The Notch-mediated circuitry in the evolution and generation of new cell lineages: the tooth model.

The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.

Diphyodont tooth replacement of Brasilodon-A Late Triassic eucynodont that challenges the time of origin of mammals.

Two sets of teeth (diphyodonty) characterise extant mammals but not reptiles, as they generate many replacement sets (polyphyodonty). The transition in long-extinct species from many sets to only two has to date only been reported in Jurassic eucynodonts. Specimens of the Late Triassic brasilodontid eucynodont Brasilodon have provided anatomical and histological data from three lower jaws of different growth stages. These reveal ordered and timed replacement of deciduous by adult teeth. Therefore, this diphyodont dentition, as contemporary of the oldest known dinosaurs, shows that Brasilodon falls within a range of wide variations of typically mammalian, diphyodont dental patterns. Importantly, these three lower jaws represent distinct ontogenetic stages that reveal classic features for timed control of replacement, by the generation of only one replacement set of teeth. This data shows that the primary premolars reveal a temporal replacement pattern, importantly from directly below each tooth, by controlled regulation of tooth resorption and regeneration. The complexity of the adult prismatic enamel structure with a conspicuous intra-structural Schmelzmuster array suggests that, as in the case of extant mammals, this extinct species would have probably sustained higher metabolic rates than reptiles. Furthermore, in modern mammals, diphyodonty and prismatic enamel are inextricably linked, anatomically and physiologically, to a set of other traits including placentation, endothermy, fur, lactation and even parental care. Our analysis of the osteodental anatomy of Brasilodon pushes back the origin of diphyodonty and consequently, its related biological traits to the Norian (225.42 ± 0.37 myr), and around 25 myr after the End-Permian mass extinction event.

Cutting blade dentitions in squaliform sharks form by modification of inherited alternate tooth ordering patterns.

The squaliform sharks represent one of the most speciose shark clades. Many adult squaliforms have blade-like teeth, either on both jaws or restricted to the lower jaw, forming a continuous, serrated blade along the jaw margin. These teeth are replaced as a single unit and successor teeth lack the alternate arrangement present in other elasmobranchs. Micro-CT scans of embryos of squaliforms and a related outgroup (Pristiophoridae) revealed that the squaliform dentition pattern represents a highly modified version of tooth replacement seen in other clades. Teeth of Squalus embryos are arranged in an alternate pattern, with successive tooth rows containing additional teeth added proximally. Asynchronous timing of tooth production along the jaw and tooth loss prior to birth cause teeth to align in oblique sets containing teeth from subsequent rows; these become parallel to the jaw margin during ontogeny, so that adult Squalus has functional tooth rows comprising obliquely stacked teeth of consecutive developmental rows. In more strongly heterodont squaliforms, initial embryonic lower teeth develop into the oblique functional sets seen in adult Squalus, with no requirement to form, and subsequently lose, teeth arranged in an initial alternate pattern.

Early development of rostrum saw-teeth in a fossil ray tests classical theories of the evolution of vertebrate dentitions.

In classical theory, teeth of vertebrate dentitions evolved from co-option of external skin denticles into the oral cavity. This hypothesis predicts that ordered tooth arrangement and regulated replacement in the oral dentition were also derived from skin denticles. The fossil batoid ray Schizorhiza stromeri (Chondrichthyes; Cretaceous) provides a test of this theory. Schizorhiza preserves an extended cartilaginous rostrum with closely spaced, alternating saw-teeth, different from sawfish and sawsharks today. Multiple replacement teeth reveal unique new data from micro-CT scanning, showing how the 'cone-in-cone' series of ordered saw-teeth sets arrange themselves developmentally, to become enclosed by the roots of pre-existing saw-teeth. At the rostrum tip, newly developing saw-teeth are present, as mineralized crown tips within a vascular, cartilaginous furrow; these reorient via two 90° rotations then relocate laterally between previously formed roots. Saw-tooth replacement slows mid-rostrum where fewer saw-teeth are regenerated. These exceptional developmental data reveal regulated order for serial self-renewal, maintaining the saw edge with ever-increasing saw-tooth size. This mimics tooth replacement in chondrichthyans, but differs in the crown reorientation and their enclosure directly between roots of predecessor saw-teeth. Schizorhiza saw-tooth development is decoupled from the jaw teeth and their replacement, dependent on a dental lamina. This highly specialized rostral saw, derived from diversification of skin denticles, is distinct from the dentition and demonstrates the potential developmental plasticity of skin denticles.

Evolutionary origins and development of saw-teeth on the sawfish and sawshark rostrum (Elasmobranchii; Chondrichthyes).

A well-known characteristic of chondrichthyans (e.g. sharks, rays) is their covering of external skin denticles (placoid scales), but less well understood is the wide morphological diversity that these skin denticles can show. Some of the more unusual of these are the tooth-like structures associated with the elongate cartilaginous rostrum 'saw' in three chondrichthyan groups: Pristiophoridae (sawsharks; Selachii), Pristidae (sawfish; Batoidea) and the fossil Sclerorhynchoidea (Batoidea). Comparative topographic and developmental studies of the 'saw-teeth' were undertaken in adults and embryos of these groups, by means of three-dimensional-rendered volumes from X-ray computed tomography. This provided data on development and relative arrangement in embryos, with regenerative replacement in adults. Saw-teeth are morphologically similar on the rostra of the Pristiophoridae and the Sclerorhynchoidea, with the same replacement modes, despite the lack of a close phylogenetic relationship. In both, tooth-like structures develop under the skin of the embryos, aligned with the rostrum surface, before rotating into lateral position and then attaching through a pedicel to the rostrum cartilage. As well, saw-teeth are replaced and added to as space becomes available. By contrast, saw-teeth in Pristidae insert into sockets in the rostrum cartilage, growing continuously and are not replaced. Despite superficial similarity to oral tooth developmental organization, saw-tooth spatial initiation arrangement is associated with rostrum growth. Replacement is space-dependent and more comparable to that of dermal skin denticles. We suggest these saw-teeth represent modified dermal denticles and lack the 'many-for-one' replacement characteristic of elasmobranch oral dentitions.

Development and evolution of dentition pattern and tooth order in the skates and rays (batoidea; chondrichthyes).

Shark and ray (elasmobranch) dentitions are well known for their multiple generations of teeth, with isolated teeth being common in the fossil record. However, how the diverse dentitions characteristic of elasmobranchs form is still poorly understood. Data on the development and maintenance of the dental patterning in this major vertebrate group will allow comparisons to other morphologically diverse taxa, including the bony fishes, in order to identify shared pattern characters for the vertebrate dentition as a whole. Data is especially lacking from the Batoidea (skates and rays), hence our objective is to compile data on embryonic and adult batoid tooth development contributing to ordering of the dentition, from cleared and stained specimens and micro-CT scans, with 3D rendered models. We selected species (adult and embryonic) spanning phylogenetically significant batoid clades, such that our observations may raise questions about relationships within the batoids, particularly with respect to current molecular-based analyses. We include developmental data from embryos of recent model organisms Leucoraja erinacea and Raja clavata to evaluate the earliest establishment of the dentition. Characters of the batoid dentition investigated include alternate addition of teeth as offset successional tooth rows (versus single separate files), presence of a symphyseal initiator region (symphyseal tooth present, or absent, but with two parasymphyseal teeth) and a restriction to tooth addition along each jaw reducing the number of tooth families, relative to addition of successor teeth within each family. Our ultimate aim is to understand the shared characters of the batoids, and whether or not these dental characters are shared more broadly within elasmobranchs, by comparing these to dentitions in shark outgroups. These developmental morphological analyses will provide a solid basis to better understand dental evolution in these important vertebrate groups as well as the general plesiomorphic vertebrate dental condition.

Ontogenetic development of an exceptionally preserved Devonian cartilaginous skeleton.

Cartilaginous vertebrate skeletons leave few records as fossils, unless mineralized. Here, we report outstanding preservation of early stages of cartilage differentiation, present in the Devonian vertebrate Palaeospondylus gunni. In large specimens of Palaeospondylus, enlarged, hypertrophic cell spaces (lacunae) are dominant in the cartilage matrix, each defined by thin mineralized matrix, where phosphorus and calcium co-occur. This is comparable to living endochondral cartilage, where cell hypertrophy and matrix mineralization mark the end of an ontogenetic process of cell growth and division before bone formation. New information from small individuals of Palaeospondylus demonstrates that the skeleton comprises mostly unmineralized organic matrix with fewer hypertrophic cell spaces, these occurring only in the central regions of each element. Only here has the surrounding matrix begun to mineralize, differing from the larger specimens in that phosphorus is dominant with little associated calcium at these earlier stages. This reflects cellular control of mineralization in living tissues through phosphate accumulation around hypertrophic cells, with later increase in calcium in the cartilaginous matrix. These features are always associated with endochondral bone development, but in the Palaeospondylus skeleton, this bone never develops. This skeletal state is thus far unique among vertebrates, with two alternative explanations: either later stages of endochondral bone development have been lost in Palaeospondylus, or, in a stepwise acquisition of the mineralized skeleton, these late stages have not yet evolved.

Evolution of developmental pattern for vertebrate dentitions: an oro-pharyngeal specific mechanism.

Classically the oral dentition with teeth regulated into a successional iterative order was thought to have evolved from the superficial skin denticles migrating into the mouth at the stage when jaws evolved. The canonical view is that the initiation of a pattern order for teeth at the mouth margin required development of a sub-epithelial, permanent dental lamina. This provided regulated tooth production in advance of functional need, as exemplified by the Chondrichthyes. It had been assumed that teeth in the Osteichthyes form in this way as in tetrapods. However, this has been shown not to be true for many osteichthyan fish where a dental lamina of this kind does not form, but teeth are regularly patterned and replaced. We question the evolutionary origin of pattern information for the dentition driven by new morphological data on spatial initiation of skin denticles in the catshark. We review recent gene expression data for spatio-temporal order of tooth initiation for Scyliorhinus canicula, selected teleosts in both oral and pharyngeal dentitions, and Neoceratodus forsteri. Although denticles in the chondrichthyan skin appear not to follow a strict pattern order in space and time, tooth replacement in a functional system occurs with precise timing and spatial order. We suggest that the patterning mechanism observed for the oral and pharyngeal dentition is unique to the vertebrate oro-pharynx and independent of the skin system. Therefore, co-option of a successional iterative pattern occurred in evolution not from the skin but from mechanisms existing in the oro-pharynx of now extinct agnathans.

No bones about it: an enigmatic Devonian fossil reveals a new skeletal framework--a potential role of loss of gene regulation.

Palaeospondylus gunni (Devonian, Scotland) is an enigmatic vertebrate, assigned to various jawless and jawed groups since its original description. New sections through the whole body allow description of a novel skeletal tissue for Palaeospondylus, comprising the entire skeleton. This tissue is mineralized cartilage and is characterized by large cell spaces embedded in minimal matrix. Bone is completely absent. Calcium phosphate mineralization has a differential topography of deposition within the cartilage that reflects a biogenic origin, despite subsequent diagenetic modification. This combination of hypertrophied cell spaces surrounded by regionalized mineralized matrix differs from all other cartilage in fossil and extant vertebrates. However, it compares most closely to gnathostome endochondral bone in early developmental stages. For example, Palaeospondylus skeletal histology differs from the Devonian agnathan Euphanerops and extant lamprey cartilage. Comparison with mineralized cartilage of armored fossil agnathans and placoderms shows the histology is not comparable to globular calcified cartilage. It also differs from that in extant chondrichthyan mineralized tesserae, which is restricted to a subperichondral zone. Amongst this diversity of calcified cartilage types we discuss various interpretations, including one that implicates tissue either in developmental stasis, before osteoblasts can deposit bone, or at a phylogenetic stage when this step has not evolved. These very different hypotheses highlight difficulties in interpreting fossil ontogenies when phylogenetic relationships are uncertain. Nevertheless, we propose that the composition of the Palaeospondylus skeleton represents a fossilized ontogenetic stage of endochondral bone, a type of bone characteristic of osteichthyan vertebrates.

Vertebrate dentitions at the origin of jaws: when and how pattern evolved.

New evidence shows that teeth evolved with a greater degree of independence from jaws than previously considered. Pharyngeal denticles occur in jawless fish and also in early gnathostomes and precede jaw teeth in phylogeny. Many of these denticles form joined polarized sets on each branchial arch; these resemble whorl-shaped tooth sets on the jaws of stem and crown gnathostomes and are proposed as homologous units. Therefore, the source of patterning of these pharyngeal denticle and tooth sets is conserved from jawless conditions. It is proposed that developmental regulatory systems, responsible for all such tooth patterns on the jaws, are co-opted from the pharyngeal region and not from the skin as classically understood. This strongly implicates embryonic endoderm as opposed to ectoderm in the genetic control of dentition patterning. New interpretations of ontogenetic data on patterning dentitions of extant sharks are proposed, together with those of osteichthyan fish. Two entirely fossil groups, placoderms and acanthodians, at the base of gnathostome phylogeny are reassessed on the basis of a new model. It is concluded that within stem group and crown group gnathostomes several different strategies, unique to each taxon, were adopted to produce different developmental models of dentition patterning from pharyngeal denticles. One shared developmental pattern is that of initiation from primordial tooth sites, independently in each dentate zone of the jaws. The new model is proposed as a framework for data on evolutionary developmental genetics.

Separate evolutionary origins of teeth from evidence in fossil jawed vertebrates.

Placoderms are extinct jawed fishes of the class Placodermi and are basal among jawed vertebrates. It is generally thought that teeth are absent in placoderms and that the phylogenetic origin of teeth occurred after the evolution of jaws. However, we now report the presence of tooth rows in more derived placoderms, the arthrodires. New teeth are composed of gnathostome-type dentine and develop at specific locations. Hence, it appears that these placoderm teeth develop and are regulated as in other jawed vertebrates. Because tooth development occurs only in derived forms of placoderms, we suggest that teeth evolved at least twice, through a mechanism of convergent evolution.

Developmental constraints conserve evolutionary pattern in an osteichthyan dentition.

The lungfish dentition is different from other osteichthyan fish because it has a characteristic and unique pattern of teeth arranged as toothplates. Growth, addition of teeth, and retention as part of a statodont dentition are determined by the initiation pattern. In adult lungfish new teeth are only added laterally to each radial row in the dentition. This is in marked contrast to marginal rows of teeth with regular, alternating replacement in most osteichthyans. We analyze development from fossil hatchling forms of the Late Devonian dipnoan Andreyevichthys and compare with those of Neoceratodus, the Australian lungfish. The specific pattern of development, unique within lungfish, is also present in the transitory, marginal, anterior dentition in both, reflecting a strongly conserved developmental pattern. These marginal teeth form but are then lost in both, so that also this program of development is conserved within lungfish for 360 million years, from the earliest known form.