Development of osteogenic and chondrogenic potentials along the mediolateral axis of the embryonic chick mandible

Archs oral Biol. Vol . 33, No . 6, pp . 443-449, 1988

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DEVELOPMENT OF OSTEOGENIC AND CHONDROGENIC POTENTIALS ALONG THE MEDIOLATERAL AXIS OF THE EMBRYONIC CHICK MANDIBLE MARY S. TYLER

Department of Zoology, University of Maine, Orono, ME 04469, U .S .A.

Summary-Various regions of the mandibular process were tested for these potentials to determine whether regional differences exist and vary with embryonic age . Mandibular processes from HH stages 17-21 .5 were cultured and grafted intact, or were subdivided into medial, mediolateral and lateral fragments and the separate regions cultured or grafted . The intact mandible from all these stages can form cartilage and membrane bone, but the 3 regions are not equally osteogenic and chondrogenic. The lateral region from all stages could form cartilage and membrane bone ; the mediolateral region could form cartilage and membrane bone but, in mediolateral fragments from HH stage 17, membrane bone was formed only in scant amounts. The medial region from HH stages 17 and 18 formed cartilage in only 50 per cent of cases and never formed membrane bone . By HH stage 20, the medial region could form membrane bone, but only in scant amounts . Medial fragments from HH stage 21 .5 formed extensive membrane bone and cartilage . The acquisition of these potentials, therefore, proceeds in a lateral-tomedial sequence, and the acquisition of an osteogenic potential lags slightly behind that of a chondrogenic potential. These findings do not indicate the mechanisms by which the two subpopulations of chondrogenic and osteogenic cells are distinguished from one another, but they give the temporal and spatial sequence in which this determination must occur .

INTRODUCTION

Within the mandibular processes of the embryonic chick, both cartilage and membrane bone form from neural crest cells which begin populating the paired processes during the second day of incubation (Noden, 1975 ; LeLievre and LeDouarin, 1975) . These paired processes fuse medially by 3 days of incubation (Hamilton, 1952), and histodifferentiation of the skeletal elements begins on the fifth day . At this time the cartilaginous matrix of Meckel's cartilage is evident (Murray, 1963 ; Tyler and Hall, 1977) ; this cartilage then forms a continuous central rod along the mediolateral axis of the mandible . Beginning on the seventh day of incubation, a series of membrane bones forms along the length of Meckel's cartilage and ultimately invests it (Fell and Robison, 1930; Tyler and Hall, 1977). Neither the mechanisms by which these osteogenic and chondrogenic lines within the mandible are delineated nor the time at which this delineation occurs are known . It is known that there are differences in the requirements for the expression of the 2 phenotypes : the formation of the membrane bones is dependent upon the presence of epithelium for a specific period prior to the onset of ossification ; chondrogenesis does not require an epithelium during this period (Tyler and Hall, 1977) . For amphibians it has been shown that the tissue interactions required for chondrogenesis occur between the neural crest cells and the pharyngeal endoderm before the arrival of the neural crest cells in the mandible (Drews, KockerBecker and Drews, 1972 ; Epperlein, 1974; Epperlein and Lehmann, 1975) . However, whether the neural crest cells arrive in the mandibular process as a homogeneous population or as a heterogeneous population with differences that distinguish the 443

osteogenic from the chondrogenic precursors is not known . The aim of my study was to determine whether the osteogenic and chondrogenic capabilities of the mandibular mesenchyme change with developmental age, and whether there are regional differences in osteogenic and chondrogenic abilities within the mandibular process . MATERIALS AND METHODS Tissue

preparation

Fertile chick eggs (Gallus dosmesticus, White Leghorn) were incubated in a forced draft incubator at 37 .5±1°C and 57 ± 2 per cent humidity . Embryos between 2.5 and 3 .5 days of incubation (Hamburger and Hamilton, 1951, stages 17-21 .5) were dissected in Tyrode's solution . Mandibular processes were either left intact or subdivided along their medial-lateral axis into one medial, two mediolateral, and two lateral pieces (see top of text Fig . 9) . Culturing procedures Intact or subdivided organs were placed either culture or onto the chorioallantoic membrane of b chick embryos . Cultured tissues were grown complex culture medium (BGJb-Fitton Jackppn modification, supplemented with 10 percent fetal ;' If serum 100 U/mI of penicillin and streptomycin 2 .5 µg/ml of fungizone ; GIBCO, Grand Island, U .S .A .) on metricel filters (5 mm discs, 0 .20 p osity, 150 pm thick) supported by stainless-st in Falcon Petri dishes . Cultures were maintai 37°C in 5 per cent CO 2 in air in a Fonna :S water-jacketed CO 2 incubator for 7 days . The medium was changed every 24-48 h . Tissues -



444

MARY S . TYLER

grafted were placed on metricel filters and inverted onto the chorioallantoic membrane of 8-day host chick embryos . This allowed the graft tissues to be in direct contact with the vascularized host tissue . Grafts were maintained for 7 days in a forced draft incubator at 37 .5 ± I `C and 57 ± 2 per cent humidity . Grafts and cultures were maintained beyond the time that chondrogenesis and osteogenesis would have occurred in vivo . In normal development, stainable cartilaginous matrix is first apparent at HH stage 27 (5-6 days of incubation), and membrane bone matrix is apparent by HH stage 34 (8 days of incubation ; Tyler and DeWitt-Stott, 1986) . Histological procedures Cultures and grafts were fixed in Bouin's fluid (Humason, 1972), dehydrated in a graded series of ethanol, cleared in toluene, and embedded in paraffin blocks. Serial sections (5µm) were stained in haematoxylin, eosin, and alcian blue (pH 2 .5-3 .0 ; Pearse, 1960) or in Van Gieson's stain (Wilsman and Van Sickle, 1971) . The results are based upon a total of 131 cultures and 86 grafts . This represents the grafts and cultures in which the surface epithelium remained in contact with the mesenchyme . As membrane bone formation in the chick mandibular process is dependent upon the presence of an epithelium until HH stage 24 (Tyler and Hall, 1977), cases in which epithelialmesenchymal contact was not maintained were not included. The reason for duplicating the culture experiments with grafts was that, as reported previously (Tyler and Hall, 1977), though the variables of the culture environment are more easily controlled, the graft supports a greater amount of growth . RESULTS

Cultured and grafted intact mandibular processes (Table I) Cartilage differentiated in grafts and in cultures of intact mandibular processes in all the stages (HH stages 17-21 .5 ; Table 1) and was found as a rod of tissue, usually centrally-located within the mesenchyme (Plate Fig . 1) . Membrane bone, however, did not form in all instances . Among the mandibles of the earliest stage (HH stage 17), membrane bone formed in only half (Table 1 ; Plate Fig . 2). In grafts and cultures of HH stage 18 mandibles, membrane bone fKrned in all but two instances (Plate Fig . 3), and in WEse of older stages (HH stages 19-21 .5), it formed Sail cases (Table 1) . Where it formed, membrane

. Occurrence of cartilage and membrane bone in T e 1 cultured and grafted chick mandibular processes e

H) 17 18

t9 20 211.5

Cartilage (per cent)

Membrane bone (per cent)

Number of grafts and cultures

100 100 100 100 100

50 86 100 100 100

8 14 4 6 IS

bone was near the cartilage, positioned between it and the epithelium . Membrane bone did not form in the absence of cartilage . Cultures and grafts of subdivided portions mandibular processes (Table 2)

of

The subdivided mandibular processes were either grown as separate pieces or were grouped with pieces

of the same region from other similarly-aged mandibular processes, thereby increasing the mass of the culture or graft . For each region of each stage, both separate and grouped pieces were grown . The presence or absence of cartilage and membrane bone in these cultures or grafts did not depend on whether the pieces had been grown separately or grouped . Skeletogenesis did vary amongst the different regions tested (Table 2). In pieces from HH stage 17 mandibles, medial fragments formed cartilage in 50 per cent (6 out of 12), and never formed membrane bone (Plate Figs 4 and 5) . Mediolateral fragments formed cartilage in all instances and formed scant amounts (fewer than 50 lacunae) of membrane bone in over half (13 out of 18 ; Plate Fig . 6). Lateral fragments formed cartilage in all instances and membrane bone with about the same frequency (12 out of 18 cases) but in more substantial amounts (greater than 200 lacunae) than mediolateral fragments . In pieces from HH stage 18 mandibles, the medial fragments developed as they did from HH stage 17 mandibles; there was chondrogenesis in 50 per cent of these, and no osteogenesis . In the mediolateral and lateral fragments, the chondrogenic abilities of the regions were similar to those of HH stage 17, forming cartilage in all instances, but the osteogenic

Table 2. Occurrence of cartilage and membrane bone in subdivided portions of embryonic chick mandibular processes grown in culture and grafted Mandibular region

Cartilage (per cent)

Number of Membrane bone grafts and (per cent) cultures

STAGE HH 17

L ML M

100 100 50

67 72 • 0

18 18 12

STAGE HH 18

L ML M

100 100

83 100 0

6 18 8

100 75 54*

24 20 13

STAGE HH 20.5 L 100 100 ML 100 M

100 100 100

4 4 4

STAGE HH 21 .5 100 L 100 ML M 100

100 83 100

10 6 5

50

STAGE HH 20

L ML M

100 100 50

Symbols : M = medial, ML=mediolateral, L=lateral . Present in scant amounts .

445

Skeletogenesis in the chick mandible abilities differed in that membrane bone formed more frequently and in greater amounts . By HH stage 20, medial fragments of the mandibular process showed chondrogenic ability in all cases (Plate Fig. 7); in 54 per cent, there was also osteogenic ability, but in five of these, membrane bone was found in only scant amounts (fewer than 50 lacunae; Fig . 7) . The chondrogenic and osteogenic abilities of the mediolateral and lateral fragments did not differ significantly from those of HH stage 18, as verified by the G test after applying Yates' correction for continuity (Sokal and Rohlf, 1981) . In mandibular processes from HH stages 20 .5 and older (HH stage 21 .5), medial, mediolateral and lateral fragments all showed chondrogenic and osteogenic abilities to a similar degree . In all instances (except one graft of a mediolateral region from HH stage 21 .5 where no membrane bone was found), both cartilage and membrane bone had formed (Plate Fig . 8) .

ML

HH STAGE 17

HH STAGE 18 ∎ E133 >'4+F iiifFS43hiYt : : :f Tim

HH STAGE 20 .x

HH STAGE 20 .5

∎

ooa

DISCUSSION

Chondrogenic and osteogenic abilities differ along the mediolateral axis of the mandibular process and change with developmental age . Both these abilities are acquired at a later stage in development in the medial region than in the more lateral regions of the mandibular process and, in all regions, the acquisition of an osteogenic ability lags slightly behind that of a chondrogenic ability . This temporal sequence is summarized in Fig . 9 . A study of a later staged embryo (HH stage 22) corroborates the finding of regional differences in osteogenic and chondrogenic abilities within the mandible (Hall, 1982) . By HH stage 22, these differences are primarily along the cephalocaudal axis, a dimension that I did not test . If the presence or absence of skeletogenesis within experimental grafts and cultures can be equated with the presence or absence of neural crest cells forming the skeletal structures, then chondrogenic and osteogenic neural crest cells must populate the mandible in a lateral-to-medial progression . An alternative explanation for the absence of membrane bone in a graft or culture is that the epithelium in these areas is not inducing osteogenesis . The results of recombination experiments on older mandibular tissues (HH stage 22) imply that there are regional differences in inductive ability within the mandibular epithelium, but these differences were exhibited along the cephalocaudal axis and not the mediolateral axis (Hall, 1982). Other recombination experiments with heterotypic tissues indicate that there is little specificity in the type or age of epithelium that is capable of inducing osteogenesis in membrane bone-forming mesenchymes (e .g . Tyler and McCobb, 1980) . Whether the neural crest cells arrive at the mandibular process as a homogeneous or as a heterogeneous population with cells predestined for either chondrogenesis or osteogenesis is still not known . Previous work suggests that at least some of the neural crest cells arrive with chondrogenic potential . Chondrogenesis within the mandible does not require a tissue interaction between the neural crest mesenchyme and the adjacent epithelium, whereas osteo-

Fig . 9 . Diagrammatic representation of the timing of the acquisition of chondrogenic (x x x) and osteogenic (000) potentials within the embryonic chick mandible along its mediolateral axis . Osteogenic and chondrogenic abilities are acquired at a later stage in the medial region than in the more lateral regions, and the acquisition of an osteogenic ability lags slightly behind that of a chondrogenic ability . M=medial region, ML=mediolateral region, L =lateral region .

genesis does (Tyler and Hall, 1977) . Furthermore, it has been shown that collagen II, which is linked with neural crest-cell commitment to chondrogenesis, does not appear in the mandible before chondrogenesis (Thorogood, Bee and von der Mark, 1986) . Instead, the neural crest cells destined to form the mandibular mesenchyme migrate through a collagen II-positive region (the basal aspects of the ventrolateral sides of the neural tube and the ventral ectoderm) before reaching the mandible . Whether or not all neural crest cells of the mandible arrive with chondrogenic potential still must be determined . If this is the case, then chondrogenesis must be inhibited in those neural crest cells which form the membrane bones . It is possible that the epithelium could do so : neural epithelium can have an inhibitory effect on chondrogenesis in the endomeninx which, although normally non-chondrog is capable of forming cartilage when the n ectoderm is removed (Tyler, 1983) . In cultures of tissues, epidermal ectoderm can inhibit cho genesis in mesenchyme lying within 200 um 6 ectoderm (Solursh, Singley and Reiter, 1981) . is suggests that an epithelially-derived factor inhi s chondrogenesis in situ in the peripheral limb meanchyme (Solursh, 1984) . If a similar mechanism ours within the mandible, then the positioning of the neural crest cells with respect to the epithelium would be critical in determining the osteogenic from the chondrogenic population of cells . This hypotletis being tested by experimentally altering the positio the epithelium with respect to the neural crest

MARY S . TYLER

446

Although it is not known how pattern formation is determined within the mandible, it is known that there is regional specificity, distinguishing mandibular arch neural crest from second branchial arch neural crest, within the premigratory cranial neural crest. When the positions of premigratory neural crest are experimentally altered so that the neural crest that normally would populate the mandibular arch populates the second branchial arch, these neural crest cells form the skeletal structures appropriate to the mandibular arch rather than those appropriate to their new location (Horstadius, 1950 ; Noden, 1983) . These findings indicate that the mechanism which distinguishes the osteogenic from the chondrogenic cells of the mandibular process is not specific to the mandibular process . The close proximity of neural crest-derived cartilage and membrane bone is not unique to the mandible, being found in other regions of the skull as well, for example around the eye (for a mapping of these regions, see Noden, 1983, Fig. 6) . In skull development, my personal observation of the general pattern is that the cartilages of a region form before its membrane bones . The pattern of chondrogenic and osteogenic potentials found in the mandible, therefore, may be typical of neural crest-derived skull elements . Acknowledgements-This investigation was supported by research grant R01 DE04859 from the National Institute of Dental Research of the National Institutes of Health. I thank Dr John Ringo for his help in statistical analyses and Dr Drew Noden for his critical review of the manuscript .

REFERENCES Drews Ul ., Kocker-Becker U . and Drews U . (1972) Die Induktion von Kiemenknorpel aus kopfneuralleistenmaterial durch prssuptive kiemendarm in der Gewebekultur and das Bewegugnsverhalten der Zellen w5hrend litter Entwicklung zu knorpel . Wilhelm Roux' Arch . 171, 17-37. Epperlein H . H. (1974) The ectomesenchymal-endodermal interaction system (EEIS) of Triturus alpestris in tissue culture . l . Observations on attachment, migration, and differentiation of neural crest cells . Differentiation 2, 151-168 . Epperlein H . H . and Lehmann R . (1975) The ectomesenchymal-endodermal interaction system (EEIS) of Triturus alpestris in tissue culture. 2. Observation on the differentiation of visceral cartilage . Differentiation 4, 159-174 . Fell H . B . and Robison R . (1930) The development and phosphatase activity in vivo and in vitro of the mandibular

skeletal tissue of the embryonic fowl . Biochem. J. 24, 1905-1921 . Hall B . K . (1982) Distribution of osteo- and chondrogenic neural crest-derived cells and of osteogenically inductive epithelia in mandibular arches of embryonic chicks. J. Embryol. exp . Morph. 68, 127-136 . Hamburger V. and Hamilton H . C . (1951) A series of normal stages in development of the chick embryo . J. Morph . 88, 49-92 . Hamilton H . L . (1952) Lillie's Development of the Chick, An Introduction to Embryology, 3rd edn. Holt, New York . HSrstadius S . (1950) The Neural Crest . Oxford University Press, London . Humason G . L. (1972) Animal Tissue Technique, 3rd edn . Freeman, San Francisco, Calif. LeLievre C. S . and LeDouarin N. (1975) Mesenchymal derivatives of the neural crest: analysis of chimeric quail and chick embryos . J. Embryol. exp . Morph . 34,125-1 .54. Murray P . D . F . (1963) Adventitious (secondary) cartilage in the chick embryo, and the development of certain bones and articulations in the chick skull . Aust . J. Zool. 11, 368-430. Noden D . (1975) An analysis of the migratory behavior of avian cephalic neural crest cells . Dev! Biol. 42, 106-130. Noden D . (1983) The role of the neural crest in patterning avian cranial, skeletal, connective and muscle tissues . Devl Biol. 96, 144-165 . Pearse A . G . E . (1960) Histochemistry, Theoretical and Applied, 2nd edn . Little, Brown, Boston, Mass . Sokal R. R . and Rohlf F . J. (1981) Biometry, 2nd edn . (W . H .) Freeman, San Francisco, Calif. Solursh M . (1984) Cell and matrix interaction during limb chondrogenesis in vitro . In : The Role of Extracellular Matrix in Development (Edited by Trelstad R . L.) pp. 277-303. (A . R.) Liss, New York . Solursh M ., Singley C . T . and Reiter R. S . (1981) The influence of epithelia on cartilage and loose connective tissue formation by limb mesenchyme cultures . Dal Biol. 86, 471-482. Thorogood P., Bee J. and Mark K . von der (1986) Transient expression of collagen type 11 at epitheliomesenchymal interfaces during morphogenesis of the cartilaginous neurocranium . Dal Biol. 116, 497-509. Tyler M . 5. (1983) Development of the frontal bone and cranial meninges in the embryonic chick : an experimental study of tissue interactions . Anal. Rec. 206, 61-70. Tyler M . S. and Hall B . K. (1977) Epithelial influences on skeletogenesis in the mandible of the embryonic chick . Anat . Rec . 188, 229-235 . Tyler M . S . and McCobb D . P. (1980) The genesis of membrane bone in the embryonic chick maxilla: epithelial-mesenchymal tissue recombination studies . J. Embryol. exp . Morph. 56, 269-281 . Tyler M . S . and DeWitt-Stott R. A. (1986) Inhibition of membrane bone formation by vitamin A in the embryonic chick mandible . Anat . Rec . 214, 193-197 . Wilsman N . J . and Van Sickle, D . C . (1971) Histochemical evidence of a functional heterogeneity in neonatal canine epiphyseal chondrocytes . Histochem . J. 3, 311-318 .

Skeletogenesis in the chick mandible

Plate l overleaf.

Plate I Fig . 1 . Mandible from an HH stage 17 embryo grown intact as a chorioallantoic membrane graft for 7 days. Cartilage (C) has formed as a central rod of tissue, but no membrane bone has formed . EP = mandibular epithelium . Haematoxylin, eosin, and alcian blue . x 44 Fig. 2 . Mandible similar to that shown in Fig . 1, except that in this case, membrane bone (MB) did form . The bone was in close proximity to the cartilage (C) and between the cartilage and the mandibular epithelium (EP) . Haematoxylin, eosin, and alcian blue. Phase contrast, x 113 Fig . 3. Mandible from an HH stage 18 embryo grown intact as a chorioallentoic membrane graft for 7 days . Cartilage (C) is present, and extensive membrane bone (MB) has formed between the cartilage and the mandibular epithelium (EP). Haematoxylin, eosin, and alcian blue . x 44 Fig . 4 . A medial fragment from an HH stage 17 mandible grown in culture for 7 days in which cartilage (C) but no membrane bone has formed . EP = mandibular epithelium. Haematoxylin, eosin, and alcian blue . x 113 Fig, 5 . A medial fragment similar to that shown in Fig . 4, except that in this culture neither cartilage nor membrane bone formed . EP = epithelium, MF = Metricel filter . Haematoxylin, eosin, and alcian blue . x 113 Fig. 6 . A mediolateral fragment from an HH stage 17 mandible grown as a chorioallantoic membrane graft for 7 days . Cartilage (C) has formed, as well as a small amount of membrane bone (MB) . Haematoxylin, eosin, and alcian blue . Phase contrast . x 113 Fig . 7. A medial fragment from an HH stage 20 mandible grown in culture for 7 days . Cartilage (C) and a sparse amount of membrane bone (MB) have formed. EP = mandibular epithelium . Haematoxylin, eosin, and alcian blue. Phase contrast . x 113 Fig . 8 . A medial fragment from an HH stage 21 .5 mandible grown as a chorioallantoic membrane graft for 7 days. Cartilage (C) is present, and extensive membrane bone (MB) has formed between the cartilage and the mandibular epithelium (EP) . Haematoxylin, eosin, and alcian blue. x44

Skeletogenesis in the chick mandible

Plate I

449