J. Embryol. exp. Morpli. Vol. 30, 3 pp. 635-646, 1973 6 3 5

Printed in Great Britain

The polarity of the dental lamina in theregenerating salamander jaw

ByHEBER T. GRAVER1

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 adultTriturus viridescens the DL is regenerated from the posterior stump tissues only. One-fourthand i mandibular jaw amputations were performed in such a manner that a short stump ofjaw, devoid of DL, remained. Larvae exhibited a posterior regrowth of the DL, while inadults 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 resultedin the DL of the inserted piece having a reversed polarity in its new position. In both larvaland adult forms, the DL of the transplant established connexions both anteriorly andposteriorly with lamina present. Transverse amputations through the inserted piece resultedin regeneration from the DL in the transplant in an anterior direction. Transplantation of asection of edentulous tissue into normal jaw tissue of the opposite side, or ttansplantation ofa section of normal tissue into the edentulous area of the opposite side resulted in no anterioroi posterior regrowth of the DL into the edentulous area.

Collectively the results indicate that no anterior-posterior polarity exists in the DL of thelarval salamander jaw, since regeneration can occur equally well in both directions. The DLof the adult salamander jaw exhibits an anterior-posterior polarity allowing for regrowth inan anterior direction only.

INTRODUCTION

Amphibians replace lost teeth from rows of reserve tooth buds lined upbehind the functional ones in order of decreasing stages of development.During development of the embryo an epithelial thickening arises in the regionof 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 dentallamina (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, asan invagination of the basal cell layer of the epidermis to form a continuousdouble-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.

636 H. T. GRAVER

The first teeth in larval urodeles develop in the epidermis, while succeedinggenerations of teeth develop from the DL. The teeth in urodele larvae are mono-cuspid but those formed after metamorphosis are bicuspid (Gaunt & Miles,1967). Functional teeth are attached to the dentary bone and arranged inmarginal rows along the dorsal edges of the mandibles.

In urodeles, jaws can regenerate after experimental amputation. The majorhistological events of jaw regeneration in the newt have been well covered byGoss & Stagg (1958) and Goss (1969). An epidermal thickening develops appro-ximately 2 weeks after removal of the distal \ of the jaw, followed by blastemaformation at 3 weeks and differentiating regenerate cartilages at 4 weeks. In the 6-week regenerating jaw, at a time when rather extensive cartilaginous mandibularregeneration has occurred, early stages of tooth production in the regenerate canbe detected as extensions of the rows of teeth in the mandibular stumps. Twodental ridges of epidermal cells, developing from the two mandibular stumps,converge medially and eventually join at the midline about 8 weeks afteramputation. Tooth buds differentiate from the innermost portions of theepidermal ridge. Before the left and right ridges meet medially, there are well-developed but unerupted teeth in the more proximal parts. These teeth areformed in association with an epidermal enamel organ or DL plus the subse-quently organized subjacent connective tissue (Goss & Stagg, 1958).

Throughout these previous studies there have been no experimental analysesof the behavior of the dental lamina during regeneration. It is not clear, forinstance, whether, in the regenerate, the lamina assumes the polarity of the jawor develops independently, or whether these relationships change in larval andadult forms. The following studies were designed to examine some of thesepossibilities.

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 larvaewere fed Tubifex and adults were given meal worms, while post-operatively nofood was given so that the operated areas were not disturbed.

Animals were anesthetized in 1:1000 MS: 222 (Sandoz) in spring water andmaintained in 1:3000 MS: 222 in full-strength Holtfreter solution during opera-tions (Stocum, 1968). Only mandibles were amputated. Post-operatively, animalswere placed in full-strength Holtfreter solution for 3 h to promote healing.

Jaw tissue removed was fixed immediately in Bouin's fixative for at least24 h and decalcified for 10 days in 5 % EDTA (ethylene diamine tetracetatedisodium salt, Fisher) in 10 % formalin. Decalcified tissues were prepared inthe usual manner for embedding in paraffin. Sections 5 jum thick were stainedwith Delafield's hematoxylin solution and light green (Humason, 1962). Photo-micrographs were taken with the Ultraphot II (Zeiss) using apochromatic

The polarity of the dental lamina 637

Fig. 1. Schematic drawings of urodele lower jaws indicating various transverseamputations and autoplastic transplantations. (A) Amputations retaining a segmentof dental lamina in the stump, aob, I jaw; aoc, ijaw; aod, whole jaw. (B) Amputa-tions 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) Amputa-tion of healed-in transplanted i normal jaw segment. (E) Transplantation of jnormal jaw segment into edentulous jaw tissue. (F) Transplantation of {- edentulousjaw segment into normal jaw tissue.

638 H. T. GRAVER

objectives and an aplanat-achromat condenser on Wratten metallographicplates.

The polarity of the dental lamina in the regenerating lower jaws of larval andadult urodeles was tested by the transverse amputation and autoplastic trans-plantation procedures illustrated in Fig. 1A-F. Regenerating jaws were preparedfor histological examination at weekly intervals.

The polarity of the dental lamina 639

Table 1. Summary of the regenerative events of the amputated whole jaw of thenewt, Triturus viridescens

Week 1 Degeneration of muscleWeek 2 Epidermal thickeningWeek 3 Blastema fully formedWeek 4 Regenerate cartilage beginning to form medial to pre-articular boneWeeks 5, 6 Regenerate cartilage formation continues toward midlineWeek 7 Bone regeneration begun as direct extension of the dentary bone. Extension of

the dental lamina occurs with bone regenerationWeek 8 Regenerate cartilages meet and fuse at midlineWeeks 9, 10 Regenerate bone meets and fuses at midline. Regenerate dental lamina com-

pleted and fused at midline.

RESULTS

Operated animals recovered rapidly from the anesthesia and displayed normalbehavior up to the time of sacrifice. Minimal bleeding occurred, and the rawstump tissues were covered with a thin layer of epithelium within 48 h post-operatively. Fig. 2 illustrates the complex nature of the normal mandibularsalamander jaw and the various tissues involved in the amputation and trans-plantation 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. viride-scens. D, Dentary bone; L, forward extent of the DL; B, regenerated dentary bone.The arrow indicates level of amputation. Schematic drawings inserted in variouscorners 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 theend. The arrow represents the direction of regeneration.Fig. 5. Horizontal section through the jaw of larval A. maculatum regenerating 7weeks from the removal of a {- jaw segment eliminating all DL in the operatedstump 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 21weeks after the removal of a ̂ jaw segment eliminating all DL in the operated stumptissues. Arrow indicates the midline area. L, Regenerate lamina does not growposteriorly; T, regenerate tooth.Fig. 8. Horizontal section through the jaws of larval A. maculatum regenerating 9weeks after the transplantation of a i normal jaw segment into normal jaw tissue.B, Regenerate dentary bone attaching transplant to stump; D, dentary bone oftransplant; L, dental laminas of stump and transplant joined together anteriorly.

640 H. T. GRAVER

Fig. 7. Schematic drawings of adult urodele lower jaws indicating regenerationfollowing 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 ofdental lamina in the stump (105 cases; Figs. 1 A, 3, 4).

Following amputation the same sequence of events of jaw regenerationoccurred as described by Goss & Stagg (1958) for the adult newt (Table 1). Jawregeneration took approximately 2 weeks longer in animals in the presentexperiments because they were kept at 16°C; Goss and Stagg grew their animalsat room temperature. Lowering the temperature slows the rate of the regenera-tive process (Balinsky, 1968). Also whole jaw amputations in this experimentinvolved proportionately more material than did the distal one-half jaw removalsin the Goss & Stagg (1958) experiments and more time was required forregrowth.

Following amputation the injured DL retracted and became establishedslightly proximal to the level of the cut. Dental lamina regeneration began bythe 7th week and was completed by the 9th—10th week (Table 1), with new toothbuds and teeth beginning to form. In £ and \ amputated adult jaws the DLalways was replaced from the posterior to the anterior in direction (Fig. 3). Inlarvae the dental epithelium was also observed entering the regenerate area atthe anterior end and growing posteriorly (Fig. 4). Thus in { and \ amputatedlower jaws of larval forms the DL was replaced from both the anterior andposterior ends of the regenerate area, while in adults the lamina was regeneratedfrom the posterior stump tissues only.

Whole jaw removal resulted in fusion of the DL in the area of the midline,

The polarity of the dental lamina 641since each side grew at about the same rate. No median symphysis was re-established and tooth buds were found regenerated on the midline itself.

Transverse amputations of 4-, \ and 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 distaljaw regeneration took place but in most cases took 2-3 weeks longer. Larval andadult regenerate jaws were usually shorter and sometimes distorted.

In 4 amputated larval jaws the DL grew posteriorly into the regenerate areaat 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 regeneratearea by 21 weeks but did not cross into it. The dental epithelium was very activeat this point. It exhibited tooth buds and small regenerate teeth. The regenerated1 jaw contained a regenerate cartilage and bone, and otherwise appeared com-pletely normal, but was devoid of DL and remained edentulous (Fig. 7 A).

In 2- amputated larval jaws, some crossing-over and posterior regrowth intothe regenerate area occurred at the midline at 6-7 weeks. In adults the DL grewto the edge of the % jaw regenerate area, accumulated there and exhibitedregenerated tooth buds and teeth (Fig. 6). The regenerated \ jaw appearedcompletely normal but again was devoid of DL and remained edentulous inadult animals maintained for 30 weeks (Fig. 7B).

Whole jaws which regenerated following amputation from larvae and adultsexhibited 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 ofthe 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 stumpborders to heal together. In larvae autografts became revascularized within2 weeks post-operatively. By 3-4 weeks regenerate cartilages formed medial tothe pre-articular bones and attached the transplant to the mandibular stumpborders both anteriorly and posteriorly. Intramembranous bone formationbegan as direct extensions of the cut ends of the dentary bones in the transplantsand in the mandibular stumps by the 5th—6th week (Fig. 8). The DL of thetransplant and the DL of the jaw sent out projections of growth both anteriorlyand posteriorly which established connexions at the different levels in thetissues by the 8th week (Fig. 8). The DL of the transplant and of the jaw nowappeared continuous and tooth germs and new teeth continued to be formed.In adults the same sequence of events occurred following transplantation, but atotal regeneration time of 10 weeks was required for the DL to establish con-nexions.

642 H. T. GRAVER

The polarity of the dental lamina 643

Transverse amputation of the healed-in 4- normal jaw segment autoplasticallytransplanted into normal jaw tissue of the opposite side (50 cases; Figs. 1D, 9, 10).

Rapid wound healing occurred in adults following amputation. Normal re-generation occurred from the amputated -4- jaw transplant but required approxi-mately 12-14 weeks to complete. By the 4th week following amputation, aregenerate cartilage formed medial to the pre-articular bone in the transplant(Fig. 9) and fused with the regenerate cartilage growing from the amputatednormal tissue of the left side at approximately 10-12 weeks. Bone regenerationwas noted as direct extensions of the dentary bone in the transplant by the 7th-8th week. Concomitant with bone regeneration, the DL regenerated in ananterior direction from the distal end of the transplant (Figs. 9, 10). No mediansymphysis was reestablished, and fusion of the DL occurred in the area of themidline at approximately 14 weeks.

Autoplastic transplantation of a 4- normal jaw segment into edentulous jaw tissueof 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 inthe transplant, and attached the transplant to the regenerate cartilage presentin the edentulous jaw segment. Both anteriorly and posteriorly intramembranousbone formation began as direct extensions of the cut ends of the dentary bonesin the transplants and in the mandibular stumps by the 5th-6th week. After 15weeks transplants did not exhibit regrowth of the DL into the adjacent edentu-lous tissue either anteriorly or posteriorly. The DL in the transplant remainednormal looking histologically, and continued producing new tooth buds andfunctional teeth.

FIGURES 9-13

Figs. 9 and 10. Horizontal sections through thejaws of adult r.v/W^ce/7.y regenerat-ing 10 weeks after the transverse amputation of a healed-in transplanted -} normaljaw segment into normal jaw tissue. L, Regenerate lamina from the distal end oftransplant; D, dentary bone of transplant; B, dentary bone of operated stump; R,regenerate cartilage; M, Meckel's cartilage. The arrows indicate the level ofamputation.Fig. 11. Horizontal section through the jaw of adult T. viridescens regenerating 10weeks after the transplantation of a I normal jaw segment into regenerated edentu-lous jaw tissue. L, dental lamina of the transplant exhibiting no regenerationanteriorly or posteriorly. The arrows mark the edges of the graft.

Figs. 12 and 13. Horizontal sections through the jaws of adult T. viridescens regenera-ting 10 weeks after the transplantation of a -)• edentulous jaw segment into normaljaw tissue. E, Edentulous area; note, no anterior or posterior regrowth of DL intothe edentulous transplant. The arrows mark the edges of the graft.

644 H. T. GRAVER

Autoplastic transplantation of a\ edentulous jaw segment into normal jaw tissueof the opposite side (65 cases; Figs. IF, 12, 13).

In adults revascularization of the autografts occurred within 2 weeks and by3-4 weeks the regenerate cartilage in the edentulous transplant was attached tothe operated stumps by the formation of regenerate cartilages medial to the pre-articular bones in the stumps. The edentulous transplant contained no pre-articular bone, only regenerate cartilage and a small amount of regenerateddentary bone. Fifteen-week regenerate jaws remained edentulous since the DLdid not exhibit regrowth anteriorly or posteriorly from the operated mandibularstumps into the adjacent edentulous tissue of the transplant (Figs. 12, 13).

DISCUSSION

The results of the experiments described herein collectively suggest that nopolarity 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 dentalepithelium was replaced equally well from both the anterior and posterior endsof the regenerate area. In \ and \ amputated mandibular jaws (retaining asegment of jaw devoid of DL), the dental epithelium was always replaced by aposterior regrowth into the regenerate area. Autoplastic transplantation of asection of normal jaw into normal jaw tissue resulted in the DL of the pieceinserted having its polarity reversed relative to the polarity of the DL in theoperated stumps. However, the transplant healed into place and the DL of thepiece established connexions both anteriorly and posteriorly with the DLalready present in the jaw and continued producing tooth buds and teeth.

The results further indicate that an anterior-posterior polarity exists in theDL of the adult T. viridescens jaw, In \ and \ amputated mandibular jaws(retaining a segment of DL in the stump), the dental epithelium was regeneratedfrom the posterior stump tissues only. In \ and \ amputated lower jaws (retain-ing 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 healinginto place. However, the joining of the DL of the transplant to the DL in theoperated mandibular stumps could be part of the healing-in phenomenon, ratherthan evidence for a lack of polarity in the adult DL. Transverse amputationsthrough the transplant resulted in regeneration from the DL in the piece in ananterior direction, indicating that the DL in the transplant had reversed itspolarity after transplantation. This conclusion is entirely plausible since Dent(1954) was able to demonstrate a reversal of the proximo-distal polarity of trans-planted regenerating forelimbs in adult newts, while Butler (1951, 1955) andDeck (1955) were also able to accomplish this in larval forms.

To test the response of a segment of DL presumably polarized in an anterior-posterior direction in an edentulous situation, a section of normal adult jaw was

The polarity of the dental lamina 645transplanted into edentulous tissue which had been regenerating 10-14 weeks.Based on the results of the previous experiments one might expect that the DLin the transplant would reverse its polarity following transplantation, and exhibitregrowth into the edentulous area in an anterior direction only. However, noanterior or posterior regrowth of the DL resulted from the transplant. Possiblythe two tissues are not temporally competent for regeneration to occur, orsome type of mechanical block exists at the junction of the healed-in normalpiece with the edentulous jaw tissue. Following transplantation of a \ jawsegment into edentulous tissue, no apical epidermal cap or blastema develop. Adifferentiating regenerate cartilage forms immediately after healing together ofthe epidermal and connective tissue elements. Possibly an accumulation of tissueelements of molecular dimensions at the healed borders between the transplantand the amputated mandibular stumps is preventing regrowth of the DL in ananterior or posterior direction.

No posterior regrowth of the DL occurred following transplantation of asection of adult edentulous tissue into normal tissue. It was not expected in thiscase, since it did not occur in previous amputation experiments. Anterior re-growth was possible but also did not occur because of (1) a temporal differencebetween the edentulous and normal tissue or, (2) a mechanical block at thehealed wound borders.

Experimental results in this investigation thus indicate that the events ofmetamorphosis initiate certain changes in regenerative ability in the adultsalamander jaw. Hormones may be causally involved in these changes. Meta-morphosis is, of course, triggered by thyroxine, a hormone which has beenshown to be antagonistic to the initiation of regeneration (Hay, 1956). AlsoSchotte & Hilfer (1957) and Schoffe & Wilber (1958) have shown that the rege-neration of amputated limbs in adult newts requires adrenal steroids, while limbregeneration in larval forms is refractory to the influence of these hormones.

There is a proximo-distal loss of regenerative ability in the tadpole leg asmetamorphosis progresses, until finally this ability is entirely lost in the adultpostmetamorphic frog. There is reason to believe that there is nothing intrinsic-ally wrong with the cells in the limbs of frogs, since these regenerative capacitiescan 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 metamor-phosis are bicuspid (Gaunt & Miles, 1967). Smith & Miles (1971) concluded intheir ultrastructural study of odontogenesis in larval and adult urodeles, that thedifferences in development between larval and adult teeth support the conceptthat there is a change in cellular activity of the internal dental epithelium whichoccurs during metamorphosis from the larval to adult urodele.

Thus, experimental results suggest that the DL of the larval urodele jawexhibits no anterior-posterior polarity, since regrowth of the dental epithelium

646 H. T. GRAYER

can proceed equally well in both directions. However, an anterior-posteriorpolarity exists in the DL of the adult salamander jaw allowing for regrowth inan anterior direction only. This change in regenerative ability may be due to achange 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 adviceand encouragement throughout the course of this investigation; to Dr Richard C. Herold forhis numerous suggestions and criticisms and for his invaluable help in the early stages of thiswork; 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 ofArts and Sciences of the University of Pennsylvania in partial fulfillment of the requirementsfor the degree of Doctor of Philosophy, 1972.

The investigation was supported by Grant 5TO1DE001-15 from the U.S.P.H.S.

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(Received 1 March 1973)