Fish: a suitable system to model human bone disorders and ... · bone disorders and discover drugs with osteogenic or osteotoxic activities ... guppy have also been used to model

DDMOD-408; No of Pages 9

DRUG DISCOVERY

TODAY

DISEASEMODELS

Fish: a suitable system to model humanbone disorders and discover drugs withosteogenic or osteotoxic activitiesVincent Laize1,3, Paulo J. Gavaia1,3, M. Leonor Cancela1,2,3,*1Centre of Marine Sciences, University of Algarve, 8005-139 Faro, Portugal2Department of Biomedical Sciences and Medicine, University of Algarve, 8005-139 Faro, Portugal

Drug Discovery Today: Disease Models Vol. xxx, No. xx 2014

Editors-in-Chief

Jan Tornell – AstraZeneca, Sweden

Andrew McCulloch – University of California, SanDiego, USA

Bone biology in animal models, including testing of bone biomaterials

This review discusses the suitability and advantages of

teleost fish for studying underlying mechanisms of

normal and pathological development and mineraliza-

tion of vertebrate skeleton, presents a selection of

zebrafish mutants and transgenic lines modeling hu-

man skeletal diseases and highlights currently available

fish systems for identifying and characterizing novel

osteogenic and osteotoxic molecules.

Introduction

The last decade has seen an exponential interest in the use of

model organisms capable of providing suitable alternatives to

mammalian models and allowing quicker and less expensive

approaches, leading to significant improvements in our

knowledge on the molecular basis of human pathologies.

In this respect, the study of skeletal diseases has not been

an exception but requires the use of vertebrates for obvious

reasons. Fish, in particular zebrafish and medaka, have been

used with a growing success due to their many similarities

with mammals in molecular pathways and mechanisms in-

volved in the onset of patterning and development of skeletal

structures. Many reports have confirmed the suitability of fish

systems to model many of the pathological defects affecting

human skeletal formation, osteoclastic bone resorption, os-

teoblastic bone mineralization and extracellular matrix

maintenance, but also to visualize the onset of normal and

Please cite this article in press as: Laize V, et al. Fish: a suitable system to model human bo

*Corresponding author.: M.L. Cancela ([email protected], [email protected])3 All authors contributed equally.

1740-6757/$ � 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddmod.201

Section editors:Oskar Hoffmann – Pharmacology and Toxicology, UZA IIPharmacy Center, Vienna, Austria.Wilhelm Hofstetter – Clinical Research Department,University of Bern, Bern, Switzerland.

abnormal skeletal structures, and the possibility of rapidly

detecting drug-induced osteogenic or osteotoxic effects in

phenotype-based assays.

Teleost fish and human skeletons are remarkably

similar

Despite some small differences that can be attributed to evo-

lutionary distance (their last common ancestor existed approx-

imately 420 million years ago), anatomic and developmental

features of teleost fish and mammalian skeletons are remark-

ably similar, with much of the skull, axial and appendicular

skeleton formed by identical bones, and with a high conserva-

tion of the developmental events and underlying mechanisms

of skeletogenesis, including the early formation of a cartilagi-

nous anlage followed by bone formation through endochon-

dral and dermal ossification [1,2]. Main differences and

similarities between fish and mammalian bone are summa-

rized in Table 1 (see [3] for a more comprehensive comparison).

The most striking difference is probably the occurrence of

acellular bone (devoid of osteocytes) and mononucleated

osteoclasts in most teleost fish species, while mammals have

exclusively cellular bone and multinucleated osteoclasts. In-

terestingly, osteocytic bone and multinucleated osteoclast

ne disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov

4.08.001 e1

http://dx.doi.org/10.1016/j.ddmod.2014.08.001


mailto:[email protected]


Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials Vol. xxx, No. xx 2014


Table 1. Features of fish and human bone: similarities and differences

Fish skeleton Human skeleton References

Endochondral and membranous ossification [1,59]

Hydroxyapatite-like calcium-phosphate crystals [1]

Bone formation by osteoblasts positive for alkaline phosphatase [5]

Bone resorption by osteoclasts positive for tartrate-resistance acid phosphatase [5]

Mesenchymal origin of osteoblasts; hematopoietic origin of osteoclasts [59]

Osteocytic and anosteocytic bone Osteocytic bone [5]

Dendritic processes mostly absent and no lacunocanalicular system Dendritic processes in a well-developed lacunocanalicular system [60–62]

Mono- and multinucleated osteoclasts Multinucleated osteoclasts [63]

Mechanosensing by osteoblasts and lining cells Mechanosensing by osteocytes [59,61,64]

Collagen I and II in bone Collagen I in bone [1,8]

Perichordal ossification of the vertebral column Endochondral ossification of the vertebral column [6]

16 types of cartilage 3 types of cartilage [8]

occur in zebrafish [4,5]. Also noteworthy is the fact that most

teleost fish ossify the vertebrae directly over the notochord,

while mammals have a cartilaginous precursor [6,7]. Fish also

have a much higher diversity of cartilage types and intermedi-

ate tissues than what is found in adult mammals [4,8,9].

Despite those differences, the availability of fish mutants

exhibiting features resembling human pathologies has con-

tributed to validate fish as a model organism to study mecha-

nisms underlying skeleton development and skeletal

disorders.

Among teleost fishes, zebrafish Danio rerio (Hamilton,

1822) has been the target of a growing interest from the

scientific community. Zebrafish is a small freshwater fish

from tropical regions of South and Southeast Asia for which

numerous models of human diseases have been identified or

developed during the last decade, leading to its recognition as

a suitable model for biomedical research (reviewed in [10]).

Zebrafish embryos develop externally and are transparent,

allowing for direct observation of embryonic development

and organ growth. Additional features such as small size (3–

4 cm long), high fecundity (mature females can lay hundreds

of non-adhesive eggs every week), short generation time

(adulthood attained in 3–4 months) and rapid development

(most body structures are visible 48 h after fertilization),

robustness (they are easy to manipulate, adapt to a wide

range of environments and can be kept in large shoals)

and availability of genetic techniques, have reinforced the

attractiveness of the zebrafish as a laboratory model. Finally

and importantly, the zebrafish genome has been almost

entirely sequenced and annotated, with most human genes

having orthologs in zebrafish [11]. In addition, it is estimated

that approximately 70% of human disease genes have func-

tional homologs in zebrafish [12]. The key regulators of bone

formation have been highly conserved, and corresponding

zebrafish orthologs share significant sequence similarities

and overlapping of expression patterns [13]. For pheno-

type-based assays related to skeletogenesis, the transparency

and external/rapid development of fish embryos is a clear


e2 www.drugdiscoverytoday.com

advantage. It is possible to monitor each stage of skeleton

development and determine at high resolution the role of

single cells in bone and cartilage formation/mineralization.

However, zebrafish is not a mammalian species and the lack

of some organs (e.g. lung or mammary gland) represents a

limitation to the modeling of some human diseases. In

addition, phenotypic characteristics of diseases caused by

orthologous genes can differ between fish and human. Be-

cause it suffered the teleost-specific whole genome duplica-

tion event, zebrafish genome contains numerous paralogous

genes that resulted in gene sub-functionalization and/or

neofunctionalization, a situation that may limit, in some

cases, the use of zebrafish as a disease model. However, this

is also true in studies using the mouse model [11,14]. Still,

zebrafish is a relevant model organism to study vertebrate

development and human diseases, and is rapidly acquiring

standard prominence worldwide as an alternative and com-

plement to rodent models, which remain the standard system

in pre-human drug tests.

Fish models of human skeletal disorders

In the last decade, there has been an increasing effort in

producing mutant and transgenic fish lines with the objective

of modeling human skeletal disorders and improving the

visualization of the skeleton. These achievements have deci-

sively contribute to facilitate studies towards uncovering

novel drugs with the potential to treat the many skeletal

pathologies that affect millions of people worldwide and

which incidence is growing due to the increase in human

life span. Human diseases such as osteogenesis imperfecta,

bone loss (osteoporosis and osteopenia), craniosynostosis,

craniofacial defects and spinal deformities (scoliosis, holos-

pondyly) are examples, among many others, of pathologies

for which fish mutants are available and being used to unveil

the corresponding changes in normal molecular pathways,

thus contributing to discover therapeutic molecules capable

of rescuing these pathologies (Table 2). Although medaka and

guppy have also been used to model human bone disorders,


Vol. xxx, No. xx 2014 Drug Discovery Today: Disease Models | Bone biology in animal models, including testing of bone biomaterials


Table 2. Example of fish models of human bone and skeletal disorders (see also [90])

Human bone/skeletal disorders (bone/skeletal phenotype) Fish model systems Affected gene(s) References

Osteogenesis imperfecta (OI)

(reduced bone density, bone fragility, skeletal deformities)

Zebrafish chihuahua (chi) mutant

Zebrafish frilly fins (frf) mutant

col1a1

bmp1

[65,66]

Osteoporosis

(reduced bone mineral density)

rankl-induced medaka rankl [67]

Glucocorticoid-induced osteoporosis (GIOP)

(reduced bone mineral density upon use of steroids)

Prednisolone-treated zebrafish [56,68]

Iron-induced osteoporosis

(reduced bone mineral density upon iron overload)

Iron-overloaded zebrafish [28]

Raine syndrome (RNS)

(increased ossification)

Zebrafish fam2b mutant fam20b [69]

Multiple osteochondromas (MO)

(cartilaginous bone tumors leading to skeletal deformities)

Zebrafish dackel (dak) mutant

Zebrafish boxer (box) mutant

Zebrafish pinscher (pic) mutant

ext2

extl3

papst1

[70,71]

Mucolipidosis II (ML-II)

(skeletal, craniofacial and joint abnormalities)

Zebrafish gnptab morphant gnptab [72]

Craniosynostosis

(premature fusion of cranial sutures)

Zebrafish dolphin (dol) and stocksteif (sst) mutants cyp26b1 [73]

Holospondyly

(fusion of vertebral centra)

Zebrafish stocksteif (sst) mutant

Retinoic acid-treated zebrafish

cyp26b1 [74]

Fibrodysplasia ossificans progressiva (FOP)

(heterotopic endochondral ossification)

Zebrafish lost-a-fin (laf) mutant acvr1/alk8 [75]

Idiopathic scoliosis

(spinal deformity)

Guppy curveback mutant Not determined [76]

Arterial calcification of infancy

(ectopic mineralization)

Zebrafish dragonfin (dgf) mutant enpp1 [16]

Holoprosencephaly (HPE)

(craniofacial defects)

Zebrafish sonic-you (syu) mutant shh [77]

Campomelic dysplasia


Zebrafish jellyfish (jef) mutant sox9a [78]

Ehlers-Danlos syndrome (EDS)


Zebrafish b4galt7 mutant b4galt7 [79]

DiGeorge syndrome (DGS)


Zebrafish van-gogh (vgo) mutant tbx1 [80]

Cranio-lenticulo-sutural dysplasia (CLSD)

(craniofacial defects and short stature)

Zebrafish crusher (cru) mutant sec23a [81]

Osteopathy related to mineral homeostasis

(failure to form mineralized bone)

Zebrafish no bone (nob) mutant entpd5 [16]

Osteopathy related to abnormal ECM deposition


Zebrafish feelgood (fel) mutant

Zebrafish man o’war (mow) mutant

Zebrafish bulldog (bul) mutant

creb3l2

uxs1

sec24d

[82–84]

Osteopathy related to delayed mineralization

(delayed vertebrae calcification)

Zebrafish bone calcification slow (bcs) mutant Not determined [85]

Hyperossification

(hyperossification and skeletal overgrowth)

Zebrafish rapunzel (rpz) mutant rpz [86]

most diseases models have been developed from zebrafish

mutants. Among the first mutants developed and character-

ized, the zebrafish chihuahua, which lacks a functional type 1

collagen gene, exhibits a skeletal dysplasia resembling human

osteogenis imperfecta characterized by extreme bone fragili-

ty. Zebrafish mutants sonic you – lacking a functional sonic

hedgehog gene, which is a key regulator of vertebrate organo-

genesis – and jellyfish – lacking a functional sox9a gene, which

is a transcription factor involved in chondrocyte differentia-

tion – exhibit skeletal dysplasia resembling human craniofa-

cial syndromes. Guppy is also a model and its mutant


curveback exhibits a skeletal dysplasia resembling idiopatic

scoliosis. Following these first mutants, many more have

been developed and characterized (Table 2) and its numbers

are continuously growing. A simple search in bibliography

using as key words ‘zebrafish’ and ‘human diseases’ can

undoubtedly show the exponential interest in zebrafish in

the last decade. Recently, zebrafish has also been proposed as

a model of excellence to study the molecular and cellular

events underlying the development of ectopic mineraliza-

tions, which are observed in a number of human pathologies

such as vascular calcification, skin calcifications, renal


www.drugdiscoverytoday.com e3



calcium stones, among others [15]. For example, the dragon-

fish mutant shows a broad range of ectopic calcifications

around perichondral bone in the craniofacial skeleton as well

as in the brain, the neural tube and the heart. On the

contrary, the no bone mutant fails to produce mineralized

bone [16].

Furthermore, the growing number of transgenic lines

expressing fluorescent proteins under the control of promo-

ters of bone-related genes such as osx (sp7), oc2, runx2, sox9,

sox10, barx1, col2 and col10 among others [17–21], provide a

plethora of in vivo tools which allow studies with a level of

morphological detail and at a temporal scale never achieved

before. Thus it can be anticipated that these improvements,

both technical and in availability of in vivo models with

improved capabilities will lead to new discoveries at a much

higher pace than before. Information on the availability of

mutant and transgenic zebrafish lines can be found at the

European Zebrafish Resource Center (EZRC; http://

www.ezrc.kit.edu) or at the Zebrafish International Resource

Center (ZIRC; http://zebrafish.org).

Fish systems to discover osteogenic drugs

Given the important advances in technology, public in gen-

eral as well as public and private decision makers expect that

these should accelerate the discovery of novel treatments,

pressing those involved in biomedical research as well as

pharma and biotech companies, to bring new drugs into

the market. Thus, economical as well as political pressures

require more and more the use of high throughput methods

to achieve the expected results. Given the advantages

highlighted above, zebrafish and medaka stand up among

other fish as models of choice to be used in biomedical

research, in particular for this quest for novel drugs suitable

to be used in bone therapeutics. Its importance can be also

measured by the growing number of articles focusing on this

issue in the last decade [10,22–26]. Here we describe available

systems used in screenings for osteogenic and mineralogenic

drugs. The success of these approaches confirms that fish, in

particular the cellular boned zebrafish, is an excellent model

for discovering therapeutic molecules for human skeletal

disorders (Fig. 1). Accordingly, Table 3 presents a number

of relevant studies which used zebrafish as an in vivo model to

uncover, following screening of various panels of drugs,

novel osteogenic and/or mineralogenic drugs, or drugs capa-

ble of reverting pathological conditions, thus proving once

more the importance of this model organism for discovering

therapeutic molecules of interest for human skeletal disor-

ders. For example, the recent screening of 320 compounds in

the zebrafish mutant Snca1 led to the identification of clem-

izole as a potential treatment for Dravet syndrome [27]; in

another study using zebrafish, deferoxamine was found to

effectively counteract iron-overload-induced inhibition of

osteogenesis [28]. Similarly, zebrafish has also been used as



a choice model to unveil the effects of pollutants and other

toxic molecules on skeletogenesis, with high relevance for

environmental monitoring and ecotoxicology.

Skeletogenesis in fish larvae

Looking at the effect of a molecule in the whole-organism is a

preferable approach since it not only allows the identification

of potential pitfalls but also the determination of therapeutic

activity, range of action and general toxicity. Fish, in partic-

ular zebrafish, embryos/larvae are small, transparent and

available in large quantities, and thus can be easily accom-

modated in 96-well plates for high-throughput screening of

molecule libraries. In zebrafish, the onset of bone formation

and mineralization occurs as early as 2 day post-fertilization

(dpf) and is first evident at 3 dpf in the cleithrum then in the

branchial and cranial skeletons [29]. Furthermore, it can be

assessed easily through whole-mount bone-specific staining

due to the transparency of the larvae at these early stages. The

fast development of the fish skeleton allows for short dura-

tion assays (few days), something difficult in traditional

mammalian models. Larvae are initially grown in petri dishes

then arrayed in 96-well plates at a density up to five fish per

well. Molecules are added directly to the liquid medium in

which the embryos/larvae develop. In wild-type strains, ossi-

fication is detected through alizarin red S staining in 6–11 dpf

zebrafish larvae [30] and quantified from the area and inten-

sity of staining determined from color analysis of ventral

pictures of the head skeleton [31] (Fig. 1b). Zebrafish trans-

genic lines using fluorescent reporters highlighting particular

structures in the skeleton can also be used to observe specific

bone elements/cells and quantify their area/number without

the need for euthanasia. For example, Tg(oc2:GFP; osx:m-

Cherry) transgenic zebrafish line (Fig. 1c), where GFP and

mCherry production is under the control of osteocalcin 2

and osterix promoters, respectively, can be used to determine

the size of the operculum or the number of pre- versus mature

osteoblasts. Similarly, col10a1:nlGFP transgenic zebrafish

line, where GFP production is under the control of collagen

10 promoter, a marker of early osteoblastic precursors, can be

used to assess events preceding mineralization in cranial and

axial skeleton [21]. In addition to its common use as a

colorant in bone research, Alizarin red S is also used as a

fluorochrome label of mineralized tissues, often as a comple-

ment to fluorescent reporters in transgenic fish ([32]; Bensi-

mon-Brito et al., in preparation). In all cases, osteogenic/

osteotoxic effects are determined from the comparative anal-

ysis of treated versus control fish.

Osteogenesis in regenerating teleost fish fin

The caudal fin of teleost fish is remarkably simple, accessible

and can be restored upon amputation or damage. Because of

these characteristics, it has logically become an excellent

system for investigating underlying mechanisms of epimorphic


http://www.ezrc.kit.edu/

http://www.ezrc.kit.edu/

http://zebrafish.org/



Please cite this article in press as: Laize V, et al. Fish: a suitable system to model human bone disorders and discover drugs with osteogenic or osteotoxic activities, Drug Discov

Skeletal deformities

Developingoperculum

Regeneratingfin rays

Mineralogenic vertebra-derived cell lines

Mineralizingscales

11 dpfCl

Nc Ps

Operculum

Tg(oc2:GFP; osx:mCherry)16 dpf

PT

Lepido-trichia

AR-Sstainednodules

5 dpa

Cb5

Op

(a)

(b)

(d) (e) (f)

(c)

Drug Discovery Today: Disease Models

Figure 1. (a) Fish systems used to study/screen molecules for osteogenic effects. (b) Ventral view of whole-mount alizarin red S (AR-S) – alcian blue (AB)

stained zebrafish larvae at 11 days post-fertilization (dpf). Calcified structures appear in red; area and intensity of staining is determined for the notochord

(Nc), operculum (Op), parasphenoid (Ps) cleithrum (Cl), ceratobranchial 5 (Cb5) and pharyngeal teeth (PT). (c) Lateral view of Tg(oc2:GFP; osx:mCherry)

transgenic zebrafish at 16 dpf. Dotted white line indicates operculum area. (d) Lateral view of AR-S stained regenerating caudal fin of a juvenile zebrafish. Black

triangle indicates the amputation plan; Dotted black line indicates regenerated area; Solid black line indicates area of new bone formation. (e) AR-S-stained

elasmoid scale from the dorsal region of a juvenile gilthead seabream (Sparus aurata L.). (f) AR-S stained mineral nodules deposited within the extracellular

matrix of gilthead seabream VSa16 cell line (osteoblast-like cells).




Table 3. Examples of fish systems to discover molecules affecting skeleton and bone formation

Molecules Fish systems Skeletal and bone effects References

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Developing medaka Impaired chondro- and osteogenesis [87]

17b-Estradiol Cultured goldfish scales Increased osteoclastic activity [58]

3-Methylcholanthrene Developing zebrafish

Regenerating zebrafish

Cultured gilthead seabream bone cells

Increased rate of skeletal deformities

Reduced de novo bone formation

Reduced ECM mineralization

[36]

Calcitonin Cultured goldfish scales Suppressed osteoclastic activity [58]

Vanadium Cultured gilthead seabream bone cells Reduced ECM mineralization [52]

Bisphenol A Cultured goldfish scales Suppressed osteoclastic and osteoblastic activites [57]

Ethyl tert butyl ether (ETBE)

Tertiary amyl methyl ether (TAME)

Developing zebrafish Craniofacial defects [88]

Vitamin D3 analogs Developing zebrafish Increased bone formation [31]

Deferoxamine Iron-overload zebrafish Rescued iron-induced osteoporosis [28]

Parathyroid hormone (PTH) Developing zebrafish Bone loss [31]

Dorsomorphin Developing zebrafish Reduced bone mineralization [89]

regeneration, that is the complete reformation of missing tissues

[33–35]. When caudal fin tissues are amputated, a thick wound

epidermis is formed at the amputation plane, then a mass of

undifferentiated mesenchymal cells, called blastema, proliferate

beneath the wound epidermis and will later differentiate into

the various cell types necessary to the faithful restoration of

missing fin structures. Full regeneration is usually achieved after

10–15 days depending on the fish species, the level of amputa-

tion and culture conditions. Although zebrafish are raised at

28 8C, assays of fin regeneration are typically performed at

33 8C to speed up regeneration process, which is well advanced

after only 5 days. Fin regeneration and de novo bone minerali-

zation is determined after alizarin red staining, imaging and

morphometric analysis of regenerated areas (Fig. 1d; see brief

protocol in [36])

Mineralogenesis in cell and scale cultures

In addition and as a complement to in vivo studies, miner-

alogenic cell lines developed from several fish species [37–39]

can be used to study more in depth mechanisms affecting

bone and cartilage cell function and metabolism and the

molecular pathways involved in various processes such as

extracellular matrix mineralization [40–46] or the mineralo-

genic effect of various molecules, for example retinoic acid

[47,48], polyunsaturated fatty acids [49], vanadate [46,50–52]

and polycyclic aromatic hydrocarbon [36]. In vitro minerali-

zation is easily detected and quantified through the alizarin

red S staining of hydroxyapatite-like crystals deposited within

the extracellular matrix of fish bone-derived cell lines upon

exposure to a mineralogenic cocktail ([37]; Fig. 1f).

While cell cultures have been used to study mechanisms of

extracellular matrix mineralization, they have limited value

in the study of cell–cell and cell–matrix interactions. Elas-

moid scales of teleost fish (Fig. 1e) are dermal bone elements

that develop late (30 dpf in zebrafish) and serve as a reservoir

of calcium [53,54]. They are small, easily accessible, abundant



(hundreds of highly similar scales per fish), transparent and

have the ability to quickly regenerate (4 weeks in zebrafish),

when lost or removed [25,55]. As for regenerating fin rays,

scales have been recently used in biomedical studies aiming

at the better understanding of mechanism of bone regenera-

tion [25,55] but also as an in vivo disease model for osteopo-

rosis studies [56]. The possibility of culturing fish scales in

vitro as a bone unit, where osteoclasts and osteoblasts cohabit

on both sides of the mineralized matrix and interact in a way

resembling in vivo conditions, has allowed their use to assess

effects of anti/pro-osteogenic molecules [57,58] and as a

valuable ex vivo system to discover new drugs affecting bone

formation and/or resorption.

Conclusions

Through the availability of mutant and transgenic lines, fish

has become an attractive model system to study bone dis-

orders. Furthermore, fish provide unique insights into under-

lying molecular mechanisms that cannot be gained in

traditional mammalian systems due to shortcomings, mor-

phological constrains and technical limitations. Fish are also

cost-effective models with the potential to identify, in a

shorter time, novel drugs to treat bone disorders as well as

screening for osteotoxic molecules. Finding new treatments

for human diseases is challenging and future works should

aim at developing novel fish systems capable of modeling

more bone disorders and at automating screening processes.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgements

This work was supported by the Portuguese Funding Agency

for Science and Technology (FCT) through AQUATOX

(PTDC/MAR/112992/2009) research project, by the Atlantic

Area Transnational Cooperation Programme of the European




Community through MARMED (2011-1/164) project, and by

the National Strategic Reference Framework (QREN I&DT)

through ZEBRAFEEDS (QREN 23000) project.

References[1] Hall BK. Bones and cartilage: developmental and evolutionary skeletal

biology. Academic Press; 2005.

[2] Javidan Y, Schilling TF. Development of cartilage and bone. Methods Cell

Biol 2004;76:415–36.

[3] Apschner A, Schulte-Merker S, Witten PE. Not all bones are created equal –

using zebrafish and other teleost species in osteogenesis research. Methods

Cell Biol 2011;105:239–55.

[4] Hall BK, Witten PE. Plasticity of and transitions between skeletal tissues in

vertebrate evolution and development. In: Anderson JS, Sues H-DD.,

editors. Major transitions in vertebrate evolution. Indiana University

Press; 2007. p. 13–56.

[5] Witten PE, Huysseune A. A comparative view on mechanisms and

functions of skeletal remodelling in teleost fish, with special emphasis on

osteoclasts and their function. Biol Rev Camb Philos Soc 2009;84:

315–46.

[6] Arratia G, Schultze H-P. Reevaluation of the caudal skeleton of certain

actinopterygian fishes: III. Salmonidae. Homologization of caudal skeletal

structures. J Morphol 1992;249:187–249.

[7] Nordvik K, Kryvi H, Totland GK, Grotmol S. The salmon vertebral body

develops through mineralization of two preformed tissues that are

encompassed by two layers of bone. J Anat 2005;206:103–14.

[8] Benjamin M. The cranial cartilages of teleosts and their classification. J

Anat 1990;169:153–72.

[9] Witten PE, Huysseune A, Hall BK. A practical approach for the

identification of the many cartilaginous tissues in teleost fish. J Appl

Ichthyol 2010;26:257–62.

[10] Lieschke GJ, Currie PD. Animal models of human disease: zebrafish swim

into view. Nat Rev Genet 2007;8:353–67.

[11] Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, et al.

The zebrafish reference genome sequence and its relationship to the

human genome. Nature 2013;496:498–503.

[12] Langheinrich U. Zebrafish: a new model on the pharmaceutical catwalk.

Bioessays 2003;25:904–12.

[13] Spoorendonk KM, Hammond CL, Huitema LFA, Vanoevelen J, Schulte-

Merker S. Zebrafish as a unique model system in bone research: the power

of genetics and in vivo imaging. J Appl Ichthyol 2010;26:219–24.

[14] Santoriello C, Zon LI. Hooked! Modeling human disease in zebrafish. J

Clin Invest 2012;122:2337–43.

[15] Hosen MJ, Vanakker OM, Willaert A, Huysseune A, Coucke P, De Paepe A.

Zebrafish models for ectopic mineralization disorders: practical issues

from morpholino design to post-injection observations. Front Genet

2013;4:74.

[16] Huitema LFA, Apschner A, Logister I, Spoorendonk KM, Bussmann J,

Hammond CL, et al. Entpd5 is essential for skeletal mineralization and

regulates phosphate homeostasis in zebrafish. Proc Natl Acad Sci U S A

2012;109:21372–77.

[17] DeLaurier A, Nakamura Y, Braasch I, Khanna V, Kato H, Wakitani S, et al.

Histone deacetylase-4 is required during early cranial neural crest

development for generation of the zebrafish palatal skeleton. BMC Dev

Biol 2012;12:16.

[18] Hammond CL, Moro E. Using transgenic reporters to visualize bone and

cartilage signaling during development in vivo. Front Endocrinol

(Lausanne) 2012;3:91.

[19] Knopf F, Hammond C, Chekuru A, Kurth T, Hans S, Weber CW, et al. Bone

regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev

Cell 2011;20:713–24.

[20] Nichols JT, Pan L, Moens CB, Kimmel CB. Barx1 represses joints and

promotes cartilage in the craniofacial skeleton. Development

2013;140:2765–75.

[21] Renn J, Buttner A, To TT, Chan SJH, Winkler C. A col10a1:nlGFP

transgenic line displays putative osteoblast precursors at the


medaka notochordal sheath prior to mineralization. Dev Biol

2013;381:134–43.

[22] Brittijn SA, Duivesteijn SJ, Belmamoune M, Bertens LFM, Bitter W, de

Bruijn JD, et al. Zebrafish development and regeneration: new tools for

biomedical research. Int J Dev Biol 2009;53:835–50.

[23] Fleming A. Zebrafish as an alternative model organism for disease

modelling and drug discovery: implications for the 3Rs [WWW

Document]; 2007, http://www.nc3rs.org.uk/news.asp?id=421.

[24] Lohr H, Hammerschmidt M. Zebrafish in endocrine systems: recent

advances and implications for human disease. Annu Rev Physiol

2011;73:183–211.

[25] Metz JR, de Vrieze E, Lock E-J, Schulten IE, Flik G. Elasmoid scales of

fishes as model in biomedical bone research. J Appl Ichthyol

2012;28:382–7.

[26] Renn J, Winkler C, Schartl M, Fischer R, Goerlich R. Zebrafish and medaka

as models for bone research including implications regarding space-

related issues. Protoplasma 2006;229:209–14.

[27] Baraban SC, Dinday MT, Hortopan GA. Drug screening in Scn1a zebrafish

mutant identifies clemizole as a potential Dravet syndrome treatment.

Nat Commun 2013;4:2410.

[28] Chen B, Yan Y-L, Liu C, Bo L, Li G-F, Wang H, et al. Therapeutic effect of

deferoxamine on iron overload-induced inhibition of osteogenesis in a

zebrafish model. Calcif Tissue Int 2014;94:353–60.

[29] Gavaia PJ, Simes DC, Ortiz-Delgado JB, Viegas CSB, Pinto JP, Kelsh RN,

et al. Osteocalcin and matrix Gla protein in zebrafish (Danio rerio) and

Senegal sole (Solea senegalensis): comparative gene and protein expression

during larval development through adulthood. Gene Expr Patterns

2006;6:637–52.

[30] Walker MB, Kimmel CB. A two-color acid-free cartilage and bone stain for

zebrafish larvae. Biotech Histochem 2007;82:23–8.

[31] Fleming A, Sato M, Goldsmith P. High-throughput in vivo screening for

bone anabolic compounds with zebrafish. J Biomol Screen 2005;10:823–

31.

[32] Mackay EW, Apschner A, Schulte-Merker S. A bone to pick with zebrafish.

Bonekey Rep 2013;2:445.

[33] Akimenko M-A, Smith A. Paired fin repair and regeneration. In: Hall BK,

editor. Fins into limbs: evolution, development, and transformation. The

University of Chicago Press; 2007. p. 152–62.

[34] Nakatani Y, Kawakami A, Kudo A. Cellular and molecular processes of

regeneration, with special emphasis on fish fins. Dev Growth Differ

2007;49:145–54.

[35] Yoshinari N, Kawakami A. Mature and juvenile tissue models of

regeneration in small fish species. Biol Bull 2011;221:62–78.

[36] Laize V, Gavaia PJ, Viegas MN, Caria J, Luis N. Osteotoxicity of 3-

methylcholanthrene in fish. Aquat Procedia 2014 [in press].

[37] Pombinho AR, Laize V, Molha DM, Marques SMP, Cancela ML.

Development of two bone-derived cell lines from the marine teleost Sparus

aurata; evidence for extracellular matrix mineralization and cell-type-

specific expression of matrix Gla protein and osteocalcin. Cell Tissue Res

2004;315:393–406.

[38] Rafael MS, Marques CL, Parameswaran V, Cancela ML, Laize V. Fish bone-

derived cell lines: an alternative in vitro cell system to study bone biology.

J Appl Ichthyol 2010;26:230–4.

[39] Vijayakumar P, Laize V, Cardeira J, Trindade M, Cancela ML.

Development of an in vitro cell system from zebrafish suitable to study

bone cell differentiation and extracellular matrix mineralization.

Zebrafish 2013;10:500–9.

[40] Fonseca VG, Rosa J, Laize V, Gavaia PJ, Cancela ML. Identification of a new

cartilage-specific S100-like protein up-regulated during endo/

perichondral mineralization in gilthead seabream. Gene Expr Patterns

2011;11:448–55.

[41] Fonseca VG, Laize V, Valente MS, Cancela ML. Identification of an

osteopontin-like protein in fish associated with mineral formation. FEBS J

2007;274:4428–39.

[42] Laize V, Pombinho AR, Cancela ML. Characterization of Sparus aurata

osteonectin cDNA and in silico analysis of protein conserved features:

evidence for more than one osteonectin in Salmonidae. Biochimie

2005;87:411–20.



http://refhub.elsevier.com/S1740-6757(14)00036-X/sbref0160



































































http://www.nc3rs.org.uk/news.asp%3Fid=421
































































[43] Rafael MS, Laize V, Florindo C, Ferraresso S, Bargelloni L, Cancela ML.

Overexpression of four and a half LIM domains protein 2 promotes

epithelial-mesenchymal transition-like phenotype in fish pre-osteoblasts.

Biochimie 2012;94:1128–34.

[44] Rafael MS, Laize V, Cancela ML. Identification of Sparus aurata bone

morphogenetic protein 2: molecular cloning, gene expression and in

silico analysis of protein conserved features in vertebrates. Bone

2006;39:1373–81.

[45] Tiago DM, Marques CL, Roberto VP, Cancela ML, Laize V. Mir-20a

regulates in vitro mineralization and BMP signaling pathway by targeting

BMP-2 transcript in fish. Arch Biochem Biophys 2014;543:23–30.

[46] Tiago DM, Laize V, Bargelloni L, Ferraresso S, Romualdi C, Cancela ML.

Global analysis of gene expression in mineralizing fish vertebra-derived

cell lines: new insights into anti-mineralogenic effect of vanadate. BMC

Genomics 2011;12:310.

[47] Conceicao N, Laize V, Simoes B, Pombinho AR, Cancela ML. Retinoic acid

is a negative regulator of matrix Gla protein gene expression in teleost fish

Sparus aurata. Biochim Biophys Acta 2008;1779:28–39.

[48] Fernandez I, Tiago DM, Laize V, Cancela ML, Gisbert E. Retinoic acid

differentially affects in vitro proliferation, differentiation and

mineralization of two fish bone-derived cell lines: different gene

expression of nuclear receptors and ECM proteins. J Steroid Biochem Mol

Biol 2014;140:34–43.

[49] Viegas MN, Dias J, Cancela ML, Laize V. Polyunsaturated fatty acids

regulate cell proliferation, extracellular matrix mineralization and gene

expression in a gilthead seabream skeletal cell line. J Appl Ichthyol

2012;28:427–32.

[50] Tiago DM, Cancela ML, Laize V. Proliferative and mineralogenic effects of

insulin, IGF-1, and vanadate in fish osteoblast-like cells. J Bone Miner

Metab 2011;29:377–82.

[51] Tiago DM, Cancela ML, Aureliano M, Laize V. Vanadate proliferative and

anti-mineralogenic effects are mediated by MAPK and PI-3K/Ras/Erk

pathways in a fish chondrocyte cell line. FEBS Lett 2008;582:

1381–5.

[52] Tiago DM, Laize V, Cancela ML, Aureliano M. Impairment of

mineralization by metavanadate and decavanadate solutions in a fish

bone-derived cell line. Cell Biol Toxicol 2008;24:253–63.

[53] Carragher JF, Sumpter JP. The mobilization of calcium from calcified

tissues of rainbow trout (Oncorhynchus mykiss) induced to synthesize

vitellogenin. Comp Biochem Physiol Part A Physiol 1991;99:

169–72.

[54] Mugiya Y, Watabe N. Studies on fish scale formation and resorption II:

effect of estradiol on calcium homeostasis and skeletal tissue resorption in

the goldfish, Carassius auratus, and the killifish, Fundulus heteroclitus. Comp

Biochem Physiol Part A Physiol 1977;57:197–202.

[55] De Vrieze E, Sharif F, Metz JR, Flik G, Richardson MK. Matrix

metalloproteinases in osteoclasts of ontogenetic and regenerating

zebrafish scales. Bone 2011;48:704–12.

[56] De Vrieze E, van Kessel MAHJ, Peters HM, Spanings FAT, Flik G, Metz JR.

Prednisolone induces osteoporosis-like phenotype in regenerating

zebrafish scales. Osteoporos Int 2014;25:567–78.

[57] Suzuki N, Hattori A. Bisphenol A suppresses osteoclastic and

osteoblastic activities in the cultured scales of goldfish. Life Sci

2003;73:2237–47.

[58] Suzuki N, Suzuki T, Kurokawa T. Suppression of osteoclastic activities by

calcitonin in the scales of goldfish (freshwater teleost) and nibbler fish

(seawater teleost). Peptides 2000;21:115–24.

[59] Shahar R, Dean MN. The enigmas of bone without osteocytes. Bonekey

Rep 2013;2:343.

[60] Cao L, Moriishi T, Miyazaki T, Iimura T, Hamagaki M, Nakane A, et al.

Comparative morphology of the osteocyte lacunocanalicular system in

various vertebrates. J Bone Miner Metab 2011;29:662–70.

[61] Totland GK, Fjelldal PG, Kryvi H, Løkka G, Wargelius A, Sagstad A, et al.

Sustained swimming increases the mineral content and osteocyte density

of salmon vertebral bone. J Anat 2011;219:490–501.

[62] Turner CH, Warden SJ, Bellido T, Plotkin LI, Kumar N, Jasiuk I, et al.

Mechanobiology of the skeleton. Sci Signal 2009;2:pt3.



[63] Witten PE, Hansen A, Hall BK. Features of mono- and multinucleated

bone resorbing cells of the zebrafish Danio rerio and their contribution to

skeletal development, remodeling, and growth. J Morphol 2001;250:197–

207.

[64] You L, Temiyasathit S, Lee P, Kim CH, Tummala P, Yao W, et al. Osteocytes

as mechanosensors in the inhibition of bone resorption due to

mechanical loading. Bone 2008;42:172–9.

[65] Fisher S, Jagadeeswaran P, Halpern ME. Radiographic analysis of zebrafish

skeletal defects. Dev Biol 2003;264:64–76.

[66] Asharani PV, Keupp K, Semler O, Wang W, Li Y, Thiele H, et al. Attenuated

BMP1 function compromises osteogenesis, leading to bone fragility in

humans and zebrafish. Am J Hum Genet 2012;90:661–74.

[67] To TT, Witten PE, Renn J, Bhattacharya D, Huysseune A, Winkler C. Rankl-

induced osteoclastogenesis leads to loss of mineralization in a medaka

osteoporosis model. Development 2012;139:141–50.

[68] Barrett R, Chappell C, Quick M, Fleming A. A rapid, high content, in vivo

model of glucocorticoid-induced osteoporosis. Biotechnol J 2006;1:

651–5.

[69] Eames BF, Yan Y-L, Swartz ME, Levic DS, Knapik EW, Postlethwait JH, et al.

Mutations in fam20b and xylt1 reveal that cartilage matrix controls

timing of endochondral ossification by inhibiting chondrocyte

maturation. PLoS Genet 2011;7:e1002246.

[70] Clement A, Wiweger M, von der Hardt S, Rusch MA, Selleck SB, Chien C-B,

et al. Regulation of zebrafish skeletogenesis by ext2/dackel and papst1/

pinscher. PLoS Genet 2008;4:e1000136.

[71] Wiweger MI, Zhao Z, van Merkesteyn RJP, Roehl HH, Hogendoorn PCW.

HSPG-deficient zebrafish uncovers dental aspect of multiple

osteochondromas. PLoS One 2012;7:e29734.

[72] Flanagan-Steet H, Sias C, Steet R. Altered chondrocyte differentiation and

extracellular matrix homeostasis in a zebrafish model for mucolipidosis II.

Am J Pathol 2009;175:2063–75.

[73] Laue K, Pogoda H-M, Daniel PB, van Haeringen A, Alanay Y, von Ameln S,

et al. Craniosynostosis and multiple skeletal anomalies in humans and

zebrafish result from a defect in the localized degradation of retinoic acid.

Am J Hum Genet 2011;89:595–606.

[74] Spoorendonk KM, Peterson-Maduro J, Renn J, Trowe T, Kranenbarg S,

Winkler C, et al. Retinoic acid and Cyp26b1 are critical regulators

of osteogenesis in the axial skeleton. Development 2008;135:

3765–74.

[75] Bauer H, Lele Z, Rauch GJ, Geisler R, Hammerschmidt M. The type I serine/

threonine kinase receptor Alk8/Lost-a-fin is required for Bmp2b/7 signal

transduction during dorsoventral patterning of the zebrafish embryo.

Development 2001;128:849–58.

[76] Gorman KF, Tredwell SJ, Breden F. The mutant guppy syndrome

curveback as a model for human heritable spinal curvature. Spine (Phila

Pa 1976) 2007;32:735–41.

[77] Schauerte HE, van Eeden FJM, Fricke C, Odenthal J, Strahle U, Haffter P.

Sonic hedgehog is not required for the induction of medial floor plate cells

in the zebrafish. Development 1998;125:2983–93.

[78] Yan Y, Miller CT, Nissen RM, Singer A, Liu D, Kirn A, et al. A zebrafish sox9

gene required for cartilage morphogenesis. Development

2002;129:5065–79.

[79] Nissen RM, Amsterdam A, Hopkins N. A zebrafish screen for craniofacial

mutants identifies wdr68 as a highly conserved gene required for

endothelin-1 expression. BMC Dev Biol 2006;6:28.

[80] Piotrowski T, Ahn D, Schilling TF, Nair S, Ruvinsky I, Geisler R, et al.

The zebrafish van Gogh mutation disrupts tbx1, which is involved in

the DiGeorge deletion syndrome in humans. Development

2003;130:5043–52.

[81] Lang MR, Lapierre LA, Frotscher M, Goldenring JR, Knapik EW. Secretory

COPII coat component Sec23a is essential for craniofacial chondrocyte

maturation. Nat Genet 2006;38:1198–203.

[82] Melville DB, Montero-Balaguer M, Levic DS, Bradley K, Smith JR,

Hatzopoulos AK, et al. The feelgood mutation in zebrafish

dysregulates COPII-dependent secretion of select extracellular

matrix proteins in skeletal morphogenesis. Dis Model Mech

2011;4:763–76.











































































































































[83] Eames BF, Singer A, Smith GA, Wood ZA, Yan Y-L, He X, et al. UDP xylose

synthase 1 is required for morphogenesis and histogenesis of the

craniofacial skeleton. Dev Biol 2010;341:400–15.

[84] Sarmah S, Barrallo-Gimeno A, Melville DB, Topczewski J, Solnica-Krezel L,

Knapik EW. Sec24D-dependent transport of extracellular matrix proteins

is required for zebrafish skeletal morphogenesis. PLoS ONE

2010;5:e10367.

[85] Xi Y, Chen D, Sun L, Li Y, Li L. Characterization of zebrafish mutants with

defects in bone calcification during development. Biochem Biophys Res

Commun 2013;440:132–6.

[86] Green J, Taylor JJ, Hindes A, Johnson SL, Goldsmith MI. A gain of function

mutation causing skeletal overgrowth in the rapunzel mutant. Dev Biol

2009;334:224–34.


[87] Dong W, Hinton DE, Kullman SW. TCDD disrupts hypural

skeletogenesis during medaka embryonic development. Toxicol Sci

2012;125:91–104.

[88] Bonventre JA, White LA, Cooper KR. Craniofacial abnormalities and

altered wnt and mmp mRNA expression in zebrafish embryos exposed

to gasoline oxygenates ETBE and TAME. Aquat Toxicol 2012;120–

121:45–53.

[89] Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, et al.

Dorsomorphin inhibits BMP signals required for embryogenesis and iron

metabolism. Nat Chem Biol 2008;4:33–41.

[90] Neuhauss SCF, Solnica-Krezel L, Schier AF, Zwartkruis F, Stemple DL,

Malicki J, et al. Mutations affecting craniofacial development in zebrafish.

Development 1996;123:357–67.





























Documents

Fish: a suitable system to model human bone disorders and ... · bone disorders and discover drugs with osteogenic or osteotoxic activities ... guppy have also been used to model