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First published online 26 March 2008
doi: 10.1242/dev.020685
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1 Functional Genomics Unit, CIC bioGUNE, Technology Park, Building 801-A, 48160
DERIO, Bizkaia, Spain.
2 Zoology Department, Stockholm University, 10691 Stockholm, Sweden.
3 Instituto de Investigaciones Biológicas Clemente Estable, Av. Italia,
3318 Montevideo, Uruguay.
4 Institut de Biologia Molecular de Barcelona, CSIC, J. Girona 18-26, 08034
Barcelona, Spain.
5 Centro de Biología Molecular Severo Ochoa, Universidad Autónoma
de Madrid, 28049 Madrid, Spain.
Author for correspondence (e-mail:
rbarrio{at}cicbiogune.es)
Accepted 4 March 2008
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Drosophila, Ecdysone, Metamorphosis, Ring gland, Smt3, Sumoylation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gill, 2005;
Heun, 2007;
Ulrich, 2005;
Verger et al., 2003).
Consequently, it affects diverse cellular processes, such as cell cycle, DNA
repair, nuclear body formation, nucleocytoplasmic transport, protein turnover
and maintenance of genomic and nuclear integrity. Although a large number of
proteins are known to be substrates for SUMO, for most of them the biological
function of sumoylation remains to be elucidated.
In yeast, insects and nematodes there is a single SUMO gene (smt3
in Drosophila), whereas in mammals three members have been identified
(Johnson et al., 1997). The
conjugation of SUMO to target proteins involves four enzymatic reactions.
First, a specific hydrolase processes the SUMO precursor into a mature form.
Second, an E1-activating enzyme activates mature SUMO. Third, during the
conjugation step, SUMO is transferred to the single E2-conjugating enzyme Ubc9
[Lesswright (Lwr) in Drosophila]. Subsequently, the covalent
interaction between SUMO and the target protein is achieved. Although Ubc9 is
able to recognise the sumoylation consensus motif in the target proteins
(Rodriguez et al., 2001),
efficient and proper modification in vivo requires E3 ligases
(Melchior et al., 2003;
Sharrocks, 2006). In addition,
SUMO conjugates are susceptible to cleavage by SUMO-specific proteases
(Hay, 2007;
Yeh et al., 2000). In
Drosophila, SUMO components are expressed during all developmental
stages (Long and Griffith,
2000), although their role in the control of development remains
unclear. Previous studies have shown a role for lwr in embryonic
patterning (Epps and Tanda,
1998). Hypomorphic mutations in lwr result in a prolonged
larval life followed by death (Chiu et al.,
2005), suggesting a role for sumoylation in development and
metamorphosis that has been largely unexplored until now.
Three major hormones regulate most aspects of post-embryonic development in
holometabolous insects: the prothoracicotropic hormone (PTTH),
20-hydroxyecdysone (20E) and the juvenile hormone (JH)
(Berger and Dubrovsky, 2005).
In Lepidoptera, PTTH, produced by a pair of neurosecretory cells located in
the dorsomedial region of the brain, is required to stimulate the synthesis of
ecdysone (E). In Drosophila, E is synthesized in the prothoracic
gland (PG) cells of the ring gland and then secreted to the hemolymph and
converted to its active form, 20E, in target tissues (for a scheme of the ring
gland in Drosophila, see Fig.
1A). Active 20E interacts with specific receptors, activates
response genes and triggers genetic programs in target tissues
(Ashburner, 1974;
Thummel, 2002). During larval
stages (or instars) periodic pulses of 20E before each larval molt act in
concert with the sesquiterpenoid JH, secreted by the corpus allatum (CA) in
the ring gland, to ensure the transition to the next larval instar. In
Manduca sexta, at the end of the last larval instar, the JH titer
drops and the peak of 20E initiates metamorphosis
(Nijhout and Williams, 1974).
However, the roles of JH and PTTH during metamorphosis are less studied in
Drosophila, where the drop of JH titer or PTTH requirement for
ecdysone production have not been demonstrated
(McBrayer et al., 2007).
In arthropods, ecdysteroids are synthesized from cholesterol or
phytosteroids. The biosynthetic pathway from cholesterol to 20E is not
completely characterised, although several members of the Halloween gene
family mediate steroid hormone biosynthesis in Drosophila
(Gilbert, 2004;
Gilbert and Warren, 2005;
Rewitz et al., 2006). The
genes phantom (phm), disembodied (dib),
shadow (sad) and shade (shd) encode
cytochrome P450 enzymes that catalyse the final four sequential hydroxylation
steps in the conversion of cholesterol to active 20E. Recently, spook
and spookier (spok) have been implicated in ecdysteroid
biosynthesis (Ono et al.,
2006), although their function is currently unknown. In addition,
a Rieske-domain protein, Neverland, has been implicated in the conversion of
cholesterol to 7-dehydrocholesterol (7dC), the first enzymatic reaction of the
pathway (Yoshiyama et al.,
2006). Little is known about the regulation of the ecdysteroid
biosynthesis enzymes and only a few transcription factors have been involved
in this pathway, including Without children (Woc)
(Warren et al., 2001;
Wismar et al., 2000), Molting
defective (Mld) (Neubueser et al.,
2005) and the β isoform of Fushi tarazu-factor1
(βFtz-f1) (Parvy et al.,
2005). Woc controls the conversion from cholesterol to 7dC, Mld is
involved in the regulation of spok, and βFtz-f1 is involved in
the transcriptional regulation of dib and phm
(Ono et al., 2006;
Parvy et al., 2005;
Warren et al., 2001). Several
other genes in Drosophila are implicated in the control of ecdysone
titers, such as ecdysoneless (ecd)
(Henrich et al., 1987),
giant ring gland (grg)
(Klose et al., 1980),
dare (Freeman et al.,
1999), giant
(Schwartz et al., 1984),
dre4 (Sliter and Gilbert,
1992) or the inositol 1,4,5,-tris-phosphate receptor
(Venkatesh and Hasan, 1997),
although for most of them their mechanism of participation in steroidogenesis
is unclear.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Mirth et al., 2005).
Information about strains not described in the text can be found in FlyBase
(http://flybase.bio.indiana.edu).
Plasmid construction and generation of transgenic strains
Knockdown experiments were performed using the GAL4/UAS system
(Brand and Perrimon, 1993). To
generate UAS-smt3i, smt3 cDNA was amplified by PCR using specific
primers (Fw 5'-GCTCTAGAGCATGCCAGCTTCAACAAGCAACCA-3' and Rev
5'-GCTCTAGAATCGATTCTTAGGGCCTGGT-3') containing XbaI sites
for cloning into pWIZ (Lee and Carthew,
2003). Transgenic lines UAS-smt3i were generated
following standard transformation procedures
(Spradling and Rubin,
1982).
Immunocytochemistry
Adults were allowed to lay eggs during 8 hours. Wandering larvae were
collected 5, 6, 11 and 15 days after egg lying (AEL), dissected in phosphate
buffered saline (PBS), fixed in 4% paraformaldehyde (PFA) for 20 minutes and
washed in PBS-Triton X-100 0.3% (PBT) three times, for 20 minutes each.
Tissues were then blocked in PBT-BSA for one hour at room temperature (RT) and
incubated with the appropriate antibodies at 4°C overnight. The following
polyclonal antibodies were used at the indicated dilution: anti-Woc, 1:500
(Raffa et al., 2005);
anti-Smt3, 1:500 (Smith et al.,
2004); anti-βFtz-f1, 1:20
(Ohno et al., 1994); anti-Phm,
1:100 (Parvy et al., 2005);
anti-Dib, 1:200 (Parvy et al.,
2005); anti-Mld, 1:200
(Neubueser et al., 2005);
polyclonal goat anti-activated-caspase-9 (Santa Cruz Biotechnology), 1:50;
rabbit anti-Sad (Abcam), 1:200; rabbit anti-HRP (Jackson ImmunoResearch),
1:600; and mouse monoclonal anti-lamin Dm0 (Developmental Studies Hybridoma
Bank), 1:10. The following day, the tissues were washed with PBT three times,
for 20 minutes each, and incubated with secondary antibodies at RT for two
hours. Fluorescent Alexa 568- and 633-conjugated secondary antibodies
(Molecular Probes) were used at a 1:200 dilution. DAPI (Roche) and
Phalloidin-TRITC (Sigma) were used at a 1:2000 dilution. Stained brains and
ring glands were mounted in Vectashield mounting medium (Roche). Confocal
images were taken with a Leica DM IRE2 microscope and images were processed
using the Leica Confocal Software and Adobe Photoshop.
Filipin and Oil Red O stainings
Ring glands were fixed in 4% PFA for 20 minutes, washed twice in PBS and
stained with 50 µg/ml of filipin (Sigma) for 1 hour or incubated in an Oil
Red O (Sigma) solution at 0.06% for 30 minutes. Samples were washed twice with
PBS before mounting in Vectashield (Roche). Pictures were taken with a Leica
DM IRE2 confocal microscope.
Quantification of lipid droplets was done on single plane confocal micrographs of Oil Red O or filipin stainings using the `Analyze particle' tool from ImageJ software. At least 10 independent micrographs were analyzed from WT and sm3i PG cells.
Rescue experiments
UAS-smt3i flies were crossed with a phm-Gal4 driver to
obtain smt3-RNAi larvae (hereinafter called smt3i).
smt3i larvae (lacking TM6B,Tb) and controls were collected
at 120 hours AEL and placed in groups of 10 individuals in new tubes
supplemented with 20E (Sigma) dissolved in ethanol at 1 mg/ml and mixed with
yeast. Control larvae were fed with yeast mixed only with ethanol.
Ecdysteroid titers and weight quantifications
Ecdysteroid levels were quantified by ELISA following the procedure
described by Porcheron et al. (Porcheron
et al., 1976), and adapted by Romañá et al.
(Romañá et al.,
1995). 20E (Sigma) and 20E-acetylcholinesterase (Cayman Chemical)
were used as the standard and enzymatic tracer, respectively. The antiserum
(Cayman Chemical) was used at a dilution of 1:50,000. Absorbance was read at
450 nm using a Multiscan Plus II Spectrophotometer (Labsystems). The
ecdysteroid antiserum has the same affinity for ecdysone and 20E
(Porcheron et al., 1976), but
because the standard curve was obtained with the latter compound, results are
expressed as 20E equivalents. For sample preparation, 15 staged larvae were
weighed and preserved in 600 µl of methanol. Prior to the assay, samples
were homogenized and centrifuged (10 minutes at 18,000 g)
twice and the resultant methanol supernatants were combined and dried. Samples
were resuspended in 50 µl of enzyme immunoassay (EIA) buffer (0.4 M NaCl, 1
mM EDTA, 0.1% BSA in 0.1 M phosphate buffer).
For weight quantification, smt3i and control larvae were collected at 5 days AEL and weighed in groups of fifty larvae. Then, smt3i larvae were placed in new tubes and weighed during the next 25 days.
Transmission electron microscopy
smt3i and wild-type wandering third-instar larvae were rinsed in
water and opened with forceps in a droplet of 0.1 M PBS, pH 7.3, on a clean
microscope slide. The brain with the attached ring gland was removed and
immersed directly in ice-cold, freshly prepared fixative containing 2.5%
glutaraldehyde and 4% PFA in 0.1 M PBS, pH 7, for six hours. The samples were
then rinsed four times, for 15 minutes each, in PBS, post-fixed for 1 hour in
an aqueous 2% solution of osmium tetroxide, rinsed in water, dehydrated in a
gradual series of ethanol and acetone, and embedded in EPON (EPON 812
embedding kit 3132, Tousimis) following the manufacturer's instructions.
Following the last infiltration step, the samples were moved to pure resin in
moulds for polymerization at 60°C for 48 hours. Semi-thin sections (around
2 µm) were cut with a glass knife, mounted on microscope slides, stained
with 0.1% boracic Toluidine Blue for histological study and to locate
appropriate sites for ultrastructural analysis. Ultra-thin sections (60 to 70
nm) were cut with a diamond knife, contrasted with lead citrate and uranyl
acetate, and observed under a JEOL JEM 1010 microscope operated at 80 kV.
Images were taken with a digital camera (Hamamatsu C4742-95). Measurements and
image processing were done with AMT Advantage CCD and Adobe Photoshop
software, respectively. Three to four larvae were analyzed from each sample
(genotype and age). smt3i samples were fixed at age 120, 144, 168 and
264 hours AEL. Control samples (WT, phm-GAL4 and UAS-smt3i)
were all fixed at 144 AEL.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Shih et al., 2002). To
investigate Smt3 function at post-embryonic stages in Drosophila, we
used the UAS/Gal4 system to drive smt3i transgene expression in
several tissues, including wing, haltere and eye imaginal dics, salivary
gland, ring gland and central nervous system, during development.
UAS-smt3i with different Gal4 lines produced strong, fully penetrant,
phenotypes. Smt3 accumulates at high levels in the nuclei of WT PG cells
(Fig. 1B), and its levels were
dramatically reduced in smt3i larvae when the phm-Gal4
driver was used (Fig. 1C). As a
result of the knockdown, smt3i animals arrested their development at
the third larval instar (L3) just before pupariation and survived for an
additional 3 weeks (Fig. 1D-F).
During this time, smt3i larvae continued feeding and gaining weight
until approximately 21 days AEL (Fig.
1F). These larvae did not present duplicated mouth hooks
(Fig. 1G,H), indicating that
previous molts were correct, and died as L3 without forming a puparium.
Interestingly, smt3 knockdown in the CA by using Aug21-Gal4
produced normal progeny with no defects in molting or metamorphosis, and gave
rise to normal adult flies. Thus, the role of smt3 on metamorphosis
seems to be due to its role within PG cells. Our results suggest an essential
role for Smt3 during post-embryonic development during initiation of the
pupariation process.
smt3i larvae show reduced ecdysteroid levels
Insect molting and metamorphosis are controlled by the hormone 20E, which
is synthesized from the precursor E produced in the PG. To determine whether
the inability of smt3i larvae to pupariate was due to reduced levels
of ecdysteroids, we measured the ecdysteroid titers in smt3i and
control larvae. At 120 hours AEL, the levels of ecdysteroids in smt3i
larvae were only slightly reduced compared with control larvae
(Fig. 2A). At 144 hours AEL,
these levels increased in the controls, probably corresponding to the level of
the 20E peak associated with pupariation, as shown by Warren et al.
(Warren et al., 2006).
However, the ecdysteroid levels remained unchanged in smt3i larvae,
suggesting that the 20E peak is absent in the knockdown animals
(Fig. 2A). During the
abnormally extended larval life of smt3i larvae there was a
progressive reduction in the ecdysteroid titer, as shown by the levels at 7
and 11 days AEL (Fig. 2A).
These results suggest that smt3i larvae are not able to produce the
ecdysteroid peak required to proceed to pupal stages.
To further demonstrate that the developmental arrest is due to reduced levels of 20E, we performed ecdysteroid-feeding rescue experiments. L3 smt3i fed with medium containing 20E pupariated within 24 hours (100%, n=30); however, these pupae were not able to develop further and died, maybe because a higher dose of 20E is required for metamorphosis (Fig. 2B). The control untreated smt3i larvae continued as L3 and died several weeks later without signs of molting (Fig. 2B). These results confirm that smt3i larvae have reduced levels of ecdysteroids, which could be the reason for their inability to pupariate.
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The reduction of ecdysteroids could reflect a massive death of PG cells in
knockdown larvae. In fact, Smt3 is necessary for cell survival in some larval
structures (Takanaka and Courey,
2005). To analyze this possibility, we used the
anti-activated-caspase-9 antibody that recognises the Drosophila
activated initiator caspase Nc (previously known as Dronc) in apoptotic cells.
At 120-144 hours AEL we did not observe features of cell death in most of the
smt3i PG cells (Fig.
3F), and we only detected active Nc in one or two cells in the PG
of some smt3i specimens (Fig.
3G). These Nc-positive cells were bigger than the other
smt3i PG cells and we called them `giant cells'
(Fig. 3C,D,G, arrows). Our
ultrastructural analysis corroborates these results, showing sporadic
apoptotic cells surrounded by non-apoptotic cells
(Fig. 3H). There may be a
progressive loss of these cells over time, perhaps by detachment from the PG
and subsequent loss into the hemolymph.
We focused on the ultrastructural changes described as typical features of
PG degeneration during metamorphosis, such as cytoplasmic fragmentation,
reduction in the amount of smooth endoplasmic reticulum (SER) and the number
of mitochondria, and amplification of autophagic vacuoles and lysosomes
(Dai and Gilbert, 1991). We
observed no change in SER or mitochondria, and no increment of autophagosomes
or lysosomes in smt3i PG cells. Therefore, our observations suggest
that the impairment of development and the reduced ecdysteroid levels in
smt3i larvae are not due to massive cell death or premature
degeneration of the PG cells at the time of puparium formation. It is likely
that the remaining levels of Smt3 in knockdown PG cells are enough to allow
cell survival (Fig.
1C'').
Variations in the levels and localisation of steroidogenic factors in smt3i larvae
Halloween genes encode for cytochrome P450 enzymes that mediate the
conversion of cholesterol to 20E. phm, dib and sad, which
encode the C25, C22 and C2 hydroxylases, respectively, are all expressed in PG
cells (Chavez et al., 2000;
Niwa et al., 2004;
Warren et al., 2002;
Warren et al., 2004). From
early to late third instar larval stages there is an upregulation of P450
enzyme expression that correlates with an increase in the ecdysteroid titers
(Warren et al., 2006). We
analysed these enzymes in 5- to 6-day-AEL smt3i larvae by
immunodetection (Fig. 4). We
did not observe changes in the expression levels or pattern of Phm, localised
in the endoplasmic reticulum (ER) of the PG cells
(Fig. 4A,A',D,D')
(Warren et al., 2004).
However, the levels of Dib, which in WT third instar larvae showed a
characteristic punctuate-like pattern corresponding to mitochondria
(Petryk et al., 2003), were
dramatically reduced in smt3i larva
(Fig. 4B,B',E,E').
We also analysed the expression pattern of Sad, expressed in WT wandering
third instar larvae in the cytoplasm with a pattern similar to Dib, as well as
in the nucleus (Fig.
4C,C'). In smt3i larvae, the nuclear accumulation
was reduced (Fig. 4F,F').
Altogether, these results show that reduced levels of Smt3 in the PG produce
changes in the expression levels of enzymes in the ecdysteroid biosynthetic
pathway.
We also analysed the transcription factors involved in the regulation of
these steroidogenic enzymes. In WT third instar larvae PG cells, Woc is
localised in the nucleus (Fig.
4G,G') and we could not detect remarkable variations in the
expression levels or the subcellular localisation of this factor in
smt3i larvae (Fig.
4J,J'). Mld is also expressed in the nucleus of WT PG cells,
in a pattern different than that of Woc
(Fig. 4H,H'). We observed
a reduction in the expression levels of Mld in the nuclei of smt3i PG
cells and, interestingly, a change in the localisation of this protein that
could now be found in the cytoplasm (Fig.
4K,K'). It has been suggested that βFtz-f1 regulates
both Dib and Phm expression (Parvy et al.,
2005), and, as shown for Dib, expression of βFtz-f1 is
drastically reduced in smt3i PG cells, in both the nucleus and the
cytoplasm (Fig.
4I,I',L,L').
If Smt3 is involved in the ecdysteroid biosynthesis pathway, we would
expect mutations in other members of the sumoylation pathway to have the same
effect on the expression and localisation of ecdysteroidogenic enzymes and
factors. We analysed the expression pattern of these in the PGs of
lwr homozygous mutants that had reached L3
(Chiu et al., 2005;
Huang et al., 2005a). Similar
to smt3i, in lwr mutant larvae we detected severe
alterations in the expression levels of Dib (not shown) and βFtz-f1
(Fig. 4O,O'), as well as
a mis-localisation of Woc and Mld (Fig.
4M-N'), confirming that smt3 knockdown alters the
sumoylation pathway in PG cells. However, the low levels of these factors
might not be sufficient to explain the reduction in the ecdysteroid titer of
smt3i larvae, as a decrease in their transcriptional levels does not
impair metamorphosis (McBrayer et al.,
2007).
|
). The
conversion of cholesterol into E involves mainly the ER and the mitochondria,
as well as the shuttling of intermediate forms between these intracellular
compartments. However, as mentioned previously, we did not observe changes in
the ER or the mitochondria in smt3i larvae
(Fig. 5A,B; data not shown).
Similar to previous observations (Aggarwal
and King, 1969; Dai et al.,
1991; King et al.,
1966), we observed in WT L3 a very high number of deep
invaginations in the membrane of PG cells facing the hemolymph, which form
channels reaching deep into the cells (Fig.
5A). These plasma membrane invaginations represent a substantial
increase in the cell surface, and are probably relevant for the efficient
uptake of lipids and the secretion necessary for the high ecdysteroid titer
characteristic of this developmental stage. In addition, we observed
elaborated interdigitations, which, overall, produced an extensive
extracellular space between the cells (see Fig. S2A in the supplementary
material). Interestingly, in smt3i PG cells there was a clear
reduction in the number and length of the invaginations of the plasma membrane
(Fig. 5B), and also a
diminution of the interdigitations (see Fig. S2B in the supplementary
material). It is difficult to assess whether the balance between the increased
size of the cells, and the reduction of the channels and interdigitations in
smt3i larvae, involves changes in the total cell surface. However,
the intracellular channels seem to be of functional relevance for ecdysteroid
secretion (Dai and Gilbert,
1991; Dai et al.,
1991) and, therefore, their reduction might be important to
understand the phenotype of smt3i larvae. Our EM analysis corroborated the abnormal morphology of the nuclei of PG cells in smt3i larvae and disclosed the formation of extraordinarily large aggregates of viral-like particles (VLPs). These particles are frequently detected in low numbers in all tissues in WT strains, although in smt3i larvae the quantitative differences were obvious (see Fig. S2B in the supplementary material). In addition to the VLPs, and associated with them, we found a high number of parallel bands of electron-dense material of unknown origin (see Fig. S2B in the supplementary material) that was absent in control larvae. In addition, smt3i PG nuclei had thickened nuclear lamina (compare Fig. 5C and 5D), although the nuclear pores seemed to be still present. In agreement with this observation, detection of lamin using Dm0 antibodies showed a thickening of the lamin layer associated with the nuclear envelope in smt3i larvae (Fig. S2C,D in the supplementary material). No other ultrastructural changes were observed. Overall, these results show that, at the ultrastructural level, the main organelles affected in smt3i larvae are the plasma membrane and the nucleus.
Ecdysone synthesis in the PG is stimulated by the brain neuropeptide PTTH
in Lepidoptera. As we used a PG-specific Gal4 to knockdown smt3, we
assume that PTTH synthesis in the neurosecretory cells of the brain is normal.
However, the reception of this signal could be compromised in smt3i
larvae as a result of the alterations in the plasma membrane. We visualised
the nerve terminals reaching the ring gland, by HRP immunostaining, in 5- to
6-day AEL larvae (Fig.
5E-F'). In WT larvae, the axons arborised and extended among
the PG cell layers as described (Fig.
5E,E'; see also Fig. S3A-B' in the supplementary
material) (Siegmund and Korge,
2001). A similar pattern was found in the PG of smt3i
larvae (Fig. 5F,F'; see
also Fig. S3D-E' in the supplementary material), although some of the
nerve endings looked slightly disorganised. Varicose nerve terminals
containing the electron-dense vesicles characteristic of neurosecretory
endings were detected by EM among the PG cells of WT and knockdown larvae, and
no obvious differences were detected among them (see Fig. S3C,F in the
supplementary material).
Lipid content is reduced in the PG cells of smt3i larvae
The reduction of plasma membrane invaginations observed in smt3i
larvae has been previously described in PG cells of the ecdysone deficient
mutant l(3)ecd1ts (Dai
et al., 1991). However, none of the other characteristic features
of l(3)ecd1ts mutants occur in smt3i PG cells,
such as accumulation of lipid droplets in the cytoplasm, disappearance of SER
or enhancement of electron-dense mitochondria. On the contrary, in 5- to
6-day-old smt3i larvae, we found a general diminution of lipid
droplets compared with WT. These droplets most likely include sterol
precursors required for ecdysteroid production. To better characterise this
observation, we used Oil Red O staining to identify the lipid droplets and
filipin staining to specifically stain non-esterified sterols. In most
smt3i PG cells, we observed a clear reduction in the number of lipid
droplets (Fig. 6A-B'') and
also a diminution of sterols (Fig.
6C,D). Only the PG cells previously described as `giant cells' had
an increased accumulation of lipid droplets
(Fig. 6B,B', arrow). We
quantified the number of lipid droplets in smt3i and WT PG cells,
excluding the `giant cells' as they were apoptotic, as shown by the active
Nc-positive staining (Fig. 3G).
Whereas each WT PG cell in a single section contained approximately 25 lipid
droplets, smt3i larvae had only 5 lipid droplets
(Fig. 6E). Interestingly, the
total lipid content in other tissues of the smt3i larvae increased
over the course of their expanded life, reflecting the reported body weight
increase (Fig. 1F; data not
shown). The quantification of filipin-stained drops gave a similar result (see
Fig. S4 in the supplementary material).
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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|
;
Nacerddine et al., 2005;
Takanaka and Courey, 2005).
smt3i larvae, after surviving the first and second molts, arrest
development specifically at the time of pupariation, giving us the possibility
to explore the role of sumoylation during metamorphosis.
The reduction in ecdysteroid titers in smt3i larvae could not be
caused by a premature degeneration of the ring gland, as we did not detect
massive cell death or an increase in lysosomes and autophagic vacuoles at the
time-point when the larvae should enter pupariation. During their abnormally
extended larval life, smt3i PGs contain apoptotic cells but never
show autophagic features characteristic of WT PG degeneration
(Dai and Gilbert, 1991).
smt3i changes in the nucleus and cytoplasm
The hypertrophy of PG cells and their nuclei found in smt3i larvae
has also been reported in ecdysteroid deficient mutants, such as mld, woc,
grg or dre4 (Klose et al.,
1980; Neubueser et al.,
2005; Sliter and Gilbert,
1992; Wismar et al.,
2000). This could reflect a compensatory mechanism triggered by
the abnormally low ecdysteroid levels common for all these genotypes.
The main organelles involved in the ecdysteroid biosynthetic pathway are
thought to be the mitochondria and the ER, and changes in these organelles
have been reported for some mutants exhibiting reduced ecdysteroids
(Dai et al., 1991;
Wismar et al., 2000). We did
not observe ultrastructural abnormalities in these structures in
smt3i larvae. However, the nucleus is affected in smt3i PGs,
showing an abnormal morphology, thickening of the nuclear lamina and
hyper-proliferation of VLPs. A similar increase in the amount of VLPs has been
found in at least one other ecdysteroid mutant, grg
(Klose et al., 1980).
smt3i PG nuclei also showed arrays of parallel electron-dense
stripes, a phenotype that increased gradually during the prolonged larval
life. These arrays of alternating electron-dense and clear material were
always tightly associated with VLPs, but the mechanism by which these bands
are formed is unknown.
Reduction of smt3 results in the thickening of the nuclear lamina
beneath the inner nuclear membrane (INM). The INM and its associated layer of
lamins have important functions, such as maintenance of the nuclear shape,
organization of the nuclear pores, chromatin and transcriptional regulation
(Heessen and Fornerod, 2007),
and the correct distribution of nuclear pore complexes
(Liu et al., 2007). As
sumoylation is crucial for the nuclear transport, smt3i larvae could
abolish the nucleo-cytoplasm transport and, therefore, could contribute to the
localisation changes observed in factors necessary for ecdysteroidogenesis.
However, this is not a general problem in smt3i PG cells, as
transport to the nucleus of some of the tested proteins was not affected (for
instance Woc). Therefore, despite the ultrastructural aberrations observed,
the protein-production machinery and nucleo-cytoplasmic transport are not
blocked.
By contrast, the reduced levels of cytoplasmic Dib or βFtz-f1, or
nuclear Mld or Sad, might contribute to the low levels of ecdysteroids in
smt3i larvae, although this might not be the only cause of impeded
pupariation, as the low transcriptional levels of these factors do not stop
entry in metamorphosis (McBrayer et al.,
2007).
Intracellular channel formation is impaired in smt3i PG cells
The severe reduction of intracellular channels and interdigitations in
smt3i PG cells might be essential to the understanding of the L3
arrest phenotype of knockdown larvae. These intracellular channels, typical of
an active gland in WT L3 (Dai et al.,
1991), could be necessary for the increased rate of ecdysone
synthesis required at this stage, perhaps because the amplification of the
interface between the PG cells and the hemolymph results in a more efficient
uptake of lipids and secretion of ecdysteroids.
|
Smt3 is necessary for cholesterol uptake in PG cells
The reduction of lipid and sterol droplets suggests a problem in
cholesterol uptake in smt3i PGs, maybe caused by the reduction of the
intracellular channels characteristic of smt3i PG cells.
Interestingly, functionally analogous structures, the microvillar channels,
seem to play an important role in lipid uptake in the adrenal gland, the
mammalian equivalent of the insect PG
(Reaven et al., 1989).
Arthropods are not able to synthesize cholesterol and depend on exogenous
cholesterol or related sterols. Receptors involved in cellular cholesterol
uptake have been described in various organisms from nematodes to mammals.
Recently, the relevance of lipoproteins and their receptors in embryonic
development and steroid hormone signalling has been reported; for example, the
delivery of cholesterol to steroidogenic tissues such as the adrenal gland
(Willnow et al., 2007).
Particularly interesting is the role of scavenger receptor class B type I
(SR-BI)-mediated cholesterol uptake, as it has been shown that SR-BI is
essential for both microvillar channel formation and HDL localisation
(Williams et al., 2002). This
receptor has been localised to caveolar rafts, plasma membrane microdomains
characterised by their elevated cholesterol content
(Martin and Parton, 2005).
These specialised regions have been implicated in different cell functions by
regulating transduction pathways.
Alternatively, the deficient cholesterol uptake and the reduction of
intracellular channels in smt3i larvae could be independent
processes. The analysis of lipid droplets in l(3)ecd1ts
(Dai et al., 1991) and
woc mutants (A.T., J.S., R.C., C.P. and R.B.) suggests that
diminution of the intracellular channels is not enough to disrupt completely
cholesterol uptake. Although both mutants show a clear reduction of plasma
membrane folding, they show a high accumulation of lipid droplets
(Dai et al., 1991;
Wismar et al., 2000).
Mutations in other factors involved in cholesterol homeostasis also show an
accumulation of cholesterol, caused by intracellular trafficking defects that
result in lethality during larval to pupal transition
(Huang et al., 2005b;
Huang et al., 2007) (for a
review, see Huang et al.,
2008).
Further studies will be required to understand the mechanism of cholesterol uptake by PG cells, how this is altered in cells that lack interdigitations, and, lastly, how this is related to deficient steroidogenesis. Alterations in the sumoylation pathway could affect steroidogenesis in other cell types and in other organisms. Our research could provide insights into physiological regulation by steroid hormones in higher organisms and into the associated pathologies.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1659/DC1
|
ACKNOWLEDGMENTS |
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Footnotes |
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|
REFERENCES |
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