Alligator Mandibular Development during Long Term Organ Culture Author(s): Mark W. J. Ferguson, Lawrence S.

Honig, Pablo Bringas and Harold C. Slavkin Source: In Vitro, Vol. 19, No. 5 (May, 1983), pp. 385-393 Published by: Society for In Vitro Biology Stable URL: http://www.jstor.org/stable/4292685 . Accessed: 05/09/2013 14:41
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IN VITRO Vol. 19, No. 5, May 1983 Inc. Association, ? 1983TissueCulture

ALLIGATOR MANDIBULAR DEVELOPMENT DURING LONG TERM ORGAN CULTURE

MARK W. J. FERGUSON,' LAWRENCES. HONIG, PABLOBRINGAS, AND HAROLDC. SLAVKIN of Belfast,Belfast,NorthernIreland(M. W. J. F.); ofAnatomy,The Queen'sUniversity Department LosAngeles, andLaboratory of SouthernCalifornia, Biology, University for Developmental (L. S. H., P. B., H. C. S.) California June23, 1982;acceptedNovember (Received 29, 1982)
SUMMARY

The development of the first branchial (mandibular) arch of the American alligator (Alligator mississippiensis) is studied in organ culture for the first time. There is extensive mandibular morphogenesis in vitro and the pattern of the differentiated elements, bones, muscles, and cartilage, is comparable to that found during normal development in ovo. In addition, we observe the development of paired lingual swellings and the formation of a small tongue consisting of fibrous, lipid containing, and muscular tissues, as found in the normal tongue. Several culture variables are examined: (a) although alligator embryos normally develop at 300 C, we successfully culture the mandibular rudiments with good short term (3 wk) results at 370 C; (b) at 300 C, we are able to culture arches for long term periods of 6 wk; (c) the mandibular arches develop well in both serum containing medium and in a chemically defined medium free from added hormones. The reptilian mandibular arches exhibit excellent, patterned, development and may have less stringent culture requirements than their avian and mammalian counterparts. Key words: organ culture; chemically defined medium; tissue culture; mandibular arch; branchial arch; alligator. We report here the organ culture of embryonic alligator mandibular arches (Fig. 1). Avian and The combination of mammal-like characteris- mouse mandibular arch rudiments have been cultics and reptilian development (1-4) in an ex- tured widely (5-8) and been shown to form bone ternal calcified egg, makes the American alligator and cartilage in vitro. Requirements for culture of (Alligator mississippiensis) a useful model for alligator tissue were unknown. Here we have experimental studies of development, especially in examined several factors: (a) In the wild, alligathe craniofacial region. For example, embryos tors develop at r,300 C; temperatures above can develop in semishell-less culture which, 340 C do not allow embryonic survival (9). Howcoupled with administration of a teratogen that ever, because we are interested in epithelialspecifically inhibits mandibular morphogenesis, mesenchymal recombination experiments involvenables in situ study of secondary palate developing the co-culturing of alligator tissues with mouse ment in living embryos. But many embryological or avian tissues, we have examined the feasibility experiments require in vitro culture especially if of organ culture at 370 C. (b) In ovo development specific hormonal effects or interspecies recom- of alligator embryos is relatively slow. The eggs, binations are to be examined. Embryonic alliga- which are laid at an advanced stage equivalent to tor tissue has not previously been cultured in a 2 d chick or 9 d mouse, require 65 d to hatchvitro. ing. Inasmuch as development in vitro would be expected to be slower than in ovo, we needed the 'To whom all correspondence should be addressed at: ability to do long term culture. Here, we successDepartment of Anatomy, The Queen's University of culture alligator mandibular arches for fully Lisburn 97 Medical Road, Belfast, Biology Centre, Belfast BT9 7BL, Northern Ireland. periods of 6 wk. (c) Most reports of chick and
INTRODUCTION

385

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386

FERGUSONET AL. ready at the 10 to 15 somite stage with head clearly demarcated and neural tube closed (10). Fertile eggs were collected and packed carefully in damp nesting media (Spartina patens) inside polystyrene insulated boxes for air transport to Los Angeles. The top surfaces of the eggs, as they lay in the nest, were marked, and eggs were always handled, transported, and incubated in this orientation. The embryo attaches to the superior surface of the shell within 12 h of egg laying and later rotation of the egg causes a high mortality. Eggs were incubated at 300 C and 100% humidity in an incubator with forced air recirculation. Organ culture. After 10 d incubation, eggs were cleaned with 70% ethanol and windowed. Under sterile conditions embryos were removed, rinsed in Hanks' balanced salt solution, and staged by examination under the stereomicroscope (10,11). Only 52 embryos at stage 10 were selected, and the mandibular arches were excised (Fig. 1). The rudiments were placed on black Millipore filters (AABP), which rested on stainless steel grids over 600 pl of media inside a humidified Grobstein organ culture dish. Explants were covered with 1% Bactoagar (Difco, Detroit, MI) to minimize ectopic keratinization. The rudiments were divided into 4 groups, each of 12 first branchial arches, which were cultured under different conditions (Fig. 1): chemically defined media (CDM) at 300 C, CDM at 370 C, CDM at 300 C with FBS, and CDM at 370 C with FBS. The

mouse mandibular culture have used semisolid media containing plasma and embryonic extracts (5), chorio-allantoic membrane grafts (6,7), or media containing serum (7). Recent evidence indicates that quail and mouse mandibular rudiments will differentiate into cartilage and osteoid in serumless, chemically defined medium, free of added hormones (8). We have used this modified Eagle's minimal essential medium for culture of the alligator tissues and compared it to medium containing fetal bovine serum (FBS). Alligators have a complex lower jaw consisting of six pairs of separate, distinct bones as well as Meckel's cartilage (Figs. 2, 3). We find that development in vitro yields well patterned differentiation similar to that seen in ovo. Despite the need for long term culture, the reptilian tissue may have less stringent requirements than avian or mammalian tissues. In addition, we observe some formation of the tongue, which has not been reported previously, in culture of the first branchial arches.
MATERIALS AND METHODS

Eggs. Female alligators each lay 30 to 40 eggs in late June (10,11). Fifteen nests in the swamps of the Rockefeller Wildlife Refuge, Louisiana, were monitored during nest construction (early June) and eggs removed from the nests within 12 h of egg laying. The day of egg laying (12 to 17 June 1981 in this study) is called incubation Day 0 and at this stage the alligator eggs are al-

FERGUSON

STAGE

10

ALLIGATOR

EMBRYO

(DAY

10)

CDM at 300C

CDM+ 10% at 30 C

FBS at

CDM
370

CDM+ 10% C at 370 C

FBS

FIG. 1. Experimental scheme.

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ALLIGATOR MANDIBULARARCHES chemically defined media is used by our laboratory for studies of mammalian dental development and consists of Eagle's minimum essential medium supplemented with 0.6 mM ascorbic acid, 0.7 mM L-glycine, 2 mM L-glutamine, and 15 mM HEPES (12) plus 1% antibioticantimycotic solution (GIBCO, Grand Island, NY). Serum containing media was identical except that it was supplemented with 10% FBS (lot 30N8101 GIBCO). Explants were cultured in air, photographed macroscopically and fixed at time zero and after 6, 14, 19, 36, and 42 d in culture. Histology. Explants were fixed either in Karnovsky's fixative for Epon embedding or scanning electron microscopy (SEM), or in 10% buffered formalin for paraffin embedding or for Alcian green wholemount staining (13). Alcian green stained wholemounts were examined and photographed while in methyl salicylate, and then were transferred to xylene for embedding in paraffin. Serial sections (5 pm) were stained with either: (a) Harris' iron hemotoxylin, eosin and Alcian blue, (b) Weigert's iron hematoxylin, Van Gieson and Alcian blue, or (c) Mallory's trichrome. Specimens fixed for SEM were postfixed in 1.3% OsO4, critical point dried from amyl acetate, mounted on aluminum stubs, sputter coated with gold, and viewed in a Cambridge Stereoscan S180. Structures of the definitive alligator lower jaw are shown for reference in Figs. 2, 3. RESULTS

387

Culture at 300 C in CDM plus FBS. The first branchial arch of the 10 d alligator consists of undifferentiated loose mesenchymal cells covered by epithelium. In culture it undergoes patterned growth and differentiation (Figs. 4-7,12,13,16). After 6 d in culture, the arches have increased in size; the midline constriction where right and left mesenchymes unite has expanded (Fig. 4 A). Little differentiation has occurred during this period except for the slight appearance of mesenchymal condensations of Meckel's cartilages (Fig. 4 B). These cartilages become prominent after 14 d culture (Figs. 5 B, 12) and continue to grow (Figs. 6 B, 7 B, 13, 16) to a size larger than ever observed in vivo (Fig. 15). The intermandibularis muscle (Fig. 15) has differentiated after 14 d culture (Fig. 12 A) and the blastemata for it and for Meckel's cartilages exhibit a comparable patterning to that seen in vivo (Fig. 15). Paired lingual swellings arise from the mandibular arches after 14 d in culture (Figs. 12 B and 19). These rudiments fuse together (Fig. 12 B, C) to form the anlage for the anterior region of the tongue. This process is identical to that seen in vivo, but the cultured tongue (Figs. 7 A, 13) is smaller than normal. By Day 36 in culture, the tongue mesenchyme has differentiated into fibrous, lipid and muscular tissue (Figs. 7 A, 13)

1 2 3

DENTARY SPLENIAL CORONOID

4 5 6

SURANGULAR ARTICULAR ANGULAR

F IG.3. Histological cross section [hemotoxylin and eosin (H&E) stained] through the lower jaw of a 4 y old alligator. Dentary (D), splenial (S) and angular (A) bones, Meckel's cartilage (M), and a tooth are visible.

lower of the bonesof the adultalligator FIG. 2. Diagram jaw.

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388

FERGUSON ET AL.

arranged in a similar fashion to that seen in vivo (Fig. 15) (14). No papillas or taste buds were observed. Blastemata for the intramandibularis muscle (Fig. 15) arise after 19 d in culture (Fig. 12 C). Intramembranous bone is present after 36 d in culture (Figs. 13, 16). The bony blastemata for the various lower jaw bones (dentary, splenial, and angular; Fig. 2) are patterned (Fig. 16) just as the blastemal patterns are in vivo (Fig. 3, 15). The bone matrix stains as if mineralized and contains osteoblasts and osteo-

cytes but no osteoclasts (Fig. 16 B). No dental structures (dental laminas, tooth germs, etc.) were observed in any explant, nor, understandably, were nerves or blood vessels present. Mitotic cells were present in both the epithelium and mesenchyme in the cultured tissue. The growth, shape changes, differentiation, and pattern formation observed in the cultured branchial arches are very similar to those occurring normally in vivo. However, development is considerably slower in vitro. Meckel's cartilages and the

FIG.4. Cultured alligator mandibular arch, 6 d at 300 C in CDM + FBS. A, Unfixed specimen; B, wholemount showing small condensation of Meckel's cartilage. FIG. 5. Arch cultured 14 d at 300 C in CDM + FBS. A, Unfixed specimen showing paired lingual swellings near the top of the arch; B, wholemount showing Meckel's cartilages. FIG.6. Arch cultured 19 d at 300 C in CDM + FBS. A, unfixed specimen; B, wholemount showing Meckel's cartilages. FIG. 7. Arch cultured 36 d at 300 C in CDM + FBS. A, Unfixed specimen showing tongue (T); B, wholemount displaying the abnormally large Meckel's cartilages (M). FIG. 8. Arch cultured 6 d at 370 C in CDM. There is little differentiation evident in the unfixed (A) or wholemount (B) specimens. FIG. 9. Arch cultured 14 d at 370 C culture in CDM. Paired lingual swellings are evident in the unfixed specimen (A) and the wholemount (B) which shows Meckel's cartilages. FIG. 10. Cultured arches after 19 d at 300 C in CDM. The unfixed specimen (A) and wholemount (B) are smaller than serum cultured counterparts (Fig. 6). FIG. 11. Arch cultured at 36 d at 300 C in CDM. Unfixed specimen (A) and wholemount (B) are of smaller size and have less cartilage than serum cultured counterparts in Fig. 7.

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ALLIGATOR MANDIBULAR ARCHES

389

FIG. 12. Histological sections through arches cultured 14 d (A,B) or 19 d (C) at 300 C in CDM + FBS. Stained with H & E and Alcian blue, the sections show Meckel's cartilages (M), paired lingual swellings (L), the intermandibularis (I) and intramandibularis (IM) muscles, and the tongue (T). These may be compared with Fig. 15. FIG. 13. Section through arch cultured 36 d at 300 C in CDM + FBS. The tongue and Meckel's cartilages are visible in the preparation stained with H & E and Alcian blue. FIG. 14. Section through an arch cultured 20 d at 370 C in CDM + FBS. The chondrocytes of Meckel's cartilages are hypertrophic; intramembranous bony blastemata are evident (arrowed). Mallory stain. FIG. 15. Coronal section through a Day 24 embryonic alligator head. Meckel's cartilages (M). Tongue (T). Intermandibularis (I) and intramandibularis (IM) muscles. Bony blastemata for the dentary (D), splenial (S), and angular (A). Mallory stain. F IG.16. Section (A) through an arch cultured 36 d at 300 C in CDM + FBS. Meckel's cartilage (M) and the bony blastemata for the dentary (D), splenial (S) and angular (A) are apparent. (Compare with Fig. 15.) Stained with Weigert, Van Giesen, and Alcian blue. A higher power view (B) of Meckel's cartilage (M), perichondrium (P) and bony blastemata for the splenial (S), stained with H & E and Alcian blue.

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390

FERGUSONET AL. tially faster 370 C counterparts. The temperature of 370 C was useful for short term culture but 300 C was superior for long term cultures. After 20 d in culture at 370 C, the chondrocytes of Meckel's cartilage became markedly hypertrophic (Fig. 14); an event that never happens in vivo where Meckel's cartilage persists throughout the life of the animal (Fig. 3). Explants cultured in chemically defined, serumless media (CDM) are much smaller than those cultured in the presence of serum (compare Figs. 8-11 with Figs. 4-7). Mitotic figures are rarely seen in either the epithelium or the mesenchyme of arches cultured in CDM for 19 d or longer and the shape of the arches is often distorted compared to that seen in vivo. Quantities of bone, cartilage, and muscle, formed by arches cultured in CDM, are less than

intermandibularis muscle blastemata appear at Day 16 in vivo, but at "Day 24" (embryonic Day 10 cultured 14 d) in vitro. Lingual swellings are apparent at Day 14 in vivo but at "Day 24" in vitro, and the intramembranous bony blastemata at Day 21 in vivo but at "Day 46" in vitro. Development in vitro at 300 C seems about half as fast as development in ovo. Other culture conditions. Explants in all culture conditions exhibited comparable differentiation although there were notable differences. In general, development proceeded faster at 370 than at 300 C (compare Figs. 14 and 13), but explants cultured at 370 C degenerated after 3 wk in culture, whereas those at 300 C were still healthy after 6 wk in culture. Consequently, the 300 C explants ultimately developed further than their ini-

FIG. 17. Section through arch cultured 14 d at 370 C in CDM. Note Meckel's cartilages (M) and compare appearance with that of the serum supplemented media (Figs. 12 A, B). H & E and Alcian blue. FIG. 18. Section through arch cultured 19 d at 300 C in CDM. Note Meckel's cartilages (M), the intermandibularis muscle (I), the swelling for the tongue (T), and mesenchymal condensations for the splenial bones (S). Compare with Fig. 12 C. H & E and Alcian blue. FIG. 19. Scanning electron micrograph of the dorsal surface of an arch cultured 36 d at 300 C in CDM. Note the midline swellings for the developing tongue (T), the shape and size of the arch. FIG. 20. Section of Meckel's cartilage (M) and blastema for the splenial bone (S) from an arch cultured 42 d at 300 C in CDM. The bony blastemata stains as if unmineralized. Compare with Figs. 13, 16 A, B. Weigert, Van Gieson and Alcian blue.

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MANDIBULARARCHES ALLIGATOR those formed by arches cultured in CDM + FBS (compare Figs. 7 and 11; 12 A, B and 19; 12 C and 18; 13, 16 A, B and 20). However, the differences are chiefly quantitative: mandibular arches cultured in chemically defined, serumless media differentiated into all of the cell types such as bone, cartilage, muscle, fibrous tissue, and lipid found in explants grown in serum containing media (Figs. 17-20) and the rudiments were patterned in all culture conditions (Figs. 19, 20). The bony blastemata of explants grown in CDM stained differently from those grown in CDM + FBS in a manner suggesting that the former were unmineralized osteoid and the latter mineralized bone.

391

DISCUSSION We have shown that alligator mandibular arches undergo differentiation, morphogenesis, and pattern formation similar to that seen in vivo when cultured in the absence of all exogenous factors, such as serum, embryonic extracts, hormones, neurotrophic, or vascular influences. In this sense, the mandibular arches represent autonomous embryonic fields. Mechanisms of development may now be investigated using heterotypic epithelial-mesenchymal recombination experiments because we have demonstrated the ability of alligator tissue to differentiate both in chemically defined media and at the nonphysiological high temperatures of 370 C (instead of 300 C). Slavkin et al. (8) reported the ability of quail and mouse mandibular arches to initiate chondrogenesis and osteogenesis within 10 d of culture in a serumless, chemically defined media. Alligator tissues are shown here to perform better in culture than their murine or avian equivalents. The specialized culture requirements of reptilian tissues may be lower. Hall (6,15) and Tyler and Hall (7) have documented an epithelial requirement for the initiation of intramembranous bone formation in the lower jaws of embryonic chicks and mice. Such a requirement should be investigated in the alligator, which not only exhibits in vitro ossification but also precise patterning of the various bony blastemata (Figs. 2,3,15). The absence of dental laminas and lingual taste buds in cultured mandibles may be due to lack of neurotrophic factors (16-20). Alternatively, the embryonic stage at the time of branchial arch excision may be too young. Neural crest cells

migrate into the alligator first branchial arch in temporally distinct waves (1), so exclusion of one wave of crest cells might deprive the explants of either dentally determined cells or some essential interaction. [For example, the pattern of tooth replacement in the rainbow trout is related to the order of migration of neural crest cells and to the local competence of the oral epithelium (21).] Kollar (22) demonstrated that the incidence of tooth germ development in cultures of mouse mandibular tissue was increased if the trigeminal ganglion was included with the explants. Experiments involving removal of alligator arches at different times and coculture of arches with trigeminal ganglia would be useful. The absence of osteoclasts probably is due to their normal origin from monocytes present in circulating blood (15). The cultured explants formed bony blastemata, which could be exploited to study factors controlling mineralization. By selectively adding various substances, such as calcium, phosphates, parathyroid hormone, vitamin D, or calcitonin, to the defined media it should be possible to investigate mineralization in a highly controlled system. Moreover, because alligators exhibit cyclical osseous growth (10) with alternating zones of vascularized, poorly organized, nonlamellar bone formed during the summer months and annuli of dense, poorly vascularized lamellar bone formed during winter hibernation (23,24), it may be possible to investigate mechanisms regulating what type of bone is formed by using in vitro explants cultured at different temperatures. In all cultured explants Meckel's cartilages were larger than in ovo; evidently, the culture conditions favor chondrogenesis. Moreover, after 20 d culture at 370 C the chondrocytes in Meckel's cartilages hypertrophy. This does not normally occur in vivo, where Meckel's cartilage persists, unmineralized, throughout the life of the alligator (Fig. 3). There have been similar findings (25,26) with invertebrate cartilages from horseshoe crab, squid, and marine snail which normally never mineralize in vivo, but do so if they are cultured at high temperatures (370 versus 200 C) in media (25,26). hydroxyapatite-metastable Perhaps the nonmineralizing alligator Meckel's cartilage is induced to ossify under high temperature culture conditions. The small size of the explants cultured in chemically defined media and the absence of mitotic figures suggests cessation of cell division similar to that observed during serum deprivation experiments in cell culture. We are interested in

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392

ET AL. FERGUSON
reproductive biology and conservation of croco-

how many cell divisions a neural crest cell must undergo before becoming either determined or differentiating into a particular phenotype. Experiments to investigate this can be designed by exploiting differences between explants cultured with and without serum. The tongues developed in vitro (under all culture conditions) were smaller than their in vivo counterparts, presumably because our cultured explants were deprived of their normal contributions from the second and third branchial arches. The morphogenesis and differentiation of the alligator tongue in chemically defined, serumless media make possible experimental study of the role of cell-cell, nerve-cell, and branchial arch-branchial interactions in lingual development.

dilians.SSARSymposium. 1982.In press.

11. Ferguson, M. W. J. The reproductive biology and embryology of the crocodilians. Gans, C.; Billett, F. S. eds. Biology of the reptilia, Vol. 14. London: Academic Press. In press. 12. Yamada, M.; Bringas, P.; Grodin, M.; MacDougall, M.; Cummings, E.; Grimmett, J.; Weliky, B.; Slavkin, H. C. Chemically-defined organ culture of embryonic mouse tooth organs: morphogenesis, dentinogenesis and amelogenesis. J. Biol. Buccale 8: 127-139; 1980. 13. Summerbell, D.; Wolpert, L. Precision of development in chick limb morphogenesis. Nature 244: 228-230; 1973. 14. Ferguson, M. W. J. The structure and development of the palate in Alligator mississippiensis.

REFERENCES 1. Ferguson, M. W. J. The value of the American alligator (Alligator mississippiensis) as a model for research in craniofacial development. J. Craniofacial. Genet. Dev. Biol. 1: 123-144; 1981. 2. Butler, G. W. On the sub-division of the body cavity in lizards, crocodiles and birds. Proc. Zool. Soc. Lond. 1889: 452-474. 3. Bellairs, A. d'A.; Attridge, J. Reptiles. London: Hutchinson University Library; 1975: 115-128. 4. Webb, G. J. W. Comparative cardiac anatomy of the reptilia. III. J. Morph. 161: 221-240; 1979. 5. Jacobson, W.; Fell, H. B. The developmental mechanics and potencies of the undifferentiated mesenchyme of the mandible. Q. J. Microsc. Sci. 82: 563-591; 1941.

Arch.OralBiol. 26:427-443;1981.

15. Hall, B. K. Developmental and cellular skeletal biology. London: Academic Press; 1978. 16. Pearson, A. A. The early innervation of the developing deciduous teeth. J. Anat. 123: 563-577; 1977. 17. Kollar, E. J.; Lumsden, A. G. S. Tooth morphogenesis: The role of the innervation during induction and pattern formation. J. Biol. Buccale 7: 49-60; 1979. 18. Farbman, A. I. The taste bud: a model system for development studies. Slavkin, H. C.; Bavetta, L. A. eds. Developmental aspects of oral biology. New York: Academic Press; 1972: 109-123. 19. Van Exan, R. J.; Hardy, M. H. A spatial relationship between innervation and the early differentiation of vibrissa follicles in the embryonic mouse. J. Anat. 131: 643-656; 1980. 20. Kennedy, J. G. The development, structure, degeneration and regeneration of taste buds in the rat. Belfast, Northern Ireland: Queen's Univ. 1981. Thesis. p. 1-206. 21. Berkovitz, B. K. B.; Moore, M. H. A longitudinal study of replacement of patterns of teeth on the lower jaw and tongue in the rainbow trout Salmo gairdneri. Arch. Oral Biol. 19: 1111-1119; 1974. 22. Kollar, E. J. The use of organ cultures of embryonic tooth germs for teratological studies. Ebert, J. D.; Marois, M. eds. Tests of teratogenicity in vitro. Amsterdam: North Holland Publishing; 1976: 303-334.

6. Hall, B. K. Tissue interactions and the initiation of

osteogenesis and chondrogenesis in the neural onic mouse as seen in isolated murine tissues and in recombinations of murine and avian tissues. J. Embyol. Exp. Morph. 58: 251-264; 1980.

crest-derived mandibular skeletonof the embry-

7. Tyler, M. S.; Hall, B. K. Epithelialinfluenceson

skeletogenesis in the mandible of the embryonic chick. Anat. Rec. 188: 229-240; 1977.

8. Slavkin, H. C.; Bringas, P.; Cummings, E.; Grodin, M. S. Initiation of quail and mouse mandibular chondrogenesis and osteogenesis in a serumless, chemically-defined medium. Calcif. Tissue Int. 34: 111-112; 1982.

of 9. Ferguson,M. W. J.; Joanen, T. Temperature

egg incubation determines sex in Alligator mississippiensis. Nature 296: 850-853; 1982.

23. De Ricqles,A. On bonehistology of fossilandliving

reptiles with comments on its functional and evolutionary significance. Bellairs, A. d'A.; Cox, B. eds. Morphology and biology of the reptiles.

10. Ferguson, M. W. J. Crocodilianembryology:an overview.Tyron, B. W.; Lang, J. W. eds. The

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Linnean Society, Symp. 3. London: Academic 25. Eilberg, R. G.; Zuckerberg,D. A. Mineralization of invertebrate Press;1976:123-150. cartilage.Calcif. Tissue Res. 19: des 24. De Buffrenil, V. Mise en ividencede l'incidence 85-90; 1975. conditionsde milieu sur la croissancede Crocodylus siamensis(Schneider,1801) et valeur des 26. Libbin, R. M.; Ozer, R.; Person, P. In vitro accumulation of mineralcomponents by invertemarques de croissance squelettiques pour de l'Ageindividuel.Arch.Zool. Exp. brate cartilage. Calcif. Tissue Res. 22: 67-75; l'ivaluation G6n. 121:63-76; 1980. 1976. We thank Mr. Ted Joanen, Mr. Larry McNease, and staff of the Rockefeller Wildlife Refuge, Louisiana, for their outstanding help in the collection and transportation of alligator eggs. Collection and specimen export was performed under U.S. Fish and Wildlife Permit PRT2-2511 and Northern Ireland Permit B/WL2/80 issued to M. W. J. F. Collaborative study was made possible by the 1981 award of a Research Travelling Scholarship to M. W. J. F. from The Wellcome Trust, for which we are grateful. This work is supported by Grant 8113610 CB from the MRC of Great Britain, Grant EP109/74/75 from the Northern Ireland Eastern Health & Social Services Board, and Grants DE-02848 and DE03569 from the National Institutes of Health, Bethesda, MD.

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