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Expression patterns of Pitx2 and Shh at early stages of axolotl odontogenesis. Expression patterns of early odontogenic markers Pitx2 and Shh on the mouth roof and floor whole mounts show initial stages of development of axolotl dentition (in case of Shh expression, the epithelium of the mouth roof was dissected from whole mounts to circumvent masking of the tooth-restricted epithelial signal by the strong Shh signal from the adjacent neural tube). At stages 37–41, Pitx2 labels tooth-competent regions while Shh is restricted to individual tooth germs and into postero-medially placed odontogenic bands (visualized as strands of Shh expression demarcated by white arrowheads). Positions of tooth germs are visible as regions of lower Pitx2 expression. (A,E,I,M) Expression pattern of Pitx2 on the mouth roof is initially restricted to its anterior part but becomes broader at later stages (parentheses). (B,F,J,K) Focal expression of Shh illustrates sequential addition of tooth germs starting from the initiator-teeth of vomerine (vom) and palatine (pal) teeth (B,F, arrows). New tooth germs are added postero-medially in the palatine tooth field (J, black arrowheads) and antero-medially in the vomerine tooth field (N, black arrowheads). (C,G,K,O) Expression pattern of Pitx2 on the mouth floor enlarges posteriorly throughout development and, at stages 40–41, the pattern separates into the prospective dentary (den) and coronoid (cor) tooth fields (K,O, parentheses). (D,H,L,P) Focal Shh expression on the mouth floor marks positions of initiator-teeth of the coronoid (D,H, arrows) and dentary fields (L,P, arrows) and the addition of new tooth germs within both tooth fields (H,L,P, black arrowheads). New tooth germs are added lingually to the initiator tooth germ in the coronoid tooth field (H,L,P, black arrowheads) and laterally from the medially positioned initiator tooth in the dentary tooth field (P, arrowheads). b1, first branchial arch; cor, coronoid tooth field; den, dentary tooth field; h, hyoid arch; m, mandibular arch; pal, palatine tooth field; vom, vomerine tooth field. Scale bar equals 500 μm.
Source publication
Vertebrate dentitions arise at various places within the oropharyngeal cavity including the jaws, the palate, or the pharynx. These dentitions develop in a highly organized way, where new tooth germs are progressively added adjacent to the initiator center, the first tooth. At the same time, the places where dentitions develop house the contact zon...
Citations
... The inner set of bones is referred to as the inner dental arcade, relative to the marginal jawbones, dentary, premaxilla and maxilla, which form the outer dental arcade. In stem osteichthyans, the inner dental arcade consists of a series of discrete domeshaped tooth plates, termed tooth cushions (Chen et al., 2017), while in crown osteichthyans the inner dental arcade contains sutured bones of distinct shapes (Clemen et al., 1998;Gardiner, 1984;Grande & Bemis, 1998;Soukup et al., 2020) that are further divided into labial and lingual sets (Zhu et al., 2019). The labial set is composed of a series of coronoids (plus the parasymphysial tooth whorl/ plate in some sarcopterygian fish) on the lower jaw and a series of dermopalatines plus the vomer and ectopterygoid on the upper jaw, probably corresponding to tooth cushion series; additionally, the prearticular on the lower jaw and entopterygoid on the palate constitute the lingual set. ...
... The axolotl coronoid displays a lingual addition of multiple tooth rows in splay mode (Soukup et al., 2020) and lingual replacement of successive teeth (Makanae et al., 2020). The coronoid of the Iberian newt is resorbed from the labial side during metamorphosis, and the multiple tooth rows are replaced by double rows and then a single row (Smirnov et al., 2020). ...
New teeth are predominantly initiated lingually or postero-lingually to the old teeth in vertebrates. Osteichthyan dentitions typically consist of linear rows of shedding teeth, but internal to the marginal jawbones osteichthyans primitively have an extra dental arcade, in which teeth are sometimes spread out into a field and not organized in rows. The tooth plates of lungfish are specialized from the jawbones of the inner dental arcade, but the teeth are arranged in radial tooth rows with new teeth added at the anterior and labial end of the rows and without shedding the old teeth, distinct from other osteichthyan dentitions. Actinopterygian teeth can be recognized by a cap of enameloid, while sarcopterygian teeth are only coated by enamel. An enameloid cap is also borne by the unicuspid larval teeth in some amphibians, but it is covered by enamel and eventually disappears in the bicuspid adult teeth. In early osteichthyans, old teeth are often not completely resorbed and shed, and the overlapping relationship of their remnants buried in the bone records the sequence of developmental events. Using synchrotron microtomography, this ontogenetic record of a coronoid tooth field of a Devonian stem actinopterygian is visualized in 3D. As a component of the inner dental arcade, the coronoid displays initial radial non-shedding tooth rows followed by radial shedding tooth rows that are later transformed into linear shedding tooth rows. The teeth are always added antero-labially and replaced labially to keep pace with the labial bone apposition and lingual bone remodeling, which causes the shift of the tooth competent zone. These provide a clue to the evolution of the radial non-shedding dentition with antero-labial tooth addition in lungfish. The tooth patterning process suggests that the superficial disorder of the tooth field is an epiphenomenon of the ever-changing local developing environment of each tooth bud: due to the retention of old tooth bases, a tooth position that has been replaced in place can at some point drift to a site between the adjacent tooth positions, splitting or merging, and then continue being replaced in situ. Primary teeth are capped by enameloid, but replacement teeth bear enamel crests without an enameloid cap. This demonstrates that the transition from enameloid capping to enamel coating through tooth replacement can happen in actinopterygians too, as one of the mechanisms for a dentition to change tooth shape. All these unexpected observations indicate that, during ontogeny, the states of dental characters, such as lingual/labial tooth initiation, linear/radial tooth rows, in situ/cross-position tooth replacement and enameloid/enamel, can be switched and the capacity to produce these characters can be suspended or reactivated; the tremendous dental diversity can thus be attributed to the manipulation in time and space of relatively few dental developmental processes.
... The germ layer origin of the epithelia involved (whether ectoderm or endoderm) has played a central role in elucidating the development and evolution of these organs, as well as in understanding their disorders. Yet germ layer boundaries have been notoriously difficult to identify, requiring most often transgenic reporter lines [5,[14][15][16][17]. This is especially the case for the oropharynx, where ectoderm and endoderm directly appose each other at multiple sites. ...
... Teeth in larval axolotls are associated with the premaxillary, vomerine and palatine bones in the upper jaw/ pharynx roof, and with the dentary and coronoid in the lower jaw [14,15,42,43]. Later in development also the maxillary acquires teeth. ...
... The tooth fields are organized into outer (premaxillary and dentary) and inner dental arcades (vomerine, palatine, and coronoid). The first teeth to develop are those from the inner arcade at stage 37. Dentary teeth start to develop at stage 40-41 only, and premaxillary teeth at stage 41 [15]. Thus, oral teeth develop long before the mouth opens. ...
Background
Previous studies have reported that periderm (the outer ectodermal layer) in zebrafish partially expands into the mouth and pharyngeal pouches, but does not reach the medial endoderm, where the pharyngeal teeth develop. Instead, periderm-like cells, arising independently from the outer periderm, cover prospective tooth-forming epithelia and are crucial for tooth germ initiation. Here we test the hypothesis that restricted expansion of periderm is a teleost-specific character possibly related to the derived way of early embryonic development. To this end, we performed lineage tracing of the periderm in a non-teleost actinopterygian species possessing pharyngeal teeth, the sterlet sturgeon (Acipenser ruthenus), and a sarcopterygian species lacking pharyngeal teeth, the axolotl (Ambystoma mexicanum).
Results
In sturgeon, a stratified ectoderm is firmly established at the end of gastrulation, with minimally a basal ectodermal layer and a surface layer that can be homologized to a periderm. Periderm expands to a limited extent into the mouth and remains restricted to the distal parts of the pouches. It does not reach the medial pharyngeal endoderm, where pharyngeal teeth are located. Thus, periderm in sturgeon covers prospective odontogenic epithelium in the jaw region (oral teeth) but not in the pharyngeal region. In axolotl, like in sturgeon, periderm expansion in the oropharynx is restricted to the distal parts of the opening pouches. Oral teeth in axolotl develop long before mouth opening and possible expansion of the periderm into the mouth cavity.
Conclusions
Restricted periderm expansion into the oropharynx appears to be an ancestral feature for osteichthyans, as it is found in sturgeon, zebrafish and axolotl. Periderm behavior does not correlate with presence or absence of oral or pharyngeal teeth, whose induction may depend on ‘ectodermalized’ endoderm. It is proposed that periderm assists in lumenization of the pouches to create an open gill slit. Comparison of basal and advanced actinopterygians with sarcopterygians (axolotl) shows that different trajectories of embryonic development converge on similar dynamics of the periderm: a restricted expansion into the mouth and prospective gill slits.
... First, while a physical link between external epithelium and oral teeth, via the mouth, is obvious, it has become apparent that pharyngeal teeth, or internal odontodes in general, always arise in the vicinity o f channels where the oropharyngeal (endodermal) epithelium meets the external (ectodermal) epithelium, be it at the pouches and ensuing gill slits (Jollie, 1968;Nelson, 1969;Oralova et al., 2020), the spiracular canal (Bjerring, 1998;Brazeau and Ahlberg, 2006), or inhalant ducts (Van der Brugghen and Janvier, 1993) (reviewed in Huysseune et al., 2022). Second, in particular taxa, dental fields have been demonstrated to be initiated close to the ectodermal/endodermal boundary (Soukup et al., 2021). Third, recent studies have discovered that the external layer o f the epidermis, the periderm, partially invades the communication channels between external (skin) and oropharyngeal epithelium, at least in the actinopterygian species for which such studies were undertaken, the zebrafish (Rosa et al., 2019;Oralova et al., 2020). ...
ABSTRACT
Teeth are defined as internal odontodes, sharing a common ancestry with skin denticles (external odontodes). The conquest of the oropharynx by odontodes, originally limited to the body surface, is now commonly accepted (the ‘outside in’ theory on tooth origins). Yet, an unanswered question is how external epithelia could have transferred odontogenic competence to internal epithelia. Here, we review the origin, structure, and fate of the tooth-forming (odontogenic) epithelia. First, we highlight recent observations supporting the importance of a physical link between external and internal epithelia for tooth initiation. Vertebrate teeth always develop from stratified epithelia, and in several taxa, the surface layer appears to have an ectodermal signature, as periderm, ‘periderm-like’, or as ‘ectodermalized endoderm’. Tooth formation in the mouth of extant vertebrates is usually restricted to odontogenic bands or dental laminae, but odontogenic potential can be awakened outside these areas. Likewise, species belonging to early branching lineages of teleosts have teeth on all pharyngeal arches, lost in more derived taxa. Both observations confirm that tooth-forming capacity was once widespread in the oropharynx. Finally, we discuss a possible relationship between retinoic acid (RA) and sonic hedgehog (SHH) for regulating tooth formation throughout the extent of the oropharynx.
... For instance, in alligators, on both dentary (Westergaard and Ferguson, 1986, PLATE I) and premaxilla (Westergaard and Ferguson, 1990, fg. 3), the frst tooth is formed around the jaw anlagen at Position 3 ( Figure 8.1C). In axolotl, the dentary tooth row is initiated from Position 1, but from Position 2 on the premaxilla (Soukup et al., 2021). The frst initiated tooth usually locates somewhere in the middle of the tooth row in typical nonmammalian osteichthyan dentitions, contradictory to what was assumed by the Zahnreihen Theory that the frst tooth anlage of reptiles was always produced at the anterior end of the jaw (Edmund, 1960, fg. ...
... The ossifcation of bone closely follows the calcifcation of teeth in the tooth patches, as well as the linear tooth row, of Mexican axolotl Ambystoma mexicanum (Soukup et al., 2021), sterlet sturgeon Acipenser ruthenus (Pospisilova et al., 2021), and threespined stickleback Gasterosteus aculeatus (Ellis et al., 2016). On the pharyngeal tooth plates of stickleback, the frst-generation teeth are generally younger peripherally. ...
... The ossifcation of bone closely follows the calcifcation of teeth in the tooth patches, as well as the linear tooth row, of Mexican axolotl Ambystoma mexicanum (Soukup et al., 2021), sterlet sturgeon Acipenser ruthenus (Pospisilova et al., 2021), and threespined stickleback Gasterosteus aculeatus (Ellis et al., 2016). On the pharyngeal tooth plates of stickleback, the frst-generation teeth are generally younger peripherally. ...
n-situ tooth replacement is a major innovation of osteichthyans, but how it evolved remains elusive. Classic models have attempted to analyze the regulation of the alternate tooth replacement relying on untested assumptions. The idealized models, however, cannot explain the large number of deviations of normal and abnormal dental development in the reality, such as the gap-filling phenomenon that causes the split or fusion of tooth families. This chapter will summarize the process components that are shared by all the odontodes. Spatiotemporal developmental shifts between teeth and the underlying skeleton are one of the key factors. Such shifts create the potential for transitioning between various dental patterns and growth modes and for producing a great diversity of dentitions simply by heterochrony. As an example, the frequent initiation of teeth coupled with the space constraint of bone could have given rise to in-situ tooth replacement, without fundamental modifications at the molecular level.
... In situ hybridization was performed on whole mounts as described previously (Soukup et al., 2021) with slight modifications. Briefly, rehydrated A. mexicanum albino (d/d) embryos were digested in 60 μg/ml Proteinase K in PBS, fixed in 4% formaldehyde + 0.2% glutaraldehyde for 30-120 min, transferred into hybridization solution (50% formamide, 1x Denhardt's, 1 mg/ml yeast RNA, 0.1% Tween-20, 10% dextran sulfate, 1x salt solution containing 0.2 M NaCl, 8.9 mM Tris-HCl, 1.1 mM Tris base, 5 mM NaH 2 PO 4 .H 2 O, 5 mM Na 2 HPO 4 and 5 mM EDTA), and incubated overnight in hybridization solution containing RNA probe (1:1,000-1: 100). ...
The asymmetric localization of biomolecules is critical for body plan development. One of the most popular model organisms for early embryogenesis studies is Xenopus laevis but there is a lack of information in other animal species. Here, we compared the early development of two amphibian species—the frog X. laevis and the axolotl Ambystoma mexicanum. This study aimed to identify asymmetrically localized RNAs along the animal-vegetal axis during the early development of A. mexicanum. For that purpose, we performed spatial transcriptome-wide analysis at low resolution, which revealed dynamic changes along the animal-vegetal axis classified into the following categories: profile alteration, de novo synthesis and degradation. Surprisingly, our results showed that many of the vegetally localized genes, which are important for germ cell development, are degraded during early development. Furthermore, we assessed the motif presence in UTRs of degraded mRNAs and revealed the enrichment of several motifs in RNAs of germ cell markers. Our results suggest novel reorganization of the transcriptome during embryogenesis of A. mexicanum to converge to the similar developmental pattern as the X. laevis.
... In the chameleon, this more lateral lamina develops into the dental glands that lubricate the teeth, while in poisonous snakes it forms the venom gland (Vonk et al., 2008;Tucker, 2010). It has been suggested that the mammalian VL and the reptilian dental gland lamina have evolved from laterally positioned DLs, similar to those that create the inner and outer dental arches in extant axolotls (Soukup et al., 2021) As the dentition became restricted to a single row of teeth in many tetrapods, the redundant DLs would have been lost or, in the case of the dental glands and VL, repurposed to create other ectodermal structures . ...
The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here we establish a molecular signature for the usually non-odontogenic VL and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the vestibular lamina, coordinating the development of the dentition and the oral cavity.
... [28][29][30][31][32][33] Recently, we showed that, in the Mexican axolotl, Pitx2 is expressed in broader regions prefiguring the positions of tooth fields while Shh is expressed focally in tooth germs. 34 Concordantly, Pitx2 in the sterlet is expressed broadly at positions of future tooth fields and only later (at around 12-14 mm TL) becomes expressed focally in the developing tooth germs ( Figure 3A The first Pitx2 expression is present just before hatching (stage 35, Figure 2A) as diffuse bands surrounding the future oral opening. These bands are allocated to prospective upper and lower jaws from stage 35 to stage 37 and represent odontogenic bands of the dermopalatine and dentary tooth fields ( Figure 2B-D). ...
Background
Sturgeons belong to an early‐branching lineage often used as a proxy of ancestor‐like traits of ray‐finned fishes. However, many features of this lineage, such as the transitory presence and the eventual loss of dentition, exemplify specializations that, in fact, provide important information on lineage‐specific evolutionary dynamics.
Results
Here, we introduce a detailed overview of the dentition during the development of the sterlet sturgeon. The dentition is composed of tooth fields at oral, palatal, and anterior pharyngeal regions. Oral fields are single‐rowed, non‐renewed and are shed early. Palatal and pharyngeal fields are multi‐rowed and renewed from the adjacent superficial epithelium without the presence of the successional dental lamina. The early loss of oral fields and subsequent establishment of palatal and pharyngeal fields leads to a translocation of the functional dentition from the front to the rear of the oropharyngeal cavity until the eventual loss of all teeth.
Conclusions
Our survey shows the sterlet dentition as a dynamic organ system displaying differential composition at different time points in the lifetime of this fish. These dynamics represent a conspicuous feature of sturgeons, unparalleled among extant vertebrates, and appropriate to scrutinize developmental and evolutionary underpinnings of vertebrate odontogenesis.
... Odontogenesis is poorly understood in amphibians, especially when compared to our understanding of tooth development in fishes and amniotes (Fraser et al. 2004;Tucker and Sharpe 2004;Thiery et al. 2017). It is not yet known if all the genes critical for tooth formation in fishes and amniotes are also expressed during the morphogenesis of teeth in amphibians (but see Soukup et al. 2021). Investigating the developmental genetics of tooth formation in the jaws of frogs may provide insights into whether a transient tooth signaling program is present in the lower jaw, providing the possible mechanism underlying the re-evolution of lost mandibular teeth in G. guentheri. ...
Dollo's law of irreversibility states that once a complex structure is lost, it cannot be regained in the same form. Several putative exceptions to Dollo's law have been identified using phylogenetic comparative methods, but the anatomy and development of these traits are often poorly understood. Gastrotheca guentheri is renowned as the only frog with teeth on the lower jaw. Mandibular teeth were lost in the ancestor of frogs more than 200 million years ago and subsequently regained in G. guentheri. Little is known about the teeth in this species despite being a frequent example of trait "re-evolution," leaving open the possibility that it may have mandibular pseudoteeth. We assessed the dental anatomy of G. guentheri using micro-computed tomography and histology and confirmed the longstanding assumption that true mandibular teeth are present. Remarkably, the mandibular teeth of G. guentheri are nearly identical in gross morphology and development to upper jaw teeth in closely related species. The developmental genetics of tooth formation are unknown in this possibly extinct species. Our results suggest that an ancestral odontogenic pathway has been conserved but suppressed in the lower jaw since the origin of frogs, providing a possible mechanism underlying the re-evolution of lost mandibular teeth.
Ambystoma mexicanum (axolotl) embryos and juveniles have been used as model organisms for developmental and regenerative research for many years. This neotenic aquatic species maintains the unique capability to regenerate most, if not all, of its tissues well into adulthood. With large externally developing embryos, axolotls were one of the original model species for developmental biology. However, increased access to, and use of, organisms with sequenced and annotated genomes, such as Xenopus laevis and tropicalis and Danio rerio, reduced the prevalence of axolotls as models in embryogenesis studies. Recent sequencing of the large axolotl genome opens up new possibilities for defining the recipes that drive the formation and regeneration of tissues like the limbs and spinal cord. However, to decode the large A. mexicanum genome will take a herculean effort, community resources, and the development of novel techniques. Here, we provide an updated axolotl‐staging chart ranging from one‐cell stage to immature adult, paired with a perspective on both historical and current axolotl research that spans from their use in early studies of development to the recent cutting‐edge research, employment of transgenesis, high‐resolution imaging, and study of mechanisms deployed in regeneration.
There are several competing hypotheses on tooth origins, with discussions eventually settling in favour of an 'outside-in' scenario, in which internal odontodes (teeth) derived from external odontodes (skin denticles) in jawless vertebrates. The evolution of oral teeth from skin denticles can be intuitively understood from their location at the mouth entrance. However, the basal condition for jawed vertebrates is arguably to possess teeth distributed throughout the oropharynx (i.e. oral and pharyngeal teeth). As skin denticle development requires the presence of ectoderm-derived epithelium and of mesenchyme, it remains to be answered how odontode-forming skin epithelium, or its competence, were 'transferred' deep into the endoderm-covered oropharynx. The 'modified outside-in' hypothesis for tooth origins proposed that this transfer was accomplished through displacement of odontogenic epithelium, that is ectoderm, not only through the mouth, but also via any opening (e.g. gill slits) that connects the ectoderm to the epithelial lining of the pharynx (endoderm). This review explores from an evolutionary and from a developmental perspective whether ectoderm plays a role in (pharyngeal) tooth and denticle formation. Historic and recent studies on tooth development show that the odontogenic epithelium (enamel organ) of oral or pharyngeal teeth can be of ectodermal, endodermal, or of mixed ecto-endodermal origin. Comprehensive data are, however, only available for a few taxa. Interestingly, in these taxa, the enamel organ always develops from the basal layer of a stratified epithelium that is at least bilayered. In zebrafish, a miniaturised teleost that only retains pharyngeal teeth, an epithelial surface layer with ectoderm-like characters is required to initiate the formation of an enamel organ from the basal, endodermal epithelium. In urodele amphibians, the bilayered epithelium is endodermal, but the surface layer acquires ectodermal characters, here termed 'epidermalised endoderm'. Furthermore, ectoderm-endoderm contacts at pouch-cleft boundaries (i.e. the prospective gill slits) are important for pharyngeal tooth initiation, even if the influx of ectoderm via these routes is limited. A balance between sonic hedgehog and retinoic acid signalling could operate to assign tooth-initiating competence to the endoderm at the level of any particular pouch. In summary, three characters are identified as being required for pharyngeal tooth formation: (i) pouch-cleft contact, (ii) a stratified epithelium, of which (iii) the apical layer adopts ectodermal features. These characters delimit the area in which teeth can form, yet cannot alone explain the distribution of teeth over the different pharyngeal arches. The review concludes with a hypothetical evolutionary scenario regarding the persisting influence of ectoderm on pharyngeal tooth formation. Studies on basal osteichthyans with less-specialised types of early embryonic development will provide a crucial test for the potential role of ectoderm in pharyngeal tooth formation and for the 'modified outside-in' hypothesis of tooth origins.
