Enamel Enameloid and the Origin of EMPs

Morphological studies of enamel and enameloid in living taxa have shown that they are different in their mode of formation. The enamel organic matrix is secreted by the ameloblasts, and contains enamel-specific proteins. In contrast, enameloid organic matrix is mostly deposited by odontoblasts and contains a large amount of collagen, but the ameloblasts contribute to its formation, too [Prostak and Skobe, 1984; Sasagawa, 1984; Prostak and Skobe, 1988; Prostak et al., 1993; Sasagawa, 1995, 2002]. However, in functional teeth, the structure of both tissues is similar, i.e. highly mineralized with only a little organic matrix left (<5%). Given the same location, the same final structure and the same evolutionary origin, most authors have considered enamel and enameloid as homologous tissues. Enamel and enameloid matrices are only partially mineralized when laid down, and their final hardness is acquired during a second stage, maturation, during which the matrix is lost through the activity of proteolytic enzymes. This process creates space, allowing mineral crystal growth to eventually achieve a highly mineralized structure. Because they are highly mineralized, enamel and enameloid are easily recognizable in the fossil record and their relationships can be traced back deep in vertebrate evolution.

The question of which tissue appeared first, enamel or enameloid, has been long debated and it is not clearly answered yet. It is, however, accepted that enamel progressively replaced enameloid during evolution in various lineages (e.g. in tetrapods) [Smith, 1995; Donoghue, 2002; Donoghue and Sansom, 2002; Donoghue et al., 2006]. Odontoblasts progressively reduced their production of loose collagenous matrix, which characterizes forming enameloid, while ameloblast activity increased with the secretion of large amounts of enamel-specific products at the dentin surface. This evolutionary 'transition' between enameloid and enamel was, in fact, probably an enamel-oid-dentin transition, as recently demonstrated in the ontogeny of caudate amphibians [Davit-Beal et al., 2007]. However, enamel did not replace enameloid in all vertebrate lineages. A particular type of enameloid is present in chondrichthyans (cartilaginous fish [Prostak et al., 1993; Sasagawa, 2002]), and this supports an ancient origin for this tissue, at least for the gnathostome lineage. Enamel and enameloid were certainly present in basal actinopterygians (ray-finned fish), as in polypterids and lepisosteids [Sire et al., 1987; Sire, 1990, 1994, 1995]. This supports the idea that enamel was already present in early osteichthyans, which also indicates an ancient origin.

Fig. 8. Chordate relationships and the origin of the mineralized skeletal elements in vertebrates (adapted from Shimeld and Holland [2000]). Chordates are deeply anchored in the Precambrian era (>700 MYA). The acquisition of a mineralized skeleton, a major event for vertebrate radiation, occurred 600-500 MYA, a period which post-dates the two genome duplications [Gu et al., 2002]. Bone and dental tissues are clearly recognized in early, jawless vertebrates, 450 MYA. Skeletal diversification in jawed vertebrates was next favored by the appearance of new genes after tandem duplication.

Fig. 8. Chordate relationships and the origin of the mineralized skeletal elements in vertebrates (adapted from Shimeld and Holland [2000]). Chordates are deeply anchored in the Precambrian era (>700 MYA). The acquisition of a mineralized skeleton, a major event for vertebrate radiation, occurred 600-500 MYA, a period which post-dates the two genome duplications [Gu et al., 2002]. Bone and dental tissues are clearly recognized in early, jawless vertebrates, 450 MYA. Skeletal diversification in jawed vertebrates was next favored by the appearance of new genes after tandem duplication.

Enamel is absent in more derived actinopterygian taxa (teleost fish), which possess enameloid only [Sasagawa, 1984; Prostak and Skobe, 1984; Sasagawa, 1995]. The large evolutionary distance between all living representatives of these chondrichthyan and actinopterygian lineages (430-420 MYA, respectively, in the fossil record: Janvier [1996]) explains why the current structure of these enameloids is so different.

Enamel and enameloid appear, therefore, to be merely grades of a hypermineralized tissue that has evolved independently in a number of vertebrate lineages [Dono-ghue, 2001]. The origin of these tissues can be traced back in early vertebrates, along with the appearance of a bony mineralized skeleton, one of the four main vertebrate character acquisitions, together with neural crest cells and their derivatives, neurogenic placodes, and an elaborate segmented brain (fig. 8). These vertebrate innovations appeared after the divergence between tunicates7 (Ciona) and craniates8 (recent genetic evidence indicates that tunicates could be closer to vertebrates than cepha-lochordates [Graham, 2004]), and probably after the divergence between craniates and vertebrates as witnessed by the fossil record. The absence of mineralized tissues in living hagfish and lampreys is probably primitive [Jan-

7 Tunicates: subphylum of chordates that feed by siphoning plankton through a filter.

8 Craniates: animals with skull.

Fig. 9. Enamel/enameloid tissues during vertebrate evolution (as reported in the fossil record), and current knowledge of the presence of EMP and SCPP genes in vertebrate lineages. Enamel-like tissues are identified in early vertebrates, the eu-conodonts, and they display a different evolutionary history in the various lineages. Enameloid was conserved in chon-drichthyan and actinopterygian lineages, but disappeared in amniotes. The early presence of enamel/enameloid tissues in vertebrate evolution strongly suggests that EMP divergence predates this time (>500 MYA). However, there is a large gap between this theoretical EMP presence in early vertebrate lineages and the current knowledge of the genes coding for these proteins, which is restricted to the tetra-pod level (350 MYA). SCPPs are known, however, from actinopterygian fish.

Jawless vertebrates


Jawed vertebrates

Actinopterygian fish




Triassic 250

Permian 300 ■ Carboniferous 350 -I

Devonian 400 j

Silurian 450

Ordovician 500

Cambrian 550

Million years





Crocodiles Birds


Enameloid, enamel

Enameloid, enamel

Conodont apparatus: Enamel-like tissue

Teeth: Enameloid, enamel

Dermal skeleton:

Enameloid, enamel

Conodont apparatus: Enamel-like tissue


vier, 1996]. Indeed, the most ancient vertebrates discovered in the Lower Cambrian of China (530 MYA), Haik-ouichthys (which looks like a hagfish) and Myllokun-mingia (which looks like a lamprey), possessed a skeleton composed of unmineralized cartilage only [Shu et al., 1999, 2003].

The first mineralized elements encountered in vertebrates are the tooth-like organs (conodont apparatus) composed of enamel-like and dentine tissue found in eu-conodonts, fossil marine vertebrates known from the Middle Cambrian (500 MYA) to the Late Triassic (230 MYA) [Sansom et al., 1992, 1994; Janvier, 1996; Dono-ghue, 1998, 2001] (fig. 9) . These minute comb-shaped denticles are located at the entrance of the pharynx (vis-cerocranium). Bone appears to be absent from these elements [Donoghue, 1998].

Enamel, or enameloid, is clearly identified in the skeleton of early jawless vertebrates (e.g. pteraspidomorphs, heterostracans, thelodonts, and 'ostracoderms') from the Early Ordovician (480 MYA) to Late Devonian (380 MYA) periods and of jawed vertebrates (early chondrichthyans and osteichthyans) [Janvier, 1996; Donoghue et al., 2006] (fig. 8). The earliest skeleton was a dermal skeleton comprising odontodes (tooth-like elements consisting of enameloid and dentine), ornamenting dermal plates composed of acellular bone [Sansom et al., 2005]. It is noteworthy that our current knowledge of early vertebrates reveals a gap of 30 million years between the appearance of the first vertebrates (530 MYA) and the first evidence of vertebrate mineralized elements (500 MYA).

It is clear that numerous gene families expanded by gene duplication in the vertebrate stem lineage (in particular gene families encoding transcription factors and signaling molecules) [Shimeld and Holland, 2000]. The acquisition of the mineralized skeleton followed the increased genetic complexity (two genome duplications and several gene duplications) which occurred early in vertebrate evolution (during the Precambrian and Cambrian periods) [Dehal and Boore, 2005; Panopoulou and Pouska, 2005]. These large scale genomic events facilitated the evolutionary success of the vertebrate lineage and, probably, led to the diversification of several members of the SCPP family. Additional tandem duplications certainly occurred during the long period of vertebrate evolution and resulted in new gene differentiation and in a further diversification of SCPPs into new biological functions (fig. 8).

The presence of enamel and enameloid tissues in early vertebrates strongly suggests that EMPs (and some other SCPPs) were present in these tissues at least 500 MYA

(fig. 9). This would mean that SCPPs diversified earlier. The hypothetical date of this diversification could be not so distant from the molecular dating of EMP origins (>600 MYA) if we consider that the duplication could have occurred long before the divergence of function/expression of the copies, and that vertebrates possessing a mineralized skeleton could have lived dozens of millions of years before any evidence of them in the fossil record. However, although structurally well-identified enameloid and enamel tissues are present in the teeth of chondrich-thyans, actinopterygians, and basal sarcopterygians, EMP genes are known in tetrapods only (fig. 9). However, this statement relates to genes only; there is evidence from immunohistochemical studies or Southern hybridization that AMEL and/or ENAM proteins could be present in sharks [Slavkin et al., 1983; Herold et al., 1989], teleost fish [Lyngstadaas et al., 1990], polypterids [Zylberberg et al., 1997] and lungfish [Satchell et al., 2000].

Whilst the data on EMP genes (mainly in model mammals) slowly accumulated over a period of approximately 15 years, the last years witnessed a rapid increase in our knowledge, mainly because of genome sequencing in numerous species, and in particular in mammals. To date eight well-covered mammalian genomes are available and seven additional genomes are provided at a low coverage level (see http:/www.ensembl.org/). The current mammalian genome project aims to add 11 mammalian species to this list in a phylogenetic perspective (http:/ www.broad.mit.edu/mammals). Therefore, within the next few months, we will have access to at least 26 mammalian genomes and, potentially, will be able to perform evolutionary analyses of any gene in the mammalian lineage. Opposite to this large covering of mammalian phy-logeny, our knowledge of non-mammalian EMPs is, unfortunately, much less advanced (fig. 10). We can see two reasons: (1) the lack of sequenced genomes and (2) the divergence of EMP sequences.

The Lack of Sequenced Genomes

In toothed reptiles (crocodiles, snakes, and lizards), there is still no sequenced genome available, although the reptilian (sauropsid) lineage is the lineage closest to mammals (fig. 10). However, AMEL sequences are available in a crocodile [Toyosawa et al., 1998], in a snake [Ishiyama et al., 1998], and in two lizards [Delgado et al., 2006; Wang et al., 2006], and AMBN has been sequenced in a crocodile [Shintani et al., 2002]. At present, there are no data on reptilian ENAM but, fortunately, we will soon have access to a lizard genome (Anolis carolinensis genome is being sequenced). However, sequencing a croco dile genome (a representative of the lineage closest to birds) would be also extremely interesting for evolutionary analyses.

In amphibians, AMEL [Toyosawa et al., 1998] and AMBN [Shintani et al., 2003] have been sequenced in the pipid frog Xenopus laevis, and an AMEL sequence is available in another frog (Rana pipiens [Wang et al., 2005a]). Moreover, sequencing of a pipid genome (Silurana tropicalis) is well advanced (fig. 10). Surprisingly, although as expected AMEL and AMBN are present in this genome, to our knowledge ENAM has not been found yet [Kawasaki and Weiss, 2006]. It is questionable whether this EMP is really absent from this frog genome. Indeed, on the one hand our evolutionary analysis indicates that ENAM is the oldest representative of the EMP family and, on the other hand, ENAM plays important roles in enamel structure and organization as illustrated by AIH2 resulting from ENAM mutations. It is also clear that pip-ids have a well-formed enamel [Sato et al., 1986]. Therefore, this 'lack' is probably related to the fact that the pipid genome is still not entirely (or correctly) assembled. One should also take into consideration that pipids are highly derived anurans and, as a consequence, EMPs could be divergent compared to more basal amphibian species. Sequencing another frog, salamander/newt or caecilian genome would be, therefore, highly informative for evolutionary analysis.

No EMP is known in basal sarcopterygians, i.e. lung-fish and coelacanth, nor in basal actinopterygians (polyp -terids and lepisosteids), and there is no sequenced genome available nor sequencing project running. However, these taxa possess enamel and they belong to lineages that are crucial to improve our understanding of EMP relationships and evolution. In contrast to this lack of data, the genome has been sequenced in four teleost species, and several SCPPs were identified. However, teleosts are derived actinopterygian lineages, and the long evolutionary distance (>420 million years) between actinop-terygians and tetrapods explains the difficulty encountered when trying to identify homology between teleost and tetrapod SCPP genes [Kawasaki et al., 2005]. For instance, no EMP gene can be related to these SCPPs.

No SCPP is known in chondrichthyans (sharks and rays). Here too, the long evolutionary distance (>430 million years) between cartilaginous fish and tetrapods could lead to problems when trying to identify homologous genes, but the syntheny conservation of SCPP genes could help [Kawasaki et al., 2005; Kawasaki and Weiss, 2006].

Fig. 10. Current knowledge of EMP genes in vertebrates. To date only two EMPs are characterized at the tetrapod level (AMBN and AMEL). ENAM is only known in mammals. The lack of data in non-mammalian lineages is clearly related to the absence of sequenced genomes. SCPP genes are identified in teleost fish, but the large evolutionary distance makes their relationships to EMPs uncertain. EMP genes on gray background are potentially accessible to sequencing. Question marks indicate lineages in which sequencing of EMP genes might be a priority to improve our understanding on their origin and evolution. * = Large DNA regions (Whole Genome Shotgun) have been sequenced in a lizard (A. carolinensis).

310 Milliun years

360 Million years

420 Million years

430 Million years

Numerous sequenced genomes


I— Crocodiles



Tetrapoda snakes

AMEL, AMBN. ENAM? No sequenced genomes

AMEL. AMBN, ENAM? No sequenced genomes4


_ Salamanders, newts, caecilians -jAmphibia

Actinopterygii n


. Frogs

■ Coelacanth Polypterids [.episosteids

Fugu, Tetraodon, Stickleback



No sequenced genomes

We 11-advanced genome sequencing (Xenopus) No sequenced genomes

No sequenced genome

Sequenced genome Sequenced genome Sequenced genomes No sequenced genomes1

The Divergence of EMP Sequences The difficulty to find EMP (and other SCPP) genes using PCR or RT-PCR resides in their variability. Indeed, except for the short N-terminal region that is relatively well conserved in each member of the family, the largest part of the sequence is variable. For instance, although they probably conserve their main function, most of the mammalian AMEL exon 6 sequences (the largest part of AMEL) cannot be accurately aligned with the homologous region in reptiles and amphibians due to numerous substitutions and indels [Sire et al., 2006]. These highly variable sequences indicate that SCPPs are intrinsically disordered proteins [Dunker et al., 2001; Kawasaki et al., 2005] and there are only a few conserved residues. Therefore, the only means to find EMPs in evolutionary distant species, such as basal sarcopterygians or actinopteryg-ians, is to study sequenced genomes or sequences of large DNA regions suspected to house these genes. For example, in a teleost fish (fugu), several SCPP genes were identified in a DNA region corresponding to the SIBLING cluster in mammals, meaning that the syntheny of the SIBLING cluster is conserved between fish and tetrapods [Kawasaki et al., 2005; Kawasaki and Weiss, 2006]. These SCPP genes were found not based on their similarity with known SCPP sequences but because they are located adjacent to SPARCL1, and because they share some structural features with tetrapod SCPPs. Fish SCPP genes are so different from tetrapod SIBLINGs that no homology could be recognized. Fish SCPP genes are expressed during tooth formation [Kawasaki et al., 2005] but one can wonder whether they play the same function as EMPs. Moreover, SIBLINGs (DSPP, DMP1, IBSP, and SPP1) are known to be expressed during tooth matrix formation in tetrapods [Fisher and Fedarko, 2003; Qin et al., 2004]. EMP genes could also be conserved in other regions of the teleost fish genome, but they remain to be discovered. Indeed, morphological studies strongly support that EMPs are present in the enamel-like tissue (ganoine) of basal actinopterygian lineages, polypterids and lepisoste-ids [Sire et al., 1987; Sire, 1994; 1995].

To date the information available for the three EMP genes largely relates to mammals and the few sequences available (or planned to be so) in other tetrapods are not sufficient to perform an evolutionary analysis at this level (fig. 10).

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