The polarity of the dental lamina in the regenerating salamander jaw

Transcription

1 J. Embryol. exp. Morpli. Vol. 30, 3 pp , Printed in Great Britain The polarity of the dental lamina in the regenerating salamander jaw ByHEBER T. GRAVER 1 From the Department of Histology and Embryology, School of Dental Medicine, and of Biology, University of Pennsylvania SUMMARY In \ and \ amputated lower jaws of larval Ambystoma maculatum the dental lamina (DL) is replaced from both the anterior and posterior ends of the regenerate area, while in adult Triturus viridescens the DL is regenerated from the posterior stump tissues only. One-fourth and i mandibular jaw amputations were performed in such a manner that a short stump of jaw, devoid of DL, remained. Larvae exhibited a posterior regrowth of the DL, while in adults the lamina accumulated at the edge of the regenerate but did not enter the new tissue. Transplantation of a section of jaw from the left to the right side of the mandible resulted in the DL of the inserted piece having a reversed polarity in its new position. In both larval and adult forms, the DL of the transplant established connexions both anteriorly and posteriorly with lamina present. Transverse amputations through the inserted piece resulted in regeneration from the DL in the transplant in an anterior direction. Transplantation of a section of edentulous tissue into normal jaw tissue of the opposite side, or ttansplantation of a section of normal tissue into the edentulous area of the opposite side resulted in no anterior oi posterior regrowth of the DL into the edentulous area. Collectively the results indicate that no anterior-posterior polarity exists in the DL of the larval salamander jaw, since regeneration can occur equally well in both directions. The DL of the adult salamander jaw exhibits an anterior-posterior polarity allowing for regrowth in an anterior direction only. INTRODUCTION Amphibians replace lost teeth from rows of reserve tooth buds lined up behind the functional ones in order of decreasing stages of development. During development of the embryo an epithelial thickening arises in the region of the future dental arch and extends along the entire free margin of the jaws. This is the primordium of the ectodermal portion of the teeth, the dental lamina (DL), which gives rise to the enamel organs of the developing tooth buds (Orban, 1953). In the normal mandibular jaw of urodeles the DL extends around the jaw, as an invagination of the basal cell layer of the epidermis to form a continuous double-layered sheet about 0-4 mm deep just internal to the functional teeth. The two walls of the DL are well defined, with some central cells between them, and the dental units form on the labial side only (Kerr, 1958). 1 Author's address: School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174, U.S.A.

2 636 H. T. GRAVER The first teeth in larval urodeles develop in the epidermis, while succeeding generations of teeth develop from the DL. The teeth in urodele larvae are monocuspid but those formed after metamorphosis are bicuspid (Gaunt & Miles, 1967). Functional teeth are attached to the dentary bone and arranged in marginal rows along the dorsal edges of the mandibles. In urodeles, jaws can regenerate after experimental amputation. The major histological events of jaw regeneration in the newt have been well covered by Goss & Stagg (1958) and Goss (1969). An epidermal thickening develops approximately 2 weeks after removal of the distal \ of the jaw, followed by blastema formation at 3 weeks and differentiating regenerate cartilages at 4 weeks. In the 6- week regenerating jaw, at a time when rather extensive cartilaginous mandibular regeneration has occurred, early stages of tooth production in the regenerate can be detected as extensions of the rows of teeth in the mandibular stumps. Two dental ridges of epidermal cells, developing from the two mandibular stumps, converge medially and eventually join at the midline about 8 weeks after amputation. Tooth buds differentiate from the innermost portions of the epidermal ridge. Before the left and right ridges meet medially, there are welldeveloped but unerupted teeth in the more proximal parts. These teeth are formed in association with an epidermal enamel organ or DL plus the subsequently organized subjacent connective tissue (Goss & Stagg, 1958). Throughout these previous studies there have been no experimental analyses of the behavior of the dental lamina during regeneration. It is not clear, for instance, whether, in the regenerate, the lamina assumes the polarity of the jaw or develops independently, or whether these relationships change in larval and adult forms. The following studies were designed to examine some of these possibilities. MATERIALS AND METHODS Ambystoma maculatum (Shaw) larvae and Triturus viridescens (Rafinesque) adults used in this work were obtained from Dr Glenn Gentry, Donelson, Tennessee, and were maintained in spring water at 16 C. Pre-operatively larvae were fed Tubifex and adults were given meal worms, while post-operatively no food was given so that the operated areas were not disturbed. Animals were anesthetized in 1:1000 MS: 222 (Sandoz) in spring water and maintained in 1:3000 MS: 222 in full-strength Holtfreter solution during operations (Stocum, 1968). Only mandibles were amputated. Post-operatively, animals were placed in full-strength Holtfreter solution for 3 h to promote healing. Jaw tissue removed was fixed immediately in Bouin's fixative for at least 24 h and decalcified for 10 days in 5 % EDTA (ethylene diamine tetracetate disodium salt, Fisher) in 10 % formalin. Decalcified tissues were prepared in the usual manner for embedding in paraffin. Sections 5 jum thick were stained with Delafield's hematoxylin solution and light green (Humason, 1962). Photomicrographs were taken with the Ultraphot II (Zeiss) using apochromatic

3 The polarity of the dental lamina 637 Fig. 1. Schematic drawings of urodele lower jaws indicating various transverse amputations and autoplastic transplantations. (A) Amputations retaining a segment of dental lamina in the stump, aob, I jaw; aoc, ijaw; aod, whole jaw. (B) Amputations ablating all dental lamina in the stump, aob, \ jaw; aoc, \ jaw; aod, whole jaw. (C) Transplantation of -\ normal jaw segment into normal jaw tissue. (D) Amputation of healed-in transplanted i normal jaw segment. (E) Transplantation of j normal jaw segment into edentulous jaw tissue. (F) Transplantation of {- edentulous jaw segment into normal jaw tissue.

4 638 H. T. GRAVER objectives and an aplanat-achromat condenser on Wratten metallographic plates. The polarity of the dental lamina in the regenerating lower jaws of larval and adult urodeles was tested by the transverse amputation and autoplastic transplantation procedures illustrated in Fig. 1A-F. Regenerating jaws were prepared for histological examination at weekly intervals.

5 The polarity of the dental lamina 639 Table 1. Summary of the regenerative events of the amputated whole jaw of the newt, Triturus viridescens Week 1 Degeneration of muscle Week 2 Epidermal thickening Week 3 Blastema fully formed Week 4 Regenerate cartilage beginning to form medial to pre-articular bone Weeks 5, 6 Regenerate cartilage formation continues toward midline Week 7 Bone regeneration begun as direct extension of the dentary bone. Extension of the dental lamina occurs with bone regeneration Week 8 Regenerate cartilages meet and fuse at midline Weeks 9, 10 Regenerate bone meets and fuses at midline. Regenerate dental lamina completed and fused at midline. RESULTS Operated animals recovered rapidly from the anesthesia and displayed normal behavior up to the time of sacrifice. Minimal bleeding occurred, and the raw stump tissues were covered with a thin layer of epithelium within 48 h postoperatively. Fig. 2 illustrates the complex nature of the normal mandibular salamander jaw and the various tissues involved in the amputation and transplantation procedures. FIGURES 2-6, 8 Fig. 2. Cross-section through the normal mandibular jaw of adult T. viridescens. L, Dental lamina and successional tooth buds; T, functional tooth; D, dentary bone. Fig. 3. Horizontal section through the 8i week regenerated jaw of adult T. viridescens. D, Dentary bone; L, forward extent of the DL; B, regenerated dentary bone. The arrow indicates level of amputation. Schematic drawings inserted in various corners of the photomicrographs indicate the type of operation that was performed. This and all subsequent photomicrographs are at the same magnification. Fig. 4. Horizontal section through the 5-week regenerated J jaw of larval A. maculatum. L, Regenerate DL growing posteriorly into the i regenerate area; R, regenerate cartilage; D, dentary bone with regenerate dentary bone forming on the end. The arrow represents the direction of regeneration. Fig. 5. Horizontal section through the jaw of larval A. maculatum regenerating 7 weeks from the removal of a {- jaw segment eliminating all DL in the operated stump tissues. L, Dental lamina growing posteriorly across the i jaw regenerate area. The arrow indicates the direction of growth. Fig. 6. Horizontal section through the jaw of adult T. viridescens regenerating 21 weeks after the removal of a ^ jaw segment eliminating all DL in the operated stump tissues. Arrow indicates the midline area. L, Regenerate lamina does not grow posteriorly; T, regenerate tooth. Fig. 8. Horizontal section through the jaws of larval A. maculatum regenerating 9 weeks after the transplantation of a i normal jaw segment into normal jaw tissue. B, Regenerate dentary bone attaching transplant to stump; D, dentary bone of transplant; L, dental laminas of stump and transplant joined together anteriorly.

6 640 H. T. GRAVER Fig. 7. Schematic drawings of adult urodele lower jaws indicating regeneration following various transverse amputations retaining a segment of jaw devoid of DL. (A) - - edentulous jaw; (B) edentulous jaw; (C) whole edentulous jaw. Transverse amputations of\, \ and whole mandibular jaws retaining a segment of dental lamina in the stump (105 cases; Figs. 1 A, 3, 4). Following amputation the same sequence of events of jaw regeneration occurred as described by Goss & Stagg (1958) for the adult newt (Table 1). Jaw regeneration took approximately 2 weeks longer in animals in the present experiments because they were kept at 16 C; Goss and Stagg grew their animals at room temperature. Lowering the temperature slows the rate of the regenerative process (Balinsky, 1968). Also whole jaw amputations in this experiment involved proportionately more material than did the distal one-half jaw removals in the Goss & Stagg (1958) experiments and more time was required for regrowth. Following amputation the injured DL retracted and became established slightly proximal to the level of the cut. Dental lamina regeneration began by the 7th week and was completed by the 9th 10th week (Table 1), with new tooth buds and teeth beginning to form. In and \ amputated adult jaws the DL always was replaced from the posterior to the anterior in direction (Fig. 3). In larvae the dental epithelium was also observed entering the regenerate area at the anterior end and growing posteriorly (Fig. 4). Thus in { and \ amputated lower jaws of larval forms the DL was replaced from both the anterior and posterior ends of the regenerate area, while in adults the lamina was regenerated from the posterior stump tissues only. Whole jaw removal resulted in fusion of the DL in the area of the midline,

7 The polarity of the dental lamina 641 since each side grew at about the same rate. No median symphysis was reestablished and tooth buds were found regenerated on the midline itself. Transverse amputations of 4-, \ an d whole mandibular jaws ablating all dental lamina in the stump (135 cases; Figs. IB, 5-6, 7A-C). Following amputation the same sequence of events characterizing more distal jaw regeneration took place but in most cases took 2-3 weeks longer. Larval and adult regenerate jaws were usually shorter and sometimes distorted. In 4 amputated larval jaws the DL grew posteriorly into the regenerate area at about 4 weeks. Fig. 5 shows the DL regenerated almost completely across the 4. regenerate area in a posterior direction by 7 weeks. In 4 amputated adult jaws the DL had grown to the edge of the regenerate area by 21 weeks but did not cross into it. The dental epithelium was very active at this point. It exhibited tooth buds and small regenerate teeth. The regenerated 1 jaw contained a regenerate cartilage and bone, and otherwise appeared completely normal, but was devoid of DL and remained edentulous (Fig. 7 A). In 2- amputated larval jaws, some crossing-over and posterior regrowth into the regenerate area occurred at the midline at 6-7 weeks. In adults the DL grew to the edge of the % jaw regenerate area, accumulated there and exhibited regenerated tooth buds and teeth (Fig. 6). The regenerated \ jaw appeared completely normal but again was devoid of DL and remained edentulous in adult animals maintained for 30 weeks (Fig. 7B). Whole jaws which regenerated following amputation from larvae and adults exhibited no dental lamina or teeth in animals maintained for 16 weeks (larvae) and 28 weeks (adults). A completely edentulous jaw was regenerated (Fig. 7C). Autoplastic transplantation of a \ normal jaw segment into normal jaw tissue of the opposite side (55 cases; Figs. 1C, 8). Animals were maintained approximately 2-3 h in 1:3000 MS: 222 in full- Strength Holtfreter solution to allow the transplant and the mandibular stump borders to heal together. In larvae autografts became revascularized within 2 weeks post-operatively. By 3-4 weeks regenerate cartilages formed medial to the pre-articular bones and attached the transplant to the mandibular stump borders both anteriorly and posteriorly. Intramembranous bone formation began as direct extensions of the cut ends of the dentary bones in the transplants and in the mandibular stumps by the 5th 6th week (Fig. 8). The DL of the transplant and the DL of the jaw sent out projections of growth both anteriorly and posteriorly which established connexions at the different levels in the tissues by the 8th week (Fig. 8). The DL of the transplant and of the jaw now appeared continuous and tooth germs and new teeth continued to be formed. In adults the same sequence of events occurred following transplantation, but a total regeneration time of 10 weeks was required for the DL to establish connexions.

8 642 H. T. GRAVER

9 The polarity of the dental lamina 643 Transverse amputation of the healed-in 4- normal jaw segment autoplastically transplanted into normal jaw tissue of the opposite side (50 cases; Figs. 1D, 9, 10). Rapid wound healing occurred in adults following amputation. Normal regeneration occurred from the amputated jaw transplant but required approximately weeks to complete. By the 4th week following amputation, a regenerate cartilage formed medial to the pre-articular bone in the transplant (Fig. 9) and fused with the regenerate cartilage growing from the amputated normal tissue of the left side at approximately weeks. Bone regeneration was noted as direct extensions of the dentary bone in the transplant by the 7th- 8th week. Concomitant with bone regeneration, the DL regenerated in an anterior direction from the distal end of the transplant (Figs. 9, 10). No median symphysis was reestablished, and fusion of the DL occurred in the area of the midline at approximately 14 weeks. Autoplastic transplantation of a 4- normal jaw segment into edentulous jaw tissue of the opposite side (75 cases; Figs. 1E, 11). In adults autografts became revascularized within 2 weeks post-operatively. By 3-4 weeks regenerate cartilages formed medial to the pre-articular bone in the transplant, and attached the transplant to the regenerate cartilage present in the edentulous jaw segment. Both anteriorly and posteriorly intramembranous bone formation began as direct extensions of the cut ends of the dentary bones in the transplants and in the mandibular stumps by the 5th-6th week. After 15 weeks transplants did not exhibit regrowth of the DL into the adjacent edentulous tissue either anteriorly or posteriorly. The DL in the transplant remained normal looking histologically, and continued producing new tooth buds and functional teeth. FIGURES 9-13 Figs. 9 and 10. Horizontal sections through thejaws of adult r.v/w^ce/7.y regenerating 10 weeks after the transverse amputation of a healed-in transplanted -} normal jaw segment into normal jaw tissue. L, Regenerate lamina from the distal end of transplant; D, dentary bone of transplant; B, dentary bone of operated stump; R, regenerate cartilage; M, Meckel's cartilage. The arrows indicate the level of amputation. Fig. 11. Horizontal section through the jaw of adult T. viridescens regenerating 10 weeks after the transplantation of a I normal jaw segment into regenerated edentulous jaw tissue. L, dental lamina of the transplant exhibiting no regeneration anteriorly or posteriorly. The arrows mark the edges of the graft. Figs. 12 and 13. Horizontal sections through the jaws of adult T. viridescens regenerating 10 weeks after the transplantation of a -) edentulous jaw segment into normal jaw tissue. E, Edentulous area; note, no anterior or posterior regrowth of DL into the edentulous transplant. The arrows mark the edges of the graft.

10 644 H. T. GRAVER Autoplastic transplantation of a\ edentulous jaw segment into normal jaw tissue of the opposite side (65 cases; Figs. IF, 12, 13). In adults revascularization of the autografts occurred within 2 weeks and by 3-4 weeks the regenerate cartilage in the edentulous transplant was attached to the operated stumps by the formation of regenerate cartilages medial to the prearticular bones in the stumps. The edentulous transplant contained no prearticular bone, only regenerate cartilage and a small amount of regenerated dentary bone. Fifteen-week regenerate jaws remained edentulous since the DL did not exhibit regrowth anteriorly or posteriorly from the operated mandibular stumps into the adjacent edentulous tissue of the transplant (Figs. 12, 13). DISCUSSION The results of the experiments described herein collectively suggest that no polarity exists in the DL of the mandibular jaw of larval A. maculatum. In and \ amputated lower jaws (retaining a segment of DL in the stump), the dental epithelium was replaced equally well from both the anterior and posterior ends of the regenerate area. In \ and \ amputated mandibular jaws (retaining a segment of jaw devoid of DL), the dental epithelium was always replaced by a posterior regrowth into the regenerate area. Autoplastic transplantation of a section of normal jaw into normal jaw tissue resulted in the DL of the piece inserted having its polarity reversed relative to the polarity of the DL in the operated stumps. However, the transplant healed into place and the DL of the piece established connexions both anteriorly and posteriorly with the DL already present in the jaw and continued producing tooth buds and teeth. The results further indicate that an anterior-posterior polarity exists in the DL of the adult T. viridescens jaw, In \ and \ amputated mandibular jaws (retaining a segment of DL in the stump), the dental epithelium was regenerated from the posterior stump tissues only. In \ and \ amputated lower jaws (retaining a segment of jaw devoid of DL), no posterior regrowth of the DL was noted. Transplantation of a section of normal jaw also resulted in the transplant healing into place. However, the joining of the DL of the transplant to the DL in the operated mandibular stumps could be part of the healing-in phenomenon, rather than evidence for a lack of polarity in the adult DL. Transverse amputations through the transplant resulted in regeneration from the DL in the piece in an anterior direction, indicating that the DL in the transplant had reversed its polarity after transplantation. This conclusion is entirely plausible since Dent (1954) was able to demonstrate a reversal of the proximo-distal polarity of transplanted regenerating forelimbs in adult newts, while Butler (1951, 1955) and Deck (1955) were also able to accomplish this in larval forms. To test the response of a segment of DL presumably polarized in an anteriorposterior direction in an edentulous situation, a section of normal adult jaw was

11 The polarity of the dental lamina 645 transplanted into edentulous tissue which had been regenerating weeks. Based on the results of the previous experiments one might expect that the DL in the transplant would reverse its polarity following transplantation, and exhibit regrowth into the edentulous area in an anterior direction only. However, no anterior or posterior regrowth of the DL resulted from the transplant. Possibly the two tissues are not temporally competent for regeneration to occur, or some type of mechanical block exists at the junction of the healed-in normal piece with the edentulous jaw tissue. Following transplantation of a \ jaw segment into edentulous tissue, no apical epidermal cap or blastema develop. A differentiating regenerate cartilage forms immediately after healing together of the epidermal and connective tissue elements. Possibly an accumulation of tissue elements of molecular dimensions at the healed borders between the transplant and the amputated mandibular stumps is preventing regrowth of the DL in an anterior or posterior direction. No posterior regrowth of the DL occurred following transplantation of a section of adult edentulous tissue into normal tissue. It was not expected in this case, since it did not occur in previous amputation experiments. Anterior regrowth was possible but also did not occur because of (1) a temporal difference between the edentulous and normal tissue or, (2) a mechanical block at the healed wound borders. Experimental results in this investigation thus indicate that the events of metamorphosis initiate certain changes in regenerative ability in the adult salamander jaw. Hormones may be causally involved in these changes. Metamorphosis is, of course, triggered by thyroxine, a hormone which has been shown to be antagonistic to the initiation of regeneration (Hay, 1956). Also Schotte & Hilfer (1957) and Schoffe & Wilber (1958) have shown that the regeneration of amputated limbs in adult newts requires adrenal steroids, while limb regeneration in larval forms is refractory to the influence of these hormones. There is a proximo-distal loss of regenerative ability in the tadpole leg as metamorphosis progresses, until finally this ability is entirely lost in the adult postmetamorphic frog. There is reason to believe that there is nothing intrinsically wrong with the cells in the limbs of frogs, since these regenerative capacities can be aroused experimentally (Rose, 1945; Polezhayev, 1946; Singer, 1951). The difficulty may lie at the tissue level as a result of postmetamorphic changes. Teeth in urodele larvae are monocuspid but those formed after metamorphosis are bicuspid (Gaunt & Miles, 1967). Smith & Miles (1971) concluded in their ultrastructural study of odontogenesis in larval and adult urodeles, that the differences in development between larval and adult teeth support the concept that there is a change in cellular activity of the internal dental epithelium which occurs during metamorphosis from the larval to adult urodele. Thus, experimental results suggest that the DL of the larval urodele jaw exhibits no anterior-posterior polarity, since regrowth of the dental epithelium

12 646 H. T. GRAYER can proceed equally well in both directions. However, an anterior-posterior polarity exists in the DL of the adult salamander jaw allowing for regrowth in an anterior direction only. This change in regenerative ability may be due to a change in the amount and types of hormones produced after metamorphosis, resulting in changes in the character of the tissues of the adult salamander jaw. The author wishes to acknowledge his gratitude to Dr Charles E. Wilde Jr. for his advice and encouragement throughout the course of this investigation; to Dr Richard C. Herold for his numerous suggestions and criticisms and for his invaluable help in the early stages of this work; and to Dr Ronald Piddington for critical reading of the manuscript. This paper forms part of a dissertation presented to the faculty of the Graduate School of Arts and Sciences of the University of Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The investigation was supported by Grant 5TO1DE from the U.S.P.H.S. REFERENCES BALINSKY, B. I. (1968). An Introduction to Embryology, 2nd ed. Philadelphia: W. B. Saunders. BUTLER, E. G. (1951). The mechanics of blastema formation and legeneration in urodele limbs of reversed polarity. Trans. N. Y. Acad. Sci. 13, BUTLER, E. G. (1955). Regeneration of the urodele forelimb after reversal of its proximodistal axis. /. Morph. 96, DECK, J. D. (1955). The innervation of urodele limbs of reversed proximo-distal polarity. /. Morph. 96, DENT, J. N. (1954). A study of the regenerates emanating from limb transplants with reversed proximo-distal polarity in the adult newt. Anat. Rec. 118, GAUNT, W. & MILES, A. (1967). Fundamental aspects of tooth morphogenesis. In Structural and Chemical Organization of Teeth, vol. i (ed. A. Miles), pp New York: Academic Press. Goss, R. J. (1969). Principles of Regeneration. New York: Academic Press. Goss, R. & STAGG, M. (1958). Regeneration of lower jaws in adult newts. J. Morph. 102, HAY, E. D. (1956). Effects of thyroxine on limb regeneration in the newt, Triturus viridescens. Bull. Johns Hopkins Hosp. 99, HUMASON, G. L. (1962). Animal Tissue Techniques. San Francisco: W. H. Freeman. KERR, T. (1958). Development and structure of some actinopterygian and urodele teeth. Proc. zool. Soc. Lond. 133, ORBAN, B. (1953). Oral Histology and Embryology. St Louis: C. V. Mosby. POLEZHAYEV, L. W. (1946). The loss and restoration of regenerative capacity in the limbs of tailless Amphibia. Biol. Rev. 21, ROSE, S. M. (1945). The effect of NaCl in stimulating regeneration of limbs of frogs./. Morph. 11, SCHOTTE, O. & HILFER, S. (1957). Initiation of regeneration in regenerates after hypophysectomy in adult Triturus viridescens. J. Morph. 101, SCHOTTE, O. & WILBER, J. (1958). Effects of adrenal transplants upon forelimb regeneration in normal and in hypophysectomized adult frogs. /. Embryol. exp. Morph. 6, SINGER, M. (1951). Induction of regeneration of forelimb of the frog by augmentation of the nerve supply. Proc. Soc. exp. Biol. Med. 76, SMITH, M. & MILES, A. (1971). The ultrastructure of odontogenesis in larval and adult urodeles: Differentiation of the dental epithelial cells. Z. Zellforsch. mikrosk. Anat. 121, STOCUM, D. L. (1968). The urodele limb regeneration blastema: a self-organizing system. I. Differentiation in vitro. Devi Biol. 18, (Received 1 March 1973)