J. Embryol. exp. Morpli. Vol. 30, 3 pp. 635-646, 1973
Printed in Great Britain
635
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.
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 6week 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
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.
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.
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
Week 2
Week 3
Week 4
Weeks 5, 6
Week 7
Degeneration of muscle
Epidermal thickening
Blastema fully formed
Regenerate cartilage beginning to form medial to pre-articular bone
Regenerate cartilage formation continues toward midline
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.
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,
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-, \ 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 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 fullStrength 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.
642
H. T. GRAVER
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 -4- jaw transplant but required approximately 12-14 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 10-12 weeks. Bone regeneration
was noted as direct extensions of the dentary bone in the transplant by the 7th8th 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.
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
The polarity of the dental lamina
645
transplanted into edentulous tissue which had been regenerating 10-14 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
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, 1972.
The investigation was supported by Grant 5TO1DE001-15 from the U.S.P.H.S.
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