Squamate egg tooth development revisited using three‐dimensional reconstructions of brown anole (Anolis sagrei, Squamata, Dactyloidae) dentition

Abstract

The egg tooth is a hatching adaptation, characteristic of all squamates. In brown anole embryos, the first tooth that starts differentiating is the egg tooth. It develops from a single tooth germ and, similar to the regular dentition of all the other vertebrates, the differentiating egg tooth of the brown anole passes through classic morphological and developmental stages named according to the shape of the dental epithelium: epithelial thickening, dental lamina, tooth bud, cap and bell stages. The differentiating egg tooth consists of three parts: the enamel organ, hard tissues and dental pulp. Shortly before hatching, the egg tooth connects with the premaxilla. Attachment tissue of the egg tooth does not undergo mineralization, which makes it different from the other teeth of most squamates. After hatching, odontoclasts are involved in resorption of the egg tooth's remains. This study shows that the brown anole egg tooth does not completely conform to previous reports describing iguanomorph egg teeth and reveals a need to investigate its development in the context of squamate phylogeny.

The egg tooth is a hatching adaptation, characteristic of all squamates. It develops from a single tooth germ, similar to the regular dentition of all the other vertebrates. This study shows that the brown anole egg tooth does not completely conform to previous reports describing iguanomorph egg teeth and reveals a need to investigate its development in the context of squamate phylogeny.

1. INTRODUCTION

The amniotic egg is considered to be one of the most important novelties in the life history of tetrapods. This adaptation assisted the evolutionary success of Amniota (including over 23 000 extant species of reptiles, birds and mammals), facilitating terrestrial reproduction in this group (Shedlock and Edwards, 2009). Amniotic eggs are protected by a calcified eggshell, which interacts with the outside world (Hirsch, 1979; Kohring, 1995). Whereas the presence of this mineralized layer is beneficial during the embryonic development of an animal, it poses a difficulty during the process of hatching (Honza et al. 2001). It is important for survival of the young hatchling to leave the egg in an appropriately short period of time and with minimal energy cost (Magrath, 1990). In order to escape from a hard‐shelled egg without excessive loss of either energy from the yolk or water from embryo tissues, in the course of evolution, different adaptations developed (Bond et al. 1986, 1988; Yoshizaki and Saito, 2002; Yasumasu et al. 2005). Such adaptations assisting hatching of terrestrial oviparous amniote embryos are the caruncle and egg tooth (Clark, 1961). Both of these structures are functional only during the process of hatching and are lost after performing their function (Smith et al. 1953; Clark, 1961). The caruncle is a keratinized structure formed from the horny epidermal thickening at the tip of the snout or beak. It is present in embryos and young individuals just after hatching of birds, crocodiles turtles, tuatara and direct developing frogs from the genus Eleutherodactylus (Clark, 1961; Wake, 1978; Hanken et al. 1992). The egg tooth is attached to the premaxilla and is considered an apomorphy of squamate reptiles (Hermyt et al. 2017). It shares general structure and development with other teeth of vertebrates (Hermyt et al. 2017). However, due to its specialized function, it shows some differences in developmental dynamics and shape in relation to more typical teeth (Smith et al. 1953; Zahradnicek et al. 2012; Hermyt et al. 2017). Knowledge of reptilian egg teeth is currently insufficient. It is known that most reptiles possess a single unpaired egg tooth, except geckos and dibamids, which have double egg teeth (Vitt and Caldwell, 2013). The presence of single or double egg teeth and germs during development is the only egg tooth character that is used in analyses of squamate phylogeny. Squamate phylogeny is still an unresolved matter. The major difference between two approaches trying to resolve squamate phylogeny—molecular and morphological—is placement of the major clade Iguania [but see Simões et al. (2018)]. Morphological analyses consider Iguania to be the sister group to all other squamates (Conrad, 2008; Gauthier et al. 2012), while molecular analyses place that group with snakes and anguimorphans far from the squamate root (Vidal and Hedges, 2005; Wiens et al. 2012; Pyron, 2016). Paired egg teeth, among other synapomorphies, were used to support close relations between dibamids and geckos in both morphological and molecular data analyses (Underwood and Lee, 2000; Townsend et al. 2004). In analyses of molecular data, the presence of a single unpaired egg tooth supported creation of the Unidentata, which comprises all squamates except geckos and dibamids which possess double egg teeth (Vidal and Hedges, 2005, 2009). Egg tooth development might be a source of more phylogenetically informative data than are currently (Zaher' and Rieppel, 1999; Caldwell, 2007) taken into account by phylogenetic analyses (Hermyt et al. 2017). Characteristics such as implantation and attachment tissue are used in phylogenetical analyses (Zaher' and Rieppel, 1999; Caldwell, 2007; LeBlanc et al. 2017). Currently, there are no data for comparative studies of those characteristics in egg teeth, which, as shown previously, differ from marginal dentition (Hermyt et al. 2017). Moreover, it was reported that iguanian lizards' egg teeth are different from egg teeth of other unidentates in terms of their shape and number of germs during development (Anan'eva and Orlov, 2013). More detailed analysis of iguanian egg teeth structure and development at the level of light microscopy, electron microscopy and X‐ray micro‐computed tomography (CT) could be a basis for future more detailed comparative studies involving a broader range of characteristics. As a representative of Iguania, in the present studies the brown anole Anolis sagrei Duméril & Bibron 1837 was chosen. The brown anole is a small diurnal lizard living a trunk‐ground lifestyle. It is native to Cuba and the Bahamas (Campbell, 1996), but has also been introduced in Grenada, Grand Cayman Island and mainland North America (Angetter et al. 2011). It belongs to the diverse Dactyloidae family with over 400 described species (Uetz, 2010). This species was selected because its embryonic development is relatively well known; it has its own developmental table (Sanger et al. 2008), is widely available in the pet trade, and yields a large amount of eggs throughout the year (Andrews, 1985).

2. MATERIALS AND METHODS

2.1. Animals

Non‐commercial brown anole A. sagrei breeding, consisting of one male and three female lizards, took place in the Department of Animal Histology and Embryology of the University of Silesia in Katowice, and was approved by the District Veterinary Officer in Katowice, Poland (PIW.ZOZ.OZ.5342.5.1/17). The brown anole is not included in the Washington Convention of 1973 (Guidelines for appropriate uses of IUCN red list), which was ratified by Poland in 1991 (Journal of Laws, No. 27 item 112 and latest regulations Journal of Laws, 2000 No. 66 item 802 and Journal of Laws, 2004 No. 112 item 1183). In accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and the Act of 15 January 2015 on the protection of animals used for scientific or educational purposes (Journal of Laws 2015 item 266), the planned studies did not require the permission of the local ethics committee.

Anoles were kept in a 35 × 35 × 60 vertical glass terrarium. The temperature in the terrarium varied from 33°C in the warmest place to 28°C in the coolest. Coconut fibre was used as a substrate. Above the tank, heat and UV lamps were set to maintain proper temperature and provide lizards with UV light. To mimic natural conditions 12‐h light/12‐h dark cycles were maintained with electrical timers. The terrarium was misted once a day to maintain humidity at ~80%, and additionally, to keep humidity at a relatively high level, pot plants were planted inside the tank. Water was provided ad libitum. Anoles were fed with crickets three times weekly. Before administration, crickets were dusted in sepia powder for reptiles and once a week in calcium and vitamin D3 powder (Exo Terra), instead in order to supplement the diet of the lizards. Female anoles laid one egg once every 1–2 weeks and buried them in the substrate. Eggs were incubated in situ at ~24°C. Development of embryos at that temperature lasted ~70 days, which is more than twice as long as at 27°C (Sanger et al. 2008). Embryos for study were isolated once every 3 months. After isolation their age was evaluated on the basis of developmental tables for A. sagrei (Sanger et al. 2008). Embryos and hatchlings were killed by cooling at 4°C for 0.5 h, followed by decapitation. Because they were small and are a warm‐climate species, the selected killing method seemed to be the most humane (Shine et al. 2015). Heads were then processed appropriately for the techniques used. For the purpose of the present study 32 anole embryos were used [20 for light microscopy (two for each analyzed developmental stage), five for scanning electron microscopy, two for transmission electron microscopy, and five for micro‐CT].

2.2. Bright field microscopy

The anoles' heads designated for light microscopy were fixed in modified Bouin's solution [15 parts saturated picric acid, five parts 40% formaldehyde and one part formic acid (Skinner, 2003)]. The material was fixed for 24 h at room temperature. Heads isolated from embryos above developmental stage 7 were decalcified in 10% formic acid aqueous solution for 1–3 weeks, depending on their size. After that, the material was dehydrated in ethanol series and embedded in paraffin wax according to the standard procedure. Then, 6‐µm sections were cut using a Leica rotary microtome (Leica RM2125RT). Sections were collected on glass slides, deparaffinized, stained with Ehrlich's haemotoxylin and eosin (H&E) and then mounted using DPX mounting medium. Micrographs were taken under an OLYMPUS BX43 light microscope with an OLYMPUS SC30 digital camera.

2.3. Scanning electron microscopy

The heads for scanning electron microscopic observations were fixed in a 1:1 mixture of 2.5% glutaraldehyde and 2.0% paraformaldehyde in a 0.1‐M phosphate buffer (Karnovsky, 1965), pH 7.4 at 4°C overnight and post‐fixed with osmium tetroxide. After fixation the material was dehydrated through an ethanol series of increasing concentration and acetone. Subsequently, the heads were critical point dried (Pelco CPD 2), mounted on aluminum stubs with double‐sided adhesive carbon tape, coated with a layer of gold (Pelco SC‐6), and observed under a Hitachi UHR FE scanning electron microscope SU 8010 (Laboratory of Scanning Microscopy, Faculty of Biology and Environmental Protection, University of Silesia in Katowice).

2.4. Transmission electron microscopy

The heads for transmission electron microscopy were fixed overnight in a mixture of 2.5% glutaraldehyde and 2.0% paraformaldehyde (1:1) in 0.1‐M phosphate buffer pH 7.4, at 4°C (Karnovsky, 1965) and post‐fixed in 1% osmium tetroxide for 2 h. After fixation the material was dehydrated through an ethanol series of increasing concentration, and acetone, then it was embedded in the Epoxy embedding medium kit (Sigma). Semi‐thin sections were mounted on glass slides and stained with methylene blue. Ultra‐thin sections were collected on copper grids, and stained with uranyl acetate and lead citrate (Reynolds, 1963). Micrographs of semi‐thin sections were taken under an OLYMPUS BX43 light microscope with an OLYMPUS SC30 digital camera. Ultra‐thin sections were analyzed under a Hitachi H500 transmission electron microscope.

2.5. X‐ray microtomography

The material was fixed in a 1:1 mixture of 2.5% glutaraldehyde and 2.0% paraformaldehyde in a 0.1‐M phosphate buffer, pH 7.4 at 4°C overnight (Karnovsky, 1965) then treated with Lugol's solution for 24 h in order to enhance tissue contrast (Degenhardt et al. 2010). The Zeiss XRadia MicroXCT‐200 system, equipped with a 90 keV/8 W tungsten X‐ray source, was used for scanning (Laboratory of Microtomography, Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland). Anole heads were scanned under voltage of 40 kV and current of 130 µA. All micro‐CT scans were performed in 70% ethanol. The acquired cross‐sectional images had pixel resolutions of 2.04 µm. 3D reconstructions were prepared with the open‐source software Drishti (Limaye, 2012).

3. RESULTS

3.1. Single egg tooth erupts in anole lizard ahead of regular dentition

The egg tooth of A. sagrei at developmental stage 12 is visible as a single flat semicircular protuberance, located medially on the surface of the rostral part of the palate. The developing egg tooth is located in a crescent‐shaped groove called the dental lamina. The dental lamina is a shallow indentation of the oral epithelium's surface at this developmental stage. At this developmental stage, the groove on both sides is smooth, without protuberances marking regular dentition. The differentiating incisive process is visible as a protuberance in the oral epithelium caudally to the differentiating egg tooth. Similar to the egg tooth germ, the developing oral teeth are covered by polyhedral epithelial cells (Figure 1A).

Figure 1.

Scanning electron micrographs of the brown anole heads at developmental stages 12, 14, 15 and of a 2‐day hatchling. (A) Scanning electron micrograph of the brown anole embryo at the developmental stage 12. Egg tooth is visible as a flat protuberance in crescent‐shaped dental lamina. (B) Scanning electron micrograph of the brown anole embryo at developmental stage 14. Egg tooth has a more triangular shape than previously. Papilla marking the development of incisive protrusion of premaxilla is visible. (C) Scanning electron micrograph of the brown anole embryo at developmental stage 15. Caudally from the incisive protrusion the developing vomer is visible. (D) Scanning electron micrograph of the brown anole embryo at developmental stage 17. The crack in the epithelium covering the egg tooth is visible showing the tip of egg tooth hard tissue. (E) Scanning electron micrograph of the brown anole 2‐day hatchling. Egg tooth remnants are visible in crack (white arrow). Regular teeth have been marked by white dotted circles. DL, dental lamina; ET; egg tooth; P, premaxillary papilla; V, vomer

At developmental stage 14 the egg tooth of the brown anole is larger than in the previously described developmental stage. It has a more triangular shape than at stage 12, and extends anteriorly beyond the upper lip. The epithelium covering the egg tooth is continuous with the palatal epithelium. On the surface, the developing regular teeth are visible as hemispherical protuberances. Protuberances of the regular dentition were significantly smaller than those of the egg tooth (Figure 1B).

At the next developmental stage (15), the differentiating egg tooth is visible as a flat wide triangle. In the stratified epithelium covering the developing egg tooth of the brown anole, the outermost layer of squamous cells is visible. At the caudo‐medial part, the developing egg tooth is covered with hexagonal cells with prominent centrally located nuclei. The shape of those nuclei ranges from spherical to spheroidal from the caudal to rostral direction of differentiating egg tooth. Laterally to the developing egg tooth the protuberances formed by regular dentition are visible. These protuberances are symmetrically located on both sides of the egg tooth, have a conical shape, and are more elongated ventrally in comparison to previous stages of development. At this stage of development, the epithelial protrusion marking vomer development is visible caudal to the protuberance of the developing incisive process (Figure 1C).

At developmental stage 17, it is apparent that most of the palate surface is smooth, with the exception of a fold in the surface of the epithelium located in close proximity to the caudal part of the developing egg tooth. The break in the epithelium covering the egg tooth is visible, showing the tip of the egg tooth hard tissue. The protuberances of the regular dentition have sharper tips (Figure 1D).

Two days after hatching, a small fissure is visible in the place where the egg tooth was attached in the preceding developmental stages. On the left side of this gap a small part of the egg tooth is visible. The protuberance marking the incisive process is located caudally. Regular teeth at this developmental stage are fully erupted (Figure 1E).

3.2. Continuous epithelial dental lamina links egg tooth to the other teeth germs

At developmental stage 9, a long, U‐shaped invagination of oral epithelium in the lip of A. sagrei extends through the whole length of the lip (Figure 2A,B). At developmental stage 12 the egg tooth has an approximately semicircular shape in the ventral view, whereas in the lateral view it is apparent that it has almost no curvature; it is only slightly curved, with its tip directed rostroventrally. It can be seen that the egg tooth and all of the developing teeth of the first generation are connected linearly through the dental lamina. The teeth of regular dentition are smaller than the egg tooth (Figure 2C,D). At developmental stage 14, the shape of the egg tooth is similar to the previously described stage. It is more elongated in the rostro‐caudal axis and is slightly more curved than in stage 12. In ventral view it reaches below the line of the lip, in contrast to the previously described stage of development (Figure 2E,F). At developmental stage 17, the egg tooth is even more elongated rostro‐caudally and curved than in previous stages. The second generation of teeth connected with the dental lamina are visible. The teeth of the second generation are smaller than teeth of the first, but neither of them extends into the oral cavity (Figure 2G,H). No change in egg tooth morphology on tomograms is observed between stages 17 and 18. The regular teeth at developmental stage 18 are significantly larger than previously and reach into the oral cavity. Anteriorly to the differentiated egg tooth two teeth of the second generation are present (Figure 2I,J).

Figure 2.

Microtomograms of brown anole heads from developmental stages 9, 12, 14, 17 and 18. Brown anole dentition development visualized with three‐dimensional micro‐computed tomography images. The whole dental lamina with enamel organs of teeth has been reconstructed. (A, B) At developmental stage 9, dental lamina throughout the whole length of the lip is visible (C, D). At developmental stages 12 and 14 (G, H), it can be seen that the developing egg tooth and regular teeth are connected linearly through the dental lamina (E, F) At developmental stage 17 the second generation of teeth can be observed. (I, J) At developmental stage 18 it can be seen that the regular teeth reach into the oral cavity. White arrow = egg tooth; asterisk =dental lamina

3.3. Differentiation of egg tooth and its attachment tissue

In transverse section through the developing brown anole embryo at developmental stage 8, undifferentiated, loosely arranged mesenchymal cells are present in most parts of the differentiating head. The margin of the differentiating head is covered with cuboidal epithelial cells. In the middle of the oral epithelium, deep invagination is visible. Laterally to the invagination, two areas of condensing mesenchyme are present. Within these areas blood cells are visible. Lateral to one of the condensing mesenchyme a developing blood vessel is present (Figure 3A).

Figure 3.

Transverse sections through brown anole embryo heads at developmental stages 8 and 9. (A) Section through the brown anole embryo at developmental stage 8. Thickening of the oral epithelium and the condensation of mesenchyme underneath it is visible. Scale bar—100 µm. (B) The oral epithelium of the brown anole embryo at the developmental stage 8. Scale bar—20 µm. (C) Section through the brown anole embryo at the developmental stage 9. Invagination of oral epithelium into mesenchyme is visible. Scale bar 50 µm. (D) Invagination of the brown anole embryo oral epithelium at developmental stage 9. Scale bar 20 µm. BV, blood vessel; Mes, mesenchyme; NP, nasal process; OE, oral epithelium; OEI, oral epithelium invagination

The epithelium of the middle invagination is thicker than the epithelium covering the remaining parts of the mouth. Within this epithelium, 2–3 layers of cuboidal cells are visible, while in the epithelium covering the other parts of the mouth, a single cell layer is present. Mesenchymal cells underlying differentiating oral epithelium in the snout of A. sagrei are loosely arranged at this developmental stage. In the transverse sections, mitotic figures are present in the cells forming the epithelial thickening in the medial part of the mouth cavity and in the mesenchymal cells located above it (Figure 3B).

Laterally, in transverse section from developmental stage 9, differentiating nasal processes are visible. Between nasal processes and the jaw, very deep epithelial invaginations are present. Mesenchymal cells are more condensed than in the previous stage except in the middle of the transverse section where they are loosely arranged. On the border dividing the condensed and loosely arranged mesenchyme differentiating blood vessels are present. Many mitotic figures can be observed at this developmental stage within the mesenchyme and oral epithelium (Figure 3C). An invagination is present in the middle of the differentiating jaw thickening of the epithelium. It consists of two to six layers of cuboidal cells. In the transverse section through the thickening in its middle part six layers are visible, while in the lateral parts there are two cell layers. The thickening consists of cuboidal cells covered with a single layer of squamous cells on the ventral part. Other parts of the oral cavity are lined with a single epithelium consisting mostly of cuboidal and single columnar cells. Around the epithelial thickening a high concentration of mesenchymal cells is visible. This concentration is similar to the mesenchymal cell concentration visible in the vicinity of the forming nasal cavity. A crescent‐shaped area is present between the epithelial thickening and the mesenchyme. It has weaker affinity to the H&E stain in relation to surrounding epithelial tissue. Mitotic figures are visible in the area of the concentrating mesenchyme, epithelial thickening, and epithelium of the mouth; however, they are not as abundant as at the previous stage of development (Figure 3D).

At the developmental stage 10 in the middle of the transverse section, a differentiating enamel organ is visible. Within the enamel organ an evagination formed by cuboidal differentiating ameloblasts is visible. Enamel organ surrounds differentiating dental papilla composed of cuboidal cells. Laterally to the differentiating egg tooth, buds of forming regular teeth are present. Dorsally several blood vessels are observed. Laterally, symmetrically arranged, closed, nasal vestibula filled with nasal plugs are present. Around the described structures still undifferentiated mesenchymal cells are present (Figure 4A).

Figure 4.

Transverse sections through brown anole embryo heads at developmental stages 10 and 11. (A) Section through the brown anole embryo at developmental stage 10. The egg tooth at the cap stage visible. Scale bar 100 µm. (B) The developing egg tooth of the brown anole at developmental stage 10, consisting of differentiating dental papilla and the enamel organ. Scale bar 20 µm. (C) Section through the brown anole embryo at developmental stage 11. Scale bar 200 µm. (D) Differentiating egg tooth at developmental stage 11. Enamel organ at that stage consists of three layers. Small amounts of dentin between enamel organ and dental papilla are visible. Scale bar 100 µm. BV, blood vessel; D, dentin; DP, dental papilla; EO, enamel organ; ET, egg tooth; J, jaw; Mes, mesenchyme; OE, oral epithelium

At developmental stage 10 the egg tooth consists of differentiating dental papilla and the enamel organ. In the cross‐section, the enamel organ epithelium, which maintains continuity with the epithelial lining of the mouth, forms a rectangular area surrounding the semicircular differentiating dental papilla. In the ventral part of this area, a small protuberance formed by the differentiating inner enamel epithelium is visible. The epithelia that surround the differentiating egg tooth, the enamel organ, and the ventral part of the dental papilla have a similar affinity for the dye. In turn, the cells forming the dorsal part of the dental papilla have a greater affinity for the dye in relation to the previously mentioned structures. Mesenchyme surrounding the developing egg tooth had a greater concentration in relation to mesenchyme located more dorsally. The cells of the dental papilla have circular nuclei. Three layers of cells are visible in the area of the forming enamel organ. Cylindrical cells in the inner layer have elongated nuclei located in the apical part of the cells. The middle part consists of six to seven layers of cuboidal cells. The outer layer consists of two layers of flattened cells. In the epithelium of the lateral part of the mouth and in the epithelium surrounding the developing egg tooth, cells with nuclei with a circular cross‐section are visible. At this stage of development, single mitotic figures are present in cells of the dental papilla, the enamel organ, surrounding mesenchyme and the epithelium lining the mouth cavity (Figure 4B).

In the cross‐sections of developmental stage 11 the dental papilla with the surrounding enamel organ has an approximately rectangular shape. Dorso‐laterally blood vessels are visible while ventrally forming mandibula with differentiating dentary bones in the centre is present (Figure 4C).

The enamel organ consists of three layers. The inner layer of the forming enamel organ is hourglass‐shaped, but without a base along the dorsal side of the embryo. In the medial part of the inner layer, differentiating ameloblasts are visible. These cylindrical cells form a single layer. Ventrally from differentiating ameloblast cells, two to three layers of squamous epithelial cells are located. In lateral parts of the inner layer condensed agglomerates of cells with spherical nuclei are visible. Along the layer these agglomerates transition into a single layer of columnar cells which in turn transition into the outer enamel epithelium consisting of a single layer of cuboidal cells. The outer enamel epithelium extends from the dorsal part of the egg to the ventral part where it connects to the epithelium of the mouth cavity. Fusiform or cuboidal cells with intercellular spaces similar in size to those in the mesenchyme are present in the area of the dental papilla. In addition, a few erythrocytes were also visible in the dental papilla. At the border between the lateral parts of the dental papilla and the inner layer of the forming enamel organ, a thin layer of connective tissue is located. On the dorsal part of the differentiating egg tooth, the mesenchyme of greater cell density than in the rest of the mouth is visible. In the vicinity of the enamel organ mesenchymal cells are more flattened than the cells located further away. At this stage of development, individual mitotic figures are visible in the cells within the inner layer of the enamel organ in the dorsal part of the egg tooth (Figure 4D).

At developmental stage 13, the differentiating egg tooth does not differ significantly from the egg tooth at stage 11 except that the differentiating premaxilla is visible dorsally to the forming egg tooth (Figure 5A). The shape of the egg tooth is wider in comparison to the previously described stage. In the close vicinity, on the dorsal side of the egg tooth, the early stage of premaxilla differentiation is visible. In the area of the dental papilla, there is a greater density of cells, and thus smaller intercellular spaces compared to the previous stage. The amount of dentine between the enamel organ and dental papilla in the cross‐section at this stage is slightly larger than in the previously described stage 11. Within the dorsal part of the egg tooth dental papilla, individual melanocytes are present (Figure 5B).

Figure 5.

Transverse sections through brown anole embryo heads at developmental stages 13 and 14. (A) Section through the brown anole embryo at developmental stage 13. The differentiating egg tooth does not differ significantly from the egg tooth at this stage, with the exception of single melanocytes present in its dorsal part. Scale bar 100 µm (B) The egg tooth of the brown anole at the developmental stage 13. (C) Section through the brown anole embryo at developmental stage 14. A thicker layer of dentine than in previous stages is visible. Scale bar 100 µm. (D) The egg tooth of the brown anole at developmental stage 14. Scale bar 50 µm. D, dentine; DP, dental pulp; EO, enamel organ; ET, egg tooth; J, jaw; Mes, mesenchyme; NC, nasal capsule; PMX, premaxilla; T, tongue; V, vestibulum

At the next stage of development the shape of the egg tooth on the cross‐section differs from the previous stage. It also resembles an hourglass but its ventral part is similar in shape to an ellipse, and not—like in the previous stage—to an isosceles trapezoid (Figure 5C). In the dental papilla, cells of various shapes, separated by relatively large intercellular spaces, are visible. Between the dental papilla and the enamel organ in the ventral part of the egg tooth, dentine is located, with a shape resembling an ellipse sharp on both sides, and invaginated medially on the ventral side. Medially in the inner epithelium of the enamel organ, high cylindrical cells are located. These cells have greater affinity for the dye than the remaining cells of the enamel organ. In lateral parts of the inner epithelium, small cells with circular or slightly ellipsoid nuclei are visible. Within the outer and middle layer of the enamel organ, cells with similar morphology are located, having circular nuclei on the cross‐section. Dorsally from the differentiating egg tooth, adjacent to the developing premaxilla, flat cells are present. In this layer, single melanocytes and their protrusions are visible. In the cells located in the area where the inner layer is connected with the outer layer, mitotic figures are noticeable (Figure 5D).

At the next developmental stage, 15, within the premaxilla, intramembranous ossification is visible. In close proximity to the ossifying premaxilla, large blood vessels are present (Figure 6A). No significant changes occurred in the structure of the egg tooth, except for the greater number of melanocytes located dorsally from the egg tooth (Figure 6B).

Figure 6.

Transverse sections through brown anole embryo heads at developmental stages 15 and 17. (A) Section through the brown anole embryo at the developmental stage 15. Many melanocytes are visible in the dorsal part of differentiating egg tooth. Scale bar100 µm. (B) The egg tooth of the brown anole embryo at developmental stage 15. Scale bar 50 µm. (C) Section through the brown anole embryo at developmental stage 17. Dentine of the egg tooth is visible in the dorsal part of the egg tooth. Scale bar 100 µm. (D) Section through the brown anole embryo at developmental stage 17, showing the caudal part of the egg tooth located directly on the premaxilla. Dorsally from the differentiating egg tooth adjacent to the developing premaxilla. Scale bar 50 µm. (E) The egg tooth of the brown anole embryo at developmental stage 17. Scale bar 50 µm. D, dentine; DP, dental pulp; EO, enamel organ; ET, egg tooth; Mes, mesenchyme; NC, nasal capsule; PMX, premaxilla; V, vestibulum

Similarly to previous stages, in developmental stage 17 the dental papilla enclosed in the dentine has the shape of an hourglass. Around nasal vestibula filled with nasal plugs, nasal capsules composed of hyaline cartilage are visible. Dorsally to the developing egg tooth, the nasal process of the premaxilla is visible, and laterally forming premaxillar alveolar plates are present. Within both parts of the premaxilla, intramembranous ossification is visible (Figure 6C). On the cross‐sections from the caudal part of the brown anole head, the egg tooth is located directly on the premaxilla (Figure 6D). Within the dental papilla of the egg tooth, a single layer of cuboid cells is visible under the dentine. In the central part of the egg tooth dental papilla, variously shaped cells are located. Intercellular spaces between cells in the centre of the dental papilla are bigger than between cells of its outer layer. Blood vessels are visible in the dental papilla. The egg tooth dentine is almost homogeneous with respect to the dye affinity and stained pink. On the medio‐ventral part of the dentine, a small invagination is present. In the enamel organ three to six cell layers are visible. Around the enamel space a single layer of cuboidal cells with dye affinity higher than any other cells of the enamel organ is located. Single pigment cells are present in the dorsal part of the brown anole egg tooth. Between the egg tooth and the premaxilla approximately three layers of flat cells are visible. These cells had high affinity for the dye. The epithelium lining the mouth consists of four cell layers at this stage of development (Figure 6E).

In the last developmental stage, 18, just before hatching, the egg tooth is wider on the cross‐section in relation to previous developmental stages. The egg tooth is composed of enamel organ, dentine and dental pulp. The dentine on the ventral side is convex at this developmental stage. Fibrous tissue connecting egg tooth to the premaxilla is visible (Figure 7A). The dorsal part of premaxilla is ossified at this developmental stage, and within its ventral part intramembranous ossification is still visible (Figure 7B). In the proximal part of the dentine, bundles of fibre entering the attachment tissue are visible (Figure 7C). Dentinal tubules are present in the dentine of the egg tooth (Figure 7D). The blood vessels and melanocytes are visible in the dental papilla. Between the dentine of the egg tooth and premaxilla, fibrous tissue is present. This fibrous tissue stains more weakly in relation to dentine and bone and darkly stained cells are enclosed inside it. All the cells of the enamel organ at this developmental stage are similar in stain affinity and morphology to the cells of epithelium lining the mouth (Figure 7A).

Figure 7.

Transverse sections through brown anole embryo head at developmental stages 18. (A) Section through the brown anole embryo at developmental stage 18 showing fully differentiated egg tooth. Scale bar 100 µm. (B) The dorsal part of the premaxilla is ossified at this developmental stage while within its ventral part intramembranous ossification is still visible. Scale bar 20 µm. (C) Attachment tissue enclosing darkly stained cells is visible. Scale bar 20 µm. (D) Dentinal tubules are visible in the dentine at that developmental stage. Scale bar 20 µm. AT, attachment tissue; D, dentine; DP, dental pulp; PMX, premaxilla; black arrow, melanocyte; white arrow, dentinal tubule

In the sections of 2‐day‐old hatchlings, egg teeth have been shed (Figure 8A). A relatively large number of melanocytes with long protrusions are visible (Figure 8B). These protrusions almost reached the oral epithelium. Multinucleated odontoclasts are located under the epithelium of the mouth and fibrous tissue previously connecting the egg tooth to the premaxilla. Pigment granules are located in the cytoplasm of some osteoclasts. This tissue can be seen to stain purple close to odontoclasts (Figure 8C). The oral epithelium consists of six to eight layers of cells. The cells in the area of the dental papilla have similar morphology as in previous stages of development. (Figure 8A).

Figure 8.

Transverse sections through brown anole 2‐day hatchling. (A) Section through the brown anole 2‐day hatchling. (B) Melanocytes are visible in the area of lost egg tooth dental papilla. Dendrites of these cells reach far into the ventral part of the palate. (C) Pigment granule filled osteoclasts are visible near the remnants of egg tooth attachment tissue. AT, attachment tissue; BV, blood vessel; OC, odontoclasts; PMX, premaxilla; black arrow, melanocyte

3.4. Transmission electron microscopy

The morphology of the egg tooth from developmental stage 18 in the semi‐thin sections is similar to that observed in the corresponding sections stained with H&E. The only exception is the presence of big oval cells—preodontoclasts—and more prominent blood vessels in dental pulp (Figure 9A).

Figure 9.

Epoxy resin sections through the brown anole embryo head at developmental stage 18. (A) Semi‐thin section through brown anole embryo at the developmental stage 18. (B) Cells of degenerating egg tooth enamel organ inner layer. Scale bar—2.5 µm. AT, attachment tissue; BV, blood vessel; D, dentin; EO, enamel organ; N, nucleus; PMX, premaxilla; pOC, preodontoclast

The population of cells comprising the the enamel organ is homogenous. These cells are irregularly shaped. Electron‐dense, irregularly shaped nuclei, with a relatively large amount of heterochromatin, are located in the central part of these cells (Figure 9B). Nuclei are surrounded by granular, electron‐dense cytoplasm. Cytoplasm is poor in organelles but contains a few mitochondria, numerous bundles of intermediate fibres and polyribosomes (Figure 10A). The cells of the superficial layer possess microvilli, that protrude into the oral cavity, on their apical side (Figure 10B). These cells are connected with each other by desmosomes and possess numerous, long finger‐like processes, interdigitated with the membranes of adjacent cells along the cell membrane. These processes contain homogenous cytoplasm without any organelles (Figure 10C). In the enamel organ, some apoptotic cells are visible. In their cytoplasm they possess large, lucent cytoplasmic vacuoles, condensed spherical and ovoid cytoplasmic fragments of varying sizes and homogeneous and heterogeneous electron densities, and membrane bound nuclear chromatin aggregated in dense mass beneath the nuclear envelope. The cytoplasm of these cells has electron density similar to that of the neighbouring cells (Figure 10D).

Figure 10.

Electronograms of brown anole embryo degenerating enamel organ cells. (A) Intermediate filaments inside the cytoplasm of cell in outer layer of degenerating enamel organ. (B) Microvilli of cells in outer layer of degenerating enamel organ. Scale bar 2.5 µm. (C) Desmosomes connecting cells within degenerating enamel organ. Scale bar 1 µm. (D) Apoptotic cell located within degenerating enamel organ. Scale bar 2.5 µm. CH, condensed chromatin; IF, intermediate filaments; N, nucleus; M, mitochondria; MV, microvilli; V, vacuole

The big oval cells located in the dental pulp have a big spherical nucleus containing a very small amount of heterochromatin and are composed mostly of homogenous chromatin with similar electron density to their cytoplasm (Figure 11A). Near their nuclei, Golgi apparatus, consisting of several sheets of lamellae surrounded by small vesicles, are present (Figure 11B). In their granular, electron‐dense cytoplasm, many mitochondria, diluted rough endoplasmic reticulum, and autophagic vacuoles are visible. These vacuoles contain degenerated fragments of mitochondria (Figure 11C) and spherical electron‐dense bodies (Figure 11D).

Figure 11.

Electronograms of brown anole embryo dental pulp cells. (A) Big oval cell located inside dental pulp of the egg tooth. Scale bar 2.5 µm. (B) Golgi apparatus of big oval cell. Scale bar 1 µm. (C) Autophagic vacuole containing mitochondrion. Scale bar 1 µm. (D) Autophagic vacuole of big oval cells containing spherical bodies. Scale bar 1 µm. (E) The odontoblasts of the brown anole egg tooth. Scale bar 1 µm. (F) Cells involved in secretion of attachment tissue matrix. Scale bar 2.5 µm. (G) Secretion vesicle inside apical cytoplasm of odontoblast. Scale bar 1 µm. AV, autophagic vacuole; D, dentine; GA, golgi apparatus; N, nucleus; M, mitochondria; RER, rough endoplasmic reticulum; SV, secretory vesicle

The odontoblasts (Figure 11D) and the cells involved in secretion of attachment tissue matrix (Figure 11E) have ultrastructure similar to each other. They possess large elongated nuclei containing single nucleoli and small clamps of electron‐dense heterochromatin. Their dark cytoplasm has similar electron density to euchromatin in nuclei and is mostly filled with a large amount of endoplasmic reticulum and some mitochondria (Figure 11D,E). In addition, electron lucent secretion vehicles are visible in their apical cytoplasm. These vesicles contain spherical granules of varying electron density (Figure 11F).

The attachment tissue is composed of thick collagen bundles oriented in various planes. Between collagen fibres small protrusions of fragments of cells containing rough endoplasmic reticulum are visible. They are irregularly shaped and contain electron‐dense homogenous cytoplasm (Figure 12A). Closer to the premaxilla irregularly shaped cells with a large amount of highly diluted rough endoplasmic reticulum are visible between fibres of attachment tissue Their relatively large nuclei contain single nucleoli and small amount of electron dense clamps of heterochromatin located near the nuclear envelope (Figure 12B). Electron‐dense borders between attachment tissue and premaxilla are penetrated by thick bundles of Sharpey's fibres. In the vicinity of Sharpey's fibres, autophagic cells and osteoblasts are present (Figure 12C). This kind of connection does not occur in the border between the attachment tissue and dentine of the egg tooth (Figure 12D).

Figure 12.

Electronograms of brown anole embryo attachment tissue. (A) Collagen fibres of attachment tissue. Scale bar 3 µm. (B) Cells with highly diluted rough endoplasmic reticulum located in attachment tissue. Scale bar 2.5 µm. (C) Sharpey's fibres penetrating the border between attachment tissue and premaxilla. Scale bar 2.5 µm. (D) Border between dentine of the egg tooth and attachment tissue. Scale bar 5 µm. AT, attachment tissue; CF, collagen fibers; D, dentin; N, nucleus; RER, rough endoplasmic reticulum

4. DISCUSSION

4.1. Egg tooth development

Brown anole egg tooth development starts before the development of regular dentition, similar to other squamates (Buchtová et al. 2013; Hermyt et al. 2017). The first visible step of egg tooth development in the brown anole is thickening of the oral epithelium in the rostral part of the palate and appears during developmental stage 8, which extends from approximately the sixth to the ninth day after oviposition (Sanger et al. 2008). This indicates that odontogenesis in A. sagrei begins relatively late in embryonic development in comparison to other species, including the grass snake (Hermyt et al. 2017). In the subsequent developmental stages, thickened epithelium invaginates, and mesenchyme located under the invagination condenses, forming the enamel organ and dental papilla, respectively. The invagination results in the formation of a U‐shaped notch in the jaw of the brown anole. Investigations of histological sections and micro‐CT scans presented in this study have not provided evidence of the existence of a second rudimentary egg tooth during any developmental stage of the brown anole, which is consistent with previous reports of its absence in iguanomorph lizards (Anan'eva and Orlov, 2013). On the basis of histological sections we found that the egg tooth enters the cap stage around developmental stage 9. In developmental stage 11 the bell stage begins. During that developmental stage, dentine in the egg tooth of A. sagrei appears. It is more prominent in the proximal sections and in the lateral area in more distal parts of the egg tooth. The developmental process of the egg tooth in A. sagrei is similar to that observed in other squamates (Hill and De Beer, 1950; Smith et al. 1953; Bandali et al. 2007; Hermyt et al. 2017), in the regular teeth of squamates (Zahradnicek et al. 2008; Richman and Handrigan, 2011; Zahradnicek et al. 2012, 2014), and in other vertebrates (Luckett, 1993; McCollum and Sharpe, 2001; Catón and Tucker, 2009; Hovorakova et al. 2018). Based on the fact that a separate layer of immature dentine could not be distinguished, mineralization of the dentine in the brown anole's egg tooth occurs relatively fast in comparison to other species in which predentine is clearly distinct from dentine (Delgado et al. 2005; Buchtová et al. 2013; Hermyt et al. 2017). The dentine of the egg tooth shortly prior to hatching is convex in the medio‐ventral part, in contrast to its previous stages of development where an invagination was observed. This might be an artifact of tissue preparation resulting from thinness of dentine during those stages. Moreover, this invagination from earlier developmental stages was also not present in scanning electron microscope microphotographs and in scans from micro‐CT.

In the course of the egg tooth differentiation the dentine thickens, blood vessels penetrate the dental pulp of the egg tooth and the three‐layered structure of the enamel organ develops. The dental pulp consists mostly of mesenchymal cells and polarized columnar odontoblasts with basal nuclei.

Starting from developmental stage 14, melanocytes and their dendrites were located in the ventral part of the developing egg tooth. Both melanocytes and cells of dental pulp originate from the neural crest (Yamazak and Hayashi, 2004; Paino et al. 2010). They were present in all subsequent developmental stages even after loss of the egg tooth. Location of these melanocytes changed in the course of development—being located increasingly deeper in the dental pulp. After egg tooth loss, their dendrites almost reached the epithelium of the oral surface. Melanocytes' dendrites are actin‐ and microtubule‐containing structures which transport melanosomes to their tip (Kippenberger et al. 1998; Scott, 2002). Moreover, melanosomes were located in the cytoplasm of osteoclasts. Cytoplasmic presence of melanosomes in osteoclasts has previously been described only in silky fowl. It was hypothesized that pigment granules were present in the osteoclast, possibly from phagocytosis of pigment‐containing osteocytes (Hirano, 1990). However, close proximity of osteoclasts to dendrites of melanocytes and absence of pigmented osteocytes in the brown anole suggest that melanosomes are transported directly from melanocytes to osteoclasts. Melanocytes were not found in grass snake teeth (Hermyt et al. 2017) and other authors also did not report their presence in egg teeth during odontogenesis in other squamate species (Hill and De Beer, 1950; Smith et al. 1953; Anan'eva and Orlov, 2013). Moreover, they are not involved in the development of regular dentition in other vertebrates (Catón and Tucker, 2009; Zahradnicek et al. 2012; Buchtová et al. 2013). Functions of melanocytes during odontogenesis are currently unknown.

The enamel organ during bell stages consists of the inner layer of columnar ameloblasts, and the outer enamel epithelium is composed of cuboidal cells and stellate reticulum, consisting of star‐shaped cells, located between the aforementioned layers. Before the appearance of dentine, the apical parts of ameloblasts and odontoblasts are in contact. Similarly, as in our previous study of the grass snake, we did not find a stratum intermedium in the enamel organ of A. sagrei egg tooth. The stratum intermedium is a layer located between the inner enamel epithelium and the stellate reticulum. It has been suggested that the inability to distinguish this layer in the investigated reptilian species is attributable to the small size of the enamel organ (Richman and Handrigan, 2011). Similar to the grass snake (Hermyt et al. 2017), despite the fact that enamel organs in egg teeth are generally larger than regular teeth De Beer, 1949; Smith et al. 1953; Brink et al. 2017), we could not find this layer in the brown anole. Many authors have noted the absence of the stratum intermedium in non‐mammalian vertebrates. Besides mammals (Johnson and Bevelander, 1957; Chiba, 1965; Kallenbach, 1978), the stratum intermedium is absent in most of the major groups of vertebrates except crocodilians (Westergaard and Ferguson, 1986, 1987; Sire et al. 2002). Absence of that layer was confirmed in fish (Huysseune and Witten, 2008; Sasagawa et al. 2008), amphibians (Kerr, 1960; Goin and Hester, 1961; Lawson, 1965) and squamates (Delgado et al. 2005; Buchtová et al. 2008, 2013) except Paroedura picta (Zahradnicek et al. 2012). The enamel organ of egg tooth degenerated and lost its three‐layered structure shortly before hatching around developmental stage 18. Its cells have similar morphology and stain affinity to the one in the epithelium lining the oral cavity at this stage of development. These changes are confirmed by transmission electron microscopic analysis. All the cells of degenerating enamel organ have similar ultrastructure. Cells with typical ultrastructural features of ameloblasts have not been found at this developmental stage. The apoptotic cells are present within the enamel organ. Apoptosis occurs at all stages of odontogenesis (Matalova et al. 2004; Matalova et al. 2012). In differentiated teeth it is associated with degradation of the eruption pathway (de Pizzol Júnior et al. 2015), and with changing of reduced enamel organ into stratified squamous epithelium, which joins the oral epithelium (Abiko et al. 1996). The egg tooth tip in the brown anole shortly before hatching is covered by very thin layer of simple squamous epithelium. The thinning of the enamel organ above the egg tooth tip might be important in its eruption. In the brown anole this change is less pronounced in the course of its development than in the previously investigated grass snake (Hermyt et al. 2017). The degenerated enamel organ in brown anoles is similar to mammalian junction epithelium, both in terms of histology and ultrastructure (Nakamura, 2018). Relatively loose intercellular junctions in that epithelium enable penetration of inflammatory cells and tissue exudate which makes it first‐line defence against pathogenic infections (Nakamura, 2018). Presumably the degenerated enamel organ, before being replaced by oral epithelium, performs a similar function in the squamate egg tooth as epithelial discontinuity caused by its shedding would be leave it vulnerable to pathogens.

The fully differentiated egg tooth has a similar shape to the egg teeth of most of the previously described species, although it is slightly more flattened dorsoventrally (Smith et al. 1953; Fioroni, 1962; Hermyt et al. 2017). Similarly, it possesses sharp cutting edges and is curved dorsoventrally. It is not congruous with previous reports describing iguanomorphs egg teeth as being cone‐shaped with a sharp tip (Anan'eva and Orlov, 2013).

We did not notice changes in egg tooth spatial orientation during its development. It faces rostroventrally through all investigated developmental stages. In contrast, snake egg teeth change from ventral to rostroventral orientation in the course of their development. It might be connected with modification of the rostral scale's shape during its development, during which a characteristic notch is formed in snakes or a different arrangement of snake embryos inside the egg (Fioroni, 1962; Hermyt et al. 2017). Rostroventral orientation of the fully developed egg tooth seems to be universal for currently investigated oviparous representatives of Unidentata. Egg teeth of oviparous species can be pointed ventrally or caudoventrally (Smith et al. 1953).

4.2. Egg tooth attachment and implantation

Tissue attaching the egg tooth to the premaxilla forms shortly before hatching. It was present only on histological slices from the last developmental stage (18) preceding hatching. Literature concerning attachment tissue development in other squamates is scarce in histological descriptions [but see Maxwell et al. (2011), Buchtová et al. (2013), Hermyt et al. (2017) and LeBlanc et al. (2017)]. Attachment tissue in the brown anole, similarly to that previously described in the grass snake, consisted of fibrous tissue with weaker stain affinity in relation to the egg tooth and premaxilla (Hermyt et al. 2017). Cells responsible for secretion of collagen matrix in attachment tissue located in dental pulp are in the same cell layer as odontoblasts and share gross morphology, stain affinity and ultrastructure (Buchtová et al. 2013). The formation of attachment tissues in mammals and probably other vertebrates involves the dental follicle (Bosshardt, 2005; Diep et al. 2009). In the case of the brown anole egg tooth the condensed mesenchyme adjacent to the developing egg tooth is restricted only to its basal part, as is the case in teeth of most amphibians and osteichtians (Davit‐Béal et al. 2006; Davit‐Béal et al. 2007). In the marginal teeth of other squamates, whole germs are surrounded by the dental follicle (Delgado et al. 2005; Buchtová et al. 2007, 2013). That localization of the dental follicle during egg tooth development might explain its acrodont implantation (described below).

In contrast to the grass snake, the matrix of brown anole attachment tissue contained darkly stained enclosed cells in the ventral part of the egg tooth—similar to the osteocyte lacunae of forming bones (Hermyt et al. 2017). Buchtová et al. (2013) described attachment tissue in Chamaeleo calyptratus as 'a layer of predentine connecting the tooth base to the membranous bone'; however, in the case of the brown anole and the previously investigated grass snake, the attachment tissue exhibited different morphology and stain affinity in relation to predentine in these species. Unchanged morphology of this tissue after loss of the egg tooth indicates that it does not undergo mineralization at any stage of its development. Among squamates, a similar kind of attachment tissue in regular dentition to that found in the egg tooth was described in various families of snakes and limbless lizards. It allows their teeth to fold posteriorly during feeding under the pressure created by hard‐bodied prey (Savitzky, 1981; Patchell and Shine, 1985; Mahler and Kearney, 2006). In the case of the egg tooth, this type of attachment is probably connected with its shedding rather than mobility. It is assumed that teeth attached by soft ligament appeared independently several times in the course of squamate evolution (Bertin et al. 2018). Currently, we suspect that its fibrous form is present in embryos of all the squamates (Hermyt et al. 2017). The brown anole is representative of the Dactyloidae, a group of reptiles characterized, among other traits, by pleurodont dentition (Nicholson et al. 2012). However, based on histological sections and 3D reconstructions from the last stage of development, the egg tooth appears to be attached to the apex of the premaxilla. Its spatial relation to that bone indicates an acrodont mode of implantation (Edmund, 1969). Acrodonty in reptiles is associated with monophyodonty (Rieppel, 2001; Smirina and Ananjeva, 2007; Tucker and Fraser, 2014), but a recent study on Opisthodontosaurus, the oldest known amniote with an acrodont dentition, showed that this mode of attachment was not always related to inhibition of tooth replacement (Haridy et al. 2018). Acrodonta (agamids and chamaeleonids) possess anterior acrodont teeth that do not undergo replacement, in contrast to the posterior part of the dentary (Zaher' and Rieppel, 1999; Dosedělová et al. 2016). Hence adaptation to extreme wear of teeth and their persistence through the whole life of the animal have been associated with acrodonty (Haridy et al. 2018; Haridy, 2018). On the other hand, the acrodont egg tooth is only a single use adaptation to assist with hatching and is lost shortly after the young individual leaves its egg. In the brown anole, similarly to most unidentates (some exceptions, e.g. Agama agama, have been reported), a successor tooth does not appear in the place of the lost egg tooth (Smith et al. 1953; Cooper, 2003; Hermyt et al. 2017).

4.3. Shedding of the egg tooth

The egg tooth appears on the slices from developmental stage 18; hence egg tooth loss most likely happens shortly before hatching of the embryo, similar to the grass snake (Hermyt et al. 2017). The egg tooth loss in A. sagrei occurs within a few days after hatching, similarly to other squamates (Trauth, 1988; Anan'eva and Orlov, 2013; Hermyt et al. 2017). Observations with the scanning electron microscope showed that the egg tooth breaks off with a horizontal fracture near the surface of the palate, leaving only a small dentinal fragment, similar to Cnemidophorus sexlineatus (Trauth, 1988). Under the oral epithelium, multinucleate odontoclasts are involved in resorption of egg tooth attachment tissue, which remains after the egg tooth loss. Odontoclasts have similar morphological and functional characteristics to the bone resorbing osteoclasts but are smaller, have a ruffled border and have fewer nuclei (Furseth, 1968; Pierce et al. 1991; Sahara et al. 1996; Sasaki, 2003). Activated odontoclasts produce acidic pH in their microenvironment which manifests on the sections as purple haematoxylin‐stained regions of attachment tissue in the vicinity of odontoclasts (Blair et al. 1989; Ne et al. 1999). These cells originate from fusion of circulating mononuclear progenitor cells residing within the pulp cavity (Sahara et al. 1996; Ne et al. 1999; Patel et al. 2010). Dental pulp of the stage 18 brown anole contained big oval cells. Their ultrastructure resembles the one of preodontoclasts (Sasaki et al. 1989). Presence of preodontoclasts before the process of hatching of the individual suggests that remodelling of the premaxilla and attachment tissue is not induced by the mechanical damage to the egg tooth but rather by endogenous signals before hatching. It is not known if odontoclasts are involved in egg tooth loss or are only responsible for resorption of egg tooth remains under the surface of the palate. The latter, along with mechanical loss of the egg tooth, can be supported by leftover dentinal fragments observed above the surface of the oral epithelium in scanning electron microscopy. These fragments possibly would not be present if the egg tooth falls off just from resorption occurring under the surface of the oral epithelium. The shedding of the egg tooth might be facilitated by uncalcified, ligamentous character of its attachment tissue, which is not as strongly connected with dentine as in squamates with ankylosed teeth. Based on the ultrastructural analyses it can be concluded that connection of the attachment tissue with the premaxilla is stronger than the connection with dentine thanks to strengthening through the presence of Sharpey's fibers.

In conclusion, the present study demonstrates structural changes occurring during the development of brown anole egg tooth and shows that squamate egg teeth can differ between species and differ from marginal teeth within the same species. The egg tooth, for the most part, resembles other teeth of vertebrates in its structure and development. However, because of the specific functions it performs, it possesses unmineralized attachment tissue, allowing its shedding after hatching and implantation on the premaxilla, which distinguishes it from regular dentition. Additionally presented results might indicate that preparation for resorption of the egg tooth precedes the process of hatching. The egg tooth of the brown anole has some characteristics in comparison to marginal teeth which might be considered ancestral. These characteristics include mode of implantation, location of dental follicle and unmineralized attachment tissue. If it is indeed a remnant of evolution, this might be another argument supporting the view that attachment tissue of squamates, historically termed 'bone of attachment', (Tomes, 1882) is homologous to periodontal ligament (LeBlanc et al. 2017). To provide more data for comparative analyses, further studies on the development and structure of this organ involving a broader range of representatives of the group are necessary. Different forces acting on the egg tooth during hatching of different species might be basis for some additional differences in terms, e.g. attachment tissue structure. It mainly concerns viviparous species due to absence of the egg shell, and geckos because they possess two differently oriented egg teeth and most of them have rigid egg shells (Pike et al. 2012).

Hermyt M, Janiszewska K, Rupik W. Squamate egg tooth development revisited using three‐dimensional reconstructions of brown anole (Anolis sagrei, Squamata, Dactyloidae) dentition. J. Anat. 2020;236:1004–1020. 10.1111/joa.13166

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article.