BIOLOGICAL Cambridge

REVIEWS Philosophical Society

Biol. Rev. (2009), 84, pp. 315–346. 315

doi:10.1111/j.1469-185X.2009.00077.x

A comparative view on mechanisms and

functions of skeletal remodelling in teleost
fish, with special emphasis on osteoclasts and
their function
P. Eckhard Witten* and Ann Huysseune
Biology Department, Ghent University, Belgium.

(Received 28 May 2008; revised 11 January 2009; accepted 22 January 2009)

ABSTRACT

Resorption and remodelling of skeletal tissues is required for development and growth, mechanical adaptation,
repair, and mineral homeostasis of the vertebrate skeleton. Here we review for the first time the current knowledge
about resorption and remodelling of the skeleton in teleost fish, the largest and most diverse group of extant
vertebrates. Teleost species are increasingly used in aquaculture and as models in biomedical skeletal research.
Thus, detailed knowledge is required to establish the differences and similarities between mammalian and teleost
skeletal remodelling, and between distantly related species such as zebrafish (Danio rerio) and medaka (Oryzias latipes).
The cellular mechanisms of differentiation and activation of osteoclasts and the functions of teleost skeletal
remodelling are described. Several characteristics, related to skeletal remodelling, distinguish teleosts from
mammals. These characteristics include (a) the absence of osteocytes in most species; (b) the absence of
haematopoietic bone marrow tissue; (c) the abundance of small mononucleated osteoclasts performing non-
lacunar (smooth) bone resorption, in addition to or instead of multinucleated osteoclasts; and (d) a phosphorus-
rather than calcium-driven mineral homeostasis (mainly affecting the postcranial dermal skeleton). Furthermore,
(e) skeletal resorption is often absent from particular sites, due to sparse or lacking endochondral ossification.
Based on the mode of skeletal remodelling in early ontogeny of all teleosts and in later stages of development of
teleosts with acellular bone we suggest a link between acellular bone and the predominance of mononucleated
osteoclasts, on the one hand, and cellular bone and multinucleated osteoclasts on the other. The evolutionary
origin of skeletal remodelling is discussed and whether mononucleated osteoclasts represent an ancestral type of
resorbing cells. Revealing the differentiation and activation of teleost skeletal resorbing cells, in the absence of
several factors that trigger mammalian osteoclast differentiation, is a current challenge. Understanding which
characters of teleost bone remodelling are derived and which characters are conserved should enhance our
understanding of the process in fish and may provide insights into alternative pathways of bone remodelling in
mammals.

Key words: resorption, remodelling, skeleton, mineral homeostasis, acellular bone, osteocytes, osteoclasts, teleost.

CONTENTS
I. Introduction ...................................................................................................................................... 316
II. A brief overview of teleost skeletal biology ...................................................................................... 317
III. Cellular mechanisms of teleost skeletal remodelling ....................................................................... 319

* Address for correspondence: Ghent University, Department of Biology, Ledeganckstraat 35, B-9000 Ghent, Belgium (E-mail:
PEckhardWitten@aol.com)

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
316 P. Eckhard Witten and Ann Huysseune

(1) Osteoclastic resorption and remodelling in teleosts .................................................................. 319

( a ) Osteoclasts: the mammalian perspective .............................................................................. 319
( b ) Teleost osteoclasts .................................................................................................................. 321
(2) Non-osteoclastic bone resorption and remodelling in teleosts .................................................. 322
(3) A possible link between bone type and osteoclast type and number? ..................................... 323
IV. Differentiation and activation of teleost osteoclasts ......................................................................... 323
(1) Multinucleated osteoclasts .......................................................................................................... 323
(2) Mononucleated osteoclasts ......................................................................................................... 325
V. The functions of teleost skeletal remodelling ................................................................................... 325
(1) Growth and morphogenesis ....................................................................................................... 325
(2) Replacement of skeletal tissues ................................................................................................... 326
(3) Mineral metabolism .................................................................................................................... 327
( a ) Calcium and phosphorus requirements ............................................................................... 327
( b ) Hormones and mineral metabolism ..................................................................................... 328
( c ) Habitat and phylogeny ......................................................................................................... 329
(4) Mechanical adaptation ............................................................................................................... 329
(5) Fracture healing, regeneration and disease-related remodelling ............................................... 330
(6) Tooth formation and replacement ............................................................................................. 331
(7) Remodelling as a lifelong process .............................................................................................. 333
VI. The evolution of skeletal remodelling .............................................................................................. 333
VII. Conclusions ....................................................................................................................................... 335
VIII. Acknowledgements ............................................................................................................................ 336
IX. References ......................................................................................................................................... 336

I. INTRODUCTION
Resorption and remodelling of the vertebrate skeleton are
essential processes for development, growth, repair,
mechanical adaptation and mineral homeostasis. Vertebrate
hard tissue structures are never permanent and thus the
skeleton is subject to constant reshaping (cartilage),
remodelling (bone) and replacement (teeth) (Hall & Witten,
2007). During ontogeny, remodelling of bone is first
triggered by the requirements of development and allome-
tric growth, later it continues as an adaptation to changes in
mechanical load. In tetrapods, skeletal remodelling is also
an important process in the animals’ mineral homeostasis.
Tooth eruption and repair of the skeleton after trauma also
require skeletal remodelling. Finally, temporary skeletal
elements that function as secondary sexual characters, such
as deer antlers or the salmon kype, require bone resorption
for shedding and/or regression (Bertram & Swartz, 1991;
Witten & Hall, 2002; Witten, Huysseune & Hall, 2003).
Similar to tetrapods the skeleton of teleost fish is subject to
remodelling and many factors that affect skeletal remodel-
ling in mammals also affect the process in teleosts. However,
skeletal remodelling in teleost fish shows several specific
differences compared to mammals. These relate to the
aquatic life of teleosts, to certain conserved traits in teleosts
(that have been lost in mammals) and to several advanced
characters that have evolved in the lineage of modern bony
fish (Metscher & Ahlberg, 1999).
The skeleton of advanced bony fish (teleosts) has recently
received increasing attention from developmental biologists
and biomedical researchers. Zebrafish (Danio rerio), medaka
(Oryzias latipes), stickleback (Gasterosteus aculeatus), and puffer-
fish (Fugu rubripes) have been well established as model
organisms (Swain, 1992; Metscher & Ahlberg, 1999; Danks
et al., 2003; Elmasri et al., 2004; Renn et al., 2006; Steinke,
Salzburger & Meyer, 2006; Mari-Beffa et al., 2007) and the
skeletal development of farmed teleost species, such as
Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus
mykiss), cod (Gadus morhua), halibut (Hippoglossus hippoglossus)
and sea bream (Sparus aurata) has received attention because
skeletal malformations are an issue of concern in fish
farming (Madsen & Dalsgaard, 1999; Boglione et al., 2001;
Lewis, Lall & Witten, 2004; Witten et al., 2006). Develop-
mental studies usually are not concerned with skeletal
development and remodelling. These studies focus on
anabolic pathways of cell and tissue differentiation and on
early events in development. Given that the skeleton,
especially the calcified skeleton, develops quite late in
ontogeny comparatively few studies tackle skeletal resorp-
tion and skeletal remodelling in teleost fish as an integral
component of skeletal development (Sire, Huysseune &
Meunier, 1990; Kahn & Partridge, 1991; Lewinson &
Kogan, 1995; Witten, Hansen & Hall, 2001; Sæle, 2002).
Thus, skeletal remodelling is best described and understood
in mammals. Still, knowledge about the differences and
similarities between mammalian and teleost skeletal remod-
elling is important to understand teleost skeletal develop-
ment, especially if teleosts are used as models in biomedical
research.
Despite evidence for skeletal remodelling in early
gnathostomes (Carroll, 1988), various observations initially
led to the view that the fish skeleton is metabolically inactive
and not subject to resorption and remodelling. These
include the fact that fish live in relatively weightless
conditions, have unlimited access to calcium from the
ambient water, an often reported absence of multinucleated
bone resorbing cells, and the failure to stimulate bone
resorption with parathyroid hormone (PTH) (Clark &

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
Skeletal remodelling in teleost fish
Flemming, 1963; Simmons, 1971; Urasa, Flik & Wendelaar-
Bonga, 1984; Ellis, Roberts & Tyler, 1985; Ingleton et al.,
1995). This view has changed recently; it is now clear that
the fish skeleton is metabolically active and that it is subject
to resorption and remodelling, despite the differences that
exist regarding the control of skeletal remodelling in
mammals and in teleosts (Taylor, 1985; Sasayama, 1999;
Huysseune, 2000; Witten et al., 2001, 2006; Witten & Hall,
2003; Witten & Huysseune, 2007).
Being the most successful and varied vertebrate group
with over 25 000 species, teleosts have conquered virtually
all aquatic habitats on the planet. Although many species
share a similar, streamlined, body shape dictated by
hydrodynamic requirements (Shadwick, 2005), teleosts are
a remarkably evolved and diverse vertebrate group (Romer,
1970; Lagler et al., 1977; Fabacher & Little, 2000).
Certainly, a long evolutionary history in an often rather
constant aquatic environment has allowed preservation of
ancestral characters such as fins and the postcranial dermal
skeleton. The anatomy and composition of the teleost
skeleton is, however, not at all ‘‘primitive’’ or less complex
compared to that of mammals (Metscher & Ahlberg, 1999).
As pointed out by Owen (1854) ‘‘more bones enter into the
formation of the skull in fishes than in any other animals;
and the composition of this skull has been rightly deemed
the most difficult problem in comparative anatomy’’,
a statement that also applies to the postcranial skeleton.
At the tissue level, teleosts confront us with an astounding
variety of skeletal tissues. Some of these are unique and
some represent persistent intermediate forms between
familiar skeletal categories (Huysseune, 2000; Hall &
Witten, 2007). To understand the function and the control
of teleost skeletal remodelling requires the proper identifi-
cation of the many tissues that constitute the teleost
skeleton. These tissues will be briefly introduced below
(Section II). Furthermore, it is important to distinguish
between traits and processes that are shared by all
vertebrates, and those that are either conserved or derived
in teleost fish (Metscher & Ahlberg, 1999). In both
mammals and in teleost fish bone remodelling involves
conserved and derived characters: characteristics of the
teleost skeleton are not always primitive nor are those of the
mammalian skeleton always derived.
II. A BRIEF OVERVIEW OF TELEOST
SKELETAL BIOLOGY
The teleost skeleton consists of two major subunits that
evolved to a large degree independently: the dermal
skeleton and the endoskeleton (Smith & Hall, 1990). The
major categories of skeletal tissues (cartilage, bone, dentine
and enamel/enameloid) and the major categories of skeletal
cells (chondroblasts, chondrocytes, osteoblasts, bone lining
cells, osteocytes, osteoclasts, odontoblasts, ameloblasts) are
present in both teleosts and mammals (Huysseune, 2000).
Apart from dentine, enamel/enameloid and bone of
attachment, that are restricted to the dermal skeleton, all
skeletal tissues and respective cells can be found in both the
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
317
teleost dermal skeleton and the endoskeleton. Compared to
mammals, additional skeletal tissue subtypes are recognised
in teleost fish as part of the regular (non-pathological, non-
regenerating) skeleton (Benjamin, 1988, 1990; Benjamin,
Ralphs & Eberewariye, 1992; Meunier & Huysseune, 1992;
Huysseune, 2000; Witten & Huysseune, 2007; Hall &
Witten, 2007). Benjamin (1990) describes seven categories
of cartilage: (a) hyaline cell cartilage, (b) zellknorpel, (c)
fibro/cell-rich cartilage, (d ) elastic/cell-rich cartilage, (e) cell-
rich hyaline cartilage, ( f ) matrix-rich hyaline cartilage, and
(g) scleral cartilage. Cartilaginous tissues are not restricted to
the endoskeleton. Secondary cartilage and chondroid
cartilaginous tissues also develop on cranial dermal bones
(Beresford, 1993; Benjamin, 1989; Huysseune, 2000; Witten &
Hall, 2002; Gillis, Witten & Hall, 2006). The best studied
teleost ‘‘intermediate skeletal’’ tissue is perhaps chondroid
bone. Chondroid bone exhibits characteristics of bone and
of cartilage and develops from osteogenic precursors
(Huysseune, 1986; Huysseune & Verraes, 1986). It is a tissue
that contains chondrocyte-like cells (devoid of cell processes)
surrounded by a matrix that has properties of bone matrix
(Beresford, 1981; Huysseune & Verraes, 1990; Huysseune &
Sire, 1990; Witten & Hall, 2002). Chondroid bone occurs in
basal teleosts with osteocyte-containing bone and in
advanced teleosts with acellular bone, and should not be
confused with cellular bone (Beresford, 1981, 1993;
Huysseune & Verraes, 1990; Huysseune & Sire, 1990;
Meunier & Huysseune, 1992; Witten & Hall, 2002; Gillis
et al., 2006). Chondroid bone can also be remodelled: for
example, in Salmo salar, chondroid bone that is part of the
lower jaw is later resorbed and remodelled into lamellar
dentary bone (Witten & Hall, 2002, 2003; Gillis et al., 2006).
The first step in the development of ‘‘regular’’ cartilage in
teleosts is condensation of mesenchymal cells (blastema stage)
that turn into closely packed prechondroblasts (Huysseune &
Sire, 1992b). These cells differentiate into chondroblasts and
finally become separated through the secretion of extracel-
lular cartilage matrix. The subsequent formation of bone is
first perichondral, leading to the formation of a bone collar
around the cartilage. This bone collar can be extended by the
formation of membranous apolamellae (Stephan, 1900;
Blanc, 1953; Bertin, 1958; Huysseune, 2000; Witten &
Huysseune, 2007). As in mammals, cartilage can be replaced
by bone through endochondral bone formation (Huysseune,
2000) but in smaller teleost species, such as medaka or
zebrafish, endochondral bone formation is rather uncom-
mon. If cartilage is removed inside a bony shaft it is typically
replaced by adipose tissue (Huysseune, 2000; Witten et al.,
2001) (Fig. 1A,B).
Structurally, bone tissue in teleost fish develops first as
woven bone. Subsequently, parallel-fibered and lamellar
bone develop in more mature individuals. In larger
individuals lamellar bone can also form osteons (Moss,
1961b; Smith-Vaniz, Kaufman & Glowacki, 1995; Witten &
Hall, 2002, 2003; Meunier, 2002). The bone of basal
teleosts contains osteocytes, as is the case for tetrapod bone,
whereas advanced teleosts possess acellular or anosteocytic
bone which has no enclosed osteocytes (Kölliker, 1859;
Moss, 1961a; Ekanayake & Hall, 1987; Meunier, 1989;
Meunier & Huysseune, 1992; Huysseune, 2000;
318 P. Eckhard Witten and Ann Huysseune

Fig. 1. (A) Endochondral bone formation in a small teleost (Astatotilapia elegans). Cartilage becomes mineralized and is removed (black
arrowheads) at both ends of a growing bone shaft (white arrowheads). The developing lumen of the bone shaft contains no bone
trabeculae but is filled with adipose tissue (black asterisk). Glycol methacrylate embedding, Toluidine Blue staining: scale bar ¼ 200 mm.
(B) Removal of cartilage by small, highly tartrate-resistant acid phosphatase (TRAP) active, chondroclasts (red) in the cichlid Oreochromis
niloticus. Glycol methacrylate embedding, TRAP staining counterstained with Haematoxylin: scale bar ¼ 25 mm. (C) Cellular bone of
a common carp (Cyprinus carpio) with bone lining cells (black arrowhead) and osteocytes (white arrowhead). Non-decalcified methyl
methacrylate embedding, Toluidine Blue staining: scale bar ¼ 20 mm. (D) Acellular bone of the cichlid Hemichromis bimaculatus. Bone
lining cells are present on the bone surface (black arrowhead) but no cells are present inside the bone matrix. Small canals (white
asterisk) that may contain blood vessels and various cell types should not be mistaken for osteocytes. Epon embedding, Toluidine Blue
staining: scale bar ¼ 10 mm. (E) Developing vertebral column in the cichlid Oreochromis niloticus. Cartilage is stained blue, mineralizing
tissues (bone and notochordal sheath) are stained red. Haemal and neural arches (white arrowheads) are preformed in cartilage. The
vertebral centra have no cartilaginous precursor. In early development, vertebral bodies (black arrowhead) are formed from the
mineralizing notochordal sheath, followed by intramembranous bone formation, without involvement of bone remodelling. Whole
mount staining with Alcian Blue and Alizarin Red: scale bar ¼ 1 mm. (F) Dorsal growth zone of two opposing vertebral body end
plates in a juvenile salmon (Salmo salar). The bone (white asterisks) is separated by the notochord, but only the notochordal sheath is
visible (black asterisk). Bone formation by osteoblasts (black arrowhead) that secrete the osteoid (white arrowhead) is purely
intramembranous. Paraffin embedding, Masson’s Trichrome staining: scale bar ¼ 20 mm.

Witten et al., 2004). Furthermore, in contrast to osteocyte-
containing bone, no cell processes penetrate acellular bone
(Fig. 1C,D). The lack of cell processes inside the matrix
distinguishes acellular bone also from typical teleost
dentine. Acellular bone bears however some resemblance
to the atubular dentine of teleost first-generation teeth and
to acellular mammalian cementum, both of which also lack
enclosed cells and are not penetrated by cell processes
(Weiss & Watabe, 1979; Ekanayake & Hall, 1987, 1988;
Meunier, 1989; Sire et al., 2002; Franz-Odendaal, Hall &
Witten, 2006). The formation of acellular bone resembles
the formation of dentine as cells never become entrapped
in the bone matrix (Huysseune, 2000). With few exceptions,
the presence or absence of osteocytes is uniform in all
elements of the teleost skeleton (see Moss, 1961b and
Meunier, 1989, for exceptions). Members of large basal
teleost groups, such as salmonids and cyprinids, with
cellular bone in the endoskeleton, can have postcranial
dermal skeletal elements (scales and fin rays) that are
acellular (Meunier, 1989). The absence of osteocytes in
advanced teleosts, cells that in mammals constitute 95% of
all bone cells (Franz-Odendaal et al., 2006), raises questions

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
Skeletal remodelling in teleost fish
about the regulation of bone remodelling in advanced
teleosts. This is because the penetration of bone and dentine
by cell processes enables osteocytes and odontoblasts to
function as stress sensors. Accordingly osteocytes are
believed to guide bone remodelling in response to
mechanical load (Burger, Klein-Nulend & Van Der Plas,
1995; Burger, Klein-Nulend & Smit, 2003; Bonewald, 2004;
Knothe Tate et al., 2004). Estimates about the percentage of
bone that is resorbed and replaced by new bone in humans
range between four and ten per cent per year (Delling &
Vogel, 1992; Manolagas, 2000). Such estimates are lacking
for teleost fish but a regular resorption and rebuilding of
scales and bony skeletal elements is well documented for
Atlantic salmon (Kacem, Meunier & Baglinière, 1998;
Persson et al., 2000; Witten & Hall, 2002, 2003).
An acellular skeletal tissue particular to all teleosts is the
mineralized notochordal sheath (Arratia, Schultze & Casciotta,
2001; Grotmol et al., 2003; Nordvik et al., 2005; Inohaya,
Takano & Kudo, 2007). Unlike other vertebrates, early
formation of teleost vertebral bodies does not start with
cartilaginous vertebral body precursors that form around the
notochord. Instead, the formation of vertebral bodies starts
with the mineralization of the notochordal sheath itself
(Fig. 1E,F). Further growth of the vertebral bodies occurs by
the apposition of bone onto the mineralized notochordal
sheath, similar to the intramembranous formation of bone
apolamellae (Ekanayake & Hall, 1988, 1991; Grotmol et al.,
2003; Nordvik et al., 2005). As a consequence, cartilage
formation and subsequent endochondral bone formation are
not part of the initial differentiation of teleost vertebral bodies
(Witten & Villwock, 1997; Nordvik et al., 2005).
While we can expect that the particular cellular
composition of the teleost skeleton affects if and how it
will remodel, a number of additional factors influence the
process. Since basic ideas about bone remodelling are
influenced by our knowledge of the processes in mammals,
it might be useful to enumerate some of the main
characteristics that distinguish teleosts from mammals with
respect to skeletal remodelling:
(1) In mammals, the bone resorbing cells, or osteoclasts,
originate from haematopoietic tissue located in the
bone marrow. The haematopoietic tissue also releases
factors that regulate osteoclast and osteoblast activities.
Such an intimate spatial relationship between bone
resorbing cells and haematopoietic cells does not exist
in teleosts as most bone marrow spaces are filled with
adipose tissue and haematopoiesis takes place in the
head kidney (Section III).
(2) The lack of osteocytes in advanced teleosts appears to
coincide with an altered morphology of bone resorb-
ing cells and an alternative mode of bone resorption.
Osteoclasts of advanced teleosts are often small,
mononucleated cells that perform non-lacunar, in
particular smooth, bone resorption (Section IV).
(3) In mammals, endochondral bone formation is a prime
cause of skeletal resorption and remodelling. Typical
endochondral ossification is, however, often lacking in
teleosts, especially in species with small individuals
such as medaka or zebrafish. In addition, in all teleosts,
vertebral bodies develop (ossify) without cartilaginous
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
319
precursors and thus initially without remodelling
(Sections V.1 and V.2).
(4) Regulation of plasma calcium content is crucial for all
terrestrial vertebrates and the skeleton is tightly
integrated into the animals’ calcium homeostasis.
Teleosts have a different approach. They obtain and
release calcium from and into the water via their gills
and the skeleton must not be used as a source or
deposit of calcium (Section V.3).
(5) In case of need of skeletal resorption, minerals are
mobilised from the extensive postcranial dermal
skeleton (scales). Minerals must not be released from
the endoskeleton (Section V.3).
(6) The lack of osteocytes in advanced teleosts implies that
bone remodelling in response to mechanical load must
be triggered by other cell types (Section V.4).
(7) The regenerative capacity of elements of the teleost
dermal skeleton (fin rays and scales) exceeds the
regenerative capacity of elements of the endoskeleton
(Section V.5).
(8) Teleosts replace their teeth throughout life, a process
that requires lifelong resorption of teeth and tooth
attachment bone and remodelling of dentigerous bone
(Section V.6).
(9) Teleosts never stop growing, and certain skeletal
elements develop rather late. Thus, growth-related
skeletal remodelling continues throughout life and
should not be mistaken for metabolism-related skeletal
remodelling (Section V.7).
III. CELLULAR MECHANISMS OF TELEOST
SKELETAL REMODELLING
Skeletal remodelling can be defined broadly as the
replacement of an existing skeletal tissue by another skeletal
tissue. A classic example is the replacement of cartilage by
bone during endochondral bone formation in mammalian
long bones. A stricter definition of bone remodelling refers to
continuous bone replacement in the adult skeleton (Parfitt,
1994). Bone remodelling usually involves the removal of bone
by osteoclasts (bone resorbing cells) and the subsequent
formation of new bone by osteoblasts (bone forming cells). To
keep the skeleton healthy and functional, coordinated action
of osteoclasts and osteoblasts is required. Given that these
cells act together, they can be considered as basic multicellular
units (BMUs) (Frost, 1969, 1987; Parfitt, 1994; Manolagas,
2000; Heino, Hentunen & Väänänen, 2002; Burger et al.,
2003; Cooper et al., 2003). Alternatively, remodelling can
follow pathways that do not involve osteoclasts.
(1) Osteoclastic resorption and
remodelling in teleosts
(a ) Osteoclasts: the mammalian perspective
In mammals (as well as in birds), osteoclasts are the principal
bone resorbing cells (Kölliker, 1873). Comprehensive reviews
320
of the development, regulation, and function of multinucle-
ated mammalian osteoclasts should be consulted for detailed
information about the biology of these cells (e.g. Chambers,
1985; Vaes, 1988; Burger & Nijweide, 1991; Blair, 1998;
Reddy & Roodman, 1998; Manolagas, 2000; Theill, Boyle &
Penninger, 2002; Boyde, 2003; Burger et al., 2003; Hall,
2005; Väänänen, 2005; Bar-Shavit, 2007). The main features
of mammalian osteoclasts are briefly summarised below to
enable us to clarify the similarities and differences between
mammalian and teleostean osteoclasts. Chondroclasts and
odontoclasts are related to osteoclasts, resorbing calcified
cartilage and dentine, respectively (Hall, 1972; Beresford,
1981; Blair, 1998; Buxton et al., 2003). Despite their different
names, environments, and regulatory mechanisms, there is
evidence that these different skeletal resorbing cells belong to
the same cell lineage (Nordahl et al., 2000; Shibata &
Yamashita, 2001; Sasaki, 2003; Helfrich, 2003).
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
P. Eckhard Witten and Ann Huysseune
Mammalian bone resorbing cells can be viewed as
specialised multinucleated macrophages. Multinucleated
mammalian osteoclasts derive from haematopoietic bone
marrow cells that also give rise to monocytes and macro-
phages (Holtrop, Cox & Glowacki, 1982). In vitro experi-
ments show that peripheral blood monocytes can fuse and
differentiate into multinucleated osteoclasts. The dendritic
cell-specific transmembrane protein (DC-STAMP) has been
identified as a critical factor for the fusion of mononucleated
cells into multinucleated osteoclasts. DC-STAMP-deficient
cells fail to fuse, but still function as mononucleated
osteoclasts (Yagi et al., 2005; Bar-Shavit, 2007). Transcrip-
tion factors and signalling molecules that are required for
macrophage differentiation such as the macrophage and B
cell-specific transcription factor PU.1 and tumor necrosis
factor receptor-associated factor 6 (TRAF6) are also
required for osteoclast development (Udagawa et al., 1990;
Skeletal remodelling in teleost fish 321

Fig. 2. (A) Multinucleated, tartrate-resistant acid phosphatase (TRAP) positive (red) giant osteoclast resorbing the kype (an antler-
like outgrowth of the lower jaw in males) of an Atlantic salmon (Salmo salar) after the spawning period. This section of the giant cell
shows about 30 nuclei (black arrowhead). Considering all three dimensions of the cell, the actual number of nuclei could be above
100. The cells’ ruffled border is clearly visible (black asterisk). Glycol methacrylate embedding, TRAP staining: scale bar ¼ 15 mm.
(B) Multinucleated TRAP-positive osteoclasts (red) on the dentary of a juvenile carp (Cyprinus carpio). The resorption lacuna and the
cells’ ruffled border are visible (black asterisk), secreted TRAP appears as a red stripe on the bone surface (black arrowheads). These
characteristics are also typical for mammalian osteoclasts. Glycol methacrylate embedding, TRAP staining counterstained with
Haematoxylin: scale bar ¼ 10 mm. (C) Mononucleated TRAP-positive osteoclasts (red) in a 20-day-old zebrafish (Danio rerio) along
the forming dentary bone (white asterisk). Meckel’s cartilage, black asterisk. Glycol methacrylate embedding, TRAP staining
counterstained with Haematoxylin: scale bar ¼ 10 mm. (D) Mononucleated osteoclasts (red) on the lower jaw of a juvenile cichlid
(Oreochromis niloticus). The cells can be flat and are connected to each other with long cell processes. This type of osteoclast is
frequently observed in advanced teleost fish with acellular bone. Acellular bone, white asterisks. Glycol methacrylate embedding,
TRAP staining counterstained with Haematoxylin: scale bar ¼ 15 mm. (E) Transmission electron micrograph showing contact
between cell processes of two mononucleated osteoclasts (black arrowhead) on the endosteal surface of a lateral line canal in the
cichlid Oreochromis niloticus. Epon embedding: scale bar ¼ 0.5 mm. (F) Flat and long osteoclasts on the endosteal surface (white
arrowhead) of a neural arch in the eel (Anguilla anguilla). The shape of the cells resembles bone lining cells rather than typical
mammalian osteoclasts. A sectioning artefact that has shifted the cells from the endosteal bone surface reveals their flat shape and
strong interconnection (black arrowheads). Spinal cord, black asterisk. Glycol methacrylate embedding, TRAP staining
counterstained with Haematoxylin: scale bar ¼ 20 mm. (G) Cross section of a growing bone tube that contains a lateral line canal
(black asterisk) in the cichlid Oreochromis niloticus. The acellular bone grows by bone resorption along the endosteal surfaces (white
arrowhead) and by bone apposition along the periosteal surfaces (black arrowhead). No multinucleated osteoclasts are visible along
the endosteal surfaces. Paraffin embedding, Azan staining: scale bar ¼ 25 mm. (H) Cross section of a growing bone tube in the
cichlid Oreochromis niloticus, similar to that shown in G revealing the presence of many small mononucleated osteoclasts by visualising
the cells’ TRAP activity. Asterisk, lateral line canal; white arrowhead: endosteal bone surface. Glycol methacrylate embedding,
TRAP staining counterstained with Haematoxylin: scale bar ¼ 25 mm.

Stenbeck, 2002). Maturation of mammalian osteoclasts
requires direct contact with bone marrow stroma cells or
with osteoblasts, as the expression of molecules by stroma
cells and osteoblasts regulates the differentiation of
osteoclasts (see BMU concept above). Key molecules are
the macrophage colony-stimulating factor (M-CSF) and
RANK ligand (RANKL). Stroma cells and osteoblasts also
release osteoprotegerin (OPG), a RANKL decoy receptor
that downregulates osteoclasts (Teitelbaum, 2000; Li, Kong &
Qi, 2006). For other cytokines and growth factors that
stimulate or inhibit osteoclasts see Teitelbaum (2000), Theill
et al. (2002), and Li et al. (2006). The differentiation and
function of mammalian osteoclasts is also under hormonal
control. Estrogen (17b-estradiol) and calcitonin (CT) are the
main osteoclast-inhibiting hormones. Parathyroid hormone
(PTH), the parathyroid hormone-related protein (PTHrP)
and Vitamin D are the main osteoclast stimulating factors.
The target cells for estrogen, PTH and Vitamin D are not
osteoclasts but osteoblasts. Subsequently, osteoblasts regu-
late osteoclasts via the above-mentioned M-CSF, RANKL
and OPG pathways (Rodan & Martin, 1981; Franz-
Odendaal et al., 2006). In mammals CT binds directly to
a receptor expressed by osteoclasts; however in non-
mammalian vertebrates CT receptors have been found to
be downregulated in osteoclasts (Dacke et al., 1993).
Like macrophages, mammalian osteoclasts are mobile
cells that migrate to the site of bone resorption. Upon
contact with the bone surface, the apical cell membrane of
osteoclasts develops a peripheral sealing zone (that conceals
the subcellular space) and a central ruffled border (multiple
deep membrane infoldings) (Parfitt, 1988; Li et al., 2006).
The central ruffled border is the area of active bone
resorption. The sealed space under the ruffled border can
be viewed as a giant extracellular phagolysosome (Baron
et al., 1993). The extracellular space is acidified by the
action of a vacuolar proton pump (H]-ATPase) (Väänänen
et al., 1990; Mattsson et al., 1994; Bastani et al., 1996;
Stenbeck, 2002). The acidified environment promotes the
dissolution of bone minerals and the function of lysosomal
enzymes. Enzymes such as cathepsins, unspecific acid
phosphatase (cleaving b-glycerophosphate) and tartrate-
resistant acid phosphatase (TRAP) break down bone matrix
components (Hammarström, Hanker & Touverud, 1971;
Lucht, 1971; Vaes, 1988; Blair, 1998; Manolagas, 2000;
Halleen & Ranta, 2001; Halleen et al., 2002; Helfrich,
2003). The participation of metalloproteinases in bone
resorption remains controversial. Everts et al. (1999) showed
that these enzymes can be present or absent, depending on
the skeletal element that is resorbed.
(b ) Teleost osteoclasts
The above model of skeletal remodelling and bone
resorption has gained general acceptance for mammals
(and birds). Typical (‘‘mammalian-like’’) multinucleated
osteoclasts and odontoclasts can be also found in teleosts,
especially in basal teleosts, such as cyprinids and salmonids
(Witten et al., 2000; Domon et al., 2004; Hall, 2005). Yet the
number of nuclei in resorbing cells of carp (Cyprinus carpio)
and salmon can be higher than in mammalian cells
(Fig. 2A). In Pacific salmon (Oncorhynchus tshawytscha),
odontoclasts may contain more than 100 nuclei (Domon
et al., 2004). Similar large osteoclasts and odontoclasts have
been observed in Atlantic salmon and in carp (Sire et al.,
1990; Witten et al., 2000; Witten & Hall, 2002). Certainly
the number of nuclei in osteoclasts differs among vertebrate
groups. In teleosts, there is large variation in number of
nuclei per osteoclast related to differences among species,

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322
ontogenetic stages, skeletal elements and physiological
conditions (Fig. 2B) (Belanger, 1977; Sire et al., 1990;
Persson, Björnsson & Takagi, 1999; Witten et al., 2001;
Roy et al., 2002; Witten & Hall, 2002; Väänänen, 2005).
Birds also have remarkably high numbers of nuclei per
osteoclast (Väänänen, 2005; Bar-Shavit, 2007). In humans
such large osteoclast nuclei numbers only occur under
pathological conditions, for example in Paget’s disease
(Manolagas, 2000; Helfrich, 2003).
As skeletal growth and remodelling always requires the
presence of osteoclasts (but see Section III.2), one may
wonder why the existence of these cells in advanced teleosts
was questioned. The reason was the often observed lack of
typical (multinucleated) osteoclasts and their traces (resorp-
tion lacunae). This is because small mononucleated
osteoclasts dominate early skeletal development in all
teleosts (Fig. 2C) and also later stages of development in
advanced teleosts with acellular bone (Fig. 2D) (Sire et al.,
1990; Witten, 1997). As these cells cannot be detected with
standard histological techniques, their identification
requires either transmission electron microscopy (TEM) or
the demonstration of the activity of the osteoclast marker
enzyme TRAP (Weiss & Watabe, 1979; Lopez et al., 1980;
Sire et al., 1990; Witten, 1997). TEM, however, only
provides information about very small skeletal segments.
Consequently, the comprehensive visualisation of skeletal
remodelling during teleost development may require the
demonstration of TRAP activity (Witten, 1997; Witten &
Villwock, 1997; Witten et al., 2000, 2001) as is also the case
for complete visualisation of osteoclasts in humans (Ballanti
et al., 1997). The complete nucleotide and amino acid
sequence of TRAP from goldfish (Carassius auratus) is
available (Azuma et al., 2007). A comparison of TRAP
from mammals and goldfish shows a completely conserved
amino acid sequence in the iron-containing active centrum
of the enzyme (Azuma et al., 2007).
TEM and enzyme detection studies have revealed that in
all teleosts, but typically in advanced teleosts, cells that
perform bone resorption can differ from ‘‘standard’’
multinucleated mammalian osteoclasts. The cells can have
an extremely flat shape, resembling bone lining cells rather
than typical osteoclasts (Weiss & Watabe, 1978, 1979;
Witten, 1997; Witten et al., 1999, 2004) (Fig. 2E,F). How
much the cells differ may depend on species, skeletal
element, and ontogenetic stage. In mammals, mononucle-
ated cells are often viewed as osteoclast precursors but in
basal and advanced teleosts, mononucleated osteoclasts are
active resorbing cells (Lopez et al., 1980; Witten, 1997).
Bone resorption in teleosts may rely solely on mono-
nucleated cells or shifts can occur from resorbing mono-
nucleated to resorbing multinucleated cells during
development (Sire et al., 1990; Witten et al., 2001).
It was shown by TRAP staining and TEM that mono-
and not multinucleated bone resorbing cells facilitate the
enlargement of bone tubes in the Nile tilapia, Oreochromis
niloticus (Witten, 1997; Witten & Villwock, 1997). The small
mononucleated osteoclasts were found to be evenly
distributed along the endosteal surfaces where the cells
perform a smooth (non-lacunar) type of bone resorption
(Fig. 2G,H) (Witten, 1997; Witten & Villwock, 1997; Witten
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
P. Eckhard Witten and Ann Huysseune
et al., 1999). Similar findings have been reported from other
cichlids (Sarotherodon melanotheron) by Weiss & Watabe (1979)
and from medaka by Ekanayake & Hall (1987) and Takano
et al. (2005). Small mononucleated osteoclasts also dominate
bone resorption in the first advanced marine teleost to be
examined for osteoclasts by demonstration of the presence
of TRAP (haddock, Melanogrammus aeglefinus) (Roy et al.,
2002; Roy, 2003). Also scale resorbing cells in salmonids are
mostly mononucleated (Persson et al., 1999) despite the fact
that salmonids, as basal teleosts with cellular (osteocyte-
containing) bone, usually possess prominent multinucleated
cells. The scales, however, are acellular and thus resemble
other acellular tissues such as acellular bone or acellular
dentine (Meunier, 1989; Sire & Akimenko, 2004). Bone
resorption by mononucleated cells is not only a character of
advanced teleosts: demonstration of the presence of TRAP
and TEM studies have revealed the presence of abundant
mononucleated osteoclasts also in basal teleosts (Sire et al.,
1990; Witten et al., 2001), in basal osteichthyans such as
Neoceratodus forsteri (Kemp, 2003), and in humans (Ballanti
et al., 1997). According to Parfitt (1988), in humans,
multinucleated osteoclasts cause deep resorption lacunae
while mononucleated osteoclasts perform smooth (non-
lacunar) resorption. This description of Parfitt (1988) fits
observations about the performance of mononucleated
osteoclasts in teleosts. Finally, in teleosts, bone resorbing
cells can be absent from particular skeletal elements
altogether, such as developing radials or developing
vertebral bodies (Ekanayake & Hall, 1987, 1988; Witten &
Villwock, 1997; Nordvik et al., 2005).
(2) Non-osteoclastic bone resorption and
remodelling in teleosts
In addition to mono- and multinucleated osteoclasts,
osteocytes in cellular-boned teleosts are capable of bone
resorption by osteocytic osteolysis. Examples derive from
European eels (Anguilla anguilla) (Lopez et al., 1980; Sbaihi
et al., 2007), salmonids (Hughes et al., 1994; Kacem &
Meunier, 2000) and carp (Witten et al., 2000). Osteocytic
osteolysis is disputed as a feature of human osteocytes
(Boyde, 1980), but it has been observed in many
mammalian species with smaller adult body size such as
bats (Doty & Nunez, 1985; Kwiecinski, 1985; Kwiecinski,
Krook & Wimsatt, 1987), hamsters (Steinberg, Singh &
Mitchell, 1981), squirrels (Haller & Zimny, 1978), rats
(Belanger, 1977; Tazawa et al., 2004), and rabbits (Zhang,
Bao & Dai, 2000). Evidence for osteocytic osteolysis also
comes from studies on the snake skeleton (Alcobendas,
Baud & Castanet, 1991). The gracile skeleton of bats
provides perhaps the best example that lacunar resorption
by multinucleated cells is not an option for thin bones: this
process could easily perforate the bone. For pregnant and
lactating Myotis lucifungus bats osteocytic osteolysis, in
addition to smooth resorption by mononucleated cells, pro-
vides a functional alternative (Kwiecinski, 1985; Kwiecinski
et al., 1987). Another resorption-related process that has
been described for teleosts is demineralization of the bone
without affecting the bone matrix, called halastatic
Skeletal remodelling in teleost fish
demineralization (Lopez, 1970; Carragher & Sumpter,
1991; Kacem & Meunier, 2003).
If we consider a broad definition of skeletal remodelling
– replacing one tissue type by another – remodelling in
teleosts occurs not only by resorption and de novo formation
but also by transdifferentiation (metaplasia) of skeletal
tissue (Beresford, 1981; Witten & Hall, 2002; Hall, 2005;
Gillis et al., 2006; Hall & Witten, 2007). All processes,
including resorption by mononucleated osteoclasts, osteo-
cytic osteolysis, halastatic demineralization and metaplasia
may also occur in mammals (humans) during normal or
pathological development (Baud, 1968; Belanger, 1967,
1969, 1977; Beresford, 1981; Chambers, 1985; Ballanti
et al., 1997; Hall, 2005). However, some mechanisms of
skeletal remodelling are more prominent and/or more
commonly observed in teleosts and are thus recognised as
a regular process that shapes the skeleton in the course of
development (Sire et al., 1990; Huysseune, 2000; Witten
et al., 2001, 2003; Hall & Witten, 2007; Witten &
Huysseune, 2007).
(3) A possible link between bone type and
osteoclast type and number?
In view of the increasing evidence for a role of osteocytes in
controlling bone remodelling and activation of osteoclasts
(Burger et al., 1995, 2003; Heino et al., 2002; Bonewald,
2004; Franz-Odendaal et al., 2006), it is tempting to hypo-
thesize that the presence of osteocytes promotes the
formation of multinucleated osteoclasts in teleosts: in the
absence of osteocytes the dominant osteoclast type would be
mononucleated. Support for this idea derives from
comparative and ontogenetic studies. Comparing the
presence and absence of osteocytes in different parts of
the teleost skeleton, Meunier (1989) reported that members
of the carp and salmon families have scales without
osteocytes, but do possess osteocytes in the endoskeleton.
In basal bony fish below the teleost level, the endoskeleton,
scales and fin rays still contain osteocytes (Geraudie, 1988;
Meunier, 1989; Witten & Huysseune, 2007). Scales in
salmon are predominantly resorbed by mononucleated
osteoclasts whereas the osteocyte-containing endoskeleton is
predominantly resorbed by multinucleated giant cells
(Persson et al., 1998, 1999, 2000; Witten & Hall, 2002,
2003). Early bone resorbing cells in teleosts are mono-
nucleated at a stage when the bone is already mineralized
but osteocytes are scarce or not yet present (Sire et al., 1990;
Huysseune, 2000; Witten et al., 2001; Meunier, 2002). In
zebrafish there is an ontogenetic progression from mono- to
multinucleated osteoclasts (Witten et al., 2001). Initially
osteoclasts are mononucleated resembling morphologically
and functionally those of advanced teleosts (Fig. 2C). Thirty
days after fertilisation, mononucleated cells are succeeded
by multinucleated osteoclasts that subsequently perform
lacunar resorption and finally become engaged in bone
remodelling. Simultaneously with the expression of multi-
nucleated osteoclasts and bone remodelling, growth-related
modelling by mononucleated osteoclasts still occurs at the
endosteal surfaces of the neural arches of zebrafish.
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
323
Further evidence for a link between the presence of
osteocytes and multinucleated osteoclasts derives from an
analysis of hyperostotic bones in advanced marine teleosts
with acellular bone (Caranx latus and Caranx hippos,
Carangidae) (Smith-Vaniz et al., 1995). Hyperostosis, the
periosteal formation of excess bone (‘‘Zuwachsknochen’’) at
spines and skull bones of adults, was formerly viewed as
a pathological process (Schlumberger & Lucke, 1948). It is
now considered part of normal late development (Smith-
Vaniz et al., 1995). An intriguing feature of hyperostotic
bones is the reappearance of osteocytes and multinucleated
osteoclasts, even if the hyperostotic bone is an outgrowth of
acellular bone. The rather early shift from acellular to
cellular bone and from mononucleated to multinucleated
osteoclasts in cellular-boned teleosts, and the late shift in
adult acellular-boned teleosts developing hyperostoses, led
Witten et al. (2004) to hypothesize that acellular bone and
mononucleated osteoclasts are not necessarily a new trait of
advanced teleosts. Rather, they may represent a hetero-
chronic shift resulting from the retention of early skeletal
characters (the absence of osteocytes and of multinucleated
osteoclasts) well into adult life.
Is there a relationship between osteoclast type and
osteoclast number? Considering the many factors that may
influence the type and the number of osteoclasts, comparing
cell numbers across species can only provide rough
indications about such a relationship. Some data are
available: in the jaws of juvenile carp there are one to
seven multinucleated TRAP-positive osteoclasts per mm of
two-dimensional (2D) endosteal bone surface whereas the
endosteal bone surfaces of the lower jaw in juvenile Nile
tilapia have up to six times more TRAP-positive mono-
nucleated cells (28 – 42 per mm 2D bone surface) (Witten,
1997; Witten et al., 2000). In juvenile haddock (acellular-
boned like O. niloticus) there are about 79- 82 small, mostly
mononucleated, osteoclasts per mm 2D bone surface (Roy
et al., 2002). These numbers may suggest that many small
mononucleated cells can functionally replace a few multi-
nucleated giant cells. The type of resorption exhibited by
mono- and multinucleated osteoclasts differs however:
multinucleated cells perform lacunar resorption, whereas
mononucleated cells are responsible for smooth resorption.
These ideas fit to Parfitt’s model about the function of
mononucleated osteoclasts (Parfitt, 1988) (see Section III.1b)
and the idea that in mammals, the amount of bone
resorption per cell nucleus is the same in multinucleated
giant cells and small oligonucleated cells (Bar-Shavit, 2007).
IV. DIFFERENTIATION AND ACTIVATION OF
TELEOST OSTEOCLASTS
(1) Multinucleated osteoclasts
Typical remodelling sequences in teleosts suggest the
coordinated action of osteoblasts and osteoclasts, according
to the BMU model proposed for mammalian bone (Peck &
Woods, 1988; Parfitt, 1988) (Fig. 3A). In zebrafish, repeated
resorption by osteoclasts after refilling of resorption lacunae
324 P. Eckhard Witten and Ann Huysseune

can be observed, a process regarded as typical remodelling
(Witten et al., 2000, 2001) (Fig. 3B). Remodelling sequences
in carp and zebrafish show multinucleated osteoclasts
performing lacunar resorption, resorption lacunae with
osteoblasts in the process of refilling lacunae, and resorption
lacunae that have been completely refilled (Fig. 3A). Several
key factors that control development of mammalian
monocytes into osteoclasts and that activate osteoclasts
(see Section III.1a) have been identified in various teleost
species. A highly conserved transcription factor PU.1 from
rainbow trout macrophages has been characterised by
Ribas et al. (2008) and a full-length orthologue of
mammalian TRAF6 has been identified in zebrafish
(Phelan, Mellon & Kim, 2005). A M-CSF from haemato-
poietic tissue of the goldfish has been characterised by
Hanington et al. (2007). RANKL has been discovered in the
genome of rainbow trout, zebrafish, and pufferfish (Glenney &
Wiens, 2007) and the RANKL decoy receptor OPG has
been isolated from the genome of medaka (Wagner et al.,
2003). It is likely that bone remodelling and the differen-
tiation of multinucleated osteoclasts in basal teleosts
depends on the same signalling pathway that activates
osteoclasts and macrophages in mammals. Conclusive
evidence for this speculation is, however, still lacking.
Ostland, McGrogan & Ferguson (1997) observed the
activation of multinucleated osteoclasts in salmonids con-
nected with an inflammatory process suggesting that
inflammation-related signalling molecules known to acti-
vate mammalian osteoclasts may also activate teleostean
osteoclasts. A similar observation was made by Takagi &
Kanoko (1995). Bone particles implanted into the muscles
of rainbow trout caused the appearance of monocyte-like
cells followed by typical TRAP-positive multinucleated
osteoclasts. Both the mineralized bone and the inflamma-
tory reaction to foreign antigens may have triggered the
development of osteoclasts. Apart from validating the
pathway of teleost osteoclast stimulation by inflammatory
factors it will be a challenge to find out how osteoclasts in
teleosts become activated in the absence of haematopoietic
tissue in the bone marrow (Figs 1A,B, 3D) precluding an
intimate spatial relationship between haematopoietic cells
and teleostean osteoclasts. The lack of haematopoietic bone
marrow tissue is an ancestral character: the first haemato-
poietic bone marrow tissue appears only in advanced
urodeles (Zapata & Amemiya, 2000). An example of how
osteoclasts can be activated without the vicinity of
haematopoietic tissue is found in the process of tooth
eruption in mammals (Marks, 1976, 1977; Marks &

Fig. 3. (A) Typical stages of bone remodelling (a-c) on the dentary of a juvenile carp (Cyprinus carpio). a, A multinucleated osteoclast
is generating a resorption lacuna. b, Osteoblasts settle inside an empty resorption lacuna and start to produce a small amount of
osteoid (white arrowhead). c, Osteoblasts have progressed in filling a former resorption lacuna; the osteoid deposited is clearly
visible (white arrowhead). Non-decalcified methyl methacrylate embedding, Toluidine Blue staining: scale bar ¼ 100 mm. (B)
Formation of a deep resorption lacuna (white arrowhead) on the palatine of an adult zebrafish (Danio rerio) (left panel); bone
resorption and filling of a resorption lacuna can occur repeatedly at the same site (black arrowheads, right panel). Adipose tissue fills
the bone marrow space (white asterisks). Epon embedding, Toluidine Blue staining: scale bar ¼ 15 mm. (C) Tartrate-resistant acid
phosphatase (TRAP), secreted by osteoclasts, persists on the bone surface of the dentary of a juvenile carp (Cyprinus carpio) and labels
previous areas of bone resorption even when osteoclasts are no longer present (white arrowhead) and osteoblasts have already
started to deposit new bone matrix (black arrowhead). Glycol methacrylate embedding, TRAP staining counterstained with
Haematoxylin: scale bar ¼ 100 mm. (D) Endochondral bone formation in a juvenile carp (Cyprinus carpio). The mineralization of the
cartilage matrix (black asterisk) is followed by the deposition of bone (white arrowheads) on mineralized cartilage remnants (black
arrowhead). Non-decalcified methyl methacrylate embedding, Toluidine Blue staining: scale bar ¼ 100 mm.

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
Skeletal remodelling in teleost fish
Schroeder, 1996). Epithelial and connective tissues that
surround the developing tooth (the dental follicle, dental
epithelium, and stellate reticulum) have been shown to
release factors required for osteoclast development and
regulation such as CSF-1, RANK, RANK-L, OPG, trans-
forming growth factor-b (TGF-b), and interleukin-1a (Il-1a)
(Wise & Lin, 1995; Marks & Schroeder, 1996; Ohazama,
Courtney & Sharpe, 2004).
(2) Mononucleated osteoclasts
Even more intriguing is the activation of the inconspicuous,
mononucleated bone resorbing cells found in advanced
teleosts with acellular bone (Weiss & Watabe, 1978, 1979;
Ekanayake & Hall, 1987; Witten, 1997; Witten & Villwock,
1997; Witten et al., 2004; Takano et al., 2005). The rarity or
reported absence of multinucleated osteoclasts and resorp-
tion lacunae in these animals, and their failure to respond to
calcium deprivation with bone resorption, led to the
perception that advanced teleosts were lacking osteoclasts
(Simmons, 1971; Urasa et al., 1984; Ingleton et al., 1995).
Experiments attempting to induce osteoclastic bone resorp-
tion in advanced teleosts by exposing the animals to
calcium-deficient conditions gave varied results (Moss,
1962; Weiss & Watabe, 1979; Taylor, 1985; Ellis et al.,
1985; Glowacki et al., 1986; Takagi & Yamada, 1991, 1992,
1993; Witten, 1997). Some failed to induce (multinucleated)
osteoclasts (Moss, 1962), while others generated mono-
nucleated cells (Weiss & Watabe, 1978, 1979; Witten, 1997).
Glowacki et al. (1986) generated multinucleated osteoclast-
like cells as a response to the implantation of devitalised rat
bone in the advanced teleost Paralabrax clathratus. Multinu-
cleated osteoclasts are known to occur in advanced teleosts
also under non-experimental conditions (Sire et al., 1990).
As discussed in Section III.3 teleosts with acellular bone can
generate multinucleated osteoclasts if lacunar resorption is
required, but the vast majority of osteoclasts in advanced
teleosts appears to be mononucleated (Witten, 1997; Witten
et al., 1999, 2004; Roy et al., 2002; Takano et al., 2005).
Despite morphological and functional dissimilarity
between typical mammalian osteoclasts and mononucleated
osteoclasts of juvenile and adult advanced teleosts the cells
share several biochemical and functional features. Like
mammalian osteoclasts and their precursors, teleost osteo-
clast precursor cells express a-napthyl acetate esterase
whereas mature cells only continue to express b-glycer-
ophosphatase, TRAP, and V]-ATPase. Like multinucleated
osteoclasts, mononucleated fish osteoclasts secrete TRAP
into the subcellular resorption space and the enzyme is
consequently detected on the bone surfaces (Ibbotson et al.,
1984; Baron et al., 1986; Hermanns, 1987; Connor et al.,
1995; Witten, 1997; Witten et al., 1999) (Fig. 3C). Thus, in
both mammals and teleosts labelling TRAP activity
identifies bone surfaces that are being actively resorbed or
have been resorbed. TRAP-staining can reveal previous
sites of bone remodelling even if the resorbing cells are no
longer present and new bone has been produced above
previously resorbed bone surfaces (Witten, 1997; Witten
et al., 2000, 2001) (Fig. 3C). The fact that small mono-
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
325
nucleated osteoclasts may not show a ruffled border
(Witten, 1997; Weiss & Watabe, 1979; Kemp, 2003) could
suggest that this morphological feature is connected to
lacunar bone resorption but is not required for continuous
smooth resorption (see also Section III.3). In a recent study
on rachitic mice Hollberg et al. (2008) found that
mammalian osteoclasts lacking a ruffled border can
effectively degrade bone matrix. Non-lacunar bone resorp-
tion by mononucleated osteoclasts without a ruffled border
is analogous to osteocytic osteolysis.
V. THE FUNCTIONS OF TELEOST
SKELETAL REMODELLING
As in other vertebrates the development and growth of the
teleost skeleton requires resorption and remodelling for
several reasons: (a) to allow enlargement of elements during
allometric growth, e.g. increasing the diameters of bone
tubes and bony arches, such as the cranial vault, jaw arches,
neural arches, and lateral line canals (Section V.1). (b) For
replacement of one skeletal tissue by another, for example
cartilage by bone, primary bone by secondary bone or
chondroid bone by lamellar bone (Section V.2). (c) Mineral
homeostasis, under particular conditions (Section V.3). (d )
To allow the skeleton to adapt to changes in mechanical
loading (Section V.4). (e) Fracture healing, regeneration and
disease-related repair (Section V.5). (f ) Replacement of
teeth, involving the partial or complete resorption of old
teeth, their attachment bone and parts of the dentigerous
bone (Section V.6). (g) Teleosts continue to grow as adults,
and therefore non-pathological and non-senescence-linked
remodelling continues throughout life (Section V.7).
(1) Growth and morphogenesis
Enlargement of the diameter of bone shafts and curved
skeletal elements is a primary reason for bone resorption
during teleost ontogeny. As bone cannot grow interstitially,
any change of tube diameter or curvature of a mineralized
skeletal element requires bone resorption on one side and
bone apposition on the other. Several studies report the
occurrence of teleost osteoclasts along growing skull bones
and endosteal bone surfaces (Huysseune & Sire, 1992a;
Sire & Huysseune, 1993; Witten, 1997; Witten & Villwock,
1997; Witten et al., 2000, 2001; Roy et al., 2002). Growth-
related bone remodelling also occurs in fin rays. In curved
soft fin rays (lepidotrichia, composed of two mineralized
hemisegments), bone resorption is assumed to facilitate
curvature (Montes et al., 1982). Spiny fin rays contain
a marrow-like cavity. This lumen increases in volume in
adult fish by resorption while new mineralized tissue is
deposited on the outside (Meunier, 2002), similar to the
enlargement of the diameter of bone shafts. Allometric
growth-related bone resorption was measured at the
endosteal surfaces of lateral line canals (Witten, 1997;
Witten & Villwock, 1997) (Fig. 2G,H). As discussed by
326
Tarby & Webb (2003), it was initially suggested that the cells
responsible for the enlargement of lateral line canals were
remnants of the placodal precursors of neuromasts. The
cells were observed to form a ‘‘connecting strand’’
responsible for the excavation of bone inside the lateral
line canal (Clapp, 1889; Merillees & Crossman, 1973).
Indeed a ‘‘connecting strand’’ of excavating cells can be
observed at endosteal bone surfaces but histochemical,
immunohistochemical and TEM studies have identified
these cells as mononucleated osteoclasts (Fig. 2E,F) (Witten,
1997; Witten & Villwock, 1997; Witten et al., 1999). Similar
‘‘connecting strands’’ of osteoclasts can be found inside the
growing neural arches of eels and zebrafish (Witten et al.,
2001). As described above, these cells perform non-lacunar
steady bone resorption and thus do not produce the typical
traces of bone resorption, such as Howship’s lacunae.
The vertebral bodies of many vertebrates (basal
osteichthyans sensu stricto and tetrapods) have a cartilaginous
anlage that is replaced by bone during ontogeny (Detwiler &
Holtzer, 1956; Hall, 1977, 2005; Arratia et al., 2001). This is
no longer the case in teleosts where the first discernible part
of a vertebral body is the mineralized notochordal sheath
(Arratia et al., 2001; Nordvik et al., 2005). Extension and
growth of vertebral bodies is then facilitated by peripheral
and terminal bone apposition (Ekanayake & Hall, 1987,
1988; Witten & Villwock, 1997; Nordvik et al., 2005)
(Fig. 1E). Moreover, and unlike other bone tubes, the
funnel-shaped halves of early vertebral bodies enlarge
initially without bone resorption, purely by terminal and
periosteal bone apposition (Witten & Villwock, 1997;
Nordvik et al., 2005). However, in later stages of Atlantic
salmon, bone remodelling and osteoclasts are regularly
observed at intravertebral spongiosa structures (Gil-Martens
et al., 2006). The neural and haemal arches require
resorption of cartilage as well as of bone during growth.
As the neural tube diameter increases, enlargement of the
neural arches follows the pattern of other bone tubes with
osteoclasts located at the endosteal surface (Fig. 2F) (Witten
et al., 2001; Roy et al., 2002).
(2) Replacement of skeletal tissues
Another primary role of skeletal resorption in teleosts is the
removal of cartilage by chondroclasts and its replacement
with another tissue such as bone, or adipose tissue. Early
chondroclasts in teleosts resemble early osteoclasts in being
often mononucleated and inconspicuous (Witten et al.,
2001). Like osteoclasts, chondroclasts can be identified
using TEM or by the demonstration of TRAP activity
(Fig. 1B). Similar to the typical situation in mammals, teleost
cartilage is resorbed once it is surrounded by a bone collar.
Often, and especially in species with small adult size (e.g.
zebrafish, medaka), removal of cartilage is not followed by
endochondral bone formation but arising bone marrow
spaces instead are filled with adipose tissue (Fig. 1A)
(Huysseune, 2000; Witten et al., 2001; Witten & Hall,
2002; Witten & Huysseune, 2007). As the removal of
cartilage starts rather late, cartilage inside some bone shafts
may persist being neither replaced by adipose tissue nor by
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
P. Eckhard Witten and Ann Huysseune
bone (Benjamin et al., 1992; Witten & Huysseune, 2007).
Where endochondral bone formation occurs (Haines, 1938)
it involves the mineralization and partial resorption of the
cartilage matrix and the presence of matrix remnants that
serve as a substratum for bone formation (Fig. 3D)
(Huysseune, 2000; Witten & Huysseune, 2007). Haines
(1934), based on observations of the scorpionfish Chelido-
nichthys capensis, and Huysseune (2000), based on observa-
tions of the cichlid Astatotilapia elegans, propose that after the
initiation of cartilage resorption, and prior to further
cartilage resorption, a thin seam of bone is deposited on
the resorption front. When the cartilage retreats further,
bone seams are left behind as bone trabeculae perpendi-
cular to the long axis of the bone. This process creates
a ladder-like construction of bone trabeculae inside the
bone tube.
One particular way in which cartilage can be remodelled
concerns the early fusion and the subdivision of the
cartilage anlagen of the teleost fin endoskeleton. Cartilage
subdivision plays a role in the differentiation of unpaired
(dorsal and anal) and paired fins. Parts of the shoulder girdle
and the radials of the pelvic and the pectoral fin also
develop by subdividing a single cartilage condensation
(Witten & Huysseune, 2007). For zebrafish a detailed
description is available of how a cartilaginous plate gives
rise to elements of the shoulder girdle and the pectoral fin.
The plate subdivides into the scapula, coracoid, mesocora-
coid, and four radials. Only distal radials are added as
separate cartilage condensations (Grandel & Schulte-
Merker, 1998). In the sea bream Sparus aurata, an advanced
teleost, the scapulocoracoid cartilage and the cartilaginous
fin plate (i.e. the precursor of the proximal radials) appear to
develop from one piece of cartilage (Faustino & Power,
1999). In other species, two cartilage anlagen exist: a
scapulocoracoid cartilage, which ossifies into the coracoid
and the scapula (which remain joined by cartilage), and a
cartilaginous blade that first cleaves centrally then dorsally
and ventrally, to form radials which also ossify (Potthoff &
Kelley, 1982; Potthoff, Kelley & Collins, 1988; Balart, 1995;
Mabee & Trendler, 1996; Cubbage & Mabee, 1996). The
way cartilage subdivides is intriguing but not well under-
stood. Crotwell & Mabee (2007) suggested that the pattern
of gene expression during the subdivision is largely
conserved between zebrafish and tetrapods. The pectoral
fin endoskeleton subdivision of zebrafish has been described
as ‘‘decomposition of cartilage matrix’’, but no apoptotic or
resorbing cells have been identified (Grandel & Schulte-
Merker, 1998). The process in teleosts appears to be similar
to the non-apoptotic process of digit separation in
amphibians but differs from amniotes, where massive cell
death occurs to transform the webbed regions of the
footplate into interdigital spaces (Cameron & Fallon, 1977;
Wood et al., 2000; Zuzarte-Luı́s & Hurle, 2002). In birds,
reptiles and mammals, macrophages are involved but their
role is believed to be restricted to the removal of apoptotic
cells. Resorption by macrophages is not considered a
primary mechanism of cartilage separation (Zuzarte-Luis &
Hurle, 2005). The absence of signs of apoptosis or
resorption in teleosts suggests that the separation of
cartilage elements may involve metaplasia: dedifferentiation
Skeletal remodelling in teleost fish
of cartilage cells and their redifferentiation into non-
cartilaginous connective tissue.
(3) Mineral metabolism
(a ) Calcium and phosphorus requirements
All vertebrates must maintain tightly regulated extra- and
intracellular calcium levels (Guerreiro et al., 2007) and in
amniotes the skeleton is highly integrated into this
regulation. Calcium is released from the skeleton through
bone resorption by osteoclasts or through osteocytic
osteolysis to compensate for low plasma calcium levels,
and calcium is deposited into the skeletal tissues to
compensate for high plasma calcium levels (Belanger,
1977; Haller & Zimny, 1978; Steinberg et al., 1981; Doty &
Nunez, 1985; Kwiecinski, 1985; Kwiecinski et al., 1987;
Alcobendas et al., 1991; Zhang et al., 2000; Tazawa et al.,
2004). The extent of involvement of the teleost skeleton in
calcium metabolism is a matter of debate. In mammals,
total plasma calcium levels range from 2.3 to 2.7 mmol l[1
(Veis, 1984). Marine and freshwater teleosts maintain total
plasma calcium levels within a similar narrow range:
typically, 2.5 mmol l[1 to 3.0 mmol l[1; marine species
appear to maintain slightly higher values than freshwater
species (Urist et al., 1972; Bone & Marshall, 1985).
Exceptionally low plasma calcium levels (1.5 – 2.0 mmol
l[1) occur in some basal freshwater bony fish (e.g. Acipenser
naccarii) although the primitive bony fish Amia calva
maintains a value of 3.0 mmol l[1 (Urist et al., 1972;
Fuentes et al., 2007). Regulation of calcium homeostasis is
under hormonal control and consequently several studies
have examined the role of calcium-regulating hormones in
bone resorption in teleosts (Lopez et al., 1980; Milet et al.,
1984; Taylor, 1985; Persson et al., 2000).
The participation of acellular bone of advanced teleosts
in calcium homeostasis is particularly interesting, as the
absence of osteocytes precludes mobilisation of calcium
from the skeleton by osteocytic osteolysis. Moss (1962)
compared fracture healing in teleosts with cellular and
acellular bone under calcium-deprived conditions. Only the
species with cellular bone (Carassius auratus, Cyprinidae)
could mineralize the fracture callus; the species with
acellular bone (Sarotherodon melanotheron, Cichlidae) formed
a fracture callus that did not mineralize. This and similar
findings did suggest that advanced teleosts with acellular
bone may not be capable of mobilising calcium from the
skeleton (Moss, 1962; Simmons, 1971; Urasa et al., 1984;
Taylor, 1985). Witten (1997) maintained juvenile cichlids
(Oreochromis niloticus) for two weeks in four different types of
media: (1) tap water supplemented with 3% sea salt, (2)
water supplemented with three times more magnesium than
calcium, (3) water without calcium and (4) regular tap water
(52 mg l[1 calcium). The experiment revealed growth-
related bone resorption but the mineral content of the water
had no influence on the number and shape of osteoclasts. A
more drastic approach to induce calcium-metabolism-
related skeletal resorption in this species was undertaken
by Takagi & Yamada (1991, 1992) who forced the fish to
regenerate half of their scales under extreme calcium-
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
327
deprivation. Under these conditions resorption of the
endoskeleton was observed, although it was conducted by
mononucleated osteoclasts. Although Takagi & Yamada
(1991, 1992) could induce skeletal resorption under very
extreme conditions, the experiment showed that advanced
teleosts do not utilise their endoskeleton as a calcium source
under physiological conditions. Indeed, teleosts have several
alternative sources of calcium, and resorption of the
endoskeleton may be used only as a last resort (Moss,
1962; Takagi & Yamada, 1991, 1992). Teleosts can obtain
calcium by intestinal absorption (the usual route for marine
teleosts) or through the gills (the usual route for freshwater
species). ATP-driven active calcium transport through the
gill epithelium functions even at low ambient calcium levels
and generally ensures sufficient calcium intake (Guerreiro
et al., 2002; Perry et al., 2003). Moreover, teleosts can resorb
their scales instead of resorbing elements of the endoskeleton.
Many studies show that in conditions of mineral shortage
the scales are preferentially resorbed (Van Someren, 1937;
Garrod & Newell, 1958; Mugiya & Watabe, 1977; Weiss &
Watabe, 1978; Carragher & Sumpter, 1991; Shearer, 1992;
Persson, Sundell & Björnsson, 1994; Persson et al., 1997,
1998, 1999, 2000; Persson, Takagi & Björnsson, 1995;
Skonberg et al., 1997; Rotllant et al., 2005).
As teleosts can obtain calcium from different sources,
calcium deficiency is uncommon in bony fish. Studies on
farmed fish show that phosphorus rather than calcium
deficiency triggers bone resorption (Roy et al., 2002; Lall &
Lewis-McCrea, 2007). Available phosphate in fresh water
and sea water is low and for proper mineralization of the
skeleton fish must rely on dietary phosphorus intake
(Halstead Tarlo, 1964; Simmons, 1971; Fenwick, 1974;
Lall & Bishop, 1977; Dacke, 1979; Vielma & Lall, 1998a,b;
Vielma et al., 1999; Avila et al., 2000; Lall, 2002; Guerreiro
et al., 2007). Consequently, a shortage of dietary phospho-
rus or starvation can cause insufficient bone mineraliza-
tion and can trigger bone resorption. Both processes may
contribute to deformities of the axial and head skeleton
(Roberts, Hardy & Sugiura, 2001; Yamada et al., 2002;
Lall, 2002; Roy et al., 2002; Roy & Lall, 2003). Persson
et al. (1998, 2000) linked scale resorption in salmon during
their upstream spawning run to the animals’ high demand
for calcium, suggesting that this could be linked to the
synthesis of calcium-rich egg yolk protein (vitellogenin).
This can only be a partial explanation, given that scale
resorption also occurs in starving upstream-running male
salmon that do not synthesise vitellogenin. In male salmon,
scale resorption is possibly triggered by the rapid growth of
some skull bones and by the development of a bony hook
at the tip of the lower jaw (an antler-like secondary sexual
character). Moreover, breeding salmon continue to replace
their teeth (Tchernavin, 1937; Vladykov, 1962; Witten,
Hall & Huysseune, 2005b; Huysseune, Hall & Witten,
2007). Male salmon that survive spawning resorb the tip of
the hook and remodel its basis into regular dentary bone
(Witten & Hall, 2002, 2003). Kacem et al. (1998) and
Kacem & Meunier (2000, 2003) linked osteoclastic
resorption, osteocytic osteolysis and halastatic loss of
minerals in pre- and post-spawning Atlantic salmon to
the animals’ calcium demand. While there is no doubt that
328
there is increased calcium demand in female Atlantic
salmon prior to spawning, males and females undergo
severe starvation during this period, and thus lack dietary
phosphorus. Therefore, and from what is known from
farmed salmon, starvation-related phosphorus deficiency
rather than calcium deficiency may trigger scale resorp-
tion in both sexes (Skonberg et al., 1997; Witten & Hall,
2003; Lall & Lewis-McCrea, 2007).
(b ) Hormones and mineral metabolism
Given that mineral metabolism in fish is under hormonal
control, and is linked to osmoregulation and dietary mineral
intake, disciplines such as fish endocrinology, fish physiol-
ogy, and fish nutrition are interested in fish skeletal
remodelling. General information about calciotropic hor-
mones, osmoregulation, and nutrition in teleosts can be
found in Urist et al. (1972), Flik & Verbost (1995), Jurss &
Bastrop (1995), Sasayama (1999), Perry et al. (2003) and
Lall & Lewis-McCrea (2007). Other studies focus on
particular hormones and vitamins that can influence teleost
bone metabolism: calcitonin (Lopez et al., 1976; Arlot-
Bonnemains & Fouchereau-Peron, 1984), stanniocalcin
(Verbost & Fenwick, 1995; Barlet et al., 1998; Amemiya,
Irwin & Youson, 2006), vitamin D (Graff et al., 2002),
thyroid hormone (Sæle et al., 2003; Okada, Tanaka &
Tagawa, 2003; Takagi et al., 1994; Sbaihi et al., 2007),
parathyroid hormone and parathyroid hormone-related
protein (Danks et al., 1993, 2003; Trivett et al., 1999;
Guerreiro et al., 2007), prolactin (Power, 2005), estrogen and
testosterone (Urasa et al., 1984; Persson et al., 2000;
Guerreiro et al., 2002), melatonin (Suzuki & Hattori,
2002; Fjelldal et al., 2004), leptin (Baker et al., 2000), and
growth hormone (Takagi et al., 1992). It has been
emphasised for calcitonin, estradiol-17b (E2) and vitamin D,
that the function of calcium-regulating factors in teleosts is
not fully understood and conflicting results have been
published (Wagner, Jaworski & Radman, 1997; Persson
et al., 1997; Lall & Lewis-McCrea, 2007). Different types of
bone (cellular versus acellular), different sites of calcium
exchange (gills versus endoskeleton), and divergent effects of
hormones on dermal and endoskeletal elements (see below)
complicate understanding of teleost calcium metabolism
(Lall & Lewis-McCrea, 2007). Below we briefly describe
how the main calciotropic hormones (calcitonin, estrogen,
PTH) and vitamin D can affect teleost bone resorption.
Salmon calcitonin is an established antiresorptive osteo-
porosis medication (Chesnut et al., 2008) and appears to
have a similar effect in fish. European eels treated with
calcitonin show reduced bone resorption and increased
bone formation (Lopez et al., 1976), and Johannsson et al.
(1998) found a dose-dependent reduction of bone resorp-
tion in calcitonin-treated rainbow trout.
The physiological role of vitamin D3 (1,25-dihydroxy
vitamin D) in fish remains to be clearly defined particularly
for marine species (Lall & Lewis-McCrea, 2007). Injection
of vitamin D3 into mature female European eels resulted in
stimulation of bone formation and a reduction of
osteoclastic resorption (Lopez et al., 1980). By contrast,
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
P. Eckhard Witten and Ann Huysseune
Rao & Raghuramulu (1999) observed that vitamin D3 or its
metabolites had no effect on calcium-phosphorus homeo-
stasis in the advanced teleost Oreochromis mossambicus
(Mozambique tilapia).
How estradiol-17b (E2) affects bone remodelling in
teleosts is difficult to understand. E2 aggravates scale
resorption in Atlantic salmon, goldfish, mummichog
(Fundulus heteroclitus), and rainbow trout (Mugiya &
Watabe, 1977; Carragher & Sumpter, 1991; Persson
et al., 1994, 2000) but has no in vivo effect on scale
resorption in sea bream (Guerreiro et al., 2002). Still,
osteoclast activity in sea bream scales was enhanced by E2
in vitro (Rotllant et al., 2005). Male goldfish react to E2 with
increased scale resorption and to calcitonin with
decreased scale resorption. These hormones, however,
have no significant effect on scale resorption in male
marine nibbler fish (Girella punctata) (Suzuki, Suzuki &
Kurokawa, 2000). The effect of E2 on osteoclasts in most
teleosts appears to be opposite to the effect of E2 in
mammals, where it is a potent inhibitor of bone resorption
(Gruber et al., 2001; Heino et al., 2002). From studies on
rainbow trout Persson et al. (1997) concluded that E2
increases scale resorption, but downregulates resorption of
the endoskeleton. This would explain the opposite effects
of E2 in teleosts and mammals and would support the
suggestion that teleosts preferentially use their scales
rather than their endoskeleton as the primary mineral
source (see Section V.3a).
In mammals PTH is a major hypercalcemic factor and
triggers osteoclast-mediated bone resorption (Hurwitz,
1996); in fish its role is still puzzling. The gill epithelium
of basal fish, viewed as the phylogenetic precursor of
tetrapod parathyroid glands, may have functions similar to
mammalian parathyroid glands (Sasayama, 1999; Loretz,
2008). Despite the detection of PTH molecules in teleosts
both with acellular and cellular bone (Danks et al., 2003;
Gensure et al., 2004) a hypercalcemic factor acting like
mammalian PTH has not been identified in fish (Guerreiro
et al., 2002; Lall & Lewis-McCrea, 2007). Studies on
lungfish (Lepidosiren paradoxa) and on the Northern Plains
killifish (Fundulus kansae) demonstrated that PTH has no
effect on fish bone resorption (Clark & Fleming, 1963;
Urist et al., 1972). Recently, Fleming, Sato & Goldsmith
(2005) published results from zebrafish larvae suggesting
that long-term exposure to extreme overdoses of PTH
reduces the mineralization of the skeleton whereas
intermittent short-term exposure to PTH has an anabolic
effect. The parathyroid hormone-related protein (PTHrP),
a hypercalcemic paracrine factor in mammals that binds
to the same receptor as PTH (Gensure, Gardella &
Juppner, 2005) is present in pufferfish and sea bream
(Power et al., 2000; Flanagan et al., 2000). A peptide that
corresponds to the amino-acid residues of the 1-38 region
of the N-terminal part of PTHrP has a hypercalcemic
effect in sea bream larvae and increases osteoclast activity
in vitro in this species (Rotllant et al., 2005; Guerreiro et al.,
2007). Fuentes et al. (2006) suggest that PTHrP functions
as a hypercalcemic factor controlling extracellular cal-
cium levels in teleosts and counteracts the action of
stanniocalcin.
Skeletal remodelling in teleost fish
(c ) Habitat and phylogeny
Given the enormous differences in mineral content between
freshwater and saltwater environments it is possible that
freshwater and marine teleosts use their skeletons differently
in the context of mineral metabolism. Several reports
suggest that the endoskeleton of freshwater species is
involved to a greater extent in mineral metabolism than is
the case for marine teleosts (Lopez, 1970; Witten et al., 2001;
Guerreiro et al., 2002; Sbaihi et al., 2007). Environment-
independent factors that determine the involvement of the
skeleton into the mineral metabolism are the type of bone
(cellular or acellular) and the animals’ phylogenetic history.
For freshwater teleosts it is important if a species belongs to
the group of primary or secondary freshwater fishes (Myers,
1938; Bermingham & Martin, 1998) (Fig. 4). For example, it
is known that the fluoride content of tooth enameloid in fish
is related to a pecies’ phylogenetic position but not to its
environment (Prostak, Seifert & Skobe, 1991; Suga, Taki &
Ogawa, 1993). Frequently studied fish that inhabit fresh
water such as zebrafish, carp, eel, and trout, represent less
advanced teleosts with an osteocyte-containing skeleton
(Group C in Fig. 4) whereas commonly used marine species
such as pufferfish, sea bream, halibut, or haddock are
advanced teleosts with a skeleton lacking osteocytes (Group
D in Fig. 4). Using the example of zebrafish (Group C in
Fig. 4) and medaka (Group E in Fig. 4), two small freshwater
species of similar appearance and widely used model
Fig. 4. Visualisation of the relationships among phylogeny,
environment, and bone type in teleosts. Basal osteichthyans
and basal teleosts (Groups A and B) have bone that contains
osteocytes, similar to mammalian bone. This type of bone was
preserved during a first wave of freshwater reinvasion by teleost
fish (‘‘primary freshwater fish’’; Group C, e.g. zebrafish).
During a long period in the marine environment acellular bone
evolved and is found in almost all advanced teleosts (Group D).
This derived character was maintained when advanced teleosts
reinvaded the fresh water (Group E; ‘‘secondary freshwater
fish’’, e.g. medaka). Consequently, teleosts that live in fresh
water (or in the marine environment) can have very different
bone types and accordingly different characteristics in respect
to bone resorption and bone remodelling. MY, million years.
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
329
organisms, it becomes apparent how different these species
are regarding their type of bone and their evolutionary
history. Zebrafish and medaka are at least 100 million years
apart and equally distant to each other as a kangooroo and
a human. The two species perform very differently when it
comes to bone resorption and bone remodelling, following
the patterns for teleosts with cellular bone (zebrafish) and
acellular bone (medaka) respectively (Ekanayake & Hall,
1987; Witten et al., 2001; Takano et al., 2005). Cichlids
provide another example of the influence of phylogenetic
history on bone remodelling and resorption. Cichlids
(Group E in Fig. 4) are advanced secondary freshwater fish
with acellular bone and are closely related to marine teleost
families such as labrids and pomacentrids (Group D in
Fig. 4). Although cichlids inhabit fresh water the endo-
skeleton is not used in mineral metabolism under
physiological conditions (Moss, 1962; Takagi & Yamada,
1991, 1992; Witten, 1997), as in their marine relatives
(Bernardi & Bucciarelli, 1999). The involvement of bone
remodelling in mineral metabolism is to a large extent
determined by the animals’ evolutionary heritage and not
by its environment.
(4) Mechanical adaptation
After Wolff (1892) published the ‘‘law of bone trans-
formation’’ it became acknowledged that the vertebrate
skeleton displays a high degree of phenotypic plasticity,
adapting its structure to changing mechanical load (see also
Bertram & Swartz, 1991; Bertram et al., 1997; Kostenuik
et al., 1999; Currey, 2003; de Ricqlès, Castanet &
Francillon-Vieillot, 2004). The teleost skeleton can display
a considerable degree of phenotypic plasticity. It adapts to
changing external forces, and the degree of morphological
plasticity in teleosts is assumed to be large and widespread
throughout many branches of the group (Greenwood, 1965;
Meyer, 1987b). Experiments have shown that the morphol-
ogy and structure of jaw bones can be altered in response to
feeding conditions (Meyer, 1987a,b; Huysseune, Sire &
Meunier, 1994; Hegrenes, 2001). Meyer (1987a, b) showed
that the type of food can alter the external shape of the
entire head in juvenile central American cichlids (Cichlasoma
managuense). Huysseune et al. (1994) demonstrated adaptive
remodelling of the pharyngeal jaw bones in response to
increased mechanical load in the cichlid Astatoreochromis
alluaudi: individuals fed with snails developed more and
stronger bone trabeculae in their pharyngeal jaws than
individuals fed a soft diet (Fig. 5A,B). The presence of
vertebral deformities in farmed teleosts also illustrates that
the internal and external shape of skeletal elements can be
changed in response to mechanical load: development of
heterotopic cartilage or heterotopic chondroid bone is
thought to be the result of excess mechanical load (Kihara
et al., 2002; Kranenbarg et al., 2005; Witten et al., 2005a)
(Fig. 5C). While a plastic response of teleost skeletal
structures to changing load regimes is expected, it is
interesting that the acellular bone of advanced teleosts also
responds to alterations in mechanical load (Meyer, 1987a,b;
Huysseune et al., 1994; Hegrenes, 2001; Kranenbarg et al.,
330 P. Eckhard Witten and Ann Huysseune

2005). In mammals, osteocytes and their meshwork of
interconnected cell processes are believed to detect strain
and mechanical load and consequently are viewed as
central components guiding bone remodelling in response
to external forces (Burger et al., 1995, 2003; Bonewald,
2004; Franz-Odendaal et al., 2006; Taylor et al., 2007). As in
mammals, osteocytes in teleosts can form an extended
meshwork via interconnected cell processes (Meunier &
Huysseune, 1992; Meunier 2002) and presumably can
function in the same way (Huysseune, 2000). However, the
number and density of osteocytes as well as the number and
length of their processes can vary to a considerable degree
according to species and location within the bone (Stephan,
1900; Blanc, 1953; Moss, 1961a; Meunier, 1983). We do not
know how osteocyte density or interconnectivity affect the
response to mechanical load. The fact that teleosts with
acellular bone also remodel their skeleton in response to
altered mechanical load strongly suggests the existence of
alternative load detection mechanisms. Osteoblasts and
bone lining cells are the obvious candidates: Vatsa et al.
(2006) showed that mammalian osteoblastic and osteocytic
cells in vitro are sensitive to mechanical load. Likewise,
mammalian odontoblasts are considered to have sensory
capacity (Magloire et al., 2004; Allard et al., 2006). The
possession of acellular bone could make advanced teleosts
such as medaka important model species to study non-
osteocyte-mediated mechanisms of load detection. At the
same time, because of the absence of osteocytes, teleosts like
medaka may have limited use as models for human bone
diseases, in contrast to recent suggestions (Wagner et al.,
2003; Nemoto et al., 2007). Zebrafish with mammalian-like
osteocyte-containing bone may indeed be a better model.
(5) Fracture healing, regeneration and
disease-related remodelling
Healing of skeletal injuries is another function of skeletal
remodelling. Moss (1962) convincingly demonstrated that
teleosts respond to jaw fractures with the formation of
a typical repair callus that can be subsequently mineralized
and remodelled. Although jaw fractures may not occur
often under natural conditions, injuries to the fins are
common and require repair and/or regeneration. The
capacity to regenerate fins is restricted to the elements of
the dermal skeleton, the fin rays (lepidotrichia); the fin
endoskeleton cannot regenerate. As shown by Misof &

Fig. 5. (A) Sagittal section through the lower pharyngeal jaw of the cichlid Astatoreochromis alluaudi fed a soft food diet. Bone
trabeculae in the medullary cavity are sparse (black asterisk). Note that tooth replacement in advanced teleosts with intramedullary
tooth development depends on bone resorption (black arrowhead). Microradiograph from ground section: scale bar ¼ 500 mm. (B)
Sagittal section through the lower pharyngeal jaw of the cichlid A. alluaudi fed on molluscs. These are crushed using the pharyngeal
teeth. Note the presence of abundant bone trabeculae (black asterisk) and dense cortical bone structures. Microradiograph from
ground section: scale bar ¼ 500 mm. (C) Development of heterotopic cartilage (black asterisk) to replace the notochordal tissues at
the dorsal growth zone of two opposing vertebral body end plates in Atlantic salmon (Salmo salar). This pathology is characterised by
osteogenic tissue (white arrowhead) that blends into chondrogenic tissue (black arrowhead). It typifies deformed and fusing vertebral
bodies, likely caused by insufficient mineralization and/or excess mechanical load. In fusing vertebral bodies the cartilage is being
remodelled into trabecular bone. See Fig. 1F for the non-pathological situation, in which cartilage is absent. Masson’s Trichrome
staining: scale bar ¼ 50 mm. (D) Vertebral fusion and complete remodelling of three fused vertebral bodies into one, almost
regularly shaped, element. The presence of three sets of neural and haemal arches (white arrowheads) indicates the number of
vertebral bodies involved in the fusion. Developmental studies have shown that this type of fusion is the endpoint of a pathology that
begins with the development of heterotopic cartilage as in C. X-ray: scale bar ¼ 5 mm.

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
Skeletal remodelling in teleost fish
Wagner (1992) for the advanced marine teleost Salaria pavo
(Blenniidae) regeneration of fin rays involves resorption of
damaged skeletal parts by osteoclasts. Resorption is
probably an integral part of fin regeneration in all teleosts,
although this process is usually not mentioned in the fin
regeneration literature (see Akimenko & Smith, 2007, and
references therein). An exception to the rule that the
endoskeletal parts of a fin do not regenerate is provided by
the glass knifefish (Eigenmannia virescens, Gymnotidae)
(Kirschbaum & Meunier, 1981). Fish of this family have
lost their caudal fin and swim using the tail, i.e. the caudal
part of the spine (Greenwood et al., 1966). If the tail is lost or
amputated, the knifefish regenerates a cartilaginous rod that
undergoes perichondral ossification. Subsequently the
cartilage is resorbed by osteoclasts/chondroclasts. True
vertebral bodies and the notochord are not regenerated.
The ossified rod, however, appears to function like the
original segmented vertebral column. The animals’ fin rays
regenerate completely, as is usually the case in teleosts
(Kirschbaum & Meunier, 1981).
Remodelling is involved not only in repair and
regeneration after trauma, it is also involved in diseases
that affect the skeleton. Skeletal malformations, especially
malformations of the vertebral column, are frequently
observed under farming conditions but have also been
reported from wild teleosts (Wunder, 1976; Watermann &
Kranz, 1992; Kvellestad et al., 2000; Kihara et al., 2002;
Hattori et al., 2003; Witten et al., 2005a; Turnbull, 2006).
Lordotic, scoliotic and kyphotic malformations and the
often recorded fusion of vertebral bodies must involve bone
resorption and bone remodelling, as a primary pathology or
in response to altered mechanical load (Kranenbarg et al.,
2005; Witten et al., 2006). There are few reports of bone
resorption connected to bone deformities. Roy et al. (2002)
observed increased resorption at the vertebral column of
haddock in association with spine malformations caused by
phosphorus deficiency and Roberts et al. (2001) found
increased bone and cartilage resorption in farmed Atlantic
salmon suffering from phosphorus deficiency. Hyperdense
vertebral bodies constitute another pathology that involves
remodelling. Increased radio-density that is due to hyper-
mineralization can be caused by heterotopic cartilage
occupying bone marrow spaces (Helland et al., 2006). The
observed resorption of such cartilage suggests that it is not
a permanent structure. An extreme case of remodelling is
the complete pathological fusion of vertebral bodies in
Atlantic salmon (Witten et al., 2006). A similar pathology
has also been observed in advanced teleosts with acellular
bone (Sawada et al., 2006a, b). In Atlantic salmon early
stages of diseased vertebral bodies resemble compressed
vertebral bodies. Both pathologies, compression and fusion,
involve the metaplastic transformation of bone-forming
cells in the vertebral growth zone into cells that produce
cartilaginous tissue instead of bone. Later this cartilage is
mineralized and remodelled into bone (Witten et al., 2005a,
2006). Remodelling of fusing vertebral bodies can result in
two or three units fusing into one vertebral body that
appears regularly shaped both externally and internally
(Fig. 5D). Remnants of notochord tissue in the intervertebral
space are also remodelled. Despite the complete fusion of
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
331
vertebral bodies, no fusion or remodelling of haemal and
neural arches occurs, suggesting that vertebral bodies and
arches, that constitute developmental modules early in
ontogeny (Holtzer, 1951; Strudel, 1953a,b; Detwiler &
Holtzer, 1956; Hall, 1977, 2005) remain as such in adult
salmon. Finally, infectious diseases can also trigger skeletal
resorption in teleosts. The parasite Myxobolus cerebralis causes
skeletal deformities and lysis of cranial cartilage (Halliday,
1973; Garden, 1992; Kelley et al., 2004). Lysis of cranial
cartilage and increased skeletal resorption resulting from
bacterial infection has been reported for salmonids infected
with Flexibacter psychrophilus (Ostland et al., 1997).
(6) Tooth formation and replacement
Teleosts, like all other non-mammalian tooth-bearing
vertebrates, display continuous and lifelong tooth formation
and replacement, accompanied by bone and tooth
resorption (Peyer, 1968; Huysseune, 1983; Huysseune &
Sire, 1992a; Witten et al., 1999, 2005b; Huysseune, 2000).
The plesiomorphic condition for osteichthyans is the
presence of teeth on the mandibular, hyoid and all five
more posterior branchial arches, as well as on the bones
that constitute the roof of the pharyngeal cavity. Thus,
many basal teleosts retain large numbers of teeth on
various skeletal elements within the buccopharyngeal cavity
(Huysseune & Sire, 1998). During teleost evolution teeth
became restricted to the mandibular arch and to the
rearmost branchial arches. In some advanced, pharyngo-
gnath, teleost families, elements of the second to fifth
branchial arches evolved into a strong second set of jaws,
the pharyngeal jaws, bearing pharyngeal teeth (Vandewalle
et al., 1994). In some species the pharyngeal teeth are
molariform and are used for grinding and crushing, similar
to the function of premolar and molar teeth in mammals
(Owen, 1845, 1854). Any tooth that is lost in a particular
position is usually replaced by a new tooth throughout the
animals’ life. In addition, growing individuals establish
new tooth positions (Huysseune & Witten, 2006, 2008;
Huysseune et al., 2007). Tooth replacement and the
establishment of new tooth positions require tooth resorp-
tion and resorption and remodelling of tooth-bearing bones.
Teleost teeth develop extra- or intramedullarly (Trapani,
2001). Teeth of the first tooth generation in all teleosts and
replacement teeth in less advanced teleosts develop on top
of tooth-bearing bones (i.e. in an extramedullary position).
In advanced teleosts replacement teeth develop inside the
jaw bones (i.e. in an intramedullary position), to a certain
extent comparable to the situation in mammals. Resorption
is an essential element of both modes of tooth formation: in
both modes the mineralized basis must first be resorbed in
order to disconnect the tooth from the bone surface
(Fig. 6A). Subsequently, osteoclasts also resorb part of the
jaw bone itself. It is thought that this resorption prepares
the attachment site for the new tooth (Van der heyden,
Huysseune & Sire, 2000). Moreover, there is evidence that
some teleosts and other non-amniotes resorb old teeth
completely as an alternative to shedding (Fig. 6B) (Shaw,
1986; Domon et al., 2004, 2006; Witten et al., 2005a;
332 P. Eckhard Witten and Ann Huysseune

Huysseune et al., 2007) perhaps to retain the minerals. In
Atlantic salmon, complete tooth resorption occurs in
periods of severe mineral deprivation (spawning migration),
during which animals also resorb their scales (Shearer,
1992; Persson et al., 1998). Partial retention of teeth has
been observed in the advanced teleost Pomatomus saltatrix
where the dentine of basal parts of old teeth is incorporated
into new jaw bone tissue (Bemis, Giuliano & McGuire,
2005).
In advanced teleosts with teeth that develop deep inside
the jaw bones (intramedullary tooth formation), bone must
be removed to accommodate the growing tooth, to allow its
outward movement through the jaw bone and to enable its
ankylosis at the top of the jaw bone (Witten et al., 1999;
Huysseune & Thesleff, 2004; Bemis et al., 2005). Osteoclasts
can be found on all endosteal bone surfaces that surround
these growing teeth (Fig. 6C) (Witten et al., 1999) (see also
Section IV.1 for osteoclast stimulation by tooth-related
tissues).
Replacement teeth are usually initiated at the tip of an
epithelial strand (successional dental lamina) that buds off
from the outer dental epithelium of the predecessor tooth
(Huysseune & Thesleff, 2004). In species with intra-
medullary tooth development the dental lamina must
penetrate the jaw bone to reach the medullary cavity and
to initiate the new tooth inside the bone. The dental
lamina penetrates the bone through a small hole
(replacement pore) located adjacent to each predecessor
tooth, as described by Bemis et al. (2005) for the advanced
teleost Pomatomus saltatrix. Subsequently the diameter of the
replacement pore decreases although later the replace-
ment tooth erupts at this site by re-enlargement of the
pore. After eruption from the medullary cavity the tooth
progresses by remodelling of the surrounding bone into the
position of the predecessor tooth. The developmental
process described by Bemis et al. (2005) for P. saltatrix
suggests that replacement pores are lost during the process
of tooth eruption and have to be re-established for each
replacement tooth. The way the replacement pore is
created has not been investigated but perhaps osteoclasts
‘‘drill’’ a canal in the bone similar to the creation of
a passage for a blood vessel through a bone (Junqueira,
Carneiro & Kelley, 1989; Meunier, 2002). In conclusion,
tooth development in advanced teleosts requires bone
resorption for: (1) the downgrowth of the dental lamina
into the bone cavity, (2) to create space for the growing and
outward moving tooth, (3) to prepare the surface to allow
the erupting tooth to attach, (4) to remove all or part of the

Fig. 6. (A) Start of resorption (black arrowhead) of a functional tooth in a zebrafish (Danio rerio) at a time when cells in the pulp
cavity are still active (black asterisk). Epon embedding, Toluidine Blue staining: scale bar ¼ 20 mm. (B) Complete resorption of the
remains of a tooth by multinucleated osteoclasts in Atlantic salmon (Salmo salar). Osteoclasts, black arrowheads; atubular dentine,
black asterisk. Our studies on tooth replacement in Atlantic salmon suggest that teeth in this species are not shed but resorbed.
Paraffin embedding, Masson’s Trichrome staining: scale bar ¼ 25 mm. (C) Intramedullary tooth formation in the cichlid Oreochromis
niloticus, a situation typical for advanced teleosts (see also Fig. 5A,B). Developing teeth and tooth anlagen (white asterisks) are
surrounded by many small tartrate-resistant acid phosphatase (TRAP) positive osteoclasts (red) as the resorption of the surrounding
bone (black asterisks) is required for the growth and the eruption of the teeth. The shape, size, and number of osteoclasts differs
considerably from the situation in salmon (cf. Fig. 6B). Glycol methacrylate embedding, TRAP staining counterstained with
Haematoxylin: scale bar ¼ 25 mm. (D) Tooth of a shark (Scyliorhinus canicula) prior to shedding. The position of the tooth (black
asterisk) is shown on the insert (black arrowhead), the dark central area represents the mineralized mandibular cartilage. Unlike in
bony fish, neither signs of tooth resorption nor signs of resorption of the bony basal plate of the tooth are observed. Sections with
Toluidine Blue (tooth) and von Kossa (insert). Non-decalcified methyl methacrylate embedding: scale bar ¼ 200 mm.

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
Skeletal remodelling in teleost fish
old tooth and (5) to move the erupting tooth into the
position of its predecessor. The latter process also occurs in
less advanced teleost species such as Atlantic salmon where
apparently it does not require bone resorption. The teeth
develop on top of the jaws and movement into the proper
position takes place before attachment to the underlying
jaw bone (Huysseune et al., 2007). The mechanism behind
extramedullary tooth movements, most prominently
observed during tooth replacement in sharks, has not
been elucidated (Peyer, 1968).
The nature of tooth-resorbing cells is still a matter of
discussion. Often bone-resorbing cells (osteoclasts) and
tooth-resorbing cells (odontoclasts) are distinguished based
on the substrate the cells resorb and on their location,
rather than on their properties. In humans (and mammals
in general), clastic cells attacking dentinal hard tissues
(cementum, dentine and enamel) and located inside the
pulp cavity are referred to as odontoclasts or dentinoclasts
(Sasaki et al., 1989; Matsuda, 1992; Sahara et al., 1992,
1994, 1996; Domon et al., 1994). Despite their different
names, the structure and ultrastructure of odontoclasts and
osteoclasts appear to be identical (Van der heyden et al.,
2000). As in mammals, odontoclasts in teleosts can be
multinucleated, can show a ruffled border and can create
deep resorption lacunae. Both mammals and teleosts have
high numbers of mononucleated odontoclasts (Domon et al.,
1994; Witten et al., 1999; Nemoto et al., 2007). Due to
continuous tooth replacement, tooth-bearing bones of
teleosts can contain high numbers of osteoclasts; they
represent, perhaps, the location with the highest osteoclast
number (Witten, 1997).
(7) Remodelling as a lifelong process
Given that teleosts grow throughout their life, remodelling
continues well into adulthood (Smith-Vaniz et al., 1995;
Persson et al., 1998, 1999; Kacem et al., 1998; Meunier,
2002; Reznick, Ghalambor & Nunney, 2002; Witten &
Hall, 2002, 2003). Due to sexual maturation, periods of
starvation or temporarily unfavourable environmental
conditions, the growth of adult fish is often intermittent
(Geffen et al., 2002). Such discontinuous growth can be
identified using growth lines in particular skeletal elements,
mostly scales and fin rays (Ferreira, Beamish & Youson,
1999; Ihde & Chittenden, 2002; Meunier, 2002). Apart
from signs of growth and growth arrest, the bone matrix of
the fin skeleton can show signs of resorption (Castanet et al.,
1993). Extensive periodic resorption also characterises the
scales of repetitive spawning Atlantic salmon. The contin-
uous remodelling of the skeleton may cause the disappear-
ance of growth marks and thus hamper age determination
(Shearer, 1992; Meunier, 2002).
Extensive and disproportionate enlargement of particular
skeletal elements is another phenomenon of late skeleto-
genesis. This can be due to the development of secondary
sexual characters, as in Atlantic and Pacific salmon
(Tchernavin, 1938; Jones, 1959; Vladykov, 1962). Witten &
Hall (2002, 2003) have shown that the kype – a secondary
sexual character consisting of a hook on the lower jaw of
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
333
male salmon – undergoes remodelling and resorption as the
structure grows and regresses.
Another example of bone enlargement in adult fish
involving resorption is hyperostotic bone (Smith-Vaniz et al.,
1995; Meunier, Bearez & Francillon-Vieillot, 1999;
Meunier et al., 2002). Hyperostosis is a localized bone
expansion through extensive membrane bone formation.
The predictability and site-specificity of hyperostosis in
a number of marine teleosts led Smith-Vaniz et al. (1995) to
propose that hyperostosis is a regular late event in the
animals’ life and not a pathological condition (osteoma), as
was suggested previously (Schlumberger & Lucke, 1948).
Equipped with the attributes of cellular bone, hyperostotic
outgrowths represent foci of extensive bone remodelling,
even in marine teleosts with an otherwise quiescent acellular
skeleton (Smith-Vaniz et al., 1995; Meunier, 2002).
Studies on aging fish suggest that resorption also appears
to be a part of normal senescence: Gerhard et al. (2002)
observed erosive changes at vertebral bodies in zebrafish at
the end of the animals’ life span.
VI. THE EVOLUTION OF SKELETAL
REMODELLING
During the ontogeny of teleost fish and other extant
vertebrates (except Chondrichthyes) osteoclasts and odon-
toclasts appear together with the first mineralized struc-
tures, whether teeth or bone (Huysseune, Van der heyden &
Sire, 1998; Witten et al., 2001). This cooccurrence, together
with the many functions of skeletal resorption raises the
possibility that skeletal resorption may have evolved
together with the first calcified structures of the dermal
skeleton. Unfortunately the fossil record does not provide
clear answers: evidence for bone resorption in early
gnathostomes is scarce (Bystrow, 1959; Donoghue &
Sansom, 2002) but resorption occasionally has been
identified in heterostracans and osteostracans (agnathans)
(Denison, 1952; Halstead Tarlo, 1964). Perhaps skeletal
resorption was not required in early vertebrates where the
dermal skeleton was composed of small (microsquamose)
finite-growing odontodes. These small odontodes were
completely shed (like teeth) and replaced by new elements,
but not resorbed (Reif, 1982). Extant chondrichthyans show
such type of squamation with placoid scales resembling the
dermal skeletal elements of heterostracans (Reif, 2006).
Interestingly extant sharks apparently do not have skeletal
resorbing cells or skeletal resorbing capacity (Fig. 6D).
Whether this is a conserved or derived trait is unclear.
Sharks do not even repair damaged cartilage, a process that
would involve at least the action of chondroclast-like cells.
The possible existence of chondroclasts in sharks is
discussed by Officer, Clement & Rowler (1995). Clement,
Officer & Dykes (1992) emphasised the absence of any
resorption in shark vertebral centra during their lifespan.
Glowacki et al. (1986) demonstrated that the leopard shark
(Triakis semifasciata) does not resorb implanted bone particles
and Ashhurst (2004) showed that the dogfish (Scyliorhinus
spp.) does not repair cartilage. Glowacki et al. (1986),
334
Ashhurst (2004) and Clement et al. (1992) found no evidence
of osteoclasts or chondroclasts. Instead, connective tissue
developed around implanted bone particles and damaged
cartilage. While studying tooth development in the dogfish
(Scyliorhinus canicula), Huysseune & Witten (2008) examined
the shedding of teeth (unpublished data). Like other
investigators (Peyer, 1968; Reif, 1982) they did not observe
any signs of macrophages or osteoclasts that could resorb
teeth or the acellular bone of the tooth basal plate prior to
shedding (Fig. 6D).
The complete shedding of dermal skeletal elements during
growth bears some resemblance with moulting, the process
used by invertebrates to enlarge their exoskeleton. By
contrast, for larger and more elaborate vertebrate endoskel-
etal and dermal skeletal elements (with the exception of teeth)
shedding is not an option. A switch from microsquamose to
macrosquameric dermal plates, the formation of a secondary
odontode layer, teeth that become ankylosed to the
mineralized jaws, or the growth of a solid head capsule
would require bone resorption. Also the enlargement of bone
tubes should always require bone resorption (Witten &
Villwock, 1997; Witten et al., 2001). However some fossil
agnathans (galeaspids and osteostracans) apparently avoided
growth-related bone resorption by developing a head capsule
only late in adulthood (Reif, 1982).
Irrespective of skeletal growth and the replacement
modes of dermal skeletal elements, resorption would have
been an inherent characteristic of the mineralized skeleton
if it has a role in mineral homeostasis. Carroll (1988)
considered several hypotheses about the function of the
early dermal skeleton: protective armour; insulation for
electroreceptors; calcium storage; and as a phosphorus
reserve. Halstead Tarlo (1964) argued that the observed
variability of dermal skeletal plates in Palaeozoic vertebrates
could indicate that the dermal skeleton functioned as
a phosphorus reservoir, since phosphorus is a limiting factor
in many ecosystems. As discussed above, the mineral
metabolism of extant teleosts would tend to support this.
Unlike calcium, that can be obtained from ambient water
via the gills, food is the main source of phosphorus in bony
fish (Simmons, 1971; Fenwick, 1974; Dacke, 1979). If
ancestral vertebrates had a similar physiology to present-
day bony fish, periods of starvation would represent periods
of phosphorus shortage. Suggestions that the early skeleton
functioned as a calcium reservoir cannot be excluded but
there is little evidence that extant teleosts suffer from
calcium deficiency under physiological conditions (Weiss &
Watabe, 1978, 1979; Witten, 1997; Roy et al., 2002). There
is, however, little evidence of bone resorption in some early
gnathostomes (Bystrow, 1959; Donoghue & Sansom, 2002)
and the absence of resorption of calcified structures in
extant chondrichthyans argues against a metabolic function
of the skeleton, unless extant chondrichthyans have
secondarily lost this capacity. Considering the trend towards
loss of osteocytes and exclusion of the skeleton from
metabolism in advanced teleosts (Fig. 4) it is not impossible
that a similar evolutionary process could have occurred in
extant chondrichthyans.
Different mechanisms of bone resorption exist and not all
resorbing cells derive from the macrophage lineage. Still, it
Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
P. Eckhard Witten and Ann Huysseune
is possible that the absence of typical multinucleated
osteoclasts in extant chondrichthyans is related to the
absence of key factors known to regulate macrophage
development and osteoclast activation in mammals (see
Section 3.1a), factors that are also present in teleosts (see
Section IV.1). However, chondrichthyans are known to
express the transcription factor PU.1 and to possess
monocytes, macrophages, an adaptive immune system,
and bone marrow-like haematopoietic tissue (Mattisson &
Fänge, 1982; Wolke, 1992, Anderson et al., 2001). Even
extant agnathans have monocytes and macrophages that
are morphologically similar to gnathostome cells and their
haematopoietic tissue is very similar to ‘bone marrow’ in
amniotes (Amemiya, Saha & Zapata, 2007). If limited or no
resorption capacity in extant chondrichthyans is a conserved
character, then development of the capacity to resorb the
skeleton could be linked to the evolution of particular
extracellular skeletal matrix molecules (Fig. 7). While the
basic skeletal tissue types and cells evolved early (Hall &
Witten, 2007) some skeletal extracellular matrix proteins
arose later, such as collagen II, SPARC (secreted protein,
acidic, cysteine-rich ¼ osteonectin) and osteocalcin (Bone
Gla Protein, BGP). Lamprey and hagfish cartilages lack
collagen type II and have instead different classes of fibrous
proteins (lamprins, myxin) (Cole & Hall, 2004; Hall &
Witten, 2007). Secretory calcium-binding phosphoproteins
(SCPP) and osteocalcin 1 arose only after the divergence of
cartilaginous and bony fish (Kawasaki, Suzuki & Weiss,
2004; Kawasaki & Weiss, 2006; Sire, Delgado & Girondot,
2006; Laize et al. 2006a,b) (Fig. 7) and both proteins play a
role in bone resorption. The Arg-Gly-Asp motif of SCPP
binds to osteoblasts and to osteoclasts via the integrin sur-
face receptor, which in turn evokes intracellular signals that
also regulate resorption (Kawasaki et al., 2004). Osteocalcin
recruits osteoclasts and osteoclast precursors to the bone
surface for resorption (Chenu et al., 1994). Several factors
are known to stimulate osteoclasts, including the presence of
non-collagenic matrix proteins, the presence of osteocytes,
and the presence of PTH. Haematopoietic tissue located in
the bone marrow is, however, not required, given that giant
osteoclasts and mononucleated osteoclasts occur in teleosts
despite the fact that they lack haematopoietic tissue in the
bone marrow.
Another question concerns the nature of early ancestral
osteoclastic cells. Mammalian and avian osteoclasts are
widely believed to possess general features of skeletal
resorbing cells: multinuclearity, a deeply folded apical cell
membrane (ruffled border), and the creation of resorption
lacunae (Chambers, 1985; Parfitt, 1988). However, skeletal
resorbing cells of extant bony fish must not express these
features and even studies on human bone reveal a majority
(61–81%) of mononucleated active osteoclasts (Evans,
Dunstan & Baylink, 1979; Chambers, 1985; Ballanti et al.,
1997). Parfitt (1988) concludes that in humans multinucle-
ated osteoclasts cause deep resorption lacunae while
mononucleated osteoclasts perform smooth bone resorp-
tion. In teleosts and in mammals resorption can be ac-
complished by mononucleated cells (Weiss & Watabe, 1978,
1979; Ekanayake & Hall, 1987; Witten, 1997; Witten &
Villwock, 1997) suggesting that these are candidates for a
Skeletal remodelling in teleost fish 335

Fig. 7. Evolution of the main skeletal tissue types and extracellular matrix components superimposed on a phylogenetic tree of
major vertebrate groups (modified after Romer, 1970). Early vertebrates (Agnatha) possessed bone, dentine and enamel/enameloid
in their dermal skeleton but no collagen type I and type II-based cartilage. Non-collagenous matrix proteins like osteocalcin 1 (OC 1)
and secretory calcium-binding phosphoproteins (SCPP) evolved only in the osteichthyan lineage. Acellular bone was already
present in some agnathans and is present in the basal plate of shark placoid scales and teeth. The acellular bone of teleosts is
however a derived feature as their ancestors (Osteichthyes and Actinopterygii) had cellular bone. The cellular bone of mammals is
a primitive (conserved) character. MGP, matrix gla protein; OC1, OC2, OC3, osteocalcin isoforms 1, 2, 3.

primitive type of bone resorbing cells. Interestingly, func-
tional osteoclasts without a ruffled border and that do not
form Howship’s lacunae have recently been described by
Kemp (2003) in the extant Australian lungfish Neoceratodus
forsteri. We observed similar mononucleated osteoclasts
together with multinucleated odontoclasts in the grey bichir
(Polypterus senegalus) (P.E. Witten & A. Huysseune, unpub-
lished data). Several arguments support the idea that
mononucleated cells may represent a primitive type of
bone resorbing cell: (a) during ontogeny multinucleated cells
arise through the fusion of mononucleated cells (Parfitt,
1988). (b) During teleost development, resorbing mono-
nucleated cells precede the appearance of multinucleated
cells (Sire et al., 1990; Witten et al., 2001). (c) In zebrafish,
mononucleated osteoclasts occur together with the first
traces of mineralized bone and teeth (Huysseune et al.,
1998; Witten et al., 2001), and are replaced only later by
multinucleated osteoclasts. (d ) Mononucleated cells are
present in basal extant osteichthyans (sarcopterygians and
actinopterygians). (e) Mononucleated osteoclasts utilise the
same enzymes for resorption as multinucleated osteoclasts
(Witten et al., 1999, 2000). (f ) Mononucleated cells resorb
scales in salmonids, skeletal elements that belong to the
phylogenetically oldest part of the animals’ mineralized
skeleton. If early bone resorbing cells were mononucleated,
responsible for a smooth type of resorption, then a lack of
recognisable multinucleated bone resorbing cells or their
traces (Howship’s lacunae) would be expected when exam-
ining primitive fossilised bone structures. In fossils, osteo-
clastic resorption is identified based on the presence of the
traces of typical multinucleated cells: resorption without
multinucleated osteoclasts, and therefore no resorption
lacunae, may not be detected (Witten et al., 2004).
VII. CONCLUSIONS
(1) The study of teleost fish reveals that an aquatic life
requires skeletal remodelling for the same reasons as in
terrestrial vertebrates: growth, replacement of skeletal tissues,
mechanical adaptation, tooth eruption and skeletal repair.
(2) Important differences exist, nevertheless, between fish
and tetrapods regarding remodelling and its control. Bone
resorption in the majority of teleost species is carried out by
small mononucleated osteoclasts instead of multinucleated
giant cells. The prevalence of mononucleated over multi-
nucleated osteoclasts appears to coincide with the evolu-
tionary disappearance of osteocytes – a derived character
within the teleost lineage. Other peculiarities of teleost bone
remodelling likely represent conserved characters as
adaptations to an aquatic life. Examples are a phosphorus-
rather than calcium-driven bone metabolism, a lack of
haematopoietic bone marrow tissue and use of the
postcranial dermal skeletal elements (scales) as a mineral
reservoir.

Biological Reviews 84 (2009) 315–346 Ó 2009 The Authors Journal compilation Ó 2009 Cambridge Philosophical Society
336
(3) A number of characteristics peculiar to skeletal
remodelling in teleost fish clearly challenge our understand-
ing of mammalian bone remodelling. The widespread
occurrence of small mononucleated osteoclasts encourages
us to reconsider the importance of mononucleated bone
resorbing cells in humans. The fact that the majority of
teleost fish regulate skeletal remodelling without an
osteocytic network and without haematopoietic bone
marrow tissue could provide a model for the study of
alternative pathways of osteoclast activation. Understand-
ing how a bone functions without osteocytes will allow
progress in our understanding of osteocyte function.
(4) Biomedical research increasingly uses species such as
zebrafish and medaka as models for studying skeletal
diseases and regeneration. Aquaculture-related research
focuses on species such as salmon or sea bream in order to
understand the cause of frequent skeletal malformations. In
all cases, skeletal remodelling is part of the observed
processes. Understanding the subdivision within teleosts
between basal species with cellular bone and advanced
species with acellular bone and their particular character-
istics regarding skeletal remodelling, is therefore of great
importance. Many teleost fish appear superficially similar
because of their streamlined body shape (e.g. zebrafish and
medaka), but their lineages diverged 100 million years ago.
For instance, salmon or zebrafish (basal teleosts, cellular
bone, ample remodelling, many multinucleated osteoclasts,
extramedullary tooth development) are very different from
medaka or sea bream (advanced teleosts, acellular bone,
sparse remodelling, many mononucleated osteoclasts, intra-
medullary tooth development).
VIII. ACKNOWLEDGEMENTS
We thank two anonymous reviewers for their very valuable
and helpful comments. We also thank our colleagues
Leonor Cancela, Brian K. Hall, Santosh Lall and Jean-
Yves Sire for inspiring discussions. A.H. acknowledges
a grant from the FWO-Vlaanderen n° 3G0040.08, P.E.W.
and A.H. acknowledge funding from the Norwegian
Research Council (Project No. 172483/s40) and from
COST Action B23, ‘‘Oral facial development and
regeneration’’.
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