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Collection Highlights

Marine Organisms as Model Systems in Biology and Medicine

Malgorzata Kloc

Medaka biology management and experimental protocols

Second Edition Kinoshita

The Zebrafish Cellular and Developmental Biology Part A

Cellular Biology 4th Edition H. William Detrich

The Zebrafish Cellular and Developmental Biology Part B

Developmental Biology 4th Edition H. William Detrich
Biomechanics in Medicine and Biology Katarzyna Arkusz

Wavelets in Medicine and Biology Akram Aldroubi

Zebrafish A Model for Marine Peptide Based Drug Screening

Saravanan Ramachandran

Fishes out of water : biology and ecology of mudskippers

1st Edition Zeehan Jaafar

Deep Sea Fishes Biology Diversity Ecology and Fisheries

1st Edition Imants G. Priede
Hiromi Hirata · Atsuo Iida Editors

Zebrafish,
Medaka, and
Other Small
Fishes
New Model Animals in Biology,
Medicine, and Beyond
Zebrafish, Medaka, and Other Small Fishes
Hiromi Hirata • Atsuo Iida
Editors

Zebrafish, Medaka,
and Other Small Fishes
New Model Animals in Biology, Medicine,
and Beyond
Editors
Hiromi Hirata Atsuo Iida
College of Science and Engineering Institute for Frontier Life
Aoyama Gakuin University and Medical Sciences
Sagamihara, Japan Kyoto University
Kyoto, Japan

ISBN 978-981-13-1878-8    ISBN 978-981-13-1879-5 (eBook)

https://doi.org/10.1007/978-981-13-1879-5

Library of Congress Control Number: 2018956132

© Springer Nature Singapore Pte Ltd. 2018

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Foreword

This book, Zebrafish, Medaka, and Other Small Fishes: New Model Animals in
Biology, Medicine, and Beyond, offers readers advanced research topics in small
fish biology. These aquatic animals have come to the forefront of biomedical
research as simple genetic models for biological studies that retain molecular, cel-
lular, and physiological similarities with humans. Consequently, the majority of
disease-causing genes have orthologs in fish, and genetic models can be used advan-
tageously for drug screening. Small fish thus offer great hopes for biology and
medicine.
The first two parts of the book describe common elements that are essential for
the development, homeostasis, and reproduction of zebrafish and medaka. The
developmental topics covered are molecular signaling in development and cancer
(Ishitani and Zou), the ontology of blood vessels (Phng), hematopoietic stem cells
(Kobayashi), and the development of sensory neurons (Ogino and Hirata). The sec-
ond part deals with homeostasis related to gravity (Chatani and Kudo), reproduction
(Kanda), and secondary sex characteristics (Ogino).
The third part focuses on the development of human disease models in zebrafish
and medaka and their use in clinical studies, including angiogenesis (Katraki-Pavlou
and Beis), myopathies (Baxter and Bryson-Richardson), dystrophies (Mitsuhashi),
scoliosis (Guo, Ikegawa, and Shukunami), and Parkinson’s disease (Uemura and
Takahashi).
The last part describes challenging studies that utilize several fish species pos-
sessing special, unique traits, such as blind cavefish (Rohner), viviparous fish (Iida),
electric fish (Kohashi), and catfish, which have an excellent sense of taste (Ikenaga
and Kiyohara).
Collectively, these reviews encompass the broad range of studies currently under
way in a variety of fish species, from cavefish to fish in space. The thought-­provoking
chapters are of general interest to a broad readership, as you will likely find a topic
of direct pertinence to your own interests in biology, biotechnology, or drug discov-
ery and at the same time discover new and fascinating subjects that can be uniquely
studied in fish. This book thus nicely illustrates the usefulness and pertinence of
modern fish biology. Quoting the concluding sentence from Neil Shubin’s book

v
vi Foreword

Your Inner Fish (Pantheon Press, 2008): “I can imagine few things more beautiful
or intellectually profound than finding the basis for our humanity, and remedies for
many of the ills we suffer, nestled inside some part of the most humble creatures that
have ever lived on our planet.” We hope this book will help convey our enthusiasm
for fish models pertinent to drier vertebrates as well.

Department of Neurosciences Pierre Drapeau

University of Montréal,
Montréal, Canada
Preface

Small fish such as zebrafish and medaka are now among the major model organisms
employed in the life sciences. As model organisms, small fish owe their use in the
life sciences to the remarkable extent of species variation that commonly presents
researchers with complementary characteristics for studies ranging from develop-
ment and regeneration to complex behaviors such as learning and memory.
Zebrafish (Danio rerio) was first introduced as a vertebrate model organism in
the 1970s by Prof. George Streisinger at the University of Oregon. Originating from
India, the zebrafish was chosen as a result of its ease of laboratory maintenance and
diurnal driven ability to produce hundreds of embryos each day that displayed a
rapid rate of external development. These attributes contributed to the use of zebraf-
ish in two large-scale mutagenesis projects in Germany and the United States in the
1990s. These and other mutagenesis projects, coupled with the development of
transgenic techniques and fluorescent proteins for live-cell imaging, solidified the
use of zebrafish in the life sciences and thereby caused an expansion of the zebrafish
research community.
By comparison, medaka (Oryzias latipes) has been a popular pet fish in Japan
since the mid-1800s. In the late 1900s, the isolation of two genetically distinct lines
and several temperature-sensitive mutants promoted the use of medaka in the life
sciences. In addition, several genetically divergent populations of medaka have
been identified. While the method of sex determination has remained enigmatic in
zebrafish, a sex chromosome and a sex-determination gene have been identified in
medaka. These and other genetic attributes have rendered medaka the second most
popular fish model in the life sciences.
Over the last decade, development of cutting-edge technologies such as next-­
generation sequencing, CRISPR/Cas9-mediated genome editing, high-resolution
imaging, and a cellular-level connectome have further fostered the use of zebrafish
and medaka in the life sciences. As many of these techniques are translatable to
other small fishes, we expect that additional fish models will be developed enabling
scientists to address research topics that are not readily assessable in zebrafish and
medaka.

vii
viii Preface

Zebrafish, Medaka, and Other Small Fishes: New Model Animals in Biology,
Medicine, and Beyond introduces readers to research topics in small fish biology
currently being investigated by a group of young and ambitious scientists. This
special book includes not only basic biology to investigate common mechanisms in
animals, but also clinical models as translational research for human diseases using
zebrafish and medaka. In addition, the other chapters deal with eccentric small fish
with highly unique traits seemingly uncommon in other species. We suggest these
are forthcoming vertebrate models to provide novel insights and significance for
basic and applied research.
It will be a great pleasure to the authors if this book aids readers in their under-
standing of small fish and the ongoing studies that utilize them, with the hope that it
encourages participation in the large and fantastic world of small fish.

Kyoto, Japan Atsuo Iida

Sagamihara, Japan Hiromi Hirata
Contents

Part I Development and Cell Biology

1 Zebrafish Wnt/β-Catenin Signaling Reporters Facilitate
Understanding of In Vivo Dynamic Regulation
and Discovery of Therapeutic Agents����������������������������������������������������    3
Tohru Ishitani and Juqi Zou
2 Endothelial Cell Dynamics during Blood Vessel Morphogenesis��������   17
Li-Kun Phng
3 Development of Hematopoietic Stem Cells in Zebrafish����������������������   37
Isao Kobayashi
4 Rohon-Beard Neuron in Zebrafish��������������������������������������������������������   59
Kazutoyo Ogino and Hiromi Hirata

Part II Homeostasis and Reproduction

5 Fish in Space Shedding Light on Gravitational Biology����������������������   85
Masahiro Chatani and Akira Kudo
6 Small Teleosts Provide Hints Toward Understanding
the Evolution of the Central Regulatory
Mechanisms of Reproduction ����������������������������������������������������������������   99
Shinji Kanda
7 Diversified Sex Characteristics Developments
in Teleost Fishes: Implication for Evolution
of Androgen Receptor (AR) Gene Function������������������������������������������ 113
Yukiko Ogino, Gen Yamada, and Taisen Iguchi

ix
x Contents

Part III Clinical Models

8 Zebrafish Angiogenesis and Valve Morphogenesis:
Insights from Development and Disease Models���������������������������������� 129
Matina Katraki-Pavlou and Dimitris Beis
9 Advances in the Understanding of Skeletal Myopathies
from Zebrafish Models���������������������������������������������������������������������������� 151
Emily Claire Baxter and Robert J. Bryson-Richardson
10 Zebrafish Models of Muscular Dystrophies
and Congenital Myopathies�������������������������������������������������������������������� 185
Hiroaki Mitsuhashi
11 Emergence of Zebrafish as a Model System
for Understanding Human Scoliosis������������������������������������������������������ 217
Long Guo, Shiro Ikegawa, and Chisa Shukunami
12 Medaka Fish Model of Parkinson’s Disease������������������������������������������ 235
Norihito Uemura and Ryosuke Takahashi

Part IV Eccentric Fish

13 “Out of the Dark” Cavefish Are Entering Biomedical Research�������� 253
Nicolas Rohner
14 Xenotoca eiseni, a Viviparous Teleost, Possesses
a Trophotaenial Placenta for Maternal Nutrient Intake���������������������� 269
Atsuo Iida
15 Mormyrid Electric Fish as a Model to Study Cellular
and Molecular Basis of Temporal Processing in the Brain������������������ 279
Tsunehiko Kohashi
16 Chemosensory Systems in the Sea Catfish, Plotosus japonicus������������ 295
Takanori Ikenaga and Sadao Kiyohara
 Part I
Development and Cell Biology
Chapter 1
Zebrafish Wnt/β-Catenin Signaling
Reporters Facilitate Understanding
of In Vivo Dynamic Regulation
and Discovery of Therapeutic Agents

Tohru Ishitani and Juqi Zou

Abstract Wnt/β-catenin signaling plays multiple roles in embryogenesis, organo-

genesis, and adult tissue homeostasis, and its dysregulation is linked to numerous
human diseases such as cancer. Although strict spatiotemporal regulation must sup-
port the multi-functionality of Wnt/β-catenin signaling, detailed mechanisms remain
unclear. In addition, Wnt/β-catenin signaling is a potential drug target candidate and
several inhibitors have been identified by in vitro screening, but none have yet been
incorporated into clinical practice. Recent studies using reporter zebrafish lines
have gradually improved our understanding of in vivo dynamic regulation of
Wnt/β-catenin signaling and have facilitated the discovery of new chemicals that
can reduce Wnt/β-catenin signaling and cancer cell viability with few side effects.
Here, we describe several new mechanisms supporting the spatiotemporal regula-
tion of Wnt/β-catenin signaling and new small molecule inhibitors, discovered
using zebrafish reporters. In addition, we discuss the potential of zebrafish signaling
reporters in both developmental biology and pharmaceutical sciences.

Keywords Wnt/β-catenin signaling · reporter · modifier · chemical inhibitor ·

anti-cancer drug

1.1 Introduction

Wnt/β-catenin signaling is an evolutionarily conserved system that controls cell

proliferation, fate specification, differentiation, survival, and death during embryo-
genesis, organogenesis, and adult tissue homeostasis (Clevers 2006; Clevers and
Nusse 2012; Logan and Nusse 2004). Dysregulation of this signaling system is

T. Ishitani (*) · J. Zou

Lab of Integrated Signaling Systems, Department of Molecular Medicine, Institute for
Molecular & Cellular Regulation, Gunma University, Maebashi, Gunma, Japan
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2018 3

H. Hirata, A. Iida (eds.), Zebrafish, Medaka, and Other Small Fishes,
https://doi.org/10.1007/978-981-13-1879-5_1
4 T. Ishitani and J. Zou

Unstimulated Stimulated Wnt

Fz LRP5/6 Fz LRP5/6
Dvl
Axin
Ub GSK3β Axin
Ub GSK3β
Ub P β-cat
β-cat β-cat
β-cat β-cat
Degradation Cytoplasm
Nucleus

co-Rs β-cat
Tcf/Lef Target gene Tcf/Lef Target gene

Fig. 1.1 The Wnt/β-catenin signaling pathway. In unstimulated conditions, levels of cytoplasmic
β-catenin are kept low by a destruction complex including GSK3β and Axin. Tcf/Lef represses the
expression of target genes by interacting with transcriptional co-repressors (co-Rs). Binding of
Wnt to the receptor Frizzled (Fz) and its co-receptor LRP5/6 activates Dvl, and then Dvl promotes
dissociation of the β-catenin destruction complex and consequently stabilizes cytoplasmic
β-catenin. As a result, β-catenin accumulates and enters the nucleus, where it forms complexes
with Tcf/Lef that activate gene expression. Ub ubiquitin, P phosphorylation, β-cat β-catenin

linked to various human diseases, including cancer, obesity, diabetes, osteoporosis,

schizophrenia, and autism (Clevers 2006; Clevers and Nusse 2012; De Ferrari and
Moon 2006; Logan and Nusse 2004). This system transduces its signal by control-
ling the levels of cytoplasmic β-catenin protein (Fig. 1.1). In unstimulated cells,
cytoplasmic β-catenin are maintained at low levels by a destruction complex that
includes glycogen synthase kinase 3β (GSK3β) and Axin. GSK3β phosphorylates
β-catenin at the N-terminal region, thereby promoting its ubiquitination by the E3
Ub ligase β-TrCP and the subsequent proteasomal degradation (Clevers 2006;
Clevers and Nusse 2012; Logan and Nusse 2004). The Tcf/Lef family of transcrip-
tion factors represses the expression of Wnt/β-catenin target genes by interacting
with transcriptional co-repressors, such as histone deacetylase 1 (HDAC1) and
Groucho (Arce et al. 2009). Wnt/β-catenin signaling is activated when the secreted
glycoprotein Wnt binds to the cell surface receptor Frizzled (Fz) and its co-receptor
LRP5/6. This Wnt-bound receptor complex then recruits the cytoplasmic protein
Dishevelled (Dvl), which in turn promotes the dissociation of the β-catenin degrada-
tion complex. This series of events results in the stabilization of cytoplasmic
β-catenin. The increased β-catenin concentration drives its migration into the
nucleus where it forms complexes with Tcf/Lef, which activates gene expression.
This core Wnt/β-catenin signaling process has been revealed through extensive
investigation using invertebrate models, mammalian cell culture, and Xenopus early
embryos over the past three to four decades. In addition, knockout mouse analyses
have contributed to our understanding of the role Wnt/β-catenin signaling plays in
animal development and disease. However, the spatiotemporal dynamics of Wnt/β-­-
catenin signaling and its regulatory mechanisms in living animals remains unclear
though recent studies using Wnt/β-catenin signaling reporter zebrafish lines have
1 Zebrafish Wnt/β-Catenin Signaling Reporters Facilitate Understanding of In Vivo… 5

gradually improved our understanding. Here, we introduce Wnt/β-catenin signaling

reporter zebrafish lines and review several studies in which new mechanisms sup-
porting the spatiotemporal regulation of Wnt/β-catenin signaling were revealed
using these reporter lines. Furthermore, we also demonstrate how reporter lines may
be useful in the exploration of new anti-cancer drugs.

1.2 Wnt/β-Catenin Signaling Reporter Zebrafish Lines

and Their Properties

Zebrafish are one of the most suitable animals for live imaging because of their opti-
cal clarity and rapid development. A transgenic zebrafish line carrying the Wnt/β-­-
catenin signaling reporter top:GFP (original name: TOPdGFP)—which contains
four copies of consensus Tcf/Lef binding sites, a c-fos minimal promoter, and a
d2EGFP reporter gene (Fig. 1.2; Dorsky et al. 2002)—has proved to be a useful tool
for understanding the regulation of in vivo Wnt/β-catenin signaling. In fact, new
domains in which Wnt/β-catenin signaling is activated and new mechanisms that
regulate Wnt/β-catenin signaling have been discovered using this reporter (some of
which are described in Sect. 1.3). However, given that fine reporter activity has only
been observed in some Wnt/β-catenin signaling-active sites in living top:GFP-­
transgenic fish using a fluorescence stereomicroscope (Fig. 1.2), attempts have been
made to improve Wnt/β-catenin signaling reporters. We generated OTM:d2EGFP
(original name: Tcf/Lef-miniP:dGFP), which comprises a destabilized green fluo-
rescent protein (d2EGFP) driven by a promoter containing six copies of Tcf/Lef
binding sites and a minimal promoter (miniP) derived from Promega pGL4 (Fig. 1.2;
Shimizu et al. 2012). Moro et al. (2012) also generated two new reporters, 7xTCF-­
Xla.Siam:GFP and 7xTCF-Xla.Siam:nlsmCherry, which express GFP or mono-
meric Cherry proteins with nuclear localization signals (nlsmCherry) under the
control of seven multimerized TCF responsive elements upstream of the minimal
promoter of the Xenopus direct β-catenin target gene, siamois (Fig. 1.2). Results
showed that both reporters were activated in almost all cells in which Wnt/β-catenin
signaling is known to be active in zebrafish, and they also revealed further new
developmental roles of Wnt/β-catenin signaling, including in the formation of the
brain-blood vessel network and gill filaments (Moro et al. 2012; Shimizu et al.
2012). These reporters are useful but should be used separately. 7xTCF-Xla.
Siam:GFP and 7xTCF-Xla.Siam:nlsmCherry are suitable for searching for new
Wnt/β-catenin signaling-active cells/areas because they express fluorescence
strongly; the half-lives of GFP and mCherry are very long (more than 24 h), so fluo-
rescence of 7xTCF-Xla.Siam:GFP and 7xTCF-Xla.Siam:nlsmCherry can be
detected in cells where Wnt/β-catenin signaling was activated in the past. On the
other hand, OTM:d2EGFP is suitable for the detection of dynamic changes during
Wnt/β-catenin signaling in vivo as the half-life of the d2EGFP protein, the fluores-
cent reporter in OTM:d2EGFP, is relatively short (approximately 2 h). In addition,
to detect “highly” dynamic signaling changes, we recently generated “the third
6 T. Ishitani and J. Zou

(1) 4xTcf/Lef BS (WT)

(3)
Poly A OTM:d2EGFP
top:GFP d2EGFP control
c-fos promoter
6xTcf/Lef BS (WT)
OTM:d2EGFP d2EGFP
minimal
promoter (miniP) Axitinib

7xTCF- 7xTcf/Lef BS (WT) siam promoter (tkP)

Xla.Siam:GFP
GFP
XAV-939
7xTCF- 7xTcf/Lef BS (WT) siam promoter (tkP)
Xla.Siam:nls
mCherry nlsmCherry

(2) BF d2EGFP
OTM:d2EGFP OTM:d2EGFP

top:GFP top:GFP

Fig. 1.2 Zebrafish Wnt/β-catenin signaling reporters. (1) Schematic diagrams of Wnt/β-catenin
signaling-reporter constructs. Tcf/Lef BS: consensus sequence of the Tcf/Lef-binding site; PolyA:
polyadenylation sequence. (2) Comparison of reporter activity in OTM:d2EGFP- and top:GFP
transgenic zebrafish embryos. Dorsal views of transgenic embryos, with the anterior side to the
left. Cells expressing d2EGFP were visualized by fluorescence microscopy (right panel). Bright-­
field (BF) images are shown in the left panel. Scale bar, 200 μm. (3) Effects of chemical inhibitors
on OTM:d2EGFP reporters. Reporter activity was shown as green. Axitinib reduces OTM:d2EGFP
expression in the midbrain and tail (red arrows), while XAV-939 completely blocks it in whole
embryos

generation of in vivo Wnt/β-catenin signaling reporter”—OTM:Eluc-CP—which

expresses destabilized emerald luciferase in response to Wnt/β-catenin activation.
Using this, we can detect the dynamic change of Wnt/β-catenin signaling activity
during brain anterior-posterior patterning (Akieda et al. in preparation).
OTM:Eluc-CP will facilitate rigorous analysis of the spatiotemporal dynamics of
Wnt/β-catenin signaling.

1.3  tudies Using the Reporter Zebrafish Revealed New

S
Regulatory Mechanisms of Wnt/β-Catenin Signaling

Mechanisms supporting the spatiotemporal dynamics of Wnt/β-catenin signaling in

living animals remain unclear, but recent studies with reporter zebrafish, described
within this section, have gradually improved our understanding.
1 Zebrafish Wnt/β-Catenin Signaling Reporters Facilitate Understanding of In Vivo… 7

1.3.1 Reck: A New Wnt Regulator in Plasma Membrane

The G protein-coupled receptor Gpr124 promotes Wnt7-mediated β-catenin signal-

ing and controls central nervous system (CNS) angiogenesis (Posokhova et al.
2015; Zhou and Nathans 2014). Vanhollebeke et al. (2015) discovered that Reck
(reversion-inducing-cysteine-rich protein with Kazal motifs), a GPI-anchored extra-
cellular protein, cooperates with Gpr124 to activate Wnt/β-catenin signaling in
zebrafish CNS angiogenesis. Knockdown of Gpr124 and Reck using morpholino
antisense oligonucleotides (MO) reduced 7xTCF-Xla.Siam: GFP reporter activity
in zebrafish CNS endothelial cells and induced identical CNS-specific vascular
defects. Reck interacted with Gpr124 in the plasma membrane and synergistically
promoted Wnt7-mediated β-catenin signaling. In addition, live imaging analyses of
genetically mosaic zebrafish revealed that Gpr124- and Reck-dependent Wnt/β-­-
catenin signaling is specifically required for endothelial tip cells during sprouting
angiogenesis in the CNS. Interestingly, knockdown of Reck and Gpr124 specifi-
cally affected CNS angiogenesis but did not produce gross morphological pheno-
types, indicating that Gpr124 and Reck are Wnt/β-catenin signaling modulators
specifically required for CNS angiogenesis.

1.3.2 Hipk2 and Nephrocystin-4: New Dvl Modulators

Homeodomain-interacting protein kinase 2 (Hipk2) was identified as a positive

regulator of Wnt/β-catenin signaling using biochemistry and Drosophila genetics
(Lee et al. 2009). However, the mechanism by which Hipk2 promotes Wnt/β-catenin
signaling and its physiological significance was unclear but is explained by our
recent study using OTM:d2EGFP. MO-mediated knockdown of Hipk2 reduced
OTM:d2EGFP activity and the protein levels of Dvl, which is a core regulator of
Wnt/β-catenin signaling. Consequently, Wnt/β-catenin signaling-mediated brain
anterior-posterior (AP) patterning and tail development in zebrafish embryos were
disturbed. Interestingly, these defects were rescued by injection of Hipk2 wild-type
or kinase-negative mutant mRNA (Shimizu et al. 2014), suggesting that Hipk2 pro-
motes Dvl protein levels and Wnt/β-catenin signaling in a kinase activity-­
independent manner, and this regulation contributes to brain and tail development.
Biochemical analyses revealed that Hipk2 promotes the binding of protein phospha-
tase 1c (PP1c) to Dvl and thus the consequent dephosphorylation of Dvl, which
prevents Itch ubiquitin E3 ligase-mediated Dvl ubiquitination and degradation to
stabilize Dvl (Fig. 1.3; Shimizu et al. 2014). Consistent with this, knockdown of
PP1c also reduced OTM:d2EGFP activity; Hipk2 MO-induced reduction of
OTM:d2EGFP activity was rescued by injection of the Dvl 3A mutant, in which
PP1c-dephosphorylation sites were substituted to alanine (Shimizu et al. 2014).
Thus, a new post-translational modification of Dvl and its roles in Wnt signal trans-
duction and embryogenesis were revealed by the reporter zebrafish studies.
 8

(1) (2)
in the absence of Hipk2-PP1c in the presence of Hipk2-PP1c
Nephrocystin-4
CK1 Hipk2
Itch Ub Dvl Dvl
P P P PP1 c Stabilization UbUb
P P P P P
UbUb Dvl
Dvl Dvl Dvl
Ub β-cat
Degradation
β-cat β-cat Tcf/Lef
target gene
Degradation
Tcf/Lef Tcf/Lef
target gene target gene

(3) PGE2 (4) (5)

PKA Ub in the absence of Nlk2 in the presence of Nlk2

Stabilization Ub
P Ub
β-cat Nlk2
Tcf/Lef
β-cat HDAC1 β-cat
Tcf/Lef
target gene Lef1 P
Nrarp Tcf/Lef Lef1 Proliferati Proliferation
NCC gene on gene gene

HSC proliferation
and survival
Proliferation

Fig. 1.3 New regulatory mechanisms of Wnt/β-catenin signaling revealed using the reporter zebrafish. (1) In the absence of Hipk2-PP1c activity, Dvl is phos-
phorylated by CK1 and consequently ubiquitinated by Itch and then degraded via proteasome. As a result, the Wnt/β-catenin target gene is inactivated. In the
presence of Hipk2-PP1c activity, Hipk2 promotes the binding of PP1c to Dvl and PP1c-mediated Dvl dephosphorylation and consequently stabilizes Dvl.
Stabilized Dvl efficiently transduces Wnt signaling and induces target gene expression. (2) Nephrocystin-4 binds to and promotes the ubiquitination and deg-
radation of Dvl. (3) PGE2 promotes PKA-mediated phosphorylation and stabilization of β-catenin and thereby stimulates HSC proliferation and survival. (4)
Nrarp blocks the ubiquitination of Lef1 and consequently promotes Lef1 stabilization and Lef1-mediated Wnt/β-catenin signaling. (5) In the absence of Nlk2
activity, HDAC1 binds to Lef1 and inhibits Lef1-mediated transcription. When Nlk2 is activated, Nlk2 phosphorylates Lef1 thereby preventing HDAC1-­
T. Ishitani and J. Zou

mediated Lef1 inhibition and allowing NPCs to proliferate

1 Zebrafish Wnt/β-Catenin Signaling Reporters Facilitate Understanding of In Vivo… 9

The NPHP4 gene encoding nephrocystin-4 is associated with nephronophthisis,

which is a hereditary nephropathy characterized by interstitial fibrosis and cyst for-
mation. Burckle et al. (2011) examined the molecular and cellular functions of
Nephrocystin-4 using zebrafish as a model. Injection of Nephrocystin-4 MO
enhanced top:GFP activity in the pronephric duct and produced pronephric cysts;
Nephrocystin-4 MO-mediated pronephric cyst formation was enhanced by co-­
injection with the MO against prickle2 gene encoding a negative regulator of Dvl.
In the mammalian kidney cell line (MDCK), exogenous Nephrocystin bound to Dvl
and reduced its protein level and Wnt/β-catenin reporter activity (Burckle et al.
2011). Based on these findings, it was concluded that Nephrocystin-4 represses the
Wnt-β-catenin pathway via Dvl degradation (Fig. 1.3) and contributes to morpho-
genesis of zebrafish pronephros.

1.3.3 Simplet and PGE-PKA Axis: New β-Catenin Modulators

Simplet was first isolated as a gene expressed in the developing CNS of medaka fish
(Deyts et al. 2005) although molecular function is unclear. Using reporter fish, Kizil
et al. (2014) showed that Simplet is required for Wnt/β-catenin signaling as it posi-
tively regulates β-catenin nuclear localization; injection of simplet MO prevented
nuclear accumulation of β-catenin. Activation of top:GFP and 7xTCF-Xla.
Siam:nlsmCherry activities and expression of Wnt/β-catenin target genes, cdx4,
tbx6, and gbx1, were also prevented in the tail bud stage of zebrafish embryos but
not in 85% epiboly stage embryos; loss of Wnt/β-catenin signaling also produced
axial defects (Kizil et al. 2014). Simplet localized into the nucleus. Overexpression
of Simplet promoted β-catenin nuclear localization but not in nuclear-localization-­
signal (NLS)-deleted mutant SimpletΔNLS positive cells; overexpression of this
mutant blocked β-catenin nuclear localization and transcriptional activity.
Biochemical analyses revealed that Simplet proteins interact directly with β-catenin
(Kizil et al. 2014). These results suggest that Simplet associates with β-catenin to
promote its nuclear localization and transcriptional activity and plays an essential
role in zebrafish axial development.
Wnt/β-catenin signaling has been implicated in the regulation of hematopoietic
stem cells (HSCs); the bioactive lipid prostaglandin E2 (PGE2) also regulates the
induction and engraftment of HSCs (Reya et al. 2003; Trowbridge et al. 2006).
Therefore, Goessling et al. (2009) focused on the relationship between PGE2 and
HSCs and discovered that PGE2 promotes Wnt/β-catenin signaling in HSC regula-
tion. Treatment of zebrafish embryos with PGE2 enhanced top:GFP activity in
HSCs and Wnt/β-catenin signaling-mediated HSC proliferation and survival, while
treatment with indomethacin, which suppresses PGE2 production, reduced them.
This suggests that PGE2 positively regulates Wnt/β-catenin signaling in HSCs.
Furthermore, treatment with the PKA chemical activator forskolin reversed
indomethacin-­induced HSC reduction, while treatment with the PKA chemical
inhibitor H89 blocked PGE2-induced HSC formation, suggesting that PGE2 regu-
10 T. Ishitani and J. Zou

lates HSCs via PKA. By carrying out assays using hematopoietic mouse ES cells,
Goessling et al. (2009) confirmed that the relationship between PGE2, PKA, and
Wnt/β-catenin signaling is also conserved in mammals. In addition, it was found
that PGE treatment promoted the phosphorylation of β-catenin at Ser-675, which is
mediated by PKA, and stabilizes β-catenin in mouse ES cells; indomethacin reduced
this phosphorylation and β-catenin protein levels (Goessling et al. 2009). Thus,
PGE2-PKA axis stimulates β-catenin stability and the consequent Wnt/β-catenin
signaling activity through β-catenin phosphorylation to promote HSC formation
(Fig. 1.3).

1.3.4 Nrarp and NLK: New Tcf/Lef Modifiers

We discovered the essential roles of Lef1 post-translational modification in embryo-

genesis using top:GFP reporter zebrafish and showed that proper control of Lef1
ubiquitination is involved in the development of neural crest cells (NCCs) (Fig. 1.3;
Ishitani et al. 2005). NCCs are pluripotent progenitors induced within the neuroepi-
thelium in vertebrate embryos. They migrate to target destinations throughout the
embryo and differentiate into diverse cell types, including sensory neurons, glia,
smooth muscle, cranial cartilage, bone cells, endocrine cells, and pigment cells. Wnt/
β-catenin signaling is known to regulate induction, migration, and differentiation of
NCCs (Yanfeng et al. 2003) and furthermore, Ishitani et al. (2005) found that Nrarp
(Notch-regulated ankyrin repeat protein), a small protein containing two ankyrin
repeats, promotes Wnt/β-catenin signaling activity in NCCs by blocking Lef1 ubiq-
uitination. Nrarp was expressed in migrating neural crest cells; Nrarp knockdown,
using MO, reduced top:GFP reporter activity in migrating NCCs and induced defects
in NCC migration and differentiation (Ishitani et al. 2005), which suggests that
Nrarp contributes to NCC development through positive regulation of Wnt/β-catenin
signaling in migrating NCCs. Biochemical analyses also showed that Nrarp blocks
the ubiquitination-proteasome-dependent degradation of Lef1 and consequently sta-
bilizes it, which promotes Wnt/β-catenin signaling in migrating NCCs (Ishitani et al.
2005). This was the first discovery of ubiquitination of Tcf/Lef family proteins.
Lef1 phosphorylation is also essential for midbrain development (Fig. 1.3).
Previous reports showed that MAP kinase-related nemo-like kinase (NLK)
­phosphorylates the Tcf/Lef family of transcription factors and activates Wnt/β-
catenin signaling in Caenorhabditis elegans (Ishitani et al. 1999; Meneghini et al.
1999) though the physiological role of NLK-mediated Tcf/Lef regulation in verte-
brates was not explained. We found that knockdown of zebrafish NLK (Nlk2)
reduced top:GFP reporter activity and proliferation of neural progenitor cells in the
developing midbrain, without gross morphological defects (Ota et al. 2012). This
suggests that Nlk2 acts as a midbrain-specific Wnt/β-catenin activator and promotes
cell proliferation in the midbrain of zebrafish. Biochemical studies revealed that
Nlk2 phosphorylates Lef1 at the conserved Thr residue, which promotes Lef1 tran-
1 Zebrafish Wnt/β-Catenin Signaling Reporters Facilitate Understanding of In Vivo… 11

scriptional activity by blocking the interaction of Lef1 with HDAC1(Ota et al.

2012). Consistent with this finding, the Nlk2 knockdown-induced reduction in
top:GFP activity was reversed by co-knockdown of HDAC1 (Ota et al. 2012). Thus,
the midbrain-specific regulation of Wnt/β-catenin signaling was revealed using
reporter fish.
It is noteworthy that inhibition of Reck, Gpr124, Nephrocystin-4, PGE2, Nrarp,
and/or Nlk2 induces defects in specific tissues, which suggests they are cell/tissue
type-specific Wnt/β-catenin signaling modifiers, but not general Wnt/β-catenin sig-
naling regulators. Such cell/tissue type-specific modifiers should support the spatio-
temporal dynamics of Wnt/β-catenin signaling, which enables Wnt/β-catenin
signaling to play diverse roles in animal development and homeostasis.

1.4 Utility of the Wnt/β-Catenin Signaling Reporter

Zebrafish for Drug Discovery

Wnt/β-catenin signaling regulates stem cell fates, and dysregulation of Wnt/β-­-

catenin signaling causes various human diseases, including cancer, mental disor-
ders, osteoporosis, and obesity. Therefore, chemical inhibitors against Wnt/β-catenin
signaling have potential as regenerative medicines and therapeutic agents; several
chemical inhibitors of Wnt/β-catenin signaling, such as XAV-939, IWR-1, IWP-2,
and ICG-001, have already been identified. XAV-939 and IWR-1 reduce β-catenin
protein levels by promoting Axin protein stabilization, and IWP-2 also blocks Wnt
secretion (Chen et al. 2009). ICG-001 blocks β-catenin binding to a histone acetyl-
transferase CREB-binding protein (CBP) and consequently prevents CREB-­
mediated β-catenin activation (Teo et al. 2005). Importantly, treatment with these
chemical inhibitors eliminates not only Wnt/β-catenin signaling activity in mam-
malian cells but also the activities of Wnt/β-catenin signaling reporters (7xTCF-­
Xla.Siam:GFP and OTM:d2EGFP) in zebrafish (Moro et al. 2012; Shimizu et al.
2012). Therefore, reporter fish were used for identification of new chemicals that
can inhibit Wnt/β-catenin signaling in vivo. In this section, we describe three chemi-
cals, the in vivo activities of which were characterized using Wnt/β-catenin signal-
ing reporter fish.

1.4.1 9 -Hydroxycanthin-6-one Promotes β-Catenin

Degradation by Activating GSK3β

Ohishi et al. (2015) screened plant extracts using reporter assays in HEK293 cells
and identified the β-carboline alkaloid 9-hydroxycanthin-6-one as a new Wnt/β-­-
catenin inhibitor. Although the direct target molecules of 9-hydroxycanthin-6-one
12 T. Ishitani and J. Zou

remain unclear, treatment with 9-hydroxycanthin-6-one activated GSK3β-

mediated phosphorylation and the consequent degradation of β-catenin. To con-
firm in vivo activity of this inhibitor, Ohishi et al. (2015) used top:GFP
Wnt/β-catenin reporter fish. Treatment with 9-hydroxycanthin-6-one reduced
top:GFP activity and expression of endogenous Wnt/β-catenin target genes,
including mitf and zic2a, and partially rescued the eyeless phenotype induced by
treatment with BIO, a GSK3β specific inhibitor (Ohishi et al. 2015), which sug-
gests that this inhibitor can block Wnt/β-catenin signaling via GSK3β regulation
in vivo.

1.4.2 PMED-1 Blocks β-Catenin Binding to CREB

Because XAV-939, IWR-1, and 9-hydroxycanthin-6-one affect the activity of the

Wnt/β-catenin core signaling system, which contributes to the homeostasis of vari-
ous tissues, these inhibitors may not only affect abnormal tissues but may also dam-
age healthy ones. In contrast, pharmacological inhibition of the cell type-specific
modulators may enable cell type-specific Wnt/β-catenin signaling regulation and
contribute to disease treatment with few side effects. Two histone acetyltransfer-
ases, CREB and p300, interact with β-catenin to activate β-catenin-mediated tran-
scription although the binding of each results in distinct effects. CBP-β-catenin
complexes positively regulate the expression of genes promoting cell proliferation
while p300-β-catenin complexes are not involved in cell proliferation (Teo and
Kahn 2010). Interestingly, ICG-001 specifically inhibits β-catenin binding to CBP,
but not to p300, and blocks β-catenin-mediated cell proliferation. In addition, ICG-­
001 is selectively cytotoxic to colon carcinoma cells because treatment with ICG-­
001 kills SW480 and HCT116 colon cancer cells, while it has no effect on
CCD-841Co normal colonic epithelial cells (Teo et al. 2005). Therefore, ICG-001
is thought to be usable for cancer treatment with few side effects. Delgado et al.
(2014) searched for chemicals that possess similar activity to ICG-001 by using in
silico analysis and zebrafish reporter assays. To identify compounds structurally
similar to ICG-001, they screened the ZINC 10 database (http://zinc.docking.org/
subsets/lead-like) and identified PMED-1 as the lead compound, with ≥70% simi-
larity to ICG-001. Similar to ICG-001, PMED-1 blocked the interaction of β-catenin
with CBP, but not with p300, and reduced the viability of hepatocellular carcinoma
(HCC) cells; it has no toxicity in human normal hepatocytes (Fig. 1.4; Delgado
et al. 2014). Results also showed that PMED-1 can block Wnt/β-catenin signaling
in vivo using OTM:d2EGFP reporter zebrafish. Interestingly, the OTM:d2EGFP
activity in PMED-1-treated zebrafish embryos was strongly inhibited from 5 to 15 h
after treatment but restored after 24 h; OTM:d2EGFP activity still continued to be
suppressed 24 h after treatment in XAV939-treated zebrafish embryos, which indi-
cates that the half-life of Wnt/β-catenin inhibitory activity of PMED-1 is shorter
than that of XAV939. Thus, it is possible to evaluate the effect on Wnt/β-catenin
signaling activity and its duration in vivo of a new Wnt/β-catenin inhibitor using
reporter fish.
1 Zebrafish Wnt/β-Catenin Signaling Reporters Facilitate Understanding of In Vivo… 13

(1) (2)
CBP
Ub
β-cat CBP β-cat Ub
Tcf/Lef Tcf/Lef Ub
Proliferation Proliferation
gene PMED-1 gene SHPRH β-cat
Stabilization
p300 p300
β-cat β-cat Axitinib
β-cat
Tcf/Lef other Tcf/Lef other
target gene
Tcf/Lef target gene
target gene

Fig. 1.4 Chemical inhibitors against Wnt/β-catenin signaling, which were characterized by
reporter fish analyses. (1) PMED-1 blocked the interaction of β-catenin with CBP but not with
p300. (2) Axitinib binds to and stabilizes the E3 ubiquitin ligase, SHPRH. Axitinib-stabilized
SHPRH promoted the ubiquitination and degradation of nuclear β-catenin

1.4.3 Axitinib Promotes β-Catenin Degradation in Nucleus

Most Wnt/β-catenin pathway mutations in cancer patients are observed in the

β-catenin gene and the APC gene, which encodes a component of the β-catenin
destruction complex. Therefore, it is important to develop drugs that target down-
stream of the destruction complex. Recently, Qu et al. (2016) identified axitinib as
such a drug; 460 Food and Drug Administration (FDA)-approved drugs were
screened to find chemicals capable of inhibiting Wnt/β-catenin signaling activation
induced by treatment with BIO, a GSK3β specific inhibitor, in HEK293 cells. Qu
et al. (2016) also confirmed that axitinib inhibits in vivo Wnt/β-catenin signaling in
OTM:d2EGFP zebrafish. Interestingly, axitinib reduced OTM:d2EGFP activity in
the developing midbrain and tail but not in the developing ear, lateral line primordia,
pectoral fin, fin fold, or cranial NCCs (Fig. 1.2; Qu et al. 2016); XAV-939 com-
pletely eliminated OTM:d2EGFP activity in the whole body (Fig. 1.2; Shimizu
et al. 2012). Results suggest that axitinib inhibits Wnt/β-catenin signaling in spe-
cific cells but not in all cells. Consistent with this idea, axitinib reduced the activities
of Wnt/β-catenin signaling and proliferation in colon cancer cells but not in normal
intestinal tissues (Qu et al. 2016), indicating that axitinib may be usable for colon
cancer treatment with few side effects. Furthermore, biochemical analyses revealed
that axitinib binds to and stabilizes the E3 ubiquitin ligase SHPRH (SNF2, histone-­
linker, PHD and RING finger domain-containing helicase). Axitinib-stabilized
SHPRH promoted the ubiquitination and degradation of nuclear β-catenin, which
was independent of the β-catenin destruction complex including APC and GSK3β
(Fig. 1.4; Qu et al. 2016). Thus, a new Wnt/β-catenin signaling inhibitor and its
mechanism of action were elucidated.

1.5 Conclusions

Numerous molecules that regulate Wnt/β-catenin signaling have been discovered

previously using invertebrate models, mammalian cell culture, and Xenopus early
embryos. It was believed that most were “general regulators” that participate in the
14 T. Ishitani and J. Zou

control of Wnt/β-catenin signaling in all cells/tissues, but their physiological roles

in vertebrates were unclear. However, recent studies using reporter zebrafish lines
have revealed cell/tissue-type specific Wnt/β-catenin signaling modifiers, such as
Reck, Gpr124, Nephrocystin-4, Nrarp, and Nlk2, which must complicate the spatio-
temporal pattern of Wnt/β-catenin signaling activity in order to play multiple roles
in animal development and homeostasis. It is also possible that parts of previously
identified regulators may also be cell/tissue-type-specific modifiers. Future studies
on previously and newly identified Wnt regulators using reporter fish will facilitate
further understanding of cell/tissue type-specific Wnt/β-catenin signaling regulation
and thereby make clear the whole picture of Wnt/β-catenin signaling regulation in
living animals.
Reporter zebrafish lines will also help the discovery of new anti-cancer drugs
that have few side effects. The chemicals that control the activity of Wnt/β-catenin
core signaling systems may affect the homeostasis of healthy tissues, while chemi-
cal inhibitors against cell/tissue-specific modulators may enable cancer tissue-­
specific regulation. It is worth noting that axitinib inhibits OTM:d2EGFP reporter
activity in a part of Wnt/β-catenin-active cells in zebrafish embryos and also reduces
the activity of Wnt/β-catenin signaling and proliferation in colon cancer cells but
not in normal intestinal tissues (Qu et al. 2016). This indicates that axitinib acts as a
Wnt/β-catenin inhibitor in specific cells and may be able to reduce colon cancer cell
activity without causing severe side effects. It also suggests that such cell/tissue-­
specific reporter inhibition could be used as an index for safe Wnt/β-catenin inhibi-
tors that can be employed for cancer therapy in anti-cancer drug screening.
In addition to Wnt/β-catenin signaling, other cell signaling pathways, including
TGF-β/BMP and Shh, are activated repeatedly and play multiple roles in animal
development and homeostasis, and dysregulation of these pathways is involved in
tumorigenesis. In addition, reporter fish lines that visualize various signaling path-
ways have been generated (Casari et al. 2014; Laux et al. 2011; Schwend et al.
2010). Therefore, a similar strategy can be implemented to investigate the in vivo
regulatory mechanisms of other cell signaling pathways and their control agents.
Thus, cell signaling reporter zebrafish are a useful tool for both investigating the
mechanisms of dynamic signaling regulation and for identifying new drugs control-
ling particular signaling pathways in specific cells/tissues.

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Chapter 2
Endothelial Cell Dynamics during Blood
Vessel Morphogenesis

Li-Kun Phng

Abstract Blood vessels, together with the heart, have a fundamental role in sup-
porting the metabolic demands of tissues not only during development but also in
adults. New blood vessels are frequently generated through angiogenesis when new
vessels emerge from pre-existing ones (Fig. 2.1a). Initially, endothelial cells (ECs)
lining an existing vessel are selected to become tip cells to spearhead the formation
of new vascular sprouts. New sprouts grow through EC proliferation and the polar-
ized collective migration of both tip and trailing stalk cells into the avascular tissue.
In order to generate a network of interconnecting vessel segments, tip cells anasto-
mose with neighboring tip cells to establish new vascular loops. Importantly, vascu-
lar sprouts develop into tubes through which oxygen, metabolites, cells, and waste
products can circulate around the body. Finally, the tubular network of blood vessels
are either maintained or, depending on the tissue requirements in which the vessels
pervade, remodeled through pruning into a more refined vascular network that car-
ries blood flow optimally to tissues (Fig. 2.1b).
Over the past few decades, many key signaling pathways that regulate blood ves-
sel development have been identified using primarily the mouse as the model organ-
ism. These include the Neuropilin (NRP)/Vascular Endothelial Growth Factor
(VEGF)/Vascular Endothelial Growth Factor Receptor (VEGFR), Jagged/Delta-­like/
Notch, Transforming Growth Factor β (TGFβ)/Bone Morphogenic Protein (BMP)
and EphrinB/EphB signaling cascades (Adams RH, Alitalo K. Nat Rev Mol Cell Biol
8:464–478, 2007; Potente M, Makinen T. Nat Rev Mol Cell Biol 18:477, 2017).
Although these studies have uncovered the fundamental principles of angiogenesis,
temporal information on the cellular dynamics of angiogenesis has been lacking due
to difficulties in performing live imaging in mouse embryos and tissues. These chal-
lenges are alleviated by the use of zebrafish, whose embryos develop ex utero, are
optically transparent and are therefore highly suited for live imaging. Combined with
recent advances in imaging techniques and the development of fluorescent biosen-
sors or reporters, it is now possible to observe the dynamics of ECs at cellular and

L.-K. Phng (*)

Laboratory for Vascular Morphogenesis, RIKEN Center for Biosystems Dynamics Research,
Kobe, Japan
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2018 17

H. Hirata, A. Iida (eds.), Zebrafish, Medaka, and Other Small Fishes,
https://doi.org/10.1007/978-981-13-1879-5_2
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