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The extinct Odontopterygiformes are the sole birds known to possess strong and sharp bony pseudoteeth, the shape and location of which are closely mimetic of real teeth. The structure of the pseudoteeth is investigated here in a late Pliocene/early Pleistocene species, Pelagornis mauretanicus, using X-ray microtomography and thin sections. The results are interpreted with regard to the pseudotooth mode of growth, and have implications concerning aspects of Pelagornis ecology. The larger pseudoteeth are hollow and approximately cone-shaped, and the smaller ones are rostro-caudally constricted. The walls of pseudoteeth are composed of bone tissue of the fibro-lamellar type, which is intensively remodeled by Haversian substitution. The jaw bones display the same structure as the pseudoteeth, but their vascular canals are oriented parallel to the long axis of the bones, whereas they are perpendicular to this direction in the pseudoteeth. There is no hiatus or evidence of a fusion between the pseudoteeth and the jaw bones. Two possible models for pseudotooth growth are derived from the histological data. The most plausible model is that pseudotooth growth began after the completion of jaw bone growth, as a simple local protraction of periosteal osteogenic activity. Pseudotooth development thus occurred relatively late during ontogeny. The highly vascularized structure and the relative abundance of parallel-fibered bone tissue in the pseudoteeth suggest poor mechanical capabilities. The pseudoteeth were most likely covered and protected by the hardened, keratinized rhamphotheca in the adult during life. The late development of the pseudoteeth would involve a similarly late and/or partial hardening of the rhamphotheca, as displayed by extant Anseriformes, Apterygiformes and some Charadriiformes. This would add support to the hypothesis of a close phylogenetic relationship between Odontopterygiformes and Anseriformes. The late maturation of the Pelagornis feeding apparatus, and hence the delayed capability for efficient prey catching, suggests that Pelagornis was altricial. [ABSTRACT FROM AUTHOR]

The article presents a study on the ovocleidin 116 (OC-116) that is found in the eggshell matrix, which is also expressed in bone cells. The researchers fertilized chicken eggs at the Institut de Selection Animale (ISA) in France, which were incubated at 37 degrees Celsius. They found that the combination of alcian blue staining and alizarin red staining allowed the detection of the first mineralized matrix at E9 in the tibia and E10 in the mandible. They also discussed the correlation between the OC-116 expression and the onset of bone mineralization.

Comparative analysis of tooth development in the main vertebrate lineages is needed to determine the various evolutionary routes leading to current dentition in living vertebrates. We have used light, scanning and transmission electron microscopy to study tooth morphology and the main stages of tooth development in the scincid lizard,Chalcides viridanus, viz., from late embryos to 6-year-old specimens of a laboratory-bred colony, and from early initiation stages to complete differentiation and attachment, including resorption and enamel formation. InC. viridanus, all teeth of a jaw have a similar morphology but tooth shape, size and orientation change during ontogeny, with a constant number of tooth positions. Tooth morphology changes from a simple smooth cone in the late embryo to the typical adult aspect of two cusps and several ridges via successive tooth replacement at every position. First-generation teeth are initiated by interaction between the oral epithelium and subjacent mesenchyme. The dental lamina of these teeth directly branches from the basal layer of the oral epithelium. On replacement-tooth initiation, the dental lamina spreads from the enamel organ of the previous tooth. The epithelial cell population, at the dental lamina extremity and near the bone support surface, proliferates and differentiates into the enamel organ, the inner (IDE) and outer dental epithelium being separated by stellate reticulum. IDE differentiates into ameloblasts, which produce enamel matrix components. In the region facing differentiating IDE, mesenchymal cells differentiate into dental papilla and give rise to odontoblasts, which first deposit a layer of predentin matrix. The first elements of the enamel matrix are then synthesised by ameloblasts. Matrix mineralisation starts in the upper region of the tooth (dentin then enamel). Enamel maturation begins once the enamel matrix layer is complete. Concomitantly, dental matrices are deposited towards the base of the dentin cone. Maturation of the enamel matrix progresses from top to base; dentin mineralisation proceeds centripetally from the dentin-enamel junction towards the pulp cavity. Tooth attachment is pleurodont and tooth replacement occurs from the lingual side from which the dentin cone of the functional teeth is resorbed. Resorption starts from a deeper region in adults than in juveniles. Our results lead us to conclude that tooth morphogenesis and differentiation in this lizard are similar to those described for mammalian teeth. However, Tomes’ processes and enamel prisms are absent. [ABSTRACT FROM AUTHOR]

AbstractSire, J.-Y. and Arnulf, I. 2000. Structure and development of the ctenial spines on the scales of a teleost fish, the cichlid Cichlasoma nigrofasciatum. — Acta Zoologica (Stockholm) 81: 139–158 Numerous teleost species possess ctenoid scales characterized by the presence of ctenial spines arranged in rows (the cteni) along their posterior, free margin. Whilst the morphology and function of the ctenial spines are similar to those of odontodes (extra-oral teeth), e.g. in armored catfish, their homology is questionable. To address this problem, we have studied ctenial spine development, structure, attachment to a bony support, and replacement with the aim of comparing these features to those described for odontodes. The ctenial spines have been studied in a growth series of the cichlid Cichlasoma nigrofasciatum, using light, scanning and transmission electron microscopy. Ctenial spines are entirely constituted of a collagen matrix. They lack a pulp cavity and, although their distal end can be in contact with the epidermal basal layer cells, they are not covered by an enameloid-like tissue. They are attached to the scale by means of a narrow strand of unmineralized collagen matrix acting as a ligament and allowing spines to be movable. The ctenial spines develop as prolongations of the external layer of the scale, a woven-fibroid collagen matrix, and subsequently grow by addition of parallel-fibred collagen matrix. New ctenial spines are added at the posterior scale border in waves that follow the same rhythm as the deposition of circuli in the anterior region. From the focus region to the scale border, the ctenial spines constitute lines in which only the most posterior ctenial spine is functional. The other spines that are no longer functional are not shed but resorbed from the top, and their attachment region mineralizes and thickens by deposition of new material. The remnants of spines constitute the main part of the superficial layer of... [ABSTRACT FROM AUTHOR]

In some living osteichthyans (e.g. the armoured catfishes) the postcranial dermal skeleton exhibits tooth-like structures (odontodes) similar to those present in the dermal skeleton of the ancient craniates. We have undertaken this work to compare odontode with tooth development, structure, attachment to a bony support and replacement. We studied the odontodes fixed on the scutes (i.e. postcranial dermal plates) in a growth series of Corydoras aeneus using light, scanning and transmission electron microscopy. Odontodes are constituted of a pulp cavity surrounded by a cone of dentine itself capped with hypermineralized substance. The pulp cavity is devoid of nerves and blood vessels and there are no odontoblastic processes in the dentine. The dentine cone is firmly attached to a circular bony protuberance of the scute surface, the pedicel or attachment bone, by means of a ligament. An odontode anlage develops as a small invagination of a dermal papilla projecting into the epidermis, the basal cell layer of which constitutes a dental epithelium. First, dentine is deposited, next the hypermineralized substance, then the ligament and attachment bone. Odontodes develop in two positions with regard to the scute surface: a primary position when new odontodes form at the posterior border of the enlarging scute; a secondary position when new odontodes replace old odontodes that have been shed during thickening of the scute. In this case, the ligament and pan of the base of the dentine cone are resorbed but not the pedicel of attachment bone, which is covered by deposition of scute matrix after the odontode has been shed. Within the scute matrix, the embedded pedicels of successive generations of odontodes are preserved, forming piles in the scutes of adult specimens. [ABSTRACT FROM AUTHOR]

The study of the formation and structure of the early teleost scale and its associated cells has been carried out on Hemichromis bimaculatus fry using in toto staining with alizarin and transmission electron microscopy techniques. Results of the study show very rapid scale formation in Hemichromis. The papilla of the scale differentiates a little in advance of the bone scale formation. No epidermal cells are involved in the constitution of the scale pocket made up of scleroblasts. In Hemichromis, as in other teleost scales, the osseous layer is the first one to be secreted by, presumably, only the scleroblasts. Then the scleroblasts specialize in their functions. Superficial ones are involved in the formation of osseous circuli; marginal scleroblasts are responsible for growth in diameter of the scale; while deep scleroblasts allow the scales to thicken owing to the progressive addition of collagen fibrils organized in a ‘plywood-like’ structure which constitutes the fibrillary plate of the scale. Mineralization occurs very rapidly within the osseous layer in the form of hydroxyapatite-like crystal deposits. The fibrillary plate is not yet mineralized in Hemichromis at the stages studied here, but presumably is later. Results obtained in Hemichromis are discussed against similar data available in the literature on teleost scale formation. [ABSTRACT FROM AUTHOR]

To develop a serum-free, chemically defined in vitro organ culture system enabling the study of epithelial-mesenchymal interactions in development and growth of fish dermal skeleton, we investigated in vitro continuation of scale regeneration in the cichlid fish Hemichromis bimaculatus. The culture medium in our system is based on Leibovitz medium (L-15) supplemented with vitamin C, additional amino acids and HEPES. With this basis medium, we examined the effects of all trans-retinoic acid, dexamethasone, and prostaglandin-E2 (PG-E2), factors known to exert an effect on development and growth of teeth and bone in mammalian culture systems, on the in vitro regeneration of scales. These effects were compared with those obtained by supplementation of the basis medium with newborn and fetal calf serum. To evaluate our culture system, the medium that allowed to mimick in the best possible way the in vivo regeneration of scales (i.e., the basis medium plus dexamethasone and PG-E2) was also tested on the in vitro development of teeth in the same fish species. Our serum-free, chemically defined organ culture system enables in vitro development and growth of both scales and teeth. With this model culture system, it is possible to evaluate the in vitro effects of hormones, growth factors, and other substances on growth and development of dermal skeleton in fish. [ABSTRACT FROM AUTHOR]

Background: The molecular bases explaining the diversity of dental tissue mineralization across gnathostomes are still poorly understood. Odontodes, such as teeth and body denticles, are serial structures that develop through deployment of a gene regulatory network shared between all gnathostomes. Dentin, the inner odontode mineralized tissue, is produced by odontoblasts and appears well-conserved through evolution. In contrast, the odontode hypermineralized external layer (enamel or enameloid) produced by ameloblasts of epithelial origin, shows extensive structural variations. As EMP (Enamel Matrix Protein) genes are as yet only found in osteichthyans where they play a major role in the mineralization of teeth and others skeletal organs, our understanding of the molecular mechanisms leading to the mineralized odontode matrices in chondrichthyans remains virtually unknown. Results: We undertook a phylogenetic analysis of the SPARC/SPARC-L gene family, from which the EMPs are supposed to have arisen, and examined the expression patterns of its members and of major fibrillar collagens in the spotted catshark Scyliorhinus canicula, the thornback ray Raja clavata, and the clawed frog Xenopus tropicalis. Our phylogenetic analyses reveal that the single chondrichthyan SPARC-L gene is co-orthologous to the osteichthyan SPARC-L1 and SPARC-L2 paralogues. In all three species, odontoblasts co-express SPARC and collagens. In contrast, ameloblasts do not strongly express collagen genes but exhibit strikingly similar SPARC-L and EMP expression patterns at their maturation stage, in the examined chondrichthyan and osteichthyan species, respectively. Conclusions: A well-conserved odontoblastic collagen/SPARC module across gnathostomes further confirms dentin homology. Members of the SPARC-L clade evolved faster than their SPARC paralogues, both in terms of protein sequence and gene duplication. We uncover an osteichthyan-specific duplication that produced SPARC-L1 (subsequently lost in pipidae frogs) and SPARC-L2 (independently lost in teleosts and tetrapods).Our results suggest the ameloblastic expression of the single chondrichthyan SPARC-L gene at the maturation stage reflects the ancestral gnathostome situation, and provide new evidence in favor of the homology of enamel and enameloids in all gnathostomes. [ABSTRACT FROM AUTHOR]

Modern birds (crown group birds, called Neornithes) are toothless; however, the extinct neornithine Odontopterygiformes possessed bone excrescences (pseudoteeth) which resembled teeth, distributed sequentially by size along jaws. The origin of pseudoteeth is enigmatic, but based on recent evidence, including microanatomical and histological analyses, we propose that conserved odontogenetic pathways most probably regulated the development of pseudodentition. The delayed pseudoteeth growth and epithelium keratinization allowed for the existence of a temporal window during which competent osteoblasts could respond to oral epithelial signaling, in place of the no longer present odontoblasts; thus, bony pseudoteeth developed instead of true teeth. Dynamic morphogenetic fields can explain the particular, sequential size distribution of pseudoteeth along the jaws of these birds. Hence, this appears as a new kind of deep homology, by which ancient odontogenetic developmental processes would have controlled the evolution of pseudodentition, structurally different from a true dentition, but morphologically and functionally similar. [ABSTRACT FROM AUTHOR]

In mammals, the matrix extracellular phosphoglycoprotein (MEPE) is known to activate osteogenesis and mineralization via a particular region called dentonin, and to inhibit mineralization via its ASARM (acidic serine-aspartate rich MEPE-associated motif) peptide that also plays a role in phosphatemia regulation. In order to understand MEPE evolution in mammals, and particularly that of its functional regions, we conducted an evolutionary analysis based on the study of selective pressures. Using 37 mammalian sequences we: (1) confirmed the presence of an additional coding exon in most placentals; (2) highlighted several conserved residues and regions that could have important functions; (3) found that dentonin function was recruited in a placental ancestor; and (4) revealed that ASARM function was present earlier, pushing the recruitment of MEPE deep into amniote origins. Our data indicate that MEPE was involved in various functions (bone and eggshell mineralization) prior to acquiring those currently known in placental mammals. [ABSTRACT FROM AUTHOR]

An evolutionary analysis of mammalian amelogenin, the major protein of forming enamel, was conducted by comparison of 26 sequences (including 14 new ones) representative of the main mammalian lineages. Amelogenin shows highly conserved residues in the hydrophilic N- and C-terminal regions. The central hydrophobic region (most of exon 6) is more variable, but it has conserved a high amount of proline and glutamine located in triplets, PXQ, indicating that these residues play an important role. This region evolves more rapidly, and is less constrained, than the other well-conserved regions, which are subjected to strong constraints. The comparison of the substitution rates in relation to the CpG richness confirmed that the highly conserved regions are subjected to strong selective pressures. The amino acids located at important sites and the residues known to lead toamelogenesis imperfectawhen substituted were present in all sequences examined. Evolutionary analysis of the variable region of exon 6 points to a particular zone, rich in either amino acid insertion or deletion. We consider this region a hot spot of mutation for the mammalian amelogenin. In this region, numerous triplet repeats (PXQ) have been inserted recently and independently in five lineages, while most of the hydrophobic exon 6 region probably had its origin in several rounds of triplet insertions, early in vertebrate evolution. The putative ancestral DNA sequence of the mammalian amelogenin was calculated using a maximum likelihood approach. The putative ancestral protein was composed of 177 residues. It already contained all important amino acid positions known to date, its hydrophobic variable region was rich in proline and glutamine, and it contained triplet repeats PXQ as in the modern sequences. [ABSTRACT FROM AUTHOR]

Evolution involves interplay between natural selection and developmental constraints. This is seen, for example, when digits are lost from the limbs during evolution. Extant archosaurs (crocodiles and birds) show several instances of digit loss under different selective regimes, and show limbs with one, two, three, four or the ancestral number of five digits. The 'lost' digits sometimes persist for millions of years as developmental vestiges. Here we examine digit loss in the Nile crocodile and five birds, using markers of three successive stages of digit development. In two independent lineages under different selection, wing digit I and all its markers disappear. In contrast, hindlimb digit V persists in all species sampled, both as cartilage, and as Sox9- expressing precartilage domains, 250 million years after the adult digit disappeared. There is therefore a mismatch between evolution of the embryonic and adult phenotypes. All limbs, regardless of digit number, showed similar expression of sonic hedgehog (Shh). Even in the one-fingered emu wing, expression of posterior genes Hoxd11 and Hoxd12 was conserved, whereas expression of anterior genes Gli3 and Alx4 was not. We suggest that the persistence of digit V in the embryo may reflect constraints, particularly the conserved posterior gene networks associated with the zone of polarizing activity (ZPA). The more rapid and complete disappearance of digit I may reflect its ZPA-independent specification, and hence, weaker developmental constraints. Interacting with these constraints are selection pressures for limb functions such as flying and perching. This model may help to explain the diverse patterns of digit loss in tetrapods. Our study may also help to understand how selection on adults leads to changes in development. [ABSTRACT FROM AUTHOR]

In humans and mice, the early development of αβ T cells is controlled by the pre-T-cell receptor α chain (ptα) that is covalently associated with the T-cell receptor β (TCRβ) chain to form the pre-T-cell receptor (pre-TCR) at the thymocyte surface. Pre-TCR functions in a ligandindependent manner through self-oligomerization mediated by pTα. Using in sillco and gene synteny-based approaches, we identified the pTα gene (PTCRA) in four sauropsid (three birds and one reptile) genomes. We also identified 25 mammalian PTCRA sequences now covering all mammalian lineages. Gene synteny around PTCRA is re- markably conserved in mammals but differences upstream of PTCRA in sauropsids suggest chromosomal rearrangements. PTCRA organization is highly similar in sauropsids and mammals. However, comparative analyses of the pTα functional domains indicate that sauropsids, monotremes, marsupials, and lagomorphs display a short pTα cytoplasmic tail and lack most residues shown to be critical for human and murine pre-TCR self-oligomerization. Chicken PTCRA transcripts similar to those in mammals were detected in immature double-negative and double-positive thymocytes. These findings give clues about the evolution of this key molecule in amniotes and suggest that the ancestral function of pTα was exclusively to enable expression of the TCRβ chain at the thymocyte surface and to allow binding of pre-TCR to the CD3 complex. Together, our data provide arguments for revisiting the current model of pTα signaling. [ABSTRACT FROM AUTHOR]

Abstract: Abnormal compressions and fusions of vertebral bodies are frequently observed in reared rainbow trout and could result from chronic and unbearable muscle pressures acting on the axial skeleton during intensive growth. Sustained swimming at moderate speeds was shown to induce many positive effects on growth and swimming performances in salmonids, but yet little is known about its effects on vertebral remodeling processes and related hormonal regulation. Rainbow trout were subjected to three different swimming speeds (0, 1.0 and 1.5body length (BL) s−1), starting one month after they were first fed (65mm) and ending when they reached 260mm in size (market-size of 275g). At the end of the experiment, 20 trout were sampled in each lot (N =60) and blood samples were taken. Vertebrae abnormalities were assessed by radiological examinations. Vertebrae from the middle axial region (V32–38) were selected to evaluate bone mineralization (BM) and total bone area (Tt-B.Ar.) on radiographed transverse sections (125±10μm). Assays were performed to evaluate mineral homeostasis (calcemia and phosphatemia), bone cell activities (alkaline phosphatase, ALP, and tartrate-resistant acid phosphatase, TRAP) and bone regulating hormones (calcitonin, CT and thyroid hormones, THs). Sustained exercise reduced the appearance of fused vertebrae, enhanced vertebral BM and decreased vertebral Tt-B.Ar., while it increased circulating CT and TH levels. No variations were observed on mineral homeostasis and bone cell activities. Increasing the swimming speed up to 1.5BL s−1 had positive effects on the vertebral skeleton, and therefore, seems to be a suitable approach to prevent aggravation of vertebral abnormalities in juvenile trout. The changes observed in vertebral features are interpreted as a compromise between the necessity to mobilize vertebral mineral ions in response to various physiological demands and to maintain vertebral strength against mechanical constraints. [Copyright &y& Elsevier]

Bone collagenous extracellular matrix provides a confined environment into which apatite crystals form. This biomineralization process is related to a cascade of events partly controlled by noncollagenous proteins. Although overlooked in bone models, concentration and physical environment influence their activities. Here, we show that collagen suprafibrillar confinement in bone comprising intra- and interfibrillar spaces drives the activity of biomimetic acidic calcium-binding polymers on apatite mineralization. The difference in mineralization between an entrapping dentin matrix protein-1 (DMP1) recombinant peptide (rpDMP1) and the synthetic polyaspartate validates the specificity of the 57-KD fragment of DMP1 in the regulation of mineralization, but strikingly without phosphorylation. We show that all the identified functions of rpDMP1 are dedicated to preclude pathological mineralization. Interestingly, transient apatite phases are only found using a high nonphysiological concentration of additives. The possibility to combine biomimetic concentration of both collagen and additives ensures specific chemical interactions and offers perspectives for understanding the role of bone components in mineralization.

The study of vertebrate ontogenies has the potential to inform us of shared developmental patterns and processes among organisms. However, fossilised ontogenies of early vertebrates are extremely rare during the Palaeozoic Era. A growth series of the Late Devonian "acanthodian" Triazeugacanthus affinis , from the Miguasha Fossil - Fish Lagerstätte , is identified as one of the best known early vertebrate fossilised ontogenies given the exceptional preservation, the large size range, and the abundance of specimens. Morphological, morphometric, histological and chemical data are gathered on a growth series of Triazeugacanthus ranging from 4 to 52 mm in total length. The developmental trajectory of this Devonian "acanthodian" is characteristic of fishes showing a direct development with alternating steps and thresholds. Larvae show no squamation but a progressive appearance of cartilaginous neurocranial and vertebral elements, and appendicular elements, whereas juveniles progress in terms of ossification and squamation. The presence of cartilaginous and bony tissues, discriminated on histological and chemical signatures, shows a progressive mineralisation of neurocranial and vertebral elements. Comparison among different body proportions for larvae, juveniles and adults suggest allometric growth in juveniles. Because of the phylogenetic position of "acanthodians", Triazeugacanthus ontogeny informs us about deep time developmental conditions in gnathostomes., Competing Interests: The authors declare there are no competing interests.

Phylogenomics is extremely powerful but introduces new challenges as no agreement exists on "standards" for data selection, curation and tree inference. We use jawed vertebrates (Gnathostomata) as model to address these issues. Despite considerable efforts in resolving their evolutionary history and macroevolution, few studies have included a full phylogenetic diversity of gnathostomes and some relationships remain controversial. We tested a novel bioinformatic pipeline to assemble large and accurate phylogenomic datasets from RNA sequencing and find this phylotranscriptomic approach successful and highly cost-effective. Increased sequencing effort up to ca. 10Gbp allows recovering more genes, but shallower sequencing (1.5Gbp) is sufficient to obtain thousands of full-length orthologous transcripts. We reconstruct a robust and strongly supported timetree of jawed vertebrates using 7,189 nuclear genes from 100 taxa, including 23 new transcriptomes from previously unsampled key species. Gene jackknifing of genomic data corroborates the robustness of our tree and allows calculating genome-wide divergence times by overcoming gene sampling bias. Mitochondrial genomes prove insufficient to resolve the deepest relationships because of limited signal and among-lineage rate heterogeneity. Our analyses emphasize the importance of large curated nuclear datasets to increase the accuracy of phylogenomics and provide a reference framework for the evolutionary history of jawed vertebrates., Competing Interests: Competing financial interests The authors declare no competing financial interests.

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene ( MMP20 ) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.

In order to improve our knowledge on the vertebral development, structure and mineralization in Acipenseriformes, we undertook a study in a growth series of reared Siberian sturgeons (Acipenser baerii) using in toto clear and stain specimens, histological and ultrastructural observations, X-ray micro-tomography, and solid state NMR analyses. Scutes were also studied to compare the tissue structure and mineralization of endoskeletal and dermal skeletal elements. This study completes and clarifies previous investigations on vertebral development and architecture in sturgeons, and brings original data on the structure of (i) the perichondral bone that is progressively deposited around the vertebral elements during ontogeny, (ii) the typical cartilage composing these elements, and (iii) the scutes. In addition we provide data on the mineralization process, on the nature of the bone mineral phase, and on the growth dynamics of the vertebral elements. Anat Rec, 300:437-449, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Well studied in mammals, amelogenesis is less known at the molecular level in reptiles and amphibians. In the course of extensive studies of enamel matrix protein (EMP) evolution in tetrapods, we look for correlation between changes in protein sequences and temporospatial protein gene expression during amelogenesis, using an evo-devo approach. Our target is the major EMP, amelogenin (AMEL) that plays a crucial role in enamel structure. We focused here our attention to an amphibian, the salamander Pleurodeles waltl. RNAs were extracted from the lower jaws of a juvenile P. waltl and the complete AMEL sequence was obtained using PCR and RACE PCR. The alignment of P. waltl AMEL with other tetrapodan (frogs, reptiles and mammals) sequences revealed residue conservation in the N- and C-terminal regions, and a highly variable central region. Using sense and anti-sense probes synthetized from the P. waltl AMEL sequence, we performed in situ hybridization on sections during amelogenesis in larvae, juveniles, and adults. We demonstrated that (i) AMEL expression was always found to be restricted to ameloblasts, (ii) the expression pattern was conserved through ontogeny, even in larvae where enameloid is present in addition to enamel, and (iii) the processes are similar to those described in lizards and mammals. These findings indicate that high variations in the central region of AMEL have not modified its temporospatial expression during amelogenesis for 360 million years of tetrapod evolution., (Copyright © 2013 Wiley Periodicals, Inc.)

Dentin matrix acidic phosphoprotein 1 (DMP1) is an acidic, highly phosphorylated, noncollagenous protein secreted during dentin and bone formation. Previous functional studies of DMP1 have revealed various motifs playing a role in either mineralization or cell differentiation. We performed an evolutionary analysis of DMP1 to identify residues and motifs that were conserved during 220 millions years (Ma) of mammalian evolution, and hence have an important function. In silico search provided us with 41 sequences that were aligned and analyzed using the Hyphy program. We showed that DMP1 contains 55 positions that were kept unchanged for 220 Ma. We also defined in a more precise manner some motifs that were already known (i.e., cleavage sites, RGD motif, ASARM peptide, glycosaminoglycan chain attachment site, nuclear localization signal sites, and dentin sialophosphoprotein-binding site), and we found five, highly conserved, new functional motifs. In the near future, functional studies could be performed to understand the role played by them.

Prenatal development in crocodilians represents a very interesting model for comparative studies. As the speed of prenatal development of crocodilians varies depending on incubation conditions, the staging of embryos and fetuses is a very important prerequisite for data correlation. To establish a background for future developmental studies on Crocodylus niloticus, we characterized its prenatal development in a collection comprising 169 animals during embryonic/incubation days 9-70. The characteristics included external morphology, head morphometry, and wet body weight determined before fixation. We documented the external morphology of prenatal Nile crocodiles in a large collection of photographs and described landmarks during the morphogenesis of the head, face and limbs. In the development of the facial processes (medial nasal, lateral nasal, maxillary), three phases could be distinguished: union, separation, reunion. At the free jaw margin, a regular series of prominences was present. The outer aspect of a prominence gave rise to a labial scale, the inner aspect to a tooth. In contrast to mammals (humans and mice), the hindlimbs of C. niloticus developed faster than the forelimbs. We also determined changes in basic measures of the head and of the wet body weight. Both morphological and morphometric characteristics showed an apparent inter-individual variability among animals of the same age. This variability decreased among animals of a similar body weight (irrespective of their age). Body weight can be considered as the most representative and complex parameter for crocodile staging reflecting the overall growth of a whole embryo/fetus.

Enamelin (ENAM) plays an important role in the mineralization of the forming enamel matrix. We have performed an evolutionary analysis of mammalian ENAM to identify highly conserved residues or regions that could have important function (selective pressure), to predict mutations that could be associated with amelogenesis imperfecta in humans, and to identify possible adaptive evolution of ENAM during 200 million years ago of mammalian evolution. In order to fulfil these objectives, we obtained 36-ENAM sequences that are representative of the mammalian lineages. Our results show a remarkably high conservation pattern in the region of the 32-kDa fragment of ENAM, especially its phosphorylation, glycosylation, and proteolytic sites. In primates and rodents we also identified several sites under positive selection, which could indicate recent evolutionary changes in ENAM function. Furthermore, the analysis of the unusual signal peptide provided new insights on the possible regulation of ENAM secretion, a hypothesis that should be tested in the near future. Taken together, these findings improve our understanding of ENAM evolution and provide new information that would be useful for further investigation of ENAM function as well as for the validation of mutations leading to amelogenesis imperfecta.

Most nonmammalian species replace their teeth continuously (so-called polyphyodonty), which allows morphological and structural modifications to occur during ontogeny. We have chosen Pleurodeles waltl, a salamander easy to rear in the laboratory, as a model species to establish the morphological foundations necessary for future molecular approaches aiming to understand not only molecular processes involved in tooth development and replacement, but also their changes, notably during metamorphosis, that might usefully inform studies of modifications of tooth morphology during evolution. In order to determine when (in which developmental stage) and how (progressively or suddenly) tooth modifications take place during ontogeny, we concentrated our observations on a single tooth family, located at position I, closest to the symphysis on the left lower jaw. We monitored the development and replacement of the six first teeth in a large growth series ranging from 10-day-old embryos (tooth I1) to adult specimens (tooth I6), using light (LM), scanning (SEM), and transmission electron (TEM) microscopy. A timetable of the developmental and functional period is provided for the six teeth, and tooth development is compared in larvae and young adults. In P. waltl the first functional tooth is not replaced when the second generation tooth forms, in contrast to what occurs for the later generation teeth, leading to the presence of two functional teeth in a single position during the first 2 months of life. Larval tooth I1 shows dramatically different features when compared to adult tooth I6: a dividing zone has appeared between the dentin cone and the pedicel; the pulp cavity has enlarged, allowing accommodation of large blood vessels; the odontoblasts are well organized along the dentin surface; tubules have appeared in the dentin; and teeth have become bicuspidate. Most of these modifications take place progressively from one tooth generation to the next, but the change from monocuspid to bicuspid tooth occurs during the tooth I3 to tooth I4 transition at metamorphosis., ((c) 2006 Wiley-Liss, Inc.)

Based on light and transmission electron-microscopic observations on erupting first-generation teeth in the zebrafish, Danio rerio, we propose a biphasic mechanism for tooth eruption: (1). formation of an epithelial crypt prior to eruption of the tooth, possibly as a result of constraints in the epithelium resulting from the growth of adjacent tooth germs, and (2). detachment of cellular interdigitations both within the pharyngeal epithelium, at the pharyngeal epithelium/enamel organ boundary, and between the outer and inner dental epithelium, resulting in the exposure of the tooth tip in the crypt, immediately after tooth ankylosis. Later, further detachment of interdigitations between the inner and the outer enamel epithelium unfolds the epithelium even more and leads to a more pronounced exposure of the tooth tip. The presence of small patches of non-collagenous matrix on the outer surface of the tooth close to where it merges with the attachment bone is interpreted as a device to prevent complete detachment of the enamel organ. The biphasic nature of the mechanism for tooth eruption is supported by observations on in vitro cultured heads. First-generation teeth develop normally and crypts are formed, as under in vivo conditions, but the teeth fail to erupt. Taken together, our observations suggest that epithelial remodelling plays a crucial role in eruption of the teeth in this model organism.