Download Ectopic expression of ecdysone oxidase impairs tissue

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Research
Cite this article: Li Z et al. 2015 Ectopic
expression of ecdysone oxidase impairs tissue
degeneration in Bombyx mori. Proc. R. Soc. B
282: 20150513.
http://dx.doi.org/10.1098/rspb.2015.0513
Received: 4 March 2015
Accepted: 1 May 2015
Subject Areas:
developmental biology, genetics,
molecular biology
Keywords:
ecdysone oxidase, Bombyx mori, CRISPR/Cas9,
Gal4/UAS, autophagy
Authors for correspondence:
Yongping Huang
e-mail: [email protected]
Anjiang Tan
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.0513 or
via http://rspb.royalsocietypublishing.org.
Ectopic expression of ecdysone
oxidase impairs tissue degeneration
in Bombyx mori
Zhiqian Li1, Lang You1, Baosheng Zeng1, Lin Ling1, Jun Xu1, Xu Chen1,
Zhongjie Zhang1, Subba Reddy Palli2, Yongping Huang1 and Anjiang Tan1
1
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology
and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032,
People’s Republic of China
2
Department of Entomology, College of Agriculture, University of Kentucky, S-225 Agriculture Science Center
North, Lexington, KY 40546, USA
Metamorphosis in insects includes a series of programmed tissue histolysis
and remolding processes that are controlled by two major classes of hormones,
juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most
active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase
(EO), a 20E inactivation enzyme, expresses predominantly in the midgut
during the early pupal stage in the lepidopteran model insect, Bombyx mori.
Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly
interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system
extended the duration of the final instar larval stage. Ubiquitous transgenic
overexpression of BmEO using the Gal4/UAS system induced lethality
during the larval–pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset
of metamorphosis was blocked. Transmission electron microscope and
LysoTracker analyses showed that the autophagy pathway in MSG is inhibited
by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the
genes involved in autophagic cell death and the mTOR signal pathway are
affected by overexpression of BmEO. Taken together, BmEO functional studies
reported here provide insights into ecdysone regulation of tissue degeneration
during metamorphosis.
1. Background
Steroid hormones, ecdysteroids (20-hydroxyecdysone, 20E, is the most active
form) regulate insect development and metamorphosis. 20E binds to the canonical nuclear receptor complex EcR/USP and regulates many cellular and
physiological processes [1 –3]. Pulses of 20E are precisely regulated by both synthesis and metabolism pathways. The biosynthesis pathway of 20E has been
well documented except for some genes involved in the so-called ‘Black Box’
[4]. By contrast, less is known about 20E metabolism. Although several genes
that function in 20E inactivation have been identified, the biological functions
of genes involved in this pathway are not well understood [3,5].
One route of 20E inactivation is through 26-hydroxylation to 26-oic acid by
the cytochrome P450 CYP18A1 [5– 7]. In Drosophila melanogaster, accumulation
of 20E after CYP18A1 knockdown repressed the expression of bFtz-F1 and
caused pupation defects [5]. By contrast, ubiquitous CYP18A1 overexpression
resulted in embryonic lethality and fat body-specific overexpression led to lethality during pupation [5]. Furthermore, CYP18A1 mediated 20E inactivation has
been identified in several lepidopteran insects, including Spodoptera littoralis,
Manduca sexta and Bombyx mori [8,9]. Distinct from CYP18A1 expression in
the peripheral tissues of many insect species, B. mori CYP18A1 is predominantly
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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The detailed information on silkworm strain, cDNA synthesis and
qRT-PCR, plasmid construction, germline transformation, RNAseq, TEM, paraffin embedding, ecdysteroids titer determination
and Western blotting is included in the electronic supplementary
material, material and methods.
3. Results
(a) Predominant expression of BmEO in the midgut
during the early pupal stage
Previous studies reported high EO activity during the
early pupal stage in many insects although the functional
significance is unknown [13]. We investigated spatial and temporal expression patterns of BmEO in three development stages
and 10 tissues using qRT-PCR. The maximum BmEO mRNA
levels were detected in the midgut dissected from day 1
pupae (P1, figure 1a). Relatively higher levels of BmEO
mRNA were also detected in the fat body (35% of the levels
in midgut) and testis (20% of the levels in midgut) dissected
from P1 (figure 1a). We further analysed BmEO mRNA levels
in the midgut and fat body from day 1 of fifth instar larvae
(L5D1) to day 9 of the pupal stage (P9). BmEO mRNA levels
in midgut dissected from larvae remained very low and
began to increase from day 1 of the prepupal stage (PP1).
During the pupal stages, BmEO mRNA levels in the midgut
(b) CRISPR/Cas9 mediated-BmEO knockout extended
the final instar larval stage
Recently, the CRISPR/Cas9 system has been demonstrated to be
effective in targeted gene knockout analysis in D. melanogaster
and B. mori [19,20], providing a powerful genetic tool for
insect functional genomics. Here, we established a binary transgenic CRISPR/Cas9 system to knockout BmEO somatically.
We created a transgenic silkworm line expressing Cas9 ubiquitously under the control of IE1 promoter (IE1-Cas9) and another
line expressing the sgRNA targeting BmEO driven by the
silkworm U6 promoter (U6-BmEOsgRNA, figure 2a). In heterozygous IE1.BmEOsgRNA offspring, genomic deletions at
the target site were detected in all examined animals by PCR
analysis (figure 2b), demonstrating that this somatic mutagenesis system was effective. IE1.BmEOsgRNA animals
(60.6%, n ¼ 33) showed larger body size than the control
animals and prolonged final instar larval stadium by 24 h (electronic supplementary material, table S1). A significant decrease
in BmEO protein levels was also detected in the midgut dissected from P4 BmEO knockout animals (figure 2c). To assess
the involvement of BmEO in 20E inactivation, ecdysteroid
titers were determined by enzyme immunoassay (EIA). A twofold increase in the ecdysteroid titers was detected in the midgut
of BmEO knockout animals on L5D4, when the endogenous
ecdysteroid levels are low (figure 2d). We also investigated the
mRNA levels of mTOR pathway genes, which are the major regulators of cell growth (figure 2e). Two positive regulators
including insulin receptor substrate (IRS) and phosohoinositide-3kinase (PI3K) were upregulated by 7.4- and 8.7-fold, respectively,
in BmEO knockout animals. On the contrary, two negative regulators tuberous sclerosis (TSC1 and TSC2) were downregulated,
respectively, to 4.8% and 0.5% of the levels in the control animals
(figure 2e).
(c) Ubiquitous overexpression of BmEO disrupted
larval –pupal transition
To further investigate BmEO biological functions, it was ubiquitously overexpressed using A3-Gal4 activator. The mRNA
levels of BmEO in A3.BmEO animals were increased by
8.3-fold in the midgut, twofold in the MSG, threefold in the
epidermis and fourfold in the fat body when compared to
that in the control UAS-BmEO animals on L5D4 (figure 3a).
A significant increase in the BmEO protein abundance was
also detected in midgut from A3.BmEO transgenic animals
during the wandering stage (figure 3b). Additionally, ecdysteroid levels in A3.BmEO silkworms decreased by 90% in
the midgut and 30% in the haemolymph during the wandering stage (figure 3c). No abnormal phenotype was observed
in the transgenic silkworms during embryogenesis and the
early larval stages. However, 86% (n ¼ 43) A3.BmEO animals arrested development prior to pupation (figure 3d).
Most of them completed head capsule slippage but failed
2
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2. Material and methods
reached the maximum at P4 and decreased again from P5 to
P9 (figure 1b). Although its relative mRNA levels in the fat
body showed a similar pattern, the maximum levels reached
were only 31.2% of that in the midgut (figure 1b). Abundant
BmEO protein was also detected during the early pupal
stage (P3 and P4) in midgut (figure 1c). These data suggested
that BmEO is predominantly expressed in the midgut during
the early pupal stage.
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expressed in the middle silk gland (MSG) and transgenic
overexpression of BmCYP18A1 leads to lethality during the
final instar larval stage [9].
Another major inactivation route of steroid hormone
is through 3-epimerization [10–12]. 20E conversion to
3-dehydroecdysone (3DE) can be catalysed by ecdysone
oxidase (EO), which could be reversed by 3-dehydroecdysone
3b-reductase [13]. The intermediate, 3DE, is converted to
3-epiecdysone by 3-dehydroecdysone 3a-reductase irreversibly
[13,14]. As one of the key enzymes in this pathway, EO was first
identified and purified from the dipteran insect Calliphora vicina.
The purified protein showed specific 3-dehydroecdysone
reductase activity for ecdysone and 20E [15,16]. In several lepidopteran insects and D. melanogaster, EO is predominantly
expressed in the midgut and is 20E-responsive [10–12,17].
However, its biological function in insect development and
metamorphosis is unknown mainly owing to lack of mutants
or transgenic insects overexpressing EO.
Here, we describe the first functional analysis of insect EO
by using both loss-of-function and gain-of-function genetic
approaches recently developed in the lepidopteran model
insect, B. mori [18,19]. We constructed a transgenic CRISPR/
Cas9 system to somatically mutate BmEO that caused an extension in the final instar larval stage. By contrast, ubiquitous
overexpression of BmEO by using transgenic Gal4/UAS
system resulted in the lethality during the larval–pupal transition. Furthermore, when BmEO was ectopically expressed
in a MSG-specific manner, MSG degeneration at the onset of
metamorphosis was severely impaired. Disruption of MSG
degeneration by overexpressing BmEO provides an excellent
model for understanding the mechanisms of hormonal
regulation of tissue degeneration in insects.
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W
P1
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P6
BmEO
a-tubulin
Figure 1. Spatial and temporal expression patterns of BmEO. (a) BmEO is expressed predominantly in the midgut during early pupal stage. The relative mRNA levels
of BmEO in 10 major tissues including the prothoracic gland (PG), fat body (FB), midgut (MG), anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland
(PSG), Malpighian tubule (MT), testis (Te), ovary (Ov) and epidermis (Epi) were determined by qRT-PCR. Three representative stages, day three of the fifth instar
(L5D3), wandering (W) and day 1 pupae (P1) were sampled. (b) The mRNA levels of BmEO in midgut and fat body from L5D1 to P9. (c) Predominant translation of
BmEO in midgut at the early pupal stage. Protein levels of BmEO were examined in midgut from L5D3 to P6. BmEO mRNA levels were normalized using the
silkworm ribosome protein 49 (Bmrp49) and the protein levels were normalized using a-tubulin. The data shown are mean+s.e.m. (n ¼ 3).
to undergo ecdysis, leaving the old cuticle covering the newly
formed pupal cuticle (figure 3d). The rest of the 14% transgenic silkworms survived pupation but died during the
pupal stage.
(d) Middle silk gland-specific overexpression of BmEO
disrupted tissue histolysis
Our previous studies showed that the 20E inactivation enzyme,
BmCYP18A1, is predominantly expressed in B. mori MSG [9].
To investigate the functional difference between BmEO and
BmCYP18A1, we overexpressed BmEO specifically in MSG
using the Ser-Gal4 driver. BmEO mRNA expression increased
to more than 1000-fold higher in the MSG of Ser.BmEO silkworms on L5D4 when compared to its levels in the MSG
from the control UAS-BmEO animals (figure 4a). There was
no significant increase detected in other five main tissues
including midgut, anterior silk gland (ASG), posterior silk
gland (PSG), fat body and epidermis. BmEO protein levels
also increased in the MSG of Ser.BmEO animals at the wandering stage (figure 4c). Increase in BmEO caused a decrease
in the ecdysteroid levels by 72% in the haemolymph and 82%
in the MSG of Ser.BmEO animals during the wandering
stage (figure 4b).
Almost all (98%, n ¼ 87) Ser.BmEO silkworms successfully
completed larval–pupal transition (electronic supplementary
material, table S1), showing a bigger size than the control
(UAS-BmEO) animals of the same age (figure 4d). Further observation revealed that MSGs were intact in Ser.BmEO pupae
at P2, when MSG was degraded and completely disappeared
in the control animals (figure 4e). This status was kept to
adult stage and the defective MSGs disappeared finally. By
contrast, the ASG and the PSG in transgenic animals
degraded normally, leaving MSG with caecum at the ends.
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(a)
4
BmEO
3
2
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ATG
TGA
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EO
sg
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EO
sg
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U6-BmEOsgRNA
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m
1>
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*
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*
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*
TS
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PI
IR
S
0
*
TS
C2
relative expression ratio
(e)
IE
U
6-
Bm
EO
EO
sg
sg
RN
A
RN
A
0
Figure 2. Loss-of-function of BmEO by binary CRISPR/Cas9 system extended the final larval stadium. (a) Schematic diagram of BmEO gene structure and the small guide
RNA (sgRNA) targeting BmEO. The line indicates the genome locus of BmEO and boxes represent the five exons. The targeting sequence was indicated and the protospacer
adjacent motif (PAM) sequence is shown in red. (b) Various types of deletion mutations in the heterozygous offspring after crossing IE1-Cas9 and U6-BmEOsgRNA transgenic silkworm lines are shown. The targeting sequence is in blue, PAM sequence is in red and the adjacent sequences are in black. The deletion size was indicated by the
number of nucleotides. (c) Decrease in BmEO protein levels detected in midgut of mutant at P4. a-tubulin was used as the control. (d) Increase in ecdysteroid levels in
midgut from L5D4 larvae detected by EIA assay. Three transgenic silkworms were used to extract ecdysteroid from the midgut. (e) Fold changes in mRNA levels of mTOR
signal pathway genes in the IE1.BmEOsgRNA transgenic silkworms at L5D4. Three transgenic silkworms were used to extract total RNA from midgut dissected from L5D4.
The data shown are mean+s.e.m. (n ¼ 3). The asterisks in (d,e) indicate statistical significance ( p , 0.05). (Online version in colour.)
Morphological observations showed an enlarged size of MSG
on L5D6 and the wandering stage, prior to MSG histolysis
in UAS.BmEO animals (figure 4f ). During the larval–pupal
transition, wild-type (WT) silkworms begin to spin and
MSG shrank rapidly in response to the rising ecdysteroid
levels. However, MSGs were kept intact until the late pupal
stage in the Ser.BmEO silkworms (figure 4f ), suggesting that
MSG histolysis was inhibited by BmEO overexpression.
Although no defects were observed during the pupal–adult
metamorphosis, neither female nor male Ser.BmEO moths
were able to mate with WT animals and therefore produced no
offspring.
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(d)
(c)
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(b)
UA
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>B
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M
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1.5
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A3>BmEO
1.0
*
0.5
*
0.5 cm
0
haemolymph
MG
Figure 3. Ubiquitous overexpression of BmEO caused an arrest in metamorphosis during larval– pupal transition. (a) BmEO mRNA levels increased to 8.3-fold in
midgut, two-fold in MSG, threefold in the epidermis and fourfold in fat body when expressed under the control of actin 3 promoter at L5D4. Three individual
biological replicates were used in qRT-PCR and the BmEO mRNA levels were normalized to silkworm Bmrp49. (b) BmEO protein showed an increase in the midgut at
the wandering stage. (c) Ecdysteroid levels decreased to 10% in the midgut and 70% in haemolymph at the wandering stage. Three individuals were used to extract
ecdysteroid and measured by EIA assay. (d ) Actin-driven BmEO (A3.BmEO) overexpression caused a metamorphosis arrest during larval –pupal transition. The
lowest panel is the control UAS-BmEO pupa and upper three are A3.BmEO animals. The asterisks in (a,c) indicate statistical significance ( p , 0.05). (Online
version in colour.)
(e) Autophagy and mTOR signalling pathway were
affected in Ser.BmEO animals
In order to understand the molecular mechanisms underlying
the phenotypes observed, MSGs from Ser.BmEO and
UAS-BmEO animals were dissected from PP2 (when MSG histolysis is initiated in WT silkworms) and subjected to RNA-seq
analysis. Among 832 differentially expressed genes (DEGs)
identified, 454 genes including BmEO were upregulated and
378 were downregulated (figure 5a). KEGG enrichment analysis revealed that autophagy and mTOR signalling were in the
top five downregulated pathways (figure 5b). As confirmed
by qRT-PCR, 13 silkworm Atg genes were downregulated in
MSG of Ser.BmEO transgenic silkworms. Among them,
Atg1, Atg6 and Atg11 decreased to 6.7%, 9.1% and 6.9% of
the control levels, respectively (figure 5c). We also examined
the expression levels of apoptosis-related genes by qRT-PCR
using RNA isolated from MSG from Ser.BmEO and UASBmEO and no significant differences were detected (electronic
supplementary material, figure S1b). These data suggest that
overexpression of BmEO in MSG may affect only autophagy.
As reported previously, the mTOR Ser/Thr kinase phosphorylates Atg13 and inhibits autophagy initiation [21]. Here,
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relative expression ratio
(a) 12
we also found that the mTOR signalling pathway is among
the most downregulated KEGG pathways. Four positively
regulated genes including BBX (one of the insulin receptor substrates), PI3K, PDK (3-phosphoinositide dependent protein kinase-1)
and Rheb (Ras homologue enriched in brain) were downregulated
to 13.5%, 15%, 35% and 42%, respectively (figure 5c). The
three negatively regulated genes: TSC1, TSC2 and mTOR
(mechanistic target of rapamycin) were upregulated by 5.3, 1.5
and 1.6-fold when compared to the levels in control (figure
5c). Although the RNA-seq results showed no significant
change in expression of 20E cascade genes, significant decrease
in their expression was detected by qRT-PCR (electronic
supplementary material, figure S1a). Among these genes,
E75B, USP, E74A and Ftz-F1 mRNA levels decreased to
27%, 9%, 5.3% and 5.4% of their levels in control animals
(electronic supplementary material, figure S1a). By contrast,
EcRB and HR3 showed similar expression levels as that in the
control (UAS-BmEO). Notably, 23 silkworm cuticle genes
including 12 with chitin binding domains, which may
function as components of the inner cuticle layer, were downregulated significantly in Ser.BmEO when compared to their
levels in control animals (electronic supplementary material,
figure S1c).
relative expression ratio
10
Se
r>
Bm
EO
(c)
UAS-BmEO
Ser>BmEO
*
12
BmEO
8
a-tubulin
6
0.010
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(d)
0.005
relative ecdysteroid titer
1.5
Ep
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G
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M
G
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1.0
(e)
0.5
*
*
0
haemolymph
MSG
(f)
L5D6
W
PP1
PP2
P1
P2
Figure 4. Inhibition of MSG histolysis in Ser.BmEO transgenic silkworms. (a) MSG-specific overexpression of BmEO detected by qRT-PCR at L5D4. (b) Decrease in
ecdysteroid levels determined by EIA assay in the haemolymph and MSG of wandering stage larvae in the Ser.BmEO transgenic silkworms. (c) BmEO protein levels
increase in MSG at the wandering stage. a-tubulin was used as the control. (d ) Increase in body size in the Ser.BmEO transgenic silkworms when compared with
the control UAS-BmEO silkworms. (e) Intact MSG in Ser.BmEO animals indicated by the green triangle. Upper panel shows MSG from Ser.BmEO and the lower
panel shows MSG from the UAS-BmEO. ( f ) Delayed histolysis of MSG from Ser.BmEO silkworms. Upper panel shows MSG from UAS-BmEO and the lower panel
shows MSG from the Ser.BmEO for each stage. For (a,b), three individual silkworms were used and the data shown are mean+s.e.m. (n ¼ 3). The scale bars in
(d – f ) stand for 0.5 cm. The asterisks in (a,b) indicate statistical significance ( p , 0.05). (Online version in colour.)
(f ) Middle silk gland-specific BmEO overexpression
inhibited autophagy in the silk gland
Silkworm silk gland was considered as the homologous tissue
of D. melanogaster salivary gland which undergoes histolysis
triggered by programmed cell death (PCD). To determine
6
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(a)
UA
SBm
EO
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whether the defects in MSG degradation could be attributed to
disruption of autophagy identified by RNA-seq, we performed
transmission electron microscopy (TEM) and LysoTracker staining on the tissues dissected from PP2 animals. TEM showed a
large number of autophagic structures in MSGs (figure 6a),
which encompassed cytoplasmic components and organelles
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no. DEGs
(a) 0
7
membrane
nucleus
integral to membrane
extracellular region
protein complex
viral caspid
intracellular
mitochondrial intermembrane space protein transporter complex
extracellular matrix
integral to endoplasmic reticulum membrane
oxidoreductase activity
structural constituent of cuticle
serine-type endopeptidase activity
zinc ion binding
catalytic activity
chitin binding
ATP binding
serine-type endopeptidase inhibitor activity
protein binding
binding
(b)
(c)
KO ID
KO term
p-value
glycine, serine and threonine metabolism
bmor00260
0.001685
regulation of autophagy
bmor04140
0.002410
TGF-beta signalling pathway
bmor04350
0.007567
mTOR signalling pathway
bmor04150
0.009627
FoxO signalling pathway
bmor04068
0.031111
6
4
2
TSC2
TSC1
Rheb
PDK
PI3K
BBX
Atg18
Atg13
Atg12
Atg11
Atg9
Atg8
Atg7
Atg5
Atg6
Atg4
Atg3
Atg2
Atg1
0
mTOR
relative expression ratio
8
Figure 5. RNA-seq analysis showed repression of autophagy and mTOR pathways in the Ser.BmEO silkworms. (a) Top 10 significantly enriched gene ontology
terms for biological process, cellular component and molecular function from the differentially expressed genes (DEGs). Log2 fold change more than or equal
to 5 and p , 0.05 were used as the cutoff criteria. (b) Top five enriched KEGG pathways of downregulated genes with p , 0.05. (c) qRT-PCR validation for
the genes associated with the regulation of autophagy pathway and mTOR signalling pathway. Three individual PP2 MSGs were used and the error bars stand
for mean+s.e.m. (Online version in colour.)
in the double membranes (inset is the enlargement of the granule indicated by green triangle) in MSG of WT animals.
However, a lower number and smaller autophagic structures
were observed in the MSG of Ser.BmEO animals (figure 6a0 ).
Interestingly, autophagy was inhibited both in the fat body
(figure 6b,b0 ) and the midgut (figure 6c,c0 ). This is probably an
indirect effect caused by circulating ecdysteroids. LysoTracker
staining of MSG sections dissected from PP2 Ser.BmEO animals showed a thinner outer silk gland cell layer when
compared with the MSG of UAS-BmEO control animals
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proteolysis
metabolic process
transport
chitin metabolic process
carbohydrate metabolic process
regulation of transcription, DNA-dependent
protein phosphorylation
protein polymerization
G-protein-coupled receptor signalling pathway
response to oxidative stress
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biological process
30
cellular component
20
molecular function
10
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TEM
LysoTracker
2 mm
(b≤)
(b¢)
5 mm
5 mm
(c¢)
(c)
2 mm
100 mm
100 mm
(c≤)
2 mm
100 mm
100 mm
(b≤¢)
100 mm
(c≤¢)
100 mm
Figure 6. Decrease in autophagic structures in the Ser.BmEO MSGs examined by the TEM and LysoTracker staining. (a) Significant levels of autophagy observed in
MSG from UAS-BmEO transgenic silkworms at PP2. The inset is the enlargement of the double membrane structure indicated by the green triangle. (a’) Less
autophagic structures were observed in the Ser.BmEO MSGs. (a’’ ) LysoTracker staining (X40) of paraffin sections of MSG from UAS-BmEO silkworms. Green triangles
show the positive staining structures in the silk gland cells of MSGs. (a’’’) Decrease in autophagy levels detected in MSGs of the Ser.BmEO silkworms. Thinner silk
gland cell layer (the outer layer of MSG) was observed. The second and third rows showed the results from the midgut and fat body, respectively. At least three
silkworms were used for each assay. Scale bars are indicated in the figures. (Online version in colour.)
(figure 6a00 ,a000 ). Furthermore, autophagic signals in both fat
body (figure 6b00 ,b000 ) and midgut (figure 6c00 ,c000 ) showed a significant decrease.
4. Discussion
(a) BmEO mediates midgut-specific 20E inactivation
The current study confirmed midgut-specific expression of
BmEO in B. mori, being consistent with its expression in other
insect species studied [10–12,17], and revealed its highest
expression during the early pupal stage, P4. We employed
loss-of-function, gain-of-function, RNA-seq and histological
methods to uncover its biological functions in B. mori.
Then employing the CRISPR/Cas9 system, we successfully
induced mutagenesis at the BmEO loci, thus providing a
promising approach for loss-of-function analysis. Depletion of
BmEO did not cause significant changes in silkworm development except for a 24 h extension of the final instar larval
stage. By contrast, overexpression of BmEO ubiquitously using
the Gal4/UAS transgenic approach induced lethality during
the larval–pupal transition. However, ubiquitous overexpression of another 20E inactivation enzyme, MSG-specifically
expressed BmCYP18A1, induced larval lethality during the
early stages of final larval instar, earlier than that observed in
BmEO overexpressed animals [9]. Considering their tissuespecificity, we also ectopically expressed BmEO in MSG,
where no BmEO expression is observed in WT animals. Interestingly, a completely different phenotype was observed in
Ser.BmEO animals in which MSG developed to a larger size
and did not degenerate right after larval–pupal transition.
However, MSG-specific overexpression of BmCYP18A1 led to
silk gland growth arrestment during the final larval instar [9].
These data suggest temporal and spatial differences in 20E inactivation by BmEO and BmCYP18A1 may go through different
routes. Since 3-epimerization catalysed by EO is reversible, inactivation of 20E by BmEO may be less drastic than by
BmCYP18A1.
Drosophila melanogaster tissues show a tissue-specific 20E
response pattern during larval–pupal metamorphosis [22].
Larval midgut cells begin to undergo autophagy during
puparium formation, when an increase in 20E levels occurs.
However, the salivary gland cells begin histolysis only
during the pre-pupal stage when the 20E levels increase
again [22,23]. These data suggest that different tissues
respond differently to circulating ecdysteroids. It has been
Proc. R. Soc. B 282: 20150513
(b)
2 mm
(a≤¢)
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(a≤)
(a¢)
(a)
8
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Studies on D. melanogaster salivary glands revealed that insect
tissue histolysis is caused by suppression of IIS/TOR pathway and activation of PCD [27 –29]. Being the homologous
organ of the D. melanogaster salivary gland, B. mori silk
gland is also a larval-specific tissue and undergoes degeneration during larval–pupal metamorphosis. As previously
reported, silkworm Atg8 and Atg12 express highly in the
ASG during metamorphosis [30], accompanied by nucleus
pyknosis, cell detachment and membrane blebbing [31]. In
addition, the markers of PCD, increase in vacuoles, acid
phosphatase activities and caspase-3 activity have been
detected in the degenerating silk glands. These data suggest
that silk gland histolysis is a result of both apoptosis and
autophagy.
Since 20E is one of the stimuli for insect metamorphosis,
direct connections between 20E and PCD have been investigated in insects [32– 34]. In silkworms, injection of the 20E
into the feeding silkworms increased expression of Atg
genes and mutation of EcR resulted in an inhibition of autophagy [35]. Here, we found that overexpression of BmEO in
MSG inhibited autophagy. qRT-PCR (figure 5c), TEM and
Data accessibility. The RNA-seq raw data was deposited on NCBI SRA
database with accession no. SRP052024.
Authors’ contributions. Z.L. performed the experiments, collected and
analysed data and wrote the paper; L.Y. performed the microinjection
and data collection; B.Z., L.L., J.X., X.C. and Z.Z. participated in the
data analysis; S.P, Y.H. and A.T designed the experiment, analysed
data and wrote the manuscript.
Competing interests. We declare we have no competing interests.
Funding. This work was supported by grants from the National Basic
Research Program of China (2012CB114101), National Science Foundation of China (31420103918, 31272037 and 31372257) and the
External Cooperation Program of BIC, Chinese Academy of Sciences
(grant no. GJHZ201305).
Acknowledgements. We thank Xiaoshu Gao, Xiaoyan Gao, Jiqin Li and
Zhiping Zhang for confocol microscopy and TEM technique supports.
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