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 Indian Academy of Sciences
Effects of mutations at the stambh A locus of Drosophila melanogaster
M. KUMAR, MINU JOSEPH and SHANTI CHANDRASHEKARAN*
Division of Genetics, Indian Agricultural Research Institute, New Delhi 110 012, India
Abstract
We report novel findings on the cytogenetic location, functional complexity and maternal and germline roles of the
stambh A locus of Drosophila melanogaster. stmA is localized to polytene bands 44D1.2 on 2R. stmA mutations are of
two types: temperature-sensitive (ts) adult and larval paralytic or unconditional embryonic or larval lethal. Twelve
alleles reported in this study fall into two intragenic complementing groups suggesting that stmA is a complex locus
with more than one functional domain. Some unconditional embryonic lethal alleles show a ‘neurogenic’ phenotype of
cuticle loss accompanied by neural hypertrophy. It is shown that embryos of ts paralytic alleles also show mild neural
hypertrophy at permissive temperatures while short exposure to heat induces severe cuticle loss in these embryos. stmA
exerts a maternal influence over heat-induced cuticle loss. Unconditional embryonic lethal alleles of stmA are also
germline lethal.
[Kumar M., Joseph M. and Chandrashekaran S. 2001 Effects of mutations at the stambh A locus of Drosophila melanogaster.
J. Genet. 80, 83–95]
Introduction
The mutation stambh A (stmA) (2–56.8 cM) in Drosophila melanogaster was discovered through its phenotype
of reversible temperature-sensitive (ts) paralysis in our
laboratory (Shyngle and Sharma 1985). Adults and larvae
homozygous for stmA1 and stmA2 are paralysed within
2–4 minutes of exposure to 38ºC temperature. When they
are brought back to 23–24ºC the flies recover to normal
behaviour within 10–12 minutes (Chandrashekaran and
Sarla 1993). Mutations isolated later included several
unconditional multiphasic lethal alleles (Kumar 1993).
Some of them were embryonic lethals and showed
pronounced hypertrophy of the embryonic dorsal neurogenic region and mild hypertrophy of the ventral
neurogenic regions (Chandrashekaran and Sarla 1993).
Neural hypertrophy was accompanied by hypotrophy of
the cuticle, closely resembling the phenotype of mutations
in genes of the Notch-Delta category collectively called
neurogenic genes (Lehmann et al. 1983). Adults homo-
*For correspondence. Email: [email protected].
Keywords.
zygous for stmA are also resistant to the neuropoison
veratridine (Chandrashekaran 1993) which causes
lethality by binding to a protein subunit of the voltagegated transmembrane sodium channels of nerve membranes
(Catterall 1986). It is unlikely that veratridine resistance in mutant flies is due to direct involvement of
stmA in the structure or function of the transmembrane
sodium channels. Reasons for doubt arise from the
observation that double mutants of stmA and para, a
structural gene for neuronal voltage-gated sodium channels in Drosophila (Loughney et al. 1989), did not show
any phenotypic interaction (Chandrashekaran and Sarla
1993).
Currently available information on the phenotypic
effects of mutations at stmA points to its role in neural
development. However, little else is known about the
genetics, molecular nature and other developmental functions of stmA. In this paper, we report several new
findings that (i) demonstrate the complex nature of the
stmA locus, (ii) provide further evidence for the role of
stmA in neuroectodermal development, and (iii) demonstrate the maternal and germline function of stmA. As a
prelude to its cloning and molecular analysis the precise
Drosophila melanogaster; stambh A; maternal effect; cuticle loss.
Journal of Genetics, Vol. 80, No. 2, August 2001
83
M. Kumar et al.
cytological location of stmA and transposon tagging are
also reported in this study.
Material and methods
Fly maintenance: Flies were grown on standard corn
meal, yeast and unrefined sugar media at 23–24ºC unless
stated otherwise.
removed by replacing the X and 3rd chromosomes by
non-P-bearing M-5 and TM6 balancers. The second
chromosome was outcrossed with a P-element-free,
multiple-marked stock al dp b pr c px sp. Recombinant
chromosomes carrying stmAP with al dp b pr were
recovered in the first step. In a second recombination
experiment the chromosomal region distal to stmA was
replaced with markers c px sp. This process almost made
sure that the new P-induced allele of stmA did not carry
any other P elements.
Fly stocks
stmA alleles: All stmA alleles originated from our own
experiments (Shyngle and Sharma 1985; Chandrashekaran
and Sarla 1993; present study).
Birm-2 and Jumpstarter: Both Birm-2 and Jumpstarter (JS)
stocks were kindly gifted by Dr William Engels. The
Birm-2 genotype carries ~ 17 natural but defective nonautonomous P elements on its second chromosome
(Robertson et al. 1988). JS is a third chromosome
carrying the P transposon ∆2-3, a stable source of
transposase. The JS stock used in this study is CyO /
Sp; ∆2-3 Sb / TM6B.
Deficiencies: The deficiencies on chromosome 2R used in
this study are listed in table 6.
+
D1
D
ovoD1: P[w ovo ] / Ms(2)bw / CyO carries the dominant
female sterile (DFS) mutation ovoD1 on chromosome 2R
at 55E (Chou et al. 1993).
Isolation of alleles: New alleles were identified on the
basis of noncomplementation with stmA2 for ts paralysis
in the F1 or with stmA12 for lethality in F2.
F2 noncomplementation for lethality over stmA12: Male flies
of the genotype al dp b pr or Canton S were irradiated
with gamma rays (30 gray; from a 60Co source) and mated
to Cy / Gla females. Cy or Gla male progeny were
individually mated to stmA12 / Pm virgins. The F2 progeny
of each vial was scored for the absence of nonbalancer
flies and putative lethal alleles were recovered over Pm.
Temperature-induced paralysis: Adult flies were placed in
thin-walled glass vials and held in a water bath at the
desired temperature. Flies that had fallen on their back
were taken as paralysed.
Scoring embryonic cuticle loss phenotypes: Cleared cuticle
preparations were made according to van der Meer
(1977). The cleared embryos were grouped into the
following five classes based on increasing loss of
continuous cuticle: class I, normal looking embryos with
continuous cuticle and head skeleton; class II, embryos
with shrunken cuticle, large dorsal hole overlying the
brain, malformed head, germband retraction often
incomplete; class III, embryos with large dorsal and
ventral cuticle holes, head skeletal structures faintly
visible; class IV, only a narrow strip of cuticle visible;
class V, no continuous cuticle.
F1 noncomplementation for ts paralysis over stmA2
EMS induced alleles: Male flies (Canton-S) were fed 0.3%
2
EMS and mated en masse to b stmA virgins. The F1 males
were tested for paralysis at 38ºC followed by recovery at
23–24ºC. The putative b+ stmA? / b stmA2 males were
outcrossed to Cy / Pm balancer-bearing females and the
new stmA-allele-bearing chromosomes recovered over Pm
(recognized as black-bodied flies because the Pm balancer
carries the marker b) and intercrossed after retesting to
confirm allelism.
Hybrid dysgenesis induced alleles: Hybrid dysgenic + / Y;
Birm-2 / CyO; ∆2-3 Sb / + males were mated to b stmA1
virgins and the progeny reared at 18ºC. Non-Cy non-Sb
male progeny were tested for paralysis at 38ºC and
recovery at 23ºC. Paralysis was confirmed over b stmA1 in
the following generation and confirmed lines were
extracted against Pm. Any P elements that might have
lodged themselves on chromosomes X and 3 were
84
Benzer’s countercurrent test: The countercurrent distribution
procedure of Benzer (1967) was used to score the
negative geotactic response of 3–4-day old adult males at
23–24ºC. Twenty males per test and 60 per genotype were
used. Flies were adapted to ambient light in test tubes for
15 min and distributed by countercurrent for their ability
to climb up the walls of the glass tube (negative geotactic
response) in five cycles of 60 sec duration. The negative
geotactic response was calculated as: (n1 × 0) + (n2 × 1)
+ (n3 × 2) + (n4 × 3) + (n5 × 4) + (n6 × 5)]/N, where n1, n2,
n3, . . . n6 denote the numbers of flies in tubes 1 . . . 6 and
N = n, the total number of flies.
Immunostaining of the embryonic nervous system: The
nervous system was stained with the neuronal-specific
mouse antibody Mab 22C10. Briefly: Dechorionated eggs
were fixed in 4% paraformaldehyde in heptane and
devitellinized in heptane–methanol. Embryos were per-
Journal of Genetics, Vol. 80, No. 2, August 2001
Mutations at stambh A in Drosophila melanogaster
meabilized with 0.1% Triton X-100 in phosphate-buffered
saline (PBS). The embryos were incubated in primary
antibody (dilution 1 : 50) overnight 4ºC. The secondary
antibody was biotinylated anti–mouse (dilution 1 : 200).
The antibody binding was visualized by horseradish
peroxidase activity (dark brown-black color) using
diaminobenzidine (5 mg per 2 ml in PBS with 0.1%
Tween 20).
Female germline clones: Clones were induced by
irradiating larvae from the cross stmA + / SM5 × SM5 /
+ [Pw+ ovoD] with gamma rays (1 gray) from a 60Co
source. The [Pw+ ovoD] chromosome carries the dominant
X-linked female-sterile mutation ovoD at 55DE on chromosome 2R (Chou et al. 1993). All stmA + / + [Pw+ ovoD]
females were mated to wild-type males and tested for egg
laying.
Results
Alleles
Four independent experiments were conducted to screen
for new stmA alleles (table 1) and 19 alleles were
recovered among approximately 70,000 chromosomes
tested. Of these, eight alleles have been lost and only 11
new alleles are being maintained in our collection. Eight
out of the 11 remaining alleles are unconditional lethals
and three are homozygous viable. Adults and larvae
homozygous for these three viable stmA alleles also get
paralysed at 38ºC like the original stmA1 (table 2). stmA
larvae also are paralysed in about 2–4 minutes (depending
upon the allele), stretch out and lose all muscle tone
on paralysis. The eight unconditional lethal alleles
showed a partially dominant lethal phenotype with
viability ranging from 30% to 50%. Lethality was multiphasic and spanned embryonic, larval and pupal stages
(data not shown).
Table 1.
Flies homozygous for the alleles stmA1, stmA2, stmAP1
and stmAP4 were paralysed within 2–3 minutes at 38ºC,
while heterozygous stmA1 / +, stmA2 / + and stmAP4 / +
flies were paralysed after a 17–24-minute exposure at
38ºC (table 2). For wild-type (Canton-S) males maintained in our laboratory, it takes 32 minutes for paralysis
at 38ºC. The paralytic behaviours of wild-type, stmA /
stmA and stmA / + flies differ greatly. Homozygous stmA
adults show a gradual slowing down of physical activity
within 30–45 seconds of heat exposure before they are
completely immobilized and fall on their backs. Canton-S
flies remain mobile even till one minute before paralysis
sets in. After they are reverted to 23ºC the paralysed
Canton-S flies revive to normal behaviour in 30 seconds
while stmA flies take over 10 minutes to begin moving
(and more than 24 hours before they resume completely
normal mobility, preening, feeding and mating behaviour).
The paralytic behaviour of stmA / + heterozygotes is very
similar to Canton-S flies in the first half of their exposure
to high temperature, after which their physical activity
slows down. Unlike Canton-S flies, which revive in 30
seconds, stmA / + heterozygotes take up to 3–4 minutes
to revive. None of the unconditional lethals showed
dominant paralytic phenotypes at 38ºC.
stmA adults show abnormal (hyperactive or hypoactive)
behaviour at temperatures of 23–24ºC (a temperature
which is otherwise defined as permissive for the paralytic
expression). stmA1 and stmAP1 adults are hypoactive
and stmA2 hyperactive. To quantify these differences in
behaviour the negative geotactic responses of adult males
were measured by Benzer’s countercurrent test. Canton-S
flies, if tapped to the bottom of a glass tube, respond by
climbing up the walls (a negative geotactic response).
Negative geotactic response scores of adult Canton-S
males was 2.26. stmA1, stmAP1 and stmP4 adults were
weakly negatively geotactic while stmA2 adults were
highly negatively geotactic (table 3). This difference in
the negative geotactic responses of the various stmA
alleles is also reflected in routine physical activity in that
Summary of stmA allele search experiments.
No. of alleles
Expt
Mutagen
1.
EMS
2.
3.
4.
P element
EMS
Gamma rays
Screening
criterion
Chromosomes
screened
Unconditional
lethal
Conditional
paralytic
Paralysis at 38ºC
over stmA1 and
recovery at 24ºC
Same as above
Same as above
Lethality over stmA12
39,000
2
1
2, 7, 12a
7827
17,280
6865
0
2
0
5
4
5
P1, P2, P3, P4, P5b
KP1, KP2, . . ., KP6c
1-2, 1-4, 2-2, 18-2, 27-1d
Alleles
a
Chandrashekaran and Sarla (1993).
P2, P3, P5 lost.
c
KP1, KP3, KP5, KP6 lost.
d
2-2 lost.
b
Journal of Genetics, Vol. 80, No. 2, August 2001
85
M. Kumar et al.
stmA2 (strongly negatively geotactic) adults are very
hyperactive while adults of stmA1, stmAP1 and stmAP4
(weakly negatively geotactic) are sluggish and almost
always flightless.
Complementation
On the basis of intragenic complementation among the
four ts paralytic and eight unconditional lethals (table 4,
figure 1) three classes of alleles could be defined. The
lethal alleles (7, 12, 18-2, 1-2 and KP2) comprise group I,
which do not complement members of groups II and III.
Group II members 27-1 and 1-4 (both unconditional
lethal) complement members of group III (KP4, 1, 2, P1
and P4, comprising unconditional lethal and viable ts
paralytic).
Table 2. Time taken (minutes; seconds) for 100% paralysis of
adult males of various heteroallelic combinations of stmA ts
paralytic alleles at a temperature of 38ºC.
+
1
2
P1
P4
+
1
2
P1
P4
32;00
20;00
4;00
17;30
3;00
3;00
32;00
2;00
2;00
2;00
24;00
3;00
3;00
2;00
3;00
Table 3. Negative geotactic responses of stmA
and wild-type adults measured by Benzer’s countercurrent test.
Allele
+
1
2
P1
P4
Response
score
Time (min; sec) taken
for 100% paralysis of
adults at 38ºC
2.26
1.26
4.12
0.3
1.6
32;00
4;00
3;00
2;00
3;00
It was previously reported that mutations leading to ts
paralysis were partial gain-of-function neomorphs, while
unconditional lethals were partial (hypomorph) or complete
loss-of-function amorphs. This conclusion was based on
the observation that ts paralytic / lethal trans heterozygous adults showed weaker paralysis than their
respective ts paralytic homozygotes (Chandrashekaran
and Sarla 1993). The paralytic phenotypes of the 32
possible viable / lethal heteroallelic and four homoallelic
combinations of stmA are shown in table 5. Compared to
their respective homozygotes, 11 heterozygotes were
weaker, eight were stronger and five were identical in
their ts paralytic phenotypes. Eight trans heterozygotes
showed complementation (wild-type phenotype) for the ts
paralytic effect. Absence of any straightforward relation
between paralytic phenotypes of paralytic / lethal heterozygotes and their respective homozygotes indicates that
stmA is not a simple locus but very likely consists of more
than one functional domain, mutations in which lead to ts
paralysis or lethality or both.
Cytological mapping
A previous study (Chandrashekaran and Sarla 1993)
revealed that flies of the genotype stmA1 / stmA1; Tp2;
3 P32 (containing two copies of stmA1 and a wild-type
copy of the 2nd chromosome region 41A to 44D4.8 on
chromosome 3) showed wild type behaviour. stmA was
therefore roughly mapped to the 41A–44D4.8 region. All
deletions spanning 41A through 41C and 42A16 through
43F8.9 were wild type over stmA. The stmA12-bearing
chromosome was associated with a visible cytological
deficiency 42A8–42B2 which could not be separated by
recombination from the stmA lethal at the time of the
study. On that basis it was concluded that stmA mapped to
the 42A16–42B2 segment. Deficiencies distal to 43F8.9
up to 44D4.8 were not tested because of the ‘tight’
association of the 42A8–42B2 deficiency with stmA12.
However, subsequently we were able to separate the lethal
stmA12 allele from the deficiency, making it essential to
Table 4. Interallelic complementation among 12 alleles of stmA. Alleles 1, 2, P1 and P4 are
conditional paralytic alleles while the rest are unconditional lethal alleles. ‘+’ indicates a wild-type
phenotype, ‘-’ indicates ts paralysis or unconditional lethality.
1
2
P1
P4
1–2
1–4
7
12
18–2
27–1
KP2
86
2
P1
P4
1–2
1–4
7
12
18–2
27–1
KP2
KP4
–
–
–
–
–
–
–
–
–
–
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+
+
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+
–
–
–
+
–
Journal of Genetics, Vol. 80, No. 2, August 2001
Mutations at stambh A in Drosophila melanogaster
redetermine the cytological location of stmA. In this study
five new deficiencies have been analysed (table 6), all with
at least one breakpoint distal to 43F8.9, which was the
distalmost breakpoint in the previous study.
Among the five deficiencies tested, two were overlapping: Df(2R)H3C1 [43F to 44D3.8] and Df(2R)H3E1
[44D1.4 to 44F12], both of which uncovered the 12 ts
paralytic and lethal stmA alleles. stmA therefore maps to
the overlapping 44D1.4 to 44D3.8 region stretching from
the proximal breakpoint 44D1.4 of Df(2R)H3E1 to the
distal breakpoint 44D3.8 of Df(2R)H3C1 (figure 2). The
breakpoints of deficiency Df(2R)44CE, according to
Flybase (http://www.flybase.bio.indiana.edu), are 44C1.2
(proximal) and 44E1.4 (distal). It is therefore deficient for
the relevant 44D1.4 to 44D3.8 region that is common to
Df(2R)H3C1 [43F to 44D3.8] and Df(2R)H3E1 [44D1.4
to 44F12]. Df(2R)44CE is therefore expected to uncover
the mutant phenotype of stmA. However, Df(2R)44CE /
stmA flies were surprisingly wild type. It can therefore
be concluded that the 44C1.2; 44E1.4 breakpoints of
Df(2R)44CE were probably determined incorrectly. A
careful study of polytene chromosomes of Df(2R)44CE /
+ larvae shows (figure 2b) that our premise was indeed
correct. Df(2R)44CE is not one continuous deficiency but
is made up of two adjacent deficiencies, one small
deficiency for the two bands 44C1.2 and another for the
bands 44D3 to 44E1.4. The polytene band 44D1.2 is
clearly present (see figure 2b) in between the two adjacent
deficiencies. 44D1.2 is the only band that is deficient
in Df(2R)H3C1 and Df(2R)H3E1 but is present in
Df(2R)44CE. On the basis of the noninclusion of stmA
in Df(2R)44CE and its inclusion in Df(2R)H3C1 and
Df(2R)H3E1, stmA is placed in the polytene bands
44D1.2 (figure 2a).
Heat-induced embryonic lethality and cuticle loss in the ts
paralytic alleles of stmA
Figure 1. Diagrammatic representation of complementation
among the stmA alleles.
It was reported earlier (Chandrashekaran and Sarla 1993)
that the unconditional lethal alleles stmA12 and stmA7
show dorsal embryonic cuticle loss overlying the brain
Table 5. Interalleleic complementation among eight unconditional lethal alleles and four ts paralytic
alleles of stmA. ‘–’ indicates ts paralysis and figures in parenthesis are time taken (min;sec) for 100%
paralysis of adult males at 38ºC, ‘+’ indicates a wild-type phenotype defined as absence of paralysis for
up to 32 minutes at 38ºC.
Homozygote
1–2
1–4
18–2
27–1
KP2
KP4
7
12
1
4;00
+
3;00
P1
2;00
P4
3;00
–
(4;30)
–
(2;00)
–
(3;00)
–
(3;00)
+
2
–
(5;00)
–
(3;00)
–
(3;00)
–
(3;00)
–
(1;40)
–
(2;10)
–
(2;45)
–
(2;45)
–
(2;30)
–
(2;00)
–
(2;00)
–
(3;00)
–
(12;00)
–
(4;00)
–
(2;15)
–
(2;20)
–
(23;00)
–
(4;00)
–
(2;15)
–
(2;20)
Table 6.
+
+
+
+
+
+
List of deficiencies on chromosome arm 2R used to map stmA.
Breakpoints
S. No.
1
2
3
4
5
Deficiency
(2R)
cn 87e
cn 9
H3C1
44CE
H3E1
Bloomington
stock numbers
224
3368
198
3643
201
Proximal
Distal
Whether
stmA+
42B4.C1
42E
43F
44C1.2*
44D1.4
43F.44A1
44C
44D3.8
44E2.4*
44F12
Yes
Yes
No
Yes
No
*Breakpoints as published in http://www.flybase.bio.indiana.edu. In this study deficiency 44CE
is shown to consist of two deficiencies: (i) for the doublet band 44C1.2 and (ii) for bands 44D3
to 44E1.4.
Journal of Genetics, Vol. 80, No. 2, August 2001
87
M. Kumar et al.
coupled with neural hypertrophy of the dorsal brain lobes
and the ventral neurogenic region. Since adults of the ts
paralytic alleles stmA1 and stmA2 show abnormal
behaviour at 23ºC and at 38ºC it is reasonable to expect
structural anomalies in the nervous system of embryos,
larvae and adults. Also, stmA embryos rarely survived
a
even brief accidental exposure to temperatures of 30ºC
and above. The present study revealed that 18-hour-old
stmA1 embryos (reared at 23ºC) show a mild hypertrophy
of the embryonic brain lobes and ventral nerve chord
(figure 3, b, d, f). Gross structural abnormalities in
neuronal connections or cuticle loss were not observed.
H3E1
44CE
H3C1
b
Figure 2. a, Polytene chromosome map of section 44 of chromosome 2 right arm (electron micrographic and
Bridges’ 1935 map), showing the position of stmA (short thick line) with respect to the breakpoints of Df(2R)H3C1
and Df(2R)H3E1 and the corrected breakpoints of Df(2R)44CE. Dark solid lines and arrows show the extent of the
deficiency; dashed line with arrowhead shows the breakpoint uncertainty. b, Polytene chromosomes of Df(2R)44CE / +
larvae showing polytene band 44D1.2 (small arrow) in between the two adjacent deficiencies 44C1.2 (two-headed
dotted arrow) and 44D3 to 44E1.4 (two-headed bold arrow).
88
Journal of Genetics, Vol. 80, No. 2, August 2001
Mutations at stambh A in Drosophila melanogaster
Given that stmA1 embryos show mild neural hypertrophy at 23ºC, it is pertinent to ask if mild expression
will be exaggerated if stmA1 embryos are exposed to
higher temperature. Morphological segregation of neuroblasts from epidermoblasts occurs during stages 8–10
(soon after three hours from egg laying at 24ºC after
completion of the ventral furrow) of embryogenesis
(Campos-Ortega 1993). The embryos of stmA1, stmA2 and
Canton-S were exposed to 31, 33 and 35ºC at two stages,
i.e. syncitial blastoderm (0–0.5 h) and cellular blastoderm
(3 h), prior to overt segregation of neuroblasts from the
neuroectoderm. In general, stmA1 and stmA2 embryos
were much more sensitive to heat treatment than the
wild-type embryos and showed high levels of heatinduced lethality (table 7). Three-hour-old embryos were
more tolerant to heat shock than freshly laid eggs.
Genotypic differences between stmA and Canton-S were
distinct at 33ºC where the mutants showed 3 5-fold
higher lethality than the wild type. Exposure to 35ºC
temperature caused nonspecific embryonic lethality,
especially in the eggs at 0 h, and was not considered for
further analysis.
Figure 3. Embryonic nervous system of 18-hour-old (stage 15) wild-type (a, c, e) and stmA1 (b, d, f) embryos reared
at 23°C. Note the enlarged and denser antennomaxillary nerve complex (amc), ventral nerve chord (vnc) and brain (b)
lobes in stmA. All embryos are oriented with anterior to the top. Photos a, e, b and f are ventral views, and c and d are
lateral views. Embryos were stained with monoclonal antibody 22C10. Magnification: a, b, c, d, 16×; e, 64×; f, 32×.
Table 7.
Heat-induced lethality in wild-type and stmA embryos.
Temperature
31°C
Age of embryo (hAEL)
33°C
Age of embryo (hAEL)
35°C
Age of embryo (hAEL)
Genotype
0
% EL (N)
3
% EL (N)
0
% EL (N)
3
% EL (N)
0
% EL (N)
3
% EL (N)
+
stm A1
stm A2
15.5 (543)
6.9 (302)
24.2 (749)
7.3 (425)
1.4 (419)
11.9 (370)
18.7 (502)
53.3 (272)
41.7 (511)
8.7 (172)
43.3 (172)
45.1 (359)
87.5 (200)
93.0 (192)
95.0 (80)
28.0 (205)
88.4 (190)
90.0 (270)
hAEL, Hours after egg laying; %EL, per cent embryonic lethality; N, number of embryos scored.
Journal of Genetics, Vol. 80, No. 2, August 2001
89
M. Kumar et al.
Since the unconditional embryonic lethal alleles stmA12
and stm7 showed cuticle loss over the neuroectodermal
region, the question was asked if heat-killed embryos of
stmA1 and stmA2 might mimic this effect. Observations on
cleared embryos of stmA1 and stmA2 showed that they did
indeed suffer from pronounced cuticle loss overlying the
embryonic brain and the ventral nerve chord (figure 4, b–
f). Cuticle loss ranged from weak to strong. The severity
of heat-shock-induced cuticle loss was quantified by
grading the embryos in five phenotypic classes: class I
(figure 4a) had near-normal embryos with intact cuticles
and class V (figure 4, e, f) comprised embryos with little
or no continuous cuticle (see Material and methods for
detailed description of each class). Heat-induced cuticle
loss in mutant stmA embryos was roughly three times that
in wild-type embryos (table 8, figure 5). Interestingly, up
to 35% of wild-type embryos also showed heat-induced
cuticle loss. However, the frequency of wild-type embryos
Figure 4. Cleared cuticle preparations of heat-killed embryos of stmA1 and stmA2 belonging to cuticle loss classes
I to V. All embryos are oriented anterior to the top and dorsal to the right. Photos a and b are in bright field optics; c, d
and f in phase contrast; and e in Nomarski optics. a, Class I embryo of stmA1; note the continuous ventral and dorsal
cuticles and normal head skeletal structures (hs) and anal structures (as); the region of the three-hour-old embryo that
gives rise to the dorsal and ventral neuroectoderm is marked by a dotted line. b, Class II embryo of stmA2 showing large
dorsal cuticle hole, reduced head skeleton and an overall reduction in the cuticle area. c and d, Class III embryos of
stmA1 and stmA2 showing large cuticle holes in the ventral and dorsal areas. e, Class IV embryo of stmA2 showing a
narrow strip of dorsal cuticle. f, Class V embryo showing no continuous cuticle.
90
Journal of Genetics, Vol. 80, No. 2, August 2001
Mutations at stambh A in Drosophila melanogaster
Table 8.
Distribution of heat-killed embryos into cuticle loss classes I to V.
% Embryos* in cuticle loss class
Age of eggs
(hAEL)
Temperature
(ºC)
0
33
3
33
Genotype
I
II
III
IV
V
+
stmA1
stmA2
+
stmA1
stmA2
79
34
23
89
44
22
21
37
61
11
16
2
0
12
2
0
30
62
0
8
4
0
2
4
0
1
0
0
8
10
hAEL, Hours after egg laying.
*Rounded off to the nearest integer.
For description of classes see Materials and methods.
in some way involved in the process that leads to cuticle
loss. We conclude that stmA is a maternally active gene
that is needed for normal neural and ectodermal
development in the embryo.
stmA+ is necessary for female germline viability
Figure 5. Frequency of heat-induced cuticleless embryos in
stmA and wild-type embryos.
with severe cuticle loss (classes II–V) was three-fold to
seven-fold lower than in stmA embryos.
We conclude from this experiment that heat-induced
embryonic lethality is associated with cuticle loss both in
stmA and in wild-type Canton-S genotypes, its severity
being much greater among stmA genotypes. These
observations demonstrate that the wild-type function of
stmA is needed postzygotically during the first three hours
of embryogenesis to ensure proper neuroectodermal
development.
Heat-induced cuticle loss is maternally influenced
It has been reported that several neurogenic genes such as
Notch, mastermind and Enhancer of split exert a maternal
influence over cell-fate choice in the neuroectoderm
(Lehmann et al. 1983; Schrons et al. 1992). To see if
stmA has a similar maternal effect, reciprocal crosses
were made between stmA1, stmA2 and Canton-S, and the
stmA / + embryos heat-shocked. The results showed that
in general the frequency of embryonic lethality and cuticle
loss (table 9) was far greater when the stmA / + embryos
were derived from stmA mothers than when derived from
wild-type mothers. stmA mothers obviously produce a
heat-sensitive product that persists in the cytoplasm and is
Maternal germline clones of stmA7 and stmA12 were
induced using mitotic recombination in the ovoD
background. The purpose of inducing maternal germline
clones of stmA embryonic lethal alleles was to ask if
(i) homozygous stmAl / stmAl clones survived and (ii) if the
clones on survival exerted any maternal influence over
postzygotic development. P[ovoD] is inserted at 55DE
while stmA is located proximally at 44D1.2 (figure 6a). In
this given situation two types of mitotic recombination
events are expected. The first category of recombination
events is expected in region I (see figure 6b) and is
expected to yield stmAl / stmAl clones. The other category
of recombination events is expected in region II between
stmA and P[ovoD] and would yield stmAl / + clones (figure
6c). We have not used any closely linked flanking marker
between stmA and P[ovoD] to distinguish event I from
event II. We have instead resorted to simple but robust
genetic testing to determine the genotype of the F1
progeny of clone-bearing females. Clone-bearing females
were crossed to wild-type males and all the F1 progeny
were individually test-crossed to b stmA1 flies. Each F2
test cross family was scored for segregation for
b stmA1 / + vs b stmA1 / stmAl flies. The F2 phenotypic
ratios determined the F1 parental genotype and thereby the
genotype of the maternal germline. The null hypothesis was simply that if the germline genotype
was stmAl / stmAl all the F1 flies would be of the
stmAl / + genotype and each F2 family would segregate for
b stmA1 / + vs b stmA1 / stmAl in a 1 : 1 ratio.
Clones in the stmA + / + P[ovoD] controls were induced
at a frequency of 6.72% (table 10). These clones represent all the mitotic recombination events between
the centromere and stmA (region I) and between
stmA and P[ovoD] (region II). Clone frequencies in
Journal of Genetics, Vol. 80, No. 2, August 2001
91
M. Kumar et al.
stmA7 + / + P[ovoD] and stmA12+ / + P[ovoD] were 2.3%
and 2.8% respectively, which are roughly half that of
the control. These low frequencies were observed
despite testing twice the number of females of
stmA7 + / + P[ovoD] and stmA12+ / + P[ovoD]. F2 progeny
testing of the adults derived from the clone-bearing
females (mated to wild-type males) showed that the clones
induced in the 13 clone-bearing females (eight from
stmA12 + / + P[ovoD] and five from stmA7 + / +P[ovoD])
were all of stmA / + genotype and none were homozygous
stmA / stmA. Data on testing of the eight germline clonebearing females derived from stmA12 + / + P[ovoD] are
Table 9. Heat-induced lethality and cuticle loss among embryos derived from
reciprocal matings involving stmA1, stmA2 and Canton-S .
0-hour-old eggs
3-hour-old eggs
Parents
(female × male)
% EL (N)
% CLE
% EL (N)
% CLE
+ × A1
A1 × +
A1 × A1
+ × A2
A2 × +
A2 × A2
+×+
22.0 (500)
91.8 (500)
89.7 (475)
41.7 (525)
48.2 (541)
54.4 (428)
29.3 (1019)
22
72
76
20
63
85
25
14.5 (359)
56.7 (466)
99.2 (495)
23.5 (375)
78.1 (375)
92.7 (301)
29.1 (760)
21
68
70
11
82
80
12
% EL, Per cent embryonic lethality; N, number of embryos studied; % CLE, per cent
cuticleless embryos.
Clone frequency
Chromosome
combination
Clone genotype
stmA7
stmA12
1,3
2,3
1,4
2,4
stmA+ / stmA+
+ ovoD / stmA+
stmA+ / + ovoD
+ ovoD / + ovoD
0
0
0
0
0
0
0
0
Chromosome
combination
1,3
2,3
1,4
2,4
Clone frequency
Clone genotype
stmA7
stmA12
stmA+ / ++
stmA ovoD / ++
stmA+ / + ovoD
2.3
0
0
0
2.81
0
0
0
stmA ovoD / + ovoD
Figure 6. a, Relative positions of stmA and P[ovoD] with respect to the centromere in mitotic chromosomes. b and c, Consequences
of mitotic recombination in region I (b) and in region II (c) with the frequencies of expected categories of clone from each of the
recombination events.
92
Journal of Genetics, Vol. 80, No. 2, August 2001
Mutations at stambh A in Drosophila melanogaster
presented in table 11 to illustrate the nature of the tests
that were done.
Eggs derived from mating the clone-bearing females
with wild-type males also showed control levels of
embryonic lethality (ranging from 3% to 6%) and did not
show any consistent pattern of cuticle loss as may have
been expected if the clones were of the stmA genotype. In
conclusion, failure to recover stmA / stmA clones indicates
that stmA7 and stmA12 are lethal to the female germline.
Discussion
This study was aimed at understanding the genetics of the
stmA locus and determining its role in fly development.
Twelve stmA alleles isolated in four different screens are
reported. The alleles fall into two phenotypic categories
of reversible ts paralytic homozygous viable (four alleles)
and unconditional lethals (eight alleles). The 12 alleles
fall into two mutually complementing groups, groups II
and III, and a third group I that does not complement
members of either group II or group III. Groups I and II
consist of unconditional lethals while group III includes ts
paralytic as well as unconditional lethals. No ts paralytic
allele belonging to group I has so far been isolated. This
might imply that stmA alleles with unconditional lethality
are mutations causing very severe impairment of gene
activity or total loss of gene function while ts paralysis is
Table 10.
Genotype
due to a less drastic change. The paralytic phenotypes of
various heteroallelic combinations (table 5) do not
however follow this simple assumption that lethality
is caused by more severe mutations than ts paralysis.
If the assumption were correct, paralytic (viable) / lethal
heterozygotes ought to have had stronger paralytic
phenotypes than their respective paralytic (viable) homozygotes. Eleven of 32 trans heterozygotes showed a
weaker paralytic phenotype than their respective
homozygotes. Interestingly stmAP1, which is recessive to
the wild type, shows weaker paralysis when heterozygous
over most lethal alleles. Absence of a simple correlation
between the phenotypes of the various trans heterozygotes and their homozygotes supports the view that the
various mutations do not fall into a single functional
group. Intragenic complementation among stmA alleles
also points towards a complex genetic nature of the locus.
Intragenic complementation is typically seen in genes that
have more than one functional domain and whose protein
products are multimeric in nature (Gepner et al. 1996;
Grant et al. 1998). It has been demonstrated that lethal
alleles of the gene shibire, which also gives rise to ts
paralytic mutations, show intragenic complementation
(Grant et al. 1998). shibire encodes the Drosophila
homologue of dynamin (a GTPase), which is known to
have multiple functional domains (Grant et al. 1998).
Molecular analysis of stmA will provide answers to
similar questions about its structure and function.
Frequency of female germline clones of stmA.
Age (hAEL)
at irradiation
% Clone bearing
females (N)
Number of
females tested
60 ± 12
84 ± 12
108 ± 12
108 ± 12
108 ± 12
4.89 (15)
6.16 (17)
6.72 (8)
2.81 (8)
2.30 (5)
307
276
119
285
215
+ / P[ovoD]
stmA12 + / + P[ovoD]
stmA7 + / + P[ovoD]
hAEL, Age at time of irradiation in hours after egg laying; N, number of clone-bearing females.
Table 11. Analysis of embryos and adult progeny of the eight germline clone-bearing females derived from irradiating stmA12
+ / + P[ovoD] larvae. The germline clone-bearing females were mated to wild-type males.
Unhatched eggs (F1)
Female
Eggs
laid
N (%)
unfertilized
N (%)
fertilized
Fertilized &
hatched eggs
(F1)
1
2
3
4
5
6
7
8
32
26
80
10
31
235
66
31
6 (18.7)
6 (23.0)
17 (17.5)
1 (10.0)
4 (12.9)
8 (3.4)
10 (15.1)
4 (12.9)
0
0
3 (3.15)
0
0
10 (4.26)
0
0
26
20
63
9
27
217
56
27
Adults
recovered
(F1)
26
18
52
8
26
198
54
25
Journal of Genetics, Vol. 80, No. 2, August 2001
F1 genotypes
F2 test cross
determined from F2
families tested
+/+
for segregation + stmA12 / +
15
15
33
7
20
103
35
20
4
9
12
2
12
42
15
3
11
6
21
5
8
61
20
17
93
M. Kumar et al.
Towards this objective, stmA has been precisely located
to the polytene map position 44D1.2, and P inserts using
wild-type P elements from a Birm-2 chromosomal source
have been generated. Birm-2 P elements do not have any
selectable markers or any plasmid rescue sequences that
simplify cloning. Attempts are in progress to generate
rescuable PlacW inserts in stmA by two methods: (i) by P
element exchange involving the Birm-2 and an X-linked
PlacW element (Sepp and Auld 1999) and (ii) by locally
mobilizing a PlacW from position 44D3.4 into stmA.
Role in development
Genetic analysis of behavioural defects through the study
of mutants of single genes in Drosophila and several other
animals has provided valuable insight into the regulation
of neural functions and development (Heisenberg 1997).
Several behavioural mutants are associated with abnormal
neural development (Hall 1982; Poodry 1990). It was
earlier suggested (Chandrashekaran and Sarla 1993) that
stmA was involved in neuroectodermal development
on the basis of observations that embryos of two
unconditional lethal alleles showed neural hypertrophy
and cuticle hypotrophy. The present study has revealed
that embryos of the ts paralytic allele stmA1 show mild
neural hypertrophy even at the permissive temperature of
23ºC. This neural abnormality at 23ºC is consistent with
the observation that stmA1 adults show abnormal
behaviour at 23ºC. No abnormalities in neural connections or cuticle loss were observed in stmA1 embryos at
this temperature. After a short heat shock at temperatures
between 31°C and 35ºC, however, stmA1 and stmA2
embryos develop a strong neurogenic phenotype showing
strong to extreme cuticle loss. It has also been found that
stmA is maternally active and is needed during early
embryogenesis between zero and three hours for proper
ectodermal differentiation.. Coinduction of ectodermal
hypotrophy and neuronal hypertrophy by heat treatment
has not yet been demonstrated experimentally. Nevertheless there are reasons to expect neuronal hypertrophy
in heat-killed embryos because embryos with zygotic
unconditional lethal alleles of stmA show strong neuronal
hypertrophy and those with conditional ts paralytic alleles
display mild hypertrophy. It is interesting that a similar
heat-induced neurogenic phenotype is seen in the ts
paralytic mutation shibire (Poodry 1990). shibire encodes
the Drosophila homologue of dynamin and is needed
for recycling of synaptic vesicles, and is unrelated
structurally and functionally to the Notch-Delta group of
neurogenic genes.
This study also shows that stmA is needed for the
viability of the maternal germline. The functions of the
neurogenic genes Notch and Delta are also required for
the viability of the female germline and participate in
cell–cell communication during oogenesis (Ruohola et al.
1991).
94
The term ‘neurogenic’ with reference to stmA is used
merely to refer to the phenotype of neural hypertrophy
and cuticle hypotrophy. In Drosophila literature the term
‘neurogenic’ is strongly linked with the first few
‘neurogenic’ genes of the Notch-Delta category, whose
mutations produce such a phenotype (Lehmann et al.
1983). The Notch-Delta neurogenic genes control the
choice of cell fate between neuroderm and ectoderm in
the early stages of Drosophila embryonic development
(among a host of other cell-fate choice decisions) by
influencing cell–cell communication. Several of them
encode epidermal-growth-factor-like proteins, transmembrane receptors and transcription factors (Campos-Ortega
1993). Using the term ‘neurogenic’ with reference to stmA
merely implies that the process of neural vs epidermal fate
might involve the function of stmA. It remains to be
demonstrated that stmA is developmentally related to the
‘neurogenic’ genes.
It has been shown previously (Chandrashekaran 1993)
that stmA adults resist the lethal effects of the sodiumchannel-binding neurotoxin veratridine at 23–24ºC. Such
resistance may be attributed to altered binding of
veratridine to voltage-gated sodium channel subunits due
to a constitutive defect in membrane-related properties of
stmA mutant flies. The observation that stmA flies show
behavioural defects and a mild neural hypertrophy at 23–
24ºC supports the idea of a constitutive neuronal defect. It
is evident that constitutive defect in the ts paralytic alleles
gets exaggerated at higher temperatures leading to cuticle
loss and death in embryos and paralysis in adults.
In conclusion, the results presented demonstrate that
stmA is a functionally complex locus. It is functional both
maternally and zygotically and is needed for normal
neuronal development in the early embryo, in normal
behavioural functions in the adult, and for the viability of
the maternal germline. Essential information to facilitate
cloning and molecular analysis have been obtained by
way of precise physical location and transposon tagging
of the gene.
Acknowledgements
I take this opportunity to thank Prof. Balram Sharma of the
Division of Genetics for critically reading this manuscript in its
final stages of preparation. We thank Dr K. S. Krishnan for
deficiency stocks, Pradip Sinha for the P[ovoD] stock, and Dr
Veronica Rodrigues for gifting us Mab 22C10 and letting us use
her laboratory.
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Received 23 May 2001; in revised form 31 August 2001
Journal of Genetics, Vol. 80, No. 2, August 2001
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