1 Department of Molecular Biology, University of South Bohemia, and Institute of
Entomology ASCR, Ceske Budejovice 37005, Czech Republic
2 Department of Biology, University of North Carolina Greensboro, Greensboro, NC
27402, USA
* Author for correspondence (e-mail: jindra{at}entu.cas.cz)
Accepted 24 February 2004
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SUMMARY |
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Key words: Steroid hormone, Ecdysone, ecdysoneless, Imaginal disc, Oogenesis, Drosophila
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Introduction |
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The major and best-studied source of ecdysone in insect larvae is the
prothoracic gland, which in Drosophila consists of the lateral lobes
of the ring gland (Dai and Gilbert,
1991). After this part of the ring gland degenerates during
metamorphosis, adult ovaries contribute to the whole body steroid titer in
Drosophila (Garen et al.,
1977
; Bownes et al.,
1984
; Bownes, 1989
;
Warren et al., 1996
). The main
role of ecdysone in adult females is to regulate vitellogenesis
(Hagedorn, 1985
;
Bownes et al., 1996
). In
addition, ecdysone has been implicated in egg chamber maturation during
mid-oogenesis (Buszczak et al.,
1999
). Inactive ecdysone conjugates are maternally deposited to
eggs and are mobilized during mid-embryogenesis by the amnioserosa
(Bownes et al., 1988
;
Kozlova and Thummel,
2003
).
Recently, several Drosophila genes involved in ecdysone
biosynthesis have been cloned. One is dare, a homolog of the human
adrenodoxin reductase that is necessary for the reduction of mitochondrial
cytochrome P450 (Cyp) enzymes (Freeman et
al., 1999). Two other genes, disembodied (dib)
and shadow (sad), encode Cyp C22-and
C2-hydroxylases, respectively, which are responsible for the final
two hydroxylation steps of ecdysone synthesis
(Chavez et al., 2000
;
Warren et al., 2002
). Ecdysone
is the final product of the ring gland, which is secreted to the hemolymph and
converted to 20E in peripheral tissues. The Cyp C20-hydroxylase
responsible for this conversion is encoded by shade (shd)
(Petryk et al., 2003
). The
dare, dib and sad genes are all expressed in the larval
lateral ring gland and in adult ovaries, and their loss-of-function phenotypes
can be fully explained as a consequence of ecdysone deficiency. Thus far, only
one steroidogenic factor that is not itself an enzyme, without
children (woc), has been identified
(Wismar et al., 2000
;
Warren et al., 2001
). This
gene encodes a zinc finger transcription factor that probably activates
expression of the cholesterol 7,8-dehydrogenase that executes the first step
of ecdysone biosynthesis. Mutations of woc affect a wide range of
tissues, suggesting that its transcriptional function is not restricted to
regulating expression of the steroidogenic enzyme. No other regulators of the
steroidogenic pathway have been identified thus far.
Among steroid-deficient Drosophila mutations,
ecdysoneless1 (ecd1) is used to study
ecdysone roles in development. The ecd1 mutation is a
recessive, temperature-sensitive allele that reduces whole-body ecdysone
titers and causes larval arrest at a restrictive temperature, 29°C
(Garen et al., 1977). The
effect of ecd1 on ecdysone production is autonomous,
because cultured ecd1 mutant ring glands fail to produce
ecdysone when upshifted to 29°C
(Henrich et al., 1987
;
Dai et al., 1991
;
Warren et al., 1996
). Ecdysone
production is also interrupted in adult ovaries upshifted to the restrictive
temperature (Garen et al.,
1977
; Redfern and Bownes,
1983
; Warren et al.,
1996
). After several days at 29°C, oogenesis pauses at the
onset of vitellogenesis; this phenotype can be reversed by lowering the
temperature (Audit-Lamour and Busson,
1981
). Transplantation experiments show that this effect of
ecd1 is autonomous to the ovary
(Garen et al., 1977
).
Developmental events disrupted in ecd1 mutants include
fat body protein synthesis (Lepesant et
al., 1978), progression of the eye-forming morphogenetic furrow
(Brennan et al., 1998
),
salivary gland glue secretion (Biyasheva et
al., 2001
) and motor neuron outgrowth
(Li and Cooper, 2001
). These
defects have been interpreted as consequences of the mutationally induced
ecdysone deficiency. However, Redfern and Bownes caution that a range of
anomalies in ecd1 adults result from an autonomous
ecd requirement for cell viability and therefore may not be
attributable to ecdysone deficiency
(Redfern and Bownes,
1983
).
It is difficult to discern which of the phenotypes result from the ecd1 mutation directly, and which are the consequence of low ecdysone titer, without knowing the primary defect in the ecdysoneless gene, whose molecular identity remained elusive for over 25 years. We report here that the ecd locus encodes a protein whose orthologs in several other species, including humans, have not yet been functionally described. The original ecd1 mutation and three non-conditional lethal alleles have been mapped and assessed for their effects. We have localized the Ecd protein to both the steroidogenic and non-steroidogenic tissues, and have demonstrated its cell-autonomous roles in imaginal discs and ovaries.
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Materials and methods |
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Genetic mapping and sequence analysis of ecd
Deficiencies Df(3L)R+R2, in the 62B-D chromosomal region that
deletes the ecd locus (Sliter et
al., 1989), and Df(3L)Aprt201, which complements the
non-conditional ecd alleles, were used to delimit the ecd
interval by a series of PCR reactions. These were performed on embryos
homozygous for either Df(3L)Aprt201 or Df(3L)R+R2 with pairs
of primers, derived from ten genes (CG17772, CG17771, CG13807, CG5714,
CG13806, CG13805, CG5717, CG13804, CG13803, CG13802) occurring between the
right breakpoints of the two deletions according to the BDGP (Berkeley
Drosophila Genome Project; Fig.
1). CG5714 was identified as ecd by genetic rescue of the
ecd mutants. Genomic DNA from embryos or larvae
homozygous for each of the ecd alleles was amplified with primers
flanking the CG5714 gene: 5'-GGTACGAAGGAGGCGGAGGG-3' and
5'-GATGAGCAAGATTCCAGGCAGCA-3'. PCR products from three independent
reactions were sequenced using the BigDye Terminator Kit (Perkin Elmer), using
these and additional internal primers to cover the entire ecd gene in
both directions.
|
Lethal phase determination
Each ecd allele was crossed with all other ecd alleles
and with the Df(3L)R+R2 deficiency. All lines were balanced with
TM3, P[w+, act-GFP]. The flies were allowed to lay eggs on
apple juice plates, supplemented with baker's yeast paste at 25°C, or at
29°C in the case of ecd1 crosses. Eggs were collected
in two-hour periods, and embryos or larvae were identified as ecd
homozygotes by the absence of the GFP-marked balancer.
Hormone feeding and titer determination
For the non-conditional ecd2 and
ecdl(3)23 mutants, 200 early-second instar larvae of each
genotype were placed in vials with a sucrose-yeast medium containing
20-hydroxyecdysone (20E) at concentrations of 1 mg/ml
(Garen et al., 1977;
Freeman et al., 1999
), 250
µg/ml, 50 µg/ml or zero, and animals progressing to the second molt or
beyond were counted. The temperature-sensitive ecd1
mutants were tested for puparium formation as third instar larvae on the same
media at 29°C. In all cases the homozygous ecd mutants were
compared with their rescued counterparts carrying the S4 construct.
Radioimmunoassay of total ecdysteroids was performed in whole-body homogenates
as described (Jindra et al.,
1994
).
Rescue with ectopic Ecd expression
A full-length ecd cDNA (GH14368; BDGP) was subcloned into the
pUAST P-element vector (Brand and Perrimon,
1993). Transgenic flies carrying the UAS-ecd construct in
the ecd2 mutant background were crossed with
ecd2 lines carrying transgenic Gal4 drivers to produce
UAS-ecd/Gal4; ecd2/ecd2. Six drivers were
tested for the ability to rescue the ecd2 lethal
phenotype: act-Gal4 (from Dr B. Edgar), ptc-Gal4
(Bloomington stock #2017), sev-Gal4 (from Dr P. Vilmos),
en-Gal4 (from Dr Y. Hiromi), Aug21 and Feb36
(Siegmund and Korge, 2001
;
Andrews et al., 2002
). All
lines were balanced with TM3, Ser, P[w+, act-GFP], so that
ecd2 homozygotes could be identified at all developmental
stages.
Generation of somatic and germline ecd clones
Mutant clones deficient for either Ecd or MBF1 (control) proteins were
generated by mitotic recombination using the FLP-FRT technique as described
(Xu and Rubin, 1993;
Theodosiou and Xu, 1998
;
Chou and Perrimon, 1996
). To
induce clones in the developing imaginal discs, w, hs-FLP;
P[w+, ub-GFP]61F FRT 80B females were mated with w; ru
ecd2 FRT 80B/TM3, P[w+, act-GFP] or with y w;
mbf1 FRT 80B males. Their progeny were heat-shocked as larvae for one
hour at 38°C, 24-36 hours after egg laying; adult females were
heat-shocked for 3 hours at 37°C to generate mutant clones in the ovarian
follicle cells. To obtain ecd-null germline clones, females w,
hs-FLP; ru ecd2 FRT3L-2A/TM6B were mated with
w; P[w+; ovoD1]3L-2X48
FRT3L-2A/TM3 males. Before reaching the second-to-third instar
transition, the progeny was heat-shocked twice for 2 hours at 38°C
(Theodosiou and Xu, 1998
).
Emerged w, hs-FLP/w; ru ecd2
FRT3L-2A/P[w+; ovoD1]3L-2X48
FRT3L-2A females were mated, examined for egg laying, and
sacrificed for immunostaining of their ovaries 3-10 days later. Alternatively,
germline clones were induced by heat shock for 1 hour at 38°C in adult
females, and were analyzed 3-7 days later.
RNA hybridization
Poly(A)+ RNA was isolated using the QuickPrep mRNA Purification
Kit (Amersham) and ecd and mbf1 transcripts were detected on
northern blots with full-length cDNA probes as described
(Uhlirova et al., 2002). The
same ecd probe, and its sense version (for control), was used for in
situ hybridization of adult ovaries (Tautz
and Pfeifle, 1989
; Buszczak et
al., 1999
); detection was with anti-DIG alkaline phosphatase and
the CBIP/NBT substrate (Roche).
ecd-lacZ expression
An ecd-lacZ reporter was constructed by cloning a 1.25 kb
ecd upstream genomic region into the pCaSpeR-AUG-ßgal vector
(Thummel et al., 1988). The
same regulatory sequence in the S4 construct was sufficient for the rescue of
ecd-null mutants. The ecd-lacZ activity was
detected in transgenic animals using a standard X-gal staining procedure.
Ecd antibodies, immunoblot and tissue staining
The central portion of Ecd (amino acids 270-429) was expressed from pET28a
(Novagen) as a hexahistidine fusion protein in the BL21-CodonPlus (Stratagene)
E. coli strain. The protein was affinity-purified on a Ni-NTA agarose
column (Qiagen) under denaturing conditions, then partially re-natured by
dialysis and used for rabbit immunization. The collected antiserum was
affinity-purified using the entire Ecd protein, produced by the yeast
EasySelect Pichia Expression Kit (Invitrogen) and immobilized on the
AminoLink Plus Coupling Gel (Pierce). For western blots, embryos or larvae
were homogenized in a denaturing sodium dodecylsulphate (SDS) buffer, and
total protein (ca. 10 µg per lane) was analyzed by 10% SDS-PAGE. Blots were
probed with the purified anti-Ecd antibody, diluted 1:5000. Detection was with
a goat HRP-conjugated anti-rabbit antibody (1:4000) and a chemiluminescent
substrate. Whole-mount immunostaining of larvae and adult gonads was performed
according to standard procedures, with antibodies diluted as follows:
anti-Ecd, 1:1000; anti-MBF1, 1:10,000 (Liu
et al., 2003); anti-Orb (4H8 DSHB), 1:30
(Lantz et al., 1994
); and
anti-FasIII (7G10 DSHB), 1:30 (Patel et
al., 1987
). Secondary antibodies conjugated with Alexa Fluor 488,
Texas-Red (Molecular Probes) and Cy3 (Amersham) were used at a dilution of
1:1000. Images were captured on Axioplan 100 and confocal LSM410 inverted
laser scanning microscopes (Zeiss).
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Results |
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The sequence of the deduced Ecd protein reveals a broad evolutionary
conservation. Putative Ecd orthologs have been found in the mosquito
Anopheles gambiae (43% overall amino acid identity), humans and mouse
(31%), zebrafish (30%), Arabidopsis thaliana (26%) and the fission
yeast Schizosaccharomyces pombe (21% identity). The human Ecd
ortholog, known as Suppressor of GCR2 (SGT1), is expressed in a wide range of
human organs (Sato et al.,
1999) and functionally rescues a mutation of GCR2, a
transcriptional regulator of glycolytic enzyme genes in the fission yeast
(Deminoff and Santangelo,
2001
). However, GCR2 is not homologous to SGT1 and thus the normal
role of SGT1 in humans is unknown. Interestingly, although several highly
conserved motifs are evident among the aligned orthologs
(Fig. 2), none of these
correspond to any known functional domain. There is a putative ATP/GTP-binding
motif (P-loop) near the C terminus of the Drosophila and
Anopheles orthologs, as recognized by the PROSITE database
(Fig. 2).
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During metamorphosis the lateral ring gland degenerates. Other organs, such as ovaries, serve as sources of adult ecdysone. In adult ovaries, Ecd protein was expressed in both the somatic follicle cells and the germline nurse cells throughout oogenesis (Fig. 6L). The signal was stronger in the nurse cells of egg chambers staged 8-10, probably because of the deposition of the Ecd protein, as well as mRNA (Fig. 6M) into the oocyte at this time. High levels of Ecd were detected in the apical part of adult testes, where the somatic and germline stem cells are localized and where spermatogenesis begins (Fig. 6N). In summary, Ecd expression was detected in the primary steroidogenic organs the larval lateral ring gland and the adult ovaries as well as in the non-steroidogenic central nervous system and imaginal discs.
Hormone feeding or ecd expression in the ring gland cannot rescue non-conditional ecd mutants
The presence of Ecd in the late-third instar ring gland is consistent with
the steroid deficiency for which ecd1 was originally
identified. The ability to induce puparium formation by feeding the
non-pupariating ecd1 larvae at 29°C with
20-hydroxyecdysone (20E) (Garen at al., 1997;
Redfern and Bownes, 1983;
Berreur et al., 1984
),
suggested that low steroid levels might be the primary cause of arrest at this
stage. To test whether the non-conditional ecd mutants could also be
rescued by dietary hormone, we fed homozygous second instar
ecd2 and ecdl(3)23 larvae with 20E.
The feeding of ecd1 larvae at 29°C served as a
positive control: 50 µg/ml and 250 µg/ml 20E doses induced pupariation
in 26 out of 100, and in 36 out of 100, ecd1 homozygotes,
respectively. By contrast, none of 600 ecd2, or 250
ecdl(3)23, larvae progressed beyond their lethal phase
when fed 20E. These results strongly imply that ecdysone deficiency alone does
not account for the second instar lethality of these mutants. In support of
this view, the whole-body ecdysteroid content was not significantly different
between ecd2/ecd2 (0.61±0.13 pg/animal)
and ecd+ (0.48±0.08 pg/animal) first instar
larvae.
To address the problem of ecd requirement directly, we have
targeted ecd expression to the steroidogenic part of the ring gland
using transgenic UAS-ecd activated by a Gal4 driver, Feb36
(Siegmund and Korge, 2001;
Andrews et al., 2002
). As was
expected from the ability of exogenous 20E to rescue pupariation of
ecd1 homozygotes at 29°C, Ecd expressed under
Feb36 allowed formation of defective puparia in around 25%
UAS-ecd, ecd2/ecd1 larvae upshifted to 29°C
(n=60). The ectopic Ecd presence in the ring gland, evident during
the second instar (Fig. 6F),
should therefore restore the impaired hormone synthesis and at least postpone
the arrest of ecd-null mutants, if disrupted ecdysone production was
the sole cause of their death. However, the Feb36-driven Ecd was
insufficient to advance UAS-ecd, ecd2/ecd2
larvae even to the second molt. By contrast, the same UAS-ecd
construct expressed under a ubiquitous actin-Gal4 driver allowed
ecd2 homozygotes to reach adulthood
(Table 2).
|
Cell-autonomous function of ecd in imaginal discs
To examine whether ecd plays a cell-autonomous role during
development of the adult, we have generated mitotic clones homozygous for the
null allele ecd2 using the FLP-FRT system. Mutant clones
of a non-essential gene, mbf1
(Liu et al., 2003), located as
ecd on the 3L chromosome arm, served as a control. For both genes,
wild-type sister clones and the heterozygous background were recognizable by
the presence of ubiquitin-driven GFP and the mini white+
gene markers, placed on the homologous chromosome. When induced early during
the first larval instar, large mbf1/ as well
as mbf1+/+ clones appeared in the adult compound eyes. By
contrast, only ecd+/+ clones were found with
ecd2 (Fig.
7A,D). The lack of ecd2/ecd2 clones
was confirmed by staining of imaginal discs, dissected from late third instar
larvae: homozygous mutant clones were only found in mbf1 but not in
ecd somatic mosaics (Fig.
7B-F). No defects were observed in the adult eyes, legs, wings or
thorax derived from the imaginal discs where
ecd2/ecd2 clones were induced. As imaginal disc
cells normally proliferate throughout larval life (Madhavan and Schneiderman,
1977), we assume that the ecd/ cells were
replaced by their ecd+ neighbors. The loss of Ecd,
however, does not seem to be immediately cell-lethal, because small
ecd/ clones could be seen in eye-antennal
imaginal discs when induced at the onset of the third instar (not shown).
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![]() |
Discussion |
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We have mapped molecular defects in the original ecd1
(Garen et al., 1977), in
ecd2 (Sliter et al.,
1989
), and in two previously undescribed alleles,
ecdl(3)23 and ecdg24. The point
mutation found in ecd1 is consistent with its hypomorphic
nature (Henrich et al., 1993
).
It converts a proline residue, conserved in all Ecd orthologs identified so
far, into serine. This substitution does not cause degradation of the Ecd
protein (Fig. 3B), or its
subcellular mislocalization in the ring gland at 29°C (not shown). The
mutation maps near the C terminus (Fig.
2), which must harbor an important function because a short
truncation in ecdl(3)23 lacking this region is
phenotypically as severe as the ecd2 mutation, removing
almost the entire protein (Fig.
3A). Although the non-conditional ecd
mutants die as second instar larvae, temperature shifts of the
ecd1 mutants suggest that Ecd is required during
embryogenesis (Kozlova and Thummel,
2003
). This early function may be executed by the maternally
supplied Ecd protein, which is still detectable in first instar
ecd homozygotes
(Fig. 3B). As the effects of
ecd2, ecdl(3)23 and ecdg24
are not worsened in hemizygous combinations with an
ecd deficiency, all of these three mutations are
likely to represent ecd-null alleles. A single transgenic
ecd+ copy rescues all ecd
mutants to adulthood, showing that the developmental and lethal defects seen
in these mutants are fully attributable to the loss of ecd
function.
Although the non-conditional ecd mutants often
die during the ecdysis to the second instar, displaying phenotypes that might
imply defective ecdysone production (Fig.
4B,C), their lethality cannot be a direct consequence of low blood
ecdysone for the following reasons. First, ecd
animals cannot be advanced to the second molt by 20E feeding, despite the fact
that similar doses of 20E are sufficient (1) to avert second instar lethality
in mutants for the steroidogenic enzyme Dare
(Freeman et al., 1999) and (2)
to induce pupariation in ecd1 larvae at 29°C. Second,
as some of the ecd animals die during the
transition to the second instar, one would expect that their ecdysone titer
would be lower from as early as the first instar. However, we have not found a
reduction of ecdysone content in first instar homozygous
ecd2 larvae. Third, although Ecd is abundant in the
lateral ring gland during the third instar, no such expression is seen at
earlier stages. By contrast, some other steroidogenic genes, such as
dib and sad, are strongly expressed in the ring gland
beginning at embryogenesis (Chavez et al.,
2000
; Warren et al.,
2002
). Finally, development of ecd2
homozygotes can be completely rescued with ubiquitous Ecd expression but not
with Ecd targeted by the Feb36-Gal4 driver to the ring gland and to
some other organs (Andrews et al.,
2002
). As Ecd presence in the ring gland cannot postpone the death
of ecd-null mutants, Ecd must be required prior to the initiation of
the second molt in some other tissues. One could be the nervous system
(Fig. 6G), because
patched-driven Ecd promotes further development of the mutants.
A cell-autonomous effect was previously demonstrated for the
ecd1 allele during differentiation of the thorax sensory
bristles (Sliter, 1989).
Unexpectedly, induction of ecd-null mitotic clones in the primordia
of the adult thorax, the wing imaginal discs, did not produce any defective
bristles. This was probably because no ecd clones
occurred in the adult epidermis. Based on the presence of twin
ecd+/+ clones in all imaginal discs and in the adult
compound eye (Fig. 7), we
conclude that the lost ecd clones were replaced by
proliferation of the surrounding ecd+ cells. Redfern and
Bownes (Redfern and Bownes,
1983
) ascribed many of the defects seen in temperature-upshifted
ecd1 mutants to autonomous cell lethality in the imaginal
discs. However, we have detected small clones of
ecd cells in imaginal discs upon induction of
recombination during early third larval instar, and
ecd clones also survived in the adult ovary. Thus,
the loss of ecd is not generally cell lethal although it reduces the
ability of the mutant cells to proliferate at the normal rate. Our mosaic
analyses provide direct evidence for a cell-autonomous, ecdysone-independent
function of ecd, which may underlie the previously described defects
in adult morphogenesis.
Clones of ecd somatic follicle cells caused
profound defects, manifest as fusions of adjacent egg chambers and leading to
duplications of the nurse cell set, in some cases with two vitellogenic
oocytes present at the opposite poles (Fig.
8D). Similar polarity defects were caused by perturbing the
Delta/Notch signaling that specifies the polar follicle cells (PFC), and by
perturbing the JAK/STAT pathway through which these cells establish proper
separation between egg chambers
(Gonzalez-Reyes and St Johnston,
1998; Grammont and Irvine,
2001
; McGregor et al.,
2002
; Torres et al.,
2003
). It remains to be tested whether the egg chamber fusions in
ecd mosaic ovaries might result from a compromised signaling by the
PFC. Follicle cells are thought to be the major site of ecdysone production in
the ovary (Lagueux et al.,
1977
; Zhu et al.,
1983
). However, it is difficult to imagine that the relatively
small ecd clones could significantly reduce the
ecdysone titer in the female. Therefore we conclude that, as in the case of
imaginal discs, the effects of ecd2 on oogenesis are
independent of free-circulating ecdysone.
Germline clones completely lacking ecd function arrest at
pre-vitellogenic stages, probably earlier than egg chambers carrying the
ovoD1 mutation, thus showing that ecd is
autonomously required for oocyte maturation. This result is consistent with
the phenotype of ecd1 mutant ovaries:
ecd1 females become sterile after a few days at 29°C,
with a majority of egg chambers at pre-vitellogenic stages
(Audit-Lamour and Busson,
1981). Interestingly, the steroidogenic enzyme Dare, and the
ecdysone response proteins EcR and E75, are similarly required in the nurse
cells for egg maturation, as germline clones mutant for these genes arrest as
pre-vitellogenic egg chambers as well
(Buszczak et al., 1999
). This
led the authors to propose that ecdysone synthesis by the germline is
necessary in an autocrine manner for the progression of oocytes to the
vitellogenic stage. As normal ecd function is required for autonomous
ecdysone production by the ovary (Garen et
al., 1977
), the pre-vitellogenic arrest of the
ecd germline clones is consistent with an autocrine
germline function.
By inducing ecd2 mutant clones in adult females, we
created mosaic egg chambers in which some nurse cells were null for
ecd, whereas others carried the ovoD1 dominant
mutation that unconditionally blocks oogenesis. Surprisingly, these
mixed-genotype egg chambers continued to mature much beyond the phase of
arrest caused by either the ecd2 or
ovoD1 mutations acting alone
(Fig. 9). This suggests a
functional rescue among the cells within the egg chamber. As nurse cells are
interconnected by ring canals, we speculate that the ecd+
ovoD1 cells and their ecd,
ovo+ sisters exchanged materials that complemented them and
consequently permitted oocyte development. In the light of the autocrine
germline hypothesis (Buszczak et al.,
1999), an intriguing possibility is that the product of the
ecd+ ovoD1 clones might be ecdysone.
Although the ecdysoneless gene encodes a protein with highly
conserved regions, we have found no data that would describe the function of
these regions and thus enlighten the mode of Ecd action. The only published
report has implicated the human ortholog of Ecd, which compensates for the
loss of an unrelated yeast protein GCR2 in transcriptional regulation
(Deminoff and Santangelo,
2001). Our antibody detects Ecd predominantly in the cytoplasm,
and thus does not directly support the possibility that Ecd acts at the level
of transcription. We have initiated yeast two-hybrid studies to address the
mechanism of Ecd action by identifying its protein partners. Until the exact
function of Ecd is known, interpretations of results obtained with the
ecdysone-deficient ecd1 mutants should consider its
non-steroidogenic effects.
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ACKNOWLEDGMENTS |
---|
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