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Background: The ability to form teeth was lost in an ancestor of all modern birds, approximately 100-80 million years ago. However, experiments in chicken have revealed that the oral epithelium can respond to inductive signals from mouse mesenchyme, leading to reactivation of the odontogenic pathway. Recently, tooth germs similar to crocodile rudimentary teeth were found in a chicken mutant. These "chicken teeth" did not develop further, but the question remains whether functional teeth with enamel cap would have been obtained if the experiments had been carried out over a longer time period or if the chicken mutants had survived. The next odontogenetic step would have been tooth differentiation, involving deposition of dental proteins., Results: Using bioinformatics, we assessed the fate of the four dental proteins thought to be specific to enamel (amelogenin, AMEL; ameloblastin, AMBN; enamelin, ENAM) and to dentin (dentin sialophosphoprotein, DSPP) in the chicken genome. Conservation of gene synteny in amniotes allowed definition of target DNA regions in which we searched for sequence similarity. We found the full-length chicken AMEL and the only N-terminal region of DSPP, and both are invalidated genes. AMBN and ENAM disappeared after chromosomal rearrangements occurred in the candidate region in a bird ancestor., Conclusion: These findings not only imply that functional teeth with enamel covering, as present in ancestral Aves, will never be obtained in birds, but they also indicate that these four protein genes were dental specific, at least in the last toothed ancestor of modern birds, a specificity which has been questioned in recent years.

Genome sequencing and gene mapping have permitted the identification of HEVIN (SPARC-Like1) as the probable ancestor of the enamel matrix proteins (EMPs), amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). We have undertaken a phylogenetic analysis to elucidate their relationships. AMEL genes available in databases, and new sequences obtained in blast searching genomes or expressed sequence tags, were compiled (22 full-length sequences), aligned, and the ancestral sequence calculated and used to search for similarities using psi-blast. Hits were obtained with the N-terminal region of AMBN, ENAM, and HEVIN. We retrieved all available AMBN (n=8), ENAM (n=3), and HEVIN (n=4) sequences. The sequences of the four proteins were aligned and analyzed phylogenetically. AMEL and AMBN are sister genes, which diverged after duplication of a common ancestor issued from ENAM. The latter derived from a copy of HEVIN. Comparisons of gene organization, amino acid sequences and location of ENAM and AMBN, adjacent on the same chromosome, suggest that AMBN is closer to ENAM than AMEL. This supports AMEL as being derived from AMBN duplication. This duplication occurred long before tetrapod differentiation, probably in an ancestral osteichthyan. The story of AMEL origin is completed as follows: SPARC-->HEVIN-->ENAM-->AMBN-->AMEL.

Amelogenin plays a crucial role in enamel structure and mineralization, but the function of its various domains is far to be understood. Evolutionary analysis seems to be a promising way to approach structure/function relationships. In this paper, we review the knowledge of amelogenin with a particular focus on what we have learnt from evolution, and we bring new data on the origin and evolution of this molecule. The comparison of amniote (reptiles and mammals) amelogenin sequences reveals that, in contrast to the well-conserved C- and N-terminal domains, the central region (most of exon 6) is highly variable. The evolutionary analysis indicates that it was created by repeated insertion of three amino acids (triplets ProXGlu or ProXX). In several mammalian lineages a new run of triplet insertions and deletions has occurred independently in a locus considered a hot spot of mutation for mammalian amelogenin. In lizard and snake amelogenin evolves rapidly. Sequence alignment reveals that several residues in the N- and C-terminal regions were kept unchanged during 250 million years (MY), proving their importance for amelogenin structure and function. This alignment permits a rapid validation of the amelogenin mutations in human. Genome sequencing and gene mapping permitted to refine the amelogenin story, in relation to the common location (chromosome 4 in human) of several genes coding for dental proteins and SPARCL1, a SPARC (osteonectin) relative. Amelogenin shares a similar organisation with these genes and a blast search in databanks indicates a strong relationship between amelogenin, ameloblastin and enamelin. Taken together these data suggest that amelogenin could have originated from either ameloblastin or enamelin, themselves being created from SPARCL1, which itself originated from a SPARC duplication, 600 millions years ago.

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 to amelogenesis imperfecta when 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.

Although molecular dating of cladogenetic events is possible, no molecular method has been described to date the acquisition of various tissues. Taking into account the specificity of the major protein in enamel in formation (amelogenin), we were able to develop such a method for enamel. Indeed, because the amelogenin protein is exclusively involved in enamel formation and mineralization and because it lacks pleiotropic effects, this protein is a good candidate to estimate the date of acquisition of this highly mineralized tissue. We searched DNA banks for similarities between the amelogenin sequence and other sequences. Similarities were found only to exon 2 of SPARC (osteonectin) in two protostomians and in eight deuterostomians, and to exon 2 of three SPARC-related deuterostomian genes (SC1, hevin, and QR1). The other amelogenin exons did not reveal significant similarities to other sequences. In these proteins, exon 2 mainly encodes the peptide signal that plays the essential role in enabling the protein to be ultimately localized in the extracellular matrix. We tested the significance of the exon 2 similarities. The observed values were always significantly higher than the expected randomly generated similarities. This demonstrates a common evolutionary origin of this exon. The phylogenetic analyses of exon 2 sequences indicated that exon 2 was duplicated to amelogenin from an ancestral SPARC sequence in the deuterostomian lineage before the duplication of deuterostomian SPARC and SC1/hevin/QR1. We were able to date the origin of the latter duplication at approximately 630 MYA. Therefore, amelogenin exon 2 was acquired before this date, in the Proterozoic, long before the so-called "Cambrian explosion," the sudden appearance of several bilateralian phyla in the fossil record at the Proterozoic-Phanerozoic transition. This sudden appearance has been often suggested to reflect intensive cladogenesis during this period. However, molecular dating of protostomian-deuterostomian divergence and of the cladogenesis among several major clades of Bilateralia lead to a different conclusion: many bilateralian clades were already present during the late Proterozoic. It has previously been proposed that these bilateralians were not mineralized and that they had low fossilization potential. Our results strongly suggest that late Proterozoic fossils possessing a mineralized tissue homologous to enamel might be found in the future.

Capture of cellular mRNA by mobile elements has been an evolutionary catalyst for the spread of genes and a cause of cancer development. Here we present evidence that an orphan gene, FAM8A1 (family with sequence similarity 8), was captured by a retrovirus, followed by multiple retrotransposition events, during primate evolution between 45 and 58 million years ago. This represents the first record of cellular mRNA transduction in humans. The human gene is localized on chromosome 6p23 with five related pseudogenes (FAM8A2P-A6P), each inserted within a human endogenous retrovirus (HERV). Only the functional FAM8A1 gene is expressed and displays a ubiquitous mRNA and a testis-specific transcript present in the haploid phase of spermatogenesis. The structural features of the FAM8A1 pseudogenes include two short sequences of similarity between the FAM8A1 mRNA and the HERV sequences at both the 5' and 3' integration sites. These hallmarks suggest an alternative model to account for the capture of FAM8A1 cellular mRNA by HERV-K, involving illegitimate recombination events at the two sites of sequence similarity during reverse transcription. Unlike previous models, which assume at least one step of retroviral integration in the genome, our model is consistent with in vitro observations showing that multiple template switches occur among packaged viral transcripts. This leads to the speculation that, in some cases, cellular mRNAs may have been captured through similar processes involved in the retroviral life cycle.

Hagfishes lack mineralized tissues and teeth. Part of a cDNA strand, allegedly from amelogenin, the major gene involved in enamel formation in mammals, has recently been cloned in a hagfish (Slavkin and Diekwish, Anat. Rec., 1996;245:131-150). This cloning is of great interest because it could change the current view about the evolution of mineralized tissues, but no phylogenetic analysis of this piece of DNA has been made by the authors. Phylogenetic analysis of this part of cDNA has been conducted using both phenetic and cladistic methods. The cDNA amplified in hagfish does not fit with a nonmammalian origin but fits well with a degraded rodent sequence. The gene cloned in hagfish is probably of mammalian origin due to contamination during PCR.

Amelogenin proteins constitute the major organic contents in forming enamel, but some previously published data indicate that the amelogenin gene could be present in several species lacking teeth or functional enamel. Therefore, amelogenin could have another, still unknown, function other than contributing to enamel formation. In order to test this hypothesis, the presence of this gene has been searched for in various vertebrates. We first compared the mammalian amelogenin sequences available in the literature to obtain the best chance of a successful detection of this gene in phylogenetically distant species. Using this analysis, we have shown that the occurrence of the amelogenin gene in the Y chromosome of primates and of an artiodactyl species is probably due to two independent duplications from genes on the X chromosome. Primers for PCR have therefore been synthesized and tested in eight species, five possessing teeth (human, two phylogenetically distant lizards, and two phylogenetically distant actinopterygians) and three lacking teeth (chicken and two phylogenetically distant turtles). The amelogenin gene has been detected in all species except those lacking teeth. This result indicates that the unique role of amelogenin in amniotes is to contribute to enamel formation.