DEVELOPMENT AND STEM CELLS
RESEARCH REPORT
959
Development 140, 959-964 (2013) doi:10.1242/dev.084590
© 2013. Published by The Company of Biologists Ltd
Analysis of RNA-Seq data reveals involvement of JAK/STAT
signalling during leg regeneration in the cricket Gryllus
bimaculatus
Tetsuya Bando1, Yoshiyasu Ishimaru2, Takuro Kida2, Yoshimasa Hamada2, Yuji Matsuoka2, Taro Nakamura2,
Hideyo Ohuchi1, Sumihare Noji2 and Taro Mito2,*
SUMMARY
In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of
dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation,
comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT
signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the
interleukin receptor Domeless (Gb’dome), the Janus kinase Hopscotch (Gb’hop) and the transcription factor STAT (Gb’Stat) were
cloned, and RNAi was performed against these genes. Gb’domeRNAi, Gb’hopRNAi and Gb’StatRNAi crickets showed defects in leg
regeneration. Blastema expression of Gb’cyclinE was decreased in the Gb’StatRNAi cricket compared with that in the control.
Hyperproliferation of blastema cells caused by Gb’fatRNAi or Gb’wartsRNAi was suppressed by RNAi against Gb’Stat. The results suggest
that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.
KEY WORDS: Regeneration, Blastema, Gryllus bimaculatus, JAK/STAT signalling, Transcriptome, Next-generation sequencer
The two-spotted cricket Gryllus bimaculatus is an emerging
model organism for regeneration research (Mito and Noji, 2009).
When we amputate the leg of the cricket nymph, a blastema forms
at the distal tip of the leg stump to restore the lost part of the leg
(Mito et al., 2002). The early blastema displays cell proliferation
and activation of several signalling pathways, such as Wg/Wnt and
EGFR, at 2 days postamputation (dpa) (Mito et al., 2002; Nakamura
et al., 2008b). However, the molecular mechanisms of blastema
formation remain to be identified.
In the present study, we analysed the global gene expression
profile during the early regeneration process to identify the
molecules involved in blastema formation. We found that the
expression levels of components of the JAK/STAT signalling
pathway were upregulated in blastemas, and that JAK/STAT
signalling was essential for leg regeneration. We conclude that
comparative transcriptome analysis could be useful to clarify the
molecular mechanisms of blastema formation.
MATERIALS AND METHODS
Animals
G. bimaculatus used in this study were reared under standard conditions
(Mito and Noji, 2009).
Quantitative PCR (q-PCR)
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama
University, 2-5-1 Shikata-cho, Kita-ku, Okayama city, Okayama, 700-8530, Japan.
2
Department of Life Systems, Institute of Technology and Science, The University of
Tokushima Graduate School, 2-1 Minami-Jyosanjima-cho, Tokushima city, 770-8506,
Japan.
The regenerating tibiae or blastemas of control or RNAi-treated nymphs
(n=9-15) were pooled into single tubes and total RNA was extracted using
the RNAqueous-Micro Kit (Ambion). Each pooled RNA was divided into
two samples and each half reverse transcribed to prepare cDNA. Each
cDNA was divided into three samples and used for q-PCR. Relative
transcript ratios in the q-PCR study were calculated from experiments
performed in triplicate and are shown as mean ± s.d. q-PCR experiments
were repeated twice for confirmation. The total number of nymphs used to
extract RNA is indicated by n. The housekeeping gene Gryllus β-actin was
used as an internal control (Bando et al., 2011a; Bando et al., 2009).
*Author for correspondence ([email protected])
Transcriptome analysis
Accepted 19 December 2012
Distal parts of regenerating legs at 0 and 24 hours postamputation (hpa),
designated as normal legs (NLs) and regenerating legs (RLs), respectively,
1
DEVELOPMENT
INTRODUCTION
Regeneration requires the recognition of tissue loss and the
subsequent restoration of the relevant structure. Several research
areas have clarified the molecular bases of tissue regeneration in
model organisms (Agata and Inoue, 2012; Tanaka and Reddien,
2011). Key molecules for the early regeneration process include
apoptotic factors and proteinases, such as matrix metalloproteinases
(MMPs) that regulate cell motility or morphology via degradation
of the cell surface and cell-cell contact (Chera et al., 2011; Maki et
al., 2010; Repiso et al., 2011).
Pluripotent stem cells or dedifferentiated multipotent cells
proliferate in response to injury to form a blastema at the tip of the
amputated stump. A blastema is a population of pluripotent
proliferating cells that can restore the lost parts of tissues. Signalling
molecules, including TGFβ, Shh, Wnt, FGF, EGF and IGF, are
expressed in the epidermis of the wound, and in some cases in
blastema cells, to regulate the proliferation and differentiation of
blastema cells via the MAPK cascade (Chera et al., 2011; Petersen
and Reddien, 2009; Repiso et al., 2011; Satoh et al., 2011; Tasaki et
al., 2011a; Tasaki et al., 2011b; Wang et al., 2009; Yazawa et al.,
2009; Yoshinari and Kawakami, 2011). The activities of MAPKs
are negatively regulated by MAPK phosphatases (Repiso et al.,
2011; Tasaki et al., 2011a; Tasaki et al., 2011b). In parallel with EGF
signalling, the Salvador/Hippo/Warts and JAK/STAT signalling
pathways regulate the proliferation of stem cells (reviewed by Jiang
and Edgar, 2011).
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were separately collected. The cDNA libraries constructed from poly(A)+
RNAs were sequenced using the Illumina Genome Analyzer IIx nextgeneration sequencer by a 50 bp single-ended process. Raw sequence data
from NLs and RLs have been deposited in the DNA Data Bank of Japan
(DDBJ) sequence read archive (accession numbers of NL and RL are
DRR001985 and DRR001986, respectively).
To construct the assembled transcripts, all of the reads obtained from the
NL and RL samples were assembled using the Velvet (EMBL) and Oases
(EMBL) de novo transcriptome assembly software packages. Each read was
mapped to the assembled transcripts using the Bowtie short-read aligner
(Johns Hopkins University). Transcript expression levels were estimated in
order to calculate the reads per kb of exons per million mapped reads
(RPKM) values. Assembled transcripts that were upregulated more than
twofold in RLs compared with NLs based on the RPKM values were
annotated with the BLASTX program against the NCBI nonredundant
protein sequence database, with an E-value cut-off of 0.001.
Functional annotation and Gene Ontology (GO) term assignment were
performed with the GO-mapping algorithm of the Blast2GO program
(Conesa et al., 2005) and the InterProScan sequence search tool (EMBL)
with default settings. The GO annotations obtained by these two programs
were merged. The GO-Slim view was used before generating multilevel
GO graphs.
Cloning and RNAi for Gryllus homologues of JAK/STAT signalling
components
Gryllus homologues of domeless, hopscotch, Stat, Socs2, Socs5, Socs7 and
su(var)2-10 [Gb’dome1/2 (Gb’dome), Gb’hop1/2 (Gb’hop), Gb’Stat1/2
(Gb’Stat), Gb’Socs2, Gb’Socs5, Gb’Socs7 and Gb’su(var)2-10,
respectively] were cloned by PCR using cDNA from the whole body of
cricket nymphs at third instar. The cDNA sequences have been deposited in
GenBank (accession numbers AB715378-AB715379 and AB761612AB761618). Preparation of dsRNA and RNAi were performed as previously
described (Bando et al., 2009). All RNAi data are given as mean ± s.d. and
represent three to five independent experiments. The total numbers of
RNAi-treated nymphs of multiple independent experiments are indicated
by n.
RESULTS AND DISCUSSION
Sequencing and assembly of the regenerating leg
transcriptome of G. bimaculatus
The early blastema is formed within 2 dpa during the leg
regeneration process, as revealed previously by cell proliferation
assays and the expression of Gb’Egfr (Mito et al., 2002; Nakamura
et al., 2008b). We studied the temporal changes in the mRNA levels
of Gb’Egfr and Gb’cyclinE by q-PCR (supplementary material Fig.
S1C). The expression of Gb’Egfr and Gb’cyclinE doubled from 12
to 132 hpa and from 24 to 132 hpa, respectively, compared with the
levels at 0 hpa (supplementary material Fig. S1C), suggesting that
EGF-mediated blastema formation starts from 24 hpa.
To uncover the molecular basis of blastema formation, we
performed a transcriptome analysis of the early blastema in G.
bimaculatus without a reference genome (supplementary material
Fig. S1D). Early blastemas of RLs at 24 hpa and the corresponding
leg part of NLs, in which the distal portion of the leg was cut off just
prior to experiments, were collected from sixth instar nymphs
(supplementary material Fig. S1A,B). The cDNAs synthesized
using RNA samples from RLs and NLs were sequenced in two
independent lanes using a next-generation sequencer.
All sequence reads from both RLs and NLs were assembled into
55,043 transcripts using transcriptome assembly software packages
(supplementary material Fig. S1, Table S1). The average length of
the assembled transcripts was 261 bp (supplementary material Fig.
S2A, Table S1). Each read obtained from the RLs and NLs was
mapped to the assembled transcripts to calculate the RPKM value
of each assembled transcript (see Materials and methods). A total of
Development 140 (5)
466 transcripts were only expressed in RLs, and the expression of
11,492 transcripts was upregulated more than twofold in RLs
(supplementary material Fig. S1D, Table S1) based on the
comparison of RPKM values between RL and NL. Because the
transcripts that were upregulated or only expressed in RLs could be
involved in blastema formation, we focused on these 11,958
transcripts for further annotation.
Annotation of upregulated transcripts in the
regenerating leg
We analysed the 11,958 transcripts using the Blast2GO program
(supplementary material Fig. S1D). In total, 2724 transcripts had
significant hits with known genes annotated by BLASTX
(supplementary material Table S1). Gryllus transcripts showed high
homologies to the orthologous genes of Pediculus humorous and
Tribolium castaneum (supplementary material Fig. S2B). Only 23%
of the transcripts were annotated because the length of the
assembled transcript obtained in this work was short and the
genome or EST data are currently insufficient in G. bimaculatus.
To obtain a general overview of the functions of the transcripts
that were upregulated during regeneration, GO terms were assigned.
Of the 2724 transcripts, 2344 were annotated with at least one GO
term (supplementary material Table S1). Frequently encountered
GO terms for biological process, molecular function and cellular
components (supplementary material Fig. S3) perhaps relate to the
fact that signalling processes transduce extracellular signals
received on the cell membrane into the nucleus via protein-protein
interactions and protein modifications.
Validation of transcriptome analysis
We searched the assembled transcripts to identify Gryllus
homologues of components known to be involved in regeneration
in other animals (Agata and Inoue, 2012; Nakamura et al., 2008a).
The assembled transcripts encoding candidate genes are listed in
Table 1 and supplementary material Tables S2-S6. A high RL/NL
RPKM value was given if the candidate gene was encoded by
several assembled transcripts.
The expression of apoptotic genes, MMPs, piwi and AGO3 was
upregulated in RLs (supplementary material Table S2), similar to
regeneration in other organisms (Chera et al., 2011; Li et al., 2011;
Maki et al., 2010; Reddien et al., 2005). Various signalling
components, including Wg/Wnt, Hh/Shh, EGF, Dpp/TGF, VEGF,
Insulin/IGF, Notch, Toll (supplementary material Table S3),
Dachsous/Fat (supplementary material Table S4) and JAK/STAT
(Table 1) were also upregulated in RLs (Agata and Inoue, 2012;
Bando et al., 2011a; Bando et al., 2009; Bando et al., 2011b;
Nakamura et al., 2008a; Nakamura et al., 2008b). Supplementary
material Tables S5 and S6 list transcription and epigenetic factors,
respectively, that were upregulated in RLs, which could be key
factors for cell fate determination and reprogramming (Maki et
al., 2010; McClure and Schubiger, 2008; Meissner, 2010; Onder
et al., 2012; Rao et al., 2009; Repiso et al., 2011; Stewart et al.,
2009).
Functional analysis of JAK/STAT signalling during
leg regeneration
Toll, Dpp/TGFβ and JAK/STAT were the top three signalling
pathways upregulated in RLs (Table 1; supplementary material
Table S3). The evolutionarily conserved JAK/STAT signalling
pathway has roles in embryogenesis, immunity and cell proliferation
(Arbouzova and Zeidler, 2006) and is involved in the proliferation
and maintenance of stem cells in Drosophila (Jiang and Edgar,
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Table 1. Transcriptional changes in JAK/STAT signalling pathway genes in the regenerating leg
RPKM value
Gene
dome
hop
Stat
Socs2
Socs5
Socs6
Socs7
su(var)2-10
csw
Product
NL
RL
RL/NL
interleukin receptor
Janus kinase
STAT protein
suppressor of cytokine signalling 2
suppressor of cytokine signalling 5
suppressor of cytokine signalling 6
suppressor of cytokine signalling 7
protein inhibitor of activated STAT
SHP2/tyrosine-protein phosphatase non-receptor type 11
19.4
5.7
8.1
10.5
35.6
6.4
2.5
34.6
29.2
49.8
7.0
30.8
68.8
44.1
16.3
14.5
26.7
88.3
2.6
1.2
3.8
6.6
1.2
2.5
5.7
0.8
3.0
RPKM, reads per kb of exons per million mapped reads; RL, regenerating leg; NL, normal leg.
1.9±0.2 and 2.1±0.2, respectively, suggesting that our transcriptome
analysis was adequate.
Two independent regions of each of the three genes were cloned
(supplementary material Fig. S4A). RNAi was performed (Fig. 1A)
to determine whether JAK/STAT signalling is involved in early
blastema formation. The relative ratios of endogenous Gb’dome,
Gb’hop and Gb’Stat transcripts were lower (P<0.01, Student’s ttest) in the respective RNAi crickets compared with the control at
5 dpa as assessed by q-PCR, indicating that RNAi had occurred
Fig. 1. RNAi phenotypes of JAK/STAT signalling components. (A) Typical phenotypes of regenerating legs (RLs) at fourth, fifth and sixth instar and
adult stages of control, Gb’dome1RNAi, Gb’hop1RNAi and Gb’Stat1RNAi crickets. NL, normal leg. Arrows and arrowheads indicate tibial spurs and tarsi,
respectively. ti, tibia; ta, tarsus; cl, claw. Scale bar: 5 mm. (B) Expression of Gb’dome, Gb’hop and Gb’Stat in RLs at 0 (set at 1), 2, 12, 24 and 132 hpa. Error
bars indicate s.d. (C) RNAi phenotypes of Gb’dome, Gb’hop and Gb’Stat were categorised into four classes based on the morphologies of RLs at sixth
instar: class I, most severe; classes II and III, milder phenotypes; class IV, normal. Total numbers of RNAi-treated nymphs are indicated by n.
DEVELOPMENT
2011; Karpowicz et al., 2010; Ren et al., 2010; Shaw et al., 2010;
Staley and Irvine, 2010). Thus, activation of JAK/STAT signalling
might be required for blastema formation.
To confirm the transcriptome data, we estimated the transcription
levels of Gryllus homologues of the interleukin receptor Domeless
(Gb’dome), the Janus kinase Hopscotch (Gb’hop) and the
transcription factor STAT (Gb’Stat) during regeneration by q-PCR.
Gb’dome, Gb’hop and Gb’Stat were upregulated at 12-24 hpa
(Fig. 1B). The relative ratios of these genes at 24 hpa were 2.1±0.2,
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(supplementary material Fig. S5C). We compared the phenotypes
obtained with two dsRNAs corresponding to different regions of
each gene. We observed a similar defect during leg regeneration
with RNAi against Gb’dome, Gb’hop or Gb’Stat (Fig. 1C;
supplementary material Fig. S5A,B). This finding indicates that the
phenotypes obtained by RNAi were not off-target effects.
In control experiments, when the metathoracic tibia of the third
instar nymph was amputated, a blastema formed at the distal end of
the amputated leg during the third to fourth instar stages. In the fifth
instar, miniaturised forms of the tibiae and tarsi were restored. In
the sixth instar and adult stages, the amputated legs restored the
missing portion to regain a nearly normal appearance (Fig. 1A).
Gb’domeRNAi, Gb’hopRNAi and Gb’StatRNAi nymphs were viable
and showed defects during leg regeneration. In the fourth instar,
Gb’domeRNAi, Gb’hopRNAi and Gb’StatRNAi nymphs showed no
obvious defects. However, tissue regeneration of the lost parts of
the legs did not occur in the fifth and sixth instar stages (Fig. 1A;
supplementary material Fig. S5A).
RNAi phenotypes were categorised into four classes at sixth
instar (Fig. 1C). The major phenotypes of each RNAi experiment
are shown in Fig. 1A. In the Gb’domeRNAi crickets, distal structures,
including tibial spurs, were regenerated. However, the lost parts of
the tarsi were not regenerated (Fig. 1A, asterisk). In Gb’hopRNAi
Development 140 (5)
crickets, the distal portion of the tibia, shown by tibial spurs, was
regenerated. The lost tarsus was regenerated as a nonsegmental
structure in miniature (Fig. 1A, arrowheads) in the sixth instar and
adult stages. In Gb’StatRNAi crickets, lost parts of the legs were not
regenerated (Fig. 1A). Given that Dome transduces signals to other
pathways, that STAT is phosphorylated by other kinases (Arbouzova
and Zeidler, 2006) and that the RNAi efficiencies against Gb’dome,
Gb’hop and Gb’Stat differed, it is conceivable that the phenotype of
Gb’hopRNAi was milder than those of Gb’domeRNAi and
Gb’StatRNAi.
We compared the lengths of the regenerated and normal tibiae at
sixth instar. In control regenerations, when tibiae were amputated at
distal (70%) positions, the lengths of the regenerated tibiae were
101±5% (n=22) of unamputated control tibia lengths
(supplementary material Fig. S5A,B). In the Gb’domeRNAi,
Gb’hopRNAi and Gb’StatRNAi nymphs, the lengths of the
regenerating tibiae were shortened by distal amputation; the
regenerated tibiae were 66±7% (n=23), 80±4% (n=22) and 64±8%
(n=19) of the control tibia lengths, respectively (P<0.01;
supplementary material Fig. S5A,B). The lengths of the regenerated
tibiae were similar to that of the amputated tibia at third instar; thus,
Gb’dome and Gb’Stat appear to regulate only blastema cell
proliferation but not allometric growth of the leg stump.
Fig. 2. Functional analyses of the role of Gb’Stat in blastema cell proliferation. (A) Morphologies of control and Gb’Stat1RNAi nymphs at sixth instar.
(B) Average body weight (mg) for control and Gb’Stat1RNAi nymphs at sixth instar. (C) Relative Gb’cyclinE expression as revealed by q-PCR in the control
(set at 100%) and Gb’Stat1RNAi nymphs. Error bars indicate s.d. (D) Morphologies of NLs and RLs of control and Gb’Stat1RNAi crickets at sixth instar.
(E) Typical blastema phenotypes of single and dual RNAi crickets at fourth instar. Asterisks indicate enlarged blastemas. (F,G) Typical phenotypes of
single and dual RNAi crickets at sixth instar (F) and adult stage (G). Scale bars: 5 mm in A; 1 mm in D-G.
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Fig. 3. RNAi phenotypes of negative regulators for JAK/STAT signalling. (A) Typical phenotypes of RLs at fourth, fifth and sixth instar and adult
stages of control, Gb’Socs2RNAi, Gb’Socs5RNAi, Gb’Socs7RNAi and Gb’su(var)2-10RNAi crickets. Scale bar: 5 mm. (B) Relative Gb’cyclinE expression as revealed by
q-PCR in control (set at 100%) and Gb’Socs2RNAi nymphs. Error bars indicate s.d.
of Gb’Stat was decreased in Gb’ftRNAi and Gb’wtsRNAi nymphs,
whereas that of Gb’dome and Gb’hop was increased (supplementary
material Fig. S6). This indicates that the JAK/STAT and
Dachsous/Fat signalling pathways may not be epistatic.
Functional analysis of negative regulators of
JAK/STAT signalling during leg regeneration
Our comparative transcriptome analysis indicates that several
negative regulators of JAK/STAT signalling were also upregulated
in RLs (Table 1). We performed RNAi against Gb’Socs2,
Gb’Socs5, Gb’Socs7 and Gb’su(var)2-10 (Fig. 3A; supplementary
material Fig. S4A,B). In all experiments, the amputated surfaces
of legs were covered by cuticle at fourth instar, and miniaturised
forms of the tarsi and tarsal claws were restored at fifth instar. In
the sixth instar and adult stages, the missing portions of the
amputated legs were restored, and a nearly normal appearance was
regained (Fig. 3A). However, the regenerating tibiae of
Gb’Socs2RNAi (110±8%) and Gb’Socs5RNAi (108±5%) crickets
were significantly lengthened, and those of Gb’su(var)2-10RNAi
(82±12%) crickets were significantly shortened (Fig. 3A;
supplementary material Fig. S5A,B).
Because SOCS negatively regulates JAK/STAT signalling
activity, we sought to clarify whether Gb’Socs2 or Gb’Socs5 could
suppress cell proliferation. The relative ratio of Gb’cyclinE mRNA
in the RLs at 5 dpa was increased in Gb’Socs2RNAi nymphs
(179±20%, n=15, P<0.01; Fig. 3B) compared with control nymphs
(100±12%, n=15) as assessed by q-PCR, suggesting that Gb’Socs2
suppresses cell proliferation in the regenerating legs, perhaps by
repressing the overactivation of JAK/STAT signalling.
In conclusion, we provide evidence that JAK/STAT signalling
promotes blastema formation and that SOCS negatively regulates
JAK/STAT signalling activity during tissue regeneration. Taken
DEVELOPMENT
To analyse the function of JAK/STAT signalling further, we
focused on the function of Gb’Stat. The Gb’StatRNAi nymphs
showed normal body morphologies and weights (111.4±15.6 mg,
n=4) compared with control nymphs (109.3±16.3 mg, n=11,
P>0.05; Fig. 2A,B), except for the morphologies of the regenerated
legs (Fig. 2D). Because the Gb’StatRNAi cricket showed defects in
leg regeneration but not in the morphology of other tissues, we
sought to clarify whether Gb’Stat promotes blastema cell
proliferation. We attempted to detect proliferating cells using EdU,
but we could not remove cuticles without damaging the regenerating
Gb’StatRNAi legs, as these were more fragile than control legs. EdU
is incorporated into S-phase cells, when cyclin E is specifically
expressed, so we determined the ratio of proliferating cells by
Gb’cyclinE expression using q-PCR as an alternative (Bando et al.,
2011a). The relative ratio of Gb’cyclinE mRNA in the blastemas at
5 dpa was decreased in Gb’StatRNAi nymphs (56±5%, n=6, P<0.01;
Fig. 2C) compared with control nymphs (100±10%, n=7). This
suggests that Gb’Stat could promote blastema cell proliferation.
We performed dual RNAi experiments against Gb’Stat with
Gb’fat (Gb’ft) or Gb’warts (Gb’wts). We previously showed that
blastemas are enlarged by hyperproliferation of blastema cells in
Gb’ftRNAi and Gb’wtsRNAi nymphs at fourth instar (Fig. 2E,
asterisks) (Bando et al., 2009). Hyperproliferation in the blastema
caused by Gb’ftRNAi or Gb’wtsRNAi was suppressed by RNAi against
Gb’Stat (Fig. 2E). The regeneration-defective phenotype caused by
Gb’StatRNAi was dominant to the abnormal regeneration phenotypes
caused by Gb’ftRNAi or Gb’wtsRNAi at the sixth instar and adult
stages (Fig. 2F,G). Therefore, Gb’Stat appears to promote blastema
cell proliferation via expression of Gb’cyclinE during Gryllus leg
regeneration.
The transcription of Gb’dome, Gb’hop and Gb’Stat was not
uniformly regulated by Dachsous/Fat signalling. The transcription
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together, these results indicate that our comparative transcriptome
analysis in the cricket system is effective in identifying genes that
are functionally involved in tissue regeneration.
Acknowledgements
We thank Itsuro Sugimura at Hokkaido System Science Co., Ltd and Yasuko
Kadomura for their technical support.
Funding
This work was supported by grants from the Ministry of Education, Culture,
Sports, Science and Technology of Japan [#23710217 to T.B.; #21770237 to
T.M.; and #22370080 and #22124003 to S.N.].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.084590/-/DC1
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