|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 21 March 2007
doi: 10.1242/dev.02835
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Group for Evolutionary Regeneration Biology, RIKEN Center for Developmental
Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
2 KAN Research Institute, Inc., Kobe MI R&D Center, 6-7-3
Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
3 Department of Biophysics, Graduate School of Science, Kyoto University,
Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
Author for correspondence (e-mail:
agata{at}mdb.biophys.kyoto-u.ac.jp)
Accepted 13 February 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Planarian, Regeneration, Central nervous system (CNS), Clathrin heavy chain, Endocytosis, AP-2, Neuronal homeostasis, Dugesia japonica
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The planarian CNS is relatively simple but well organized,
consisting of several functional and structural domains, as defined by the
discrete expression of three otd/Otx-related homeobox genes
and a complex set of other regulatory genes
(Umesono et al., 1997;
Umesono et al., 1999;
Cebrià et al., 2002b;
Cebrià et al., 2002c;
Mineta et al., 2003;
Nakazawa et al., 2003;
Koinuma et al., 2003;
Inoue et al., 2004).
Furthermore, planarians possess a remarkable regenerative ability that derives
from pluripotent somatic stem cells (Agata
and Watanabe, 1999;
Saló and Baguñà,
2002; Agata et al.,
2003; Reddien and
Sánchez Alvarado, 2004), whereby even a tiny fragment cut
from most parts of the body can regenerate the entire body with a functional
nervous system within 5 days (Inoue et
al., 2004). To identify genes involved in functional brain
regeneration, we systematically cloned genes expressed in the regenerating
brain (Nakazawa et al., 2003;
Mineta et al., 2003) and
analyzed their expression timing during brain regeneration
(Cebrià et al., 2002c).
Those studies revealed that planarian brain regeneration consists of at least
five steps: blastema formation, brain induction, patterning, neural circuit
formation and functional recovery. Several candidate genes involved in each
step have been identified. Djnlg, a planarian noggin homologue, and
DjvlgA, a vas-related gene, are the earliest genes found to
be involved in blastema formation (Ogawa
et al., 2002; Shibata et al.,
1999). The second-earliest gene is nou-darake, a
dominant-negative-type fibroblast growth factor receptor (FGFR) that is
activated in only the anterior blastema within 24 hours after amputation to
form a brain rudiment (Cebrià et
al., 2002a). DjotxA, DjotxB and Djotp,
brain-specific homeobox genes, are activated in the brain rudiment 36 hours
after amputation and act to create patterns in the brain
(Umesono et al., 1997;
Umesono et al., 1999). After
patterning, netrin and several immunoglobulin superfamily neural cell
adhesion molecule genes (IgCAMs) are activated in the brain approximately 72
hours after amputation to form neural circuits
(Cebrià et al., 2002b;
Cebrià and Newmark,
2005; Fusaoka et al.,
2006). Finally, 5 days after amputation, a functional brain is
recovered (Inoue et al.,
2004). The structure and function of the CNS depend on precisely
controlled interactions among the neural cells, but the mechanisms of
intercellular interaction required to construct and maintain the functional
brain during brain regeneration are unclear. Here, we demonstrate that the
clathrin heavy chain gene (DjCHC) of a planarian,
Dugesia japonica, was required for proper brain regeneration and
investigate at what point it acts during brain regeneration.
Clathrin-mediated membrane trafficking plays an important role in cell
signaling and is involved in numerous cell functions, including nutrient
uptake, regulation of the number of signaling receptors on the cell surface
and the recycling of synaptic vesicles at nerve terminals (Takei and Haucke,
2002; Murthy and DeCamilli,
2003; Le Roy and Wrana,
2005). Functional analyses of clathrin genes has been performed in
cultured cells, fungi, trypanosomes and yeast, and have provided insights into
the functions of clathrins and their interactions with other proteins in cell
signaling (Seeger et al., 1992; Ruscetti
et al., 1994; Niswonger and
O'Halloran, 1997b; Wettey et
al., 2002; Motley et al.,
2003; Allen et al.,
2003; Hinrichsen et al.,
2003). However, much remains unknown about the developmental,
molecular and cellular processes that determine the onset and maintenance of
intercellular communication and the role of such communication in
physiological responses of the nervous system. Here, by in vitro and in vivo
analyses, we show that DjCHC-RNA interference (RNAi) inhibits
neuronal survival, and neurite outgrowth and maintenance, and that
DjCHC is required for proper CNS regeneration and homeostasis.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cDNA cloning of DjCHC, Djmu-1 and Djmu-2
cDNA clones (Dj_aH_000_01290HH, Dj_aH_507_P09, Dj_aH_402_I20, Dj_aH_302_F09
and Dj_aH_316_I10) encoding the respective proteins (DjCHC, Djmu-1, Djmu-2,
Djunc13A and DjsbpA) were first identified in a previously constructed
database of expressed sequence tags (ESTs)
(Mineta et al., 2003). The
longest cDNA of DjCHC was obtained by the stepwise dilution method
(Watanabe et al., 1997) from a
cDNA library constructed from poly A(+) RNA of whole planarians in the ZAPII
vector (Stratagene). The sense primer
5'-TGATCATCGAAACTTACAAAACC-3' and antisense primer
5'-ATACCCATATGTGCTCGTTCTAA-3' corresponded to the sequence of
Dj_aH_000_01290HH. The positive cDNA clone with the longest insert was
re-cloned into pBluescript SK(-) (Stratagene).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out using 20 ng/ml of
digoxygenin (DIG)-labeled riboprobes (Roche), as previously described
(Umesono et al., 1997;
Agata et al., 1998).
Preparation of antibody against DjCHC
A fragment of Dj_aH_000_01290HH (amino acids 1190-1462) was inserted into
pQE30 vector (Qiagen) and the construct was introduced into E. coli
strain JM109. The fusion protein with a histidine tag was induced with
isopropyl-ß-D-thiogalactopyranoside and purified according to the
supplier's protocol (Qiagen). For further purification, the gel slice
containing the target protein was cut out after SDS-PAGE, and the purified
protein was collected using an electroeluter (Amicon). The purified
recombinant DjCHC protein was injected with Titer Max Gold adjuvant (CytRx)
into mice (Balb/c) three times at intervals of 1 month. The antiserum against
the fusion protein was prepared according to a standard procedure
(Orii et al., 2002).
Whole-mount immunostaining
Whole-mount immunostaining was performed as described previously
(Cebrià et al., 2002c).
Planarians were stained using mouse antibodies: 1/2000 anti-planarian
synaptotagmin (anti-DjSYT) (Tazaki et al.,
1999); 1/2000 anti-DjCHC; 1/2000 anti-planarian G-protein ß
subunit (anti-DjGß) (AB245430); 1/5000 VC-1 monoclonal antibody
(Sakai et al., 2000); and
1/200 anti-phospho histone H3 antibody (anti-H3P)
(Hendzel et al., 1997)
(Upstate Biotechnology), all of which were diluted in 10% goat serum in 0.1%
Triton X-100-containing PBS (TPBS).
RNA interference
Double-stranded RNA (dsRNA) was synthesized essentially as previously
described (Sánchez Alvarado and
Newmark, 1999). Control animals were injected with dsRNA for green
fluorescence protein (GFP), a protein not found in planarians. At 4 hours
after the third injection, planarians were amputated immediately posterior to
the auricles and pharynxes, and the resulting pieces were used for various
assays.
BrdU labeling and detection in RNAi-treated planarians
BrdU labeling was performed by microinjection basically as described
previously (Newmark and Sánchez
Alvarado, 2000). RNAi-treated planarians (RNAi planarians) were
prepared as described above and then cut into three pieces. At 3 hours after
amputation, 10 mg/ml of BrdU was injected once into the intestinal tract of
the trunk-piece. At either 4 or 48 hours after the injection, planarians were
fixed and BrdU was detected immunohistochemically
(Agata et al., 1998).
FACS and the culturing of sorted primary neurons
Fluorescence-activated cell sorting (FACS) and the culturing of planarian
neural cells were performed basically as previously described
(Asami et al., 2002). In total,
50 RNAi planarians were cut into head, trunk and tail pieces. After 4 days,
the regenerated heads of the trunk and tail pieces (total of 100 pieces) were
collected and used for FACS. The collected cells were maintained statically in
a humidified incubator at 22°C for 3 days in vitro (DIV).
Immunocytochemistry
Cultured cells were fixed in 4% PFA, 5/8 Holtfreter's solution for 60
minutes at 4°C, washed three times with TPBS and blocked in 10% goat serum
in TPBS for 30 minutes at 4°C. The cells were then incubated with 1/2000
diluted mouse anti-DjCHC, mouse anti-DjSYT, mouse anti-DjGß or hamster
anti-planarian 14-3-3
(whose signal was distributed throughout the whole
cell; AB245429; used as counterstaining) for 60 minutes at 4°C. The
samples were washed with 10% goat serum in TPBS for 5 minutes three times and
signals were detected with 1/400 Alexa Fluor 488-conjugated goat anti-mouse
IgG(H+L) (Invitrogen) or 1/400 Alexa Fluor 546-conjugated goat anti-hamster
IgG(H+L) (Invitrogen) diluted 1/400 in 10% goat serum in TPBS for 60 minutes
in the dark. After the samples were washed three times with TPBS, cell nuclei
were labeled with Hoechst 33342 (Invitrogen). Fluorescence was detected with
an LSM 510 confocal microscope (Carl Zeiss).
TUNEL assay of regenerating planarian neural cells
The head-abundant cell fraction on day 4 of regeneration (R4HAC) was
cultured and collected at 0, 1, 2 or 3 DIV, and TUNEL reactions were performed
(Hwang et al., 2004). The
signal was enhanced using 1/400 Alexa Fluor 488-conjugated anti-fluorescein
(Invitrogen). Immunostaining was performed using 1/400 hamster anti-planarian
14-3-3 for counterstaining and 1/400 Alexa Fluor 546-conjugated
anti-hamster IgG(H+L) (Invitrogen) as a counterstain.
Statistical evaluation
Quantitative data were analyzed by one-way analysis of variance (ANOVA) and
the statistical significance of differences between test results was
determined by Student's t-test; P values greater than 0.05
were taken as not significant (NS). For in vitro analysis, data from at least
eight images were averaged.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and named DjCHC. Sequencing analysis revealed that
DjCHC has five highly conserved functionally distinct domains - the
globular N-terminal domain, knee region, distal domain, proximal domain and
C-terminal end (Ybe et al.,
1999; Fotin et al.,
2004; Wilbur et al.,
2005) - and seven clathrin heavy-chain repeat (designated
CHCR1-CHCR7) motifs (residues 546-1583) (see Fig. S1A in the supplementary
material) (Ybe et al., 1999;
Wilbur et al., 2005).
Phylogenetic analysis using the full-length DjCHC showed high homology of this
protein to the clathrin heavy chains (CHCs) of other eukaryotes (see Fig. S1B
in the supplementary material).
Expression of DjCHC mRNA and proteins
Intact planarians were examined for the expression pattern of
DjCHC. In agreement with previous reports, whole-mount in situ
hybridization showed strong and ubiquitous expression of DjCHC
transcripts (Fig. 1A). In
X-ray-irradiated planarians, weaker DjCHC signals were detected than
in intact planarians, suggesting that DjCHC was expressed in various
tissues, including in stem cells (Fig.
1B). To analyze the expression pattern of DjCHC protein,
anti-DjCHC antibody was produced. DjCHC protein was also detected
ubiquitously, but was detected especially strongly in the spongy region
composed of the neurites of the neural cells
(Fig. 1D). Confocal microscopy
of the brain revealed much higher expression of DjCHC protein in the CNS
compared with in other organs (Fig.
1E). Thus, the DjCHC gene might play a major role in the
planarian CNS. Expression of the DjCHC gene within the newly formed
blastema was first detected at day 2 of regeneration as a weak signal in two
clusters of cells, which probably corresponded to the primordium of the
inverted U-shaped brain region (Fig.
1G).
|
|
)
and anti-DjGß, which stains the lateral branches of the brain, revealed
that DjCHC- RNAi-treated planarians could not regenerate their brain
normally (Fig. 2C), which was
confirmed by the fact that they could not recognize the direction of light and
they hardly moved (see Fig. S2C in the supplementary material). To determine
whether DjCHC gene function is specific to the brain, we investigated
the effects of DjCHC-RNAi in other tissues and organs by using
molecular markers, including a tail-specific homeobox gene, DjAbd-Ba
(Nogi and Watanabe, 2001).
Interestingly, the homeobox gene was expressed with the normal spatial
pattern, but the regenerating ventral nerve cords (VNCs) in the posterior
blastema were strongly disorganized (Fig.
3A). No disorganization was detected in the newly formed pharynx
(Fig. 3B) or in the
regenerating intestinal duct or body-wall musculature
(Nogi and Levin, 2005;
Orii et al., 2002)
(Fig. 3C,D).
|
;
Reddien et al., 2005b;
Salvetti et al., 2005).
Therefore, we speculated at first that the phenotype of the
DjCHC-RNAi-treated planarians was caused by a stem cell defect,
because DjCHC is expressed ubiquitously (including in stem cells). To
test this possibility, we compared the phenotypes of planarians in which the
stem cells were ablated by X-ray irradiation
(Wolff and Dubois, 1948;
Baguñà et al.,
1989), DjCHC-RNAi-treated planarians and planarians that
had undergone both treatments. It is known that the cell number in planarians
is determined by the balance between cell proliferation and cell death, and
even starved planarians can maintain cell proliferation while their body is
shrinking in size via `tissue homeostasis'
(Baguñà and Romero,
1981; Sánchez Alvarado,
2006). We investigated the phenotype of
DjCHC-RNAi-treated planarians whose stem cells were destroyed by
X-ray irradiation. Although, in control and X-ray-irradiated planarians, we
could not find any defects through 14 days after irradiation or
RNAi-treatment, CNS atrophy could be detected in DjCHC-RNAi-treated
planarians from 5 day after RNAi-treatment, and the number of CNS-regressed
planarians progressively increased thereafter until almost all planarians
showed head atrophy by 14 days after RNAi-treatment
(Fig. 4A,B). Furthermore,
ablation of the stem cells by X-irradiation enhanced the CNS atrophy of the
DjCHC-RNAi-treated planarians, perhaps because it caused a decrease
in the cell number in the CNS
(Baguñà and Romero,
1981; Hwang et al.,
2004). These results support the notion that the CNS atrophy
caused by DjCHC-RNAi treatment was due to effects on differentiated
neuronal cells rather than on stem cells.
To investigate the stem cells of DjCHC-RNAi-treated planarians in
greater detail, the cell division and migration of stem cells were assessed
using BrdU (Newmark and Sánchez
Alvarado, 2000). The number and distribution of BrdU-positive stem
cells (which might migrate to the blastema) did not differ between control and
DjCHC-RNAi-treated planarians at 4 or 48 hours after BrdU injection
(Fig. 4C). Furthermore,
analysis of the number of mitotic stem cells using anti-H3P
(Newmark and Sánchez Alvarado,
2000) in uncut DjCHC-RNAi-treated planarians 14 days
after RNAi treatment showed that H3P-positive cells were present in
DjCHC-RNAi-treated planarians and survived for over 3 weeks even
though the CNS was atrophied (Fig.
4D, and see Fig. S2B in the supplementary material). There were
around 130 H3P-positive cells in an 8-mm-long planarian in both control
(130.3±6.5 cells) and DjCHC-RNAi-treated (128.2±7.0
cells) planarians (not significantly different)
(Fig. 4D,E). On the other hand,
in planarians at 14 days after X-ray irradiation, when half of the treated
animals had died (see Fig. S2B in the supplementary material), there were no
H3P-positive cells and the CNS was normal
(Fig. 4D,E). However, Reddien
et al. (Reddien et al., 2005b)
reported that a planarian homolog of the piwi gene was involved in
supporting the generation of cells that promote regeneration and homeostasis,
but not the division or migration of stem cells; we therefore performed
further histological analyses using several molecular markers under
DjCHC-knockdown conditions to identify the stage at which
DjCHC is required for proper brain formation during head
regeneration.
|
)],
for DjvlgA expressed in the early blastema and stem cells
(Shibata et al., 1999), and
for nou-darake (ndk) expressed in the predicted brain region
in the anterior blastema (Cebrià et
al., 2002a). Djnlg, DjvlgA and ndk were detected
in DjCHC-RNAi-treated planarians as in control planarians
(Fig. 5A-C), indicating that
DjCHC was not required for the formation of the blastema or brain
rudiment early in regeneration.
Moreover, expression analysis was performed using the DjotxA,
DjotxB and Djotp genes, which are thought to be involved in
patterning of the brain 48 hours after decapitation
(Umesono et al., 1997;
Umesono et al., 1999). The
expression patterns of these genes were not grossly different between control
and DjCHC-RNAi-treated planarians, suggesting that brain patterning
was not affected by DjCHC-RNAi
(Fig. 5D,E).
Silencing of DjCHC perturbs the proper formation of the CNS after patterning and cell differentiation
Immunostaining using anti-DjSYT and anti-DjGß 72 hours after
amputation revealed conspicuous abnormalities of the regenerating brain.
Positive signals were detected in the newly formed blastema region
(Fig. 5F), but the brain
architecture was disorganized. Although the regenerated brain was not properly
organized, some normal features, such as an inverted U-shaped structure and
lateral branches, were observed. Staining of visual cells with specific
antibody against visual neurons (VC-1)
(Sakai et al., 2000) revealed
that eye regeneration was also perturbed. Planarian eye regeneration can be
divided into three steps: formation of two visual-cell clusters in the dorsal
side of the anterior blastema; projection of the left and right visual neurons
to the opposite ones; and connection of the optic nerves onto the brain
(Inoue et al., 2004). Although
the differentiation of visual neurons was not inhibited by DjCHC
silencing, projection of left and right visual neurons to the opposite ones
and connection of the optic nerves onto the brain were inhibited
(Fig. 5G), implying that
DjCHC might be required for the projection of axons during CNS
regeneration.
|
). The
DjCHC-RNAi-treated HAC contained DjCHC protein (data not shown) due
to the fact that RNAi does not affect already expressed proteins. The RNAi
technique, combined with the strong regenerative ability of planarians, makes
it possible to disrupt the gene expression specifically in newly formed
differentiated structures (T. Takano, J. Pulvers, T. Inoue, H. Tarui, H.
Sakamoto, K.A. and Y. Umesono, unpublished). We used the newly formed head of
DjCHC-RNAi-treated planarians to collect the head-abundant cell
fraction on day 4 of regeneration (R4HAC) by FACS. This method is optimal for
adjusting the start point of the RNAi effect, and therefore to perform
time-course analysis. Fig. 6A shows a comparison of the FACS profiles of the body and head fractions. The specific cell fraction designated R4HAC (Fig. 6A, black ellipse in middle panel) was detected in cells from the head but not from the body (Fig. 6A, left panel). R4HAC accounted for approximately 10% of the total cells in the head fraction in both control (10.9±2.9%) and DjCHC-RNAi-treated (9.3±3.5%) planarians (not significantly different) (Fig. 6B). Over 95% of the collected control and DjCHC-RNAi-treated R4HAC expressed the neural marker DjSYT (data not shown). Immunostaining of R4HAC at 0 days in vitro (DIV) using anti-DjCHC showed that 90.6±1.4% of control R4HAC were positive for DjCHC protein. DjCHC-RNAi treatment drastically reduced the percentage of DjCHC-protein-positive cells to 23.5±4.6%, and even positive cells expressed lower levels of DjCHC protein (Fig. 6C).
R4HAC began to extend neurites soon after culturing (Fig. 6D). Although the neurites of some cells regressed after 1 DIV, those of other cells showed additional elongation and branching by 3 DIV. In the cultures of DjCHC-RNAi-treated R4HAC, projection of neurites was observed, but almost all of the extended neurites regressed during culturing. The neurites of the DjCHC-RNAi-treated R4HAC (11.0±1.6 µm) were shorter than those of the control R4HAC at 1 DIV, and the length was further shortened to 2.1±0.3 µm at 3 DIV (Fig. 6F). The percentage of neurite-extending cells in DjCHC-RNAi-treated R4HAC was similarly decreased (Fig. 6E). Thus, neuronal cells of DjCHC-RNAi-treated planarians could not progressively extend the growth of or maintain their neurites.
To further evaluate the molecular features of DjCHC-RNAi-treated
R4HAC, immunocytochemical analysis was performed using antibodies against
DjCHC and DjSYT. DjCHC protein was strongly detected in neurites in R4HAC, but
was not detected in DjCHC-RNAi-treated R4HAC, at 3 DIV. More than 95%
of the control R4HAC at 3 DIV still expressed DjSYT protein, which was
accumulated in neurites, consistent with the expression pattern in vivo (Figs
2,
3 and
4)
(Tazaki et al., 1999). DjSYT
expression was detected at least until 3 DIV
(Fig. 6G), suggesting that the
DjCHC-RNAi-treated R4HAC might maintain their neural identity.
DjCHC-silencing induces neuronal apoptosis
To investigate how DjCHC mediates neuronal cell survival, the
TdT-mediated dUTP nick-end labeling (TUNEL) assay
(Hwang et al., 2004) was
performed on R4HAC to detect apoptosis. DjCHC-RNAi treatment induced
an increase in apoptosis (Fig.
7A-D). The degree of neuronal apoptosis was also quantified by
measuring the percentage of TUNEL-positive cells among the total cells, and
the data showed that the increase peaked at 3 DIV, when over 80% of the
neurons were either dying or dead (Fig.
7A,B,E) and exhibited typical features of apoptosis
(Fig. 6G,
Fig. 7A-D). To examine whether
DjCHC-silencing specifically caused apoptosis in
DjCHC-protein-negative cells, we performed double TUNEL staining and
immunostaining using anti-DjCHC. In the DjCHC-positive cell fraction, there
was no significant difference in the fraction of TUNEL-positive cells between
the control and DjCHC-RNAi-treated R4HAC, whereas, in the
DjCHC-negative cell fraction, DjCHC-RNAi treatment markedly elevated
the fraction of TUNEL-positive cells (Fig.
7C,D,F,G). Furthermore, all TUNEL-positive cells in the
DjCHC-RNAi-treated R4HAC lacked neurites
(Fig. 7B). Thus, dysfunction of
the DjCHC gene might induce cell-autonomous apoptosis in neuronal
cells.
|
; Wu,
2004). When we knocked-down the planarian unc13 homolog
(Djunc13A; AB281585) planarian syntaxin-binding protein homolog
(DjsbpA; AB281586) and the Djsyt genes involved in synaptic vesicle
transmission, we could not find any abnormalities in the regeneration or
maintenance of CNS architecture, although behavioral abnormalities were
detected in these gene-knockdown planarians
(Fig. 8A). This suggested that
clathrin-mediated synaptic-vesicle recycling might not be important for the
regeneration and maintenance of the CNS.
|
).
Thus, it was difficult to distinguish whether CNS abnormalities were caused by
exocytic or endocytic dysfunction. To address the function of DjCHC
necessary for neuronal survival, we compared the effects of DjCHC
with those of the adaptor protein complex-1 (AP-1) gene,
whose protein product is found on the TGN and endosomes, and the AP-2
gene, which is specifically involved in endocytosis
(Boehm and Bonifacino, 2001;
Robinson, 2004). First, we
isolated the planarian mu-1 gene, encoding a subunit of
AP-1, and the mu-2 gene, encoding a subunit of
AP-2, and named them Djmu-1 and Djmu-2,
respectively (Fig. 8B). They
are expressed ubiquitously (Fig.
8C,D). Then, we performed loss-of-function analysis by RNAi. Like
DjCHC-RNAi-treated planarians, Djmu-2-RNAi-treated
planarians could not regenerate their brain properly, although they showed a
relatively weak phenotype (Fig.
8E). By contrast, no gross effects were detected in the brain
regeneration of Djmu-1-RNAi-treated planarians. Moreover, gene
knockdown of the 
and 
1 genes, which encode the other
subunits of AP-1 (Hirst and
Robinson, 1998) had no effect on CNS regeneration (data not
shown). No additive phenotype was seen when double knockdown of
Djmu-1 and Djmu-2 or of DjCHC and Djmu-2
was performed (Fig. 8E,F).
These data suggest that AP-2- and clathrin-mediated endocytosis, but not
protein trafficking from the TNG via AP-1, are essential for proper CNS
regeneration and maintenance in planarians. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wilbur et
al., 2005).
Clathrin silencing in the planarian prevented proper regeneration and
maintenance of the CNS (Fig.
2). The headless or head-atrophied animals resulting from ablation
of endocytic components did not die, because planarians have extraordinary
regenerative potential. This feature of planarians was advantageous for
analyzing the phenotype during the process of CNS formation and maintenance in
vivo. Although the DjCHC transcripts were expressed ubiquitously, the
DjCHC protein was strongly accumulated in neurites of CNS neurons
(Fig. 1). Furthermore, gene
knockdown of DjCHC without the regeneration process revealed that the
CNS atrophied independently of stem cells
(Fig. 4). These data imply that
DjCHC might have an important role in neuronal cells in the formation
and maintenance of the proper brain architecture. Notably, a tail, pharynx and
intestinal tract were regenerated in the head- and tail-pieces of
DjCHC-RNAi-treated planarians
(Fig. 3A-C), indicating that
intercalary regeneration, the principal manner of regeneration, generally
occurred normally in DjCHC-depleted planarians
(Kato et al., 1999;
Agata et al., 2003). We could
find no defects caused by silencing of DjCHC except for those in the
CNS, despite the ubiquitous expression of this gene.
Clathrin dysfunction perturbs CNS formation after patterning and cell differentiation
We could find no deficiency in stem cell division, differentiation or
migration, as indicated by BrdU, H3P and FACS analysis. Histological analysis
using several genes involved in different steps of CNS formation revealed that
blastema formation, brain induction, patterning and cell differentiation
occurred normally until 48 hours after decapitation in
DjCHC-RNAi-treated planarians
(Fig. 5). At 72 hours after
decapitation, DjCHC-RNAi-treated planarians could express a
postmitotic neural-marker protein and could form a disorganized brain
(Fig. 5F). Furthermore,
although clathrin dysfunction did not disturb the differentiation or the
distribution of visual neurons, projection of left and right visual neurons to
the opposite ones and the connection of the optic nerves onto the brain were
defective (Fig. 5G), implying
that DjCHC is not needed for cell differentiation of the brain and
eye, but rather for the projection of the axons and formation of the proper
CNS. Thus, clathrin is essential for the construction of the proper
architecture of the CNS after patterning and neuronal cell differentiation,
and before neural circuit formation during CNS regeneration, as well as for
the maintenance of the proper architecture of the CNS after synaptic
connection in planarians (Fig.
9).
|
; Motley et
al., 2003). The combination of RNAi and the in vitro analysis
described here should prove useful for the functional characterization of
neural genes at the cellular level during the regeneration and maintenance of
the planarian CNS. At 3 DIV, almost none of the DjCHC-RNAi-treated
R4HAC could lengthen or maintain their neurites, which seemed to be regressed
but not retracted (Fig. 6D-F).
These observations are consistent with the features of RNAi-treated planarians
in vivo and suggest that DjCHC-silenced neurons could extend some
neurites, but could not further extend or maintain these neurites, and seemed
to undergo cell death after the completion of differentiation.
Interestingly, although the percentage of neurite-extending cells and the
neurite length per cell of the control R4HAC peaked by 1 DIV and were
subsequently reduced, the length of the neurite-extending cells tended to be
slightly increased (from 30.5±1.9 µm at 1 DIV to 41.5±6.5
µm at 3 DIV) (data not shown). It is tempting to speculate that some
mechanisms related to appropriate synaptic connections or trophic factors
might be important for neurite outgrowth and maintenance in this organism
(Vaudry et al., 2002;
Howe and Mobley, 2005). In
contrast to the findings in control R4HAC, both the percentage of
neurite-extending cells and neurite length of DjCHC-RNAi-treated
R4HAC progressively decreased during culturing, indicating that dysfunction of
clathrin-mediated endocytosis might accelerate neurite regression
(Fig. 6E,F). Previous reports
suggested that neurotrophic factors such as nerve growth factor are produced
and released in target tissues to activate receptors on the presynaptic
elements of innervating neurons, thereby signaling to regulate the neuronal
survival of these neurons at nerve terminals involved in mediating endocytosis
(Casaccia-Bonnefil et al.,
1999; Ye et al.,
2003; Yano and Chao,
2004; Howe and Mobley,
2005).
|
). However, clathrin-depleted HeLa cells do not show any
apparent increase in apoptosis (Motley et
al., 2003; Hinrichsen et al.,
2003). These discrepancies will be resolved by further analyses
revealing the details of the mechanism of endocytosis in each cell type.
Clathrin-mediated endocytic signals are essential for proper CNS regeneration
Besides clathrin, one of the most important components of clathrin-mediated
endocytosis is AP-2, which binds both clathrin and cytoplasmic tyrosine-based
signals on transmembrane proteins (Boehm
and Bonifacino, 2001;
Robinson, 2004). AP complexes
involved in cargo selection for inclusion into coated vesicles in the late
secretory and endocytic pathways are widely distributed among eukaryotes
(Boehm and Bonifacino, 2001;
Boehm and Bonifacino, 2002).
Our finding that the disruption of endocytic genes caused defective CNS
regeneration, but disruption of an exocytic gene, Djmu-1, did not,
suggested that clathrin-mediated endocytosis at the plasma membrane, not
protein trafficking at the TNG, is necessary for CNS formation
(Fig. 8E). This is consistent
with our finding that the DjSYT and DjGß proteins were detected in
neurites in DjCHC-RNAi-treated planarians, indicating that protein
trafficking occurred without the DjCHC gene
(Fig. 2C,
Fig. 5F). However, the
possibility that AP-1-independent alternative factors in the clathrin-coated
pits at the TGN are involved in proper CNS formation and maintenance could not
be completely ruled out.
Interestingly, the gene-knockdown animals for DjCHC and
Djmu-2 did not show exactly the same phenotypes: DjCHC-RNAi
treatment caused a more-severe phenotype than Djmu-2-RNAi treatment.
The distribution of 
-adaptin and epsin and the cellular morphologies
are different in CHC-siRNA- and mu-2-siRNA-treated HeLa cells
(Motley et al., 2003;
Hinrichsen et al., 2003).
Furthermore, clathrin-mediated endocytosis can still occur in the absence of
AP-2, and AP-2 may not be essential for the recruitment and assembly of
clathrin at the plasma membrane (Motley et
al., 2003; Hinrichsen et al.,
2003; Conner and Schmid,
2003). Our data, together with these previous findings, lead us to
speculate that clathrin, AP-2 and alternative adaptors may co-assemble at the
plasma membrane, bringing cargo with different types of internalization
signals into the coated pit. In the absence of AP-2, alternative adaptors are
still recruited onto the plasma membrane and co-assemble with clathrin
(Mishra et al., 2001;
Mishra et al., 2002;
Motley et al., 2003;
Hinrichsen et al., 2003;
Sever, 2003). Furthermore,
simultaneous silencing of Djmu-1 and Djmu-2 or
DjCHC and Djmu-2 did not cause additive effects on CNS
formation (Fig. 8E,F),
indicating that the more-severe phenotype of DjCHC- RNAi-treated
planarians was probably not caused by inhibition of both endocytosis and
trafficking at the TNG. In planarians, alternative adaptors in addition to
AP-2 in the clathrin-coated pits mediating endocytosis may be necessary for
proper CNS regeneration and maintenance.
In summary, our data demonstrate that clathrin-mediated endocytosis is essential for neuronal cell survival, and for neurite outgrowth and maintenance, after the completion of neuronal differentiation to enable the formation as well as the maintenance of the proper functional CNS in planarians in vivo (Fig. 9). Studies of this enormously interesting organism will advance our understanding of the physiological roles, and the cellular and molecular mechanisms of intercellular communication and cellular signaling in maintaining the proper function of the nervous system.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1679/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Faculty of Science, Kumamoto University, 2-39-1, Kurokami,
Kumamoto, 860-8555, Japan
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agata, K. and Watanabe, K. (1999). Molecular and cellular aspects of planarian regeneration. Semin. Cell Dev. Biol. 10,377 -383.[CrossRef][Medline]
Agata, K., Soejima, Y., Kato, K., Kobayashi, C., Umesono, Y. and Watanabe, K. (1998). Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers. Zool. Sci. 15,433 -440.[Medline]
Agata, K., Tanaka, T., Kobayashi, C., Kato, K. and Saitoh, Y. (2003). Intercalary regeneration in planarians. Dev. Dyn. 226,308 -316.[CrossRef][Medline]
Allen, C., Goulding, D. and Field, M. (2003). Clathrin-mediated endocytosis is essential in Trypanosoma brucei. EMBO J. 22,4991 -5002.[CrossRef][Medline]
Asami, M., Nakatsuka, T., Hayashi, T., Kou, K., Kagawa, H. and Agata, K. (2002). Cultivation and characterization of planarian neuronal cells isolated by fluorescence activated cell sorting (FACS). Zool. Sci. 19,1257 -1265.[CrossRef][Medline]
Baguñà, J. and Romero, R. (1981). Quantitative analysis of cell types during growth, degrowth and regeneration in the Planarians Dugesia mediterranea and Dugesia tigrina. Hydrobiologia 84,181 -194.[CrossRef]
Baguñà, J., Saló, E. and Romero, R. (1989). Effects of activators and antagonists of the neuropeptides substance P and substance K on cell proliferation in planarians. Int. J. Dev. Biol. 33,261 -266.[Medline]
Boehm, M. and Bonifacino, J. (2001). Adaptins:
the final recount. Mol. Biol. Cell
12,2907
-2920.
Boehm, M. and Bonifacino, J. (2002). Genetic analyses of adaptin function from yeast to mammals. Gene 286,175 -186.[CrossRef][Medline]
Casaccia-Bonnefil, P., Gu, C. and Chao, M. (1999). Neurotrophins in cell survival/death decisions. Adv. Exp. Med. Biol. 468,275 -282.[Medline]
Cebrià, F. and Newmark, P. A. (2005).
Planarian homologs of netrin and netrin receptor are required for proper
regeneration of the central nervous system and the maintenance of nervous
system architecture. Development
132,3691
-3703.
Cebrià, F., Kobayashi, C., Umesono, Y., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T., Itoh, M., Taira, M., Sánchez Alvarado, A. et al. (2002a). FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. Nature 419,620 -624.[CrossRef][Medline]
Cebrià, F., Kudome, T., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T. and Agata, K. (2002b). The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system. Mech. Dev. 116,199 -204.[CrossRef][Medline]
Cebrià, F., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T. and Agata, K. (2002c). Dissecting planarian central nervous system regeneration by the expression of neural-specific genes. Dev. Growth Differ. 44,135 -146.[CrossRef][Medline]
Conner, S. and Schmid, S. (2003). Differential
requirements for AP-2 in clathrin-mediated endocytosis. J. Cell
Biol. 162,773
-779.
Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S., Kirchhausen, T. and Walz, T. (2004). Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432,573 -579.[CrossRef][Medline]
Fusaoka, E., Inoue, T., Mineta, K., Agata, K. and Takeuchi,
K. (2006). Structure and function of primitive immunoglobulin
superfamily neural cell adhesion molecules: a lesson from studies on
planarian. Genes Cells
11,541
-555.
Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106,348 -360.[CrossRef][Medline]
Hinrichsen, L., Harborth, J., Andrees, L., Weber, K. and
Ungewickell, E. (2003). Effect of clathrin heavy chain- and
alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins
and receptor trafficking in HeLa cells. J. Biol. Chem.
278,45160
-45170.
Hirst, J. and Robinson, M. S. (1998). Clathrin and adaptors. Biochem. Biophys. Acta 1404,173 -193.[Medline]
Howe, C. and Mobley, W. (2005). Long-distance retrograde neurotrophic signaling. Curr. Opin. Neurobiol. 15,40 -48.[CrossRef][Medline]
Hwang, J., Kobayashi, C., Agata, K., Ikeo, K. and Gojobori, T. (2004). Detection of apoptosis during planarian regeneration by the expression of apoptosis-related genes and TUNEL assay. Gene 333,15 -25.[CrossRef][Medline]
Inoue, T., Kumamoto, H., Okamoto, K., Umesono, Y., Sakai, M., Sánchez Alvarado, A. and Agata, K. (2004). Morphological and functional recovery of the planarian photosensing system during head regeneration. Zool. Sci. 21,275 -283.[CrossRef][Medline]
Kato, K., Orii, H., Watanabe, K. and Agata, K. (1999). The role of dorsoventral interaction in the onset of planarian regeneration. Development 126,1031 -1040.[Abstract]
Koinuma, S., Umesono, Y., Watanabe, K. and Agata, K. (2003). The expression of planarian brain factor homologs, DjFoxG and DjFoxD. Gene Expr. Patterns 3, 21-27.[CrossRef][Medline]
Le Roy, C. and Wrana, J. (2005). Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat. Rev. Mol. Cell Biol. 6,112 -126.[CrossRef][Medline]
Mineta, K., Nakazawa, M., Cebrià, F., Ikeo, K., Agata, K.
and Gojobori, T. (2003). Origin and evolutionary process of
the CNS elucidated by comparative genomics analysis of planarian ESTs.
Proc. Natl. Acad. Sci. USA
100,7666
-7671.
Mishra, S., Agostinelli, N., Brett, T., Mizukami, I., Ross, T.
and Traub, L. (2001). Clathrin- and AP-2-binding sites in
HIP1 uncover a general assembly role for endocytic accessory proteins.
J. Biol. Chem. 276,46230
-46236.
Mishra, S., Keyel, P., Hawryluk, M., Agostinelli, N., Watkins, S. and Traub, L. (2002). Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor. EMBO J. 21,4915 -4926.[CrossRef][Medline]
Motley, A., Bright, N., Seaman, M. and Robinson, M.
(2003). Clathrin-mediated endocytosis in AP-2-depleted cells.
J. Cell Biol. 162,909
-918.
Murthy, V. and DeCamilli, P. (2003). Cell biology of the presynaptic terminal. Annu. Rev. Neurosci. 26,701 -728.[CrossRef][Medline]
Nakazawa, M., Cebrià, F., Mineta, K., Ikeo, K., Agata, K.
and Gojobori, T. (2003). Search for the evolutionary origin
of a brain: planarian brain characterized by microarray. Mol. Biol.
Evol. 20,784
-791.
Newmark, P. and Sánchez Alvarado, A. (2000). Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev. Biol. 220,142 -153.[CrossRef][Medline]
Niswonger, M. and O'Halloran, T. (1997a). Clathrin heavy chain is required for spore cell but not stalk cell differentiation in Dictyostelium discoideum. Development 124,443 -451.[Abstract]
Niswonger, M. and O'Halloran, T. (1997b). A
novel role for clathrin in cytokinesis. Proc. Natl. Acad. Sci.
USA 94,8575
-8578.
Nogi, T. and Watanabe, K. (2001). Position-specific and non-colinear expression of the planarian posterior (Abdominal-B-like) gene. Dev. Growth Differ. 43,177 -184.[CrossRef][Medline]
Nogi, T. and Levin, M. (2005). Characterization of innexin gene expression and functional roles of gap-junctional communication in planarian regeneration. Dev. Biol. 287,314 -335.[Medline]
Ogawa, K., Ishihara, S., Saito, Y., Mineta, K., Nakazawa, M., Ikeo, K., Gojobori, T., Watanabe, K. and Agata, K. (2002). Induction of a noggin-like gene by ectopic DV interaction during planarian regeneration. Dev. Biol. 250, 59-70.[CrossRef][Medline]
Orii, H., Ito, H. and Watanabe, K. (2002). Anatomy of the planarian Dugesia japonica I. The muscular system revealed by antisera against myosin heavy chains. Zool. Sci. 19,1123 -1131.[CrossRef][Medline]
Reddien, P. and Sánchez Alvarado, A. (2004). Fundamentals of planarian regeneration. Annu. Rev. Cell Dev. Biol. 20,725 -757.[CrossRef][Medline]
Reddien, P. W., Bermange, A. L., Murfitt, K. J., Jennings, J. R. and Sánchez Alvarado, A. (2005a). Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev. Cell 8, 635-649.[CrossRef][Medline]
Reddien, P. W., Oviedo, N. J., Jennings, J. R., Jenkin, J. C.
and Sánchez Alvarado, A. (2005b). SMEDWI-2 is a
PIWI-like protein that regulates planarian stem cells.
Science 310,1327
-1330.
Robinson, M. (2004). Adaptable adaptors for coated vesicles. Trends Cell Biol. 14,167 -174.
