drosophila
DDC decarboxylates DOPA to DA (Lunan
and Mitchell 1969) (Figure 1) and 5-hydroxytryptophan to serotonin (5-hydroxytryptamine), but not tyrosine to tyramine
1 7 8 The Journal of Heredity 199687(3)
(Livingstone and Tempel 1983). DA is required to effect the sclerotlzation of the
cuticle by being further metabolized to Nacetyl DA (NADA) and N-8-alanyldopamine (NBAD), which, after oxidation to their
respective quinones by diphenol oxidase,
crosslink cutlcular proteins and chitin
(Figure 1). In adults and white prepupae,
95% of the DDC activity is located in the
epidermis (Lunan and Mitchell 1969;
Scholnick et al. 1983), and - 5 % is found
in the central nervous system, where it
produces the neurotransmitters DA and
serotonin.
More than 50 Ddc mutations have been
Isolated (Lindsley and Zimm 1992; Wright
et al. 1982). Most are recessive lethals having an effective lethal phase (ELP) at the
end of embryogenesis when actively moving larvae with unpigmented cephalopharyngeal apparatuses and denticle belts are
unable to hatch (Wright and Wright 1978).
However, many larvae hemizygous for null
alleles, e.g., the 2.3-kb intragenic deletion
Ddc*7 over Df(2L)TW130, when released
from the egg membranes, will continue to
develop to the third instar and even complete metamorphosis getting to the late
pharate adult stage (Valles and White
1986; Wright 1987a). Genotypes that produce adults with drastically reduced DDC
activities (<5% of wild type) effect an "escaper" phenotype characterized by in-
complete pigmentation and sclerotlzation
of the cuticle (Wright et al. 1976a) (Figure
4). Developmental times may be prolonged by as many as 5 days, and mutant
puparia are easily scored by abnormal melanization at each end of the greenish-gray
pupa case (Figure 5). Newly eclosed
adults with 0.5-2% DDC activity are virtually pigmentless with thin, straw-colored
macrochaetae, whereas those with somewhat more DDC activity, 3-5%, have pigmented macrochaetae and clearly darken
upon aging a few hours (Figure 4). Their
wing axillae become heavily melanized,
similar to the phenotype of the mutant
speck, and their leg joints also become melanized (Figure 4). This is, perhaps, due to
the phenol oxidase wound reaction
brought on by the rupture of weakened
cuticle and/or the oxidation of extremely
high DOPA pools produced by the metabolic block (Black et al. 1987).
The time- and tissue-specific expression
of Ddc during development has been extensively examined (Beall and Hirsh 1984,
1986, 1987; Budnik and White 1988; Clark
et al. 1986; Gietz and Hodgetts 1985; Hodgetts et al. 1986; Konrad and Marsh 1987;
Kraminsky et al. 1980; Lundell and Hlrsh
1994; Marsh and Wright 1980; Marsh et al.
1985; Scholnick et al. 1983; Valles and
White 1988), and the effects of mutations
on expression of Ddc have also been in-
\
Figure 4. Ddc?2 homozygous females shifted from 18°C to 30°C at pupariation. Newly hatched female on the right
and on the left a female ~24 h after hatching. The phenotypes are typical for flies with 5-10% dopa decarboxylase
activity, i.e., sufficient activity for pigmentation of the bristles in the young fly and for the older fly to darken as
it aged. In the older fly, note the heavy melanization of the axillae of the wings and the joints of the legs, which
probably arises as a wound reaction to the rupture of weak cuticle along with extremely high DOPA pools.
pools a prominent electroactive compound is missing in intragenic complementing heteroallelic heterozygous adults
(amd"'/amdHSg). However, it has not been
possible to reproduce these results (Homyk T, personal communication). Although the precise function of the amd
gene product is not yet apparent, it seems
highly likely that it is a decarboxylase, because the derived amino acid sequence
(Eveleth and Marsh 1986b; Marsh et al.
1986) shows a 45% sequence identity with
D. metanogaster and rat DDC, 39% with
both D. melanogaster and rat histidine decarboxylase (HDC), and 37% with periwinkle tryptophan decarboxylase (Burg et al.
1993; Jackson 1990). All five of these enzymes are pyridoxal phosphate (PLP)-dependent decarboxylases with identical putative PLP binding sites, amd also has a
putative PLP binding site, which, with the
exception of one amino acid, is identical
to that in the above five decarboxylases.
Diphenol oxidase A2 subunit (Dox-A2).
The Dox-A2 gene is located in the distal
subcluster —63 kb from Ddc (Figure 1).
Dox-A2 function is necessary for the production of the A2 component of the complex phenol oxidase enzyme and is probvestigated (Bishop and Wright 1987; Bud- lethals. Null allele homo- and hemizygotes
ably the A2 structural gene (Pentz et al.
nik and White 1987; Budnik et al. 1986,
of amd die as normally pigmented larvae
1986). It is the first insect phenol oxidase
1989; Estelle and Hodgetts 1984a,b; Valles
both prior to and just after ecolsion from
and White 1986, 1990). Hirsh's group has
the egg. In addition to having necrotic, ex- gene to be cloned and sequenced, and the
encoded putative protein product is
studied the transcriptional regulation of
truded anal organs, these larvae burst
the Ddc gene, including the regulation of
very easily when manipulated, suggesting unique among currently known phenol oxidases (Pentz and Wright 1991). Geiger
its tissue-specific alternate splicing in the
incomplete sclerotization of the colorless
and Mitchell (1966) and Seybold et al.
hypoderm versus the central nervous sys- body wail cuticle (Wright 1977). Electron
(1975) have characterized this complex
tem (Bray et al. 1988, 1989; Hirsh 1989; micrographs indicate that the anal organ
Hirsh et al. 1986; Johnson and Hirsh 1990; defect arises from the incomplete sclero- enzyme system as being made up of at
least three protein components, Al, A2,
Johnson et al. 1989; Morgan et al. 1986; tization of the cuticular suture between
Scholnick et al. 1986; Shen and Hirsh 1994;
anal organ cells and the surrounding epi- and A3, all of which are activated by a reShen et al. 1993). None of these aspects
dermal cells (Wang and Marsh 1995). Kon- action cascade involving at least three additional proteins. Pre-S interacts with S-acon the regulation of Ddc expression will be rad et al. (1993) established a requirement
tivator to yield S, and then S acts on P to
covered here.
for amd* activity during oogenesis by
produce P', which interacts with the A
making homozygous amd mutant germline
alpha methyldopa hypersensitive (amd).
components
to yield active phenol oxiand
follicle
cell
clones.
These
activities
As heterozygotes, amorphic mutations of
dase.
The
activated
enzyme complex utilwere
inferred
to
be
involved
in
vitelline
amd (amd/+~) are hypersensitive to the diizes both monophenol and diphenol subetary administration of the DDC analog in- membrane biosynthesis, because feeding
strates. The three A components can be
females aMD caused them to lay eggs with
hibitors a-methyldopa (aMD) (Sparrow
2
separated from one another by gel electrodefective
vitelline
membranes
(Konrad
et
and Wright 1974) and N'(DL-seryl)-N phoresis of homogenates prior to activaal. 1993).
(2,3,4-trihydroxybenzyl)hydrazine (Wright
tion.
After electrophoresis, the gels are
et al. 1976a), and resistance to dietary
amd heterozygotes (omd/+) and amd
first
incubated
in an activator solution and
aMD is directly correlated with amd* gene intragenic complementing heteroallic hetthen
in
a
suitable
substrate solution. If the
HSS
dosage: the more doses of the amd* gene, erozygotes (amd"'I&md ) do not affect
substrate is oxidized, pigment is depositthe more resistant (Marsh and Wright
DDC nor DA acetyl transferase in any way ed in the gel at the A component sites.
1986; Wright et al 1976a). This relationship whatsoever: neither as adults nor as white
Monophenol substrates are oxidized prisuggests that the in vivo function of the
prepupae, and neither in the epidermis
marily by the Al component and diphenol
AMD protein product is inhibited by the
nor in the central nervous system (Hunt- substrates, including DOPA, DA, NADA,
binding of the modified catecholamine
ley 1978; Wright 1977; Wright and Wright
and NBAD (Figure 1) by the A2 and A3
aMD.
1978; Wright et al. 1976a, 1981a).
components (for methods, see Pentz et al.
Thirty-nine amd mutations have been
Black et al. (1987) infer that amd activity
1986; Rizki et al. 1985; Warner et al. 1975).
isolated (Lindsley and Zimm 1992; Wright
is necessary for colorless sclerotization.
et al. 1982), most of which are recessive They reported that in catecholamine
Four Dox-A2 recessive lethal alleles and
Wright • Ddc Gene Cluster Mutants 1 7 9
have in vivo any functional phenol oxidase
activity at all, i.e., for the Al and A3 components to be active in vivo also. The pigment deposited during embryogenesis in
the mouth parts and denticle belts of DoxA2 homozygotes may be due to the presence of a maternal component, protein, or
mRNA, or be due to independent Al or A3
component activity. No experiments have
been done to verify these possibilities.
One mutant allele, Dox-AZ"", reduces the
viability of hemizygotes to 60% of controls, and both females and males that
eclose are sterile.
Mutations Affecting Catecholamine
Pool Levels During Development
Besides Ddc and Dox-A2,fiveother genes
within the Ddc gene cluster dramatically
affect in vivo catecholamine pool levels
during prepupal and pupal development
(Homyk T, Mclvor WE, and Wright TRF, unpublished data). However, the molecular
function of these five loci is not understood; therefore, it is not known if these
genes encode enzymes. Mutations in these
genes, Be, Be, Cc, Ce, and brat, all exhibit
severe cuticle abnormalities, all develop
melanotic psuedotumors, and, with the exception of Be, all affect female fertility (Table 1).
l(2)37Bc (Be). Twenty-nine Be alleles
were recovered and characterized (StaFigure 5. (A) Left: Wild-type pupa. Right: Ddcf!/Ddc"! pupa raised at 25°C. The yellow-green color of Ddc pupae thakis DG and Wright TRF, unpublished
has made it possible to score them reliably vis-a-vis wild-type pupae (Scholnick et al. 1983). (B) Middle: Wild-type
data). Amorphic mutations of the Be gene
pupa. Left and right: l(2)37Bg'/Df(2L)TW130 pupae. The mutant pupae are consistently larger and darker. The as homozygotes or hemizygotes die as
abnormal air space visible in the posterior end of the mutant pupa on the left is found in many, but not all, flg'
pupae. (Q Middle: Wild-type pupa. Left and right: l(2)37Bc"/Df(2L)TW130 pupae. The mutant pupae are darker, stunted first instar larvae. Numerous hyparticularly at the posterior end. In the central region of the mutant on the right, the initial stage of abnormal
pomorphic alleles permit homo- and hemmelanization is apparent. (D) The same three pupae shown in C, but 24 h later. The mutant melanization in the
izygotes to survive to later stages in decentral region of both Be" pupae is readily seen. Some of this melanization arises from oxidized hemolymph exuded
velopment, even becoming pharate adults
from ruptured abdomen.
and eclosing as adults. Any one hypomorphic allele can cause mortality at any
developmental
stage from late embryos
one male-female sterile allele have been
Hemizygotes, Dox-A2"/Df(2L)TW130, of
isolated (Lindsley and Zimm 1992; Pentz
all the lethal alleles of Dox-A2 die during through pharate adults. In addition, both
et al. 1986; Wright et al. 1982). The lethal
the first larval instar, have normally pig- amorphic and hypomorphic alleles effect
some lethality as heterozygotes, Bc"/+ or
alleles as heterozygotes over the CyO bal- mented mouth parts and denticle belts,
ancer chromosome reduce diphenol oxi- and show no cuticular abnormalities. Bc/CyO, i.e., semi-dominant lethality (Stathakis DG, personal communication). Hetdase activities to 47-79% of wild type but
However, the dead larvae never turn
have no effect on monophenol oxidase ac- black. A rare Dox-A2l homozygous mutant eroallelic heterozygous combinations of
many Be alleles exhibit a complex pattern
tivity. Pool sizes of DOPA, DA, and NADA
individual survived to the pharate adult
of negative and positive complementation
are significantly elevated in a mixed pop- stage and was released alive from the
(Stathakis DG, personal communication).
ulation of embryos collected from a Doxpupa case by dissection. The mutant was
x
A2 jCyO stock in comparison to suitable
completely unpigmented with bristles and
Measurement of catecholamine pools in
controls (Pentz et al. 1986). This indicates
cuticle being totally colorless (see colored
the later stages of embryogenesis in
that Dox-A2 function is necessary to oxi- illustration in Pentz et al. 1986). This mu- amorphic allele hemizygotes, Bcl2/Df, redize these substrates to their respective
tant never developed melanization in the veal a dramatic 500% (fivefold) increase in
quinones (Figure 1). Dox-A2/+ heterozy- joints of the legs, axillae of the wings, or
DA and NADA pools vis-a-vis wild-type
gotes reduce only the A2 component ac- even when the very weak cuticle of the ab- controls (Stathakis D, personal communitivity after separation of the A compo- domen eventually ruptured. The fact that
cation). The fact that tyrosine pools renents in polyacrylamide gels, implicating
this D0X-A21 homozygote never tanned or main normal suggests that tyrosine hyDox-A2 as the structural locus for this
melanized suggests that normal A2 com- droxylase activity may be increased sigcomponent.
ponent must be present at this stage to
nificantly in these mutant embryos. That
1 8 0 The Journal of Heredity 1996:87(3)
Table 1. Summary of Ddc cluster mutant phenotype*
Location and locf
Distal subcluster
hk(6)
l(2)37Be(5)
l(2)37Bc (29)
l(2)37Ba(\2)
l(2)3TBb(\l)
Dox-A2(5)
Effective*
lethal phase
None
E
E,UP
P
L
L
Distal scattered gene!i
l(2)37Bg(2)
P
l(2)37Bd(9)
L
Proximal subcluster
amd (39)
Ddc (53)
l(2)37Cc(25)
l(2)37Cb (32)
l(2)37Cd(\\)
l(2)37Ca (44)
l(2)37Ce (8)
l(2)37Cg(4-)
E,L
E.L.P
L
L
Catecholamlne*
pools
Melanotic
pseudotumors
Incomplete
cuticle
Incomplete Abnormal
sclerotlpigmenzatlon
tation
nd
A
A
+
NoYesYes
Yes
No
No
No
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
No
No
fs(3)
mfs(l)
No
mfs(l)
Yes
No
Yes
Yes
No
No
Yes
No
fs
No
Yes
Yes
Yes
No
No
No
No
No
No
Yes
Yes
No
Yes
Yes
Yes
No
Yes
Yes
No
No
Yes
Yes
No
Yes
Yes
Yes
No
fs
No
fs(2)
mis
fs
mfs
fs
fs
Yes
No
Yes
No
11
Yes
No
10
mfs
is (15)
13
+
A
A
+
+
A
A
+
L
L
P
+
+
A
L
+
No
Yes
Yes
A
nd
8
Yes
No
11
Proximal scattered genes
6raf(30)
P
fs(2)TW-l (15)
None
Total (340)
Yes
8
Yes
Fertility'
effects
• Number of alleles In parentheses.
*E = embryonic; L = larval; P - pupal.
' A — catecholamlne pools perturbed; + = wild-type pools; nd = not determined.
'fs = female sterility; mfs = male and female sterility; number of fs alleles Isolated In parentheses.
* Yes = mutant phenotype found; No = mutant phenotype not found; nd = not determined.
DDC activity is not rate limiting and DOPA
pools are unchanged is consistent with
this interpretation. Thus, the BC protein
may play a fundamental role in regulating
tyrosine hydroxylase, the rate-limiting enzyme in this pathway. Note that NBAD and
NBANE have not been detected in either
Bcn[Di hemizygote or wild-type embryos.
The hypomorphic alleles of Be produce
an array of pleiotropic mutant phenotypes
later in development, with Be'1 being the
prime example (Stathakis DG, personal
communication). Variable degrees of promiscuous melanization occur in Be11 homozygotes, hemizygotes (Bc"/Df), and
heterozygotes (Be1'/CyO). In third instar
larvae and occasionally in second instars,
either all or only a few salivary gland cells
become melanized. In some larvae, melanization can also be seen in the mid-dorsal
region, probably in the lymph glands,
which can then spread throughout the hemolymph. These larvae never pupariate.
Many other Be" mutant larvae do pupariate, producing pupal cases that are initially darker than wild type (Figure 5).
Upon ageing these pupal cases become
even darker, and most of them exhibit
large irregular regions of internal melanization (Figure 5). Of those Be mutants that
die subsequent to pupariation, some never pupate (i.e., the imaginal discs don't
evert); others pupate and produce relatively normal cuticle on the head and thorax but exhibit extensive regions of incomplete cuticle formation on the abdomen
(Figure 6D). Usually large areas of the incomplete cuticle are heavily melanized
(Figure 6D). Be mutant adult escapers often have a few abdominal segments with
incomplete cuticle, and frequently large
melanotic tumors are apparent internally
(Stathakis DG, personal communication).
Three Be female sterile alleles have been
isolated that, at 25°C, have little or no effect on viability as Be*/Df(2L)TW130 hemizygotes, but two of the three are cold-sensitive lethals at 18°C.
Consistent with observations of promiscuous melanization is the determination
that homozygotes, hemizygotes, and heterozygotes of Be hypomorphic alleles not
only significantly increase the levels of diphenol oxidase activity in activated extracts of whole third instar larvae, prepupae, and pupae, but also accelerate activation with a distinctly shorter lag period
(Stathakis DG, personal communication).
Normally, without the addition of activator
extracted from another time period in development, there is no activation of phenol oxidase in extracts of wild-type 4-8-h
prepupae (Geiger and Mitchell 1966; Pentz
et al. 1986). This brief hiatus in activation
potential is completely missing in extracts
of Be" female prepupae and is significantly delayed and foreshortened in extracts
of Be11 male prepupae (Stathakis DG, personal communication). These results suggest that in Be" larvae and pupae diphenol oxidase is activated promiscuously at
random times in development and randomly in tissues where it is not normally
activated, and that this is reflected in promiscuous melanization and the random
timing of lethality.
The analysis of catecholamine pools
during the first 72 h after pupariation of
Be" hemizygotes showed dramatic increases at various times during this period (Homyk T, Mclvor WE, and Wright TRF,
unpublished data). The time course for
NADA pools in Be hemizygotes exhibited
peaks that were 500-1,000% higher than
wild-type controls. NBAD and NBANE
peaks were up 200-600%, DA up 300%, but
DOPA remained unchanged. Although tyrosine pools have not been measured in
these mutant pupae, the elevation of these
pools may be due to increased tyrosine
hydroxylase activity in Be mutants. It is
not clear just why diphenol oxidase activity is increased and its activation accelerated in Be mutants. Perhaps the flc+ product regulates the activity and/or activation
of diphenol oxidase as well as tyrosine hydroxylase. It is unlikely that the high levels
of these catecholamines, substrates normally oxidized by the diphenol oxidase to
their quinones, are involved in feedback
regulation of the enzyme, because other
mutations with higher pool levels, e.g.,
brat, do not exhibit the expected promiscuous melanization syndrome.
Analysis of the amino acid sequence of
the Be protein derived from the sequence
of a full-length cDNA (Stathakis DG, personal communication) indicates that the
Be protein may be a transmembrane protein, but no informative homologous proteins have been found in the data bases.
l(2)37Be (Be). Of the five Be alleles isolated, four are amorphic mutations that,
as hemizygotes, die during embryogenesis, producing active, unhatched larvae
exhibiting no obvious abnormalities.
Hemizygotes of the hypomorphic allele
Be3 often produce mutant pharate adults
with an incomplete cuticle. This mutant
phenotype is almost identical to those
produced by hypomorphic alleles of Be
and Ba (Figure 6, compare A with B and
D). Numerous Be3 hemizygotes eclose as
completely normal, fertile adults.
Melanotic pseudotumors are found in
Be?/DF(2L)TW130 mutant pharate adults
Wright • Ddc Gene Ouster Mutants 1 8 1
B
D
I
Figure 6. (A) Dissected l(2)37Be'IDf(2L)TW130 pharate adult. The cuticle on some of the abdominal segments has not completed differentiation. Melanotic pseudotumors
are apparent. This is a hypomorphic allele phenotype. Amorphic Be allele hemizygotes die as embryos. (B) Dissected l(2)37Ba2/Df(2L)TWI30 pharate adult. The cuticle has
not completed differentiation on any of the abdominal segments. One large melanotic tumor is present. This is an amorphic allele phenotype, because Ba is a late pupal
lethal. (Q Dissected l(2)37Ce'/Df(2L)TWI30 pharate adult. The cuticle on the abdomen has not completed differentiation. The wrinkled appearance of the exposed, undifferentiated cuticle is different from that of Be3 and Ba2. A very large melanotic tumor is visible. This is an example of the Ce amorphic phenotype, Ce being a late pupal lethal.
(D) Dissected l(2)37Bc"/Df(2L)TW130 pharate adult. The ruptured abdomen arises in an area where cuticle formation is incomplete, resulting in leakage of hemolymph that
melanizes. The melanization is apparent through the undamaged pupa case at the time of imaginal disk eversion and darkens as development proceeds. This is a hypomorphic
allele phenotype, because Be amorphic allele hemizygotes die as collapsed first instar larvae that do not melanize at all. (E) Dissected l(2)37Bg'/Df(2L)TW130 pupa 50-60 h
postpupariation. There is a complete lack of cuticle differentiation except perhaps over the genital disk region. The eye-antennal disk has everted, but the leg and wing disks
have not. This is the hypomorphic allele phenotype, because amorphic Bg alleles, cub, are embryonic lethal. (F) Dissected l(2)37Bcf/l(2)37Bd> pharate adult. The differentiated
cuticle is absent over regions of the head, thorax, and abdomen. Melanotic tumors are apparent. This hypomorphic allele phenotype is also produced by partially complementing heteroallelic heterozygotes. Amorphic Bd allele hemizygotes die as larvae. (G) Adult l(2)bral'/l(2)braf partially complementing heteroallelic heterozygote >1 day
old. This escaper phenotype is identical to the l(2)37Cc"/l(2)37Cc° permissive temperature phenotype. The deep lateral thoracic creases and uninflated wings are symptoms
of a partially collapsed thorax that probably results from muscle tension on incompletely sclerotinized cuticle. (H) Adult l(2)37Cc/J'/Df(2L)TWI30 hemizygote that eclosed at
25°C unaided. The obviously distorted thorax is probably due to tension created by the indirect flight muscles on incompletely sclerotinized cuticle. The uninflated wings
are diagnostic of a partially collapsed thorax. Note the reduced pigmentation on the head.
(Figure 6A), and, during early pupal development in these hemizygotes, catecholamine pools are perturbed. DOPA
pools are 30% of wild-type controls, and
DA, NADA, and NBAD pools are down to
-50%, but the NBANE pool is up to 150%
of wild type (Table 2) (Homyk T, Mclvor
WE, and Wright TRF, unpublished data).
l(2)37Cc (Cc). Amorphic alleles of Cc as
hemizygotes or homozygotes are larval
1 8 2 The Journal of Heredity 1996:87(3)
lethals. As long as 192 h after egg collection at 25°C, late second instar and small
early third instar Cc'°/Df(2L)130 larvae
can be found crawling about in the food.
Upon dissection, no abnormalities were
seen, and the imaginal discs appeared to
be appropriately sized for larvae at that
stage.
At least nine of the 26 Cc alleles are classified as hypomorphic on the basis that an
allele-dependent fraction of Cc/Df(2L>
fjTW130 heterozygotes survives to eclose
as adults. Besides extended developmental times, many of these "escapers," as
well as some complementing heteroallelic
heterozygotes (Cc/Cc?), exhibit mutant
phenotypes indicative of incompletely
sclerotized cuticle, i.e., partially collapsed
thoraces characterized by abnormally
deep dorsal lateral sutures and uninflated
Table 2. Ddc eltuter mutant effects on prepnpal and papal catecholamtne pools
Location, loci,
allele
Distal subcluster
ft*
l(2)37Be het
l(2)37Bc hy
l(2)37Ba am
l(2)37Bb ts
Dox-A2 am
Distal scattered genes
l(2)37Bg am
l(2)37Bd hy
Proximal subcluster
amd het
Ddc am
l(2)37Cc hy
1(2)370} ts
l(2)37Cd ts
l(2)37Ca ts
/(2J37G: am
/62J37CS ts
Prox. scattered genes
brat am
fcf2J7TV-;
DOPA
DOPAMINE
NADA
NBAD
NBANE
nd
30%
nd
50%
300%
+
+
nd
50%
500-1,000%
+
nd
50%
200-600%
nd
150%
200-600%
+
+
+
+
+
+
+
+
nd
nd
rr
Tt
rr
+
+
+
+
+
20-30*
10,000%
300%
+
0
+
0
120%
120%
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
200-300%
+
+
+
+
200%
nd
400%
nd
400%
nd
+
nd
+
nd
75%
+
+
+
+
+
0
0
150%
150%
nd = not determined; % = % of appropriate controls; het = heteroallellc heterozygotes: Be'/Be3, amdH'lamdtm
(adults); hy - hypomorphlc allele hemlzygotes: Be", B&, Cc'5; am = amorphlc allele hemlzygotes: Bo", D0X-A21
(embryos), Bg1, Ddc*", Ce3, brat"; ts •= ts allele hemlzygotes shifted to 29°C' Bb>, CV, OP, Co", Cg-'.
wings. The phenotype is identical to that
depicted in Figure 6G for brat escapers. Besides Cc and brat, escapers of seven of the
other clustered lethals express a very similar phenotype (Table 1). In addition, other Cc escapers without the deep thoracic
creases do inflate their wings, which remain fragile, opaque, and somewhat wrinkled—also indicative of incomplete sclerotization. Furthermore, many of the escapers have thin scutellar and dorsocentral macrochaetae, i.e., a mild Minute-Wke
phenotype, and some, e.g., CcPj
DftfUjbh"0, have large abdominal melanotic tumors.
Relevant to incompletely sclerotized cuticle and melanotic tumors in some Cc escapers, catecholamine pool profiles during the 24-h period following pupariation
from Ccl5/DfC2L)TW130 hemizygotes show
significant differences in DOPA pool levels
from wild-type controls, exhibiting a 300%
increase (Homyk T, Mclvor WE, and
Wright TRF, unpublished data). Other catecholamine pool levels are also aberrant,
but to a lesser extend. NBAD and NBANE
pools are elevated 50%, whereas DA and
NADA are up 20%. How these changes in
catecholamine pools are related to the
mutant escaper phenotypes described
above is not clear.
All of Cc hemizygous (Cc/Df) female and
male escapers tested were sterile except
for Cc5 females. Individuals from 34 different heteroallelic heterozygous combinations that eclosed as adults were tested
for both female and male fertility. Fourteen of the combinations were both female
and male sterile, eight were neither female
nor male sterile, 11 were male sterile but
female fertile, and only one combination
was female sterile but male fertile. Although many of the hemizygous and heterozygous genotypes reduce viability, it is
clear that Cc* function is required for both
female and male fertility, with the latter
having a more stringent requirement. Females from some of the Cc genotypes laid
no eggs at all, and yet others did lay eggs
that did not develop. Females homozygous for Cd1', actually a cold-sensitive
semilethal allele, are completely sterile,
only laying collapsed eggs. One nonleaky
recessive lethal allele, Cc", also produces
a dominant female-sterile phenotype.
Most females carrying one wild-type dose
of Cc* and Cc13 lay no eggs, but a few females lay some eggs that do not develop,
remaining completely white. Examination
of the ovaries shows that the ovarioles
contain numerous follicles with inappropriate numbers of cystocytes: either with
too few or with too many. Yolk production
is also significantly reduced. The addition
of another wild-type allele to a genome
carrying Cc13, i.e., Cc" plus two doses of
Cc*, ameliorates the dominant female sterility but does not completely reverse it.
This is the case when the additional wildtype allele is provided either by three different duplications, Dp(2;Y)H3, Dp(2;l)AT,
or CyO,Dp(2;2)M(2)m*, or by different P
element-mediated transgenes carrying
the same fragment of DNA that rescues Cc
lethality.
Genomic and cDNAs of Cc have been sequenced (Eveleth and Marsh 1986a). The
published sequence has subsequently
been extensively corrected and shows
74% amino acid identity (Marsh JL, personal communication) with the rat prohibitin protein (Nuell et al. 1991), an antiproliferative factor. Microinjection of synthetic prohibitin mRNAs into normal human fibroblasts blocks 70% of the cells
from entering S phase of the cell cycle,
and microinjection of prohibitin antisense
oligonucleotides stimulates cells to divide
(McClung et al. 1992). The human prohibitin gene (99.6% identical to the rat gene
at the amino acid level) is located on chromosome 17q21, a locus associated with 23
sporadic breast cancers, and in four of
them mutations have been identified in
the prohibitin gene (Sato et al. 1992). Just
how this relates to the Cc amorphic lethal,
hypomorphic cuticular, and female-sterile
mutant phenotypes is not clear, particularly since Cc protein has been shown to
be associated with the mitochondrial inner membrane (Konrad KD and Marsh JL,
personal communication). The Cc transcript is expressed in early and late embryos, late third instar larvae, and adults
(Eveleth and Marsh 1986a).
l(2)37Ce (Ce). Eight alleles of Ce have
been recovered. Hemizygotes of amorphic
alleles of Ce die as pharate adults, most
with incomplete abdominal cuticle formation (Figure 6C). The wrinkled appearance
of the undifferentiated cuticle is different
from that of other mutants in the Ddc cluster, e.g., Be3 (Figure 6A) and Ba2 (Figure
6B). Occasionally, a Ce1 hemizygous pharate adult was found with incomplete formation of the cuticle on the thorax and
head as well as the abdomen: a mutant
phenotype which approaches in severity
that depicted for Bd'/Bd4 in Figure 6F.
Some Ce\ Ce*, and Ce8 hemizygous pharate adults have patches of pigmented cuticle, either with and without bristles and
hairs on the abdomen. Others have reduced numbers of complete and incomplete segments invested with pigmented
endocuticle. These are usually the most
posterior two or three segments. The mutant phenotype of incomplete tergites and
sternites is much more extreme than the
etching produced by extreme bobbed mutants.
Melanotic tumors, variable in size, are
present in most Ce hemizygous pharate
adults. A very large melanotic tumor is ev-
Wright • Ddc Gene Ouster Mutants 1 8 3
ident in the Ce1 pharate adult depicted in
Figure 6C. NBAD pools in Ce5 hemizygous
16-18-h-old pupa are 200-300% higher
than wild-type controls. All the other catecholamine pool are not perturbed. All
combinations of complementing heteroallelic heterozygous females and males are
fertile. However, although transplanted
ovaries from late third instar Ce5 hemizygous larvae attach to wild-type host oviducts during metamorphosis, they do not
produce fertile eggs (McCrady and Tolin
1994). Even though the follicles mature, no
vitellogenesis takes place in the Ce5 transplanted ovaries. These results indicate
that wild-type Ce function is required for
female fertility and that, in complementing
heteroallelic heterozygotes, sufficient activity is produced to promote fertility.
gion from which the optic lobes develop.
Pieces of brat mutant larval brains transplanted into adult female host abdomens
were found to grow in an unrestrained and
invasive manner, eventually filling the entire abdomen with neuroblasts, ganglion
mother cells, and neurons (Gateff E, personal communication). Proliferating cells
are also found in the thorax, where they
are seen to invade the indirect flight muscles. This is true for brat14 hemizygous
brains and brat11 and brat" homozygous
brains. Homo- or hemizygotes of brat1* and
brat" are 100% lethal, whereas 45-55% of
brat" hemizygotes eclose as adults at
25°C. Transplanted imaginal discs and ventral nerve cords from mutant larvae do
not show tumorous growth. On the other
hand, transplanted pieces of braf1 prepupal and pupal brains do show tumorous
growth in primary and secondary hosts,
indicating that the hormonal state of the
donor is not a factor in determining the
capacity for tumorigenesis. Serial transplants of brain tissue from bratu indicate
that the transplanted cells have as yet not
progressed into a state of immortality
(Hanratty W and Hankins GR, unpublished
data).
t(2)brain tumor (brat) [l(2)37Cf]. Most
lethality of brat hemizygotes occurs at the
pharate adult stage, although death of
hemizygotes may occur at any time from
embryogenesis on throughout development. Of the 29 alleles isolated to date,
nine are leaky with >10% hemizygote viability at 22°C, with another eight alleles
permitting 1-9% of the hemizygotes to
eclose as adults. The most striking mutant
phenotype of brat homozygotes and hemiMany of the brat hemizygous adult "eszygotes is the grossly enlarged brain hemi- capers" appear normal, whereas others
spheres of late third instar larvae. The
exhibit the incomplete sclerotizatlon pheventral nerve cord and imaginal discs are
notype, illustrated in Figure 6G by the hetunaffected. Brain enlargement is not ap- eroallelic heterozygote bratl/braf, where
parent in larvae until late in the third in- the thorax is deformed by the contraction
star. All 23 alleles examined as homo- and/
of the indirect flight muscles. Some brat
or hemizygotes show gross neuroblasto- hemizygous pharate adults show incommas. Surprisingly, the 23 alleles include
plete cuticle formation on the head, thotemperature-sensitive and female-sterile
rax, and abdomen; an extreme mutant
alleles at permissive temperatures for le- phenotype identical to that depicted in
thality. Depending on the allele, the size of
Figure 6F Because some brat mutants also
the enlarged brains can be quite constant
have melanotic tumors, catecholamine
or very variable, with some alleles produc- pools were surveyed in brat™ hemizygous
ing brains 10 times the normal volume.
pupae. Sixteen to 20 h after pupariation
There is no correlation between the le- mutant DA pools were up 200% vis-a-vis
thality of an allele and the amount of over- controls, and NADA and NBAD pools were
growth of the brain produced. To date, no
up 400%. At this crucial stage of developevidence of metastases has been seen in
ment, catecholamine metabolism is clearthird instar larvae. In spite of the huge
ly perturbed.
brains, most larvae proceed to pupariate
Female fertility has been tested for the
and pupate, eventually dying as pharate
164
heteroallelic heterozygous combinaadults. Often the entire enlarged brain
tions
that are not 100% lethal. Only four
does not fit in the head, and part is
highly viable combinations (65-95%) were
squeezed into the thorax. Sections of
normally fertile. Male fertility also showed
brat1* hemizygous mutant and wild-type
a
correlation of high fertility with high vicontrol wandering third instar larvae
ability,
brat males do not demonstrate norshow that brat™ mutant brains are indeed
mal
mating
behavior, and preliminary evisignificantly larger, being twice as long in
dence
indicates
that females also do not
the anterior-posterior axis as those in conmate
normally.
Whether
or not either sex
trol larvae. The excess growth is predomcan
see
or
whether
their
ERGs are abnorinately, if not exclusively, located in the
mal
has
yet
to
be
determined.
Three alposterior portion of the brain, in the releles with relatively high viabilities but
1 8 4 The Journal of Heredity 1996:87(3)
with very low female fertility have been
designated as female-sterile alleles (Hankins 1991).
The sequence of brat* has not been informative about its function, except indicating that it is a 99-kD hydrophilic protein
with two putative NTP-binding sites (Hankins 1991).
Mutations Affecting the Formation,
Sclerotizatlon, and Pigmentation of the
Cuticle
Mutations in seven genes within the Ddc
gene cluster show extensive cuticle malformations, but have little effect on in vivo
catecholamine pool levels during pupation. However, these seven genes do exhibit cuticular phenotypes characteristic
of mutations in the Ddc gene cluster (Table 1). These eight genes are Ba, Bb, Bg
Bd, Cd, Ca, and Cg.
t(2)37Ba (Ba). Eleven of the 12 Ba alleles isolated are lethal mutations which
can produce mutant pharate adult phenotypes that are almost identical to those
produced by Be hypomorphic hemizygotes and very similar to those found
among Be hypomorphic hemizygotes (Figure 6, compare A, B, and C). Amorphic allele hemizygotes, e.g., Bcfi/Df and flaVDf,
die as pharate adults unable to eclose.
Most of these are completely normal, with
only a few showing the mutant phenotype
of incomplete cuticle formation, particularly over the abdomen. This mutant phenotype, depicted in Figure 6B, is more frequently found among hypomorphic allele
hemizygotes such as Sa2/Df and Bff'/Df. Individuals hemizygous for these hypomorphic alleles also eclose as completely
normal adults, except that both females
and males are sterile. This result is consistent with the isolation of the 12th Ba
allele, Ba", hemizygotes for which are
completely viable but female or male sterile. Although melanotic pseudotumors are
found in Ba mutant pharate adults (Figure
6B), catecholamine pools during early pupal development are not perturbed (Homyk T, Mclvor WE, and Wright TRF, unpublished data).
l(2)37Bb (Bb). Except for only one pharate adult, all Bb hemizygotes investigated—Bb1, Bb9, and Bb",—died as third instar larvae. Six days after egg collection,
when essentially all wild-type siblings had
pupariated, Bb/Df(2L)TW130 hemizygotes
were still second instar or very small third
instar larvae. Eight-day-old Bb hemizygous
larvae were the size of mid third instar
wild-type larvae, which upon dissection
had very small second instar-sized ima-
ginal discs and very small salivary glands
and fat bodies with small cells. The brain,
VNS, gut, Malpighian tubules, and gonads
were the size expected for mid third instar
larvae. The mutant larvae were still active
on day 12, but remained immobile in the
food on day 14, exhibiting no attempt to
pupariate. The one exceptional Bb9/
Df(2L)TW130 hemizygote was alive when
dissected as a pharate adult on day 12 and
exhibited the deformed thorax phenotype
characteristic of incomplete sclerotization
of the cuticle. Except that the thorax was
more severely depressed, the phenotype
was identical to that of the brat hemizygote depicted in Figure 6G.
Bd* hemizygotes that were shifted from
the permissive temperature to the restrictive temperature at pupariation had completely normal prepupal and pupal catecholamine pools, and the adult females
and males that eclosed were fertile.
l(2)37Bg (Bg). In spite of extensive
screening for recessive lethals in the Ddc
region, only two Bg alleles have been isolated, both of which as hemizygotes produce almost identical mutant phenotypes.
Third instar larvae only begin to pupariate
at 168 h after egg laying, and, although
most have pupariated by 192 h, a few are
still crawling about. The mutant larvae are
—25% larger than control siblings, forming
distinctly larger than normal pupa cases,
which are a darker tan than wild-type pupae (Figure 5). Numerous Bg2 hemizygous
third instar larvae have very small melanotic tumors rarely seen in Bg1 hemizygous larvae, but most Bg' pupae have one
large melanotic tumor. Usually, both Bg1
and Bg2 hemizygous pupae occupy only
one-half to two-thirds of the pupa case at
the anterior end, the remaining one-third
to one-half presumably being filled with
air. Occasionally, it is the posterior end
that is occupied.
When dissected 10 days after egg laying,
Bg hemizygous pupae show no evidence
of differentiated adult cuticle (Figure 6E).
Except for a few very small knobs on the
head, the only melanized cuticle present
is a small spot in the region where the genitalia should develop. The pupae appear
to have a defined head, but no eyes, ocelli,
antennae, proboscis, or any bristles are
evident. No structures that derive from
the thoracic imaginal discs (wings, halteres, legs, notum, scutellum, etc.) are
found. Although the abdominal region
shows evidence of segmentation, no sternites or tergites with their accompanying
bristles and hairs develop. The cuticle encompassing the entire pupa (except in the
genital region) appears to be identical to
the undifferentiated cuticle found in pharate adult mutants of many of the other
genes of this cluster, e.g., Be, Ba, Be, Ce,
and Bd, illustrated in Figure 6. Dissected
6- and 8-day-old Bg crawling third instar
larvae have normal sized imaginal discs,
CNSs, salivary glands, guts, Malpighian tubules, and fat bodies. However, Bg hemizygous third instar ovaries, when transplanted into wild-type larval hosts, have
not been recovered, neither attached nor
unattached to the oviducts of the metamorphosed host, suggesting that Bg* activity is required for the continued development of the ovary (McCrady and Tolin
1994). Recently, a mutation isolated in a
screen for mitotic mutants, currant bun
(cub), has been shown to be allelic to Bg
(Ohkura H and Glover D, personal communication). It is possible that cub, which
plays an important role in mitotic progression, is an amorphic allele and Bg' and Bg2
are hypomorphic alleles.
Bg' has a minimal effect, if any, on four
of the five monitored catecholamine pools
in early pupae, with the DOPA, DA, NADA,
and NBANE pools decreased an insignificant 10%. However, the NBAD pool is
down to 75% of wild-type controls.
l(2)37Bd (Bd). Most Bd2 and Bd6 hemizygotes never pupariate, remaining in the
food as second and third instar larvae,
even after 8 and 9 days after egg laying.
However, a few hemizygotes begin to pupariate alter 8-11 days after egg laying.
Most of these pupate and continue to develop into pharate adults that do not
eclose. Almost all exhibit the complete
lack of differentiated cuticle on the abdomen, along with reduced cuticle formation
on the thorax and head (Figure 6F). The
phenotype is much more severe than that
produced by other mutations in the Ddc
cluster, e.g., Be, Be, Ba, and Ce. In spite of
this drastic lack of differentiated cuticle,
Bd6 hemizygotes do not perturb early pupal catecholamine pools (Homyk T and
Wright TRF, unpublished data).
Significantly more Bd1 hemizygotes and
Bd7/Bd4 heteroallelic heterozygotes pupate, producing pharate adults with the
same mutant phenotype, which is often
less severe. In fact, many of the Bd'/Bd*
heterozygotes eclose as adults that fall to
mate or produce offspring. However, third
instar Bd'/Bd* ovaries, when transplanted
into wild-type host larvae, attach to the
host oviducts during metamorphosis and
proceed to develop into completely fertile
ovaries, producing fertilizable eggs that
complete development to eclose as adults
(McCrady and Tolin 1994).
l(2)37Cd (Cd). Hemizygotes of amorphic
alleles of Cd die as normal sized, mature
third instar larvae unable to pupariate or
as pseudopupae with hardened cuticles
but that have not contracted into the wildtype pupal form. Most of the larvae and
pseudopupae have one large, hard melanotic pseudotumor, usually located in the
posterior fifth of the individual. Some have
several smaller tumors. The imaginal discs
in dissected 6- and 7-day-old hemizygous
larvae are very small: perhaps one-eighth
to one-half the size of discs in mature wildtype third instar larvae. The mutant CNSs
and salivary glands are also one-third to
one-half normal size at this time. Later, 10and 11-day-old larvae with melanotic tumors are still alive, with very small imaginal discs but now with normal sized CNSs
and salivary glands. By day 12, most of the
hemizygotes have died, with some forming complete psuedopupae, whereas others have only a few central segments
sclerotinized.
Most hypomorphic and temperaturesensitive allele hemizygotes (Cd5, Cd9, and
Ctf") at permissive temperatures complete development to the pharate adult
stage without forming melanotic tumors.
Although morphologically normal, most of
these pharate adults fail to eclose. Most of
these unhatched pharate adults fail to develop the brown coloration characteristic
of sclerotinized cuticle and, therefore, appear gray, with normally melanized bristles and hairs and slightly lighter than normal abdominal stripes. A small percentage
of hemizygotes [Ctf1 (20%), C(P (4%)] and
heteroallelic
heterozygotes [OP/Cd*
(22%), CcPICcP (12%), C&/QP (3%)] eclose
at 25°C as adults with a unique phenotype.
The overall body size is smaller than nonmutant siblings, body color is lighter, but
the abdominal bands are normally black,
bristles are thinner (like a weak Minute),
the dorsal side of the abdomen is markedly flattened, and ventrally female abdomens appear foreshortened (like Tb Ch*).
The wings appear not to be completely
sclerotinized, with delicate wavy edges.
Heteroallelic heterozygous females (Of I
Ca5, Qfi/Cd2, Ctf/Cd1, C&IC&, Ccf'/Ccf) and
Cd" hemizygous females were completely
female sterile. However, some CdP'ICd*
and Ccf'/Cd3 females were fertile. The fertility of heteroallelic heterozygous males
(Qf'ICtf and Cd>ICd* raised at 25°C) was
extremely variable, ranging from 100% of
the males being fertile to 100% being sterile (Cd-'/Cd1 6 6). Ccf hemizygotes that
Wright • Ddc Gene Cluster Mutants 1 8 5
were shifted from the permissive temperature to the restrictive temperature at pupariation had completely normal prepupal
and pupal catecholamine pools.
l(2)37Ca (Ca). The Ca locus is, with the
possible exception of Ddc, the most mutable locus in the Ddc cluster, with 44 alleles having been recovered to date (see
list in Lindsley and Zimm 1992). Hemizygotes of amorphic alleles of Ca (e.g., Ca*)
die as larvae, with no obvious mutant phenotype. Some individuals hemizygous for
the temperature-sensitive allele, CcP2,
raised at 25°C die as pharate adults with
almost no differentiated cuticle, producing
a mutant phenotype almost identical to
that of the Bd'/Bd* pharate adult depicted
in Figure 6F. Other Ca"2 hemlzygotes
eclose at 25°C, with obviously abnormal
thoracic cuticles (Figure 6H), presumably
due to the distortion of incompletely
sclerotized cuticle by the contraction of
indirect flight muscles. In these individuals, usually the wings are incompletely inflated as a result of the partially collapsed
thorax. Less severely affected Cat*2 hemizygous adults exhibit a mutant phenotype
identical to that shown in Figure 6G by
brut1/brat1 heteroallelic heterozygotes and
by Cd" homozygotes at 25°C. Other CcP2
hemizygous and Ca2/Ca16 adults are normal except they appear to be darker and
have thin, weak MinuteAike thoracic and
scutellar bristles. This is true for five additional Ca2/Cax heteroallelic heterozygous
combinations. Adult abdominal mosaic
patches of hemizygous Ca* tissue appear
to be normal except all the socketed abdominal bristles in the patches are quite
small, approximately one-third as long as
neighboring wild-type bristles (Bishop CP,
personal communication). No melanotic
tumors have been found in Ca mutants,
nor is there evidence that catecholamine
pools are perturbed in Ca"' hemizygous
prepupae and pupa maintained at 29°C after pupariation.
Mutations for Which Effects on the
Formation, Sclerotlzation, or
Pigmentation of the Cuticle Have Not
Been Found
l(2)37Cg (Cg). Hemizygotes of amorphic
alleles of Cg do not complete larval development. Seven days after egg laying, Cg1
hemizygous first and second instar larvae
are still found in the food. In Cg2 hemizygous cultures, some of the larger larvae
have melanotic tumors, but catecholamine pools of Cg*' hemizygous pupae
raised at 29°C after pupariation are not
perturbed; neither have effects on the for-
186 The Journal of Herecfity 1996:87(3)
mation, sclerotization, and pigmentation
of the cuticle been found in Cg"' hemizygous pupae and adults. Most transplanted
Cg"' hemizygous ovaries (10/12) were not
recovered after metamorphosis of the
wild-type hosts, and the two that were recovered were small and not attached to
the host's oviducts (McCrady and Tolin
1994). This indicates that Cg* function is
required for the normal development of
ovaries.
l(2)37Cb (Cb). Noncomplementing,
amorphic alleles of Cb, when hemizygous,
are lethal: as larvae that are unable to pupariate and exhibit no obvious mutant
phenotype. Thirteen of the 33 alleles isolated to date show intragenic complementation with heteroallelic heterozygotes
hatching as adults. These heterozygotes
are completely normal, although certain
combinations are female sterile. Similarly,
temperature-sensitive allele hemizygotes
(Cb"7 and Cb3), which eclose at subrestrictive temperatures, are completely normal
and fertile. Catecholamine pools of these
heteroallelic heterozygous adults are also
completely normal (Homyk T and Wright
TRF, unpublished data). There is no evidence that Cb mutations affect formation,
pigmentation, or sclerotization of the cuticle in any way.
The 675 bp of partial sequence of Cb
(Danner DB, personal communication)
coding for 225 amino acids in a 194-amino
acid overlap region show 37.6% amino
acid sequence identity and 79.4% conserved amino acid sequence similarity to
a yeast pre-mRNA splicing factor RNA helicase, PRP16. Also in a 154-amino acid
overlap region, it shows 27.3% identity
and 84.4% conserved amino acid similarity
to another yeast pre-mRNA splicing factor
RNA helicase, PRP2. There is no evidence
suggesting that Cb has or has not a similar
function in Drosophila.
fs(2)TWl (fsTWl). The most proximal
locus identified in Ddc cluster is a gene in
which all 15 mutant alleles recovered are
completely female sterile and are completely male fertile as hemizygotes. None
of the alleles reduce viability nor produce
a visible mutant phenotype (Hankins
1991). All fsTWl hemizygous females lay
numerous eggs, none of which develop at
all. No cleavage nuclei are found, and it is
not evident that the eggs are fertilized
(Wright et al. 1981a). The sterility is ovary
autonomous, because transplanted ovaries produce no progeny (Wright et al.
1981a). The female sterility phenotype of
the first allele, fsTWl1, was initially attributed to the Ddc-' allele (Wright et al.
1981a). Subsequently, the two mutations,
Ddc"' and fsTWl1, induced in the same
chromosome in the Ddc cluster were separated by crossing-over, and it was determined that Ddc"' did not affect female fertility (Wright TRF, unpublished data).
Discussion
Functional Relatedness of Genes in the
Ddc Cluster
Catecholamine Metabolism. It has been established that at least two of the 18 genes
in the cluster encode enzymes involved in
catecholamine metabolism: Ddc for DOPA
decarboxylase and Dox-A2 for a subunit of
phenoloxidase. Furthermore, that the
AMD protein is also enzymatically involved in biogenic amine metabolism is indicated by the fact that amd/+ heterozygotes are hypersensitive to the dietary administration of the DDC analog inhibitors
aMD and N'(DL-seryl>NH2,3,4-trihydroxybenzyl)hydrazine, and that resistance to
dietary aMD is directly correlated with
amd+ gene dosage. This inference is substantiated by the observation that the cuticle of hemizygous amd embryos is very
weak. Because amd shows high sequence
similarity with Ddc, including the pyridoxal phosphate binding site, AMD is undoubtly a decarboxylase, perhaps tyrosine
decarboxylase or aspartate decarboxylase. The latter produces B-alanine, an integral component of the sclerotlzing agent
N-B-alanyldopamine (NBAD). The former
decarboxylates tyrosine to tyramine, a
precursor to the neurotransmitter octopamine in insects, but a role for tyramine
in sclerotization has yet to be reported.
In addition, catecholamine pools are significantly perturbed in pupae hemizygous
for mutations in five more of the 18 genes:
Be, Be, Cc, Ce, and brat (Table 2) (Homyk
T, Mclvor WE, and Wright TRF, unpublished data). This raises the distinct possibility that these five genes are also directly involved in catecholamine metabolism, either encoding catalytic or regulatory proteins.
Of the other 10 genes, eight—Ba, Bb, Bg,
Bd, Cb, Cd, Ca, and Cg—have been assayed
for perturbations in pupal catecholamine
pools. Among these, only Bg showed a
marginal decrease in the NBAD pool,
whereas pupal pools for the other genes
were completely normal, hk and fs(2)TWl
mutants were not assayed.
It is important to note that amorphic alleles of only Ba, Ce, and brat have pupal
effective lethal phases, making it possible
to survey catecholamine pools in hemizy-
gous pupae with little or no gene activity.
Because amorphic alleles of Be and probably Bg are embryonic lethals and
amorphic alleles of Be, Bb, Bd, Cc, Cb, Cd,
Ca, and Cg are larval lethals, it was necessary to assay catecholamine pools in ts
allele hemizygotes raised at 29°C after pupariation or in hypomorphic allele hemizygotes or in complementing heteroallelic
heterozygotes that had enough residual
activity to develop into pupae. Many of
these may have had sufficient gene activity to obviate observing differences in the
pools. Because wild-type alleles of almost
all of these genes are active late in embryogenesis (Stathakis et al. 1995), when
there are significant pools of DOPA, DA,
and NADA but not NBAD (Wright 1987a;
Stathakis DG, unpublished data), amorphic allele hemizygotes of all of these
genes should be surveyed at this stage. To
date, only hemizygous Be embryos (Stathakis DG, unpublished data) and mixed
genotypes of Dox-A2 embryos (Pentz et al.
1986) have been assayed for catecholamines, and in both cases important differences were found (see above). The use of
an odd mutation (2-8) that modifies segmentation makes it possible to identify
and collect late embryos hemizygous for
null mutations in the Ddc cluster (Stathakis DG, unpublished data).
adults dissected from their pupal cases reveals a remarkable similarity among the
abnormalities found. Various mutant genotypes of eight of the 18 genes show various degrees of incomplete cuticle formation (Figure 6A-F). Most often, the cuticle
formation is incomplete over the abdomen
but apparently complete on the thorax,
head, and legs. However, examples were
frequently found in which the cuticle over
the thorax and head was also incomplete
(Figure 6F). This incomplete cuticle phenotype was found among amorphic allele
hemizygotes of Ba, Bg, Cg and brat, hypomorphic allele hemizygotes and partially complementing heteroallelic heterozygotes of Be, Bd, and Ca. The fact that incomplete cuticle formation is observed
most frequently over the abdomen may be
related to the fact that during normal development cuticle formation occurs last
over the abdomen (Walter et al. 1991). Mutations in four of these eight genes—Be,
Be, Ce, and brat-^significantly modify catecholamine pools during metamorphosis.
What aspects of cuticle formation are interrupted in these mutations is not clear.
The epidermis secretes a thin outer epicuticle made up of four primary layers—
cement, wax, cuticulin, and protein—and
secretes an inner, lamellar procuticle,
which makes up the bulk of the cuticle
(Fristrom and Fristrom 1993; Hepburn
Because catecholamine pools have not
1985; Martinez-Arias 1993). During develbeen assayed in amorphic allele hemizy- opment, the procuticle differentiates into
gotes of Ba, Bb, Bg Bd, Cb, Cd, Ca, and Cg, three distinct layers: an exocuticle, a methe possibility that some of these genes
socuticle, and an endocuticle (Hepburn
are also involved in catecholamine metab1985). The exocuticle lies just beneath the
olism has not yet been eliminated.
epicuticle, undergoes extensive sclerotiMelanotic Pseudotumors. Mutations in 11 zation, and contains deposits of melanin
of the 18 genes effect the production of
and other pigments. The innermost, lamelanotic pseudotumors at some stage of
mellar endocuticle is not well sclerotized
development (Table 1). Eight of these 11—
and lacks pigments. The mesocuticle apBe, Be, Bg amd, Ddc, Cc, Ce, and bratpears to be a transition zone between the
perturb catecholamine pools. Among the
endocuticle and exocuticle. Superficial exother three—Ba, Cd, and Cg— no differ- amination of the mutant, incompletely
ences in catecholamine pools were noted
formed cuticles suggests that the pigin hypomorphic hemizygous pupae. Dox- mented and sclerotized exocuticle has not
A2 is the only gene involved in catechol- been differentiated from the procuticle.
amine metabolism that does not produce This suggestion has yet to be substantimelanotic pseudotumors. The absence in
ated by electron microscopy and other exDox-A2 hemizygotes of phenoloxidase ac- perimental manipulations. It is important
tivity, a prerequisite for any melanization,
to note, however, that there are areas on
precludes the production of melanotic
these mutants, even within the abdominal
pseudotumors. Because there is such a
region (Figure 6A), where cuticle develhigh degree of correlation between muta- opment is complete, including melanin detions effecting perturbations in catechol- position and bristle formation. This indiamine pools and the formation of mela- cates that the mutations do not completenotic pseudotumors, the latter may be di- ly block the synthesis of some component
agnostic of the former.
of the cuticle in the whole organism, but
Incomplete Formation of Cuticle. Inspec- rather an insufficient amount of one or
tion of the morphological effects of muta- more key components is being made or
tions of genes in the cluster on pharate
the use of the components is being temporally or spatially misregulated.
Incomplete Sclerotization. Adult hemizygous or heteroallelic heterozygous escapers for mutations in eight of the 18 genes
exhibit a very similar phenotype that we
have inferred to be due to incomplete
sclerotization of the thoracic cuticle. The
phenotype is characterized by the presence of deep lateral thoracic creases and
uninflated or partially inflated wings
symptomatic of a deformed or partially
collapsed thorax (Figure 6G). The weak
cuticle is probably deformed by contractions of the indirect flight muscles inserted into it, and the collapsed thorax prevents hemolymph from being pumped into
the wings to inflate them. In some extreme
cases, distortion of the thoracic cuticle is
particularly obvious (Figure 6H). Mutations in Be, Ba, Bb, Cc, Cd, Ca, Ce, and brat
have been found to produce this incomplete sclerotization phenotype. Of these,
mutations in Be, Ba, Ca, Ce, and brat also
exhibit incomplete formation of cuticle,
and Be, Cc, Ce, and brat mutations perturb
catecholamine pools during metamorphosis.
In Table 1, amd and Ddc mutations have
also been designated as effecting incomplete sclerotization for the following reasons. Hemizygous amd embryos produce
a very weak larval cuticle, and the mouthhooks of Ddc hemizygous embryos are insufficiently sclerotized to permit the actively moving embryos to tear the embryonic membranes in order to hatch. In addition, the melanization of the leg joints
and wing axillae in adult Ddc escapers has
been attributed to breakage of weak, incompletely sclerotized cuticle. This means
that 10 of the genes in the cluster affect
sclerotization of the cuticle in some way.
Pigmentation. Mutations in 10 of the 18
genes affect pigmentation of embryos, pupal cases, pharate adults, and adult escapers in a variety of ways. Reduced levels of
Ddc and Dox-A2 activity can result in the
development of essentially colorless pharate adults and adult escapers with little or
no brown pigmentation in the cuticle (for
Ddc, see Figure 4; for Dox-A2, see Figure 2
in Pentz et al. 1986), and in extreme examples even the macrochaetae are colorless or straw colored, with no deposition
of melanin. Embryos hemizygous for
amorphic Ddc alleles have light brown or
completely unpigmented mouthparts and
denticle belts. Mutations in three of the
genes—Be, Bg, and Ddc—can produce pupal cases that are darker than wild type
(Figure 6), and various genotypes of mu-
Wnght • Ddc Gene Ouster Mutants 187
tations in six genes—Ba, Cc, Cd, Ca, Ce,
and brat—have produced adult escapers
that are also darker than wild type (a
darker brown). As one might expect, a majority (six and possibly seven) of these 10
genes has been shown to perturb catecholamine pools during pupal development (Table 1).
Female Sterility. Evidence that the activity of 13 of the 18 genes in the cluster is
required for complete female fertility
comes from four sources. First, femalesterile alleles were isolated in eight of the
genes: 15 fs(2)TWl alleles; three Be and
three brat fs alleles; one fs allele each in
Ba, Dox-A2, Cb, and Ca; and two alleles in
Cc, one of which is a dominant Fs. Second,
adult escapers hemizygous for hypomorphic and temperature sensitive alleles
and complementing heteroallelic heterozygous escapers were tested for fertility,
with the results indicating that the activities of Ba, Be, Cc, Cb, Cd, Ca, Ce, and brat
were necessary for female fertility. Third,
McCrady and Tolin (1994) transplanted
hemizygous mutant larval ovaries into
wild-type larval hosts to determine if the
ovaries would develop normally enough
to produce progeny. They found that
transplanted hemizygous Bg and Cg ovaries did not grow at all and that hemizygous Ce ovaries grew and attached to the
host oviducts but failed to produce fertile
eggs. Finally, the production of mutant
clones established the requirement for
amd activity in both the germline and follicle cells in vitelline membrane biogenesis during oogenesis (Konrad et al. 1993).
Although the effects of the above female-sterile genotypes on oogenesis have
not been thoroughly investigated, superficial observations do not reveal a common mutant phenotype, and only arnds vitelline membrane defect might be attributed to abnormal crosslinking of proteins,
i.e., sclerotization. Interestingly, five of the
above female-sterile genotypes also effect
male sterility. These include mutations in
Ba, Dox-A2, Ca, Cb, and brat.
Why Are These Functionally Related
Genes Clustered?
Coordinate Regulation? If one accepts the
premise that many of the genes in the Ddc
cluster are functionally related, one must
then ask why they are clustered. The obvious explanation is that at least some of
the genes are subject to global coordinate
control. Although a temporal survey of
mRNA levels (Stathakis et al. 1995) revealed 13 of the 21 transcription units are
expressed at different levels throughout
188 Tne Journal oJ Heredity 1996:87(3)
development, all the genes were active
late in embryogenesis and may be subject
to coordinate regulation at this stage in
development. Also, because the activity of
at least 13 of the genes is required for female fertility, a subset may be subject to
coordinate regulation during oogenesis.
However, that these genes need not be located in the subclusters in order to function is indicated by the fact that genomic
DNA fragments encompassing only one, or
at the most two, genes relocated out of the
subclusters to ectopic sites by P elementmediated transformation can function well
enough to rescue mutations in all the essential genes. However, not all these transformed genes may be functioning optimally. Significant variability was observed in
the efficiency of rescue by identical constructs inserted into different ectopic locations. Thus, transcription levels may be
dependent on local enhancers. Furthermore, drastically reduced levels of gene
activity may be sufficient to effect rescue,
for it has already been established that
<10% wild-type DDC activity is sufficient
to ensure the complete rescue of Ddc mutations (Wright et al. 1982). Thus, the possibility of global regulation of the genes in
the subclusters should not yet be completely rejected.
Evolutionary Conservation. Although in
D. virilis and D. pseudoobscura the proxi-
mal and distal subclusters are separated
from each other by significant segments of
the chromosome, the integrity of the subclusters is conserved (Wright TRF, unpublished data). That is, the same genes are
located in the same subclusters, and in D.
virilis the arrangement of the genes relative to one another in both subclusters is
identical to the arrangement in D. melanogaster. Of even greater significance is
the fact that the two subclusters are also
conserved in the mosquito Anopheles
gambiae, with only a minor rearrangement
(inversion) of two of the genes in the distal subcluster (Romans P, personal communication). One is, therefore, led to believe that some functional rationale must
underlie the clustering of these genes.
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Received April 20, 1995
Accepted November 8, 1995
Corresponding Editor Stephen J. O'Brien