In the brain of the silkworm Bombyx mori, two pairs of lateral neurosecretory cells LNCs produce the prothoracicotropic hormone PTTH [1,2].. Two EcR isoforms, EcR-A and EcR-B1, and two U
Trang 1producing neurosecretory cells of the Bombyx mori brain
An indication of the master cells of insect metamorphosis
Monwar Hossain1, Sakiko Shimizu2, Haruhiko Fujiwara3, Sho Sakurai1,2and Masafumi Iwami1,2
1 Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Japan
2 Division of Biological Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan
3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
The insect brain is the center of developmental control
In the brain of the silkworm Bombyx mori, two pairs
of lateral neurosecretory cells (LNCs) produce the
prothoracicotropic hormone (PTTH) [1,2] The peptide
PTTH stimulates the prothoracic glands to synthesize
and release ecdysone [3] The active form of ecdysone,
20-hydroxyecdysone (20E), controls many
physiologi-cal and developmental processes of insect molting and
metamorphosis [4] Beside PTTH, the brain produces
many neurosecretory hormones that orchestrate
devel-opmental processes It is also the target organ of 20E,
effecting dynamic morphological changes and
rear-rangement of the neural network during processes such
as the formation of the optic lobe and mushroom body
differentiation during metamorphosis [5,6] Hence, the analysis of 20E-induced gene expression in the brain is
of critical importance in understanding insect develop-ment
The 20E signal is mediated intrinsically through binding with its heterodimeric nuclear receptor consist-ing of the ecdysone receptor (EcR) and ultraspiracle (USP) [7,8] The 20E–receptor complex directly induces several early genes and the products of these genes activate late effector genes that control stage- and tissue-specific developmental responses to 20E [9] Two EcR isoforms, EcR-A and EcR-B1, and two USP iso-forms, usp-1 and usp-2, which have been identified in Bombyx [10–13], are involved in specific responses to
Keywords
Bombyx mori; brain; ecdysone receptor;
metamorphosis; prothoracicotropic
hormone-producing neurosecretory cell
Correspondence
M Iwami, Division of Life Sciences,
Graduate School of Natural Science and
Technology, Kanazawa University,
Kanazawa 920-1192, Japan
Fax: +81 76 2646251
Tel: +81 76 2646255
E-mail: masafumi@kenroku.kanazawa-u.ac.jp
(Received 18 May 2006, revised 22 June
2006, accepted 26 June 2006)
doi:10.1111/j.1742-4658.2006.05398.x
The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear recep-tors, the ecdysone receptor (EcR) and ultraspiracle (USP) Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses
in the developing cells of the silkworm Bombyx mori Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B mori brain Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the pro-thoracicotropic hormone (PTTH)- producing cells (PTPCs) In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs As PTTH controls ecdysone secre-tion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis
Abbreviations
20E, 20-hydroxyecdysone; EcR, ecdysone receptor; LNC, lateral neurosecretory cell; MEF2, myocyte enhancer factor 2; OLC, optic lobe cell; NaCl⁄ P i ⁄ Tween, phosphate buffered saline containing Tween; PTPC, PTTH-producing neurosecretory cell; PTTH, prothoracicotropic hormone; TCC, tritocerebral cell; USP, ultraspiracle.
Trang 220E EcR-A expression regulates neuronal maturation,
while EcR-B1 expression controls neuronal regression
both in Drosophila melanogaster [14,15] and Manduca
sexta [16] Mutational analysis of EcR isoforms in
Drosophila identified several different lethal phases,
including developmental arrest late in embryogenesis,
failure of pupariation [17,18], and disruption of
neur-onal remodeling [18] Although the effect of ecdysone
on neurons of the central nervous system has been
extensively studied in Drosophila and Manduca, little
information is available on the role of EcR expression
in the brain In the present study, we examined EcR-A
and EcR-B1 expression in the Bombyx brain and
demonstrate stage- and cell-specific expression of EcR
isoforms, which is especially prominent in
PTTH-producing cells (PTPCs) These results provide an
insight into ecdysone receptor function in the brain
during larval-pupal-adult development
Results
Expression of EcR-A and EcR-B1 in the Bombyx
brain
Day-2 fifth instar B mori larvae were injected with
1 lg 20E (+20E) or Ringer’s solution ()20E), and
RNA was extracted from the brains 2 h after the
injec-tions RT-PCR revealed that both EcR-A and EcR-B1
were weakly expressed in the absence of 20E, and
that 20E up-regulated the expression of both genes
(Fig 1A) EcR-A was shown to be expressed at a
slightly higher level than EcR-B1 following induction
by 20E No transcript was amplified after 25 cycles of
RT-PCR for both +20E and )20E samples
Expres-sion of the control housekeeping gene RpL32 showed
no difference between +20E and )20E samples after
25 and 35 cycles of RT-PCR
To examine spatial expression of EcR isoforms,
we carried out RT-PCR on RNA purified from the
anterior and the posterior sections of B mori brains
(Fig 1B) Intense EcR-A expression was observed exclusively in the anterior part while no expression was detected in the posterior part after 35 cycles of RT-PCR (Fig 1C) Strong EcR-B1 expression was also detected in the anterior part of the brain and weaker expression was detected in the posterior section, indi-cating that both genes exhibit a spatial-specific pattern
of expression RpL32 expression did not differ between the two parts of the brain
Cell-specific expression of EcR isoforms as revealed by in situ hybridization
The spatial specificity of EcR-A and EcR-B1 expres-sion was also determined by whole-mount in situ hybridization of larval and pupal brains using EcR iso-form-specific probes EcR-A expression was detected exclusively in two pairs of lateral neurosecretory cells (LNCs) in day-2 (Fig 2A) and day-7 (Fig 2C) fifth instar larval brains The location of the EcR-A-positive cells was assumed to be the same as that of PTTH-producing cells (PTPCs), as shown in Fig 2E To con-firm this, we performed in situ hybridization using a mixture of EcR-A and PTTH probes The hybridiza-tion signal was again detected exclusively in the two pairs of LNCs (Fig 2G), indicating that EcR-A and PTTH mRNAs were colocalized in PTPCs of the lar-val brain EcR-B1 expression was also detected exclu-sively in the same LNCs in day-2 (Fig 2B) and day-7 (Fig 2D) fifth instar larval brains, while EcR-B1 and PTTH expression was shown to colocalize in PTPCs
of the larval brain (Fig 2H)
In the pupal brain, EcR-A expression was observed
in PTPCs (Fig 2I,L) and in several tritocerebral cells (TCCs) (Fig 2K) and optic lobe cells (OLCs) (Fig 2J) EcR-B1 expression was similarly detected in PTPCs (Fig 2F), TCCs, and OLCs (Fig 3E) but at a lower level than EcR-A In addition to the brain, faint EcR-A expression was detected in the subesophagal ganglion (Fig 2K) Both EcR isoforms are therefore
Fig 1 EcR-A and EcR-B1 are induced by 20E and are predominantly expressed in the anterior of day-2 fifth instar larval brains (A) RT-PCR analysis of EcR-A and EcR-B1 expression following injection of 20E (+20E) or Ringer’s solution ( )20E) The number of PCR cycles is indica-ted above the panel The housekeeping gene RpL32 was used as a control (B) Total RNA was extracindica-ted either from the anterior (A) or pos-terior (P) part of the brains and analyzed by RT-PCR to assess the spatial distribution of gene expression (C) RT-PCR analysis of EcR expression using RNA from the anterior (A) and posterior (P) part of the brains The number of PCR cycles is indicated above the panel.
Trang 3expressed exclusively in PTPCs at the larval stage and
in PTPCs, TCCs, and OLCs in the pupal stage of
B moridevelopment
Immunohistochemical confirmation of EcR-A and
EcR-B1 cell-specific expression
To confirm the in situ hybridization results, we
performed double-labeled immunohistochemistry with
anti-EcR-A and anti-EcR-B1 sera, and anti-PTTH IgG
on the brains of day-2 and day-7 fifth instar larvae
and day-2 pupae EcR-A and PTTH expression was
detected exclusively in two pairs of LNCs in day-2 fifth
instar larvae (Fig 3A, left) The merged image shows
that EcR-A and PTTH expression colocalizes in
PTPCs This colocalization was also observed in day-7
larval and day-2 pupal brains (Fig 3B,C, left) The
same results were obtained for EcR-B1 (Fig 3A–C,
right), confirming that both isoforms are colocalized in PTPCs in the brain
In the pupal brain, EcR-A and EcR-B1 expression was observed in TCCs and weakly in OLCs in addi-tion to PTPCs (Fig 3D,E, also Fig 2I,J) It is note-worthy that EcR-B1 fluorescence was strongest just beneath the cell membrane of TCCs (Fig 3F) and was weaker in the axons, while EcR-A fluorescence was more diffuse throughout the cytoplasm (Fig 3D, also Fig 3A), indicating that the distribution of the two isoforms differed slightly EcR-A and EcR-B1 expression was also detected in the subesophagal gan-glion (Fig 3D,E)
Discussion
In the present study, we have shown for the first time that EcR-A and EcR-B1 are up-regulated by 20E
D C
H G
I
J
K
L
Fig 2 Localization of EcR-A, EcR-B1 and
PTTH mRNAs by whole-mount in situ
hybridization EcR-A and EcR-B1 mRNA was
detected in day-2 (A,B) and day-7 (C,D) fifth
instar larval brains and day-2 pupal brains
(F,I) For fifth instar day-2 specimens, brains
were dissected 2 h after 20E injection.
PTTH mRNA was detected in day-2 fifth
instar larval brains (E) (G) EcR-A and PTTH
as well as (H) EcR-B1and PTTH probes
were used simultaneously for hybridization
of day-2 larval brains (A–E,G,H) show the
anterior portion of the larval brain EcR-A
expression in the pupal brain (I) is magnified
in panels J–L, where OLCs (J, white
arrows), TCCs (K, white arrows) and PTPCs
(L, white arrows) are shown in green, blue,
and black boxes, respectively The blue
arrows in K indicate faint signals in the
sub-esophagal ganglion Scale bar ¼ 100 lm.
Trang 4exclusively in PTPCs in the B mori larval brain The
spatial specificity of EcR-A and EcR-B1 expression
was determined by in situ hybridization and confirmed
by immunohistochemistry using EcR isoform-specific
antibodies
Expression of EcR-A and EcR-B1 in PTPCs has not
been reported previously Recently, Vafopoulou et al
[19] used immunohistochemistry to demonstrate EcR
expression in the medial neurosecretory cells of
Rhodn-ius prolixus The postembryonic development of
Rhodnius and Bombyx is strikingly different Unfed
Rhodnius larvae exist in an arrested developmental
state caused by an ecdysteroid deficiency [20], which is
immediately met by the consumption of a blood meal,
thus initiating development No such phenomenon
exists in Bombyx larvae, and it is possible that the
difference in 20E-responsive cells between the two insects reflects the different developmental systems Our study indicates that the expression level of
EcR-A is slightly higher than that of EcR-B1, as previously observed during the neuronal maturation of Drosophila and Manduca [16] The developmental function of EcR-A is distinct from those mediated by the EcR-B1 and EcR-B2 isoforms in Drosophila An EcR-A mutant
is arrested during early to mid-pupal development, indicating that EcR-A is required for the formation of the basic pupal body plan prior to the differentiation
of most adult structures [15] By contrast, an EcR-B1 mutant is lethal at the first and second larval molts [18] and fails to undergo pupariation [17]
PTPCs are the only cells to express EcR isoforms in the Bombyx larval brain, and these cells also express
A
B
C
D
F
E
Fig 3 Immunohistochemical localization of EcR-A, EcR-B1, and PTTH (A) Day-2 larval, (B) day-7 larval and (C) day-2 pupal brains For fifth instar day-2 specimens, brains were dissected 6 h after 20E injection Anti-EcR-A, anti-EcR-B1, and anti-PTTH were used as primary antibodies A FITC-conjugated anti-rabbit secondary antibody was used to detect EcR-A or EcR-B1, and a Texas-red conjugated anti-mouse secondary antibody was used to detect PTTH The middle panel (A–C) shows a merged (yellow) image of the upper (green, EcR-A or EcR-B1) and lower (red, PTTH) panels (D) EcR-A and (E) EcR-B1 expression in day-2 pupal brains White arrows in (D) and (E) indicate the EcR-A- and EcR-B1-producing cells in the tritocerebral region of the pupal brain, respectively Red arrows indicate the
EcR-A-or EcR-B1-producing cells in the subesopha-gal ganglion, respectively A magnified image of the red box in (E) is shown in (F), where EcR-B1 expression is indicated by white arrows Scale bar ¼ 100 lm.
Trang 5usp isoforms (MDM Hossain & M Iwami,
unpub-lished data) As the 20E signal is transduced via the
EcR–USP complex, it can be concluded that PTPCs
are the only cells to respond to 20E at the
transcrip-tional level, and these cells therefore play important
roles in 20E-dependent larval-pupal metamorphosis
PTTH controls the hemolymph 20E level by
stimula-ting ecdysone synthesis and coordinastimula-ting its release
from the prothoracic glands EcR-A and EcR-B1
expression in the PTPCs suggests that 20E modulates
PTTH expression through a feedback loop Beside its
prothoracicotropic effect, PTTH may act as a growth
factor as it shares a common ancestor with the
verteb-rate growth factor superfamily peptides such as nerve
growth factor, transforming growth factor, and
plate-let-derived growth factor [21] It also enhances the
syn-thesis of several short-lived proteins that mediate a
variety of extracellular signals [22,23] Despite the first
demonstration of PTTH almost three decades ago in
LNCs in Manduca [24] and later in Bombyx [1,2],
the molecular mechanisms of PTTH production and
release are still at an early level of understanding,
although these are of critical importance in insect
developmental control [25]
Since the work of Truman [26], it has been believed
that PTTH release is controlled by a circadian clock in
the brain [27–29] The close association between clock
cells and PTPCs in the brain protocerebral region is
seen in the three divergent genera Rhodnius,
Dro-sophila, and Bombyx, suggesting that there are routes
of communication between these two cell populations
[30,31] The association with clock cells and the
responsiveness of PTPCs for ecdysteroidogenesis
sug-gests that 20E influences PTTH release from PTPCs
through neurons that provide input to clock cells
[31,32] The PTTH titer in Bombyx larval hemolymph
is, however, not exclusively controlled by photoperiod
and⁄ or circadian clock mechanisms [27,28], as
protho-racicostatic hormones have been shown to influence
ecdysteroidogenesis and ecdysteroid release from the
PTTH-stimulated prothoracic gland [33,34] At the
transcriptional level, Bombyx PTTH is regulated by
trans-regulatory factors such as myocyte enhancer
fac-tor 2 (MEF2) [32] In the brain, MEF2 is expressed at
an elevated level in PTPCs and corazonin-like
immu-noreactive-LNCs Over-expression of MEF2 increases
PTTHexpression in Bombyx brain [32], while
up-regu-lation of MEF2 by 20E was reported in adult
Dro-sophilamyoblasts [35] An emerging hypothesis is that
MEF2 is regulated by 20E, which in turn modulates
PTTHexpression
In addition to PTPCs, EcR-A and EcR-B1
expres-sion was detected in the tritocerebrum and the optic
lobe of day-2 pupae: a crucial stage for the morpholo-gical and neurolomorpholo-gical reorganization of the brain 20E induces differentiation of the optic lobe and adult eye
in Manduca [5,36], while EcR is expressed during the puparium stage in the optic lobe of Drosophila [16] In the present study, EcR expression in B mori OLCs is consistent with these findings In the tritocerebrum, the number and position of EcR-A-producing cells differed from EcR-B1-producing cells The intracellular local-ization of EcR-A was also distinct from that of EcR-B1: EcR-A was localized in the cytoplasm while EcR-B1 was beneath or close to the cell membrane of PTPCs and TCCs The intracellular distribution of EcR-B1 could indicate a nongenomic role for 20E in these cells [37]
The present results clearly show that EcR isoform expression in the brain is exclusive to the PTPCs until pupation This indicates that PTPCs are the master cells during larval-pupal metamorphosis and that they control ecdysteroidgenesis At the pupal stage, the number of cells expressing EcR isoforms increases to include TCCs and OLCs This unique expression pro-file indicates the importance of EcR isoform expression
in TCCs and OLCs during larval-pupal metamorpho-sis, although the roles of the individual isoforms in these cells remain to be elucidated
Experimental procedures
Animals and hormones
obtained from Ueda Sanshu (Ueda, Japan), and larvae were reared on an artificial diet (Silkmate II, Nihon
days, consisting of a photophase followed by a scotophase Fifth instar day-2 and day-7 larvae and day-2 pupae were studied 20E (Sigma, St Louis, MO, USA) was dissolved in ethanol and its concentration was determined
stock solution was evaporated and dissolved in insect
RNA isolation and semiquantitative RT-PCR
Total RNA was isolated from whole brains 2 h after the injection of 20E (1 lg per larva) (+20E) or insect Ringer’s
expression in the brain, total RNA was isolated from the anterior and posterior sections of brains RNA was purified
method [39] with minor modifications [40]
Trang 6One microgram total RNA was reverse-transcribed in a
20 lL reaction using 100 U ReverTra Ace (Toyobo, Osaka,
accord-ing to the manufacturer’s instructions After reverse
tran-scription, the reaction was stopped by heating the solution
five-fold with water PCR primers were designed according
to the nucleotide sequences: EcR-A 5¢-TGGAGCTGAAA
CACGAGGTGGC-3¢ and 5¢-TCCCATTAGGGCTGTAC
GGACC-3¢, EcR-B1 5¢-ATAACGGTGGCTTCCCGCTG
CG-3¢ and 5¢-CGGTGTTGTGGGAGGCATTGGTA-3¢,
and RpL32 5¢-GAGGACGAAGAGATTTATCAGGCA-3¢
and 5¢-CGAAGAGACACCATGAGCGAT-3¢ PCR was
Noni-det P40, 0.2 mm each dNTPs, 0.5 mm each primer, 0.25 U
Canada), and 1 lL synthesized cDNA The reaction was
subjected to 25 or 35 cycles of amplification in a
thermo-cycler (GeneAmp PCR System 9700, Applied Biosystems,
bro-mide staining There was no amplification without reverse
transcriptase even at 35 cycles of PCR (data not shown),
indicating the specificity of EcR-A and EcR-B1 mRNA
amplification
In situ hybridization
Whole-mount in situ hybridization of brains was performed
as described previously [41] After dissecting, brains were
washed with 10 mm phosphate buffered saline, pH 7.4
for 40 min The brains were then incubated in a solution of
tem-perature for 20 min The brains were washed three times
EcR-A probe (5¢-TCCCATTAGGGCTGTACGGACC-3¢)
or EcR-B1 probe (5¢-CGGTGTTGTGGGAGGCATTG
GTA-3¢) and ⁄ or PTTH probe (5¢-GTACACAAACACGCC
ACGCTGACG-3¢) 3¢-labeled with digoxigenin using a
Dig-labeled kit (Roche Diagnostics, Mannheim, Germany)
followed by a 2 h incubation with a 1 : 500 diluted alkaline
Diagnostics) at room temperature Color development
potent inhibitor of endogenous alkali phosphatases After dehydration with ethanol, the brains were clarified with methyl salicylate and observed with a microscope (BX-50F, Olympus, Tokyo, Japan) Negative controls omitted the labeled probes, and no signal was detected (data not shown)
Anti-peptide serum for Bombyx EcR isoforms
GHP(15–29)C-COOH] [13] (Accession no D87118) and an
MSSG(94–112)-COOH] [12] (Accession no D43943) were synthesized, HPLC purified, and used to immunize rabbits (Sawady Technology, Tokyo, Japan) Handling of rabbits was performed according to regulation and guidelines of the local authority The anti-peptide serum titers for EcR-A and EcR-B1, determined using an enzyme-linked immuno-sorbent assay, were 4700 and 119 700, respectively
Immunohistochemistry
Double-labeled fluorescent immunohistochemistry was used
to detect EcR-A, EcR-B1, and PTTH expression Brains
facili-tate the penetration of antibodies through the brain sheath [2] The pretreated brains were incubated with rabbit
serum (1 : 300) and mouse anti-Bombyx PTTH monoclonal IgG (1 : 100) [2] overnight The brains were then incubated
anti-bodies, mouse anti-rabbit IgG conjugated with fluorescein isothiocyanate (Cappel, Aurora, OH, USA) at a dilution of
1 : 400 for EcR-A and EcR-B1, and goat anti-mouse IgG conjugated with Texas-Red (Cappel) at a dilution of 1 : 400
Fluorescence was detected using a fluorescence microscope (BX-50F, Olympus) using NIBA filters for fluorescein isoth-iocyanate and WIY filters for Texas-Red Negative controls
Tween20 (data not shown) The absence of detectable flo-rescence in these controls demonstrated the specificity of the reaction Images were processed with Adobe Photoshop (Adobe Systems Inc, San Jose, CA, USA)
Trang 7We are grateful to Dr A Mizoguchi for the gift of
the anti-PTTH antibody This work was supported by
Grants-in-Aid for Scientific Research (15580039 and
18380040) from the Japan Society for the Promotion
of Science
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