Development of cartilage and bone tissues of the anterior part of the mandible in cichlid fishA light and TEM study.код для вставкиСкачать
THE ANATOMICAL RECORD 233:357-375 (1992) Development of Cartilage and Bone Tissues of the Anterior Part of the Mandible in Cichlid Fish: A Light and TEM Study ANN HUYSSEUNE AND JEAN-YVES SIRE Laboratorium uoor Morfologie en Systematiek der Dieren, B-9000 Gent, Belgium (A.H.) and Uniuersitk Paris 7, Laboratoire d‘Anatomie comparee, UA CNRS 1137, 75251 Paris Cedex 05,France (J.-Y.S.) ABSTRACT The present paper presents ultrastructural details of chondrogenesis of Meckel’s cartilage and of ossification of its associated peri- and parachondral bones in a teleost fish, the cichlid Hemichromis bimaculatus. We have distinguished four stages during chondrogenesis, each of which is characterized by specific cellular and matrix features: blastema, primordium, differentiated cartilage and cartilage surrounded by perichondral bone. The blastema is characterized by prechondroblasts and the lack of cartilage matrix; the primordium by chondroblasts and the onset of secretion of matrix of fibrillar and granular nature; differentiated cartilage is characterized by chondrocytes and larger amounts of typical hyaline cartilage matrix. Once perichondral bone is laid down, the chondrocytes show degenerative features but not true hypertrophy. Differentiation of the cartilage cells is attended with cytoplasmic changes indicative of an increasing secretory activity. There is a regional calcification of the cartilage matrix by fusion of calcospherites. Chondrogenesis of the symphyseal area is continuous with that of the rami but starts slightly later. Formation of perichondral bone at the cartilage surface is attended with the deposition of a transitional zone apparently containing a mixture of the two matrices. The role of the perichondral cells is discussed and it is proposed that they may contribute to the formation of the two matrices. The transitional zone may then result either from a diffusion process or from the simultaneous deposition of elements of the two matrices. Growth of the cartilage is argued to be largely the result of matrix secretion, except in the symphyseal area where appositional growth probably occurs until the region is completely covered by perichondral bone. This paper provides a basis for further studies on the developmental interactions between cartilage, bone and teeth during mandibular development in cichlids. o 1992 Wiley-Liss, Inc. Previous studies on the development of the pharyngeal jaws in Cichlidae, a family of highly evolved teleost fishes, have suggested that these organs develop from branchial arches as a result of interactions between cartilage, bone and teeth (Huysseune, 1983, 1989). It has been suggested that teeth play a considerable role in the morphogenesis of these jaws (Huysseune, 1989). An unanswered question was whether such interactions would also play a role during development of the lower jaw (mandible) in these fishes, or whether they are limited to specialized organs such as pharyngeal jaws. Huysseune (1990) has previously shown that the larval cichlid mandible contains almost all the skeletal elements which make up the mandible of adult cichlids: a rod of cartilage (Meckel’s cartilage) along with, on each side of the head, two dermal bones (dentary and angular) and three out of the four perichondral bones of the adult (mentomeckelian, articular and retro-articular). The two ossifications in the anterior part of the mandible (the perichondral mentomeckelian bone and the parachondral dentary bone) develop separately, the former in front of the latter. They fuse 0 1992 WILEY-LISS. INC afterwards to form the compound dento-mentomeckelium (Huysseune, 1990). The ontogenetic and phylogenetic significance of these two ossifications has been discussed elsewhere (Huysseune, 1990). Although the mandible is one of the best studied skeletal organs in birds and mammals from a developmental viewpoint, studies on the mandible in fish are rare. Descriptive studies are those of Haines (1937) on various teleosts, de Beer (19371, Verraes (1973) and Francillon (1974, 1977) on Salmo and Ismail (1979) on Haplochromis. The tissue interactions required to permit chondrogenesis of Meckel’s cartilage and development of the mandibular bones and tooth tissues from neural crest precursors in higher vertebrates have been the subject of numerous studies (see Hall, 1983, 1987; Lumsden, 1987 for reviews). In contrast, very little attention has been paid to the mutual relationships between differentiated cartilage, bones and teeth, and how these tissues interact to build up the adult Received May 7, 1991; accepted September 24, 1991. A. HUYSSEUNE AND J.-Y. SIRE 358 _/--->'I / sa _ _ Fig. 1. Lateral, slightly oblique view of a cleared specimen of Hernichromis bimaculatus at 7 days post-hatching showing the position of the two rami of Meckel's cartilage (Mc) and the symphyseal area (sa). cp = coronoid process; pq = palatoquadrate. Bar = 0.5 mm. lower jaw (the studies of Cusimano-Carollo [19721 and Cassin and Capuron 119791 being some of the few exceptions). A preliminary study of the formation of the lower jaw and of the mandibular dentition in cichlids has indicated that important developmental relationships seem to exist between Meckel's cartilage, developing tooth germs and the anlage of the perichondral (mentomeckelian) and parachondral (dentary) bones (Huysseune, 1990). In order to make a detailed morphological analysis of the tissue relationships involved in mandibular development, knowledge of the ultrastructure of each of these tissues was required. However, in our search for such data, we were confronted with a considerable lack of studies on the fine structure of developing or even of mature cartilage, perichondral and dermal bones in teleosts. We therefore decided to make a detailed descrip- b bc cb cc m Mc ob pa PC Pe Pec Pq Pr RER sa Y Abbreviations blastema buccal cavity chondroblast chondrocyte coronoid process Golgi region cartilage matrix Meckel's cartilage osteoblast parachondralbone peripheral cell perichondral bone perichondral cell palatoquadrate primordium rough endoplasmic reticulum symphyseal area yolk sac tion, using light and transmission electron microscopy, of the skeletal tissues involved in mandibular development during early postembryonic life. This provides us with a basis to study in detail, in a subsequent paper, the relationships between cartilage, bone and teeth. MATERIALS AND METHODS Laboratory-bred larvae of the substrate-brooding cichlid Hemichromis bimaculatus, ranging from 4.0 mm total length (TL) a t one day post-hatching (stage with the initial prechondrogenic condensation) to 4.7 mm TL a t seven days (stage prior to cartilage resorption), were processed for light and transmission electron microscopical observations as follows. Specimens were fixed in a mixture of 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 hours at room temperature. Most specimens were decalcified by adding 0.1 M EDTA to the fixative solution for 5 days at 4°C. The decalcifying solution was changed daily. After fixation, the specimens were rinsed in 0.1 M cacodylate buffer with 10% sucrose, then postfixed for 2 hours in 1%Os04 solution in 0.1 M cacodylate buffer to which 8%sucrose was added. After rinsing in the same buffer, the specimens were dehydrated in a graded series of ethanol and embedded in epon. One-pm-thick and thin transverse sections were cut on a Reichert OMU-3 ultratome. Thick sections were stained with toluidine blue. Thin sections were contrasted with uranyl acetate and lead citrate and viewed in a 201 Philips Electron Microscope operating a t 80 kV. Computer-aided reconstructions (cf. Vanden Berghe et al., 1986) were prepared of the anterior portion of the mandible (including Meckel's cartilage, the perichondral mentomeckelian bone and the parachondral dentary bone) viewed under various angles. TEM OF CICHLID MANDIBULAR DEVELOPMENT Figs. 2-4. Blastema stage of the rami of Meckel’s cartilage at one day post-hatching. Fig. 2. One-km-thick transverse section showing the two ovalshaped blastemata (b), located at both sides of the mediosagittal plane. bc = buccal cavity; y = yolk sac. Bar = 50 pm; x 275. 359 and surrounded by approximately two layers of peripheral cells (pc). Note the distinct limit between both cell populations (*I. Bar = 5 pm; x 3,600. Fig. 4. Detail of a prechondroblast. Arrows point to short desmo= Golgi region. Bar = 1 pm; x 9,000. somes. G Fig. 3. Thin section in a blastemal region. The blastema (b) is composed of closely adjoining pyramidal-shaped cells (prechondroblasts) RESULTS Meckel’s Cartilage and Perichondral Bone During postembryonic development in cichlids, each Blastema stage hemimandible is built up by a curved cartilaginous rod At one day post-hatching, two small oval-shaped or ramus, Meckel’s cartilage. The two rami merge into blastemata (about 18 pm high) are present a t both one another in the mediosagittal plane, forming the sides of the mediosagittal plane (Fig. 2). They lie amid so-called symphyseal area. Posteriorly, the rod pre- a mesenchymal cell population that occupies the region sents a dorsally directed process (coronoid process), between the buccal epithelium and the ventral epidercaudal to which the rod articulates with the cartilagi- mis. They represent the future anterior portions of each rod of Meckel’s cartilage. The blastema is characnous palatoquadrate (Fig. 1). The present description covers the development of terized by a small number of cells (about 10 on a secthe anterior portion of the mandible and deals with: (i) tion) which are closely packed and separated from the the anterior part of each ramus of Meckel’s cartilage surrounding cells (peripheral cells) by a narrow but and its perichondral bone (i.e., the mentomeckelian distinct space. bone), (ii) the cartilaginous symphysis, and (iii) the At the ultrastructural level, the cells of the blastema parachondral bone (i.e., the dentary bone) which lies (referred to as prechondroblasts) show a pyramidal along each ramus posterior to the perichondral bone. shape with their tip towards the center of the blastema The descriptions are based on examination of trans- (Fig. 3). They are about 6 pm wide and 8 pm high. verse sections. However, because of the curvature of Their nucleus is slightly eccentric, being pushed toMeckel’s cartilage (Fig. l), such sections cut each rod wards the broader base. The cytoplasm contains a few cisternae of rough endoplasmic reticulum (RER) and more or less obliquely. 360 A. HUYSSEUNE AND J.-Y. SIRE moderately developed Golgi regions in addition to numerous free ribosomes, small vesicles and mitochondria (Fig. 4). The prechondroblasts are joined to each other by short desmosomes. The narrow intercellular spaces are generally devoid of any material. The wider spaces surrounding the blastema show no signs of extracellular products either (Fig. 3). Peripheral cells, smaller than the prechondroblasts, are roughly rectangular and circumferentially arranged around the blastema in one or two layers (Fig. 3). These cells are loosely organized with hardly any matrix in the intercellular spaces. Their nucleo-cytoplasmic ratio is higher than that of the blastemal cells. Short desmosomes link the peripheral cells. Prirnordiurn stage Figure 5 shows the two primordia of Meckel’s cartilage of a more advanced specimen at one day after hatching. The chondroblasts are organized in a highly ordered fashion within each primordium (which is about 20 pm high). A distinct intercellular space delimits the primordium. The section was taken slightly obliquely and the animal’s right primordium is cut in a more mature region. Figures 6 and 7 present an overview of the left and right primordium, respectively. In the left primordium (Fig. 6), many chondroblasts still show the pyramidal shape, and form approximately two rows. Several of these cells show a long process that penetrates the space between opposite adjacent cells. Other chondroblasts have a more elongated shape with a centrally placed nucleus. In a slightly more mature region (Fig. 7), almost all the cells have an elongated shape. Examination of serial sections shows that these chondroblasts are more or less polygonal in the third plane. They take the entire height of the cell column so as to form only one cell row. In both primordia, chondroblasts closely adjoin each other, leaving hardly any intercellular space in between (Figs. 6, 7). They are linked to each other by occasional short desmosomes. At two days post-hatching, this cellular arrangement is even more pronounced (Fig. 8). The chondroblasts, on transverse sections (we should recall that Meckel’s cartilage in such sections is cut obliquely), are extremely flattened (in average 15 pm high and 1.5 pm wide) and are packed like coins in a pile in a direction parallel to the long axis of the rod. At this stage, the cells are slightly separated from each other so that narrow intercellular spaces appear, especially between the apical parts of the cells. However, occasional short desmosomes still link the cells. The chondroblasts in the latter two stages are characterized by numerous mitochondria, free ribosomes, a well-developed network of RER, mostly located in the apical parts of the cells, and Golgi regions (Figs. 9,101. The outline of the cells is smooth except for the apical part which shows a slightly scalloped surface with local electron-dense concavities. The space that surrounds the chondroblasts in the primordium of advanced one-day-old specimens is approximately 0.5 pm wide (1.0 pm in the more mature primordium) (Figs. 6, 7). It houses loosely dispersed fine fibrils (approximately 12 nm in diameter) amid small amounts of granular material (Fig. 9). At two days, the space that separates the chondroblasts from the peripheral cells is wider (approximately 1.5 pm in Fig. 8) but the amount of extracellular matrix has not increased considerably (Fig. 10). Extracellular material is also visible in the intercellular spaces, mainly in the apical region of the chondroblasts. Some of this material lies close to the electron-dense thickenings of the cell membrane. Transverse sections at one day post-hatching show the polygonal peripheral cells to be smaller and to contain relatively less cytoplasm than the chondroblasts within the primordium (Figs. 6, 7). At two days, they are more flattened and organized more or less distinctly in one layer around the cartilage (Fig. 8). Despite their difference in shape, peripheral cells of both stages resemble each other in the amount and content of their cytoplasm: many free ribosomes, several mitochondria, some Golgi regions, vesicles, but a weakly developed RER (Fig. 9). Patches of extracellular material face electron-dense thickenings of the peripheral cell membrane. Differentiated cartilage At three days post-hatching, Meckel’s cartilage is about 25 pm high and is easily recognizable as typical cartilage by purple staining of the matrix with toluidine blue. Its cells can now be regarded as chondrocytes and the cells that surround the cartilage as perichondral cells (Fig. 11). The chondrocytes lie well apart, although the intercellular spaces are small. The cells are elongated (about 20.0 pm high and 2.5 pm wide) and present a clearly scalloped borderline (Fig. 12). A well-developed network of RER cisternae, several Golgi regions and Figs. 5-10. Primordium stage of the rami of Meckel’s cartilage in advanced specimens at one day post-hatching (Figs. 5, 6, 7, 9)and at two days (Figs. 8, 10). Fig. 5. One-pm-thick slightly oblique section showing right and left primordium (pr) (left and right side of the picture, respectively) in a slightly different stage of development. bc = buccal cavity; y = yolk sac. Bar = 25 pm; x 380. Fig. 6. Left primordium. Some chondroblasts (cb) show a pyramidal shape, others are elongated. Note the space (*I that has been formed between the chondroblasts and the peripheral cells (pc). Arrowheads point towards long cell processes penetrating between adjacent chondroblasts. Arrows point to short desmosomes. Bar = 5 p,m; x 3,300. Fig. 7. Right primordium. In this more mature stage, all the chondroblasts (cb) have acquired a n elongated shape. Arrows point towards short desmosomes. pc = peripheral cell. Bar = 5 pm; x 3,300. Fig. 8. Maturing cartilage at two days post-hatching, characterized by very elongated chondroblasts (cb) and the appearance of intercellular spaces. Note the widening of the space between chondroblasts and peripheral cells (pc). Arrow points towards a desmosome. Bar = 5 pm; x 3,300. Fig. 9. Detail of the peripheral cartilage matrix of the same specimen as shown in Figure 7. Arrows point to patches of extracellular matrix facing the plasmalemma of the chondroblasts (cb) and of the peripheral cells (pc). m = cartilage matrix. Bar = 1 pm; x 15,000. Fig. 10. Detail of the peripheral cartilage matrix at a more mature stage (same specimen as in Fig. 8). Intercellular spaces and peripheral zone have noticeably enlarged (compare to Fig. 9). cb = chondroblast; G = Golgi region; m = cartilage matrix; RER = rough endoplasmic reticulum. Bar = 1 p,m; x 15,000. TEM OF CICHLID MANDIBULAR DEVELOPMENT Figs. 5-10. 361 362 A. HUYSSEUNE AND J.-Y. SIRE Figs. 11-13. Differentiated cartilage a t three days post-hatching. Fig. 1l.One-pm-thick transverse section of one ramus showing typical cartilage composed of elongated chondrocytes and well-developed matrix. The cartilage is surrounded by flattened perichondral cells (pec). At its lateroventral side, the anlage of the parachondral bone (pa) is lined by large, strongly basophilic osteoblasts. bc = buccal cavity. Bar = 25 pm; X 550. Fig. 12. The chondrocytes show a well-developed network of dilated cisternae of rough endoplasmic reticulum (RER) and Golgi regions (GI. The intercellular spaces are filled with fibrillar and granular material. m = cartilage matrix. Bar = 500 nm; x 18,000. Fig. 13. The cartilage matrix (m) is composed of thin fibrils and dark small granules. The density of the fibrillar material increases close to the perichondral cells (pee). Bar = 250 nm; x 40,000. ~ Figs. 14-21. Rami of Meckel’s cartilage with perichondral bone a t four days (Figs. 14-20) and seven days (Fig. 21) post-hatching. Fig. 14. One-pm-thick transverse section of the two rami. The cartilage surface shows a thin layer of translucent matrix (arrows). Most of the chondrocytes still have a viable appearance. Bar = 25 pm; x 420. Fig. 15. One-Fm-thick transverse section, showing the same area in a slightly more developed specimen compared to that shown in Figure 14. A distinct layer of perichondral bone (pe) surrounds the cartilage. Most of the chondrocytes show a necrotic aspect. Bar = 25 pm; x 400. Fig. 16. First stage of degenerating chondrocytes (lateral side of the ramus) with onset of perichondral bone formation (pe). Chondrocytes (cc) either show an electron-dense cytoplasm and vesicles of various size and density or a n electron-lucent cytoplasm. m = cartilage matrix; pec = perichondral cell. Bar = 5 pm; x 3,300. Fig. 17. Advanced stage of degeneration of the chondrocytes (medial side of the ramus). The cell membrane of the necrotic chondrocytes (cc) has been disrupted and the cells have lost most of their content. A well-developed perichondral bone layer (pe) surrounds the cartilage (m). pec = perichondral cell. Bar = 5 pm; x 3,300. Fig. 18. Detail of the chondrocytes (cc) and the cartilage matrix (m) in the first stage of degeneration of the chondrocytes (cf. Fig. 16). pec = perichondral cell. Bar = 1 pm; x 9,000. Fig. 19. Detail of the peripheral zone of the cartilage and of the perichondral bone anlage (pe) during degeneration. Typical cartilage matrix (m) (1) grades into a loosely organized fibrillar zone with slightly thicker fibrils and concurrent decrease of the number of dense particles (2) and then into a band composed of densely packed fibrils embedded in an electron-dense background substance (3). The latter is separated from the perichondral cells (pec) by a n electron-lucent fibrillar zone (4). Bar = 500 nm; x 30,000. Fig. 20. Perichondral bone in a n advanced stage similar to that presented in Figure 17. The same four layers can be recognized as in Figure 19 but zones (2) (transitional zone) and (3) (perichondral bone proper, pe) have appreciably thickened. Note the increase in electrondense background substance in zone 2. m = cartilage matrix. Bar = 500 nm; x 30,000. Fig. 21. Undecalcified specimen at seven days post-hatching. Only the perichondral bone matrix (pe, zone 3) is mineralized and appears as a heavily electron-dense layer. Zone 4 is not prominent. m = cartilage matrix. Bar = 500 nm; x 30,000. TEM O F CICHLID MANDIBULAR DEVELOPMENT Figs. 14-21. 363 364 A. HUYSSEUNE AND J.-Y. SIRE mitochondria are their most prominent characteristics. The RER has largely dilated cisternae filled with electron-dense material. Cellular contacts are no longer visible. The chondrocytes are separated by spaces ranging from 0.2 to 1.0 pm wide, containing moderate amounts of fine fibrillar and granular material (Fig. 12). The space between the chondrocytes and the perichondral cells is about 3.0 pm wide and is filled with a meshwork of fine fibrils (12 nm in diameter) amid which electron-dense granules are dispersed (Fig. 13). Adjacent to the perichondral cells, the matrix is dense over a thickness of about 0.5 pm. This denser aspect is largely due to an increase in number of fibrils rather than to an increase in electron-dense background. The perichondral cells are elongated and flattened (approximately 0.3 pm high). Their cytoplasm is poor in organelles and contains predominantly free ribosomes (Fig. 13). Cartilage with perichondral bone Two specimens a t four days post-hatching have been used to show two different stages of maturation of Meckel's cartilage (in both the latter is approximately 25 pm high) (Figs. 14,151. In the less mature stage, the cartilage is surrounded by a thin layer of translucent matrix which is covered by rectangular, elongated perichondral cells (Fig. 14). The chondrocytes lie clearly separated from each other. Most of them show a viable aspect, whereas some show initial stages of degeneration. In a more mature stage, the cartilage is surrounded by a distinct layer of perichondral bone while most chondrocytes show a necrotic, degenerative aspect (Fig. 15). The former perichondral cells are less elongated and more cuboidal. In the two stages, the chondrocytes still are about 20 pm high and lie approximately 1.5 pm apart. They present various stages of degeneration (Figs. 16,171. In the less degenerated chondrocytes, the nucleus, RER cisternae and mitochondria are relatively well preserved (Figs. 16, 18). The RER cisternae are still filled with electron-dense material and the cell membrane is not disrupted. Often the cell is separated from the surrounding matrix by an empty space about 0.5 pm wide. In the more degenerated stage (Fig. 17), the cell content has almost completely disappeared except for the nucleus and some RER. The cell membrane has been disrupted. In both stages, the cartilage matrix is evenly distributed both between and around the cells (Figs. 16, 17). In some areas, the matrix is arranged concentrically around the cells. At the periphery of the less mature cartilage, the matrix is more electron-dense and consists of patches which coalesce to form a continuous layer (0.5 pm wide in average) along the lateral margin (Fig. 16). In the more mature stage, this layer is well-developed (1.5 pm wide in its thickest part) and continuous all around the cartilage (Fig. 17).This layer corresponds to the first perichondral bone deposit visible in the light microscope (Fig. 15). From the center to the periphery of the cartilage, there is a progressive change in the composition and organization of the matrix in function of the degree of maturation. As in the previous stages, the typical matrix in the major part of the cartilage consists of ran- domly disposed fine unbanded fibrils through which electron-dense particles are dispersed (Figs. 18, 19 [zone 11). Towards the perichondral cells, the aspect of the matrix changes. Numerous thicker and straight fibrils appear to be superimposed upon typical cartilage matrix (Fig. 19, zone 2). Near the perichondral cells, the matrix is composed of densely packed fibrils similar to those appearing in zone 2, embedded in an electron-dense background substance (zone 3, Fig. 19). This organization is still more pronounced in the more degenerated cartilage (Fig. 20). There is a broad transition zone (zone 2 on Fig. 20) in which the fine cartilage fibrils (about 12 nm in diameter) and dark granules appear to be intermingled with thicker fibrils (about 20 nm in diameter). The latter fibrils are the only type found in zone 3. In some regions, these fibrils are oriented roughly parallel to the cartilage surface. None of the fibrils in these two zones appears to be banded. Immediately below the perichondral cells is a narrow zone occupied by possibly banded collagen fibrils without electron-dense background substance (Fig. 20, zone 4). In an undecalcified specimen at seven days posthatching, the transition zone (zone 2) between cartilage matrix proper and perichondral bone (zone 3) is devoid of any mineral (Fig. 21). In contrast, zone 3 (perichondral bone matrix proper) is completely mineralized. Figs. 22-29. Symphyseal region of Meckel's cartilage. Fig. 22. Thin section in the blastemal region in a late one day posthatching specimen. Prechondroblasts are roughly polygonal and the cells look less organized than those in the rod blastema (see Figs. 3,4). Arrow points towards a cilium. Bar = 5 pm; x 3,600. Fig. 23. One-pm-thick transverse section of the primordium stage, two days post-hatching. The mediosagittal plane is indicated by arrows. There is a n onset of concentric arrangement of chondroblasts in this area. bc = buccal cavity. Bar = 25 pm; x 500. Fig. 24. Thin section of the symphyseal region shown in Figure 23. Chondroblasts (cb) are separated by fairly large intercellular spaces filled with fibrillar and granular material. Peripheral cells (pc) are not well-delimited from the chondroblasts. m = cartilage matrix. Bar = 1 pm; x 7,700. Inset: Detail of the cartilage matrix (m) in the symphyseal area. cb = chondroblast. Bar = 500 nm; x 30,000. Fig. 25. One-pm-thick transverse section of the symphyseal area at three days post-hatching. bc = buccal cavity. Bar = 25 pm; x 450. Fig. 26. Detail of the symphyseal cartilage shown in Figure 25 with dispersed rounded or triangular-shaped chondrocytes (cc). Note the concentric arrangement of the cartilage matrix (m) around the chondrocytes, and the increase of matrix density (arrows) close to the perichondral cell (pec) layer. Bar = 2 pm; x 6,000. Fig. 27. One-pm-thick transverse section of the symphysis at four days post-hatching. Laterally, the symphysis flattens down towards the rod-shaped part of Meckel's cartilage, which is covered in this area by perichondral bone (pe). Bar = 25 pm; x 400. Fig. 28. Detail of Figure 27, showing the center of the symphysis to the right of the picture, with viable chondrocytes, and first stage of degeneration of the chondrocytes (cc) near the center of the symphysis. m = cartilage matrix. Bar = 5 pm; x 3,300. Fig. 29. Detail of the peripheral region of the symphyseal cartilage at four days post-hatching. Note the dense territorial organization of the matrix (m) and the electron-lucent zone separating the latter from the perichondral cells (pec).cc = chondrocyte. Bar = 1 pm; x 14,000. TEM OF CICHLID MANDIBULAR DEVELOPMENT Figs. 22-29. 365 366 A. HUYSSEUNE AND J.-Y. SIRE The former perichondral cells facing regions with lit- domly dispersed cells embedded in a purple staining tle deposits of perichondral bone matrix are roughly matrix (Fig. 25). At the periphery, it is lined by one or flattened (approximately 1.7 pm high), whereas they two layers of elongated perichondral cells at the buccal show a more rectangular shape (approximately 3.5 pm side and three to four layers at the epidermal side. high) in regions facing a well-developed perichondral Ultrastructurally, the cartilage cells are typical chonbone anlage (Figs. 16, 17). Both types have a well-de- drocytes with a clearly scalloped borderline (Fig. 26). veloped cytoplasm equipped with large amounts of The fibrillar component of the cartilage matrix is arRER. At no time do cells become trapped into the ma- ranged concentrically around the chondrocytes in such trix of the newly formed perichondral bone. The latter a way as to roughly delimit a territory. The territorial boundary has a denser aspect due to an increase in always presents a smooth surface. In subsequent stages, the fine structure of Meckel’s number of fibrils, particularly where it meets the pericartilage remains unchanged until resorption starts chondral cells. Between the latter and the territorial (i.e., after one week post-hatching). One pm thick sec- boundary, there is often a small amount of extracellutions of specimens at five, six, and seven days after lar matrix which again contains a more unorganized hatching reveal a mere thickening of the perichondral and looser meshwork of fibrils (Fig. 26). bone. Symphysis In this section, the description will deal essentially with the characteristics of the symphyseal area of Meckel’s cartilage which differ from those of its rodshaped part. Cartilage with perichondral bone At four days post-hatching, the height of the symphyseal area has considerably increased (up to 50 pm) concomitant with an important increase in number of cells (Fig. 27). The symphysis is characterized by viable chondrocytes in the medio-sagittal region and gradually degenerating ones towards the cartilage rod, Blastema stage which in this area is covered by perichondral bone. In early one day post-hatching specimens, the symThe viable chondrocytes are at most about 8 pm physeal blastema is not yet visible, indicating that across and contain moderate amounts of RER, several chondrogenesis lags slightly behind in the medio-sag- mitochondria, and well-developed Golgi regions (Fig. ittal plane. In late one-day specimens, the symphyseal 28). The degenerating cells on either side are wider and blastema has appeared and is characterized by a larger have the same characteristics as the moderately degennumber of more loosely packed cells as compared to the erated chondrocytes previously described for the rodrod blastema. The blastemal boundary is less well-de- shaped part of Meckel’s cartilage (electron-lucent cytofined and the blastemal cells gradually merge with the plasm with prominent RER and mitochondria, surrounding mesenchymal cells (Fig. 22). The prechonwell preserved cell membrane; compare Figs. droblasts are polygonal, about 7 pm in diameter, and relatively have no preferential orientation. Their nucleus takes a 28,In16). the medio-sagittal region, the territorial cartilage large part of the cell volume and usually houses one or matrix is separated from the perichondral cells by an two prominent nucleoli. Their cytoplasm contains electron-lucent zone (1.5 pm wide in its thickest part) RER, Golgi regions, mitochondria and numerous free 29). ribosomes. Peripheral cells are not clearly distinguish- (Fig. Unlike the rod-shaped part of Meckel’s cartilage, the able. Cilia are frequently observed in mesenchymal symphyseal region is not affected by resorption for sevcells surrounding the blastema, or in blastemal cells eral weeks. proper (Fig. 22). Primordium stage At two days post-hatching, the symphyseal region (15 pm high in average) houses rounded to rectangularly-shaped chondroblasts that are organized in about one to two layers and that are surrounded by chondroblasts with another orientation a t the cartilage periphery (Figs. 23, 24). The symphyseal chondroblasts (approximately 12 pm high and 6 pm wide) differ from those in the rod by their shape, but their fine structure and that of the matrix are similar (compare Figs. 10, 24, 24 inset). However, the intercellular spaces are generally wider in the symphysis, and cellular junctions are not present. This matrix is as well-developed between the cells as in the peripheral region. The peripheral cells (approximately 6 pm wide and 2.5 pm high) are smaller than the chondroblasts of the symphysis proper and resemble the chondroblasts located at the cartilage periphery (Fig. 24). Calcified cartilage At seven days post-hatching, blue staining globules can be observed in the cartilage matrix on one-pmthick sections. Although they are located throughout the matrix, they show a preferential distribution a t the periphery close to the perichondral bone (Fig. 30). The globules are most abundant in the symphyseal region, and decrease in numbers posterior to this region. U1trastructurally, the globules are seen to be either isolated or to form large clusters (Fig. 31). Isolated globules have different sizes but are a t most about 1pm in diameter (Fig. 32). In the center of most globules, an electron-lucent circular spot, about 0.25 pm in diameter, is clearly visible (Figs. 31-33). The periphery and center of this spot are often electron-dense. Crystals radiate from the surface of the globule. The clusters are frequently seen to merge without demarcation with the mineralization front along the perichondral bone (Fig. 33). Patches of electron-dense background substance Differentiatedcartilage occur within the cartilage matrix ahead of the minerAt three days post-hatching, the symphysis (approx- alization front. In some areas, calcified globules are imately 20 pm high) is constituted of seemingly ran- seen to be located amidst cellular debris (Fig. 33). TEM O F CICHLID MANDIBULAR DEVELOPMENT Figs. 30-33. Calcified cartilage in the symphyseal area of a specimen at seven days post-hatching. Fig. 3O.One-pm-thick section through the rear part of the symphy sis showing globules (arrows) dispersed in the cartilage matrix. Bar = 25 pm; x 440. Fig. 31. Low power electron micrograph of the right part of the symphysis, clearly showing isolated (arrows) and coalesced (arrowheads) globules. cc = chondrocyte; m = cartilage matrix; pe = perichondral bone. Bar = 5 pm; x 2,200. 367 arrangement of the crystals in the globules. The highly fibrillar matrix (m) between the globules may be due to the proximity of perichondral bone in this region beyond the plane of section. Bar = l pm; x 15,000. Fig. 33. Detail of the coalesced globules visible on Figure 31. The latter are continuous with the perichondral bone (pe). Electron-dense patches of background substance (arrow) are located ahead of the mineralization front within the cartilage matrix (m). Note the presence of calcified globules in a region of cellular debris (star). Bar = 500nm; x 22,500. Fig. 32. High magnification of some calcified globules, two of which are coalescing (arrow). Note the electron-lucent center and the radial Parachondral Bone The relative positions of the perichondral and parachondral bones with regard to the cartilage are given schematically in Figure 34. At two days post-hatching, the anlage of the parachondral bone is visible along the lateroventral side of Meckel’s cartilage as a slightly elongated tiny spicule approximately 8 km separated from it (Fig. 35). At the ultrastructural level, the bone anlage (approximately 2.0 km in its thickest part) is covered on both sides by a layer of osteoblasts clearly distinguishable from the surrounding mesenchymal cells (Fig. 36). The osteoblasts have abundant cytoplasm containing predominantly well-developed RER, Golgi regions and secretory granules. The cell membrane frequently pre- sents thin finger-like processes penetrating into the bone matrix. The latter presents a regional variation in electron-density (but see below). The osteoblasts facing the cartilage are separated from the latter only by one layer of flattened perichondral cells. At this level, there is no perichondral bone. At three days post-hatching the parachondral bone anlage stretches further laterally and ventrally below the cartilage (Fig. 11). A well-defined population of strongly basophilic cells surrounds the anlage. There are only minor ultrastructural changes with regard to the previous stage. The osteoblasts are more flattened and overlap each other on the side facing the cartilage (Fig. 37). They are more regularly aligned, defining in this way a bone lamella of approximately 368 A. HUYSSEUNE AND J.-Y. SIRE D’ A’ mentomeckelium I Fig. 34. Schematical drawing based on computer-aided reconstruction of a specimen at four days post-hatching showing the relative positions of the mentomeckelian, i.e., perichondral bone (pel and of the dermal dentary, i.e., parachondral bone (pa) with regard to the cartilage (Mc). On the left the cartilage is shown exposed. Arrows indicate the level of sections (labeled A to D) through the cartilage (left) and through the cartilage with bone (right). Bar = 50 km. homogeneous thickness. At its lateral edge, the bone lamella is surrounded by cuboidal or pear-shaped 0steoblasts (Fig. 38). Small patches of bone matrix are found in the lateral prolongation of the bone lamella. The cartilage still has no perichondral bone collar in this area. Slight differences in fine details are observed in more advanced three-day-old specimens. They especially concern the matrix and will be described below. The different stages of maturation of the matrix can be illustrated in several regions of the bone of the same Figs. 35-43. Development of the parachondral bone. Fig. 35. One-km-thick transverse section a t two days post-hatching, showing the position of the anlage of the parachondral bone (pa). Bar = 25 pm; x 500. Fig. 36. Detail of Figure 35. The parachondral bone (pa) appears at some distance from the cartilage matrix (m) which lacks perichondral bone in this area. The bone matrix is covered on each side by a layer of well-developed osteoblasts (ob). pec = perichondral cell. Bar = 1 pm; x 9,000. Fig. 37. Detail of Figure 11.At three days post-hatching, the parachondral bone (pa) is present as a plate-like structure lined on each side by a layer of elongated osteoblasts (ob). The bone matrix is more or less homogeneously electron-dense (compare with Figure 36). A layer of perichondral cells (pec) separates the unossified cartilage matrix (m) from the osteoblasts. Bar = 1 km; x 9,000. Fig. 38. The lateral boundary of the parachondral bone (pa) shown in the previous picture is surrounded by large cuboidal or pear-shaped osteoblasts (ob) but patches of bone matrix can be seen at a distance from this margin (arrow). Bar = 2 pm; x 6,000. Fig. 39. First stage of matrix deposition during parachondral bone (pa) formation. Scarce collagen fibrils and numerous rounded parti- cles are the first elements seen in the intercellular space between the two layers of osteoblasts (oh). Bar = 500 nm; x 30,000. Fig. 40. In a more advanced stage, the collagen fibrils and rounded particles of the central area of the bone matrix (pa) lie embedded within a finely granular, electron-dense background substance. ob = osteoblast. Bar = 500 nm; x 30,000. Fig. 41. Final stage of bone maturation. A dense matrix occupies the entire intercellular space except in some narrow marginal zones where the collagen fibril meshwork is loose and the background substance is lacking (arrowhead). ob = osteoblast; pa = parachondral bone. Bar = 500 nm; x 30,000. Fig. 42. One-hm-thick transverse section of Meckel’s cartilage at late three days post-hatching, anterior to the region of parachondral ossification described previously. Here, the parachondral bone (pa) merges with the perichondral bone (arrow). Bar = 25 km; x 500. Fig. 43. Detail of the zone of fusion between perichondral (pel and parachondral (pa) bones shown in Figure 42. The two matrices are alike and present a denser aspect than the parachondral bone described previously. m = cartilage matrix; ob = osteoblast. Bar = 1 pm; x 10,000. Figs. 35-43. A. HUYSSEUNE AND J.-Y. SIRE 370 A B C D ckel's cartilage growth. A = blastema; B = eai-y Fig. 44. Interpretative scheme of the dynamics of primordium; C = late primordium; D = differentiated cartilage. See Discussion for further details. Bar = 5 pm. specimen (Figs. 39-41). Figure 39 shows the initial stage of matrix formation of the parachondral bone. The intercellular space is electron-lucent and houses few randomly dispersed collagen fibrils (15 to 20 nm in diameter) along with numerous rounded particles of various diameters (mostly about 40 nm, but up to 100 nm) resembling transversely cut cell prolongations. In a further stage, the central part of the intercellular TEM OF CICHLID MANDIBULAR DEVELOPMENT space is occupied by dispersed collagen fibrils and rounded particles, both embedded in a finely granular, electron-dense background substance (Fig. 40). In a more mature stage, the intercellular space is filled with typical woven bone matrix characterized by randomly dispersed, packed collagen fibrils and dense background substance obscuring much of the rounded particles (Fig. 41). In some marginal regions, the collagen fibrils are loosely disposed and the electrondense background is lacking. In well-developed specimens at three days posthatching, the parachondral bone merges anteriorly at the level of the third tooth germ with the perichondral bone or with apolamellae arising from it (Fig. 42). The matrix of both the perichondral and parachondral bone consists of randomly dispersed collagen fibrils and fine granular material (Fig. 43). As for perichondral bone, no cells are being trapped into the parachondral bone matrix. DISCUSSION Light microscopical studies dealing with the development of teleost cartilage are rare. Stephan (1900), Blanc (1953), Haines (1937) and Francillon (1974, 1977) present histological aspects of cartilage development in teleosts. However, only the latter two authors deal with Meckel’s cartilage, although not with very early stages. As far as we are aware, this is the first ultrastructural study on chondrogenesis in a bony fish. The only ultrastructural study known to us on “fish” cartilage development deals with the development of the adult piston cartilage in lampreys, a tissue unlike other vertebrate cartilages (Armstrong et al., 1987). Comparisons with the few existing ultrastructural studies of (mature) fish cartilage are not relevant because they deal either with non-hyaline cartilages (e.g., Wright and Youson, 1982, 1983; Benjamin, 1988, 1989a,b; Benjamin and Sandhu, 1990) or with tissues intermediate between cartilage and bone (e.g., chondroid bone, Huysseune and Sire, 1990). As far as we know, the only ultrastructural study of Meckel’s cartilage in a cold-blooded vertebrate is that of Thomson (1986) in Xenopus laeuzs. Not only does premetamorphic Meckel’s cartilage in Xenopus resemble invertebrate cartilage, but early postmetamorphic cartilage shows several features unlike those observed in the cichlid mandible (e.g., solitary cells exhibiting degenerative features). Because of the lack of ultrastructural data on fish cartilage and on its differentiation, we have to refer largely to studies on chondrogenesis in warm-blooded vertebrates for comparison. Chondrogenesk of Meckel’s Carfikge The observations presented in this study on the differentiation of chondrocytes in Meckel’s cartilage of the cichlid Hemichromis bimaculatus can be summarized and interpreted schematically as in Figure 44. We have followed Thorogood (1983) in designating a condensation of cells destined to form an individual skeletal element and showing no detectable cartilage matrix at the EM level, as a “blastema.” The blastema becomes a “primordium” once cartilage matrix is detectable ultrastructurally. At the onset, pyramidal-shaped prechondroblasts 371 form approximately two cell layers pointing with their apex towards the center of the blastema (Fig. 44A). Next, cells of opposite sides of the blastema diverge from one another, whereby their apices prolong into elongated cell processes, penetrating between the two opposite cells (Fig. 44B). In a third phase, these cell processes widen and the cells gradually start to secrete matrix (Fig. 44C). Finally, the chondrocytes further differentiate, thereby secreting large amounts of matrix (Fig. 44D). During this process, the cytoplasm of the chondroblasts, later chondrocytes, shows an increasing state of differentiation, as shown by the lowering of the nucleo-cytoplasmic ratio, and the increase in numbers of cell organelles associated with secretory activity. While the chondroblasts flatten, they become oriented in a plane perpendicular to the long axis of the rod. This resembles the arrangement of the chondrogenic cells during long bone formation in the chick limb bud (Rooney et al., 1984). The authors ascribe this cell orientation to the fact that initially there is a greater dilation in the radial direction and because of this, cells orient in that direction. Because we lack data on longitudinal growth of the rod, we cannot fully support this hypothesis. Our observations indicate that the time needed for chondroblasts to differentiate into mature chondrocytes is about two days. This is comparable to the rate of chondrogenesis in Meckel’s cartilage of the rat: one day for differentiation from primordium to differentiated chondrocytes (Granstrom et al., 1988). Furthermore, our results indicate that the different regions of Meckel’s cartilage in Hemichromis birnaculatus do not mature at the same rate; e.g., differentiation of the symphysis lags behind but catches up soon. This is in agreement with the development of Meckel’s cartilage in mice (Richman and Diewert, 1987). Our results convincingly show that the differentiation of cartilage is continuous in the symphyseal region. Therefore, when the two rami are seen to be discontinuous a t some later stage during ontogeny, this must be the result of processes that take place after initial chondrogenesis. The continuous chondrification of the two rami of Meckel’s cartilage has been reported previously (e.g., de Beer, 1937) but not a t the ultrastructural level. In contrast with Meckel’s cartilage, the ceratobranchials of the fifth arch, which make up the lower pharyngeal jaws in cichlids, chondrify completely separately (unpublished data). The increasing differential state of the cartilage cells during chondrogenesis (lowering of nucleo-cytoplasmic ratio, development of RER and Golgi regions) resembles the cellular changes reported for mammalian chondrogenesis (e.g., Godman and Porter, 1960; Silberberg et al., 1964; Sheldon, 1983). It is striking that what we consider to be a fully differentiated chondrocyte (e.g., our Fig. 121, would be called a very young chondroblast by others (Silbermann and Lewinson, 1978, Fig. 6). This is related to the relatively small amounts of matrix. Mature chondrocytes, as a result of their mode of formation, typically are flattened cells. Their characteristic ultrastructural features are: their scalloped cell surface, a large centrally placed nucleus, well-developed Golgi regions and RER, and several mitochondria. The mature flattened chondrocytes resemble the chon- 372 A. HUYSSEUNE AND J.-Y. SIRE drocytes described a t the light microscopical level in the flattened cell zone in epiphyseal cartilages of fish (Haines, 1934, 1938, 1942; Meunier, 1979) and of warm-blooded vertebrates (e.g., Howlett, 1979). The chondrocytes in Meckel’s cartilage of H . bimaculatus however, seem to lack one feature which is often held to be characteristic for such cells, namely deposits of glycogen (e.g., Godman and Porter, 1960; Silberberg et al., 1964, 1966, 1976; Silva and Hart, 1967; Holtrop, 1972; Silbermann and Frommer, 1974; Silbermann and Lewinson, 1978). The presence of glycogen in chondrocytes is indicative of the state of maturation of the cells and related to the synthesis of matrix polysaccharides (Knese, 1979). It may be that the chondrocytes examined are too young to exhibit significant amounts of glycogen (we should recall the small amount of matrix even differentiated cartilage possesses) and that glycogen possibly accumulates at later stages when more matrix surrounds the chondrocytes. Alternatively, it has been claimed that permanent cartilage (as is a large part of Meckel’s cartilage in cichlids) contains little glycogen (but see Knese, 1979). A third possibility is that the presence of glycogen has often been related to mineralization of the matrix. The limited calcification of Meckel’s cartilage is meaningful in this respect. The chondrocytes show profound changes in cellular morphology once the perichondral bone is being deposited and although these changes could normally be collectively termed “degenerative changes,” the cells do not show true hypertrophy in the sense of considerable enlargement. There is evidence, however, from later developmental stages, that the cell lacunae do enlarge and therefore gradually acquire a hypertrophic aspect, The association of hypertrophy with perichondral ossification is well-known from “epiphyses” in fish (Haines, 1938) and from long bone development in higher vertebrates (e.g., Pechak et al., 1986). However, initial osteogenic activity, e.g., in the developing chick limb, is claimed not to require chondrocytic hypertrophy (Osdoby and Caplan, 1981). Similarly, our results indicate that perichondral ossification precedes socalled degenerative changes in the chondrocytes. The degeneration of the cells has usually been ascribed as the result of poor nutrient and oxygen supply through the bone, but recently, with the use of improved processing techniques, evidence is growing that shows that the hypertrophic cells do in fact not die (Arsenault et al., 1988; Hunziker and Herrmann, 1990) and may even transform into other cell types (Yoshioka and Yagi, 1988; Roach and Shearer, 1989). The composition of the cartilage matrix (fibrils and granules probably representing proteoglycans) resembles that of typical cartilage matrix of higher vertebrates (Scott and Pease, 1956; Godman and Porter, 1960; Sheldon, 1983). The lacunar space which is observed around chondrocytes a t a mature stage probably presents a n artifact. Arsenault et al. (1988), Arsenault (1990) and Hunziker and Herrmann (1990) provide ample evidence that improved fixation techniques show the absence of the lacunar space around the mature or degenerating chondrocyte. Our results clearly show the appearance in the cartilage matrix of isolated globules or calcospherites in which crystals are radially orientated (i.e., spheritic mineralization, IZlrvig, 1951; 1967; Francillon-Vieillot et al., 1990). Our observations indicate that the mineralization extends by fusion of calcospherites either to one another or to the mineralization front which has formed on the inside of the perichondral bone, or directly by progression of this mineralization front. The presence of calcified nodules is a feature of calcifying regions of fetal bone, dentine and cartilage in mammals (Boyde and Jones, 1972; Ali, 1983; Bonucci and Motta, 1990). Calcospherites have been frequently reported during calcification of selachian cartilage (Kemp and Westrin, 1979; Peignoux-Deville et al., 1982; Bordat, 1988) and, a t the light microscopical level only, in teleosts (Meunier, 1979). Formation of Perichondral Bone The formation of perichondral bone deserves particular attention. At the light microscopical level, a n acellular lamella of perichondral bone becomes visible only when appreciable changes have already taken place ultrastructurally at the cartilage periphery. These changes include: (1)the deposition of a layer of loosely organized bone-like fibrils but which still contains granules (probably proteoglycans) (zone 21, and (2) the deposition of a dense fibrillar bone-like matrix without proteoglycan granules but with a dense interfibrillar background substance (zone 3). Mineralization takes place in the latter zone, representing the perichondral bone proper. These gradual changes clearly elicit the question of the role of the perichondral cells. Our observations suggest that these cells seem to change their metabolic properties as the process of chondrogenesis goes on. At the blastema stage, when they are still called peripheral cells, they are probably little active. In the primordium stage, they present a more important secretory activity, but then again, these cells, called now perichondral cells, seem to go through a phase of limited activity once the cartilage is differentiated. In contrast, the perichondral cells seem to resume a n important secretory activity concomitant with the appearance of the first perichondral bone matrix at the cartilage periphery. These events strongly suggest that perichondral cells could be involved in cartilage matrix deposition prior to synthesis of perichondral bone matrix. The important, yet unsolved, issue is which signal may trigger such a switch in secretory activity of the perichondral cells. The evidence brought here seems to fully document a n earlier discussion of, e.g., 0rvig (1951) and de Ricqles (1979) on the functional change of a perichondrium into a periosteum. We insist on the use of the term perichondral rather than periosteal bone for zone 3, both in a topographical and in a histological sense (i,e., the juxta-chondral bone of de RicqlBs, 1979). Periosteal bone should then be reserved to the bone deposited from zone 4 outwards (but see de Ricqles, 1979, for a more circumstantial discussion on terminology problems). The presence of a transitional zone (zone 2) (apparently containing elements of the two types of matrices) between cartilage and perichondral bone is seen a s further evidence for a n alternating state of activity of the perichondral cells. The presence of such a zone of gradual transition is also suggested in the perichondral TEM OF CICHLID MANDIBULAR DEVELOPMENT bone in the branchial arch of the eel (Lopez et al., 1978, Fig. 8). Whether the transitional zone in cichlids contains a mixture of bone-specific (type I) collagen and cartilage-specific (type 11)collagen remains to be demonstrated. Von der Mark et al. (1976) report the presence of matrix containing a mixture of type I and type I1 collagen in at least three different areas in chick long bone development, and suggest this to be due either to overgrowth of type I collagen matrix by expanding cartilage (however, this is in areas where cartilage adjoins soft type I collagen-containing connective tissue, hence irrelevant to our case) or to diffusion of collagen type I into the unmineralized cartilage matrix. Although diffusion of matrix components seems the most likely of these two explanations, in the case of ossification of Meckel’s cartilage in H. bimaculatus, another hypothesis could be that cells synthesize elements of both matrices simultaneously. Is this evidence for a metaplastic way of bone formation? Because the transitional zone forms de novo either as the result of diffusion or of simultaneous deposition of elements of the two matrices, there is no metaplasia of cartilage into bone. There has long been a controversy on the possible occurrence of metaplasia in teleosts. Blanc (1953), in his histological description of the different modes of bone formation in teleosts, regards perichondral ossification in the branchial arches of bony fish as an example where it is not clear whether one is dealing with metaplastic or neoplastic bone. His doubt is reinforced by the reexamination in Salmo irideus (S.gairdneri) of the “tissu mixte,” reported by Stephan (1900) to be present in the branchial arches of Gadidae and of Esox, and in various other instances. In the case of the “tissu mixte,” the cartilage grades into a tissue with sparser and more elongated cells amid a calcified matrix. Blanc (1953) considers this to be an intermediate tissue and suggests its presence to be the result of a progressive rather than a sudden change of the perichondrium into a periost. On the other hand, Benjamin (1989b) states that a direct transformation of cartilage into bone is a common process in lower vertebrates. Is this mode of perichondral ossification linked to the acellular nature of the bone matrix in cichlids? The perichondral bone of Meckel’s cartilage in larval and juvenile cellular-boned fishes such as carp (Cyprinus carpio) is virtually acellular and occasional osteocytes lie at a distance from the cartilage surface (unpublished data). It seems likely, therefore, that a similar process of perichondral ossification may be expected in cellular-boned fishes. The peripheral cells present at a stage when perichondral bone is being deposited, as well as cells depositing parachondral bone, without doubt can be termed osteoblasts. Their ultrastructure fits in with the description of teleost osteoblasts by Lopez et al. (1978). Riehl (1978), Weiss and Watabe (1979) and Wendelaar Bonga et al. (1983). We have not been able to distinguish the two stages of osteoblasts described by Lopez et al. (1978) during perichondral ossification of the branchial arches in the eel (AnguzEla anguilla). From the electron micrographs presented by Lopez et al. (1978), it would appear that the mineralization front is very broad. This, along with the fact that the bone they describe is cellular (and their second type of osteoblasts already partially embedded in osteoid) can 373 account for the differences between our observations and those of Lopez et al. (1978). Dynamics of Cartilage Growth The data presented in this study allow us to draw a hypothesis on the mechanism of growth of Meckel’s cartilage of H . bimaculatus, and possibly of cichlids in general. According to Hinchliffe and Johnson (1983), cartilage growth may be achieved in one of four ways: cell recruitment from the surrounding tissues, cell division, cell enlargement and matrix secretion. At the blastemal stage in H . bimaculatus, the prechondroblasts are well-delimited from the peripheral mesenchyme, indicating that there is probably no cell recruitment to increase the size of the blastema. In warm-blooded vertebrates too, cell recruitment from the surrounding mesenchyme is considered to be an unlikely mechanism for blastemal growth (Thorogood, 1983). At a later stage, the perichondral cells are separated from the centrally located chondroblasts (later chondrocytes) by a zone of matrix which contains no cells. This leads us to assume that there is no appositional growth in larval stages. A possible exception to this is the symphyseal area, where neither the blastema, nor the later stages of the symphysis show distinct boundaries with the surrounding mesenchyme, and where apposition is assumed to allow growth of the symphyseal area both in a dorso-ventral and rostrocaudal direction. Moreover, it is suggested that, through incorporation of cells in the symphysis, adjacent cells may be gradually displaced towards the ramus. In this way, the symphysis could provide an additional means for increase in length of Meckel’s cartilage up to a stage where the symphysis becomes entirely surrounded by bone. Prior t o and during cartilage differentiation, there is only very little evidence for cell divisions, and none, once perichondral bone is laid down. In contrast, the increase in number of cell layers in the ramus caudal to the anterior perichondral ossification as well as morphological evidence for recent divisions (unpublished data), indicates that cell divisions may play a considerable role in the growth of the ramus posterior to the ossified region. Labeling with 3H-thymidine is currently being carried out to quantify cell divisions in the mandible. Haines (1934) reported mitotic divisions in the flattened cell zone of the branchial bones of teleosts and therefore regarded this zone as a means for providing interstitial growth. Cell enlargement as a possible factor contributing to cartilage is usually important only in the case of hypertrophy of the chondrocytes. Hypertrophic chondrocytes in chick epiphyseal cartilage are reported to be about 20 times larger than cells of the rounded cell zone (Rooney et al., 1984). No true hypertrophy is observed in Meckel’s cartilage of Hemichromis bimaculatus and therefore cell enlargement is supposed to contribute only to a small extent to the cartilage growth. Obviously, the most prominent way in which the volume increase is achieved in the studied region of Meckel’s cartilage must be by matrix secretion, both among the chondrocytes and at the periphery of the cartilage, which again raises the question of the participation of the peripheral cells to matrix secretion. The assumed 374 A. HUYSSEUNE AND J.-Y. SIRE important contribution of matrix secretion confirms a previous quantitative study on branchial cartilage growth in another cichlid (Huysseune et al., 1988). In this study, matrix volume increase was shown to be the most important factor contributing to cartilage growth. In Xenopus, where architectural changes in Meckel’s cartilage during the period of metamorphic climax occur in three phases, matrix increase is important in phase I11 only (Thomson, 1987). In the first two phases, changes in shape of the cartilage are mainly due to a balance between cell division and death, while the amount of matrix remains essentially unchanged (Thomson, 1987). In conclusion, the present ultrastructural description of the development of cartilage anlagen, transformation of perichondrium to periosteum and deposition of bone in a teleost mandible illustrates forms of skeletal development that have been neglected in recent lines of skeletal research. Our findings reveal both similarities and differences between teleosts and other vertebrates and therefore demonstrate the relevance of such studies in a phylogenetic perspective. The present study hopefully provides an adequate basis to study the induction and differentiation of the teleost mandible. It has provided us with a basis to examine the relationships between cartilage, bone and teeth during further morphogenesis of the cichlid mandible, and which will be the subject of a second paper. ACKNOWLEDGMENTS The authors are greatly indebted to Prof. W. Verraes (Ghent) and Prof. A. de Ricqles, Dr. F.-J. Meunier and Dr. L. Zylberberg (Paris) for their valuable comments on the manuscript. The authors also thank Mrs. F. Allizard and G. De Wever for excellent technical assistance. This study was carried out within the frame of an International Program of Cooperation between Belgium (Ministerie van de Vlaamse Gemeenschap) (A.H.) and France (CNRS/DRCI) (J.-Y S.).Financial support was provided by grant 2.9005.90 of the Belgian Fund for Joint Basic Research. A.H. is a Senior Research Assistant of the Belgian National Fund for Scientific Research. LITERATURE CITED Ali, S.Y. 1983 Calcification of cartilage. In: Cartilage. Vol. 1. Structure, Function and Biochemistry. B.K. Hall, ed. Academic Press, New York, London, pp. 343-378. Armstrong, L.A., G.M. Wright, and J.H. Youson 1987 Transformation of mucocartilage to a definitive cartilage during metamorphosis in the sea lamprey, Petromyzon marinus. J. Morph., 194:l-21. Arsenault, A.L. 1990 The ultrastructure of calcified tissues: Methods and technical problems. In: Ultrastructure of Skeletal Tissues. E. Bonucci and P.M. Motta, eds. Kluwer Academic Publishers, Dordrecht (The Netherlands), pp. 1-18. Arsenault, A.L., F.P. 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