NEAP/DUSP26 suppresses receptor tyrosine kinases and regulates neuronal development in zebrafish
NEAP/DUSP26 suppresses receptor tyrosine kinases and regulates neuronal development in zebrafish
OPEN Expression of neuroendocrine-associated phosphatase (NEAP, also named as dual specificity phosphatase 26, [DUSP26]) is restricted to neuroendocrine tissues. We found that NEAP, but not its phosphatase-defective mutant, suppressed nerve growth factor (NGF) receptor TrkA and fibroblast growth factor receptor 1 (FGFR1) activation in PC12 cells upon NGF stimulation. Conversely, suppressing NEAP expression by RNA interference enhanced TrkA and FGFR1 phosphorylation. NEAP was capable of de-phosphorylating TrkA and FGFR1 directly in vitro. NEAP-orthologous gene existed in zebrafish. Morpholino (MO) suppression of NEAP in zebrafish resulted in hyper-phosphorylation of TrkA and FGFR1 as well as abnormal body postures and small eyes. Differentiation of retina in zebrafishes with NEAP MO treatment was severely defective, so were cranial motor neurons. Taken together, our data indicated that NEAP/DUSP26 have a critical role in regulating TrkA and FGFR1 signaling as well as proper development of retina and neuronal system in zebrafish.
Proteins of the nerve growth factor (NGF) family, also known as neurotrophins (NTs), are evolutionary conserved
factors with numerous functions. NTs (including NGF, brain-derived growth factor [BDGF], NT3, and NT4/5)
stimulate signals through receptor tyrosine kinases (RTKs, including TrkA, B, and C) and/or p75NTR, a tumor
necrosis factor receptor family member. These two distinct classes of receptors are preferentially activated by
mature NT and unprocessed pro-NT, respectively1?3. Due to the earlier identification, more information is known
about the NGF-induced signaling and its biological effects mediated by TrkA and p75NTR1. Sympathetic neurons
express both p75NTR and TrkA; while pro-NGF activates p75NTR and induces cell death, mature NGF activates
both p75NTR and TrkA to promote cell survival1, 3. Signaling events triggered by Trk receptors are similar to
those of other RTKs; however, Trk-containing endosomes delivering the activated signaling complex to the
distant somas through retrograde axonal transportation is a unique feature in nerve cells2, 4.
Phosphorylation is a prominent post-translational modification in proteins and its levels are balanced by
actions of kinases and phosphatases. Dual-specificity phosphatases (DUSPs) are particularly interesting among all
protein phosphatases5?7. DUSPs, defined by sequence homology to MAP kinase phosphatases (MKPs), are named
for their ability to de-phosphorylate both serine/threonine and tyrosine residues. Some DUSP members have
been classified as atypical DUSPs due to the presence of a conserved DUSP phosphatase domain while lacking
other recognizable domains and showing little or no activity against major MAPKs5?7.
We previously identified an atypical DUSP named neuroendocrine-associated phosphatase (NEAP), also
named as DUSP26 or MKP-88?10. We found that NEAP/DUSP26 is specifically expressed in neuroendocrine
cells/tissues and does not inactivate MAPKs9. NEAP/DUSP26 suppresses NGF-induced PC12 differentiation
through down-regulating the phosphatidylinositide-3 kinase (PI3K)/Akt pathway and impairs PC12 cell growth
through suppressing EGFR expressions by WT1-mediated transcriptional suppression9, 11. Nevertheless, other
reports find that NEAP/DUSP26 is capable of inactivating p38-MAPK8, 12. NEAP/DUSP26 dephosphorylates p53
and inhibits its tumor suppressor activity in neuroblastoma cells13. Adenylate kinase 2 (AK2) has been shown to
enhance NEAP/DUSP26 activity against FADD and suppress cell growth14. A recent report shows that NEAP/
DUSP26 promotes A?42 production through enhancing axonal transportation of amyloid precursor protein
In this study we found that NEAP/DUSP26 expression decreased TrkA and FGFR1 phosphorylation induced
by NGF and, conversely, knockdown of NEAP enhanced TrkA and FGFR1 activation. NEAP/DUSP26
dephosphorylated TrkA and FGFR1 directly. Suppression of NEAP/DUSP26 expression in zebrafish embryos also caused
activation of TrkA and FGFR1 as well as defects in retinal and neuronal development. Our data supported a
critical role of NEAP/DUSP26 in regulating RTK signaling in neuronal system.
NEAP decreases NGF-, but not heregulin-, induced Akt activation in PC12 cells. We previously
showed that NEAP down-regulated NGF-induced Akt activation specifically without affecting other signaling
pathways, such as ERK, JNK, and p38-MAPK. However, further examinations showed that PI3K/Akt pathway
was not directly targeted by NEAP9. To know whether NEAP could down-regulate the PI3K/Akt pathway in
response to different stimuli, we treated PC12-cDNA, NEAP, and NEAP-C152S (phosphatase-inactive mutant)
cells with either NGF or heregulin and examined for Akt phosphorylation. We found that although NEAP
suppressed NGF-induced Akt phosphorylation at Ser 473, it did not decrease heregulin-induced Akt
phosphorylation as much in the same PC12 cell system (Fig.?1). This result indicated that, instead of targeting PI3K/Akt,
NEAP must have acted on a molecule(s) more upstream in the TrkA signaling pathway.
NEAP suppresses TrkA and FGFR1 phosphorylation in PC12 cells upon NGF stimulation. The
results shown in Fig.?1 prompted us to examine whether NGF receptor TrkA was directly targeted by NEAP.
We found that TrkA phosphorylations on Tyrs 490 and 674/675 were decreased by NEAP, but not by the
phosphatase-inactive mutant (NEAP-C152S), upon NGF stimulation. Phosphorylation of FRS2 (at Tyrs 196 and
436), an adaptor molecule mediating TrkA signaling16, was also decreased in the presence of NEAP (Fig.?2a).
FGFR1 is known to be activated by NGF signaling through receptor cross talk and autocrine effect17. We found
that NGF-induced FGFR1 phosphorylation on Tyr 653/654 was also decreased by NEAP expression in PC12 cells
Suppressing NEAP expression by the RNA interference method decreased the levels of both TrkA and FGFR1
(Fig.?3a). In this reversed experiment, both NGF-induced TrkA and FGFR1 Tyr phosphorylation (normalized
by receptor levels) were elevated in PC12-shNEAP cells in comparison to those in PC12-pSuper control cells
(Fig.?3a). TrkA is down-regulated by ubiquitination and degradation upon ligand stimulation and
phosphorylation18, 19. In light of the low level of TrkA in PC12-shNEAP cells, we examined whether lack of NEAP caused a
quicker turnover of TrkA protein. Upon inhibition of protein synthesis by puromycin, NGF-induced TrkA
degradation indeed was faster in PC12-shNEAP cells in comparison to that in PC12-pSuper control cells (Fig.?3b).
Our data clearly indicated that TrkA, or its closely associated signaling molecule(s), was targeted by NEAP in the
PC12 cell system.
NEAP de-phosphorylates TrkA and FGFR1 directly in vitro. Tyr residues 674 and 675 are located
at the activating loop of the TrkA kinase domain and are important for NGF-induced biological functions20.
Phosphorylation(s) at the activating loop is a common feature for kinase activation and the similar dual Tyr
phosphorylation were found in the activating loops of FGFR and IGFR family members, but not in EGFR (Fig.?4a)21, 22.
In light of the data in the PC12 cell system, we tested whether TrkA, FGFR1, and IGFR1 were direct
substrates of NEAP in vitro. As shown in Fig.?4b, NEAP, but not NEAP-C152S mutant, dephosphorylated TrkA
on Tyr 674/675 in vitro. The addition of GST-AK2, a previously reported NEAP-associated protein14, enhanced
the de-phosphorylation ability (Fig.?4b). Similarly, NEAP could de-phosphorylate FGFR1 either in the
presence or absence of AK2 (Fig.?4c). However, only a minimal de-phosphorylation effect on IGFR1 was observed
under the same condition (Fig.?4d). The data of in vitro reactions showed that TrkA and FGFR1 were directly
de-phosphorylated by NEAP.
NEAP is expressed in the central nerve system of zebrafishes. We then used zebrafish as an
experimental system to study the biological role of NEAP in vivo, since NEAP orthologous gene can be found in
zebrafish and its protein was highly conserved (with sequence 73% homologous and 64% identical to human NEAP) in
comparison to those of rodents and human (Fig.?5a). NEAP mRNA was detectable in the zebrafish eggs at 0 h post
fertilization (hpf). The expression levels of NEAP increased evidently at 48 hpf, which is later than the
development of neuromeres (at 18 hpf) and the distinctive morphogenesis of central nerve system (CNS) at 24 hpf23, in
the zebrafish embryos (Fig.?5b). Using the whole mount in situ hybridization (WMISH) assay, we detected NEAP
mRNA expression most strongly in the brain of zebrafish embryos (Fig.?5c). NEAP mRNA could also be detected
in zebrafish retina (Fig.?5c, lower right panel). These results were consistent with our previous report showing that
NEAP is preferentially expressed in human neuroendocrine tissues9.
Suppression of NEAP leads to hyper-phosphorylation of TrkA and FGFR1. We then suppressed
NEAP expression in zebrafish embryos using the morpholino (MO) approach. As shown in Fig.?6a, a MO
targeting the fish NEAP mRNA decreased the expression of NEAP but not that of a homologous DUSP23. Knockdown
of NEAP was associated with increased phosphorylation of TrkA and FGFR1 in zebrafish embryos, suggesting
that these two receptor tyrosine kinases (RTKs) were regulated by NEAP in vivo (Fig.?6b). In general, NEAP MO
injection had various effects on embryos. Some but not all of the injected fishes showed abnormal body curvature
(Fig.?6c). However, all of the NEAP MO morphants had significantly smaller head (468 ? 48 vs. 550 ? 9 ?m,
p < 0.001; Fig.?6d) and smaller eyes (172 ? 44 vs. 260 ? 27 ?m, p < 0.001; Fig.?6e) in comparison to those of fish
embryos treated with control scrambled (SC) MO.
Zebrafishes lacking NEAP have developmental defects in retina and neuronal system. The
smaller eyes and heads in NEAP MO morphants were intriguing. Hematoxylin & eosin staining and microscopic
examinations showed that the NEAP MO caused defective retinal development. Unlike the zebrafishes with SC
MO had differentiated and layered retina, NEAP MO morphants? retina containing cells without distinctive layers
even at 72 hpf (Fig.?7a). Also, the eye lens of NEAP morpholino-treated fishes were less developed than that of
the control group (Fig.?7a). This result indicated that NEAP had important functions in regulating retina and eye
Additionally, smaller heads suggested the development of central nerve system (CNS) was defective in
zebrafishes lacking NEAP. We first examined the development of cranial motor neurons using the islet-GFP transgenic
fishes, because their GFP-labeled neurons can be visualized through emitting green fluorescence24. In NEAP
MO/islet-GFP morphants, which has smaller heads, cranial motor neurons (showed in green) did not show long
axonal extensions in comparison to those in control group (Fig.?7b). Consistent with this, whole mount in situ
fluorescence staining using an anti-acetylated tubulin antibody showed a similar result that there was less axonal
structures in NEAP MO-injected zebrafishes (Fig.?7c). These collective data indicated that NEAP was essential for
normal eye and CNS development in zebrafishes.
Previously we found that NEAP/DUSP26 suppressed NGF-induced PI3K/Akt activation in PC12 cells
without affecting MAP kinase pathways. When ectopically expressed in H1299 cells, NEAP/DUSP26 also failed to
inhibit the activation of all MAPKs9. However, we also do not find any evidence that NEAP acts on PI3K or Akt
directly9. Moreover, NEAP/DUSP26 failed to suppresses heregulin-induced Akt activation in PC12 cells,
suggesting that, instead of being an Akt suppressor, NEAP/DUSP26 must act on TrkA or TrkA-associated signaling
molecules. Using antibodies generated against phosphorylated motifs of TrkA, we found that Tyr-phosphorylation
levels of TrkA were decreased in PC12 expressing NEAP/DUSP26, but not its phosphatase-defective mutant.
Phosphorylation of FRS2, an adaptor molecule required for proper TrkA signaling16, was also decreased in the
presence of NEAP/DUSP26. It is a bewildering question that why expression of NEAP specifically affected PI3K/
Akt pathway but not other TrkA-downstream signaling pathways and we do not have a definite answer at this
point. One possible explanation is that all MAPK pathways are consisted of multiple layers of kinases and the
initial suppressive effect of NEAP/DUSP26 is diminished by the amplification nature of kinase cascades.
Other than TrkA, we found NGF-induced FGFR1 phosphorylation was also suppressed by NEAP/DUSP26.
FGFR1 activation through autocrine production is critical for proper neurite outgrowth in PC12 cells upon NGF
treatment17. However, the immediate response of FGFR1 phosphorylation to NGF stimulation implicated that cross
talk between TrkA and FGFR1 existed in the PC12 context (Fig.?2). We noticed that the dual Tyr-phosphorylation
motifs in the activating loops are conserved among the neurotrophin receptor and FGFR families. It is
possible that sequence homology among these receptors allowed selective substrate recognition by NEAP/DUSP26.
Nevertheless, despite the result that?NEAP/DUSP26 directly de-phosphorylated TrkA and FGFR1 in vitro, it did not
de-phosphorylate IGFR1 whose activating loop also has the conserved dual Tyr-phosphorylation motif. The
mechanism determines that substrate selectivity of NEAP/DUSP26 is very intriguing. It has been shown that adenylate
kinase 2 (AK2) interacts with NEAP/DUSP26 and facilitates the de-phosphorylation of FADD14. We have tested
the combination of AK2 and NEAP/DUSP26 in vitro to see whether AK2 has a role in mediating NEAP/DUSP26?s
effect on RTKs and did find an enhancing NEAP/DUSP26 activity against pTrkA in the presence of AK2. However,
we did not detect the NEAP-AK2 interaction in PC12 cells by various methods. Therefore, what determines the
substrate selectivity of NEAP/DUSP26 against RTKs in neuronal cells remained unclear. Moreover, despite detecting a
direct de-phosphorylation of FGFR1 by NEAP in vitro, we cannot conclude whether NEAP decreased NGF-induced
FGFR1 phosphorylation through direct, indirect, or the combination of both mechanisms at this point.
The NEAP/DUSP26-orthologous gene was found in zebrafish and was highly expressed in the central nerve
system. The noticeable curved body posture and smaller eyes and heads in NEAP/DUSP26 morpholino-injected
embryos indicated that this gene was important in the developmental process. Defective differentiation of retina and
failure in axonal extension of cranial motor neurons further supported the role of NEAP/DUSP26 in the neuronal
system. Suppression of NEAP/DUSP26 by morpholino injection up-regulated the Tyr phosphorylation levels of
TrkA and FGFR1 in zebrafish embryos, which was consistent with our finding in vitro. However, we cannot
conclude that the developmental defects were exclusively caused by aberrant RTK signaling in light of the fact that other
known NEAP/DUSP26?s substrates also have important roles in neuronal cell differentiation and survival12?14, 25.
Moreover, TrkA is known to be a substrate of other protein Tyr phosphatases (PTPs)26, 27. It is also possible that other
PTPs can compensate, in parts, the loss of NEAP/DUSP26 in vivo. NEAP/DUSP26 expression was not paralleled
with the initial development of nerve system and had the highest expression at a later developmental stage in
zebrafish embryos. Interestingly, NEAP/DUSP26 expression is also highest in differentiated PC12 cells with well formed
neurites, indicating a role of NEAP in mature neuronal cells. More detailed analyses of genetically modified animal
models will facilitate the understanding of the biochemical and biological function of NEAP/DUSP26.
Cell culture and transfection. Establishment of permanent PC12 cell lines, including PC12-cDNA4,
PC12-NEAP, PC12-C152S, PC12-pSuper and PC12-shNEAP (previously named PC12-NEAPi) were described
previously9. Parental PC12 and its derivative cell lines were maintained as monolayer cultures in a 100-mm
culture dish in Dulbecco?s modified Eagle?s medium (DMEM) supplemented with 10% horse serum (HS), 5% heat
inactivated fetal bovine serum (FBS), penicillin (50 unit/ml) and streptomycin (50 ?g/ml). H1299-derivative cells
were cultured in RPMI medium supplemented with 10% FCS plus penicillin and streptomycin and were
transfected using the Lipofectamine 2000 reagent.
Plasmid constructions. Rat TrkA-coding DNA fragment was PCR amplified using specific primers and a
PC12 cDNA pool as a template. The TrkA fragment was then inserted between BamHI and EcoRI sites in pcDNA4/
TO/myc-His B vector (Invitrogen, Carlsbad, CA). Recombinant His-tagged NEAP was constructed by inserting
the NEAP-coding sequence between the Bam HI and Xho I sites of pHis-4T-3 vector. AK2 DNA fragment was
PCR-amplified using specific primers and a H1299 cDNA pool as a template. Recombinant GST-AK2 expressing
vector was constructed by inserting the AK2-coding sequence between the Bam HI and Xho I sites of pGST-4T-3
vector. Zebrafish NEAP coding sequence (reference Genbank number XM_694337) was PCR-amplified, using a
zebrafish cDNA pool as a template, and was then inserted between BamHI and Xho I sites of the pCMV-Tag2B
vector, which contains T3 and T7 promoters for the synthesis of sense and antisense probes, respectively.
Reagents and antibodies. Anti-Flag (M2), anti ?-actin, and anti-acetylated tubulin monoclonal antibodies
were purchased from Sigma (St. Louis, MO, USA). Anti-NEAP/DUSP26 antibody was purchased from GeneTex
(Hsinchu, Taiwan). Anti-phospho-Akt (S473), pTrkA (Y490 and Y674/675), anti-pFGFR1(Y653/654), anti-pFRS2
(Y196 and Y436), anti-pIGFR1(Y), and anti-IGFR1 antibodies were purchased from Cell Signaling Technology
(Beverly, MA, USA). Anti-Akt, anti-TrkA, anti-FGFR1, anti-GST antibodies and peroxidase-conjugated
anti-mouse and anti-rabbit IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). NGF was purchased from R & D System (Minneapolis, MN, USA) and heregulin was purchased from
Calbiochem (San Diego, CA, USA).
Cell extract preparation and immunoblotting analysis. Whole cell lysate was prepared by suspending
2 ? 106 cells in 200 ?l of lysis buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% triton-X 100, 0.5% deoxycholate,
0.1% SDS, 2 ?g/ml leupeptin, 5 ?g/ml aprotinin, 1 mM PMSF, 1 mM DTT, and 1 mM Na3VO4). Immunoblotting
assays were performed as described previously9.
Immuno-precipitation (IP) and phosphatase assays. Target proteins in cell extracts were precipitated
by incubation with a specific antibody plus protein A/G-agarose beads in 1 ml of lysis buffer with continuously
rotation at 4 ?C for 2 h. The precipitates were washed three times with lysis buffer. IP complexes for phosphatase
assays were washed twice more with phosphatase reaction buffer (50 mM Tris (pH 7.0), 50 mM Bis-Tris, 100 mM
sodium acetate, and 10 mM DTT). The immune-complex was then mixed with 100?l of phosphatase reaction buffer
containing 20 ?g/ml of GST-NEAP or GST-NEAP (C152S), in the presence or absence of GST-AK2 (20 ?g/ml).
The phosphatase reaction was performed at 37?C for 1 h, and then terminated by adding SDS sampling buffer. The
reaction mixtures were heated at 95 ?C for 5 min and analyzed by SDS-PAGE plus immunoblotting blot analyses.
Zebrafish morpholino (MO) injection and embryo examination. Zebrafish NEAP MO and scram
bled MO were purchased from Gene Tools, LLC (Philomath, OR), dissolved in sterilized ddH2O (17.39 ?g/?l),
and stored at ?20 ?C. For microinjection, MOs were prepared in phosphate buffered saline with 0.05% phenol
red and then were injected into embryos of one-cell stage with PV820 Pneumatic PicoPump (World Precision
Instruments, Inc., Sarasota, FL). After injection, embryos were incubated in egg water (60 ?g sea salt/ml distilled
water) supplemented with 0.003% 1-phenyl-2-thiourea (PTU) at 28 ?C to prevent pigmentation. Embryo
development was examined at 1.5, 4, 8, 20, 24, 48 and 72 hpf.
Whole mount in situ hybridization (WMISH) and immune-fluorescence (IF) staining. Embryos
were fixed in 4% paraformaldehyde overnight at 4 ?C and dehydrated in methanol at ?20 ?C. WMISH procedure
was performed according to the protocol described by Thisse B and Thisse C on ZFIN (http://zfin.org) with some
modifications. In brief, embryos were rehydrated gradually with PBST (1x PBS, 0.1% Tween 20). For 96-hpf
embryos, they were digested with 25 ?g/ml proteinase K (Sigma-Aldrich) in PBST for 30 minutes at room
temperature. After post-fixed with 4% paraformaldehyde, the embryos were incubated in HYB+ (50 ?g/ml heparin,
500 ?g/ml wheat germ tRNA and HYB? containing 50% formamide, 5x SSC and 0.1% Tween 20) which
contained 100 ng DIG-labeled antisense (or sense) RNA probe at 65 ?C overnight. Embryos were then washed with
75%, 50% and 25% HYB?/2x SSC for 10 minutes, 2x SSC for 10 minutes, two times with 0.2x SSC for 30 minutes,
75% 0.2x SSC/25% PBST, 50% 0.2x SSC/50% PBST, 25% 0.2x SSC/75% PBST for 5 minutes at 65 ?C and PBST
for 10 minutes and transferred to blocking buffer (2 mg/ml BSA, 2% sheep serum in PBST) at least for 1 hour.
Embryos were incubated in 1?5000 anti-DIG-AP (Roche) in blocking buffer overnight at 4 ?C. After 6 washes
with PBST for 15 minutes and 3 washes with alkaline Tris buffer for 5 minutes, bound antibody was detected
by SIGMA FAST? BCIP/NBT. After staining, labeled embryos were mounted in 90% glycerol and examined
on an Olympus stereomicroscope (SZX-ILLD100, Tokyo, Japan). The images were captured using an Olympus
DP70 digital microscope camera and processed by Helicon Focus software. Whole mount IF staining using the
anti-acetylated tubulin antibody was performed as described previously28.
RNA extraction and RT-PCR. The RNA extraction was performed using the Trizol reagent (Thermo Fisher
Scientific) in combination with the MagNA Lyser Green Beads (Roche Diagnostics, Indianapolis, IN) according
to the instructions of the manufacturer. Reverse transcription of cDNAs were performed using the ProtoScript
first strand cDNA synthesis kit (New England Biolabs, Beverly, MA) and using 1 ?g of total RNA as templates.
PCR reactions were performed and the reaction products were analyzed using agarose gel electrophoresis.
Nucleotide sequences. The nucleotide sequences used in this study are listed below.
Rat TrkA forward: 5?-GCGGAATTCGCCACCATGCTGCGAGGCCAGCGGCAC,
TrkA reverse: 5?-GCGCTCGAGATGCCCAGAACGTCCAGGTAACTC,
NEAP forward: 5?-GCGGGATCCACCATGGCCCGCTTCTCCCGGAG,
NEAP reverse: 5?-GCGGTCGACATTGCTTCCAGACCCTGCCGCAG,
AK2 forward: 5?-GCGAGATCTACCATGGCTCCCAGCGTGCCAGC,
AK2 reverse: 5?-GCGCTCGAGATGATAAACATAACCAAGTCTTTACATGTGG,
fNEAP cDNA forward: 5?-GCGGGATCCACCATGGCGTTTATGTCCAGATTGTCTC
fNEAP cDNA reverse: 5?-GCGCTCGAGATTGTGGTGCTGCGGCTGCTG
fDUSP23 forward: 5?-GTGCGCCCACATTTGAGCAGATC,
fDUSP23 reverse: 5?-CTGCACAATCATTTGTTCCTGCTCTC,
zebrafish 18S rRNA forward: 5?-CCGCAGCTAGGAATAATGGA,
zebrafish 18S rRNA reverse: 5?-CATCGTTTACGGTCGGAACT,
fNEAP morpholino sequence is 5?-GAGACAATCTGGACATAAACGCCAT,
fNEAP scrambled morpholino is 5?-CCATAATGGACATGAGATCACAACG.
Data availability statement.
All data generated or analyzed during this study are included in this pub
We thank the Taiwan Zebrafish Core Facility at NHRI for the assistance in zebrafish-related studies as well as the
NHRI Optical Biology Core and Pathology Core for the assistance in performing experiments. This work was
supported by grants from National Health Research Institutes (MG-104 and 105-PP-03 to YRC), the Ministry
of Science and Technology, Taiwan (MOST105-2320-B-400-018-MY3 to YRC), and from the Ministry of Health
and Welfare, Taiwan (Advance Medical Plan, 106-0324-01-10-07).
C.H.Y., Y.J.Y., J.Y.W. performed experiments, analyzed the data and wrote the paper. Y.W.L., Y.L.C., H.W.C.,
C.M.C. conducted parts of the experiments. Y.J.C. and C.H.Y. contributed to the zebrafish experiments and paper
writing. Y.R.C. designed and directed the study and wrote the paper.
Supplementary information accompanies this paper at doi:10.1038/s41598-017-05584-7
Competing Interests: The authors declare that they have no competing interests.
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