Witten and Huysseun, 2009

Biol. Rev. (2009), 84, pp. 315–346. 315doi: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 ...................................................................................................................................... 316II. 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:[email protected])

Biological Reviews 84 (2009) 315–346 � 2009 The Authors Journal compilation � 2009 Cambridge Philosophical Society

Cambridge Philosophical Society

BIOLOGICALREVIEWS

(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 .............................................................................................. 333VII. Conclusions ....................................................................................................................................... 335

VIII. Acknowledgements ............................................................................................................................ 336IX. References ......................................................................................................................................... 336

I. INTRODUCTION

Resorption and remodelling of the vertebrate skeleton areessential processes for development, growth, repair,mechanical adaptation and mineral homeostasis. Vertebratehard tissue structures are never permanent and thus theskeleton is subject to constant reshaping (cartilage),remodelling (bone) and replacement (teeth) (Hall & Witten,2007). During ontogeny, remodelling of bone is firsttriggered by the requirements of development and allome-tric growth, later it continues as an adaptation to changes inmechanical load. In tetrapods, skeletal remodelling is alsoan important process in the animals’ mineral homeostasis.Tooth eruption and repair of the skeleton after trauma alsorequire skeletal remodelling. Finally, temporary skeletalelements that function as secondary sexual characters, suchas deer antlers or the salmon kype, require bone resorptionfor shedding and/or regression (Bertram & Swartz, 1991;Witten & Hall, 2002; Witten, Huysseune & Hall, 2003).Similar to tetrapods the skeleton of teleost fish is subject toremodelling and many factors that affect skeletal remodel-ling in mammals also affect the process in teleosts. However,skeletal remodelling in teleost fish shows several specificdifferences compared to mammals. These relate to theaquatic life of teleosts, to certain conserved traits in teleosts(that have been lost in mammals) and to several advancedcharacters that have evolved in the lineage of modern bonyfish (Metscher & Ahlberg, 1999).

The skeleton of advanced bony fish (teleosts) has recentlyreceived increasing attention from developmental biologistsand biomedical researchers. Zebrafish (Danio rerio), medaka(Oryzias latipes), stickleback (Gasterosteus aculeatus), and puffer-fish (Fugu rubripes) have been well established as modelorganisms (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 theskeletal development of farmed teleost species, such asAtlantic salmon (Salmo salar), rainbow trout (Oncorhynchusmykiss), cod (Gadus morhua), halibut (Hippoglossus hippoglossus)and sea bream (Sparus aurata) has received attention becauseskeletal malformations are an issue of concern in fishfarming (Madsen & Dalsgaard, 1999; Boglione et al., 2001;Lewis, Lall & Witten, 2004; Witten et al., 2006). Develop-mental studies usually are not concerned with skeletaldevelopment and remodelling. These studies focus onanabolic pathways of cell and tissue differentiation and onearly events in development. Given that the skeleton,especially the calcified skeleton, develops quite late inontogeny comparatively few studies tackle skeletal resorp-tion and skeletal remodelling in teleost fish as an integralcomponent 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 understoodin mammals. Still, knowledge about the differences andsimilarities between mammalian and teleost skeletal remod-elling is important to understand teleost skeletal develop-ment, especially if teleosts are used as models in biomedicalresearch.

Despite evidence for skeletal remodelling in earlygnathostomes (Carroll, 1988), various observations initiallyled to the view that the fish skeleton is metabolically inactiveand not subject to resorption and remodelling. Theseinclude the fact that fish live in relatively weightlessconditions, have unlimited access to calcium from theambient water, an often reported absence of multinucleatedbone resorbing cells, and the failure to stimulate boneresorption with parathyroid hormone (PTH) (Clark &

P. Eckhard Witten and Ann Huysseune316


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 thatthe fish skeleton is metabolically active and that it is subjectto resorption and remodelling, despite the differences thatexist regarding the control of skeletal remodelling inmammals 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 groupwith over 25 000 species, teleosts have conquered virtuallyall aquatic habitats on the planet. Although many speciesshare a similar, streamlined, body shape dictated byhydrodynamic requirements (Shadwick, 2005), teleosts area remarkably evolved and diverse vertebrate group (Romer,1970; Lagler et al., 1977; Fabacher & Little, 2000).Certainly, a long evolutionary history in an often ratherconstant aquatic environment has allowed preservation ofancestral characters such as fins and the postcranial dermalskeleton. The anatomy and composition of the teleostskeleton is, however, not at all ‘‘primitive’’ or less complexcompared to that of mammals (Metscher & Ahlberg, 1999).As pointed out by Owen (1854) ‘‘more bones enter into theformation of the skull in fishes than in any other animals;and the composition of this skull has been rightly deemedthe most difficult problem in comparative anatomy’’,a statement that also applies to the postcranial skeleton.At the tissue level, teleosts confront us with an astoundingvariety of skeletal tissues. Some of these are unique andsome represent persistent intermediate forms betweenfamiliar skeletal categories (Huysseune, 2000; Hall &Witten, 2007). To understand the function and the controlof teleost skeletal remodelling requires the proper identifi-cation of the many tissues that constitute the teleostskeleton. These tissues will be briefly introduced below(Section II). Furthermore, it is important to distinguishbetween traits and processes that are shared by allvertebrates, and those that are either conserved or derivedin teleost fish (Metscher & Ahlberg, 1999). In bothmammals and in teleost fish bone remodelling involvesconserved and derived characters: characteristics of theteleost skeleton are not always primitive nor are those of themammalian skeleton always derived.

II. A BRIEF OVERVIEW OF TELEOSTSKELETAL BIOLOGY

The teleost skeleton consists of two major subunits thatevolved to a large degree independently: the dermalskeleton and the endoskeleton (Smith & Hall, 1990). Themajor categories of skeletal tissues (cartilage, bone, dentineand enamel/enameloid) and the major categories of skeletalcells (chondroblasts, chondrocytes, osteoblasts, bone liningcells, osteocytes, osteoclasts, odontoblasts, ameloblasts) arepresent in both teleosts and mammals (Huysseune, 2000).Apart from dentine, enamel/enameloid and bone ofattachment, that are restricted to the dermal skeleton, allskeletal tissues and respective cells can be found in both the

teleost dermal skeleton and the endoskeleton. Compared tomammals, additional skeletal tissue subtypes are recognisedin 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 categoriesof 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 tothe endoskeleton. Secondary cartilage and chondroidcartilaginous tissues also develop on cranial dermal bones(Beresford, 1993; Benjamin, 1989; Huysseune, 2000; Witten &Hall, 2002; Gillis, Witten & Hall, 2006). The best studiedteleost ‘‘intermediate skeletal’’ tissue is perhaps chondroidbone. Chondroid bone exhibits characteristics of bone andof cartilage and develops from osteogenic precursors(Huysseune, 1986; Huysseune & Verraes, 1986). It is a tissuethat 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 inbasal teleosts with osteocyte-containing bone and inadvanced teleosts with acellular bone, and should not beconfused with cellular bone (Beresford, 1981, 1993;Huysseune & Verraes, 1990; Huysseune & Sire, 1990;Meunier & Huysseune, 1992; Witten & Hall, 2002; Gilliset al., 2006). Chondroid bone can also be remodelled: forexample, in Salmo salar, chondroid bone that is part of thelower jaw is later resorbed and remodelled into lamellardentary bone (Witten & Hall, 2002, 2003; Gillis et al., 2006).

The first step in the development of ‘‘regular’’ cartilage inteleosts is condensation of mesenchymal cells (blastema stage)that turn into closely packed prechondroblasts (Huysseune &Sire, 1992b). These cells differentiate into chondroblasts andfinally become separated through the secretion of extracel-lular cartilage matrix. The subsequent formation of bone isfirst perichondral, leading to the formation of a bone collararound the cartilage. This bone collar can be extended by theformation of membranous apolamellae (St�ephan, 1900;Blanc, 1953; Bertin, 1958; Huysseune, 2000; Witten &Huysseune, 2007). As in mammals, cartilage can be replacedby bone through endochondral bone formation (Huysseune,2000) but in smaller teleost species, such as medaka orzebrafish, endochondral bone formation is rather uncom-mon. If cartilage is removed inside a bony shaft it is typicallyreplaced by adipose tissue (Huysseune, 2000; Witten et al.,2001) (Fig. 1A,B).

Structurally, bone tissue in teleost fish develops first aswoven bone. Subsequently, parallel-fibered and lamellarbone develop in more mature individuals. In largerindividuals lamellar bone can also form osteons (Moss,1961b; Smith-Vaniz, Kaufman & Glowacki, 1995; Witten &Hall, 2002, 2003; Meunier, 2002). The bone of basalteleosts contains osteocytes, as is the case for tetrapod bone,whereas advanced teleosts possess acellular or anosteocyticbone which has no enclosed osteocytes (Kolliker, 1859;Moss, 1961a; Ekanayake & Hall, 1987; Meunier, 1989;Meunier & Huysseune, 1992; Huysseune, 2000;

Skeletal remodelling in teleost fish 317


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 matrixdistinguishes acellular bone also from typical teleostdentine. Acellular bone bears however some resemblanceto the atubular dentine of teleost first-generation teeth andto acellular mammalian cementum, both of which also lackenclosed 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 entrappedin the bone matrix (Huysseune, 2000). With few exceptions,the presence or absence of osteocytes is uniform in allelements of the teleost skeleton (see Moss, 1961b andMeunier, 1989, for exceptions). Members of large basalteleost groups, such as salmonids and cyprinids, withcellular bone in the endoskeleton, can have postcranialdermal skeletal elements (scales and fin rays) that areacellular (Meunier, 1989). The absence of osteocytes inadvanced teleosts, cells that in mammals constitute 95% ofall bone cells (Franz-Odendaal et al., 2006), raises questions

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



about the regulation of bone remodelling in advancedteleosts. This is because the penetration of bone and dentineby cell processes enables osteocytes and odontoblasts tofunction as stress sensors. Accordingly osteocytes arebelieved to guide bone remodelling in response tomechanical load (Burger, Klein-Nulend & Van Der Plas,1995; Burger, Klein-Nulend & Smit, 2003; Bonewald, 2004;Knothe Tate et al., 2004). Estimates about the percentage ofbone that is resorbed and replaced by new bone in humansrange between four and ten per cent per year (Delling &Vogel, 1992; Manolagas, 2000). Such estimates are lackingfor teleost fish but a regular resorption and rebuilding ofscales and bony skeletal elements is well documented forAtlantic salmon (Kacem, Meunier & Bagliniere, 1998;Persson et al., 2000; Witten & Hall, 2002, 2003).

An acellular skeletal tissue particular to all teleosts is themineralized notochordal sheath (Arratia, Schultze & Casciotta,2001; Grotmol et al., 2003; Nordvik et al., 2005; Inohaya,Takano & Kudo, 2007). Unlike other vertebrates, earlyformation of teleost vertebral bodies does not start withcartilaginous vertebral body precursors that form around thenotochord. Instead, the formation of vertebral bodies startswith the mineralization of the notochordal sheath itself(Fig. 1E,F). Further growth of the vertebral bodies occurs bythe apposition of bone onto the mineralized notochordalsheath, similar to the intramembranous formation of boneapolamellae (Ekanayake & Hall, 1988, 1991; Grotmol et al.,2003; Nordvik et al., 2005). As a consequence, cartilageformation and subsequent endochondral bone formation arenot part of the initial differentiation of teleost vertebral bodies(Witten & Villwock, 1997; Nordvik et al., 2005).

While we can expect that the particular cellularcomposition of the teleost skeleton affects if and how itwill remodel, a number of additional factors influence theprocess. Since basic ideas about bone remodelling areinfluenced by our knowledge of the processes in mammals,it might be useful to enumerate some of the maincharacteristics that distinguish teleosts from mammals withrespect to skeletal remodelling:

(1) In mammals, the bone resorbing cells, or osteoclasts,originate from haematopoietic tissue located in thebone marrow. The haematopoietic tissue also releasesfactors that regulate osteoclast and osteoblast activities.Such an intimate spatial relationship between boneresorbing cells and haematopoietic cells does not existin teleosts as most bone marrow spaces are filled withadipose tissue and haematopoiesis takes place in thehead kidney (Section III).

(2) The lack of osteocytes in advanced teleosts appears tocoincide 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, inparticular smooth, bone resorption (Section IV).

(3) In mammals, endochondral bone formation is a primecause of skeletal resorption and remodelling. Typicalendochondral ossification is, however, often lacking inteleosts, especially in species with small individualssuch as medaka or zebrafish. In addition, in all teleosts,vertebral bodies develop (ossify) without cartilaginous

precursors and thus initially without remodelling(Sections V.1 and V.2).

(4) Regulation of plasma calcium content is crucial for allterrestrial vertebrates and the skeleton is tightlyintegrated into the animals’ calcium homeostasis.Teleosts have a different approach. They obtain andrelease calcium from and into the water via their gillsand the skeleton must not be used as a source ordeposit of calcium (Section V.3).

(5) In case of need of skeletal resorption, minerals aremobilised from the extensive postcranial dermalskeleton (scales). Minerals must not be released fromthe endoskeleton (Section V.3).

(6) The lack of osteocytes in advanced teleosts implies thatbone remodelling in response to mechanical load mustbe triggered by other cell types (Section V.4).

(7) The regenerative capacity of elements of the teleostdermal skeleton (fin rays and scales) exceeds theregenerative capacity of elements of the endoskeleton(Section V.5).

(8) Teleosts replace their teeth throughout life, a processthat requires lifelong resorption of teeth and toothattachment bone and remodelling of dentigerous bone(Section V.6).

(9) Teleosts never stop growing, and certain skeletalelements develop rather late. Thus, growth-relatedskeletal remodelling continues throughout life andshould not be mistaken for metabolism-related skeletalremodelling (Section V.7).

III. CELLULAR MECHANISMS OF TELEOSTSKELETAL REMODELLING

Skeletal remodelling can be defined broadly as thereplacement of an existing skeletal tissue by another skeletaltissue. A classic example is the replacement of cartilage bybone during endochondral bone formation in mammalianlong bones. A stricter definition of bone remodelling refers tocontinuous bone replacement in the adult skeleton (Parfitt,1994). Bone remodelling usually involves the removal of boneby osteoclasts (bone resorbing cells) and the subsequentformation of new bone by osteoblasts (bone forming cells). Tokeep the skeleton healthy and functional, coordinated actionof osteoclasts and osteoblasts is required. Given that thesecells act together, they can be considered as basic multicellularunits (BMUs) (Frost, 1969, 1987; Parfitt, 1994; Manolagas,2000; Heino, Hentunen & Vaananen, 2002; Burger et al.,2003; Cooper et al., 2003). Alternatively, remodelling canfollow pathways that do not involve osteoclasts.

(1) Osteoclastic resorption andremodelling in teleosts

(a ) Osteoclasts: the mammalian perspective

In mammals (as well as in birds), osteoclasts are the principalbone resorbing cells (Kolliker, 1873). Comprehensive reviews



of the development, regulation, and function of multinucle-ated mammalian osteoclasts should be consulted for detailedinformation 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; Vaananen, 2005; Bar-Shavit, 2007). The main featuresof mammalian osteoclasts are briefly summarised below toenable us to clarify the similarities and differences betweenmammalian and teleostean osteoclasts. Chondroclasts andodontoclasts are related to osteoclasts, resorbing calcifiedcartilage and dentine, respectively (Hall, 1972; Beresford,1981; Blair, 1998; Buxton et al., 2003). Despite their differentnames, environments, and regulatory mechanisms, there isevidence that these different skeletal resorbing cells belong tothe same cell lineage (Nordahl et al., 2000; Shibata &Yamashita, 2001; Sasaki, 2003; Helfrich, 2003).

Mammalian bone resorbing cells can be viewed asspecialised multinucleated macrophages. Multinucleatedmammalian osteoclasts derive from haematopoietic bonemarrow 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 anddifferentiate into multinucleated osteoclasts. The dendriticcell-specific transmembrane protein (DC-STAMP) has beenidentified as a critical factor for the fusion of mononucleatedcells into multinucleated osteoclasts. DC-STAMP-deficientcells fail to fuse, but still function as mononucleatedosteoclasts (Yagi et al., 2005; Bar-Shavit, 2007). Transcrip-tion factors and signalling molecules that are required formacrophage differentiation such as the macrophage and Bcell-specific transcription factor PU.1 and tumor necrosisfactor receptor-associated factor 6 (TRAF6) are alsorequired for osteoclast development (Udagawa et al., 1990;



Stenbeck, 2002). Maturation of mammalian osteoclastsrequires direct contact with bone marrow stroma cells orwith osteoblasts, as the expression of molecules by stromacells and osteoblasts regulates the differentiation ofosteoclasts (see BMU concept above). Key molecules arethe macrophage colony-stimulating factor (M-CSF) andRANK ligand (RANKL). Stroma cells and osteoblasts alsorelease osteoprotegerin (OPG), a RANKL decoy receptorthat downregulates osteoclasts (Teitelbaum, 2000; Li, Kong &Qi, 2006). For other cytokines and growth factors thatstimulate or inhibit osteoclasts see Teitelbaum (2000), Theillet al. (2002), and Li et al. (2006). The differentiation andfunction of mammalian osteoclasts is also under hormonalcontrol. Estrogen (17b-estradiol) and calcitonin (CT) are themain 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 notosteoclasts but osteoblasts. Subsequently, osteoblasts regu-late osteoclasts via the above-mentioned M-CSF, RANKLand OPG pathways (Rodan & Martin, 1981; Franz-Odendaal et al., 2006). In mammals CT binds directly toa receptor expressed by osteoclasts; however in non-mammalian vertebrates CT receptors have been found tobe downregulated in osteoclasts (Dacke et al., 1993).

Like macrophages, mammalian osteoclasts are mobilecells that migrate to the site of bone resorption. Uponcontact with the bone surface, the apical cell membrane ofosteoclasts develops a peripheral sealing zone (that concealsthe subcellular space) and a central ruffled border (multipledeep membrane infoldings) (Parfitt, 1988; Li et al., 2006).The central ruffled border is the area of active boneresorption. The sealed space under the ruffled border canbe viewed as a giant extracellular phagolysosome (Baron

et al., 1993). The extracellular space is acidified by theaction of a vacuolar proton pump (H]-ATPase) (Vaananenet al., 1990; Mattsson et al., 1994; Bastani et al., 1996;Stenbeck, 2002). The acidified environment promotes thedissolution of bone minerals and the function of lysosomalenzymes. Enzymes such as cathepsins, unspecific acidphosphatase (cleaving b-glycerophosphate) and tartrate-resistant acid phosphatase (TRAP) break down bone matrixcomponents (Hammarstrom, 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 boneresorption remains controversial. Everts et al. (1999) showedthat these enzymes can be present or absent, depending onthe skeletal element that is resorbed.

(b ) Teleost osteoclasts

The above model of skeletal remodelling and boneresorption has gained general acceptance for mammals(and birds). Typical (‘‘mammalian-like’’) multinucleatedosteoclasts 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 thenumber 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 (Domonet al., 2004). Similar large osteoclasts and odontoclasts havebeen observed in Atlantic salmon and in carp (Sire et al.,1990; Witten et al., 2000; Witten & Hall, 2002). Certainlythe number of nuclei in osteoclasts differs among vertebrategroups. In teleosts, there is large variation in number ofnuclei per osteoclast related to differences among species,

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 cellshows about 30 nuclei (black arrowhead). Considering all three dimensions of the cell, the actual number of nuclei could be above100. 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 thecells’ ruffled border are visible (black asterisk), secreted TRAP appears as a red stripe on the bone surface (black arrowheads). Thesecharacteristics are also typical for mammalian osteoclasts. Glycol methacrylate embedding, TRAP staining counterstained withHaematoxylin: scale bar ¼ 10 mm. (C) Mononucleated TRAP-positive osteoclasts (red) in a 20-day-old zebrafish (Danio rerio) alongthe forming dentary bone (white asterisk). Meckel’s cartilage, black asterisk. Glycol methacrylate embedding, TRAP stainingcounterstained 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 isfrequently 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 contactbetween cell processes of two mononucleated osteoclasts (black arrowhead) on the endosteal surface of a lateral line canal in thecichlid Oreochromis niloticus. Epon embedding: scale bar ¼ 0.5 mm. (F) Flat and long osteoclasts on the endosteal surface (whitearrowhead) of a neural arch in the eel (Anguilla anguilla). The shape of the cells resembles bone lining cells rather than typicalmammalian osteoclasts. A sectioning artefact that has shifted the cells from the endosteal bone surface reveals their flat shape andstrong interconnection (black arrowheads). Spinal cord, black asterisk. Glycol methacrylate embedding, TRAP stainingcounterstained 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 (whitearrowhead) and by bone apposition along the periosteal surfaces (black arrowhead). No multinucleated osteoclasts are visible alongthe endosteal surfaces. Paraffin embedding, Azan staining: scale bar ¼ 25 mm. (H) Cross section of a growing bone tube in thecichlid Oreochromis niloticus, similar to that shown in G revealing the presence of many small mononucleated osteoclasts by visualisingthe 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.



ontogenetic stages, skeletal elements and physiologicalconditions (Fig. 2B) (B�elanger, 1977; Sire et al., 1990;Persson, Bjornsson & Takagi, 1999; Witten et al., 2001;Roy et al., 2002; Witten & Hall, 2002; Vaananen, 2005).Birds also have remarkably high numbers of nuclei perosteoclast (Vaananen, 2005; Bar-Shavit, 2007). In humanssuch large osteoclast nuclei numbers only occur underpathological conditions, for example in Paget’s disease(Manolagas, 2000; Helfrich, 2003).

As skeletal growth and remodelling always requires thepresence of osteoclasts (but see Section III.2), one maywonder why the existence of these cells in advanced teleostswas questioned. The reason was the often observed lack oftypical (multinucleated) osteoclasts and their traces (resorp-tion lacunae). This is because small mononucleatedosteoclasts dominate early skeletal development in allteleosts (Fig. 2C) and also later stages of development inadvanced teleosts with acellular bone (Fig. 2D) (Sire et al.,1990; Witten, 1997). As these cells cannot be detected withstandard histological techniques, their identificationrequires either transmission electron microscopy (TEM) orthe demonstration of the activity of the osteoclast markerenzyme TRAP (Weiss & Watabe, 1979; Lopez et al., 1980;Sire et al., 1990; Witten, 1997). TEM, however, onlyprovides information about very small skeletal segments.Consequently, the comprehensive visualisation of skeletalremodelling during teleost development may require thedemonstration of TRAP activity (Witten, 1997; Witten &Villwock, 1997; Witten et al., 2000, 2001) as is also the casefor complete visualisation of osteoclasts in humans (Ballantiet al., 1997). The complete nucleotide and amino acidsequence of TRAP from goldfish (Carassius auratus) isavailable (Azuma et al., 2007). A comparison of TRAPfrom mammals and goldfish shows a completely conservedamino acid sequence in the iron-containing active centrumof the enzyme (Azuma et al., 2007).

TEM and enzyme detection studies have revealed that inall teleosts, but typically in advanced teleosts, cells thatperform bone resorption can differ from ‘‘standard’’multinucleated mammalian osteoclasts. The cells can havean extremely flat shape, resembling bone lining cells ratherthan typical osteoclasts (Weiss & Watabe, 1978, 1979;Witten, 1997; Witten et al., 1999, 2004) (Fig. 2E,F). Howmuch the cells differ may depend on species, skeletalelement, and ontogenetic stage. In mammals, mononucle-ated cells are often viewed as osteoclast precursors but inbasal and advanced teleosts, mononucleated osteoclasts areactive 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 duringdevelopment (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 theenlargement of bone tubes in the Nile tilapia, Oreochromisniloticus (Witten, 1997; Witten & Villwock, 1997). The smallmononucleated osteoclasts were found to be evenlydistributed along the endosteal surfaces where the cellsperform a smooth (non-lacunar) type of bone resorption(Fig. 2G,H) (Witten, 1997; Witten & Villwock, 1997; Witten

et al., 1999). Similar findings have been reported from othercichlids (Sarotherodon melanotheron) by Weiss & Watabe (1979)and from medaka by Ekanayake & Hall (1987) and Takanoet al. (2005). Small mononucleated osteoclasts also dominatebone resorption in the first advanced marine teleost to beexamined for osteoclasts by demonstration of the presenceof TRAP (haddock, Melanogrammus aeglefinus) (Roy et al.,2002; Roy, 2003). Also scale resorbing cells in salmonids aremostly mononucleated (Persson et al., 1999) despite the factthat salmonids, as basal teleosts with cellular (osteocyte-containing) bone, usually possess prominent multinucleatedcells. The scales, however, are acellular and thus resembleother acellular tissues such as acellular bone or acellulardentine (Meunier, 1989; Sire & Akimenko, 2004). Boneresorption by mononucleated cells is not only a character ofadvanced teleosts: demonstration of the presence of TRAPand TEM studies have revealed the presence of abundantmononucleated osteoclasts also in basal teleosts (Sire et al.,1990; Witten et al., 2001), in basal osteichthyans such asNeoceratodus forsteri (Kemp, 2003), and in humans (Ballantiet al., 1997). According to Parfitt (1988), in humans,multinucleated osteoclasts cause deep resorption lacunaewhile mononucleated osteoclasts perform smooth (non-lacunar) resorption. This description of Parfitt (1988) fitsobservations about the performance of mononucleatedosteoclasts in teleosts. Finally, in teleosts, bone resorbingcells can be absent from particular skeletal elementsaltogether, such as developing radials or developingvertebral bodies (Ekanayake & Hall, 1987, 1988; Witten &Villwock, 1997; Nordvik et al., 2005).

(2) Non-osteoclastic bone resorption andremodelling in teleosts

In addition to mono- and multinucleated osteoclasts,osteocytes in cellular-boned teleosts are capable of boneresorption by osteocytic osteolysis. Examples derive fromEuropean eels (Anguilla anguilla) (Lopez et al., 1980; Sbaihiet al., 2007), salmonids (Hughes et al., 1994; Kacem &Meunier, 2000) and carp (Witten et al., 2000). Osteocyticosteolysis is disputed as a feature of human osteocytes(Boyde, 1980), but it has been observed in manymammalian species with smaller adult body size such asbats (Doty & Nunez, 1985; Kwiecinski, 1985; Kwiecinski,Krook & Wimsatt, 1987), hamsters (Steinberg, Singh &Mitchell, 1981), squirrels (Haller & Zimny, 1978), rats(B�elanger, 1977; Tazawa et al., 2004), and rabbits (Zhang,Bao & Dai, 2000). Evidence for osteocytic osteolysis alsocomes from studies on the snake skeleton (Alcobendas,Baud & Castanet, 1991). The gracile skeleton of batsprovides perhaps the best example that lacunar resorptionby multinucleated cells is not an option for thin bones: thisprocess could easily perforate the bone. For pregnant andlactating Myotis lucifungus bats osteocytic osteolysis, inaddition to smooth resorption by mononucleated cells, pro-vides a functional alternative (Kwiecinski, 1985; Kwiecinskiet al., 1987). Another resorption-related process that hasbeen described for teleosts is demineralization of the bonewithout affecting the bone matrix, called halastatic



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 inteleosts occurs not only by resorption and de novo formationbut also by transdifferentiation (metaplasia) of skeletaltissue (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 metaplasiamay also occur in mammals (humans) during normal orpathological development (Baud, 1968; B�elanger, 1967,1969, 1977; Beresford, 1981; Chambers, 1985; Ballantiet al., 1997; Hall, 2005). However, some mechanisms ofskeletal remodelling are more prominent and/or morecommonly observed in teleosts and are thus recognised asa regular process that shapes the skeleton in the course ofdevelopment (Sire et al., 1990; Huysseune, 2000; Wittenet al., 2001, 2003; Hall & Witten, 2007; Witten &Huysseune, 2007).

(3) A possible link between bone type andosteoclast type and number?

In view of the increasing evidence for a role of osteocytes incontrolling 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 theformation of multinucleated osteoclasts in teleosts: in theabsence of osteocytes the dominant osteoclast type would bemononucleated. Support for this idea derives fromcomparative and ontogenetic studies. Comparing thepresence and absence of osteocytes in different parts ofthe teleost skeleton, Meunier (1989) reported that membersof the carp and salmon families have scales withoutosteocytes, but do possess osteocytes in the endoskeleton.In basal bony fish below the teleost level, the endoskeleton,scales and fin rays still contain osteocytes (G�eraudie, 1988;Meunier, 1989; Witten & Huysseune, 2007). Scales insalmon are predominantly resorbed by mononucleatedosteoclasts whereas the osteocyte-containing endoskeleton ispredominantly 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 mineralizedbut osteocytes are scarce or not yet present (Sire et al., 1990;Huysseune, 2000; Witten et al., 2001; Meunier, 2002). Inzebrafish there is an ontogenetic progression from mono- tomultinucleated osteoclasts (Witten et al., 2001). Initiallyosteoclasts are mononucleated resembling morphologicallyand functionally those of advanced teleosts (Fig. 2C). Thirtydays after fertilisation, mononucleated cells are succeededby multinucleated osteoclasts that subsequently performlacunar resorption and finally become engaged in boneremodelling. Simultaneously with the expression of multi-nucleated osteoclasts and bone remodelling, growth-relatedmodelling by mononucleated osteoclasts still occurs at theendosteal surfaces of the neural arches of zebrafish.

Further evidence for a link between the presence ofosteocytes and multinucleated osteoclasts derives from ananalysis of hyperostotic bones in advanced marine teleostswith acellular bone (Caranx latus and Caranx hippos,Carangidae) (Smith-Vaniz et al., 1995). Hyperostosis, theperiosteal formation of excess bone (‘‘Zuwachsknochen’’) atspines and skull bones of adults, was formerly viewed asa pathological process (Schlumberger & Lucke, 1948). It isnow considered part of normal late development (Smith-Vaniz et al., 1995). An intriguing feature of hyperostoticbones is the reappearance of osteocytes and multinucleatedosteoclasts, even if the hyperostotic bone is an outgrowth ofacellular bone. The rather early shift from acellular tocellular bone and from mononucleated to multinucleatedosteoclasts in cellular-boned teleosts, and the late shift inadult acellular-boned teleosts developing hyperostoses, ledWitten et al. (2004) to hypothesize that acellular bone andmononucleated osteoclasts are not necessarily a new trait ofadvanced teleosts. Rather, they may represent a hetero-chronic shift resulting from the retention of early skeletalcharacters (the absence of osteocytes and of multinucleatedosteoclasts) well into adult life.

Is there a relationship between osteoclast type andosteoclast number? Considering the many factors that mayinfluence the type and the number of osteoclasts, comparingcell numbers across species can only provide roughindications about such a relationship. Some data areavailable: in the jaws of juvenile carp there are one toseven multinucleated TRAP-positive osteoclasts per mm oftwo-dimensional (2D) endosteal bone surface whereas theendosteal bone surfaces of the lower jaw in juvenile Niletilapia 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, mostlymononucleated, osteoclasts per mm 2D bone surface (Royet al., 2002). These numbers may suggest that many smallmononucleated cells can functionally replace a few multi-nucleated giant cells. The type of resorption exhibited bymono- and multinucleated osteoclasts differs however:multinucleated cells perform lacunar resorption, whereasmononucleated cells are responsible for smooth resorption.These ideas fit to Parfitt’s model about the function ofmononucleated osteoclasts (Parfitt, 1988) (see Section III.1b)and the idea that in mammals, the amount of boneresorption per cell nucleus is the same in multinucleatedgiant cells and small oligonucleated cells (Bar-Shavit, 2007).

IV. DIFFERENTIATION AND ACTIVATION OFTELEOST OSTEOCLASTS

(1) Multinucleated osteoclasts

Typical remodelling sequences in teleosts suggest thecoordinated action of osteoblasts and osteoclasts, accordingto the BMU model proposed for mammalian bone (Peck &Woods, 1988; Parfitt, 1988) (Fig. 3A). In zebrafish, repeatedresorption by osteoclasts after refilling of resorption lacunae



can be observed, a process regarded as typical remodelling(Witten et al., 2000, 2001) (Fig. 3B). Remodelling sequencesin carp and zebrafish show multinucleated osteoclastsperforming lacunar resorption, resorption lacunae withosteoblasts in the process of refilling lacunae, and resorptionlacunae that have been completely refilled (Fig. 3A). Severalkey factors that control development of mammalianmonocytes into osteoclasts and that activate osteoclasts(see Section III.1a) have been identified in various teleostspecies. A highly conserved transcription factor PU.1 fromrainbow trout macrophages has been characterised byRibas et al. (2008) and a full-length orthologue ofmammalian TRAF6 has been identified in zebrafish(Phelan, Mellon & Kim, 2005). A M-CSF from haemato-poietic tissue of the goldfish has been characterised byHanington et al. (2007). RANKL has been discovered in thegenome of rainbow trout, zebrafish, and pufferfish (Glenney &Wiens, 2007) and the RANKL decoy receptor OPG hasbeen 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 teleostsdepends on the same signalling pathway that activatesosteoclasts and macrophages in mammals. Conclusiveevidence for this speculation is, however, still lacking.

Ostland, McGrogan & Ferguson (1997) observed theactivation of multinucleated osteoclasts in salmonids con-nected with an inflammatory process suggesting thatinflammation-related signalling molecules known to acti-vate mammalian osteoclasts may also activate teleosteanosteoclasts. A similar observation was made by Takagi &Kanoko (1995). Bone particles implanted into the musclesof rainbow trout caused the appearance of monocyte-likecells followed by typical TRAP-positive multinucleatedosteoclasts. Both the mineralized bone and the inflamma-tory reaction to foreign antigens may have triggered thedevelopment of osteoclasts. Apart from validating thepathway of teleost osteoclast stimulation by inflammatoryfactors it will be a challenge to find out how osteoclasts inteleosts become activated in the absence of haematopoietictissue in the bone marrow (Figs 1A,B, 3D) precluding anintimate spatial relationship between haematopoietic cellsand teleostean osteoclasts. The lack of haematopoietic bonemarrow tissue is an ancestral character: the first haemato-poietic bone marrow tissue appears only in advancedurodeles (Zapata & Amemiya, 2000). An example of howosteoclasts can be activated without the vicinity ofhaematopoietic tissue is found in the process of tootheruption 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 osteoclastis generating a resorption lacuna. b, Osteoblasts settle inside an empty resorption lacuna and start to produce a small amount ofosteoid (white arrowhead). c, Osteoblasts have progressed in filling a former resorption lacuna; the osteoid deposited is clearlyvisible (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); boneresorption and filling of a resorption lacuna can occur repeatedly at the same site (black arrowheads, right panel). Adipose tissue fillsthe bone marrow space (white asterisks). Epon embedding, Toluidine Blue staining: scale bar ¼ 15 mm. (C) Tartrate-resistant acidphosphatase (TRAP), secreted by osteoclasts, persists on the bone surface of the dentary of a juvenile carp (Cyprinus carpio) and labelsprevious areas of bone resorption even when osteoclasts are no longer present (white arrowhead) and osteoblasts have alreadystarted to deposit new bone matrix (black arrowhead). Glycol methacrylate embedding, TRAP staining counterstained withHaematoxylin: scale bar ¼ 100 mm. (D) Endochondral bone formation in a juvenile carp (Cyprinus carpio). The mineralization of thecartilage matrix (black asterisk) is followed by the deposition of bone (white arrowheads) on mineralized cartilage remnants (blackarrowhead). Non-decalcified methyl methacrylate embedding, Toluidine Blue staining: scale bar ¼ 100 mm.



Schroeder, 1996). Epithelial and connective tissues thatsurround the developing tooth (the dental follicle, dentalepithelium, and stellate reticulum) have been shown torelease factors required for osteoclast development andregulation 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 advancedteleosts 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 orreported absence of multinucleated osteoclasts and resorp-tion lacunae in these animals, and their failure to respond tocalcium deprivation with bone resorption, led to theperception 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 tocalcium-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 ratbone in the advanced teleost Paralabrax clathratus. Multinu-cleated osteoclasts are known to occur in advanced teleostsalso under non-experimental conditions (Sire et al., 1990).As discussed in Section III.3 teleosts with acellular bone cangenerate multinucleated osteoclasts if lacunar resorption isrequired, but the vast majority of osteoclasts in advancedteleosts appears to be mononucleated (Witten, 1997; Wittenet al., 1999, 2004; Roy et al., 2002; Takano et al., 2005).

Despite morphological and functional dissimilaritybetween typical mammalian osteoclasts and mononucleatedosteoclasts of juvenile and adult advanced teleosts the cellsshare several biochemical and functional features. Likemammalian osteoclasts and their precursors, teleost osteo-clast precursor cells express a-napthyl acetate esterasewhereas mature cells only continue to express b-glycer-ophosphatase, TRAP, and V]-ATPase. Like multinucleatedosteoclasts, mononucleated fish osteoclasts secrete TRAPinto the subcellular resorption space and the enzyme isconsequently 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, inboth mammals and teleosts labelling TRAP activityidentifies bone surfaces that are being actively resorbed orhave been resorbed. TRAP-staining can reveal previoussites of bone remodelling even if the resorbing cells are nolonger present and new bone has been produced abovepreviously resorbed bone surfaces (Witten, 1997; Wittenet al., 2000, 2001) (Fig. 3C). The fact that small mono-

nucleated osteoclasts may not show a ruffled border(Witten, 1997; Weiss & Watabe, 1979; Kemp, 2003) couldsuggest that this morphological feature is connected tolacunar bone resorption but is not required for continuoussmooth resorption (see also Section III.3). In a recent studyon rachitic mice Hollberg et al. (2008) found thatmammalian osteoclasts lacking a ruffled border caneffectively degrade bone matrix. Non-lacunar bone resorp-tion by mononucleated osteoclasts without a ruffled borderis analogous to osteocytic osteolysis.

V. THE FUNCTIONS OF TELEOSTSKELETAL REMODELLING

As in other vertebrates the development and growth of theteleost skeleton requires resorption and remodelling forseveral reasons: (a) to allow enlargement of elements duringallometric growth, e.g. increasing the diameters of bonetubes and bony arches, such as the cranial vault, jaw arches,neural arches, and lateral line canals (Section V.1). (b) Forreplacement of one skeletal tissue by another, for examplecartilage by bone, primary bone by secondary bone orchondroid bone by lamellar bone (Section V.2). (c) Mineralhomeostasis, under particular conditions (Section V.3). (d )To allow the skeleton to adapt to changes in mechanicalloading (Section V.4). (e) Fracture healing, regeneration anddisease-related repair (Section V.5). ( f ) Replacement ofteeth, involving the partial or complete resorption of oldteeth, their attachment bone and parts of the dentigerousbone (Section V.6). (g) Teleosts continue to grow as adults,and therefore non-pathological and non-senescence-linkedremodelling continues throughout life (Section V.7).

(1) Growth and morphogenesis

Enlargement of the diameter of bone shafts and curvedskeletal elements is a primary reason for bone resorptionduring teleost ontogeny. As bone cannot grow interstitially,any change of tube diameter or curvature of a mineralizedskeletal element requires bone resorption on one side andbone apposition on the other. Several studies report theoccurrence of teleost osteoclasts along growing skull bonesand 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 curvedsoft fin rays (lepidotrichia, composed of two mineralizedhemisegments), bone resorption is assumed to facilitatecurvature (Montes et al., 1982). Spiny fin rays containa marrow-like cavity. This lumen increases in volume inadult fish by resorption while new mineralized tissue isdeposited on the outside (Meunier, 2002), similar to theenlargement of the diameter of bone shafts. Allometricgrowth-related bone resorption was measured at theendosteal surfaces of lateral line canals (Witten, 1997;Witten & Villwock, 1997) (Fig. 2G,H). As discussed by



Tarby & Webb (2003), it was initially suggested that the cellsresponsible for the enlargement of lateral line canals wereremnants of the placodal precursors of neuromasts. Thecells were observed to form a ‘‘connecting strand’’responsible for the excavation of bone inside the lateralline canal (Clapp, 1889; Merillees & Crossman, 1973).Indeed a ‘‘connecting strand’’ of excavating cells can beobserved at endosteal bone surfaces but histochemical,immunohistochemical and TEM studies have identifiedthese 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 thegrowing neural arches of eels and zebrafish (Witten et al.,2001). As described above, these cells perform non-lacunarsteady bone resorption and thus do not produce the typicaltraces of bone resorption, such as Howship’s lacunae.

The vertebral bodies of many vertebrates (basalosteichthyans sensu stricto and tetrapods) have a cartilaginousanlage that is replaced by bone during ontogeny (Detwiler &Holtzer, 1956; Hall, 1977, 2005; Arratia et al., 2001). This isno longer the case in teleosts where the first discernible partof a vertebral body is the mineralized notochordal sheath(Arratia et al., 2001; Nordvik et al., 2005). Extension andgrowth of vertebral bodies is then facilitated by peripheraland terminal bone apposition (Ekanayake & Hall, 1987,1988; Witten & Villwock, 1997; Nordvik et al., 2005)(Fig. 1E). Moreover, and unlike other bone tubes, thefunnel-shaped halves of early vertebral bodies enlargeinitially without bone resorption, purely by terminal andperiosteal bone apposition (Witten & Villwock, 1997;Nordvik et al., 2005). However, in later stages of Atlanticsalmon, bone remodelling and osteoclasts are regularlyobserved at intravertebral spongiosa structures (Gil-Martenset al., 2006). The neural and haemal arches requireresorption of cartilage as well as of bone during growth.As the neural tube diameter increases, enlargement of theneural arches follows the pattern of other bone tubes withosteoclasts located at the endosteal surface (Fig. 2F) (Wittenet al., 2001; Roy et al., 2002).

(2) Replacement of skeletal tissues

Another primary role of skeletal resorption in teleosts is theremoval of cartilage by chondroclasts and its replacementwith another tissue such as bone, or adipose tissue. Earlychondroclasts in teleosts resemble early osteoclasts in beingoften mononucleated and inconspicuous (Witten et al.,2001). Like osteoclasts, chondroclasts can be identifiedusing TEM or by the demonstration of TRAP activity(Fig. 1B). Similar to the typical situation in mammals, teleostcartilage 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 byendochondral bone formation but arising bone marrowspaces instead are filled with adipose tissue (Fig. 1A)(Huysseune, 2000; Witten et al., 2001; Witten & Hall,2002; Witten & Huysseune, 2007). As the removal ofcartilage starts rather late, cartilage inside some bone shaftsmay persist being neither replaced by adipose tissue nor by

bone (Benjamin et al., 1992; Witten & Huysseune, 2007).Where endochondral bone formation occurs (Haines, 1938)it involves the mineralization and partial resorption of thecartilage matrix and the presence of matrix remnants thatserve 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 theinitiation of cartilage resorption, and prior to furthercartilage resorption, a thin seam of bone is deposited onthe 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 createsa ladder-like construction of bone trabeculae inside thebone tube.

One particular way in which cartilage can be remodelledconcerns the early fusion and the subdivision of thecartilage anlagen of the teleost fin endoskeleton. Cartilagesubdivision plays a role in the differentiation of unpaired(dorsal and anal) and paired fins. Parts of the shoulder girdleand the radials of the pelvic and the pectoral fin alsodevelop by subdividing a single cartilage condensation(Witten & Huysseune, 2007). For zebrafish a detaileddescription is available of how a cartilaginous plate givesrise 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 asseparate cartilage condensations (Grandel & Schulte-Merker, 1998). In the sea bream Sparus aurata, an advancedteleost, the scapulocoracoid cartilage and the cartilaginousfin plate (i.e. the precursor of the proximal radials) appear todevelop from one piece of cartilage (Faustino & Power,1999). In other species, two cartilage anlagen exist: ascapulocoracoid cartilage, which ossifies into the coracoidand the scapula (which remain joined by cartilage), and acartilaginous blade that first cleaves centrally then dorsallyand ventrally, to form radials which also ossify (Potthoff &Kelley, 1982; Potthoff, Kelley & Collins, 1988; Balart, 1995;Mabee & Trendler, 1996; Cubbage & Mabee, 1996). Theway cartilage subdivides is intriguing but not well under-stood. Crotwell & Mabee (2007) suggested that the patternof gene expression during the subdivision is largelyconserved between zebrafish and tetrapods. The pectoralfin endoskeleton subdivision of zebrafish has been describedas ‘‘decomposition of cartilage matrix’’, but no apoptotic orresorbing cells have been identified (Grandel & Schulte-Merker, 1998). The process in teleosts appears to be similarto the non-apoptotic process of digit separation inamphibians but differs from amniotes, where massive celldeath occurs to transform the webbed regions of thefootplate into interdigital spaces (Cameron & Fallon, 1977;Wood et al., 2000; Zuzarte-Luıs & Hurle, 2002). In birds,reptiles and mammals, macrophages are involved but theirrole is believed to be restricted to the removal of apoptoticcells. Resorption by macrophages is not considered aprimary mechanism of cartilage separation (Zuzarte-Luis &Hurle, 2005). The absence of signs of apoptosis orresorption in teleosts suggests that the separation ofcartilage elements may involve metaplasia: dedifferentiation



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- andintracellular calcium levels (Guerreiro et al., 2007) and inamniotes the skeleton is highly integrated into thisregulation. Calcium is released from the skeleton throughbone resorption by osteoclasts or through osteocyticosteolysis to compensate for low plasma calcium levels,and calcium is deposited into the skeletal tissues tocompensate for high plasma calcium levels (B�elanger,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 incalcium 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 totalplasma calcium levels within a similar narrow range:typically, 2.5 mmol l[1 to 3.0 mmol l[1; marine speciesappear to maintain slightly higher values than freshwaterspecies (Urist et al., 1972; Bone & Marshall, 1985).Exceptionally low plasma calcium levels (1.5 – 2.0 mmoll[1) occur in some basal freshwater bony fish (e.g. Acipensernaccarii) although the primitive bony fish Amia calvamaintains a value of 3.0 mmol l[1 (Urist et al., 1972;Fuentes et al., 2007). Regulation of calcium homeostasis isunder hormonal control and consequently several studieshave examined the role of calcium-regulating hormones inbone resorption in teleosts (Lopez et al., 1980; Milet et al.,1984; Taylor, 1985; Persson et al., 2000).

The participation of acellular bone of advanced teleostsin calcium homeostasis is particularly interesting, as theabsence of osteocytes precludes mobilisation of calciumfrom the skeleton by osteocytic osteolysis. Moss (1962)compared fracture healing in teleosts with cellular andacellular bone under calcium-deprived conditions. Only thespecies with cellular bone (Carassius auratus, Cyprinidae)could mineralize the fracture callus; the species withacellular bone (Sarotherodon melanotheron, Cichlidae) formeda fracture callus that did not mineralize. This and similarfindings did suggest that advanced teleosts with acellularbone may not be capable of mobilising calcium from theskeleton (Moss, 1962; Simmons, 1971; Urasa et al., 1984;Taylor, 1985). Witten (1997) maintained juvenile cichlids(Oreochromis niloticus) for two weeks in four different types ofmedia: (1) tap water supplemented with 3% sea salt, (2)water supplemented with three times more magnesium thancalcium, (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 waterhad no influence on the number and shape of osteoclasts. Amore drastic approach to induce calcium-metabolism-related skeletal resorption in this species was undertakenby Takagi & Yamada (1991, 1992) who forced the fish toregenerate half of their scales under extreme calcium-

deprivation. Under these conditions resorption of theendoskeleton was observed, although it was conducted bymononucleated osteoclasts. Although Takagi & Yamada(1991, 1992) could induce skeletal resorption under veryextreme conditions, the experiment showed that advancedteleosts do not utilise their endoskeleton as a calcium sourceunder physiological conditions. Indeed, teleosts have severalalternative sources of calcium, and resorption of theendoskeleton may be used only as a last resort (Moss,1962; Takagi & Yamada, 1991, 1992). Teleosts can obtaincalcium by intestinal absorption (the usual route for marineteleosts) or through the gills (the usual route for freshwaterspecies). ATP-driven active calcium transport through thegill epithelium functions even at low ambient calcium levelsand generally ensures sufficient calcium intake (Guerreiroet al., 2002; Perry et al., 2003). Moreover, teleosts can resorbtheir scales instead of resorbing elements of the endoskeleton.Many studies show that in conditions of mineral shortagethe scales are preferentially resorbed (Van Someren, 1937;Garrod & Newell, 1958; Mugiya & Watabe, 1977; Weiss &Watabe, 1978; Carragher & Sumpter, 1991; Shearer, 1992;Persson, Sundell & Bjornsson, 1994; Persson et al., 1997,1998, 1999, 2000; Persson, Takagi & Bjornsson, 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 onfarmed fish show that phosphorus rather than calciumdeficiency triggers bone resorption (Roy et al., 2002; Lall &Lewis-McCrea, 2007). Available phosphate in fresh waterand sea water is low and for proper mineralization of theskeleton 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; Guerreiroet al., 2007). Consequently, a shortage of dietary phospho-rus or starvation can cause insufficient bone mineraliza-tion and can trigger bone resorption. Both processes maycontribute 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). Perssonet al. (1998, 2000) linked scale resorption in salmon duringtheir upstream spawning run to the animals’ high demandfor calcium, suggesting that this could be linked to thesynthesis of calcium-rich egg yolk protein (vitellogenin).This can only be a partial explanation, given that scaleresorption also occurs in starving upstream-running malesalmon that do not synthesise vitellogenin. In male salmon,scale resorption is possibly triggered by the rapid growth ofsome skull bones and by the development of a bony hookat the tip of the lower jaw (an antler-like secondary sexualcharacter). Moreover, breeding salmon continue to replacetheir teeth (Tchernavin, 1937; Vladykov, 1962; Witten,Hall & Huysseune, 2005b; Huysseune, Hall & Witten,2007). Male salmon that survive spawning resorb the tip ofthe hook and remodel its basis into regular dentary bone(Witten & Hall, 2002, 2003). Kacem et al. (1998) andKacem & Meunier (2000, 2003) linked osteoclasticresorption, osteocytic osteolysis and halastatic loss ofminerals in pre- and post-spawning Atlantic salmon tothe animals’ calcium demand. While there is no doubt that



there is increased calcium demand in female Atlanticsalmon prior to spawning, males and females undergosevere starvation during this period, and thus lack dietaryphosphorus. Therefore, and from what is known fromfarmed salmon, starvation-related phosphorus deficiencyrather 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 hormonalcontrol, and is linked to osmoregulation and dietary mineralintake, disciplines such as fish endocrinology, fish physiol-ogy, and fish nutrition are interested in fish skeletalremodelling. General information about calciotropic hor-mones, osmoregulation, and nutrition in teleosts can befound in Urist et al. (1972), Flik & Verbost (1995), Jurss &Bastrop (1995), Sasayama (1999), Perry et al. (2003) andLall & Lewis-McCrea (2007). Other studies focus onparticular hormones and vitamins that can influence teleostbone 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-relatedprotein (Danks et al., 1993, 2003; Trivett et al., 1999;Guerreiro et al., 2007), prolactin (Power, 2005), estrogen andtestosterone (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), andgrowth hormone (Takagi et al., 1992). It has beenemphasised for calcitonin, estradiol-17b (E2) and vitamin D,that the function of calcium-regulating factors in teleosts isnot fully understood and conflicting results have beenpublished (Wagner, Jaworski & Radman, 1997; Perssonet al., 1997; Lall & Lewis-McCrea, 2007). Different types ofbone (cellular versus acellular), different sites of calciumexchange (gills versus endoskeleton), and divergent effects ofhormones on dermal and endoskeletal elements (see below)complicate understanding of teleost calcium metabolism(Lall & Lewis-McCrea, 2007). Below we briefly describehow 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 tohave a similar effect in fish. European eels treated withcalcitonin show reduced bone resorption and increasedbone 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-dihydroxyvitamin D) in fish remains to be clearly defined particularlyfor marine species (Lall & Lewis-McCrea, 2007). Injectionof vitamin D3 into mature female European eels resulted instimulation of bone formation and a reduction ofosteoclastic resorption (Lopez et al., 1980). By contrast,

Rao & Raghuramulu (1999) observed that vitamin D3 or itsmetabolites had no effect on calcium-phosphorus homeo-stasis in the advanced teleost Oreochromis mossambicus(Mozambique tilapia).

How estradiol-17b (E2) affects bone remodelling inteleosts is difficult to understand. E2 aggravates scaleresorption in Atlantic salmon, goldfish, mummichog(Fundulus heteroclitus), and rainbow trout (Mugiya &Watabe, 1977; Carragher & Sumpter, 1991; Perssonet al., 1994, 2000) but has no in vivo effect on scaleresorption 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 withincreased scale resorption and to calcitonin withdecreased scale resorption. These hormones, however,have no significant effect on scale resorption in malemarine nibbler fish (Girella punctata) (Suzuki, Suzuki &Kurokawa, 2000). The effect of E2 on osteoclasts in mostteleosts appears to be opposite to the effect of E2 inmammals, where it is a potent inhibitor of bone resorption(Gruber et al., 2001; Heino et al., 2002). From studies onrainbow trout Persson et al. (1997) concluded that E2

increases scale resorption, but downregulates resorption ofthe endoskeleton. This would explain the opposite effectsof E2 in teleosts and mammals and would support thesuggestion that teleosts preferentially use their scalesrather than their endoskeleton as the primary mineralsource (see Section V.3a).

In mammals PTH is a major hypercalcemic factor andtriggers osteoclast-mediated bone resorption (Hurwitz,1996); in fish its role is still puzzling. The gill epitheliumof basal fish, viewed as the phylogenetic precursor oftetrapod parathyroid glands, may have functions similar tomammalian parathyroid glands (Sasayama, 1999; Loretz,2008). Despite the detection of PTH molecules in teleostsboth with acellular and cellular bone (Danks et al., 2003;Gensure et al., 2004) a hypercalcemic factor acting likemammalian PTH has not been identified in fish (Guerreiroet al., 2002; Lall & Lewis-McCrea, 2007). Studies onlungfish (Lepidosiren paradoxa) and on the Northern Plainskillifish (Fundulus kansae) demonstrated that PTH has noeffect on fish bone resorption (Clark & Fleming, 1963;Urist et al., 1972). Recently, Fleming, Sato & Goldsmith(2005) published results from zebrafish larvae suggestingthat long-term exposure to extreme overdoses of PTHreduces the mineralization of the skeleton whereasintermittent short-term exposure to PTH has an anaboliceffect. The parathyroid hormone-related protein (PTHrP),a hypercalcemic paracrine factor in mammals that bindsto 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 thatcorresponds to the amino-acid residues of the 1-38 regionof the N-terminal part of PTHrP has a hypercalcemiceffect in sea bream larvae and increases osteoclast activityin vitro in this species (Rotllant et al., 2005; Guerreiro et al.,2007). Fuentes et al. (2006) suggest that PTHrP functionsas a hypercalcemic factor controlling extracellular cal-cium levels in teleosts and counteracts the action ofstanniocalcin.



(c ) Habitat and phylogeny

Given the enormous differences in mineral content betweenfreshwater and saltwater environments it is possible thatfreshwater and marine teleosts use their skeletons differentlyin the context of mineral metabolism. Several reportssuggest that the endoskeleton of freshwater species isinvolved to a greater extent in mineral metabolism than isthe 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 theskeleton 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 tothe group of primary or secondary freshwater fishes (Myers,1938; Bermingham & Martin, 1998) (Fig. 4). For example, itis known that the fluoride content of tooth enameloid in fishis related to a pecies’ phylogenetic position but not to itsenvironment (Prostak, Seifert & Skobe, 1991; Suga, Taki &Ogawa, 1993). Frequently studied fish that inhabit freshwater such as zebrafish, carp, eel, and trout, represent lessadvanced teleosts with an osteocyte-containing skeleton(Group C in Fig. 4) whereas commonly used marine speciessuch as pufferfish, sea bream, halibut, or haddock areadvanced teleosts with a skeleton lacking osteocytes (GroupD in Fig. 4). Using the example of zebrafish (Group C inFig. 4) and medaka (Group E in Fig. 4), two small freshwaterspecies of similar appearance and widely used model

organisms, it becomes apparent how different these speciesare regarding their type of bone and their evolutionaryhistory. Zebrafish and medaka are at least 100 million yearsapart and equally distant to each other as a kangooroo anda human. The two species perform very differently when itcomes to bone resorption and bone remodelling, followingthe patterns for teleosts with cellular bone (zebrafish) andacellular bone (medaka) respectively (Ekanayake & Hall,1987; Witten et al., 2001; Takano et al., 2005). Cichlidsprovide another example of the influence of phylogenetichistory on bone remodelling and resorption. Cichlids(Group E in Fig. 4) are advanced secondary freshwater fishwith acellular bone and are closely related to marine teleostfamilies such as labrids and pomacentrids (Group D inFig. 4). Although cichlids inhabit fresh water the endo-skeleton is not used in mineral metabolism underphysiological conditions (Moss, 1962; Takagi & Yamada,1991, 1992; Witten, 1997), as in their marine relatives(Bernardi & Bucciarelli, 1999). The involvement of boneremodelling in mineral metabolism is to a large extentdetermined by the animals’ evolutionary heritage and notby its environment.

(4) Mechanical adaptation

After Wolff (1892) published the ‘‘law of bone trans-formation’’ it became acknowledged that the vertebrateskeleton displays a high degree of phenotypic plasticity,adapting its structure to changing mechanical load (see alsoBertram & Swartz, 1991; Bertram et al., 1997; Kostenuiket al., 1999; Currey, 2003; de Ricqles, Castanet &Francillon-Vieillot, 2004). The teleost skeleton can displaya considerable degree of phenotypic plasticity. It adapts tochanging external forces, and the degree of morphologicalplasticity in teleosts is assumed to be large and widespreadthroughout 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 tofeeding conditions (Meyer, 1987a,b; Huysseune, Sire &Meunier, 1994; Hegrenes, 2001). Meyer (1987a, b) showedthat the type of food can alter the external shape of theentire head in juvenile central American cichlids (Cichlasomamanaguense). Huysseune et al. (1994) demonstrated adaptiveremodelling of the pharyngeal jaw bones in response toincreased mechanical load in the cichlid Astatoreochromisalluaudi: individuals fed with snails developed more andstronger bone trabeculae in their pharyngeal jaws thanindividuals fed a soft diet (Fig. 5A,B). The presence ofvertebral deformities in farmed teleosts also illustrates thatthe internal and external shape of skeletal elements can bechanged in response to mechanical load: development ofheterotopic cartilage or heterotopic chondroid bone isthought to be the result of excess mechanical load (Kiharaet al., 2002; Kranenbarg et al., 2005; Witten et al., 2005a)(Fig. 5C). While a plastic response of teleost skeletalstructures to changing load regimes is expected, it isinteresting that the acellular bone of advanced teleosts alsoresponds to alterations in mechanical load (Meyer, 1987a,b;Huysseune et al., 1994; Hegrenes, 2001; Kranenbarg et al.,

Fig. 4. Visualisation of the relationships among phylogeny,environment, and bone type in teleosts. Basal osteichthyansand basal teleosts (Groups A and B) have bone that containsosteocytes, similar to mammalian bone. This type of bone waspreserved during a first wave of freshwater reinvasion by teleostfish (‘‘primary freshwater fish’’; Group C, e.g. zebrafish).During a long period in the marine environment acellular boneevolved and is found in almost all advanced teleosts (Group D).This derived character was maintained when advanced teleostsreinvaded the fresh water (Group E; ‘‘secondary freshwaterfish’’, e.g. medaka). Consequently, teleosts that live in freshwater (or in the marine environment) can have very differentbone types and accordingly different characteristics in respectto bone resorption and bone remodelling. MY, million years.



2005). In mammals, osteocytes and their meshwork ofinterconnected cell processes are believed to detect strainand mechanical load and consequently are viewed ascentral components guiding bone remodelling in responseto external forces (Burger et al., 1995, 2003; Bonewald,2004; Franz-Odendaal et al., 2006; Taylor et al., 2007). As inmammals, osteocytes in teleosts can form an extendedmeshwork via interconnected cell processes (Meunier &Huysseune, 1992; Meunier 2002) and presumably canfunction in the same way (Huysseune, 2000). However, thenumber and density of osteocytes as well as the number andlength of their processes can vary to a considerable degreeaccording to species and location within the bone (St�ephan,1900; Blanc, 1953; Moss, 1961a; Meunier, 1983). We do notknow how osteocyte density or interconnectivity affect theresponse to mechanical load. The fact that teleosts withacellular bone also remodel their skeleton in response toaltered mechanical load strongly suggests the existence ofalternative load detection mechanisms. Osteoblasts andbone lining cells are the obvious candidates: Vatsa et al.(2006) showed that mammalian osteoblastic and osteocyticcells in vitro are sensitive to mechanical load. Likewise,mammalian odontoblasts are considered to have sensory

capacity (Magloire et al., 2004; Allard et al., 2006). Thepossession of acellular bone could make advanced teleostssuch as medaka important model species to study non-osteocyte-mediated mechanisms of load detection. At thesame time, because of the absence of osteocytes, teleosts likemedaka may have limited use as models for human bonediseases, in contrast to recent suggestions (Wagner et al.,2003; Nemoto et al., 2007). Zebrafish with mammalian-likeosteocyte-containing bone may indeed be a better model.

(5) Fracture healing, regeneration anddisease-related remodelling

Healing of skeletal injuries is another function of skeletalremodelling. Moss (1962) convincingly demonstrated thatteleosts respond to jaw fractures with the formation ofa typical repair callus that can be subsequently mineralizedand remodelled. Although jaw fractures may not occuroften under natural conditions, injuries to the fins arecommon and require repair and/or regeneration. Thecapacity to regenerate fins is restricted to the elements ofthe dermal skeleton, the fin rays (lepidotrichia); the finendoskeleton 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. Bonetrabeculae in the medullary cavity are sparse (black asterisk). Note that tooth replacement in advanced teleosts with intramedullarytooth 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 pharyngealteeth. Note the presence of abundant bone trabeculae (black asterisk) and dense cortical bone structures. Microradiograph fromground section: scale bar ¼ 500 mm. (C) Development of heterotopic cartilage (black asterisk) to replace the notochordal tissues atthe dorsal growth zone of two opposing vertebral body end plates in Atlantic salmon (Salmo salar). This pathology is characterised byosteogenic tissue (white arrowhead) that blends into chondrogenic tissue (black arrowhead). It typifies deformed and fusing vertebralbodies, likely caused by insufficient mineralization and/or excess mechanical load. In fusing vertebral bodies the cartilage is beingremodelled into trabecular bone. See Fig. 1F for the non-pathological situation, in which cartilage is absent. Masson’s Trichromestaining: scale bar ¼ 50 mm. (D) Vertebral fusion and complete remodelling of three fused vertebral bodies into one, almostregularly shaped, element. The presence of three sets of neural and haemal arches (white arrowheads) indicates the number ofvertebral bodies involved in the fusion. Developmental studies have shown that this type of fusion is the endpoint of a pathology thatbegins with the development of heterotopic cartilage as in C. X-ray: scale bar ¼ 5 mm.



Wagner (1992) for the advanced marine teleost Salaria pavo(Blenniidae) regeneration of fin rays involves resorption ofdamaged skeletal parts by osteoclasts. Resorption isprobably an integral part of fin regeneration in all teleosts,although this process is usually not mentioned in the finregeneration literature (see Akimenko & Smith, 2007, andreferences therein). An exception to the rule that theendoskeletal parts of a fin do not regenerate is provided bythe glass knifefish (Eigenmannia virescens, Gymnotidae)(Kirschbaum & Meunier, 1981). Fish of this family havelost their caudal fin and swim using the tail, i.e. the caudalpart of the spine (Greenwood et al., 1966). If the tail is lost oramputated, the knifefish regenerates a cartilaginous rod thatundergoes perichondral ossification. Subsequently thecartilage is resorbed by osteoclasts/chondroclasts. Truevertebral bodies and the notochord are not regenerated.The ossified rod, however, appears to function like theoriginal segmented vertebral column. The animals’ fin raysregenerate completely, as is usually the case in teleosts(Kirschbaum & Meunier, 1981).

Remodelling is involved not only in repair andregeneration after trauma, it is also involved in diseasesthat affect the skeleton. Skeletal malformations, especiallymalformations of the vertebral column, are frequentlyobserved under farming conditions but have also beenreported 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 theoften recorded fusion of vertebral bodies must involve boneresorption and bone remodelling, as a primary pathology orin response to altered mechanical load (Kranenbarg et al.,2005; Witten et al., 2006). There are few reports of boneresorption connected to bone deformities. Roy et al. (2002)observed increased resorption at the vertebral column ofhaddock in association with spine malformations caused byphosphorus deficiency and Roberts et al. (2001) foundincreased bone and cartilage resorption in farmed Atlanticsalmon suffering from phosphorus deficiency. Hyperdensevertebral bodies constitute another pathology that involvesremodelling. Increased radio-density that is due to hyper-mineralization can be caused by heterotopic cartilageoccupying bone marrow spaces (Helland et al., 2006). Theobserved resorption of such cartilage suggests that it is nota permanent structure. An extreme case of remodelling isthe complete pathological fusion of vertebral bodies inAtlantic salmon (Witten et al., 2006). A similar pathologyhas also been observed in advanced teleosts with acellularbone (Sawada et al., 2006a, b). In Atlantic salmon earlystages of diseased vertebral bodies resemble compressedvertebral bodies. Both pathologies, compression and fusion,involve the metaplastic transformation of bone-formingcells in the vertebral growth zone into cells that producecartilaginous tissue instead of bone. Later this cartilage ismineralized and remodelled into bone (Witten et al., 2005a,2006). Remodelling of fusing vertebral bodies can result intwo or three units fusing into one vertebral body thatappears regularly shaped both externally and internally(Fig. 5D). Remnants of notochord tissue in the intervertebralspace are also remodelled. Despite the complete fusion of

vertebral bodies, no fusion or remodelling of haemal andneural arches occurs, suggesting that vertebral bodies andarches, that constitute developmental modules early inontogeny (Holtzer, 1951; Strudel, 1953a,b; Detwiler &Holtzer, 1956; Hall, 1977, 2005) remain as such in adultsalmon. Finally, infectious diseases can also trigger skeletalresorption in teleosts. The parasite Myxobolus cerebralis causesskeletal deformities and lysis of cranial cartilage (Halliday,1973; Garden, 1992; Kelley et al., 2004). Lysis of cranialcartilage and increased skeletal resorption resulting frombacterial infection has been reported for salmonids infectedwith Flexibacter psychrophilus (Ostland et al., 1997).

(6) Tooth formation and replacement

Teleosts, like all other non-mammalian tooth-bearingvertebrates, display continuous and lifelong tooth formationand replacement, accompanied by bone and toothresorption (Peyer, 1968; Huysseune, 1983; Huysseune &Sire, 1992a; Witten et al., 1999, 2005b; Huysseune, 2000).The plesiomorphic condition for osteichthyans is thepresence of teeth on the mandibular, hyoid and all fivemore posterior branchial arches, as well as on the bonesthat constitute the roof of the pharyngeal cavity. Thus,many basal teleosts retain large numbers of teeth onvarious skeletal elements within the buccopharyngeal cavity(Huysseune & Sire, 1998). During teleost evolution teethbecame restricted to the mandibular arch and to therearmost branchial arches. In some advanced, pharyngo-gnath, teleost families, elements of the second to fifthbranchial arches evolved into a strong second set of jaws,the pharyngeal jaws, bearing pharyngeal teeth (Vandewalleet al., 1994). In some species the pharyngeal teeth aremolariform and are used for grinding and crushing, similarto the function of premolar and molar teeth in mammals(Owen, 1845, 1854). Any tooth that is lost in a particularposition is usually replaced by a new tooth throughout theanimals’ life. In addition, growing individuals establishnew tooth positions (Huysseune & Witten, 2006, 2008;Huysseune et al., 2007). Tooth replacement and theestablishment 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 andreplacement teeth in less advanced teleosts develop on topof tooth-bearing bones (i.e. in an extramedullary position).In advanced teleosts replacement teeth develop inside thejaw bones (i.e. in an intramedullary position), to a certainextent comparable to the situation in mammals. Resorptionis an essential element of both modes of tooth formation: inboth modes the mineralized basis must first be resorbed inorder to disconnect the tooth from the bone surface(Fig. 6A). Subsequently, osteoclasts also resorb part of thejaw bone itself. It is thought that this resorption preparesthe attachment site for the new tooth (Van der heyden,Huysseune & Sire, 2000). Moreover, there is evidence thatsome teleosts and other non-amniotes resorb old teethcompletely as an alternative to shedding (Fig. 6B) (Shaw,1986; Domon et al., 2004, 2006; Witten et al., 2005a;



Huysseune et al., 2007) perhaps to retain the minerals. InAtlantic salmon, complete tooth resorption occurs inperiods of severe mineral deprivation (spawning migration),during which animals also resorb their scales (Shearer,1992; Persson et al., 1998). Partial retention of teeth hasbeen observed in the advanced teleost Pomatomus saltatrixwhere the dentine of basal parts of old teeth is incorporatedinto new jaw bone tissue (Bemis, Giuliano & McGuire,2005).

In advanced teleosts with teeth that develop deep insidethe jaw bones (intramedullary tooth formation), bone mustbe removed to accommodate the growing tooth, to allow itsoutward movement through the jaw bone and to enable itsankylosis at the top of the jaw bone (Witten et al., 1999;Huysseune & Thesleff, 2004; Bemis et al., 2005). Osteoclastscan be found on all endosteal bone surfaces that surroundthese growing teeth (Fig. 6C) (Witten et al., 1999) (see alsoSection IV.1 for osteoclast stimulation by tooth-relatedtissues).

Replacement teeth are usually initiated at the tip of anepithelial strand (successional dental lamina) that buds offfrom 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 andto initiate the new tooth inside the bone. The dentallamina penetrates the bone through a small hole(replacement pore) located adjacent to each predecessortooth, as described by Bemis et al. (2005) for the advancedteleost Pomatomus saltatrix. Subsequently the diameter of thereplacement pore decreases although later the replace-ment tooth erupts at this site by re-enlargement of thepore. After eruption from the medullary cavity the toothprogresses by remodelling of the surrounding bone into theposition of the predecessor tooth. The developmentalprocess described by Bemis et al. (2005) for P. saltatrixsuggests that replacement pores are lost during the processof tooth eruption and have to be re-established for eachreplacement tooth. The way the replacement pore iscreated has not been investigated but perhaps osteoclasts‘‘drill’’ a canal in the bone similar to the creation ofa passage for a blood vessel through a bone (Junqueira,Carneiro & Kelley, 1989; Meunier, 2002). In conclusion,tooth development in advanced teleosts requires boneresorption for: (1) the downgrowth of the dental laminainto the bone cavity, (2) to create space for the growing andoutward moving tooth, (3) to prepare the surface to allowthe 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 pulpcavity are still active (black asterisk). Epon embedding, Toluidine Blue staining: scale bar ¼ 20 mm. (B) Complete resorption of theremains 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 Oreochromisniloticus, a situation typical for advanced teleosts (see also Fig. 5A,B). Developing teeth and tooth anlagen (white asterisks) aresurrounded by many small tartrate-resistant acid phosphatase (TRAP) positive osteoclasts (red) as the resorption of the surroundingbone (black asterisks) is required for the growth and the eruption of the teeth. The shape, size, and number of osteoclasts differsconsiderably from the situation in salmon (cf. Fig. 6B). Glycol methacrylate embedding, TRAP staining counterstained withHaematoxylin: scale bar ¼ 25 mm. (D) Tooth of a shark (Scyliorhinus canicula) prior to shedding. The position of the tooth (blackasterisk) is shown on the insert (black arrowhead), the dark central area represents the mineralized mandibular cartilage. Unlike inbony fish, neither signs of tooth resorption nor signs of resorption of the bony basal plate of the tooth are observed. Sections withToluidine Blue (tooth) and von Kossa (insert). Non-decalcified methyl methacrylate embedding: scale bar ¼ 200 mm.



old tooth and (5) to move the erupting tooth into theposition of its predecessor. The latter process also occurs inless advanced teleost species such as Atlantic salmon whereapparently it does not require bone resorption. The teethdevelop on top of the jaws and movement into the properposition takes place before attachment to the underlyingjaw bone (Huysseune et al., 2007). The mechanism behindextramedullary tooth movements, most prominentlyobserved during tooth replacement in sharks, has notbeen elucidated (Peyer, 1968).

The nature of tooth-resorbing cells is still a matter ofdiscussion. Often bone-resorbing cells (osteoclasts) andtooth-resorbing cells (odontoclasts) are distinguished basedon the substrate the cells resorb and on their location,rather than on their properties. In humans (and mammalsin general), clastic cells attacking dentinal hard tissues(cementum, dentine and enamel) and located inside thepulp 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 differentnames, the structure and ultrastructure of odontoclasts andosteoclasts appear to be identical (Van der heyden et al.,2000). As in mammals, odontoclasts in teleosts can bemultinucleated, can show a ruffled border and can createdeep resorption lacunae. Both mammals and teleosts havehigh numbers of mononucleated odontoclasts (Domon et al.,1994; Witten et al., 1999; Nemoto et al., 2007). Due tocontinuous tooth replacement, tooth-bearing bones ofteleosts can contain high numbers of osteoclasts; theyrepresent, perhaps, the location with the highest osteoclastnumber (Witten, 1997).

(7) Remodelling as a lifelong process

Given that teleosts grow throughout their life, remodellingcontinues 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 ofstarvation or temporarily unfavourable environmentalconditions, the growth of adult fish is often intermittent(Geffen et al., 2002). Such discontinuous growth can beidentified using growth lines in particular skeletal elements,mostly scales and fin rays (Ferreira, Beamish & Youson,1999; Ihde & Chittenden, 2002; Meunier, 2002). Apartfrom signs of growth and growth arrest, the bone matrix ofthe fin skeleton can show signs of resorption (Castanet et al.,1993). Extensive periodic resorption also characterises thescales 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 particularskeletal elements is another phenomenon of late skeleto-genesis. This can be due to the development of secondarysexual characters, as in Atlantic and Pacific salmon(Tchernavin, 1938; Jones, 1959; Vladykov, 1962). Witten &Hall (2002, 2003) have shown that the kype – a secondarysexual character consisting of a hook on the lower jaw of

male salmon – undergoes remodelling and resorption as thestructure grows and regresses.

Another example of bone enlargement in adult fishinvolving resorption is hyperostotic bone (Smith-Vaniz et al.,1995; Meunier, Bearez & Francillon-Vieillot, 1999;Meunier et al., 2002). Hyperostosis is a localized boneexpansion through extensive membrane bone formation.The predictability and site-specificity of hyperostosis ina number of marine teleosts led Smith-Vaniz et al. (1995) topropose that hyperostosis is a regular late event in theanimals’ life and not a pathological condition (osteoma), aswas suggested previously (Schlumberger & Lucke, 1948).Equipped with the attributes of cellular bone, hyperostoticoutgrowths represent foci of extensive bone remodelling,even in marine teleosts with an otherwise quiescent acellularskeleton (Smith-Vaniz et al., 1995; Meunier, 2002).

Studies on aging fish suggest that resorption also appearsto be a part of normal senescence: Gerhard et al. (2002)observed erosive changes at vertebral bodies in zebrafish atthe end of the animals’ life span.

VI. THE EVOLUTION OF SKELETALREMODELLING

During the ontogeny of teleost fish and other extantvertebrates (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, togetherwith the many functions of skeletal resorption raises thepossibility that skeletal resorption may have evolvedtogether with the first calcified structures of the dermalskeleton. Unfortunately the fossil record does not provideclear answers: evidence for bone resorption in earlygnathostomes is scarce (Bystrow, 1959; Donoghue &Sansom, 2002) but resorption occasionally has beenidentified in heterostracans and osteostracans (agnathans)(Denison, 1952; Halstead Tarlo, 1964). Perhaps skeletalresorption was not required in early vertebrates where thedermal skeleton was composed of small (microsquamose)finite-growing odontodes. These small odontodes werecompletely shed (like teeth) and replaced by new elements,but not resorbed (Reif, 1982). Extant chondrichthyans showsuch type of squamation with placoid scales resembling thedermal skeletal elements of heterostracans (Reif, 2006).Interestingly extant sharks apparently do not have skeletalresorbing 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 thatwould involve at least the action of chondroclast-like cells.The possible existence of chondroclasts in sharks isdiscussed by Officer, Clement & Rowler (1995). Clement,Officer & Dykes (1992) emphasised the absence of anyresorption in shark vertebral centra during their lifespan.Glowacki et al. (1986) demonstrated that the leopard shark(Triakis semifasciata) does not resorb implanted bone particlesand Ashhurst (2004) showed that the dogfish (Scyliorhinusspp.) does not repair cartilage. Glowacki et al. (1986),



Ashhurst (2004) and Clement et al. (1992) found no evidenceof osteoclasts or chondroclasts. Instead, connective tissuedeveloped around implanted bone particles and damagedcartilage. While studying tooth development in the dogfish(Scyliorhinus canicula), Huysseune & Witten (2008) examinedthe shedding of teeth (unpublished data). Like otherinvestigators (Peyer, 1968; Reif, 1982) they did not observeany signs of macrophages or osteoclasts that could resorbteeth or the acellular bone of the tooth basal plate prior toshedding (Fig. 6D).

The complete shedding of dermal skeletal elements duringgrowth bears some resemblance with moulting, the processused by invertebrates to enlarge their exoskeleton. Bycontrast, 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 tomacrosquameric dermal plates, the formation of a secondaryodontode layer, teeth that become ankylosed to themineralized jaws, or the growth of a solid head capsulewould require bone resorption. Also the enlargement of bonetubes should always require bone resorption (Witten &Villwock, 1997; Witten et al., 2001). However some fossilagnathans (galeaspids and osteostracans) apparently avoidedgrowth-related bone resorption by developing a head capsuleonly late in adulthood (Reif, 1982).

Irrespective of skeletal growth and the replacementmodes of dermal skeletal elements, resorption would havebeen an inherent characteristic of the mineralized skeletonif it has a role in mineral homeostasis. Carroll (1988)considered several hypotheses about the function of theearly dermal skeleton: protective armour; insulation forelectroreceptors; calcium storage; and as a phosphorusreserve. Halstead Tarlo (1964) argued that the observedvariability of dermal skeletal plates in Palaeozoic vertebratescould indicate that the dermal skeleton functioned asa phosphorus reservoir, since phosphorus is a limiting factorin many ecosystems. As discussed above, the mineralmetabolism of extant teleosts would tend to support this.Unlike calcium, that can be obtained from ambient watervia the gills, food is the main source of phosphorus in bonyfish (Simmons, 1971; Fenwick, 1974; Dacke, 1979). Ifancestral vertebrates had a similar physiology to present-day bony fish, periods of starvation would represent periodsof phosphorus shortage. Suggestions that the early skeletonfunctioned as a calcium reservoir cannot be excluded butthere is little evidence that extant teleosts suffer fromcalcium deficiency under physiological conditions (Weiss &Watabe, 1978, 1979; Witten, 1997; Roy et al., 2002). Thereis, however, little evidence of bone resorption in some earlygnathostomes (Bystrow, 1959; Donoghue & Sansom, 2002)and the absence of resorption of calcified structures inextant chondrichthyans argues against a metabolic functionof the skeleton, unless extant chondrichthyans havesecondarily lost this capacity. Considering the trend towardsloss of osteocytes and exclusion of the skeleton frommetabolism in advanced teleosts (Fig. 4) it is not impossiblethat a similar evolutionary process could have occurred inextant chondrichthyans.

Different mechanisms of bone resorption exist and not allresorbing cells derive from the macrophage lineage. Still, it

is possible that the absence of typical multinucleatedosteoclasts in extant chondrichthyans is related to theabsence of key factors known to regulate macrophagedevelopment and osteoclast activation in mammals (seeSection 3.1a), factors that are also present in teleosts (seeSection IV.1). However, chondrichthyans are known toexpress the transcription factor PU.1 and to possessmonocytes, macrophages, an adaptive immune system,and bone marrow-like haematopoietic tissue (Mattisson &Fange, 1982; Wolke, 1992, Anderson et al., 2001). Evenextant agnathans have monocytes and macrophages thatare morphologically similar to gnathostome cells and theirhaematopoietic tissue is very similar to ‘bone marrow’ inamniotes (Amemiya, Saha & Zapata, 2007). If limited or noresorption capacity in extant chondrichthyans is a conservedcharacter, then development of the capacity to resorb theskeleton could be linked to the evolution of particularextracellular skeletal matrix molecules (Fig. 7). While thebasic skeletal tissue types and cells evolved early (Hall &Witten, 2007) some skeletal extracellular matrix proteinsarose later, such as collagen II, SPARC (secreted protein,acidic, cysteine-rich ¼ osteonectin) and osteocalcin (BoneGla Protein, BGP). Lamprey and hagfish cartilages lackcollagen type II and have instead different classes of fibrousproteins (lamprins, myxin) (Cole & Hall, 2004; Hall &Witten, 2007). Secretory calcium-binding phosphoproteins(SCPP) and osteocalcin 1 arose only after the divergence ofcartilaginous and bony fish (Kawasaki, Suzuki & Weiss,2004; Kawasaki & Weiss, 2006; Sire, Delgado & Girondot,2006; Laiz�e et al. 2006a,b) (Fig. 7) and both proteins play arole in bone resorption. The Arg-Gly-Asp motif of SCPPbinds to osteoblasts and to osteoclasts via the integrin sur-face receptor, which in turn evokes intracellular signals thatalso regulate resorption (Kawasaki et al., 2004). Osteocalcinrecruits osteoclasts and osteoclast precursors to the bonesurface for resorption (Chenu et al., 1994). Several factorsare known to stimulate osteoclasts, including the presence ofnon-collagenic matrix proteins, the presence of osteocytes,and the presence of PTH. Haematopoietic tissue located inthe bone marrow is, however, not required, given that giantosteoclasts and mononucleated osteoclasts occur in teleostsdespite the fact that they lack haematopoietic tissue in thebone marrow.

Another question concerns the nature of early ancestralosteoclastic cells. Mammalian and avian osteoclasts arewidely believed to possess general features of skeletalresorbing cells: multinuclearity, a deeply folded apical cellmembrane (ruffled border), and the creation of resorptionlacunae (Chambers, 1985; Parfitt, 1988). However, skeletalresorbing cells of extant bony fish must not express thesefeatures 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 whilemononucleated 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



primitive type of bone resorbing cells. Interestingly, func-tional osteoclasts without a ruffled border and that do notform Howship’s lacunae have recently been described byKemp (2003) in the extant Australian lungfish Neoceratodusforsteri. We observed similar mononucleated osteoclaststogether with multinucleated odontoclasts in the grey bichir(Polypterus senegalus) (P.E. Witten & A. Huysseune, unpub-lished data). Several arguments support the idea thatmononucleated cells may represent a primitive type ofbone resorbing cell: (a) during ontogeny multinucleated cellsarise through the fusion of mononucleated cells (Parfitt,1988). (b) During teleost development, resorbing mono-nucleated cells precede the appearance of multinucleatedcells (Sire et al., 1990; Witten et al., 2001). (c) In zebrafish,mononucleated osteoclasts occur together with the firsttraces of mineralized bone and teeth (Huysseune et al.,1998; Witten et al., 2001), and are replaced only later bymultinucleated osteoclasts. (d ) Mononucleated cells arepresent in basal extant osteichthyans (sarcopterygians andactinopterygians). (e) Mononucleated osteoclasts utilise thesame enzymes for resorption as multinucleated osteoclasts(Witten et al., 1999, 2000). ( f ) Mononucleated cells resorbscales in salmonids, skeletal elements that belong to thephylogenetically oldest part of the animals’ mineralizedskeleton. If early bone resorbing cells were mononucleated,responsible for a smooth type of resorption, then a lack ofrecognisable multinucleated bone resorbing cells or theirtraces (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 thetraces of typical multinucleated cells: resorption withoutmultinucleated osteoclasts, and therefore no resorptionlacunae, may not be detected (Witten et al., 2004).

VII. CONCLUSIONS

(1) The study of teleost fish reveals that an aquatic liferequires skeletal remodelling for the same reasons as interrestrial vertebrates: growth, replacement of skeletal tissues,mechanical adaptation, tooth eruption and skeletal repair.

(2) Important differences exist, nevertheless, between fishand tetrapods regarding remodelling and its control. Boneresorption in the majority of teleost species is carried out bysmall mononucleated osteoclasts instead of multinucleatedgiant cells. The prevalence of mononucleated over multi-nucleated osteoclasts appears to coincide with the evolu-tionary disappearance of osteocytes – a derived characterwithin the teleost lineage. Other peculiarities of teleost boneremodelling likely represent conserved characters asadaptations to an aquatic life. Examples are a phosphorus-rather than calcium-driven bone metabolism, a lack ofhaematopoietic bone marrow tissue and use of thepostcranial dermal skeletal elements (scales) as a mineralreservoir.

Fig. 7. Evolution of the main skeletal tissue types and extracellular matrix components superimposed on a phylogenetic tree ofmajor vertebrate groups (modified after Romer, 1970). Early vertebrates (Agnatha) possessed bone, dentine and enamel/enameloidin 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 alreadypresent in some agnathans and is present in the basal plate of shark placoid scales and teeth. The acellular bone of teleosts ishowever a derived feature as their ancestors (Osteichthyes and Actinopterygii) had cellular bone. The cellular bone of mammals isa primitive (conserved) character. MGP, matrix gla protein; OC1, OC2, OC3, osteocalcin isoforms 1, 2, 3.



(3) A number of characteristics peculiar to skeletalremodelling in teleost fish clearly challenge our understand-ing of mammalian bone remodelling. The widespreadoccurrence of small mononucleated osteoclasts encouragesus to reconsider the importance of mononucleated boneresorbing cells in humans. The fact that the majority ofteleost fish regulate skeletal remodelling without anosteocytic network and without haematopoietic bonemarrow tissue could provide a model for the study ofalternative pathways of osteoclast activation. Understand-ing how a bone functions without osteocytes will allowprogress in our understanding of osteocyte function.

(4) Biomedical research increasingly uses species such aszebrafish and medaka as models for studying skeletaldiseases and regeneration. Aquaculture-related researchfocuses on species such as salmon or sea bream in order tounderstand the cause of frequent skeletal malformations. Inall cases, skeletal remodelling is part of the observedprocesses. Understanding the subdivision within teleostsbetween basal species with cellular bone and advancedspecies with acellular bone and their particular character-istics regarding skeletal remodelling, is therefore of greatimportance. Many teleost fish appear superficially similarbecause of their streamlined body shape (e.g. zebrafish andmedaka), but their lineages diverged 100 million years ago.For instance, salmon or zebrafish (basal teleosts, cellularbone, ample remodelling, many multinucleated osteoclasts,extramedullary tooth development) are very different frommedaka 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 valuableand helpful comments. We also thank our colleaguesLeonor Cancela, Brian K. Hall, Santosh Lall and Jean-Yves Sire for inspiring discussions. A.H. acknowledgesa grant from the FWO-Vlaanderen n° 3G0040.08, P.E.W.and A.H. acknowledge funding from the NorwegianResearch Council (Project No. 172483/s40) and fromCOST Action B23, ‘‘Oral facial development andregeneration’’.

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