|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online June 8, 2006
doi: 10.1242/10.1242/dev.02428
1 Department of Integrated Biosciences, Rm201, Graduate School of Frontier
Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562,
Japan.
2 Laboratory of Insect Genome, National Institute of Agrobiological Sciences,
Owashi 1-2, Tsukuba, Ibaraki 305-8643, Japan.
Authors for correspondence (e-mail:
ryusuke-niwa{at}umin.ac.jp;
kataoka{at}k.u-tokyo.ac.jp)
Accepted 5 May 2006
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Bombyx mori, Cholesterol, Drosophila melanogaster, Ecdysone, Growth, Molting, Prothoracic gland, Rieske, Ring gland, Steroidogenesis
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
-ecdysone) and
20-hydroxyecdysone (20E), plays central roles in development, especially in
guiding transition from one developmental stage to the next via molting and
metamorphosis (Thummel, 2001
;
Gilbert et al., 2002;
Thummel and Chory, 2002).
During larval and pupal development of insects, ecdysone is synthesized from
dietary cholesterol or phytosterols via a series of hydroxylation and
oxidation steps in the small endocrine organ called the prothoracic gland (PG)
(Gilbert et al., 2002).
Studies using Drosophila have identified several molecules that
are involved in ecdysteroid biosynthesis in the PG. For example, ecdysone
synthesis is regulated by itpr, which encodes inositol
1,4,5-trisphosphate receptor (Venkatesh
and Hasan, 1997; Venkatesh et
al., 2001); dare, which encodes adrenodoxin reductase
(Freeman et al., 1999);
ecdysoneless (ecd), which encodes an evolutionally conserved
protein with no known motifs (Warren et
al., 1996; Gaziova et al.,
2004); and without children, which encodes a putative
transcriptional regulator (Wismar et al.,
2000; Warren et al.,
2001). Ras-dependent signaling cascade and insulin-dependent PG
cell growth are also essential for the ecdysone production and/or release
(Caldwell et al., 2005;
Mirth et al., 2005).
Recently, five hydroxylase genes that are essential for ecdysteroid
biosynthesis have been identified in Drosophila. All of the
hydroxylase genes, Cyp306a1/phantom (phm),
Cyp302a1/disembodied (dib), Cyp315a1/shadow
(sad), Cyp314a1/shade (shd) and
Cyp307a1/spook (spo), are named the Halloween genes
and encode cytochrome P450 mono-oxygenases
(Chávez et al., 2000;
Warren et al., 2002;
Petryk et al., 2003;
Niwa et al., 2004;
Warren et al., 2004;
Namiki et al., 2005). A
combination of molecular and biochemical experiments have shown that Phm, Dib,
Sad and Shd play pivotal roles in the final four steps of ecdysteroidogenesis,
namely the conversion of 5 ß-ketodiol to 20E
(Gilbert and Warren, 2005).
Orthologs of the Halloween P450 genes have also been identified in the
silkworm Bombyx mori and the tobacco hornmoth Manduca sexta.
The expression patterns of these lepidopteran hydroxylase genes are spatially
restricted to the PG and are temporally correlated with the ecdysteroids titer
during larval development (Niwa et al.,
2004; Warren et al.,
2004; Namiki et al.,
2005; Niwa et al.,
2005; Rewitz et al.,
2006). The identification of these genes provides the basis for
investigating the regulation of insect hormone production in more detail. For
example, it has been shown that the expression of phm and
dib is regulated by the ßFTZ-F1 transcription factor in
Drosophila (Parvy et al.,
2005). Similarly, molting defective, which encodes a
putative transcription factor in Drosophila, influences the
expression level of some Halloween P450 genes
(Neubueser et al., 2005). In
Bombyx, the expression of the dib ortholog is significantly
induced by steroidogenic neuropeptide prothoracicotropic hormone in cultured
PGs (Niwa et al., 2005).
Although the enzymes involved in the final biochemical steps of ecdysteroid
biosynthesis are relatively well characterized, little is known about the
molecules involved in earlier steps
(Gilbert and Warren, 2005).
Dietary cholesterol (C) is first converted to 7-dehydrocholesterol (7dC) by
7,8-dehydrogenation in the endoplasmic reticulum. Conversion of the 7dC to the
4-diketol constitutes the so called `black box'.
Subsequently, cytosolic 5 ß-reduction and microsomal 3 ß-reduction
steps convert the 4-diketol to the 5 ß-ketodiol.
Further identification and characterization of these `early' ecdysteroidogenic
genes are important for understanding the mechanisms by which
ecdysteroidogenesis and developmental timing are precisely controlled in
arthropods.
To facilitate identification and characterization of components responsible
for ecdysteroid biosynthesis, we have used Bombyx to identify genes
predominantly expressed in the PG (Niwa et
al., 2004; Namiki et al.,
2005; Niwa et al.,
2005; Yamanaka et al.,
2005). Based on gene expression analysis using Bombyx
cDNA microarrays that we have previously performed
(Niwa et al., 2004), we now
describe a novel gene named neverland (nvd), the expression
of which is specifically enriched in ecdysone-synthesizing tissues, including
the PG. We show that loss of nvd function in the PG causes growth
arrest at the larval stages, and this phenotype is rescued by application of
20E or 7dC. Our results suggest that Nvd plays a pivotal role in the
metabolism of cholesterol and steroid intermediates during
ecdysteroidogenesis. Considering that the nvd gene family is
evolutionally conserved, we propose that the nvd family of proteins
is an essential regulator of steroid biosynthesis in various animal phyla.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
SHOWA F1
hybrid), have been described previously
(Niwa et al., 2004). All
D. melanogaster flies were reared on a standard agar-cornmeal medium
at 25°C under a 12-hour light/12-hour dark photoperiod unless otherwise
specified. 2-286-GAL4 (Timmons et
al., 1997; Andrews et al.,
2002), AUG21-GAL4
(Siegmund and Korge, 2001) and
AKH-GAL4 (Lee and Park,
2004) were kindly provided from C. S. Thummel, G. Korge and J. H.
Park, respectively. wocrgl
(Wismar et al., 2000) was
generous gift from J. T. Warren and L. I. Gilbert.
breathless-GAL4 (Shiga
et al., 1996), elavc155-GAL4
(Luo et al., 1994) and
teashirt-GAL4 (Shiga et
al., 1996) were provided from Genetic Strains Research Center in
National Institute of Genetics, Japan. scabrous-GAL4
(Klaes et al., 1994) and
decapentaplegic-GAL4
(Staehling-Hampton et al.,
1994) were obtained from the Bloomington stock center.
Lsp2-GAL4 (Cherbas et al.,
2003) and pGawB5015 were provided from the
Drosophila Genetic Resource Center, Kyoto Institute of Technology.
Although a previous study shows that a GAL4 in
pGawB5015 is active in lymph gland and hematopoietic cells
(Sinenko et al., 2004), the
GAL4 was also expressed in the larval ring gland containing the PG
cells (see Fig. S1 in the supplementary materials; J.-B. Peyre and T. Aigaki,
personal communication). All flies were maintained in yw background.
Cyo[y+] and TM3[y+] were used as
balancers.
Molecular cloning
Because an EST clone prgv0382 from the Bombyx EST project
(Mita et al., 2003) lacked the
5 ' region of full-length cDNA of Bombyx neverland
(nvd-Bm), the 5 ' end of nvd-Bm cDNA was obtained by
the 5 ' Rapid Amplification of cDNA ends (5 ' RACE) method using
the GeneRacer Kit (Invitrogen). The first 5 ' RACE product was amplified
with a gene-specific primer (5 '-GGGCAGAAGTAAGGAGCGCCATCTCTGTG-3')
and GeneRacer 5 ' Primer. Then we performed the nested PCR with another
gene-specific primer (5 '-CCGCTGTAAAGAAGCCAATTAAGGTGGCGC-3') and
GeneRacer 5 ' nested primer. The nucleotide sequence of Drosophila
neverland (nvd-Dm) was identified from the Drosophila
EST database (GenBank Accession Number BT021261). The cDNA containing the
entire open reading frame (ORF) for nvd-Dm was amplified by PCR from
wild-type Drosophila ring gland-derived cDNA using the following
primers: forward, 5 '-ATGACGAGCTACAGTTTATTTTGGATGTC-3'; reverse, 5
'-CTACCAACCAATATTGGTTGCTTCAG-3 '. The DNA sequence data of
nvd-Bm and nvd-Dm were deposited in GenBank (AB232986 and
AB232987, respectively).
Reverse transcription (RT)-PCR
For analyzing tissue expression pattern of nvd-Bm, total RNA was
derived from tissue of second-day wandering (W1) fifth instar larvae of
Bombyx as described (Niwa et al.,
2004). Specific primers for nvd-Bm (forward, 5
'-AGATGGCGCTCCTTACTTCTG-3'; reverse, 5
'-TCAGACACTTGGTCACTCCATC-3') were used. The expression level of
nvd-Dm was examined by RT-PCR using the following specific primers:
forward, 5 '-CGAGCTACAGTTTATTTTGGATGTCATTGC-3'; reverse, 5
'-GGGCATATAACACAGTCGTCAGC-3 '. Quantitative RT-PCR was performed
as described (Niwa et al.,
2005). rpL3 and rp49 were used as loading
controls for nvd-Bm and nvd-Dm, respectively
(Foley et al., 1993;
Matsuoka and Fujiwara,
2000).
Northern and in situ hybridization
Digoxigenin (DIG)-labeled RNA probes were synthesized using the DIG RNA
labeling kit (Roche) and T3 or T7 RNA polymerase (Invitrogen). For the
nvd-Bm and nvd-Dm probes, prgv0382 and a pBluescript
containing nvd-Dm (nvd-Dm-pBluescript), respectively, were
used as templates. Northern blot and hybridization using a DIG-labeled probe
was performed as described (Charles et al.,
1999). In situ hybridization was performed as described
(Lehmann and Tautz, 1994;
Buszczak et al., 1999;
Niwa et al., 2004).
UAS vector construction and generation of transgenic strains
Overexpression studies and RNAi experiments using hairpin double-stranded
RNAs was performed using GAL4/UAS system
(Brand and Perrimon, 1993). The
construct for nvd-Dm overexpression was generated by ligation of a
BamHI/XhoI fragment isolated from
nvd-Dm-pBluescript into the BglII/XhoI site of
pUAST vector (Brand and Perrimon,
1993). To carry out a transgenic RNAi
(Kennerdell and Carthew,
2000), we generated two distinct UAS-nvd-Dm-Inverted
Repeat constructs, designated UAS-nvd-Dm-IR-1 and
UAS-nvd-Dm-IR-2. UAS-nvd-Dm-IR-1 was a genomic-cDNA fusion construct
(Kalidas and Smith, 2002)
comprising the 488-1131 bp region of the nvd-Dm gene. This genomic
fragment contained a part of the fourth exon downstream of an intrinsic
BspHI site, the entire fourth intron and a NotI site at the
3 ' end (see Fig. S2 in the supplementary materials). These fragments
amplified by PCR were ligated into pUAST. To make UAS-nvd-Dm-IR-2 (a
cDNA-cDNA RNAi construct), the 77-757 bp region was amplified and cloned into
pUAST as a tail-to-tail inverted repeat (see Fig. S2 in the supplementary
material). The Drosophila transformants were obtained using standard
protocols.
Phenotypic characterization of nvd-Dm RNAi animals
To assess the phenotype of nvd-Dm RNAi individuals, yw;
UAS-nvd-Dm-IR males of each RNAi strain were crossed with yw;
2-286-GAL4/TM3[y+] females and 0-12 hours after egg laying (AEL)
eggs were collected. The animals were reared on agar-apple juice plates with
yeast paste. nvd-Dm RNAi progeny carrying both UAS and
GAL4 transgenes (yw; UAS-nvd-Dm-IR/+; 2-286-GAL4/+) were
distinguished from the others not carrying either UAS or
GAL4 by the yellowish color of their mouth hooks and denticle belts,
because all of UAS and GAL4 strains were balanced by
balancers with y+ marker. Progeny carrying either
UAS or GAL4 transgene alone were used as a control. Control
and nvd-Dm RNAi larvae were transferred to fresh plates 36±6
hours AEL. After 48 hours AEL, we checked the lethality and phenotype of
larvae every 24 hours. Larvae were staged by the morphology of the mouth hook
as described (Roberts and Standen,
1998).
Ecdysteroid titer measurements
Control and nvd-Dm RNAi larvae were collected 30-42 hours AEL and
stored in 1.5 ml tubes that were weighed before and after the addition of
larvae in order to determine the weight of the larvae. Sample preparation for
ecdysteroid titer measurement was done as described
(Bialecki et al., 2002). The
amounts of ecdysone were measured using the ecdysteroid radioimmunoassay (RIA)
as described (Mizoguchi et al.,
2001). As 20-hydroxyecdysone (20E, Sigma) was used as the
standard, the ecdysteroid amount was expressed in 20E equivalents.
Ecdysteroid feeding experiments
Control and nvd RNAi larvae were collected 30±6 hours AEL
and placed on agar-apple juice plates. These larvae were fed on yeast paste
with a final concentration of 1 mg/ml or zero 20E in 3.3% ethanol 30-42, 54-66
and 72 hours AEL in order to stimulate the hormone pulse that triggers the
molt (Bialecki et al., 2002;
Warren et al., 2006).
Lethality was scored at 12-hour intervals. Cholesterol (C) and
7-dehydrocholesterol (7dC) were generous gifts from Y. Fujimoto. Control and
nvd-Dm RNAi larvae were also fed on yeast paste containing 0.5% wet
weight of C and 7dC (50 mg dry yeast, 95 µl water, 5 µl of 100% ethanol
and 0.75 mg of each ecdysteroid intermediate)
(Warren et al., 2001) from 30
hours AEL until all animals had emerged or died. The experiment using 7dC was
carried out under constant dark conditions because 7dC is unstable in
light.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). We have compared gene expression profiles of the PGs
from first day of fifth instar (V0) larvae, in which ecdysone is not produced,
with that of second-day of wandering (W1) larvae, in which ecdysone is
actively synthesized. The microarray data have yielded 1883 out of 5760
nonredundant cDNA clones showing at least a twofold increased expression
(Niwa et al., 2004). Of these
clones, prgv0382 (hereafter referred to as nvd-Bm) showed a 4.7-fold
higher expression in the PGs of second-day wandering (W1) larvae than those of
first-day fifth instar (V0) larvae. Consistent with the microarray data,
Northern blot analysis revealed that a change in mRNA expression level of
nvd-Bm was positively correlated with the changes of hemolymph
ecdysteroid titers during Bombyx development
(Fig. 1A)
(Satake et al., 1998;
Mizoguchi et al., 2001).
Furthermore, expression of nvd-Bm in various tissues of the W1
Bombyx larvae was examined by RT-PCR. nvd-Bm was expressed
predominantly in the PG, while weak nvd-Bm was also detected in brain
and malpighian tubules (Fig.
1B). These results suggest that nvd-Bm is involved in the
temporal regulation of ecdysteroid biosynthesis in the PG.
|
). The putative
nvd family of proteins also have a highly conserved domain in the
C-terminal region that contains a mononuclear, non-heme iron-binding motif
(E/D-X3-D-X2-H-X4-H), which is thought to be
involved in oxygen binding (Mason and
Cammack, 1992). The primary structure of the Nvd proteins, which
has both the Rieske domain and the non-heme iron-binding motif, is typically
conserved in class IA terminal oxygenases from prokaryotes
(Jiang et al., 1996), such as
KshA (3-ketosteroid 9 -hydroxylase) of Rhodococcus
erythropolis (van der Geize et al.,
2002) and PrnD (aminopyrrolnitrin dioxygenases) of Pseudomonas
fluorescens (Kirner et al.,
1998). In the case of Nvd proteins from Bombyx, Drosophila, C.
elegans and zebrafish, putative transmembrane regions are predicted in
their N-terminal regions (Fig.
2A). Only one homolog existed in the genome of each of these
eukaryotic species. We could not find nvd orthologs from mammalian or
plant genomes using a standard BLAST search.
The Drosophila homolog of neverland (nvd-Dm) was
not originally annotated in the Drosophila euchromatin genome
database. Our BLAST search revealed that nvd-Dm is composed of six
exons distributed over 76 kb and located on the third chromosome
heterochromatin scaffold (Hoskins et al.,
2002). The 5 ' region of nvd-Dm overlapped with a
predicted gene designated CG40050 that might have been mis-annnotated
in FlyBase (see Fig. S3 in the supplementary materials).
Drosophila neverland is expressed in tissues that synthesize steroid hormones
To analyze the expression pattern of nvd-Dm during
Drosophila development, in situ RNA hybridizations were carried out
on embryos, larvae and adult ovaries. We detected no nvd-Dm mRNA in
unfertilized eggs, suggesting that there is no maternal contribution of
nvd-Dm (Fig. 3A), as
was previously noted for phm, dib and sad
(Chávez et al., 2000;
Warren et al., 2002;
Niwa et al., 2004;
Warren et al., 2004).
Moreover, no nvd-Dm expression was detected at the blastoderm,
gastrulation, germ band elongation and retraction stages
(Fig. 3A,B; see Fig. S4 in the
supplementary materials). nvd-Dm expression was first seen at stage
14 in the primordia of the ring gland, which contains Drosophila PG
cells (see Fig. S4 in the supplementary material). nvd-Dm expression
in the ring gland became higher at stage 15, and continued through the
remainder of embryogenesis (Fig.
3C). nvd-Dm mRNA was also expressed specifically in the
ring gland at the larval stage (Fig.
3D,E). In the ring gland, this expression was exclusively observed
in the PG, but not in the corpus allatum or corpus cardiacum cells
(Fig. 3D,D').
nvd-Dm expression in the PG was downregulated after ecdysis from the
second instar to the third instar, and then was significantly upregulated at
the late third instar larval stage (Fig.
3F). These results suggest that the transcriptional activity of
nvd correlates with ecdysone titer changes during the larval molting
cycle, similar to that of phm, dib, sad and Start1
(Drosophila homolog of StAR)
(Roth et al., 2004;
Gilbert and Warren, 2005).
In adult females, nvd-Dm mRNA was expressed in the nurse cells of
developing egg chambers (Fig.
3G-I). The nurse cells are considered to be the source of
ecdysteroids in adult females (Riddiford,
1993; Gilbert et al.,
2002) and a number of ecdysteroidogenic genes, such as dare,
Start1 and ecdysoneless, are known to be expressed in the nurse
cells (Freeman et al., 1999;
Gaziova et al., 2004;
Roth et al., 2004). These
results indicate that the expression of nvd-Dm is enriched in tissues
that synthesize steroid hormones.
RNAi of Drosophila neverland in the PG causes larval arrest
To assess the importance of nvd-Dm during development, we examined
phenotypes of loss- or gain-of-nvd-Dm function in developing flies.
As nvd-Dm is located in heterochromatin region (see Fig. S3 in the
supplementary materials), it is difficult to isolate or create genetic
mutations within the nvd-Dm locus by genetic mutant screens or
homologous recombination techniques. We therefore examined the effects of
overexpression or knock down of nvd-Dm in developing flies using the
GAL4/UAS system (Brand and Perrimon,
1993). In a wild-type background, overexpression of
nvd-Dm using any of GAL4 drivers in
Table 1 had no visible effect
on development (data not shown). To knock down nvd-Dm in developing
flies, we used transgenic RNA interference (RNAi), known to be an effective
method of degrading endogenous target mRNAs in Drosophila
(Kennerdell and Carthew, 2000;
Kalidas and Smith, 2002). We
established transgenic lines in which double-stranded RNA molecules
corresponding to nvd-Dm mRNA were generated using an inverted repeat
construct under the control of the UAS promoter
(UAS-nvd-Dm-IR; see Fig. S2 in the supplementary materials). We found
that all of the RNAi animals failed to develop into adults when the
UAS lines were crossed with two GAL4 lines,
2-286-GAL4 and pGawB5015, in which GAL4
transgenes are active in the PG cells
(Table 1). By contrast, the
progeny resulting from crosses between UAS-nvd-Dm-IR flies and
GAL4 lines, in which the GAL4 transgenes are not active in
the PG, were fully viable (Table
1), consistent with the expression pattern of nvd-Dm in
wild-type animals. This lethal phenotype by PG-GAL4 strains was
observed using two distinct nvd-Dm RNAi constructs
(UAS-nvd-Dm-IR-1 and UAS-nvd-Dm-IR-2), which target
different regions of nvd-Dm mRNA
(Table 1; see Fig. S2 in the
supplementary material). The reduction of nvd-Dm mRNA level in the
RNAi larvae was confirmed by RT-PCR (Fig.
4A). These results suggest that nvd-Dm plays an essential
role in the PG during fly development.
|
|
48 hours after egg laying
(AEL), nvd-Dm RNAi animals showed apparent growth arrest in body size
compared with control animals (Fig.
4E-G). To examine whether larval molting occurs in nvd-Dm
RNAi animals, the larval stages were determined by stage-specific size and
morphology of mouth hook (Roberts and
Standen, 1998). By 48 hours AEL, both the control and RNAi animals
had the small, one-toothed mouth hook that is characteristic of the first
instar larvae (Fig. 4H,I,M,N).
Forty-eight hours AEL, the control animals underwent the first molt and showed
the second instar larva-type mouth hook with two to five teeth
(Fig. 4O,P). After 72 hours
AEL, the control animals molted to the third instar larvae and possessed
larger mouth hooks with numerous small teeth
(Fig. 4Q). By contrast,
nvd-Dm RNAi animals still retained the mouth hook of the first instar
larva-type, even 96 hours AEL (Fig.
4J-L). At around 72 hours AEL, most of the nvd-Dm RNAi
animals gradually ceased feeding and became stationary. Ninety-eight percent
(54/55) of these animals died before 108 hours AEL as first instar larva, as
judged from the mouth-hook morphology (Fig.
4B, Fig. 6I). We
noted that all of the nvd-Dm RNAi animals failed to show the
double-mouth-hook phenotype that frequently occurs in mutants with defects in
ecdysone signaling (Li and Bender,
2000; Gaziova et al.,
2004), implying that loss of nvd-Dm function prevents
larval growth prior to the initiation of the molting process. These results
suggest that nvd-Dm plays a pivotal role in larval development in
Drosophila. On this basis of the prolonged first instar larval
phenotype, we named this gene `neverland', which is the fictional
island featured in J. M. Barrie's play Peter Pan, where children
cease to age.
|
;
Sullivan and Thummel, 2003;
Warren et al., 2006). Organic
extracts prepared from nvd-Dm RNAi animals had a significantly
reduced level of ecdysteroid compared with extracts from control animals
(Fig. 5), indicating that
nvd-Dm activity is required for ecdysteroid biosynthesis in PG. To address whether the larval arrest of nvd-Dm RNAi animals is due to reduced ecdysteroid titers, we attempted to rescue the larval arrest phenotype by feeding ecdysteroids to nvd-Dm RNAi animals. When newly-hatched nvd-Dm RNAi animals were fed yeast paste supplemented with active ecdyseroid (20E) 30-42 hours AEL in the first instar period, the arrest phenotype was rescued and the animals grew to the second instar larvae. However, the animals later died as prolonged second instar larvae (Fig. 6A,B,I). Similarly, when the rescued second instar larvae were fed the 20E-containing food during 54-66 hours AEL in the second instar period, they molted into the third instar larvae but failed to become pupae (Fig. 6C,D,I). Furthermore, when the rescued third instar animals received food with 20E after 78 hours AEL, these RNAi animals were partially rescued and developed into pupae. A number of the pupae completed metamorphosis and emerged as fertile adults (Fig. 6E,I), although almost all died within one week of eclosion. Like the nvd-Dm animals without 20E, second and third instar larvae fed with 20E for 12 hours during each instar also showed the prolonged larval phenotype, surviving for 3-4 days after each molting. In spite of the prolonged phenotype, both the rescued second and third instar larvae did not overgrow, and their body sizes were almost the same as mature second and third instar wild-type larvae, respectively (Fig. 6A,C). We confirmed that PG cells looked normal in the prolonged nvd-Dm RNAi larvae (see Fig. S5 in the supplementary materials), indicating that the nvd-Dm RNAi phenotype is not due to loss of PG cells. Taken together, these results suggest that nvd-Dm is essential for insect molting, metamorphosis and body growth throughout development via the regulation of ecdysteroid biosynthesis in the PG.
|
|
). C and 7dC are involved in the first step of
ecdysteroidogenesis in the PG; 7dC is synthesized from C by
7,8-dehydrogenation by an uncharacterized enzyme
(Grieneisen et al., 1993;
Gilbert et al., 2002;
Gilbert and Warren, 2005).
When 7dC was added to the food, all nvd-Dm RNAi animals were
completely rescued and developed into adults
(Fig. 6F,I). The food with 7dC
also restored the ecdysone titer in nvd-Dm RNAi animals
(Fig. 5), suggesting that the
nvd-Dm mutants do not have a defect in the conversion steps from 7dC
to ecdysone in its PG. However, nvd-Dm RNAi animals raised on food
containing excessive C were partially rescued; about 47% (18/38) of these
animals molted to the second instar larvae
(Fig. 6G,H,I), but no
nvd-Dm RNAi animal was observed to molt to the third instar larvae.
It should be noted that although the nvd-Dm RNAi larvae rescued by C
arrested in the second instar larvae, they survived for about 3 weeks and
their body size reached almost the same size as wild-type third instar larvae
(Fig. 6G). These results
strongly suggest that the Nvd proteins act upstream of 7dC production in the
ecdysteroid synthesis pathway in the PG. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
),
Dare (Freeman et al., 1999)
and Start1 (Roth et al.,
2004).
|
; Moróy et
al., 1988; Sullivan and
Thummel, 2003). The expression levels of the Halloween genes are
strongly correlated with embryonic ecdysteroid titers, and mutations in any of
these genes cause ecdysteroid deficiency in the embryo
(Gilbert and Warren, 2005;
Namiki et al., 2005). By
contrast, nvd-Dm expression is not detected at this early embryonic
stage, suggesting that nvd-Dm is not necessary to regulate ecdysone
production during embryogenesis. Although it is known that maternally
synthesized ecdysteroids are provided in Drosophila eggs
(Bownes et al., 1988), it has
been unclear what type of ecdysteroid precursor is required for ecdysone
production during embryogenesis
(Chávez et al., 2000).
Identifying the exact steps in which Nvd is involved, as discussed below,
would shed light on how ecdysone synthesis varies across different
developmental stages.
Molecular functions of the neverland family of proteins
The Nvd proteins have strong similarities to class IA oxygenases, which
have both a consensus [2Fe-2S] Rieske-type domain and a domain that is highly
similar to the proposed mononuclear non-heme Fe (II) binding motif
(Mason and Cammack, 1992).
Among the enzymes in this class, the protein encoded by Rhodococcus
kshA functions in steroid hydroxylation
(van der Geize et al., 2002),
raising the possibility that the Nvd proteins could catalyze oxidation or
hydroxylation reactions in the ecdysteroid biosynthesis pathway.
In which step of ecdysteroid biosynthesis does Nvd function? Considering
the results of our rescue experiments with cholesterol (C) and
7-dehydrocholesterol (7dC), it is likely that the Nvd protein might play a
role in transport and/or metabolism of cholesterol and/or its derivatives.
This idea is also supported by the recently reported phenotype of
Drosophila NPC1 mutants (Fluegel
et al., 2005; Huang et al.,
2005). Drosophila NPC1 is an ortholog of mammalian
Niemann-Pick type C disease genes that are well known to encode
cholesterol-binding proteins (Chang et al.,
2005). NPC1 mutants show a larval arrest phenotype
similar to that of the nvd-Dm RNAi animals discussed above, and this
phenotype is rescued to varying degrees by feeding with 20E, C or 7dC.
Our feeding experiments reveal that food containing 7dC completely rescues the nvd-Dm RNAi phenotype, indicating that Nvd acts upstream of 7dC synthesis. Given the fact that such complete rescue activity is not observed when food supplemented with excessive C was used, it is possible that Nvd might function in the conversion of C to 7dC. This hypothesis is consistent with the following points.
). ).
It is possible, however, that Nvd itself does not directly catalyze C to
7dC. Previous biochemical studies have shown that the conversion of C to 7dC
is mediated by an enzyme with cytochrome P450 characteristics under the
control of the Zn-finger protein Without Children (Woc)
(Grieneisen et al., 1993;
Warren et al., 1995;
Warren et al., 2001). Our
RT-PCR analysis did not detect a significant difference of nvd-Dm
expression level between woc mutant and wild type (see Fig. S6 in the
supplementary materials). We also tested whether the Nvd proteins can catalyze
cholesterol and cholesterol derivatives (22-hydroxycholesterol,
25-hydroxycholesterol and 7dC) using a S2 cell system previously described in
biochemical studies of Halloween P450s
(Warren et al., 2002;
Niwa et al., 2004). However,
no metabolites have yet been detected (data not shown).
Although the proposed function for Nvd on the conversion from C to 7dC is
most likely, we should point out that food containing excessive C partially
rescues the defect of nvd-Dm RNAi animals. This raises the
possibility that the partial rescue activity by C might be due to a
hypomorphic nature of the nvd-Dm RNAi. In nvd-Dm RNAi
animals, we detect a significantly reduced but specific level of
nvd-Dm mRNA (Fig. 4A)
and substantial 20E (Fig. 5),
suggesting that the nvd-Dm RNAi animals might still possess low
levels of Nvd activity and thus these animals may be able to produce small
amounts of 7dC from C. An alternative explanation is that Nvd would function
in the `black box' in which 7dC is converted to the
4-diketol by an uncharacterized mechanism, because 7dC is a
slightly unstable chemical and it is possible that the rescuing activity of
7dC might be caused by a contaminant oxidated in the food. In order to
determine the function of Nvd in more detail, isolation of genetic null
mutants of nvd and the biochemical properties of Nvd proteins will
need to be investigated.
The functional role of the Neverland proteins in other organisms
Our current studies do not address the functional role of the Nvd proteins
in organisms other than insects. Notably, a nvd ortholog is found in
the C. elegans genome. According to recent advances in taxonomy, both
insects and nematodes belong to Ecdysozoa, which are characterized by molting
(ecdysis) behavior (Aguinaldo et al.,
1997). In C. elegans, it has been suggested that
uncharacterized steroid hormones play a role in both molting and dauer
formation (Thummel, 2001;
Entchev and Kurzchalia, 2005).
Therefore, we postulate that Nvd may be important in controlling proper larval
development in nematode species. Indeed, during the revision of this paper, it
has been demonstrated that the daf-36 gene, which encodes the C.
elegans homolog of nvd, is required to bypass the C.
elegans dauer diapause (Rottiers et
al., 2006). It is thought that daf-36 mutants cannot
produce a steroid-like hormone that prevents dauer formation, suggesting that
nvd/daf-36 is also essential for a steroid-like synthesis pathway in
nematodes. daf-36 mutants are also shown to displays adult aging
phenotypes (Rottiers et al.,
2006). As reduced 20E in Drosophila is associated with
expanded lifespan (Tu et al.,
2002; Simon et al.,
2003; Colombani et al.,
2005), it would be intriguing to explore the role of nvd
on insect aging.
Interestingly, no ortholog of nvd has been found in genomes of mammalian species, while nvd orthologs are conserved in other chordates, such as ascidian, zebrafish and chicken. This suggests that nvd orthologs might have been lost during mammalian evolution. Currently, we do not know the exact differences of steroid biosynthesis pathways between mammals and other animals. Future studies on the nvd family could provide new insight into the similarities and differences between steroid hormone biosynthesis among animal clades.
Supplementary materialSupplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/13/2565/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Aguinaldo, A. M., Turbeville, J. M., Linford, L. S., Rivera, M.
C., Garey, J. R., Raff, R. A. and Lake, J. A. (1997).
Evidence for a clade of nematodes, arthropods and other moulting animals.
Nature 387,489
-493.[CrossRef][Medline]
Andrews, H. K., Zhang, Y. Q., Trotta, N. and Broadie, K.
(2002). Drosophila sec10 is required for hormone
secretion but not general exocytosis or neurotransmission.
Traffic 3,906
-921.[CrossRef][Medline]
Bialecki, M., Shilton, A., Fichtenberg, C., Segraves, W. A. and
Thummel, C. S. (2002). Loss of the ecdysteroid-inducible E75A
orphan nuclear receptor uncouples molting from metamorphosis in
Drosophila. Dev. Cell
3, 209-220.[CrossRef][Medline]
Bownes, M., Shirras, A., Blair, M., Collins, J. and Coulson,
A. (1988). Evidence that insect embryogenesis is regulated by
ecdysteroids released from yolk proteins. Proc. Natl. Acad. Sci.
USA 85,1554
-1557.
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.[Abstract]
Buszczak, M., Freeman, M. R., Carlson, J. R., Bender, M.,
Cooley, L. and Segraves, W. A. (1999). Ecdysone response
genes govern egg chamber development during mid-oogenesis in
Drosophila. Development
126,4581
-4589.[Abstract]
Caldwell, P. E., Walkiewicz, M. and Stern, M.
(2005). Ras activity in the Drosophila prothoracic gland
regulates body size and developmental rate via ecdysone release.
Curr. Biol. 15,1785
-1795.[CrossRef][Medline]
Chang, T. Y., Reid, P. C., Sugii, S., Ohgami, N., Cruz, J. C.
and Chang, C. C. (2005). Niemann-Pick type C disease and
intracellular cholesterol trafficking. J. Biol. Chem.
280,20917
-20920.
Charles, J. P., Shinoda, T. and Chinzei, Y.
(1999). Characterization and DNA-binding properties of GRF, a
novel monomeric binding orphan receptor related to GCNF and betaFTZ-F1.
Eur. J. Biochem. 266,181
-190.[Medline]
Chávez, V. M., Marques, G., Delbecque, J. P., Kobayashi,
K., Hollingsworth, M., Burr, J., Natzle, J. E. and O'Connor, M. B.
(2000). The Drosophila disembodied gene controls late
embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates
embryonic ecdysone levels. Development
127,4115
-4126.[Abstract]
Cherbas, L., Hu, X., Zhimulev, I., Belyaeva, E. and Cherbas,
P. (2003). EcR isoforms in Drosophila: testing
tissue-specific requirements by targeted blockade and rescue.
Development 130,271
-284.
Chitwood, D. J. (1999). Biochemistry and
function of nematode steroids. Crit. Rev. Biochem. Mol.
Biol. 34,273
-284.[CrossRef][Medline]
Colombani, J., Bianchini, L., Layalle, S., Pondeville, E.,
Dauphin-Villemant, C., Antoniewski, C., Carre, C., Noselli, S. and Leopold,
P. (2005). Antagonistic actions of ecdysone and insulins
determine final size in Drosophila. Science
310,667
-670.
Entchev, E. V. and Kurzchalia, T. V. (2005).
Requirement of sterols in the life cycle of the nematode Caenorhabditis
elegans. Semin. Cell Dev. Biol.
16,175
-182.[CrossRef][Medline]
Fluegel, M. L., Parker, T. J. and Pallanck, L. J.
(2005). Mutations of a Drosophila NPC1 gene confer
sterol and ecdysone metabolic defects. Genetics
172,185
-196.
Foley, K. P., Leonard, M. W. and Engel, J. D.
(1993). Quantitation of RNA using the polymerase chain reaction.
Trends Genet. 9,380
-385.[CrossRef][Medline]
Freeman, M. R., Dobritsa, A., Gaines, P., Segraves, W. A. and
Carlson, J. R. (1999). The dare gene: steroid
hormone production, olfactory behavior, and neural degeneration in
Drosophila. Development
126,4591
-4602.[Abstract]
Gaziova, I., Bonnette, P. C., Henrich, V. C. and Jindra, M.
(2004). Cell-autonomous roles of the ecdysoneless gene
in Drosophila development and oogenesis.
Development 131,2715
-2725.
Gilbert, L. I. and Warren, J. T. (2005). A
molecular genetic approach to the biosynthesis of the insect steroid molting
hormone. Vitam. Horm.
73, 31-57.[Medline]
Gilbert, L. I., Rybczynski, R. and Warren, J. T.
(2002). Control and biochemical nature of the ecdysteroidogenic
pathway. Annu. Rev. Entomol.
47,883
-916.[CrossRef][Medline]
Grieneisen, M. L., Warren, J. T. and Gilbert, L. I.
(1993). Early steps in ecdysteroid biosynthesis: evidence for the
involvement of cytochrome P-450 enzymes. Insect Biochem. Mol.
Biol. 23,13
-23.[CrossRef][Medline]
Hoskins, R. A., Smith, C. D., Carlson, J. W., Carvalho, A. B.,
Halpern, A., Kaminker, J. S., Kennedy, C., Mungall, C. J., Sullivan, B. A.,
Sutton, G. G. et al. (2002). Heterochromatic sequences in a
Drosophila whole-genome shotgun assembly. Genome
Biol. 3,RESEARCH0085
.[Medline]
Huang, X., Suyama, K., Buchanan, J., Zhu, A. J. and Scott, M.
P. (2005). A Drosophila model of the Niemann-Pick
type C lysosome storage disease: dnpc1a is required for molting and
sterol homeostasis. Development
132,5115
-5124.
Jiang, H., Parales, R. E., Lynch, N. A. and Gibson, D. T.
(1996). Site-directed mutagenesis of conserved amino acids in the
alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron
coordination sites. J. Bacteriol.
178,3133
-3139.
Kalidas, S. and Smith, D. P. (2002). Novel
genomic cDNA hybrids produce effective RNA interference in adult
Drosophila. Neuron
33,177
-184.[CrossRef][Medline]
Kennerdell, J. R. and Carthew, R. W. (2000).
Heritable gene silencing in Drosophila using double-stranded RNA.
Nat. Biotechnol. 18,896
-898.[CrossRef][Medline]
Kirner, S., Hammer, P. E., Hill, D. S., Altmann, A., Fischer,
I., Weislo, L. J., Lanahan, M., van Pee, K. H. and Ligon, J. M.
(1998). Functions encoded by pyrrolnitrin biosynthetic genes from
Pseudomonas fluorescens. J. Bacteriol.
180,1939
-1943.
Klaes, A., Menne, T., Stollewerk, A., Scholz, H. and Klambt,
C. (1994). The Ets transcription factors encoded by the
Drosophila gene pointed direct glial cell differentiation in
the embryonic CNS. Cell
78,149
-160.[CrossRef][Medline]
Kraminsky, G. P., Clark, W. C., Estelle, M. A., Gietz, R. D.,
Sage, B. A., O'Connor, J. D. and Hodgetts, R. B. (1980).
Induction of translatable mRNA for dopa decarboxylase in Drosophila:
an early response to ecdysterone. Proc. Natl. Acad. Sci.
USA 77,4175
-4179.
Lee, G. and Park, J. H. (2004). Hemolymph sugar
homeostasis and starvation-induced hyperactivity affected by genetic
manipulations of the adipokinetic hormone-encoding gene in Drosophila
melanogaster. Genetics
167,311
-323.
Lehmann, R. and Tautz, D. (1994). In
situ hybridization to RNA. Methods Cell Biol.
44,575
-598.[Medline]
Li, T. and Bender, M. (2000). A conditional
rescue system reveals essential functions for the ecdysone receptor
(EcR) gene during molting and metamorphosis in Drosophila.
Development 127,2897
-2905.[Abstract]
Link, T. A. (1999). The structures of Rieske
and Rieske-type proteins. Adv. Inorg. Chem.
47, 83-157.
Luo, L., Liao, Y. J., Jan, L. Y. and Jan, Y. N.
(1994). Distinct morphogenetic functions of similar small
GTPases: Drosophila Drac1 is involved in axonal outgrowth and
myoblast fusion. Genes Dev.
8,1787
-1802.
Mason, J. R. and Cammack, R. (1992). The
electron-transport proteins of hydroxylating bacterial dioxygenases.
Annu. Rev. Microbiol.
46,277
-305.[CrossRef][Medline]
Matsuoka, T. and Fujiwara, H. (2000).
Expression of ecdysteroid-regulated genes is reduced specifically in the wing
discs of the wing-deficient mutant (fl) of Bombyx mori.
Dev. Genes Evol. 210,120
-128.[CrossRef][Medline]
Mirth, C., Truman, J. W. and Riddiford, L. M.
(2005). The role of the prothoracic gland in determining critical
weight for metamorphosis in Drosophila melanogaster. Curr.
Biol. 15,1796
-1807.[CrossRef][Medline]
Mita, K., Morimyo, M., Okano, K., Koike, Y., Nohata, J.,
Kawasaki, H., Kadono-Okuda, K., Yamamoto, K., Suzuki, M. G., Shimada, T. et
al. (2003). The construction of an EST database for
Bombyx mori and its application. Proc. Natl. Acad. Sci.
USA 100,14121
-14126.
Mizoguchi, A., Ohashi, Y., Hosoda, K., Ishibashi, J. and
Kataoka, H. (2001). Developmental profile of the changes in
the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx
mori: correlation with ecdysteroid secretion. Insect Biochem.
Mol. Biol. 31,349
-358.[CrossRef][Medline]
Moróy, P., Kaufmann, G. and Dübendorfer, A.
(1988). Embryonic ecdysteroids of Drosophila
melanogaster. J. Insect Physiol.
34,633
-637.[CrossRef]
Namiki, T., Niwa, R., Sakudoh, T., Shirai, K., Takeuchi, H. and
Kataoka, H. (2005). Cytochrome P450 CYP307A1/Spook: a
regulator for ecdysone synthesis in insects. Biochem. Biophys. Res.
Commun. 337,367
-374.[Medline]
Neubueser, D., Warren, J. T., Gilbert, L. I. and Cohen, S.
M. (2005). molting defective is required for
ecdysone biosynthesis. Dev. Biol.
280,362
-372.[CrossRef][Medline]
Niwa, R., Matsuda, T., Yoshiyama, T., Namiki, T., Mita, K.,
Fujimoto, Y. and Kataoka, H. (2004). CYP306A1, a cytochrome
P450 enzyme, is essential for ecdysteroid biosynthesis in the prothoracic
glands of Bombyx and Drosophila. J. Biol.
Chem. 279,35942
-35949.
Niwa, R., Sakudoh, T., Namiki, T., Saida, K., Fujimoto, Y. and
Kataoka, H. (2005). The ecdysteroidogenic P450
Cyp302a1/disembodied from the silkworm, Bombyx mori, is
transcriptionally regulated by prothoracicotropic hormone. Insect
Mol. Biol. 14,563
-571.[Medline]
Parvy, J. P., Blais, C., Bernard, F., Warren, J. T., Petryk, A.,
Gilbert, L. I., O'Connor, M. B. and Dauphin-Villemant, C.
(2005). A role for ßFTZ-F1 in regulating ecdysteroid titers
during post-embryonic development in Drosophila melanogaster.
Dev. Biol. 282,84
-94.[CrossRef][Medline]
Petryk, A., Warren, J. T., Marques, G., Jarcho, M. P., Gilbert,
L. I., Kahler, J., Parvy, J. P., Li, Y., Dauphin-Villemant, C. and O'Connor,
M. B. (2003). Shade is the Drosophila P450 enzyme
that mediates the hydroxylation of ecdysone to the steroid insect molting
hormone 20-hydroxyecdysone. Proc. Natl. Acad. Sci. USA
100,13773
-13778.
Rewitz, K. F., Rybczynski, R., Warren, J. T. and Gilbert, L.
I. (2006). Identification, characterization and developmental
expression of Halloween genes encoding P450 enzymes mediating ecdysone
biosynthesis in the tobacco hornworm, Manduca sexta.
Insect Biochem. Mol. Biol.
36,188
-199.[Medline]
Riddiford, L. M. (1993). Hormones and
Drosophila development. In The Development of Drosophila
melanogaster (ed. M. Bate and A. Martinez-Arias), pp.899
-939. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press.
Roberts, D. B. and Standen, G. N. (1998). The
elements of Drosophila biology and genetics. In
Drosophila: A Practical Approach (ed. D. B. Roberts),
pp. 1-54. Oxford: Oxford University
Press.
Roth, G. E., Gierl, M. S., Vollborn, L., Meise, M., Lintermann,
R. and Korge, G. (2004). The Drosophila gene
Start1: a putative cholesterol transporter and key regulator of
ecdysteroid synthesis. Proc. Natl. Acad. Sci. USA
101,1601
-1606.
Rottiers, V., Motola, D. L., Gerisch, B., Cummins, C. L.,
Nishiwaki, K., Mangelsdorf, D. J. and Antebi, A. (2006).
Hormonal control of C. elegans dauer formation and life span by a
Rieske-like oxygenase. Dev. Cell
10,473
-482.[Medline]
Satake, S., Kaya, M. and Sakurai, S. (1998).
Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in
the last-larval stadium of the silkworm, Bombyx mori: role of low
ecdysteroid titer in larval-pupal metamorphosis and a reappraisal of the head
critical period. J. Insect Physiol.
44,867
-881.[CrossRef][Medline]
Shiga, Y., Tanaka-Matakatsu, M. and Hatashi, S.
(1996). A nuclear GFP/ß-galactosidase fusion protein as a
marker for morphogenesis in living Drosophila. Dev. Growth
Diff. 38,99
-106.
Siegmund, T. and Korge, G. (2001). Innervation
of the ring gland of Drosophila melanogaster. J. Comp.
Neurol. 431,481
-491.[CrossRef][Medline]
Simon, A. F., Shih, C., Mack, A. and Benzer, S.
(2003). Steroid control of longevity in Drosophila
melanogaster. Science
299,1407
-1410.
Sinenko, S. A., Kim, E. K., Wynn, R., Manfruelli, P., Ando, I.,
Wharton, K. A., Perrimon, N. and Mathey-Prevot, B. (2004).
Yantar, a conserved arginine-rich protein is involved in Drosophila
hemocyte development. Dev. Biol.
273, 48-62.[CrossRef][Medline]
Staehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A.
H. and Hoffmann, F. M. (1994). Specificity of bone
morphogenetic protein-related factors: cell fate and gene expression changes
in Drosophila embryos induced by decapentaplegic but not
60A. Cell Growth Differ.
5, 585-593.[Abstract]
Sullivan, A. A. and Thummel, C. S. (2003).
Temporal profiles of nuclear receptor gene expression reveal coordinate
transcriptional responses during Drosophila development.
Mol. Endocrinol. 17,2125
-2137.
Thummel, C. S. (2001). Molecular mechanisms of
developmental timing in C. elegans and Drosophila.
Dev. Cell 1,453
-465.[CrossRef][Medline]
Thummel, C. S. and Chory, J. (2002). Steroid
signaling in plants and insects - common themes, different pathways.
Genes Dev. 16,3113
-3129.
Timmons, L., Becker, J., Barthmaier, P., Fyrberg, C., Shearn, A.
and Fyrberg, E. (1997). Green fluorescent
protein/ß-galactosidase double reporters for visualizing
Drosophila gene expression patterns. Dev.
Genet. 20,338
-347.[CrossRef][Medline]
Tu, M. P., Yin, C. M. and Tatar, M. (2002).
Impaired ovarian ecdysone synthesis of Drosophila melanogaster
insulin receptor mutants. Aging Cell
1, 158-160.[CrossRef][Medline]
van der Geize, R., Hessels, G. I., van Gerwen, R., van der
Meijden, P. and Dijkhuizen, L. (2002). Molecular and
functional characterization of kshA and kshB, encoding two
components of 3-ketosteroid 9 -hydroxylase, a class IA monooxygenase,
in Rhodococcus erythropolis strain SQ1. Mol.
Microbiol. 45,1007
-1018.[CrossRef][Medline]
Venkatesh, K. and Hasan, G. (1997). Disruption
of the IP3 receptor gene of Drosophila affects larval metamorphosis
and ecdysone release. Curr. Biol.
7, 500-509.[CrossRef][Medline]
Venkatesh, K., Siddhartha, G., Joshi, R., Patel, S. and Hasan,
G. (2001). Interactions between the inositol
1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting
in Drosophila. Genetics
158,309
-318.
Warren, J. T., Rybczynski, R. and Gilbert, L. I.
(1995). Stereospecific, mechanism-based, suicide inhibition of a
cytochrome P450 involved in ecdysteroid biosynthesis in the prothoracic glands
of Manduca sexta. Insect Biochem. Mol. Biol.
25,679
-695.[Medline]
Warren, J. T., Bachmann, J. S., Dai, J. D. and Gilbert, L.
I. (1996). Differential incorporation of cholesterol and
cholesterol derivatives into ecdysteroids by the larval ring glands and adult
ovaries of Drosophila melanogaster: a putative explanation for the
l(3)ecd1 mutation. Insect Biochem. Mol. Biol.
26,931
-943.[CrossRef][Medline]
Warren, J. T., Wismar, J., Subrahmanyam, B. and Gilbert, L.
I. (2001). Woc (without children) gene
control of ecdysone biosynthesis in Drosophila melanogaster.
Mol. Cell. Endocrinol.
181, 1-14.[CrossRef][Medline]
Warren, J. T., Petryk, A., Marques, G., Jarcho, M., Parvy, J.
P., Dauphin-Villemant, C., O'Connor, M. B. and Gilbert, L. I.
(2002). Molecular and biochemical characterization of two P450
enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster.
Proc. Natl. Acad. Sci. USA
99,11043
-11048.
Warren, J. T., Petryk, A., Marques, G., Parvy, J. P., Shinoda,
T., Itoyama, K., Kobayashi, J., Jarcho, M., Li, Y., O'Connor, M. B. et al.
(2004). Phantom encodes the 25-hydroxylase of
Drosophila melanogaster and Bombyx mori: a P450 enzyme
critical in ecdysone biosynthesis. Insect Biochem. Mol.
Biol. 34,991
-1010.[CrossRef][Medline]
Warren, J. T., Yerushalmi, Y., Shimell, M. J., O'Connor, M. B.,
Restifo, L. L. and Gilbert, L. I. (2006). Discrete pulses of
molting hormone, 20-hydroxyecdysone, during late larval development of
Drosophila melanogaster: correlations with changes in gene activity.
Dev. Dyn. 235,315
-326.[CrossRef][Medline]
Wismar, J., Habtemichael, N., Warren, J. T., Dai, J. D.,
Gilbert, L. I. and Gateff, E. (2000). The mutation
without children(rgl) causes ecdysteroid deficiency in
third-instar larvae of Drosophila melanogaster. Dev.
Biol. 226,1
-17.[CrossRef][Medline]
Yamanaka, N., Hua, Y. J., Mizoguchi, A., Watanabe, K., Niwa, R.,
Tanaka, Y. and Kataoka, H. (2005). Identification of a novel
prothoracicostatic hormone and its receptor in the silkworm Bombyx
mori. J. Biol. Chem.
280,14684
-14690.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  N. Fielenbach and A. Antebi C. elegans dauer formation and the molecular basis of plasticity Genes & Dev., August 15, 2008; 22(16): 2149 - 2165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Talamillo, J. Sanchez, R. Cantera, C. Perez, D. Martin, E. Caminero, and R. Barrio Smt3 is required for Drosophila melanogaster metamorphosis Development, May 1, 2008; 135(9): 1659 - 1668. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
