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Development of cartilage and bone tissues of the anterior part of the mandible in cichlid fishA light and TEM study.

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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
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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.
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