広島大学学位請求論文

Mechanistic analysis of position-dependent fin
regeneration in zebrafish(ゼブラフィッシュにおける位置特異的ヒレ再生機構の解析)2018 年
広島大学大学院理学研究科
生物科学専攻

髙山和也

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目次
1. 主論文Mechanistic analysis of position-dependent fin regeneration in zebrafish(ゼブラフィッシュにおける位置特異的ヒレ再生機構の解析)
髙山和也
2. 公表論文(1) Leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are
required for position-dependent regeneration
Kazuya Takayama, Akihiko Muto, Yutaka Kikuchi
Scientific Reports, 2018;8(1):8278

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主論文
Mechanistic analysis of position-dependent fin
regeneration in zebrafish

Kazuya Takayama

Department of Biological Science,
Graduate School of Science,
Hiroshima University
20183

-Contents-

Chapter 1. General introduction.................................................................................................. 5
References ......................................................................................................................... 10
Chapter 2. Leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are
required for position-dependent regeneration. ............................................................................ 12
Abstract ......................................................................................................................... 13
Introduction ................................................................................................................... 14
Results ........................................................................................................................... 16
DISCUSSION ................................................................................................................ 21
Materials and Methods .................................................................................................. 24
Acknowledgements......................................................................................................... 27
References ...................................................................................................................... 28
FIGURES ...................................................................................................................... 32
Supplementary figures ................................................................................................... 46
Chapter 3. General discussion .................................................................................................. 60
References ...................................................................................................................... 63
Acknowledgement .......................................................................................................... 65

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Chapter 1. General introduction

5General introduction
Unlike mammals, some animals, like the adult teleost and urodele amphibians, cancompletely regenerate their appendages after amputation [1]. Although mice have agood capacity for organ regeneration, the ability to regenerate is limited [2]. In contrast,
the teleost and urodele amphibians have this regenerative capacity throughout theirlifespan. What is the difference between the mammal and some other animals that havea great capacity for organ regeneration? Understanding of this fascinating phenomenon
more deeply may be beneficial in the medical field because the mechanism may be
applicable to disease management and healing in the future.
As a potential model system for studying regeneration, the zebrafish has a lot of great
advantages [3]. Firstly, they can regenerate organs relatively quickly throughout theirlifespan, from the larvae stage to adulthood. Secondly, the sequence of their genome is
known, so we can use the data set, forward genetic approaches, and some transgenics
method. Finally, they can regenerate different kinds of organs such as the fin, brain,
spinal cord, retina, and heart. Although each regeneration is induced each different
system, but there are many common systems in same time.
The zebrafish has become the predominant model, because of these advantages. In
most cases of fin regeneration research, we use the caudal fin because we can easily
amputate it and observe the process of regeneration [4]. Zebrafish fins can be observed
simply as being a construction of bone rays, which provide the fin shape and structure.
Bone rays are composed of two sets of hemi-rays that are segmented into each bone.
The fin ray bones are thought to be regenerated via direct ossification rather than celldifferentiation from some premature cells, while other cells are structured in sometissues by fibroblasts, nerves, blood vessels, pigment, and mesenchymal tissues.
There are 3 steps in caudal fin regeneration [5][6][7]: wound healing, blastema
formation, and regenerative outgrowth. Fin regeneration usually takes only 2 weeks tocomplete.
1. Wound healing: 12 hours post amputation (hpa)
Following fin amputation, epidermal cells cover the amputation plane within an hour,while some cells migrate to the plane. At the same time, the cells migrate to below theamputation plane and start forming the premature cells, called the blastema.

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2. Blastema formation: 12-36 hpa
From 12 hpa to 36 hpa, changed premature cells, the blastema, begin to have acapacity for cell proliferation. The blastema is a highly proliferative mass of
undifferentiated mesenchymal cells from which several cell types will differentiate inorder to form the missing structures. This is derived from existing cells before
amputation.
3. Regenerative outgrowth: 36 hpa~
Regenerative outgrowth is induced by cell proliferation and dedifferentiation from theblastema. Gradually, the fin grows back its original shape, completing the regeneration
around 14 days. Some of the blastema cells aligning the epithelial tissue start todifferentiate into the scleroblasts, a process which depends on each signal arising fromthe apical epithelial cap (AEC). Within the following 3 to 14 days, the interactions
between the AEC and the blastema assure the outgrowth of the regenerating fin andduring this process, all cell types are redifferentiated and a new caudal fin is established.
The cell mass has been proposed to play a critical role in the regulation of gene
expression pattern and outgrowth throughout the fin regeneration due to the gene
expressions of the many growth factors, which are implicated in important signaling
pathways to induce the blastema cells, such as Wnt/b-Catenin Signaling, Fibroblast
growth factors, Insulin-like growth factors, and Sonic hedgehog.
Recently, many researchers have tried to reveal the mechanism of regeneration, andthey have reported new and important factors [8]. These include a lot of growth factors,
immune system related factors, epigenetic modification related factors, and other factors
involved in intercellular signaling pathways. However, a lot of details about theregeneration are still unknown and nobody can induce organ regeneration directly in thehuman body in the medical field.
One of the unsolved points in regeneration research is how the amputated fin
regenerates until the original shape is completed. This phenomenon is thought to be
induced by some critical factors. Many researchers, including me, believe thismechanism can be explained by the general concept of positional memory [9].
In the regeneration fin, the original appendages' size, shape, and some pattern isrestored. The regeneration requires that spared cells sustain or recall the information
encoding pattern. But almost all factors have not been implicated as a key factor of theposition-dependent regeneration during appendage regeneration. Recent work has
produced cellular and molecular models for blastema formation and regenerative7

outgrowth during fin regeneration, but the mechanism of storing the original shape’s
information is still largely unknown. One paper about the mechanism has been reported
in zebrafish fin regeneration [10]. During fin regeneration, the induction of cellproliferation mediated by Fgf signaling is different between the proximal and distal
positions of the amputation plane. This induction starts at around 48 hpa and the latestage of regeneration follows this induction. Complete regeneration of the fin depends
on the amputation position. However, nobody knows when this induction starts andwhat factor creates the difference.
In our laboratory, a previous has reported that the mechanistic target of rapamycin(mTORC1) signaling is the one of the earliest inducted signaling pathways after fin
amputation [12] and I have found that the pathway is related to the position-dependent
induction in the zebrafish fin regeneration. Additionally, I have discovered the signaling
pathway is regulated by v-ATPase/lysosomal acidification during fin regeneration andthe amino acid transporter is also required for this regeneration. In this chapter, I show
the distribution of lysosome acidification mediated by amino acid transporter during fin
regeneration. Finally, in chapter 3, I discuss how the new mechanism is involved inposition-dependent zebrafish fin regeneration.

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Figure. The process of fin regeneration
Following amputation, epidermal cells migrate to cover the injured plane and form the woundepidermis. The inter-ray cells de-differentiate and migrate to the under wound epidermis until 24
hpa. The de-differentiated cells begin to proliferate and form blastema. Formed blastema
proliferate and re-differentiate, and regenerative outgrowth is induced. Finally, regeneration iscompleted for two weeks.

9References
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Gemberling, M., Bailey, T. J., Hyde, D. R. & Poss, K. D. The zebrafish as amodel for complex tissue regeneration. Trends in Genetics 29, 611-620,
doi:10.1016/j.tig.2013.07.003 (2013).
Gonzalez-Rosa, J. M., Burns, C. E. & Burns, C. G. Zebrafish heart regeneration:
15 years of discoveries. Regeneration 4, 105-123, doi:10.1002/reg2.83 (2017).
Hirose, K., Shiomi, T., Hozumi, S. & Kikuchi, Y. Mechanistic target of
rapamycin complex 1 signaling regulates cell proliferation, cell survival, anddifferentiation in regenerating zebrafish fins. Bmc Developmental Biology 14,
doi:10.1186/s12861-014-0042-9 (2014).
Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M. & Poss, K. D. Fgf signaling
instructs position-dependent growth rate during zebrafish fin regeneration.
Development 132, 5173-5183, doi:10.1242/dev.02101 (2005).
Monteiro, J. et al. V-ATPase Proton Pumping Activity Is Required for Adult
Zebrafish Appendage Regeneration. Plos One 9,
doi:10.1371/journal.pone.0092594 (2014).
Nechiporuk, A. & Keating, M. T. A proliferation gradient between proximal andmsxb-expressing distal blastema directs zebrafish fin regeneration. Development
129, 2607-2617 (2002).
Pfefferli, C. & Jazwinska, A. The art of fin regeneration in zebrafish.
Regeneration 2, 72-83, doi:10.1002/reg2.33 (2015).
Rabinowitz, J. S. et al. Transcriptomic, proteomic, and metabolomic landscapeof positional memory in the caudal fin of zebrafish. Proceedings of the National
Academy of Sciences of the United States of America 114, E717-E726,
doi:10.1073/pnas.1620755114 (2017).
Sahara, M., Santoro, F. & Chien, K. R. Programming and reprogramming ahuman heart cell. Embo Journal 34, 710-738, doi:10.15252/embj.201490563(2015).
Sehring, I. M., Jahn, C. & Weidinger, G. Zebrafish fin and heart: what's special
about regeneration? Current Opinion in Genetics & Development 40, 48-56,
doi:10.1016/j.gde.2016.05.011 (2016).
Simoes, M. G. et al. Denervation impairs regeneration of amputated zebrafish
fins. Bmc Developmental Biology 14, doi:10.1186/s12861-014-0049-2 (2014).
Tanaka, E. M. & Reddien, P. W. The Cellular Basis for Animal Regeneration.
Developmental Cell 21, 172-185, doi:10.1016/j.devcel.2011.06.016 (2011).
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Tzahor, E. & Poss, K. D. Cardiac regeneration strategies: Staying young atheart. Science 356, 1035-1039, doi:10.1126/science.aam5894 (2017).

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Chapter 2. Leucine/glutamine and v-ATPase/lysosomal
acidification via mTORC1 activation are required for
position-dependent regeneration.

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Abstract
In animal regeneration, control of position-dependent cell proliferation is crucial for thecomplete restoration of patterned appendages in terms of both, shape and size. However,detailed mechanisms of this process are largely unknown. In this study, we identified
leucine/glutamine and v-ATPase/lysosomal acidification, via mechanistic target of
rapamycin complex 1 (mTORC1) activation, as effectors of amputation plane-dependent
zebrafish caudal fin regeneration. mTORC1 activation, which functions in cellproliferation, was regulated by lysosomal acidification possibly via v-ATPase activity at
3 h post amputation (hpa). Inhibition of lysosomal acidification resulted in reduced
growth factor-related gene expression and suppression of blastema formation at 24 and48 hpa, respectively. Along the proximal-distal axis, position-dependent lysosomal
acidification and mTORC1 activation were observed from 3 hpa. We also report that
Slc7a5 (L-type amino acid transporter), whose gene expression is position-dependent, isnecessary for mTORC1 activation upstream of lysosomal acidification during fin
regeneration. Furthermore, treatment with leucine and glutamine, for both proximal anddistal fin stumps, led to an up-regulation in cell proliferation via mTORC1 activation,
indicating that leucine/glutamine signaling possesses the ability to change the positiondependent regeneration.
Our findings reveal that leucine/glutamine and vATPase/lysosomal acidification via mTORC1 activation are required for positiondependent zebrafish fin regeneration.

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Introduction
Complete regeneration (in terms of both shape and size) of the patterned appendages isobserved in some vertebrates such as fish and salamanders [1,2]. This restorative ability
is achieved via cellular properties that control cell proliferation and patterning depending
on regional-specific information, also known as positional memory [3-6]. To date, threefactors (transmembrane receptor Prod1 [7,8], retinoic acid (RA) [5], and Hand2 [9]) havebeen proposed as effectors of the positional memory in salamander limbs, amphibianlimbs, and zebrafish pectoral fins, respectively. The expression of Prod1 and hand2
exhibits a gradient along the proximal-distal (P-D) axis in salamander limbs 8 andzebrafish pectoral fins 9, respectively, while no gradient of the RA signaling components
has been reported in salamander limbs or zebrafish fins prior to injury. Although a
recent report identified many genes, proteins, and metabolites via omics analyses in
addition to Prod1, RA, and Hand2 along the P-D axis of the zebrafish caudal fin 10, thedetailed molecular regulatory mechanisms of position-dependent regeneration are stilllargely unknown.
Signaling molecules, such as transforming growth factor- (TGF-), Wnt, fibroblast
growth factor (FGF), Notch, Hedgehog, insulin-like growth factor (IGF), RA, andmechanistic target of rapamycin (mTOR) have been determined to be necessary for
appendage regeneration [11,12]. Among them, the mTOR is well known to sense
environmental cues (growth factors, nutrients, and the cellular energy change) and control
the cell growth and metabolism [13]. mTOR belongs to a serine/threonine protein
kinase family and exists as two distinct complexes-mTOR complex1 (mTORC1) and 2(mTORC2) [13]. In the mTORC1 signaling pathway, growth factors, intracellular andenvironmental stresses (e.g. energy, oxygen, and DNA damage), and amino acids are
known to be upstream regulators [14,15]. mTORC1 regulates cell growth via protein,lipid, and nucleotide synthesis, and it is also known to inhibit autophagy [14,15]. The
amino acids, especially leucine, glutamine, and arginine, activate mTORC1 signaling via
a lysosomal amino acid transporter (SLC38A9) and a RAS-related GTP-binding protein(Rag)/Regulator/vacuolar-type proton transporter H+-ATPase (v-ATPase) complex[14,15]. A previous report proved that the expression of v-ATPase demonstrated
position-dependency and that the v-ATPase activity was necessary for the expression ofaldehyde dehydrogenase 1 family, member A2 (aldh1a2) (a type of RA synthetase) during
zebrafish caudal fin regeneration [16]. However, the function of the amino acids and
SLC38A9/Rag/Regulator/v-ATPase complex in the position-dependent appendage
regeneration process has not yet been reported.
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Zebrafish caudal fins are complex appendages that are composed of epidermal cells,
segmented bony rays, blood vessels, fibroblast-like mesenchymal cells, and nerve axons.
Using zebrafish caudal fin regeneration systems, we have previously showed thatmTORC1 signaling regulated cell proliferation, cell survival, and differentiation inzebrafish fin regeneration downstream of the IGF and Wnt pathways [17]. However,
the regulation of mTORC1 activity via upstream regulators other than growth factors
during fin regeneration has not yet been analyzed. In this study, we explored the
upstream regulators of mTORC1 and identified the axis of leucine/glutamine signalingv-ATPase/lysosomal acidification via mTORC1 activation as an effector of positiondependent zebrafish caudal fin regeneration.

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Results
mTORC1 activation is not controlled by IGF and Wnt signaling at 3 h post
amputation
We have previously reported that mTORC1 activation was regulated by both IGF and
Wnt signaling pathways at 24 and 48 h post amputation (hpa) during zebrafish fin
regeneration by using pharmacological inhibitors, NVP-ADW742 (an IGF-1R inhibitor)
and IWP-2 (a Wnt/-catenin inhibitor) [17]. However, amino acids and environmental
stresses have been known to function as the upstream regulators of mTORC1 in addition
to growth factors [14,15]. To explore the involvement of the upstream regulators otherthan growth factors in mTORC1 regulation, we carefully observed the expression ofphosphorylated S6 kinase (p-S6K; activated form of S6K), a downstream target ofmTORC1, during fin regeneration while IGF or Wnt signaling was inhibited. The pS6K fluorescence intensities per area that consist of the whole regenerates and 500 m
below the amputation plane were significantly reduced via rapamaycin (rapa) treatment(Fig. 1C-D’, G, H-I’, L, M-N’, and Q). On the other hand, the p-S6K fluorescenceintensities in IGF or Wnt signaling inhibited-fin stumps remained unchanged at 3 hpa(Fig. 1C, C’, and E-G), but were significantly reduced at 6 and 12 hpa when compared tothose in the DMSO-treated fin stumps (Fig. 1H, H’, J-L, M, M’, and O-Q). The resultsof p-S6K fluorescence intensities at 3 and 12 hpa observed by immunohistochemical
staining were further supported by western blotting analysis (Fig. S1), indicating thatmTORC1 activation is not under the control of IGF and Wnt signaling at 3 hpa during fin
regeneration.
Lysosomal acidification, possibly through v-ATPase, is required for mTORC1
activation
Previous studies using cell culture systems reported that the mTORC1 activation wasclosely linked to the SLC38A9/Rag/Regulator/v-ATPase complex on the lysosomal
surface [13-15]. Interestingly, one of components in the lysosomal protein complex, vATPase, is required for zebrafish fin regeneration [16]. Therefore, we first examined
the activity of the proton transporter, v-ATPase, during fin regeneration with the help of
the LysoTracker, which is a fluorescent dye for labelling acidic lysosomes. Highfluorescent signals of the LysoTracker were observed at 3 hpa at the amputation plane,
which gradually decreased until 12 hpa (Fig. S2), showing the acidification of lysosomesduring fin regeneration. To examine the relationship between the activation of v-ATPaseand mTORC1, we further examined the functional inhibition of v-ATPase by using two
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different pharmacological inhibitors, Concanamycin A (ConcA) [18] and Bafilomycin A1(BafA1) [19]. We found that the p-S6K fluorescence intensity and p-S6K protein level
were significantly reduced in each inhibitor-treated fin stump at 3 hpa byimmunohistochemistry and western blotting, respectively (Fig. 2B-F). Lysosomal
acidification at the amputation plane was suppressed by ConcA or BafA1 treatment at 3
hpa (Fig. 2G-J), suggesting that one of the mTORC1 regulators is a v-ATPase that
regulates lysosomal acidification at this regeneration stage.
To evaluate the lysosomal acidification on mTORC1 activation, we next examined
the inhibition of lysosomal acidification via treatment of ammonium chloride (NH4Cl) or
chloroquine (CQ), both of which are the weak bases and function to increase thelysosomal pH [20]. The reagent treatment lead to a significant reduction of the p-S6K
fluorescence intensity and p-S6K protein level at 3 hpa by immunohistochemistry andwestern blotting, respectively (Fig. 3B-F). We confirmed that lysosomal acidification
was blocked in NH4Cl- or CQ-treated fin stumps at 3 hpa using LysoTracker (Fig. S3)
and apoptosis was not increased by the reagent treatment at 3 and 24 hpa (Fig, S4). The
expression of growth factor-related genes (wnt10a, igf2b, aldh1a2, and fgf20a) [11,12]
was significantly downregulated at 24 hpa in NH4Cl- or CQ-treated fin stumps (Fig. 3G).
To further investigate the relationship between p-S6K and lysosomal acidification, we
double stained fin stumps using the LysoTracker and p-S6K antibody. Approximately
55 % of the p-S6K-positive cells were LysoTracker-positive in the fin stumps at 3 hpa(Fig. 3H-K), suggesting an association between mTORC1 activation and lysosomal
acidification. Collectively, these results showed that lysosomal acidification is required
to activate mTORC1 possibly via v-ATPase activity.
Lysosomal acidification and mTORC1 activation are dependent on the amputation
plane along the P-D axis
A previous report showed that the expression of atp6v1e1b, a v-ATPase family gene,and the H+ efflux in the proximal regenerates were higher than those in the distal
regenerates at 12 hpa [16]. These results prompted us to analyze whether theacidification of lysosome was dependent on the position of the amputation plane along
the P-D axis. LysoTracker fluorescence was detected at both proximal and distal
amputation planes from 1 hpa (Fig. 4B-E’’). The fluorescence intensities at the proximal
regions were significantly higher than those at the distal regions, and this difference influorescence intensities remained until 6 hpa (Fig. 4F). Cryosections under the confocal
microscope showed that the fluorescent LysoTracker-positive cells were observed in theepidermis and fin rays of proximal and distal fin stumps at 3 hpa (Fig. 4G and H).
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Moreover, the ratio of the fluorescent LysoTracker-positive cell number/total cell number(RLT) within the area that consist of the whole regenerates and 500 m below theamputation plane in proximal stumps was significantly higher than that in distal stumps
at 3 hpa (Fig. 4I). Because lysosomal acidification is required for mTORC1 activation,
the position-dependence of p-S6K- and proliferating cell nuclear antigen (PCNA, a makerfor proliferative cells [21])-positive cells was also examined during fin regeneration.
We found that p-S6K fluorescence intensity and p-S6K protein level in the proximal
regions were significantly higher than those in the distal regions during fin regeneration
at 3 and 12 hpa by immunohistochemistry and western blotting, respectively (Fig. 5A-F).
Consistent with previous results that reported the regulation of cell proliferation via
mTORC1 [17], the ratios of PCNA-positive cell number/total cell number (RPTs) within
the area that consist of the whole regenerates also showed dependence on the amputation
plane at 36 and 48 hpa (Fig. 5G-K). Our results suggest that position-dependent
lysosomal acidification may cause the differences in mTORC1 activation between theproximal and distal amputation planes.
L-type amino acid transporter 1 is required for mTORC1 activation
Previous studies implicated branched-chain amino acids, especially leucine, and their Ltype amino acid transporters (LATs) as effective activators of mTORC1 [22-25].
Because there are five LATs (LAT1; Slc7a5, LAT2; Slc7a8a and Slc7a8b, LAT3; Slc43a1aand Slc43a1b) in zebrafish as per the zebrafish database, we first examined the expressionof these LATs at 0, 1, 3, 6, and 12 hpa. Among them, slc7a5 showed the highest increasein expression levels at 3 hpa, and its expression in the proximal region was significantly
higher than that in the distal region (Fig. 6A and Fig. S5). Therefore, we further
performed vivo-morpholino oligo nucleotide (MO)-mediated slc7a5 knockdown
experiments during fin regeneration. We first confirmed that a previously reported
slc7a5-MO [26] blocked the exon1 splicing of slc7a5 pre-mRNA detected via
quantitative real-time PCR (qPCR) (Fig. S6). Formation of blastema, a heterogeneous
population of progenitor cells, was suppressed in slc7a5-MO-injected fin regenerates
when compared to in standard MO (st-MO)-injected fin regenerates at 48 hpa (Fig. 6CF). Consistent with the suppressed blastema formation, p-S6K fluorescence intensity,
p-S6K protein level, and expression of growth factor-related genes were significantly
reduced in slc7a5-MO-injected fins (Fig. 6G-K). We further found via LysoTracker
analysis that lysosomal acidification was significantly reduced in slc7a5-MO-injected finstumps when compared to in st-MO-injected fin stumps (Fig. 6L-N). In addition to
knockdown experiments, we have used a Slc7a5-specific inhibitor, JPH203 [27], to block18

the cellular entry of amino acids.
Similar to vivo-MO mediated knockdown
experiments, blastema formation at 48 hpa, the p-S6K fluorescence intensity, p-S6K
protein level, and lysosomal acidification by LysoTracker were significantly reduced at 3
hpa in JPH203-treated fin stumps when compared to those in DMSO-treated fins (Fig.
S7). These our findings suggest that amino acid signaling through the Slc7a5 transporter
is involved in mTORC1 activation possibly via lysosomal acidification during fin
regeneration.
Treatment with leucine and glutamine increases mTORC1 signaling and cellproliferation in both proximal and distal fin stumps
A recent study showed that mTORC1 signaling stimulated by leucine and glutamine
rescues defects associated with Roberts Syndrome in zebrafish [28]. Based on thisfinding, we wondered if the amino acids, leucine and glutamine, were involved in
mTORC1 activation during fin regeneration. To explore this possibility, we carried out
leucine and/or glutamine treatment in fin-amputated fish. Fin stumps after leucine or
glutamine treatment showed no increased mTORC1 activation (Fig. S8), whereas cotreatment with leucine and glutamine (LG) significantly increased the p-S6K fluorescenceintensity at 3, 6, 12 hpa and p-S6K protein level at 3 hpa in both proximal and distal finstumps (Fig. 7B-G). In addition, although LysoTracker fluorescence intensities were
not increased by leucine or glutamine treatment (Fig. S9B-K), they showed an LGconcentration-dependent increase in LG-treated fin stumps compared to control finstumps at 3 hpa (Fig. S9L-P). Consistent with increase of the p-S6K fluorescenceintensities, RPTs in both proximal and distal regenerates were increased in LG-treated fin
regenerates at 36 hpa (Fig. 7H-L). We further found that LG treatment significantly
increased the expression of growth factor-related genes, except wnt10a, at 24 hpa (Fig.
7M). These results showed that LG signaling had the ability to change positiondependent cell proliferation via mTORC1 activation during fin regeneration. Because
it is known that amino acid signaling is one of upstream regulators of mTORC1 via the
SLC38A9/Rag/Regulator/v-ATPase complex [13-15], we further tested the hierarchicalrelationship between LG signaling and v-ATPase/lysosomal acidification. The p-S6K
fluorescence intensity and p-S6K protein level were significantly increased by LGtreatments at 3 hpa; however, this increased level of mTORC1 signaling was suppressedby the co-treatment with ConcA or NH4Cl (Fig. 7N-T). Furthermore, up-regulation of
LysoTracker fluorescence intensity by LG treatment was annulled by slc7a5-MO
injection (Fig. S10). Results of the LG signal activation experiments, combined withthe slc7a5 knockdown data, suggest that LG and their transporter, Slc7a5, are upstream
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regulators of mTORC1 activity via v-ATPase/lysosomal acidification.

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DISCUSSION
One of the most important processes in regeneration is the complete restoration of lost
tissues or organs by cellular factors that regulate position-dependent cell proliferation andpatterning (positional memory). Because the mTORC1 signaling pathway plays acentral role in cell proliferation via the production of proteins, lipids, and nucleotides, andsuppression of catabolic pathways such as autophagy, it is a potent candidate effector of
position-dependent regeneration. In this study, we arrived at four conclusions. First,
lysosomal acidification, possibly through v-ATPase activity, is required for mTORC1
activation. Second, both lysosomal acidification and mTORC1 activation are dependent
on the amputation plane along the P-D axis. Third, an amino acid transporter Slc7a5 isnecessary for mTORC1 activation and the expression of growth factor-related genes.
Fourth, leucine and glutamine, which functions upstream of v-ATPase/lysosomal
acidification, have the ability to increase cell proliferation via mTORC1 activation in bothproximal and distal fin regenerates. Based on our results, we propose a molecularpathway that leads to the position-dependent cell proliferation during zebrafish caudal fin
regeneration (Fig. S11). Leucine and glutamine are proximally enriched in unamputatedfins as shown previously [10]. Leucine/glutamine signaling via Slc7a5 initiates vATPase activity in a position-dependent manner and causes lysosomal acidification.
The position-dependent v-ATPase activity/lysosomal acidification activates the mTORC1
signaling, which leads to cell proliferation and upregulation of growth factor-related gene
expression directly or indirectly.
In addition to leucine/glutamine, mTORC1 senses and responds to various stresses,
such as energetic/metabolic stress and oxidative stress, but most of these stresses are
repressive [29]. In contrast, a previous report using a cell culture system revealed thatoxidative stress activates mTORC1 through modulating the TSC1/TSC2-Rheb GTPasepathway [30]. Oxidative stress is one of main regulation factors for zebrafish cauda fin
regeneration: reactive oxygen species (ROS), such as H2O2, production was gradually
increased at the amputation plane by 16 hpa [31], and nerves and H2O2 levels controlledeach other in a positive feedback loop [32]. Moreover, the spatial and temporal
localization of ROS is closely similar to that of lysosome acidification. These previous
data, combined with our results, suggest that oxidative stress might be one of upstream
regulators for mTORC1 activation.
Double staining experiments with p-S6K and LysoTracker showed that approximately
55 % of p-S6K-positive cells were fluorescent LysoTracker-negative at 3 hpa (Fig. 3HK).

A possible explanation for this result is that lysosomal acidification probably
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occurred before 3 hpa and therefore, LysoTracker fluorescence had already disappearedat 3 hpa. Another possibility is that upstream environmental signals of mTORC1 otherthan amino acids and growth factors, such as oxygen and energy, activate mTORC1
within 3 hpa. It would be pertinent to explore these upstream environmental signals thatfunction in mTORC1 activation immediately after fin-amputation.
Recently, three amino acids (leucine, glutamine, and arginine) were implicated in theactivation of mTORC1 through various amino acid sensing mechanisms [15,25,33].
Some amino acid sensing mechanisms for mTORC1 activation have been reported:
binding of cytosolic leucine and Sestrin2, or cytosolic arginine and Cellular Arginine
Sensor for mTORC1 (CASTOR1), prevents Sestrin2-GAP activity towards Rags2(GATOR2), or CASTOR1-GATOR2 interaction via GATOR1 inactivation, leading toincreased Rag-dependent mTORC1 signaling, respectively [15,25,33]; glutamine andleucine activate mTORC1 via glutaminolysis and -ketoglutarate production upstream ofRag [34]; mTORC1 is activated by glutamine via adenosine diphosphate ribosylation
factor-1 dependent on v-ATPase [35]. In this study, we showed that individual leucine
or glutamine treatments had no effect on mTORC1 activation during fin regeneration,
whereas treatment of both leucine and glutamine activated mTORC1 signaling. There
are two possible explanations for this result; one is that both amino acids are required for
mTORC1 activation as in the glutaminolysis and -ketoglutarate production pathway, as
mentioned above; while the other possibility is that, because Lat1 (Slc7a5) and 4F2hc(Slc3a2) are bi-directional transporters 36, leucine is transported into the cytosol insteadof the glutamine efflux. A recent study revealed that Lat1/4F2hc is recruited bylysosomal-associated transmembrane protein 4b (LAPTM4b) to the lysosome [37].
This recruitment leads to uptake of leucine into the lysosome and is required for mTORC1
activation depending on v-ATPase activity. These previous results are consistent with
our results that show the axis of leucine/glutamine signaling-v-ATPase/lysosomal
acidification is associated with mTORC1 activation. Further studies are needed toelucidate how leucine and glutamine activate mTORC1 during fin regeneration.
Previous reports revealed that the H+ pump v-ATPase activity is necessary forregeneration of Xenopus larval tail [38] and zebrafish caudal fin [16]. In Xenopus larvae,v-ATPase mRNA and protein are expressed in the regeneration bud, and its protein is
localized in the cell-membrane of the bud cells [38]. The v-ATPase activity changes
membrane voltage in the bud cells via the H+ efflux and repolarization by its activity isnecessary for tail outgrowth [38]. Like the Xenopus larval tail, H+ efflux via v-ATPaseis required for blastema cell proliferation in zebrafish caudal fin regeneration at 24 and48 hpa [16].

In addition to the previous study results, we also found that lysosomal
22acidification, possibly through v-ATPase, is required for activation of mTORC1 signaling
within 3 hpa. To date, only one study reported that lysosomal pH functions not only inmTORC1 activation, but also in its deactivation via protein degradation in osteoclasts[39]. It is possible that lysosomal pH itself may affect the amino acid transportation into
the lysosome, amino acid sensing in the lysosome, or formation of the Rag/Regulator/vATPase complex formation on the lysosome membrane. Our results combined withprevious data showed that v-ATPase has dual functions in both lysosomal pH control andplasma membrane voltage for zebrafish caudal fin regeneration at different regeneration
stages.
However, the most important finding of this study is that leucine/glutamine and vATPase/lysosomal acidification via mTORC1 activation are required for the positiondependent fin regeneration. A recent report showed by metabolomic analyses that many
specific amino acids are proximally or distally enriched in uninjured zebrafish caudal fin
along the P-D axis. Both leucine and glutamine were identified as proximally-enrichedamino acids [10]. In addition, the expression of atp6v1e1b, a component of the vATPase complex, in the proximal regenerates was higher than that in the distal regenerates[16]. In this study, we also showed that the expression of slc7a5, encoding L-type aminoacid transporter, in the proximal regenerates was higher than that in the distal regenerates
at 3 hpa, and that treatment with leucine and glutamine lead to the upregulation of PCNA
expression in the distal regenerates at 36 hpa.
Furthermore, inhibition ofleucine/glutamine signaling and lysosomal acidification resulted in downregulation of
growth factor gene expression, such as wnt10a, igf2b, aldh1a2, and fgf20a.
Cumulatively, these results suggest that the axis of leucine/glutamine signaling-vATPase/lysosomal acidification via mTORC1 activation is a potent candidate effector of
the position-dependent regeneration along the P-D axis at the early stage of fin
regeneration (within 3 hpa). Moreover, differences in cell proliferation along the P-D
axis are regulated by mTORC1 or possibly by growth factors downstream of this axis.

23

Materials and Methods
Zebrafish husbandry, drug treatments, LysoTracker staining, amino acidtreatments, and morpholino knockdown
All zebrafish experiments were conducted in strict accordance with relevant nation andinternational guidelines: ‘Act on Welfare and Management of Animals’ (Ministry ofEnvironment of Japan), and were approved by the Hiroshima University Animal ResearchCommittee (Permit Number: F17-2). For caudal fin amputation, adult zebrafish(AB/Tüebingen strain) were anesthetize using Tricaine (Sigma-Aldrich) and the caudal
fins were amputated with razor blades along the dorsoventral axis. The proximal or
distal amputations were performed as described previously [16]; for distal amputation,
one or two ray segments were cut before the first ray bifurcation, and for proximal
amputation, two segments distal to the most posterior scale covering the fin base were
cut. In case amputation position is not specified, caudal fins were amputated at theproximal position.
Rapamycin (2.4 M, LC Laboratories), NVP-ADW742 (5 M, AdooQ Biosciences),
IWP-2 (10 M, Promega), CQ (4.5 mM, Sigma-Aldrich), NH4Cl (5 mM, Sigma-Aldrich),
and JPH203 (40 M, Chemescene) were used as specific inhibitors. All inhibitors,
except CQ and NH4Cl, were dissolved in DMSO, with a 0.1% final DMSO concentrationin fish water. The control fish were maintained in fish water (for CQ and NH4Cl) orwith 0.1% DMSO (for rapamycin, NVP-ADW742, IWP-2, and JPH203) at 28.5 ˚C andthis water containing respective drug was replaced daily. These treatments started 12 h
before fin amputation. Two fish were placed in 50 ml of fish water containing respective
drug and during the drug treatment, the fish were fed once a day. ConcA (200 nM, Santa
Cruz Biotechnology) or BafA1 (200 nM, Cell Signaling) were dissolved in 100 % DMSO
and diluted in Danieau medium 16. This diluted solution was injected into amputatedfins at 1 hpa.
For LysoTracker staining, fin-amputated adult fish were incubated in LysoTracker Red
DND-99 (ThermoFisher Scientific) solution diluted in fish water (1:1000) at 28.5 ˚C for
15 min before live imaging. To observe longitudinal ray sections, 2 nl of the diluted
LysoTracker solution (1 M) was injected directly into the 2nd, 3rd, and 4th fin rays fromthe dorsal and ventral amputated fins below the amputation plane. For leucine andglutamine treatment, the fish were incubated in the fish water containing 5, 15, and 25
mM L-leucine (Sigma-Aldrich) and/or L-glutamine (Sigma-Aldrich). The fish watercontaining of the two amino acids was replaced every 12 h.
For MO knockdown experiments, we used vivo-MO targeted against slc7a5 and24

standard control vivo-MO (st-MO) (GeneTools, LLC) as following: slc7a5 vivo-MO(slc7a5-MO) 5’-AGGTAACAGTTTACTTACGTATACA-3’ [26], st-MO 5’CCTCTTACCTCAGTTACAATTTTATA-3’. slc7a5-MO (1 mM) or st-MO (1 mM)
was injected into each inter-ray 3 h before fin amputation, and then every 12 h before
observation or harvest, as described previously [40].
Immunohistochemical staining and detection of cell death
The amputated fins were fixed overnight in 4% paraformaldehyde in 0.1 M phosphatebuffered saline at 4 ˚C, embedded in Tissue-Tek O.C.T compound (Sakura Finetek), andcryosectioned to 14 m thickness by using a Leica CM3050S [17]. The following
primary antibodies were used: anti-PCNA mouse monoclonal antibody at 1:1000 (Sigma,#P8825) and phospho-S6 ribosomal protein (Ser240/244) rabbit polyclonal antibody at
1:300 (Cell Signaling, #2215). The following secondary antibodies were used: Alexa
Fluor® 488 goat anti-rabbit IgG antibody at 1:500 (Invitrogen, Life Technologies Corp.)
and Alexa Fluor® 594 goat anti-mouse IgG antibody at 1:500 (Invitrogen, Life
Technologies Corp.). 4',6-diamidino-2-phenylindole (DAPI) was used for nuclei
staining at a concentration of 1:1000. To detection apoptotic cells, we performed
TUNEL staining using an In situ cell death detection kit (Roche, #11684795910)
according to the manufacturer’s instructions. The images were captured using an
Olympus FV1000-D confocal microscope with the same exposure times using the
FluoView software. Eight optical sections per one cryosection along the z-axis weretaken in 0.67 m intervals, and the captured images were analyzed using ImageJ (NIH).
qPCR analyses
Extraction of total RNA from regenerating fins was performed as described previously
41. Five independent qPCR experiments of ten genes (wnt10a, igf2b, aldh1a2, fgf20a,
slc7a5, slc7a8a, slc7a8b, slc43a1a, slc43a1b, and ribosomal protein L13a (rpl13a)) forgene expression and MO efficacy was performed in duplicates using the Thermal Cycler
Dice Real-Time System II and SYBR Premix Ex Taq II (TAKARA) as described
previously [42]. Zebrafish rpl13a was used as a reference gene. The qPCR primers
are listed in Table S1.
LysoTracker image analyses
Fluorescent LysoTracker images were acquired using a Leica MZ FLIII microscope andPenguin 600CL cooled CCD camera (Pixera). As described previously [43], average
LysoTracker fluorescence intensity was measured using the ImageJ software (NIH) with25

the following formula: [integrated intensity in the whole amputation plane area (W 500
μm × L full-length of the amputation plane) – mean fluorescence intensity of background].
Western blotting
Western blotting was performed as described previously [44]. Fins were lysed in 2 ×
SDS-sample buffer (125mM Tris-HCl (pH 6.8), 4% SDS, 20% Glycerol, 0.01%
bromophenol blue, 2% 2-mercaptoethanol) containing a protease inhibitor cocktail(Roche, #11836153001) and a phosphatase inhibitor cocktail (Nacalai, #07574-61).
Total proteins (5 g) were separated by SDS-PAGE and transferred to a polyvinylidene
fluoride (PVDF) membrane. The PVDF membranes were incubated overnight withprimary antibodies: anti-γTubulin (Sigma-Aldrich, #T6557) at 1:20000, ribosomal
protein S6 Antibody (C-8) (Santa Cruz Biotechnology, #sc-74459) at 1:1000, and antiphospho-S6 ribosomal protein (Ser240/244) (Cell signaling, #2215) at 1:1000. For
detection, horseradish peroxidase-coupled secondary antibodies: goat anti-mouse IgGHRP at 1:10000 (Santa Cruz Biotechnology, #sc-2055) and mouse anti-rabbit IgG-HRP
at 1:10000 (Santa Cruz Biotechnology, #sc-2357), and the ECL Prime Western Blotting
Detection Reagent (GE Healthcare, #RPN2232) were used, according to the
manufacturer’s instructions.

A densitometric analysis of the western blotting was

performed using ImageJ, with Tubulin for normalization.
Statistical analyses
All experiments were performed independently more than three times and the results
were reported as means ± Standard Error of Mean (SEM). More than 100 cells for distal
amputation and 500 cells for proximal amputation were used for quantification offluorescence intensity (for p-S6K and LysoTracker) or quantification of RLT, RPT, andpercentage of TUNEL+ cells. Statistical significance was determined by using theStudent’s t-test.

p values of ≤0.05 were considered to be statistically significant.

26

Acknowledgements
We thank Institute for Amphibian Biology in Hiroshima University for cryostat
sectioning and qPCR; the members of Kikuchi and Atsushi Suzuki laboratories in
Hiroshima University for helpful discussion and critical comments. This study was
supported by grants from Grant-in-Aid for Scientific Research from the JSPS (KAKENHI
JP15J05983) to K.T.

27

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31

FIGURES

Figure 1. Activation of S6K was not inhibited by pharmacological inhibitors of IGF
or Wnt signaling at 3 hpa.
(A) Scheme of inhibitor treatments for rapamycin (a mTORC1 inhibitor), NVPADW742 (an IGF-1R inhibitor), and IWP-2 (a Wnt/-catenin inhibitor) from -12 to 12
hpa. (B) Bright-field images of longitudinal ray sections of wild-type (WT) stumps (0and 1-12 hpa) and fin regenerates (36 hpa). Black and red dotted lines indicate theamputation planes (amp) and the border between epidermis and blastema, respectively.
(C-F’, H-K’, and M-P’) Longitudinal ray sections of DMSO- or inhibitors-treated WT fin
stumps that were immunohistochemically stained with an antibody against p-S6K (green)
at 3, 6, and 12 hpa (n = 5). 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)
fluorescent signal (grayscale; pseudo color) indicates the presence of nuclei.
Representative images used for quantification are shown in G, L, and Q along with a32highly magnified view.

White dashed lines indicate the amputation planes. Scale bars:

50 m (C-F, H-K, and M-P) and 25 m (C’-F’, H’-K’, and M’-P’). (G, L, and Q)
Quantification of the p-S6K fluorescence intensities per area that consist of the whole
regenerates and 500 m below the amputation plane (including the intra-ray, epidermal,
and wound epidermal cells) in rapamycin-, NVP-ADW742-, or IWP-2-treated fin stumps
at 3 (G), 6 (L), and 12 (Q) hpa (n = 5). n.s.: not significant. ***p < 0.001 by Student’s
t test. Error bars represent the standard error.

33

Figure 2. v-ATPase activity is required for the S6K activation.
(A) Experimental scheme. DMSO, ConcA, or BafA1 solution was injected into theamputated fins at 1 hpa. The red two-headed-arrow indicates LysoTracker treatment,
which was applied 15 min before observation. (B-E) Longitudinal ray sections andquantification of p-S6K fluorescence intensities per area that consist of the whole
regenerates and 500 m below the amputation plane in DMSO-, ConcA-, or BafA1treated fin stumps at 3 hpa; p-S6K and nuclei were visualized by immunohistochemical
staining and DAPI staining, respectively (n = 5). Representative images (B-D’) used forquantification are shown in E. White dashed lines indicate the amputation planes.
Scale bars: 50 m (B-D) and 25 m (B’-D’). ***p < 0.001 by Student’s t test. Error
bars represent the standard error. (F) Western blotting analysis of Tubulin, S6K, and pS6K in the DMSO-, ConcA-, or BafA1-treated fin stumps at 3 hpa (n = 6). Tubulin
serves as a loading control. Numbers below each lane show the level of p-S6K in
ConcA-, or BafA1-treated fin stumps relative to that in DMSO-treated fin stumps at 3 hpa
normalized to loading control. (G-I’) Images of bright-field and fluorescence (red)microscopy, and quantification of the LysoTracker fluorescence intensity (see Materials
34and Methods) of DMSO-, ConcA-, or BafA1-treated fin stumps (n = 8). Black boxed
areas in G-I are enlarged in G’-I’, respectively. The LysoTracker fluorescenceintensities in red boxed areas were measured (G-I’). Representative images (G’-I’) used
for quantification are shown in J. White dashed lines indicate the amputation planes.
Scale bars: 1 mm (G-H) and 500 m (G’-H’). ***p < 0.001 by Student’s t test.
bars represent the standard error.

35

Error

Figure 3. Requirement of lysosomal acidification in the S6K activation.
(A) Experimental scheme. NH4Cl or chloroquine (CQ) was treated from -12 to 3 or
24 hpa (B-G). LysoTracker solution was injected into the amputated fins 15 min before
fixation (H-K).
(B-E) Longitudinal ray sections and quantification of p-S6K
fluorescence intensities per area that consist of the whole regenerates and 500 m below
the amputation plane in control, NH4Cl-, or CQ-treated fin stumps at 3 hpa; p-S6K andnuclei were visualized by immunohistochemical staining and DAPI staining, respectively(n = 5). Representative images (B-D’) used for quantification are shown in E. White
dashed lines indicate the amputation planes. Scale bars: 50 m (B-D) and 25 m (B’D’). ***p < 0.001 by Student’s t test. Error bars represent the standard error. (F)Western blotting analysis of Tubulin, S6K, and p-S6K in the DMSO-, NH4Cl-, or CQtreated fin stumps at 3 hpa. Tubulin serves as a loading control. Numbers below eachlane show the level of p-S6K in NH4Cl-, or CQ-treated fin stumps relative to that in
DMSO-treated fin stumps at 3 hpa normalized to loading control. (G) The relative
expression of growth factor-related genes in NH4Cl- or CQ-treated fin stumps by qPCR
at 24 hpa.

***p < 0.001, ** p < 0.05 by Student’s t test.
36

Error bars represent thestandard error.

(H-K) Longitudinal ray sections and quantification of p-S6K+ and

LysoTracker fluorescence+ cells in area that consist of the whole regenerates and 500 m
below the amputation plane at 3 hpa; p-S6K (green), nuclei (grayscale), and lysosomal
acidification (red) were visualized by immunohistochemical staining, DAPI, and
LysoTracker (Lyso.), respectively (n = 8). White boxed areas in H are enlarged in I-J’,
respectively. Representative images (H-J’) used for quantification are shown in K.
Arrowheads in I’ and J’ indicate LysoTracker fluorescence and p-S6K double positive
cells. Scale bars: 50 m (H) and 25m (I-J’). A pie chart shows that 55.4 % of pS6K+ cells are the LysoTracker fluorescence positive cells (K).

37

Figure 4. Position-dependent lysosomal acidification during fin regeneration.
(A) Experimental scheme. Red two-headed-arrows indicate LysoTracker treatment,
which was applied 15 min before observation. For cryosections, LysoTracker solution
was injected into the amputated fins 15 min before fixation. (B-E’’) Bright-field andfluorescent images of LysoTracker-treated fins at 1, 3, 6, and 12 hpa (n = 5). Black
boxed areas in B-E are enlarged in B’-E’ and B’’-E’’, respectively. The LysoTracker
fluorescence intensities in red boxed areas were measured (B-E’’). Representative
images (B’-E’’) used for quantification are shown in F. White dashed lines indicate theamputation planes. Scale bars: 3 mm (B-E) and 500 m (B’-E’ and B’’-E’’). (F)Quantification of Lysotracker fluorescence intensities at the proximal and distal positionsat 1, 3, 6, and 12 hpa (n = 5). n.s.: not significant.
38***p < 0.001, ** p < 0.05 by

Student’s t test. Error bars represent the standard error. (G-I) Longitudinal ray
sections and quantification of ratios of LysoTracker fluorescence-positive cellnumber/total cell number (RLTs) within 500 m of the amputation plane at 3 hpa (n = 5).
Representative images (G and H) used for quantification are shown in I. Arrowheads
indicate LysoTracker fluorescence-positive cells (red). Scale bars: 50 µm (G and H)
n.s.: not significant. ***p < 0.001 by Student’s t test. Error bars represent the standard
error.

39

Figure 5. The position-dependent activation of S6K during fin regeneration.
(A-E) Longitudinal ray sections (3 and 12 hpa) and quantification of p-S6K
fluorescence intensities per area that consist of the whole regenerates and 500 m below
the amputation plane (3, 6, 12, and 18 hpa) of proximal and distal positions in WT fins;p-S6K and nuclei were visualized by immunohistochemical staining and DAPI staining,respectively (n = 8). Representative images (A-D’) used for quantification are shown in
E. White dashed lines indicate the amputation planes. Scale bars: 50 m (A-D) and25m (A’-D’). ***p < 0.001 by Student’s t test. Error bars represent the standard error.
(F) Western blotting analysis of Tubulin, S6K, and p-S6K in WT fin stumps at 3 and 12
hpa. Tubulin serves as a loading control. Numbers below each lane show the level of
p-S6K in proximal fin stumps relative to that in distal fin stumps at 3 or 12 hpa normalized
to loading control, respectively. (G-J) Longitudinal ray sections of proximal and distal
fins in WT fins at 36 and 48 hpa; PCNA and nuclei were visualized by40

immunohistochemical staining and DAPI staining, respectively (n = 5). Representative
images (G-J) used for quantification are shown in K. White dashed lines indicate theamputation planes. Scale bars: 50 m (G-J). (K) Ratios of PCNA-positive cellnumber/total cell number (RPTs) in whole regenerates at 36 and 48 hpa (n = 5). ***p <0.001 by Student’s t test. Error bars represent the standard error.

41

Figure 6. Slc7a5 is required for S6K activation and lysosomal acidification.
(A) The relative expression of slc7a5 in proximal and distal fin regenerates at 0, 1, 3,
6, and 12 hpa by qPCR analyses. (B) Scheme of vivo-MO mediated knockdown
experiments from -3 to 48 hpa. A red two-headed-arrow indicates LysoTracker
treatment, which was applied 15 min before observation. Syringes indicate vivo-MO
injection. (C-F) Outgrowth of fin regenerates and quantification of their length after
being injected with standard vivo-MO (st-MO) or slc7a5 vivo-MO (slc7a5-MO) at 48
hpa (n = 5).
The boxed areas in C are enlarged in D and E, respectively.
Representative images (C-E) used for quantification are shown in F. Black dashed lines
indicate the amputation planes. Scale bars: 1 mm (C) and 250 m (D and E). ***p <42

0.001 by Student’s t test. Error bars represent the standard error. (G-I) Longitudinal
ray sections and quantification of p-S6K fluorescence intensities per area that consist of
the whole regenerates and 500 m below the amputation plane in st-MO or slc7a5-MO
injected fin stumps at 3 hpa; p-S6K and nuclei were visualized by immunohistochemical
staining and DAPI staining, respectively (n = 8). Representative images (G-H’) used
for quantification are shown in I. Scale bars: 50 m (G and H) and 25 m (G’-H’).
***p < 0.001 by Student’s t test. Error bars represent the standard error. (J) Western
blotting analysis of Tubulin, S6K, and p-S6K in st-MO or slc7a5-MO injected fin stumps
at 3 hpa (n = 5). Tubulin serves as a loading control. Numbers below each lane show
the level of p-S6K in st-MO injected fin stumps relative to that in slc7a5-MO injected finstumps at 3 hpa normalized to loading control. (K) The relative expression of growth
factor-related genes in st-MO- or slc7a5-MO-injected fins by qPCR at 24 hpa. ***p <0.001. Error bars represent the standard error. (L-N) Images of bright-field andfluorescence microscopy, and quantification of LysoTracker fluorescence intensities inst-MO- or slc7a5-MO-injected fin stumps at 3 hpa (n = 5). Black boxed areas in L and
M are enlarged in L’ and M’, respectively. The LysoTracker fluorescence intensities in
red boxed areas were measured (L-M’). Representative images (L’-M’) used forquantification are shown in N. Scale bars: 1 mm (L and M) and 500 m (L’ and M’).
***p < 0.001. Error bars represent the standard error.

43

Figure 7. Leucine and glutamine treatment activates mTORC1 signaling via vATPase activity and lysosomal acidification.
(A) Experimental scheme.
(B-F) Longitudinal ray sections (3 hpa) andquantification of p-S6K fluorescence intensities per area that consist of the whole
regenerates and 500 m below the amputation plane (3, 6, and 12 hpa) of proximal anddistal positions in control or LG-treated fin regenerates; p-S6K and nuclei were visualized
by immunohistochemical staining and DAPI staining, respectively (n = 6).
44

Representative images (B-E) used for quantification are shown in F. White dashed lines
indicate the amputation planes. Scale bars: 50 m. ***p < 0.001 by Student’s t test.
Error bars represent the standard error. (G) Western blotting analysis of Tubulin, S6K,
and p-S6K in proximally and distally amputated LG-treated fin stumps at 3 hpa.
Tubulin serves as a loading control. Numbers below each lane show the level of p-S6K
in proximally or distally amputated LG-treated fin stumps relative to that in proximallyor distally amputated control fin stumps at 3 hpa normalized to loading control,
respectively. (H-L) Longitudinal ray sections and RPT quantification in proximally anddistally amputated control or LG-treated fin regenerates at 36 hpa; PCNA and nuclei werevisualized by immunohistochemical staining and DAPI staining, respectively (n = 5).
Representative images (H-K) used for quantification are shown in L. White dashed lines
indicate the amputation planes. Scale bars: 50 m. ***p < 0.001 by Student’s t test.
Error bars represent the standard error. (M) The relative expression of growth factorrelated genes in control and LG-treated fin regenerates by qPCR at 24 hpa. n.s.: notsignificant. ***p < 0.001 by Student’s t test. Error bars represent the standard error.
(N-S) Longitudinal ray sections and quantification of p-S6K fluorescence intensities perarea that consist of the whole regenerates and 500 m below the amputation plane incontrol, LG-treated, LG-treated and DMSO-injected (LG + DMSO), LG-treated and
ConcA-injected (LG + ConcA), or LG- and NH4Cl-treated (LG + NH4Cl) fin stumps at
3 hpa; p-S6K and nuclei were visualized by immunohistochemical staining and DAPI
staining, respectively (n = 5). Representative images (N-R) used for quantification are
shown in S. White dashed lines indicate the amputation planes. Scale bars: 50 m.
***p < 0.001 by Student’s t test. Error bars represent the standard error. (T) Western
blotting analysis of Tubulin, S6K, and p-S6K in control, LG-treated, LG + DMSO, LG+ ConcA, or LG + NH4Cl fin stumps at 3 hpa (n=6). Tubulin serves as a loading
control. Numbers below each lane show the level of p-S6K in LG-treated, LG + DMSO,
LG + ConcA, or LG + NH4Cl fin stumps relative to that in control fin stumps at 3 hpa
normalized to loading control.

45

Supplementary figures

Figure S1. Western blot analysis of rapamycin-, NVP-ADW742-, or IWP-2-treated
fin stumps.
Western blot analysis of Tubulin, S6K, and p-S6K in rapamycin-, NVP-ADW742-,
or IWP-2-treated fin stumps at 3 and 12 hpa (n = 5). Tubulin serves as a loading control.
Numbers below each lane show the level of p-S6K in rapamycin-, NVP-ADW742-, orIWP-2-treated fin stumps relative to that in DMSO-treated fin stumps at 3 or 12 hpa
normalized to loading control, respectively.

46

Figure S2. Lysosomal acidification during fin regeneration.
(A) Experimental scheme. Red two-headed-arrows indicate LysoTracker treatment,
which was applied 15 min before observation. (B-F’) Images of bright-field andfluorescence microscopy, and quantification of LysoTracker fluorescence intensities at 0,
3, 6, 12, and 24 hpa (n = 5). Black boxed areas in B-F are enlarged in B’-F’, respectively.
The LysoTracker fluorescence intensities in red boxed areas were measured (B-F’).
Representative images (B’-F’) used for quantification are shown in G.

White dashedlines indicate the amputation planes. Scale bars: 1 mm (B-F) and 500 m (B’-F’). n.s.:
not significant. ***p < 0.001 by Student’s t test. Error bars represent the standard
error.

47

Figure S3. Lysosomal acidification in NH4Cl- and CQ-treated fins.
(A) Experimental scheme.
A red two-headed-arrows indicates LysoTracker
treatment, which was applied 15 min before observation. (B-E) Images of bright-field
and fluorescence microscopy, and quantification of LysoTracker fluorescence intensities
in control, NH4Cl-, or CQ-treated fins at 3 hpa (n = 8). Black boxed areas in B-D are
enlarged in B’-D’, respectively. The LysoTracker fluorescence intensities in red boxed
areas were measured (B-D’). Representative images (B’-D’) used for quantification are
shown in E. White dashed lines indicate the amputation planes. Scale bars: 1 mm (BD) and 500 m (B’-D’).
standard error.

***p < 0.001 by Student’s t test. Error bars represent the48

Figure S4. Detection of apoptotic cells by NH4Cl- or CQ-treatment.
(A) Experimental scheme. NH4Cl or chloroquine (CQ) was treated from -12 to 3 or
24 hpa.

(B-G) Longitudinal ray sections and quantification of TUNEL positive

cells/total cell number per area that consists of the whole regenerates and 500 m below
the amputation plane in control, NH4Cl-, or CQ-treated fin stumps at 3 and 24 hpa;
apoptotic cells and nuclei were visualized by immunohistochemical staining and DAPI
staining, respectively (n = 5). Representative images (B-G) used for quantification are
shown in H. White dashed lines indicate the amputation planes. Scale bars: 50 m (BG). n.s.: not significant. Error bars represent the standard error.

49

Figure S5. Expression of LATs genes during fin regeneration.
The relative expression of the four LATs genes (slc7a8a, slc7a8b, slc43a1a, andslc43a1b) at 0, 3, 6, and 12 hpa by qPCR. Error bars represent the standard error.

50

Figure S6. slc7a5 pre-mRNA splicing was blocked by vivo-MO.
(A) Scheme of the slc7a5 pre-mRNA regions analyzed for splicing (boxed, exons;lines, introns; arrows, primers). (B) Spliced PCR products of slc7a5, obtained by qPCR,
were significantly reduced in slc7a5-MO-injected fins when compared to in st-MOinjected fins by qPCR. ***p < 0.001 by Student’s t test. Error bars represent thestandard error.

51

Figure S7. S6K activation and lysosomal acidification are inhibited by JPH203.
(A) Scheme of JPH203 (Slc7a5-specific inhibitor) treatment from -12 to 3 or 48 hpa.
A red two-headed-arrow indicates LysoTracker treatment, which was applied 15 min
before observation. (B-D) Outgrowth of fin regenerates and quantification of theirlength in control or JPH203-treated fin regenerates at 48 hpa (n = 5). Representative
images (B and C) used for quantification are shown in D. Black dashed lines indicate
the amputation planes. Scale bars: 250 m. ***p < 0.001 by Student’s t test. Error
bars represent the standard error. (E-G) Longitudinal ray sections and quantification of
p-S6K fluorescence intensities per area that consists of the whole regenerates and 500 m
below the amputation plane in DMSO- or JPH203-treated fin regenerates at 3 hpa; p-S6K
and nuclei were visualized by immunohistochemical staining and DAPI staining,respectively (n = 5). Representative images (E-F’) used for quantification are shown in
G. White dashed lines indicate the amputation planes. Scale bars: 50 m (E and F)
and 25 m (E’ and F’). ***p < 0.001 by Student’s t test. Error bars represent thestandard error. (H) Western blot analysis of Tubulin, S6K, and p-S6K in the DMSOor JPH203-treated fin stumps (n = 6). Tubulin serves as a loading control. Numbers
52below each lane show the level of p-S6K in JPH203-treated fin stumps relative to that in
DMSO-treated fin stumps at 3 hpa normalized to loading control. (I-K) Images ofbright-field and fluorescence microscopy, and quantification of LysoTracker fluorescenceintensities in DMSO- or JPH203-treated fins at 3 hpa (n = 8). Black boxed areas in I
and J are enlarged in I’ and J’, respectively. The LysoTracker fluorescence intensities in
red boxed areas were measured (I-J’).
Representative images (I’-J’) used forquantification are shown in K. White dashed lines indicate the amputation planes.
Scale bars: 1 mm (I and J) and 500 m (I’ and J’).
Error bars represent the standard error.

53***p < 0.001 by Student’s t test.

Figure S8. Leucine or glutamine treatment has no effect on S6K activation.
(A) Experimental scheme of leucine or glutamine treatment from -12 to 3 hpa. (BE) Longitudinal ray sections and quantification of p-S6K fluorescence intensities per areathat consists of the whole regenerates and 500 m below the amputation plane in control,
leucine-, or glutamine-treated fin regenerates at 3 hpa; p-S6K and nuclei were visualized
by immunohistochemical staining and DAPI staining, respectively (n = 5).
Representative images (B-D’) used for quantification are shown in E. White dashedlines indicate the amputation planes. Scale bars: 50 m.
bars represent the standard error.

54

n.s.: not significant.

Error

Figure S9. LG treatment up-regulates lysosomal acidification in a concentrationdependent manner.
(A) Experimental scheme of leucine and/or glutamine treatment from -12 to 3 hpa.
A red two-headed-arrow indicates LysoTracker treatment, which was applied 15 min
before observation. (B-P) Images of bright-field and fluorescence microscopy, andquantification of LysoTracker fluorescence intensities in fin stumps at 3 hpa (n = 6).
Black boxed areas in B-E, G-J, and L-O are enlarged in B’-E’, G’-J’, and L’-O’,
respectively. The LysoTracker fluorescence intensities in red boxed area were measured(B-E’, G-J’, and L-O’). Representative images (B-E’, G-J’, and L-O’) used forquantification are shown in F, K, and P. White dashed lines indicate the amputation
planes. Scale bars: 1 mm (B-E, G-J, and L-O) and 500 m (B’-E’, G’-J’, and L’-O’).
***p < 0.001 by Student’s t test. Error bars represent the standard error.
55

Figure S10. Upregulation of lysosome acidification by LG treatment is blocked by
Slc7a5 knockdown.
(A) Experimental scheme of LG treatment from -12 to 3 hpa. A red two-headedarrow indicates LysoTracker treatment, which was applied 15 min before observation.
A syringe indicates vivo-MO injection at -3 hpa. (B-D’) Images of bright-field andfluorescence microscopy, and quantification of LysoTracker fluorescence intensities inst-MO-, LG-treated and st-MO-injected (LG + st-MO), or LG-treated and slc7a5-MOinjected (LG + slc7a5-MO) fin stumps at 3 hpa (n = 6). Black boxed areas in B-D are
enlarged in B’-D’, respectively. The LysoTracker fluorescence intensities in red boxed
areas were measured (B-D’). Representative images (B’-D’) used for quantification are
shown in E. Scale bars: 1 mm (B-D) and 500 m (B’-D’).
represent the standard error.

56***p < 0.001.

Error bars

Figure S11. A proposed model of position-dependent fin regeneration in zebrafish.
For discussion, see text. Leucine and glutamine are proximally enriched inunamputated fins.
The position-dependency of slc7a5 expression, lysosomal
acidification, and mTORC1 activity is found in this study (marked by red boxes).

57

Table S1Primer sequences 5' to 3'
Gene nameForward primer

Reverse primer

Ref.

wnt10aATTCACTCCAGGATGAGACTTCATA

GTTTCTGTTGTGGGCTTTGATTAG

1

igf2b

GCAGGTCATTCCAGTGATGC

TCTGAGCAGCCTTTCTTTGC

2

aldh1a2

GCTTACCTTGCTACCCTGGAGTCCAATGGGCTCATGTCTGGTGAG

This study

fgf20aGGACCACAGCAGATTTGGTATATTGG

CAGCTTTTCAGATCCGTACAGTTCGC

This study

slc7a5GGAGATCGGCAAAGGTGATACGCTCAATCATCTCCTCTGTGAC

This study

slc7a8a

GCAGGGTTTCTGCGATTATGGCAAGTCAGCAGCAACAAGCAG

This study

slc7a8b

CAGCAAGAAGAGCAGGAATGG

GTTTGGAAGTCTCTCTGTGCC

This study

slc43a1aCGTAACGTCACGGTTAGCACTTCTTCACCAGTAGCATTCACTGTC

This study

slc43a1b

GGCAGCATCAGGAGAAGAAGTGCGAACACAGCAGGTTCTCCAAC

This study

rpl13aTCTGGAGGACTGTAAGAGGTATGC

AGACGCACAATCTTGAGAGCAG

3

AGGCATCTTGAACCCTTGTG

4

qRT-PCR

checking morpholino efficacy
slc7a5TGAAACTGTGGATCGAGCTG

58

References[1] Bouzaffour, M., Dufourcq, P., Lecaudey, V., Haas, P. & Vriz, S. Fgf and Sdf-1
pathways interact during zebrafish fin regeneration. PLoS One 4, e5824,
doi:10.1371/journal.pone.0005824 (2009).
[2] Chablais, F. & Jazwinska, A. IGF signaling between blastema and wound epidermis
is required for fin regeneration. Development 137, 871-879, doi:10.1242/dev.043885(2010).
[3] Hirose, K., Shimoda, N. & Kikuchi, Y. Transient reduction of 5-methylcytosine and5-hydroxymethylcytosine is associated with active DNA demethylation during
regeneration of zebrafish fin. Epigenetics 8, 899-906, doi:10.4161/epi.25653 (2013).
[4] Chung, J. et al. The mTORC1/4E-BP pathway coordinates hemoglobin production
with L-leucine availability. Sci Signal 8, ra34, doi:10.1126/scisignal.aaa5903 (2015).

59

Chapter 3. General discussion

60General discussion
Positional memory is of one the unsolved issues in the regeneration field. How isregeneration regulated such that the organ achieves its original formation? It is an
important working as well as initiation of regeneration especially in vivo regeneration
therapy. By resolving this, I believe that this can possibly be applied directly in the therapy
of function defective organs in the future.
In this research, we applied approaches to elucidate its mechanism using a simplified
experimental system on zebrafish fins. Furthermore, we clarified new factors throughscreening experiments using various inhibitors and drugs, revealing a part of themechanism in positional memory. In this study, it was revealed that amino acids, which
is important in our daily life activity, have a critical function in the process of tail fin
regeneration. Previously, it was reported that the mTORC1 pathway influences various
factors involved in regeneration from the early stage during zebrafish tail regeneration
process. However, the activation of the mTORC1 pathway could be confirmed
immediately after amputation when the induction of growth factor had not started, soother functions of mTORC1 pathway could be expected. What is upstream of themTORC1 pathway in the early stage of fin regeneration? According to the results of thisstudy, it was revealed that incorporation of amino acids induced the activation of themTORC1 pathway immediately just after fin amputation.
Further research revealed that the overtaking of amino acids, particularly leucine which
is one of the branched amino acids, had been greatly induced in the early stage ofregeneration with the mediation of the transporter. This branched chain amino acid had
been known as an important factor in our life [1]. Compared with other amino acids,
leucine is rapidly incorporated into the body and is known to greatly affect muscle repair
because the it directly regulates the expression of eukaryotic translation initiation factor
4E binding protein 1 (4E-BP1) through the mTOR pathway. In recent years, many athletes
are beginning to incorporate it in muscle enhancing supplement foods [2]. This meansthat taking in leucine, along with other amino acids, is an easier way to improve thecondition in our body than other therapies. These amino acids have already been reported
to affect differentiation not only in the muscle cell [3], but also in the pancreatic cell [4],neuronal cell [5], and so on [6][7]. However, the mechanism of the amino acid effect has
been unknown especially in developmental stage and cell differentiation [8][9].
In addition, fin regeneration with leucine and glutamine amino acid treatment was
attempted, but no fin elongation was observed. It was because cell death occurred during
the fin regeneration with long time amino acid treatment. In general, the amino acid,61

especially leucine, enters the metabolic pathway with the branched transferase andchanges various important molecules [10]. One of these processes is the N-acetyl-1leucine induced cell death via the p53 pathway, which occurs not only in cancer cells but
also in some somite cells [11]. In fact, it had been confirmed that the gene expression of
the branching type amino acid modification enzyme, BCAT, rapidly upregulated theexpression immediately after fin amputation during regeneration. These results impliedthat the metabolic pathways, N-acetyl-1-leucine related pathway, were activated, and thecell death signaling pathway had been activated in same time. Additionally, it had beenreported that the mTORC1 pathway was activated even in cell death induced cells andthese reports suggest that the next step is to induce fin regeneration with inhibition of thecell death. If the cell death pathway will be inhibited in fin regeneration with leucine andglutamine treatment, what will happen during fin regeneration? Can we observe fin
growth which implies regeneration activation? There is a way to inhibit the cell deathpathway in zebrafish model system by using the inhibitor of some caspases or the mutant
in p53. I believe it is possible to activate the regeneration, but the experiment has not been
done yet.
Recently, regenerative therapy are hot topics especially by using and in vitro system.
There are different systems in in vitro therapy, ES cells [12], iPS cells [13], and other cell
culture system [14][15][16]. If these systems can be applied to our therapy, many people
who have diseases and defects in the organ or tissue will benefit from these. However,these systems still have risks as much as benefits. The most important problem is the highrisk of carcinoma cells to develop and they have to be clear for our clinical trial. Many
researchers are trying to find a safer way to apply this therapy. Now, we know in vivo
regenerative therapy can possibly make therapy application safer. If we can directly
induce organ or tissue regeneration in our body, will these therapies be rejected by the
body or will carcinoma develop? Of course, nobody can answer these questions, but weknow the risk is lower in in vivo than in vitro therapy because we do not need to transplant
the cells from outside and use the cells derived from others. Additionally, applying thetreatment inside the body might be less expensive and efficient. I believe that in vivo
therapy will take the place of the iPS cell or regeneration therapy in the near future. If we
can directly induce the whole organ or tissue in the body and use an easy treatment, likea 2 type amino acid treatment, many people can be treated more effectively.

62

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Brosnan, J. T. & Brosnan, M. E. Branched-chain amino acids: Enzyme andsubstrate regulation. Journal of Nutrition 136, 207S-211S (2006).
Cossu, G. et al. Lancet Commission: Stem cells and regenerative medicine.
Lancet 391, 883-910, doi:10.1016/s0140-6736(17)31366-1 (2018).
Csobonyeiova, M., Polak, S., Zamborsky, R. & Danisovic, L. iPS cell
technologies and their prospect for bone regeneration and disease modeling: A
mini review. Journal of Advanced Research 8, 321-327,
doi:10.1016/j.jare.2017.02.004 (2017).
Garcia-Espinosa, M. A., Wallin, R., Hutson, S. M. & Sweatt, A. J. Widespreadneuronal expression of branched-chain aminotransferase in the CNS:
implications for leucine/glutamate metabolism and for signaling by amino acids.
Journal of Neurochemistry 100, 1458-1468, doi:10.1111/j.14714159.2006.04332.x (2007).
Kimbrel, E. A. & Lanza, R. Pluripotent stem cells: the last 10 years.
Regenerative Medicine 11, 831-847, doi:10.2217/rme-2016-0117 (2016).
Mansfeld, J. et al. Branched-chain amino acid catabolism is a conserved
regulator of physiological ageing. Nature Communications 6,
doi:10.1038/ncomms10043 (2015).
Mao, A. S. & Mooney, D. J. Regenerative medicine: Current therapies andfuture directions. Proceedings of the National Academy of Sciences of theUnited States of America 112, 14452-14459, doi:10.1073/pnas.1508520112(2015).
Nyman, S., Sopanen, T. & Mikola, J. REGULATION OF DEVELOPMENT OFLEUCINE UPTAKE ACTIVITY BY GLUTAMINE IN THE SCUTELLUMOF GERMINATING BARLEY-GRAIN. Plant Physiology 73, 135-141,
doi:10.1104/pp.73.1.135 (1983).
Pedroso, J. A. B., Zampieri, T. T. & Donato, J. Reviewing the Effects of lLeucine Supplementation in the Regulation of Food Intake, Energy Balance, andGlucose Homeostasis. Nutrients 7, 3914-3937, doi:10.3390/nu7053914 (2015).
Pereira, M. G. et al. Leucine Supplementation Improves Skeletal Muscle
63

12

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Regeneration after Cryolesion in Rats. Plos One 9,
doi:10.1371/journal.pone.0085283 (2014).
Rachdi, L., Aiello, V., Duvillie, B. & Scharfmann, R. L-Leucine Alters
Pancreatic beta-Cell Differentiation and Function via the mTor Signaling
Pathway. Diabetes 61, 409-417, doi:10.2337/db11-0765 (2012).
She, P. X. et al. Obesity-related elevations in plasma leucine are associated withalterations in enzymes involved in branched-chain amino acid metabolism.
American Journal of Physiology-Endocrinology and Metabolism 293, E1552E1563, doi:10.1152/ajpendo.00134.2007 (2007).
Suryawan, A., Orellana, R. A., Fiorotto, M. L. & Davis, T. A. TRIENNIAL
GROWTH SYMPOSIUM: Leucine acts as a nutrient signal to stimulate protein
synthesis in neonatal pigs. Journal of Animal Science 89, 2004-2016,
doi:10.2527/jas.2010-3400 (2011).
Tabar, V. & Studer, L. Pluripotent stem cells in regenerative medicine:challenges and recent progress. Nature Reviews Genetics 15, 82-92,
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64

Acknowledgement
I would like to gratefully hank my supervisor Prof. Yutaka Kikuchi (Hiroshima
University) for his critical instruction, encouragement and patience this thesis. I also
thank Akihiko Muto, and Shunya Hozumi (Hiroshima University) for their technicaladvise, valuable discussion, and helpful instruction; Dr Atushi Szuki, all member ofAtushi Suzuki laboratory for helpful discussion and critical comments; and the memberof our laboratory for supporting the experiments.

65