2.2. Tail Regeneration in the Tuatara Represents a Case of Regengrow
Like numerous species of lizard, the tuatara possesses autonomous fracture planes in the tail
[35][54][55]. The histological analysis of autotomous tail vertebrae of
Sphenodon, designated as pygous vertebrae by Seligmann et al.
[56], showed that the splitting or fracture plane contains small cells resembling blood elements of the bone marrow and additional flat perichondrial cells or even chondrocytes/chondroblasts in continuity with the fibro-cartilaginous tissue of the vertebral bone at the splitting surface (
Figure 2A–C). The fibro-cartilage is contacted at the fracture plane by numerous connective fibrils in separated pre-fracture and post-fracture vertebral bodies. The fibrils give rise to 15–30 µm thick fibrous bundles crossing the peri-vertebral adipose tissue and are in continuation with the inter-muscle septa (
Figure 2D,E). The latter terminate in the dermis and contact the basement lamella of the scales, thereby forming the autotomous planes of the tuatara tail
[55], as observed also in lizards
[35][57].
Figure 2. Histological images (stained with Haematoxylin-Eosin in (
A–
C), and Palmgreen stain in (
D,
E)) of the caudal vertebrae of a normal tail in the tuatara (inset in Figure
A, Bar, 2 mm). (
A), image showing the spinal cord, meninx (artifactually dislocated after sectioning), and ventrally the vertebral body with the intra-vertebral fracture plane indicated (arrow), which has artifactually been separated during sectioning. Bar, 100 μm. (
B), closer view of fracture plane with fragments of blue-stained cartilaginous cells (arrows), probably dislocated from the articular surfaces during sectioning. Bar, 100 μm. (
C), close-up of the intra-vertebral splitting plane where numerous fibro-cartilaginous/connective cells (arrowheads) are present in continuation with the periosteum (arrow). Bar, 50 μm. (
D), peri-vertebral region rich in fat cells and loose connective showing two fibrous bundles (arrows) connecting pre- and post-vertebral bodies at the fracture plane to the inter-muscle connective septa (arrowheads). The image represents the plane of autotomy of the tail, which continues (not shown) into the dermis to externally reach the scales. Bar, 100 μm. (
E), detail of the fibrous bundles (arrows) connected to the pre-vertebral and post-vertebral bodies at the fracture or splitting autotomous plane. Bar, 50 μm.
Legends: bm, bone marrow; bo, vertebral bone; crt, cartilage; fp, fracture plane; mx, meninx; pt, post-vertebral body (more caudal) at the fracture plane; pv, pre-vertebral body (more rostral) at the fracture plane; sc, spinal cord; vb, vertebral body.
Note: All micrographs are based on material obtained in 1988 and 1989 through a permit issued by Mr Ian Govey of the New Zealand Department of Conservation and Dr Mike Thompson of Victoria University, Wellington (New Zealand). The material was used in Alibardi and Meyer-Rochow
[52][55] and all subsequent publications on tuatara by these authors.
The microscopic observation of the cells present in the fracture plane of the vertebrae indicates that they resemble those described for lizards
[35][57], which demonstrates the presence of stem/pro-cartilaginous cells in this region where also 5BrdU-Long Retaining Labelled cells (LRC) have been observed
[58]. The presence of putative stem elements that can give rise to new cartilaginous cells explains the production of a large cartilaginous tube after the tail is autotomized along the fracture (autotomous) plane. The presence of remaining cartilaginous/chondroblast cells within the caudal vertebrae of
Sphenodon, suggests that cartilage cells for tail regeneration could also derive from the inter-vertebral region when the amputation occurs at this level or after vertebral ablation
[56].
In the study by Alibardi and Meyer-Rochow
[52], during the first 4 years and 5 months of life (47 months in regeneration + 5 months since they hatched = 53 months of life in total), three young tuatara grew about 25% (snout-vent length, from 7.5 cm to 10.6 cm) during which time they were autotomized two times to study the regeneration of their tails (
Figure 3). The first tail autotomy was performed on number 1 specimen at about 5 months after birth, and the sample was collected at 5 months of regeneration (=10 months of age); a second sample came from another individual at 7 months of regeneration (=12 months of age), and 3 samples represented 10 months of regeneration (2 re-amputated and one at the first amputation, but all at an age of the tuatara of 15 months). After 37 months from the last amputation, the average body lengths in 2 juveniles were15.5 cm (with an expected snout-vent length of 16.3 cm, the individuals were therefore 0.8 cm shorter than had been expected). This value corresponded to a loss of 5% total body length but to 24.6% of tail growth due to the 2 repetitive regenerations (
Figure 3, the graph). Because of the long duration of tail regeneration in the tuatara, it is likely that in addition to the initial wound healing, blastema formation, and the differentiation of small muscle segments and an axial cartilage, the slow process of growth contributed to the apparent regeneration of the tail
[52][59][60]. Tail re-regeneration was also studied in the skink
Egernia kingii by Barr et al.
[60]: it was present in 17.2% across three populations and the authors concluded that “the ability to re-regenerate may minimise the costs to an individual’s fitness associated with tail loss, efficiently restoring ecological functions of the tail”. Although slower in the tuatara
[61] than in the aforementioned skink, re-regeneration of the tail in tuatara may provide similar benefits.
Figure 3. Growth of the regenerating tail during 47 months associated with body regeneration in three tuatara (indicated by asterisk of different coloration), received initially at 3 months of age, autotomized at 5 months of age at about half-distal length of the tail, measured and sampled at different periods thereafter (see text for further explanations). The ordinate refers to the growth of the regenerate, while the abscissa refers to time.
If we now compare this finding with a previous study on regenerated tails in
Sphenodon, in which it was calculated that juveniles grow at about 1.14 cm/year
[55], we have to note that in our study we observed a much lower value, namely 0.79 cm/year, based on snout-vent length, which increased by 3.1 cm in 47 months (3.92 years). This corresponded to an about 31% lost growth by comparison to 1.14 cm/year, and is therefore much higher than our calculated 5% and 24.6% figures. Despite the different absolute percentages of body length loss in the two examples, it appears that repeated tail regeneration did influence normal growth in
Sphenodon. Since the bodies of these juveniles grew about 25% in length in 47 months (about 4 years), this suggests that the tissues observed in the regenerated tails must have also grown at a similar rate.
The influence of somatic growth over the years affects all tissues including those in the regenerating tail
[61] and especially muscles and axial cartilage, a process that is indicative of regengrow (defined earlier in this paper and in Alibardi
[24]). Although small muscles are regenerated during the first year following amputation
[52], large muscle masses are observed in the longer regenerated tails of mature individuals
[55] and present study], and they are equally regenerated by juvenile stages during the years following tail loss. These large masses of muscle and cartilaginous tissues have also been observed in two adult individuals that we have analysed, a male and a female, which presented long-term regenerated tails of 2.8 and 10.4 cm. An older juvenile with a regenerating tail of 1.7 cm, produced from a previous regenerated tail, also showed a complex tissue organization in the new tail
[51][59][62]: see next paragraph.
Wounds to the limb, like in lizards may heal, but as with lizards, they do not lead to a regenerate in
Sphenodon. This was also observed after accidental injury where a juvenile lost the anterior left foot but, after 3–15 months only developed a pale and scaled-over scar covered by very small scales (lower inset in
Figure 4A). Although digits also do not regenerate
[56], if a toe clip (a method for identifying individuals that is now generally avoided) is performed incorrectly only just beneath the base of the claw, then the tissue and claw do sometimes regenerate with a stub claw (A. Cree, personal communication, 2021). Other cases of wound healing are not known or have not been described microscopically in
Sphenodon and New Zealand veterinarians, who from time to time have to treat tuatara, have a saying that “tuatara get sick and recover on tuatara time”, meaning that recovery is very, very slow (M. Jolly, personal communication 2021).
Figure 4. Gross aspect (
A) and histology of regenerating tail ((
B–
E), Toluidine blue stain). (
A), juvenile of about 4 years of age with regenerated tail (arrowhead). Bar, 1 cm. In the upper inset (Bar, 1 mm) a blastema of about 2 months is shown. The scar (arrowhead) depicted in (A) lower inset formed after about 3 months following limb amputation. (
B), blastema at about 3 months with a loose connective covered by a thick wound epidermis. Bar, 10 μm. This schematic inset shows a blastema with the regions shown in (
B–
E). (
C), proximal area of cross-sectioned conical blastema of 10 months post-autotomy showing three pro-muscle aggregates (arrows) separated by forming connective septa. A dense dermis is present beneath the cornified epidermis. Bar, 50 μm. (
D), detail of a muscle bundle at 10 months post-autotomy. The arrow indicates a myotube in cross-section. Bar, 10 μm. (
E), cross sectioned central cartilaginous cylinder surrounding the ependymal canal in proximal regions of a cone of 7 months post-autotomy. Arrows point to the outer and inner perichondria. Bar, 50 μm.
Legends: bl, blastema; cs, connective septa (forming intermuscle); de, dermis; ep, ependymal canal; mx, meninge; nt, normal tail (stump containing vertebrae and spinal cord); rca, regenerated cartilage; we, wound (regenerating) epidermis.
Note: All micrographs are based on material obtained in 1988 and 1989 through a permit issued by Mr Ian Govey of the New Zealand Department of Conservation and Dr Mike Thompson of Victoria University, Wellington (New Zealand). The material was used in Alibardi and Meyer-Rochow
[52][59][60][62].
The question, of course, arises what the benefits to tuatara could have been to evolve and maintain the ability to autotomize its tail and grow a new one, even if the regrowth takes a very long time
[60]. The high frequency of tail regeneration, estimated by one of us (LA) on the basis of field observations on Stephens Island to be around 80%, suggests that the loss of the tail is a common event in this species. These days tail loss is probably primarily due to predation attempts of adult individuals on juveniles, attacks by large centipedes such as
Cormocephalus rubriceps on very small tuatara, and fighting during courtship among adults
[61]. Predation by flighted predators such as harriers and kingfishers is also known and until its extinction in 1914, predation by the Laughing Owl
Ninox (Sceloglaux) albifacies most likely occurred as well.
But in the past—for at least 20 million years—tuatara had to cope with more formidable predators, among them being large flightless birds such as
Aptornis otidiformis in the North Island and
A. defossor in the South Island known as adzebills
[63]. These extinct predators appear to have been by and large diurnally active
[63] and that may have been a factor why tuatara, despite possessing an eye dominated by photoreceptive cones characteristic of diurnal species Meyer-Rochow et al.
[64], became a largely nocturnal predator with vision adapted to very low light intensities
[65][66]. It is obvious that the nocturnal lifestyle, however, did not eliminate the need for tail autotomy, but whether regeneration has always been as slow as it is now or was faster in the past is difficult to ascertain.
2.3. Histology of Regenerating and Regenerated Tails in the Tuatara
A summary of our earlier studies is presented in this paper, but more detailed information on specific aspects of the processes involved in regenerating different tissues is available from our previously published and more detailed analyses
[51][58][59][62][67][68][69][70]. After tail autotomy or amputation the stump heals very slowly and to complete re-epithelialization (at 23–24 °C) about 1 month is required. A regenerative blastema is visible from about 2–3 months (
Figure 4A, upper inset), but in the following months from then on the stump grows considerably more slowly (
Figure 3). At about 3 years (37 months) from the second amputation, the tail has grown, but is much shorter than the original, often with a club-like shape (
Figure 4A). An amputated limb forms a scar after 3 months from the amputation, but it does not grow into a limb even after 10–15 months, and does not regenerate any further (
Figure 4, lower inset).
Microscopic investigations show that the wound (regenerating) epidermis covering the blastema of 1–2 mm is multilayered and forms an initially soft corneous layer, while underneath loose connective tissue is present containing mainly fibroblasts, blood vessels, sparse nerves, and blood cells (Figure 4B). In regenerating cones of 3–6.5 mm at 5–10 months also some pro-muscle aggregates are recognizable in the more proximal regions close to the original tail. Here they form, just like in lizards, 12–16 very small muscle groups identifiable in cross section, each one made up of a limited number of myotubes (15–30; Figure 4C,D). Most of the regenerating cones at 10–15 months post-amputation are composed of connective tissue containing fibrocytes and numerous collagen fibrils with irregular orientation. In the central part of the cones of 3–6.5 mm in length (Figure 3), a cartilaginous cylinder is formed that shows flat chondroblasts at the external and internal periphery, as observed in longitudinal and cross-sections (Figure 4E).
Inside the cartilaginous tube a loose meninx with numerous blood vessels and a simple spinal cord are regenerated (Figure 4E and Figure 5A). At 5 months of regeneration the spinal cord is formed by ependymal cells, most of which appear as elongated tanicytes terminating into the external basal lamina. The pale spaces among tanicytes are occupied with axons and neuropilar elongations, while rare glial and neuronal cells are present. Among the fibrous connective tissue located outside the cartilaginous cylinder, various amyelinic and myelinated nerves are present, and their terminations reach the apex of the regenerating tail at 5–10 months of tail regeneration (Figure 5B,C). Numerous fat cells are formed in the proximal regions of regenerating 3–6.5 mm large cones.
Figure 5. Histology of regenerating tail (
A,
B,
F) Toludine blue stain; (
D,
E) Palmgreen stain). (
A) cross-sectioned ependymal tube showing the elongation of ependymal tanicytes ending on the basement membrane (arrows). Arrowheads point to glial cells detached from the ependymal epithelium. Bar, 10 μm. (
B), cross-sectioned myelinated (arrows) nerve at 10 months regeneration. Bar, 10 μm. The schematic drawing shows the indicated positions of the figures. (
C), longitudinal section of myelinated nerve 10 months post-autotomy. Bar, 10 μm. (
D), cross section of the cartilaginous tube in a long regenerated tail of unknown age. Arrows indicate intra-cartilaginous areas of calcification/degeneration. Arrowheads indicate the fibrous connective contacting the perichondrium. Bar, 50 μm. (
E) longitudinal section of axial cartilage in an old regenerate of unknown age. The outer perichondrium is indicated by arrowheads. Bar, 20 μm. The upper inset (Bar, 10 μm) details the pseudostratified ependymal epithelium. The lower inset (Bar, 50 μm) instead shows the apical end of the cartilaginous tube, close to the connective tissue of the tip of the regenerated tail. (
F), numerous fat cells (arrows) are present around the cartilaginous tube. Bar, 20 μm.
Legends: ca, regenerated cartilage; cc, central canal; cnt, connective (fibrous) tissue; e, epidermis of neogenic apical scale; ep, ependymal epithelium; epi, epinevrio; mx, meninx; np, areas occupied from axons and neuropile; nt, normal tail (stump);rca, regenerated cartilage; rm, regenerating muscles/myomeres.
Note: All micrographs are based on material obtained in 1988 and 1989 through a permit issued by Mr Ian Govey of the New Zealand Department of Conservation and Dr Mike Thompson of Victoria University, Wellington (New Zealand). The material was used in Alibardi and Meyer-Rochow
[52][58][59][62].
After 37 months from the last collection (the last sampling done on 2 out of the initial 3 specimens), the regenerated tail appeared to consist mainly of irregular dense connective tissue, with large accumulations of fat cells around the central cartilaginous tube where calcification is predominantly noticeable among internal isogenic groups (
Figure 5D–F). Flat chondroblasts likely forming a perichondrium are seen in the inner and outer periphery of the cartilage, while only tanicytes with a pseudostratified organization are present in the regenerated spinal cord. Segmental muscles remain limited in these regenerated tail of about 3 years, but the 2–3 amputations carried on during this period (
Figure 3) may have stimulated excessive fibrosis. This is also indicated by the histological analyses of regenerated tails from older specimens of unknown age
[52].
In a smaller individual, a regenerating tail of 1.7 cm shows small muscle aggregates formed by multinucleated myotubes that exhibit a “leaf-like shape” in the flat plane of the section, as myotubes are attached with a central connective myoseptum and 2 external myosepta (
Figure 6A,B). Very large myotomes are instead observed in the other two large specimens of unknow age, possessing long regenerated tails (2.8 and 10.4 cm), as has also been illustrated in other cases (see Figures 10 and 11 in:
[55]). Old regenerate/regengrow tails also contain large deposits of peri-cartilaginous fat, and more externally substantial muscle bundles with a high innervation score, as observed using the Palmgreen silver staining method for nerve fibres. The latter staining evidences an external innervation of myotubes, starting from nerve fibres crossing the external and internal connective septa and penetrating inside the myotubes or the muscle fibres in more proximal myomeres of the regenerated/regengrow tail (
Figure 6D,E).
Figure 6. Histology of regenerating tail of unknown age (
A,
B) Haematoxylin-eosin stain; (
C–
E), Palmgreen stain). (
A), mid-apical region showing a distinctly cellular cartilage with outer and inner peripheries (arrows) representing the perichondrium. Bar, 50 μm. The schematic drawing shows the indicative positions of the following figures. (
B), detail on a forming, leaf-like myomere, with an axial intermuscle connective (arrows) and external limiting connective septa (arrowheads). Bar, 10 μm.(
C), tangential longitudinal section of the axial cartilage surrounded by fat connective tissue in an old (age unknown) regenerating tail. Arrows indicate the fibrous layer in contact with the perichondrium. Bar, 50 μm. (
D), detail showing nerve fibres coursing within the outer connective septum (arrows) and other nerves entering the myofibres (arrowheads). Bar, 10 μm. (
E), additional details of nerve endings from peripheral (arrow) and central myoseptum (double arrow) entering myofibres (arrowheads). Bar, 10 μm.
Legends: cal, beginning of cartilage calcification; ep, ependyma; fat, connective tissues rich in fat cells; mx, meninx; my, multinuclear myotubes/myofibres; rca, regenerated cartilaginous tube; v, blood vessels.
Note: All micrographs are based on material obtained in 1988 and 1989 through a permit issued by Mr Ian Govey of the New Zealand Department of Conservation and Dr Mike Thompson of Victoria University, Wellington (New Zealand). The material was used in Alibardi and Meyer-Rochow
[52][58][62].
Large segmental myomeres occupy extensive areas of regenerated tail at 2.5–10 cm from the tip of the new tail. They give rise to over 20 muscle bundles in cross section whose dimensions decrease from proximal regions toward the apex (
Figure 7A, inset). In his study, Ali
[55] observed about 40 bundles of “regenerated” muscles, which did, however, exhibit smaller dimension than the original intrinsic and extrinsic tail muscles. As indicated above, it is likely that these large segmental muscles derive from a long process of growth superimposed on the initial regeneration of pro-muscle aggregates, observed in the early regenerating tail. The structure of the large myomeres maintains the original leaf-like shape in a flat plane, with a thick, central connective septum and two peripheral septa to which the muscle fibres are attached (
Figure 7A–C). Although the precise three-dimensional shape of these segmental muscles remains un-determined, it appears that the terminal cones of one muscle segment insert into at least one, perhaps even two, successive myomeres, forming an acute zig-zag conformation (
Figure 7A,B,D,E; see also:
[55]). In both longitudinal and cross sections of the regenerated tail, numerous variably thick inter-muscle connective septa contact the fibrous periosteum of the cartilaginous tube (
Figure 7E,F). This anatomical connection or arrangement suggests that muscle and axial skeleton are mechanically integrated, and even that muscle contraction (after nerve impulse registration in the regenerated/grown muscles) is transmitted to the axial skeleton, although the tail in tuatara appears quite stiff and little capable of bending.
Figure 7. Images derived from different areas of old regenerated tails of unknown age (
A–
D), Haematoxylin-Eosin stain; (
E,
F), Palmgren stain). (
A), detail of regenerated muscles with outer myoseptum (arrows) and inner myoseptum (arrowhead). Bar, 50 μm. The schematic drawing shows the positions of the following figures. (
B), detail to show the leaf-like organization of regenerated muscles. The arrow shows a muscle cone of a myomer that is inserted in the following myomer through the central myoseptum. Bar, 50 μm. (
C), detail of multinucleated muscle fibres attached to the central and lateral (arrow) myosepta. Bar, 10 μm. (
D), cross sectioned muscle bundles close to the tail stump showing the central myoseptum (arrowhead), the lateral myosepta (arrows) that are in continuation (double arrow) with a fibrous bundle connected to the central cartilaginous tube. Bar, 50 μm. (
E), other details showing more clearly the fibrous connections (arrows) between intermuscle connective tissue and the circular fibrous tissues (arrowhead) contacting the cartilaginous tube. Bar, 50 μm. (
F), longitudinal section showing numerous fibrous bundles (arrows) connecting the cartilaginous tube with surrounding tissues, including muscles (here not included/visible). Bar, 50 μm.
Legends: cmi, central connective myoseptum; de, dermis; my, myofibres; nt, normal tail (stump); pc, pericartilaginous connective tissue; rca, regenerating cartilage; sca, scales (neogenic).
Note: All micrographs are based on material obtained in 1988 and 1989 through a permit issued by Mr Ian Govey of the New Zealand Department of Conservation and Dr Mike Thompson of Victoria University, Wellington (New Zealand). The material was used in Alibardi and Meyer-Rochow
[51][58].