DEVELOPMENTAL DYNAMICS 244:955–972, 2015 DOI: 10.1002/DVDY.24293

RESEARCH ARTICLE

Expression of SPARC and the Osteopontin-like Protein During Skeletal Development in the Cichlid Fish Oreochromis mossambicus a

Jochen Weigele,1,2* Tamara A. Franz-Odendaal,2 and Reinhard Hilbig1 1

Zoological Institute, University of Stuttgart-Hohenheim, Stuttgart, Germany Department of Biology, Mount Saint Vincent University, Halifax, Nova Scotia, Canada

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2

Background: Bones are mainly composed of calcium hydroxyapatite and a proteinous matrix. In this study, we focus on the bone matrix proteins, the fish osteopontin orthologous protein (osteopontin-like protein; OP-L) and SPARC, because the current knowledge regarding their expression is fragmentary or contradictory. Results: We first provide a comprehensive and detailed description of skeletal development in the cichlid fish Oreochromis mossambicus. Following this, we analyzed the expression pattern of OP-L and SPARC in detail during development. OP-L expression was only found in tissues that undergo ossification (i.e., developing bones and teeth). Furthermore, we show that there is a fundamental difference in cartilage formation of the splanchnocranium and all other cartilages, concerning SPARC expression. Significantly, we show that the initial calcification of cranial bones occurs simultaneously with the expression of OP-L and SPARC in the osteoblast-like cells, which appear early in development. Conclusions: The difference in SPARC expression during chondrogenesis of the splanchnocranium is likely based on its different evolutionary history compared with the dermatocranium and chondrocranium. Moreover, our results suggest a co-occurrence of the initial calcium deposition and bone matrix protein expression during osteogenesis. Overall, this study enhances our understanding of fish skeletal development and evolution. Developmental Dynamics C 2015 Wiley Periodicals, Inc. 244:955–972, 2015. V Key words: Oreochromis mossambicus; SPARC; osteopontin-like protein; bone matrix protein Submitted 9 March 2015; First Decision 13 May 2015; Accepted 16 May 2015; Published online 22 May 2015

Several proteins are involved in the formation of vertebrate bones – these include collagenous and noncollagenous proteins. The major noncollagenous bone matrix proteins identified in mammals are osteopontin (OPN), osteonectin (SPARC), and osteocalcin. Among these, OPN is thought to play a major role in mammalian osteogenesis and is involved in diverse functions, such as the formation and remodeling of calcified tissues and the activity of hypertrophic chondrocytes. OPN is a phosphorylated sialic acid rich protein (Prince and Butler, 1983) containing an adhesive amino acid sequence motif that can interact with several integrins (Liaw et al., 1995). It was originally isolated from bone marrow (Franzen and Heinegård, 1985) and is expressed in osteoblasts, osteocytes, and also in osteoclasts (Weinreb et al., 1990; Merry et al., 1993; Hirakawa et al., 1994; Takano-Yamamoto et al., 1994). It is thought that osteopontin plays major roles in the formation, remodeling and resorption of mammalian bone tissue (Butler, 1989; Denhardt and Guo, 1993; McKee et al., 1993; Terai et al., 1999). It is also involved in tooth development where

it is present in cementoblasts and cementocytes (Bronckers et al., 1994). However, several studies report that osteopontin can also be detected in mammalian soft tissues such as the inner ear, kidney, placenta, gallbladder, blood, brain, and testis, where it serves different functions (Nomura et al., 1988; Hudkins et al., 1999; Shin et al., 1999; Luedtke et al., 2002). In contrast to mammalian OPN the orthologous protein in fish, the osteopontin-like protein (OP-L), is mainly expressed in calcified tissues (bones and teeth, Laue et al., 2006; Kawasaki and Weiss, 2006; Fonseca et al., 2007; Kawasaki, 2009; Venkatesh et al., 2014). Some researchers describe its expression restricted to only a few soft tissues (Fonseca et al., 2007), whereas others report it present in several soft tissues: e.g., ovary (during ovulation), testis, and in small amounts in the kidney, gills, brain, and skin of trout (Bobe and Goetz, 2001). Moreover, OP-L seems to function specifically in cardiac tissue during zebrafish development (Just et al., 2011; Huang et al., 2012; Banjo et al., 2013). The inconsistency among these studies might be due to the focus on selected developmental stages along with the focus on specific tissues and the analysis methods used for adult tissues.

*Correspondence to: Jochen Weigele, Mount Saint Vincent University, Department of Biology, 126, 166 Bedford Highway, Halifax, Nova Scotia, Canada b3m2j6. E-mail: [email protected]

Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy. 24293/abstract C 2015 Wiley Periodicals, Inc. V

Introduction

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Fig. 1. Early appearance of bone calcification, cartilages, and OP-L expression in selected developmental stages (st). At stage 13, calcification in the skull bones (maxillary, dentary, ceratohyale, the cleithrum and fin ray primordia of the caudal fin occurs. At stage 14, calcification in the vertebrae (arrowheads), the hypural and urohyal is present. At stage 15, calcification in the fin rays of the dorsal, anal and pectoral fins and at stage 23, calcification in the pelvic fin is present. At stage 13, all cartilages of the fish skull are present (Meckel’s cartilage, cartilage of the branchial arches, ceratohyal, and auditory capsule), also the cartilage of the neural and haemal arches of the vertebrae, scapula-coracoid and the urohyal. This is followed by the cartilages of the hypurals at stage 14 and radials at stage 15. OP-L expression in the branchiostegal rays (arrowhead) and caudal fin is observed at stage 13, followed by the fin rays of the dorsal, pectoral and anal fin at stage 17 and the pelvic fin at stage 23. SPARC expression was not shown due to the expression in the dermis, which is overlying the labeling in the bones. Scale bars ¼ 2 mm.

SPARC on the other hand is thought to be a multifunctional protein that has a high affinity for calcium hydroxyapatite (Ca5(PO4)3(OH)) and promotes crystal formation when it is bound to collagen (Termine et al., 1981a, 1981b; Brekken and Sage, 2001). It is dynamically expressed in skeletal and nonskeletal tissues from early development to adulthood (Est^ev~ao et al., 2006; Renn et al., 2006; Rolland et al., 2008). Moreover SPARC supports the extracellular matrix, mediates the activities of a wide range of growth factors (Brekken and Sage, 2001), and functions mainly in cell–matrix interactions (Gilmour et al., 1998; Bradshaw et al., 2002, 2003; Brekken et al., 2003; Delany et al., 2003; Eckfeld et al., 2005; Rolland et al., 2008), and it especially plays a role in the calcification of fish otoliths, bones, teeth, and scales. In fish, SPARC is involved in the mineralization of perichondral and dermal bones (Renn et al., 2006; Rotllant et al., 2008; Li et al., 2009). However, the interpretation of whole-mount in situ hybridizations of SPARC in bones is often difficult in fish because of the expression of SPARC in the epidermis and the connective

tissues that overlie the more internally located bones such as the parasphenoid (Li et al., 2009). Holland et al. (1987) and Salonen et al. (1990) reported that SPARC was expressed in the ameloblasts and odontoblasts of fully developed teeth. It is also expressed in fish scales, another mineralized tissue. Here SPARC is expressed in the scale-forming cells, the scleroblasts (Lehane et al., 1999; Redruello et al., 2005; Renn et al., 2006; Iimura at al., 2012), which undergo mineralization. In summary, SPARC can be found in nearly every organ/tissue during fish development (including brain, kidney, liver, heart, gill, spleen, skin, musculature, eye, ear, bones, tooth, notochord, and cartilage; Renn et al., 2006). A continuous detailed spatial expression analysis of OP-L and SPARC during fish embryonic and larval development is currently missing. Here we focus our study on a cichlid fish, the mozambique tilapia (Oreochromis mossambicus). This fish-taxon was chosen as the experimental animal because cichlids have a long evolutionary history (Meyer et al., 1990), exhibit remarkable

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Fig. 2. Cartilage and bone development in the skull of Oreochromis mossambicus in selected developmental stages (lateral view). Cartilage development stained by alcian blue, calcification by alizarin-complexone, and mineralized bone tissue by alizarin red. ac, auditory capsule; ag, angulary; at, articulary; ba, branchial arches; boc, basioccipital; bp, basal plate; brs, branchiostegal rays; ch, ceratohyal; cl, cleithrum; dn, dentary; e, eye; ect, ectopterygoid; ep, ethmoid plate; epb, epiphyseal bar; f, frontal bone; hm, hyomandibular; iop, interopercular bone; mc, Meckel’s cartilage; mx, maxillary; ns, nasal septum; oa, occipital arch; op, opercular bone; ot, otoliths; pop, preopercular bone; pq, palatoquadrate; ps, parasphenoid; px, premaxillary; qu, quadrate. sc, scleral cartilage; sco, scapula-coracoid; soc, supraoccipital; sop, subopercular bone; t, trabecula carnii; tc, travecula communis; tma, taeniae marginales anterior; tmp, taeniae marginales posterior. Scale bars ¼ 50 mm.

Fig. 3. Schematic diagram depicting the constitution of the splanchnocranium (A) at developmental stage 15 and dermatocranium (B) at stage 23. Abbreviations as in Figure 6.

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Fig. 4. Expression of OP-L in the development of the pectoral girdle and vertebrae. A: Dorsal view of head (stage 13), early appearance of the cleithrum (white arrowheads) stained by alizarin-complexone, pair of otoliths (ot), and the basal plate (bp) are visible. B,C: Frontal sections of the lateral occipital. Initial expression of OP-L (B, arrowhead) and SPARC (C, arrowhead) in the cleithrum, and the basisphenoid (bs) and yolk (y) are shown. D: Frontal section of the heart region, expression of OP-L in the cleithro-coracoid (arrowheads) extends anteriorly between the hypobranchial muscle (hym) and ventrolateral atrium (at). E–G: OP-L and SPARC expression in the developing vertebrae. E,F: Frontal sections of the developing vertebrae, lateral part, early appearance of OP-L (E) and SPARC (F) in osteoblast-like cells (arrowheads). Note the position of the notochord (nc) and spinal cord (sc). G: Lateral view of the tail region, expression of OP-L in the posterior vertebrae. The expression is located median of the haemal- (white arrowheads) and corresponding neural arch (black arrowheads). Scale bars ¼ 100 mm.

morphological, behavioral, and ecologic diversity, and are well known examples of explosive evolution and adaptive radiation (e.g., Fryer and Iles, 1972; Kocher, 2004). In particular, the skull of the cichlid fish, including its oral jaws exhibits great diversity due to adaptation of feeding behaviors to various environments (e.g., Fryer and Iles, 1972; Liem, 1991). The vertebrate skull as a composite structure is highly complex and originates from three different phylogenetic sources, the splanchnocranium, chondrocranium, and the dermatocranium. We analyzed the complete skeletogenesis, with focus on skull development in relation to the evolutionary background of these three cranial regions and compared these with several post cra-

nial bones. Moreover, we correlate these data with the initial expression of the bone matrix proteins OP-L and SPARC.

Results Early Appearance of the Fish Skeleton The first cartilage detected during development of the cichlid Oreochromis mossambicus is the basal plate of the chondrocranium at late stage 11 just before hatching. All the main cartilages of the chondrocranium and splanchnocranium are present at stage 12. The early cartilaginous skull is almost completely

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Fig. 5. Summary of data for the axial skeleton and fins during cichlid fish development. Grey: OP-L; green: SPARC; blue: cartilage (alcian blue); yellow: calcification (alizarin-complexone); red: mineralized bone tissue (alizarin red).

formed by stage 13 (Fig. 1). These cartilages enlarge and undergo partial endochondral ossification as development proceeds (Fig. 1). Calcification starts at stage 13 in bones of the dermatocranium (maxillary, mx; premaxillary, px; parasphenoid, ps; opercular bone, op; branchiostegal rays, brs), splanchnocranium (dentary, dn), pectoral girdle (cleithrum, cl), and in the fin ray primordia of the caudal fin (Fig. 1). During the later phases of stage 13 of cichlid development, further calcification in these bones and in the bones of the chondrocranium and axial skeleton continues. At stage 14, the first calcification of the vertebrae is detected followed by the formation of the epurals (eu) and hypurals (hu) of the caudal fin (cf). This is followed by the fin rays of the pectoral fin (pf) at stage 15 of cichlid development and the fin rays of the dorsal (df) and anal fin (af) at stage 17. By stage 23, the fin rays of the pelvic fin (plf) are also calcified (Fig. 1). The first evidence of endochondral ossification (i.e., cartilage replacement) is observed at stage 17 and this process continues throughout further development. Cartilage formation in the splanchno- and chondrocranium mainly takes place from stage 12 onward and by stage 13 all major cartilages have developed. After further growth of the cartilages, perichondral ossification replaces the cartilages from

stage 17 onward (Fig. 2). The splanchnocranium of vertebrates supports the gills and in addition, some of these elements contribute to the jaw and the hyoid apparatus (Fig. 3A). Here we investigate most of the bones of the splanchnocranium, including Meckel’s cartilage (mc), the quadrate, the articular (at), the epipterygoid, the hyomandibular (hm), the symplectic, the interhyal, the ceratohyal (ch), the hypohyal, the basihyal, the pharangobrachyal, the epibrachial, the ceratobrachial, the hypobrachial, and the branchial arches (ba). All bony elements of the splanchnocranium develop rapidly and attach to their respective underlying cartilages between stage 13 and 14 in Oreochromis mossambicus (Fig. 2). The chondrocranium is located beneath the brain and also supports it. It is generally made up of the occipital bones, the mesethmoid bone, the ethmoid bones, the sphenoid bones, and the bones of the otic capsule. The calcification of the chondrocranium starts at stage 14 in the basioccipital (boc) and is followed by the sphenotic bone of the otic capsule (stage 16) and finally the supraoccipital bone (soc) at stage 20 (Fig. 2). The bones of the dermatocranium form most of the outer casing of the skull and are composed of dermal bones (Fig. 3B). It encases most of the chondrocranium, together with the contributions from the splanchnocranium. Of interest, this region develops in a different temporal series. The bones of the dermatocranium are divided into the bones of the braincase (premaxillary, px; maxillary, mx; nasals; lacrimal; prefrontal; postfrontal; postorbital; jugal; intertemporal; supratemporal; tabular; squamosal; quadrojugal; frontal, (f) parietal; postparietal; vomer; palatine; ectopterygoid, (ect); pterygoid; parasphenoid, ps), in the bones of the mandible (dentary, dn; splenials; angular, ag; surangular, prearticular and the coronoids), and in the bones of the opercular series (branchiostegal rays, brs; preopercular, pop; interopercular, iop; opercular, op and subopercular, sop). In general, skull formation and the appearance of cartilages and bones of Oreochromis mossambicus are broadly similar to the previously described skull formation of the Nile tilapia O. niloticus (Fujimura and Okada, 2008).

Expression of OP-L and SPARC During Axial Skeletal Development The expression of OP-L (Fig. 1) and SPARC appear together within the calcification of the bony skeleton elements. The first expression of OP-L is detected at stage 13 in the cleithrum (cl), a dermal part of the pectoral girdle, at the same stage as the first calcification is observed (Figs. 4A,B, 5). At stage 14, SPARC expression appears and the first mineralized bony tissue is observed (Figs. 4C, 5). As development proceeds, the cleithrum fuses to the coracoids and this complex expands posteriomedially. These bones express OP-L at stage 20 (Fig. 4D). In the developing vertebrae, expression of OP-L and SPARC emerges at stage 14 in flattened osteoblast-like cells at the same locations where calcification staining is observed (Figs. 4E,F, 5). At the end of stage 25, in the posterior vertebrae, OP-L expression starts dorsally and ventrally of the vertebral bodies corresponding to the haemal neural arches (Fig. 4G). This expression was likely induced by shear force in this region during swimming movement. At hatching (stage 12), diffuse staining of OP-L expression is present in osteoblasts of the mesenchyme at the origin of the bony fin rays of the caudal fin (Fig. 6A). During further development (stage 13 up to stage 23), OP-L expression and also SPARC

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Fig. 6. Expression of OP-L and SPARC in the development of the segmented fin rays (lepidotrichia). A: Lateral view of the caudal fin at stage 12, expression of OP-L in the mesenchyme of the fin rays (black arrowhead), white arrowhead: notochord. B,C: Lateral view of the caudal fin at stage 12, association of OP-L (B) and SPARC (C) with developing fin rays. The first developing actinotrichium (A1) is located in the middle of the caudal fin. All following actinotrichia emerge alternately dorsal and ventral (A2–A7). D: Transverse section of the caudal fin. Expression of OP-L located laterally of the actinotrichia (white arrowheads). E,F: Expression pattern in the final arranged caudal fin at stage 15. Expression of OP-L (E) and SPARC (F) is restricted to the lepidotrichia. G,H: Back view of the dorsal fin, OP-L expression located in the mesenchyme of the lepidotrichia (arrowheads). Back view (G) and section (H) of the dorsal fin, OP-L expression is located in the mesenchyme of the lepidotrichia (arrowheads), laterally of the differentiating actinotrichia. I: Section of a fully developed lepidotrichium (pectoral fin), OP-L is located within the lepidotrichia (arrowheads) and only partly in the external mesenchyme. Scale bars ¼ 100 mm.

expression (after stage 13) are associated with both lateral sides of each single actinotrichia of the caudal fin (Fig. 6B,C). This expression is located in the osteoblasts that cover the developing actinotrichia (Fig. 6D). The first developing actinotrichium (A1) is located in the middle of the caudal fin. All following actinotrichia emerge alternately dorsal and ventral of A1 and the respective successor is slightly shorter than the proceeding one. OP-L and SPARC expression precedes the formation of these structures (Fig. 6B,C). The development of the actinotrichia in the caudal fin is completed at stage 15 (Fig. 6E,F). The developmental sequence

in the other fins starts with the pectoral fin at stage 15 and is followed by the dorsal (Fig. 6G–I) and anal fin (at stage 16) and the pelfic fin at stage 23 (Figs. (1 and 5)). The maturation of the actinotrichia is characterized by the formation of two bony hemisegments, which can be stained early during osteoblast formation by OP-L (Fig. 5). Furthermore, in all actinotrichia ossification starts at the respective proximal part and proceeds to the distal part; this process is accompanied by the expression of OP-L and SPARC. During and after completion of this process, no further changes in OP-L expression are observed.

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Fig. 7. Expression of SPARC during cartilage formation of the splanchnocranium. A: Lateral view of the head region at stage 10, SPARC expression (stained by whole-mount in situ hybridization) in the mesenchyme of the splanchnocranium (arrowheads). B: Transversal section of the anterior head region, SPARC expression in the ventral (arrowheads) and lateral parts of the developing splanchnocranium. C: High magnification showing the first detectable prechondroblasts (arrowhead) in the lateral mesenchyme. Scale bars ¼ 100 mm.

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Expression of OP-L and SPARC During Skull Development SPARC is expressed in the developing skull during early chondrogenesis and later during osteogenesis. The first expression of SPARC associated with chondrogenesis occurs at stage 10 in the mesenchyme of the developing splanchnocranium (Fig. 7A), with stronger staining ventrally compared with dorsally and laterally (Fig. 7B,C). The expression of developing cartilages is scattered and shows no distinct expression pattern in relation to defined structures (including Meckel’s cartilage, the quadrate, the hyomandibular, sympleptic, and branchial arches). Moreover, the first preosteoblasts are histologically detectable within the mesenchyme including with SPARC expression at stage 10 (Fig. 7C). At stage 12, expression of SPARC has disappeared in the chondrogenic mesenchyme of the splanchnocranium with the development of chondrocytes, which no longer express SPARC. In the early chondrogenesis of all other skull elements, no association with SPARC was observed, not even in the chondrocranium. OP-L expression was detectable first, followed by the presence of SPARC expression, which was expressed concurrently with calcification. Only after this, is the mineralized bony tissue detected by alizarin red staining. The initial expression of OP-L and then SPARC in developing bones occurs in osteoblast-like cells, which appear together with the underlying cartilages. The cartilage is later mostly replaced by bone by means of endochondral ossification. Our data strongly suggests that the bone matrix is produced by these osteoblast–like cells (compare Figs. 8 and 9). In the quadrate, OP-L is initially expressed at stage 13 in the osteoblast-like cells, again followed by SPARC expression and calcification in the next stage of development, stage 14 (Fisg. 8A–C, 9). In both the hyomandibular and the ceratohyal, OP-L, SPARC, and calcification emerge together at stage 14 in osteoblast-like cells of the hyomandibular bone (Figs. 8D–F,G–I, 9). In the branchial arches, OP-L occurs together with the initial calcification (at stage 13) in the osteoblast-like cells of the first, second, and third branchial arch (Figs. 8J,K, 9). These cells partly surround the branchial cartilages. The initial SPARC expression is detected in the arches one stage later (Figs. 8L, 9). Beginning at stage 14, the branchial arches start differentiating into the basiobranchial cartilage and the ceratobranchial cartilage. In the basiobranchial bones (the most ventral elements) and in the cera-

tobranchials, OP-L is first expressed at stage 14 and is followed by SPARC and calcification at stage 15 again in the osteoblastlike cells (Figs. 8M–R, 9). Additionally, we observed strong expression of OP-L and SPARC in the basiobranchials and ceratobranchials after stage 17 (Fig. 10L). In these bones, the expression of SPARC and OP-L is clearly associated with tooth development. Moreover, OP-L is also observed in various osteoclasts (Fig. 10L). Expression in the oral and pharyngeal teeth is discussed later (Fig. 10A–K). Most bones of the chondrocranium appear relatively late in development (after stage 25). Of particular interest here are the three earliest appearing bones (namely the basioccipital, sphenotic, and supraoccipital bone; Figs. (11 and 12)). The first expression of OP-L associated with the basioccipital bone is detected at stage 14, together with the first calcification. This is shortly followed by SPARC at stage 15, and the first detectable mineralized bone tissue at stage 16 (Figs. 11A–C, 12). The initial calcification of the sphenotic bone is detected at stage 16 together with the first SPARC expression. Only in the next stage is OP-L detected and the first mineralized bone present (Figs. (2 and 11)D–F, 12). In the supraoccipital bone, the expression of OP-L and calcification first appear at stage 20, followed by SPARC and mineralized bone tissue later (Figs. 11G–I, 12). In the developing dermatocranium, initial OP-L and SPARC expression is detected together with the onset of calcification while the mineralized bony tissue follows rapidly (compare Figs. (13 and 14), and 15). The first expression of OP-L and SPARC in the maxillary bone appears at stage 12 in the mesenchyme where osteoblasts are forming. This process advances rapidly and the first calcification also appears at this stage (Figs. 13A–C, 14). The first evidence of bony tissue is detected shortly after this process at stage 13 (Fig. 2). The premaxillary and dentary form simultaneously with the maxillary bone and have similar OP-L and SPARC expression patterns (Figs. (2 and 13)A–F, 14). In the angular bone, OP-L and SPARC expression and the initial calcification are all detected at stage 14 (Figs. 13J–L, 14). In contrast to the previous bones, the bony tissue appears only later at stage 16 (Fig. 14). The parasphenoid is a dermal bone in the palatine and forms slightly later than the other described bones. The first mesenchymal expression of SPARC is visible at the end of stage 12) where the trabecular cumunis split into the paired trabecular

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Fig. 8. Appearance of OP-L and SPARC expression and bone calcification in selected bones of the splanchnocranium in selected developmental stages. Calcification column: Localization and calcification of the corresponding bones (arrowheads) in lateral (A,D,J,P) and ventral view (G,M). OP-L and SPARC column: Initial expression in the corresponding bones (arrowheads) on transversal sections. Quadrate bone: OP-L (B) and SPARC (C) expression (arrowheads) is located lateral of the underlying palatoquadrate cartilage in the osteoblast-like cells. Hyomandibular bone: Lateral of the hyomandibular cartilage OP-L (E) and SPARC (F) expression (arrowheads) is detected in the osteoblast-like cells. Ceratohyal bone: The ceratohyal cartilage is enclosed by the osteoblast-like cells, which are OP-L (H) and SPARC (I) positive (arrowheads). Branchial arch: OP-L expression (K, arrowhead) and SPARC (L, arrowhead) expression, both detected in the osteoblast-like cells enclosing the cartilage. Basiobranchial bones: Section of the basiobranchialia, arrowheads marks the OP-L (N) and SPARC (O) expression of the basiobranchialia. Ceratobranchial bone: Section of the lateral palatine, the OP-L (Q) and SPARC (R) positive osteoblast-like cells (arrowheads) enclose the ceratobranchial cartilage). *, artifact; atr, atrium: hc, head chorda; lap, lapillus; sag, sagitta. Scale bars ¼ 100 mm.

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gated than the previous one. This pattern is consistent until the fifth ray emerges (br5). At this stage of development, the expression in br6 and br7 starts more posterior and causes a decrease in length compared with (br5) (Fig. 15F–I). With further development, the branchiostegal rays B6 and B7 fuse with the subopercular bone (br6) and opercular bone (br7) (Fig. 15J). The first mineralized bone tissue of the branchiostegal rays appears at stage 13, and these are the first ossified structures, together with the opercular bone (Fig. 15A). In the preopercular bone SPARC expression is first detectable at stage 14, followed by OP-L and calcification at stage 15 and bony tissue at stage 16 (Figs. (14 and 15)B). The subopercular bone arises out of the branchiostegal ray 6 and can be distinguished by OP-L and SPARC expression together with calcification from stage 14 onward (Figs. (14 and 15)B). SPARC expression and calcification of the interopercular bone appears at stage 15, whereas OP-L expression is first distinguishable from stage 17 onward coinciding with the appearance of the mineralized bone (Figs. (2 and 14)). With further development, OP-L and SPARC expression associated with the opercular bones appears as streaks within the opercule (Fig. 15K,L).

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Development of Teeth (OP-L and SPARC Expression)

Fig. 9. Summary of data for the splanchnocranium during cichlid fish development. Grey: OP-L; green: SPARC; blue: cartilage (alcian blue); yellow: calcification (alizarin-complexone); red: mineralized bone tissue (alizarin red).

carnii. At stage 13, SPARC expression is rapidly followed by OPL expression, calcification, and subsequently by the formation of bony tissue (Figs. (2 and 13)G–I, 14). In the frontal bone, SPARC together with calcification are first detected at stage 15, this is followed by OP-L at stage 16 and completely mineralized bony tissue at stage 17 (Figs. (2 and 13)M–O, 14). The opercular bone is the first bone that appears during skull development. Here the expression of OP-L and SPARC arise simultaneously with calcification, at stage 12 (Figs. (14 and 15)C,D). Later in development, this bone fuses with the seventh branchiostegal ray (Fig. 15J). The expression of OP-L and SPARC in the branchiostegal rays emerges at stage 13. The most ventral branchiostegal ray appears first, expressing OP-L and SPARC together (Fig. 15E,F). The other branchiostegal rays (br2–br7) develop dorsally of the first one and each ray becomes more elon-

The first step of tooth development is characterized by the formation of the enamel organ and the dermal papilla. Both the enamel organ and the dermal papilla express SPARC (Fig. 10C). At stage 13, the cells of the enamel organ differentiate into ameloblasts that secrete enamel and the cells within the dermal papilla differentiate into odontoblasts, which secrete dentine. While SPARC is weakly expressed in the enamel organ and the dermal papilla, it is strongly expressed in the ameloblasts (Fig. 10D). Following the secretion of dentine and enamel, calcification is detectable in the pharyngeal teeth (at stage 14) and then in the teeth of the upper and lower jaw (stage 15) (Fig. 10A,B). In contrast to SPARC, OP-L first occurs during tooth development in the cementoblasts at stage 14. Besides the OP-L expression in the teeth, the mesenchyme of the alveolar bone also expresses this marker (Fig. 10E,F). Strong SPARC staining in cementocytes appears at stage 15 and continues to be expressed during the entire tooth development (Fig. 10G,H). At stage 20, the OP-L expressing region of the cementocytes enlarges to over two thirds of the tooth length (Fig. 10J). Later in development (until stage 25), this expression domain decreases to the lowest part of the teeth, toward the tooth root (Fig. 10K). Similar to the cementoblasts, the ameloblasts initially strongly express SPARC. This expression is later reduced (at stage 16) and finally disappears at stage 17 (Fig. 10H). OP-L is not observed during the entire development of the amelocytes. In the fully developed odontocytes, SPARC expression appears at stage 15 (Fig. 10G). Later at stage 16, the staining in the odontocytes decreases and remains at a low level until stage 25 (Fig. 10H). OP-L expression in the odontocytes is first observable at stage 17 at a low level (Fig. 10I) and remains at this low level up to stage 25. In addition to the odontocytes, OP-L is expressed in the adamantine reticulum between the odontocytes and the alveolar mesenchyme (Fig. 10K).

Expression of OP-L and SPARC During Scale Development Scales are ossified dermal structures that arise from folds in the dermis. In O. mossambicus, SPARC is initially expressed (at stage 9) ubiquitously in the dermis with no spatial restriction but with

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Fig. 10. OP-L and SPARC expression and calcification during teeth development. Dorsal view of the head at stage 14 (A) and lateral view of the jaw complex (B) at stage 15, alizarin-complexone staining, arrowheads indicate the initial teeth calcification. C–K: Transversal sections of the developing pharyngeal teeth at selected stages, located in the ceratobranchials and basiobranchials. C,D: SPARC expression of in the developing enamel organ (black arrowhead, C) and the dermal papilla (white arrowhead, C), in the first ameloblasts (black arrowhead, D), and odontoblasts (white arrowhead, D). E,F: OP-L expression in the teeth located in the alveolar bone (arrowheads, E), is restricted to the cementoblasts (arrowhead, F). G: SPARC expression in the cementocytes (white arrowheads) and odontocytes (black arrowheads). H: Reduction of SPARC expression in the odontocytes, remainin in the cementocytes (arrowhead). I–K: Expression of OP-L in the odontoblasts and odontocytes (black arrowheads, I, white arrowhead indicate cementocytes). J: Enlargement of the OP-L expression region in the cementoblasts (white arrowhead), which increases in further development (white arrowhead, K). Expression of OP-L (K) in the adamantine is still detectable (black arrowhead). L: Post stage 16, OP-L expression in the ceratobrachials (arrowheads), Scale bars ¼ 50 mm. at: atrium.

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EXPRESSION OF SPARC AND OP-L IN CICHLID 965

Fig. 11. Appearance of OP-L and SPARC expression and bone calcification of selected bones of the chondrocranium in characteristic developmental stages. Calcification column: Localization and calcification of the corresponding bones (arrowheads) in lateral view (A,D,G). Basioccipital bone: Frontal section of the hindbrain, OP-L (B) and SPARC (C) expression in the osteoblast-like cells (arrowheads) of the basioccipital. Sphenotic bone: Transversal section of the lateral inner ear region, OP-L (E) and SPARC (F) expression in the osteoblast-like cells (arrowheads) of the sphenotic bone between the epidermis and the cartilage of the auditory capsule. Supraoccipital bone: Frontal section of the dorsal hindbrain, OP-L (H) and SPARC (I) expression (arrowheads) is detected between the epidermis and the cartilagous brain capsule. *, artifact; lap, lapillus; sag, sagitta. Scale bars ¼ 200 mm.

a clear anterior–posterior progression (Fig. 16A). Long after this initial expression, SPARC disappears from all nonscaled parts of the dermis of the head and fins and becomes restricted to the scale primordia (Fig. 16B). SPARC expression persists only in the scale primordia during postlarval development. OP-L expression and calcification are not observed during the whole of larval development (up to stage 25) and only takes part in scale development during postlarval development.

Discussion Cichlid fishes are an important aquaculture species and are also important models for research on vertebrate physiology, behav-

ior, and evolutionary biology (Lee et al., 2010). To date the genetic resources for tilapia are relatively well developed (Lee et al., 2010). This includes a microsatellite-based genetic map (Lee et al., 2005), a physical map based on BAC fingerprints (Katagiri et al., 2005), expressed sequence tag resources that rivals for other model fish species (Rise et al., 2004; Li et al., 2007; Lee et al., 2010) and the Broad Institute (USA) has released the first version of the tilapia genome. In particular, the oral jaws of cichlids are of special interest in the light of their explosive adaptive radiation (Fryer and Iles, 1972; Kocher, 2004). In general, the schema of skull development characteristic for teleosts is also followed in Oreochromis mossambicus with the specifics for O. niloticus noted by Fujimura and Okada (2008) (Aumonier,

966 WEIGELE ET AL.

of evolution. Not surprisingly, OP-L, as an exclusive calcification marker (for bones and teeth), was not detected during chondrogenesis.

Calcification Occurs Simultaneously With OP-L and SPARC Expression in Most of the Dermatocranium

Developmental Dynamics

Fig. 12. Summary of data for the chondrocranium during cichlid fish development. Grey: OP-L; green: SPARC; blue: cartilage (alcian blue); yellow: calcification (alizarin-complexone); red: mineralized bone tissue (alizarin red).

1941a, 1941b; Kang et al., 2004). During cichlid development, the expression of bone matrix proteins (SPARC, OP-L, osteocalcin) occurs during the osteoblast-cell aggregation phase (condensation phase) and during the formation of matrices for the composition of connective tissues. It is known that their expression patterns differ both temporally and spatially during development; however, no one has yet described the continuous detailed spatial expression analysis of OP-L and SPARC during fish embryonic and larval development until now.

SPARC Expression Preceeds Chondrogenesis in the Splanchnocranium We were able to confirm that SPARC expression precedes chondrogenesis in the splanchnocranium, the oldest part of the vertebrate skull, in the cichlid mouth breeder (Oreochromis mossambicus). This is similar to the gilthead seabream (Sparus auratus; Est^ev~ao et al., 2006) and zebrafish (Danio rerio; Rotllant et al., 2008), but unlike medaka (Oryzias latipes) in which SPARC was not found postcranially (Renn et al., 2006). It is known that SPARC is part of a cascade of genes that regulate cartilage morphogenesis in zebrafish (Rotllant et al., 2008). It is directly involved in differentiation of cells within pharyngeal precartilage condensations and functions downstream of the gene sox9a and upstream of col2a1a in cartilage differentiation (Rotllant et al., 2008). From these results, it was concluded that SPARC may function as a transcription factor. Our results support this function for SPARC during chondrogenesis. SPARC was not detected in the developing cartilages of the chondrocranium nor in the axial skeleton; this is in keeping with results from medaka (Renn et al., 2006). Based on these findings, it appears that there could be an ancient relationship between the paleocranium sensu neurocranium (which became adapted to contribute, reinforce, and protect the brain in more advanced teleosts) and the axial skeleton. Moreover, these results suggest an independent evolution of the splanchnocranium and all its derivative cartilages. It remains, however, unclear why two independent evolutionary trajectories with the same target, namely cartilage, developed over the course

It was previously proposed that calcification starts after bone matrix protein expression (Renn et al., 2006; Li et al., 2009). This conclusion was based on the results derived from alizarin red staining for calcified tissues. Here we show that that OP-L and SPARC typically appear together with the initial calcium deposition as evidenced by live alizarin-complexone staining. Typically one to two stages later, the calcified tissues are visible by means of alizarin red S staining. This is similar to zebrafish (Li et al., 2009). Therefore, we conclude that calcification occurs simultaneously with OP-L and SPARC expression in nearly all the bones of the dermatocranium (9 of 11). For the entire data set of bones analysed 18 out of 27 bones express SPARC.

OP-L and SPARC Are Expressed Before Calcification in the Fin Rays (Lepidotrichia) Lepidotrichia are bony, bilaterally paired, segmented fin rays found in bony fishes. They develop around actinotrichia as part of the dermal exoskeleton, and appear as a series of disks stacked on top of one other. In the developing caudal fin, we found that OP-L and SPARC expression domains emerge first, followed by calcification. These findings are in agreement with analysis concerning the differentiating bony lepidotrichia in the caudal fin of rainbow trout (Oncorhynchus mykiss; Geraudie and Landis, 1982). Here the OP-L and SPARC expression domains indicate differentiating osteoblasts, which will later form the bony hemisegments of the lepidotrichia. These domains are located laterally of the nonmineralized proteinaceous rays, the actinotrichia. During the further growth of fins, the actinotrichia extend in parallel with the expression domains of OP-L and SPARC, which mark the development of lepidotrichia.

Osteoclasts and Osteoblast-Like Cells Remodel the Fish Skeleton It has been assumed that the OP-L protein plays a similar role in the bones of fish that osteopontin plays in mammals (Fonseca et al., 2007). In mammalian bones, osteopontin regulates calcium-phosphate crystal distribution during mineralization of the mineralized bone tissue and it additionally mediates the activity of osteoclasts (Beck et al., 2000; Ito et al., 2004; Pampena et al., 2004; Gericke et al., 2005). Here, we show that OP-L is also expressed in the rare multinucleated osteoclasts of fish. Furthermore, OP-L and SPARC are expressed in the flattened osteoblastlike cells, which line the cartilaginous elements. These findings are consistent with the previously described perichondral OP-L domains undergoing endochondral ossification (Laue et al., 2006). We were able to confirm previous findings in Avaron et al. (2006) regarding the early occurrence of osteoblast-like cells, which later in the course of development build the endoskeleton. Little is known about these cells (which are also called periost cells or spindle-shaped osteoblast by other authors; Wendelaar Bonga et al., 1983; Witten, 1997). These cells line of fish bones express OP-L and we show here, that they emerge together with

Developmental Dynamics

EXPRESSION OF SPARC AND OP-L IN CICHLID 967

Fig. 13. Appearance of OP-L and SPARC expression and bone calcification of selected bones of the dermatocranium in characteristic developmental stages. Calcification column: Localization and calcification of the corresponding bones (arrowheads) in lateral (A,D,J,M) and dorsal (G) view. OP-L and SPARC column: Initial expression in the corresponding bones (arrowheads) on transversal sections. Maxillary and premaxillary bone: Sections of the developing lateral jaw complex. Initial expression of OP-L (B) and SPARC (C) in the mesenchyme of the maxilary and praemaxilary (arrowheads). Dentary bone: Sections of the developing lower jaw. E: First detectable OP-L expression in mesenchyme of the dentary (arrowhead). F: Frontal section of the anterior head, arrowhead indicates the first SPARC expression. Parasphenoid row: Sections of the palatine, OP-L (H, arrowhead) and SPARC (I, arrowhead) expression in the mesenchyme of the parasphenoid. The trabecula carnii and head chorda (hc) are given for orientation. Angular row: Sections of the lower jaw, OP-L expression of the angulary (arrowhead, K) besides Meckel’s cartilage and SPARC expression (arrowhead, L) in the height of the palatoquadrate cartilage. Frontal bone row: Sections of the dorsal head in the mid region of the eye. OP-L (N, arrowhead) and SPARC (O, arrowhead) expression in the developing frontal bone. Scale bars ¼ 100 mm.

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therefore provide some insight into how fish bones grow and are remodeled without the presence of alkaline phosphatase positive osteoblasts lining the bony surfaces, and/or without osteocytes embedded in the bone (as in acellular bones).

OP-L and SPARC Are Expressed in All Cell Types in Developing Teeth

Developmental Dynamics

OP-L expression was detected during tooth development in the developing cementocytes and odontocytes, similar to that of mammalian osteopontin (Takano-Yamamoto et al., 1994). Osteopontin especially plays a role in the formation of the cellular cement, and it was assumed that osteopontin is involved in cell attachment during cementogenesis in mammals (Takano-Yamamoto et al., 1994). Based on our results, we hypothesize that OP-L plays a similar role in fish. Moreover, in line with previous investigations, OP-L (Kawasaki, 2009) and osteopontin (Mets€aranta et al., 1989; Fujisawa et al., 1993; Qin et al., 2001) are present in dentine at low levels. OP-L was additionally expressed in the adamantine reticulum between the odontocytes and the alveolar mesenchyme and may also be involved in cell attachment; OP-L was not expressed in the adamantocytes. In contrast to OP-L, SPARC is involved in the initial step of tooth development, the formation of the enamel organ and the dermal papilla. Therefore SPARC may have two distinct functions in tooth development, one in the initial formation and differentiation of cells in the tooth tissues and the second in the process of calcification. SPARC expression in the odontoblasts and ameloblasts is in full agreement with the current literature (Holland et al., 1987; Salonen et al., 1990), but significantly, we also detect SPARC expression in cementoblasts and cementocytes. Therefore, SPARC occurs in all three cell types that secrete calcified tooth substances (in contrast to OP-L).

Expression of OP-L in Nonmineralized Soft Tissues In contrast to Bobe and Goetz (2001), we provide direct evidence that OP-L is not expressed in the ovary, testis, intestine, stomach, spleen, gills, and brain from stage 10 to 25 during cichlid fish embryonic and early larval development. Bobes and Goetz’s findings in the ovary and testis of adult trout may indicate that OP-L is only expressed in functional organs and not during developmental stages. Moreover, we also could not confirm the occurrence of OP-L during cardiac development for O. mossambicus in contrast to zebrafish, where it regulates cell migration during atrioventricular canal formation (Just et al., 2011; Huang et al., 2012; Banjo et al., 2013). However, we confirmed the reports of Fonseca et al. (2007), that OP-L is not expressed in the intestine, stomach, spleen, and brain of teleost fishes.

Experimental Procedures Fig. 14. Summary of data for the dermatocranium during cichlid fish development. Grey: OP-L; green: SPARC; yellow: calcification (alizarincomplexone); red: mineralized bone tissue (alizarin red).

the underlying cartilage and produce the mineralized bone tissue. These cells do not express alkaline phosphatase typical of more mature osteoblasts and osteocytes (Witten, 1997). Our results

Taxonomy and Animals The cichlid fish (Oreochromis mossambicus, Perciformes, Labroidei, Cichlidae) used in this study were obtained from our stock at the Institute of Zoology of the University of Hohenheim, Germany. All experimental procedures were approved by the animal care and health officer of the University of Hohenheim, according to the guidelines of the “Bundesamt f€ ur Veterin€arwesen.” The staging follows Anken et al. (1993) and all experimental animals

Developmental Dynamics

EXPRESSION OF SPARC AND OP-L IN CICHLID 969

Fig. 15. Appearance of OP-L and SPARC expression and bone calcification of opercular series. Lateral view of the head, calcification stained by alizarin complexone at stage 13 (A) and 15 (B), arrowheads indicate the branchiostegal rays. Transversal section of the opercule, arrowheads mark the Initial expression of SPARC (C) and OP-L (D) of the opercular bone. E: Frontal section of the opercule, arrowheads indicate the SPARC expression in the branchiostegal rays. F–J: Lateral view of the opercule, OP-L, and SPARC expression stained by whole-mount in situ hybridizations. Arrowheads indicate the SPARC and OP-L expression of the branchiostegal rays (br1–7). J: The branchiostegal ray br6 and br7 follow later in development integrated into the subopercular bone (br6) and opercular bone (br7). K,L: Transversal section of the fully developed opercule, arrowheads indicated the OP-L (K) and SPARC (L) expression. e, eye. Scale bars ¼ 100 mm.

were anesthetized with tricain-s (Western Chemical Inc., WA) using standard procedures. Tissue processing for in situ hybridization was performed using 4% paraformaldehyde/phosphate

buffered saline (PBS) fixation for 12 hr at 4 C. The samples were then processed through a graded dilution series to 100% methanol and stored at –20 C for further use.

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Fig. 16. Scale development. A: Lateral view of the fish embryo, SPARC is located in the dermis of the head region. B: Restriction of SPARC to the scale primordia (arrowheads mark two of the primordia) and dermal parts (*), which undergo scaled in the postlarval development (stage 25). Scale bars ¼ 300 mm.

Developmental Dynamics

Cloning and Sequencing Cichlid OP-L and SPARC Gene Fragment For primer design, fish (e.g., zebrafish, medaka, or fugu) cDNA and/or genomic sequences were aligned. Typically four degenerated primers were selected and were used in different combinations in a standard polymerase chain reaction (PCR) and nestedPCR. The PCRs were performed on cDNA and carried out under standard conditions. The products of the correct size were R -T Easy Vector (Promega, Madison, WI) and cloned in the pGEMV sequenced on both strands. Successful PCR-primers for SPARC were 50 -GGG AAC CAA GGC CA-30 ; 50 -ACT CCT CCA GGG CGA TGT A-30 and for OP-L for the first PCR 50 -TGC TCT T(CT)G C(AC)A C(AG)G TCC TC-30 ; 50 -CCT GGC TCT CAA C(AGCT) CAG30 and for the second PCR 50 -CAG AGA GCT C(AT)G AAG AA(GC) TGG T-30 ; 5-GGT GCT GGT GTC CTC CTC-30 . The entire sequenced cDNA inserts were deposited into NCBI GenBank with the accession numbers: OP-L (JN854244), SPARC (HQ667765).

In Situ Hybridization Techniques Digoxigenin labeled riboprobes were generated from partial R -T easy plasmid using either SP6 or T7 cDNA clones in pGEMV Polymerases (Promega) and a linearized vector. The transcription was carried out for 2 hr at 37 C. The riboprobes were diluted in 50% formamide after purification and stored at –80 C until required. Both sense and antisense riboprobes were synthesized; the sense riboprobes served as negative controls. The following whole-mount in situ hybridization was performed according to Belo et al. (1997). For detailed analyses of the expression patterns cryostat sections were cut as follows. The stored specimens were rehydrated in PBS and decalcified in a solution of 8% ethylenediaminetetraacetic acid (Roth) in PBS two times for 45 min at 4 C. The samples were cryoprotected in a 1% natrium-carboxymethylcellulosis (Roth) / PBS solution, overnight (14 hr). Following these procedures, the samples were frozen in Leica OCT Cryocompound “tissue freezing medium” (Leica Microsystems, Bensheim, Germany) in –80 C n-methylbutane and stored at –80 C. Sectioning was carried out using a GM3050 cryostat (Leica Microsystems) and sections were adhered to Superfrost Plus microscope slides (Menzel-Glaeser, Braunschweig, Germany). In situ hybridization on 14-mm-thick tissue sections was performed according to Schwarzenbacher et al. (2004).

Whole-Mount Skeletal Staining The whole-mount cartilage and bone staining were performed following standard methodology according to Potthoff (1984) with certain modifications. The stored samples were rehydrated using a descending methanol series (75%, 50%, and 25%) in 0.5 SSC solution (saline-sodium citrate buffer). To remove pigmentation, the samples were bleached in 0.5 SSC, 4% hydrogen peroxide solution for 140 min under cold-light source. The bleached samples were then washed intensively in PBS and dehydrated using an ascending methanol series (25%, 50%, 75%, and 100%). Samples were stored in acetone to remove fat for 3 days. For cartilage staining, the samples were equilibrated in 100% and 75% ethanol and then stained using Alcian blue staining solution (0.1% Alcian blue, Sigma, Schnelldorf, Germany; 70% ethanol; 30% glacial acetic acid) for 3 hr. Samples were washed in 70% ethanol and rehydrated using a descending ethanol series (75%, 50%, and 25%) and cleared in a 0.25%, 0.5%, and 1% KOH solution for 20 min each. Samples were neutralized by rinsing in saturated sodium borate solution twice for 10 min, and then digested in 1% trypsin (Roth) saturated sodium borate solution until the tissue cleared. Finally, samples were rinsed in 1% KOH solution and stored in 25% ethanol. For alizarin red S staining of bones, samples were equilibrated in 100% ethanol and rehydrated through a descending ethanol series (75%, 50%, and 25%), cleared with KOH and digested with trypsin as described above. Afterward, the bones were stained in alizarin red staining solution (0.02% alizarin red, Chroma-Gesellschaft, Stuttgart, Germany; 1% KOH) overnight (14 hr). Finally the samples were rinsed in 1% KOH solution and stored.

Calcification Staining by Alizarin-Complexone The whole-mount alizarin-complexone staining was performed following standard methodology (Beier et al., 2004). Live animals were incubated in a 0.01% alizarin-complexone (Sigma, #395278-1) solution overnight and then rinsed in fresh water for 3 hr. Afterward, the experimental animals were deeply anaesthetized with tricain-s and stored in 100% ethanol until required. For clearing, the stored samples were rehydrated by means of a descending series and cleared in a 1% KOH solution for 2 hr. After clearing, the samples were washed in PBS for 15 min and then stored in 25% ethanol until required.

EXPRESSION OF SPARC AND OP-L IN CICHLID 971

Photography and Image Analysis The analysis of the whole-mount in situ hybridization and cartilage staining results was carried out using a stereomicroscope (SteREO Discovery.V12, Zeiss), and images were taken with a digital camera (Aciocam Hrc, Zeiss). The histological sections were captured using a photo-microscope (Axioskop 2 mot plus; camera: Aciocam Hrc, both Zeiss). The whole-mount alizarin-complexone stained samples were analyzed using a fluorescence stereomicroscope (MZ FLIII; filter: excitation filter 535 nm, beam splitter 555 nm, barrier filter OG580; both Leica; camera: Axiocam MRC, Zeiss). Axiovision rel. 4.7 software (Zeiss) was used for data acquisition in all cases.

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