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Towards the origins of vertebrate mineralization: the contribution of large scale transcriptome sequencing – JAWS

Towards the origins of mineralization in vertebrates

Skeleton mineralization (bones and teeth) was a crucial event in vertebrate diversification. However, although fossil records reveal that early mineralized vertebrates possessed enamel, dentin and bone, current genomic data do not allow finding the genes encoding proteins controlling mineralization of bone and teeth in all vertebrate lineages.

Evolutionary story of mineralizing proteins in vertebrates

Going back in geological periods towards the origins of mineralization of bone and tooth tissues of early vertebrates and understanding how mineralization tools evolved in the various lineages (sharks, rays, bony ray fish, tetrapods) are the main objective of this study. In order to reach this objective the only transcriptome sequencing could bring us data that are lacking on mineralizing protein genes of the current non mammalian vertebrate lineages.Indeed, in contrast to more than 60 mammalian genomes sequenced or in course of, the total of sequenced genomes in all non mammalian lineages is less than 30.

We have selected representative species for the main vertebrate lineages (one crocodile, lizard, salamander, lungfish, bichir, gar pike, several teleosts, shark and ray) and extracted RNA from jaws in order to sequence the transcriptome (i.e. all transcripts of genes that were expressed in the jaw at a given time) in which tooth and bone genes were present. These genes are looked for in the transcriptomes using in silico approaches and their structure and composition are analyzed through an evolutionary context in order to identify events that have occurred during long lasting geological periods.

The 6 first months of our project allowed to obtain the jaw transcriptome of crocodile, lungfish and salamander, and start to analyze the data. To date we can tell that numerous mineralizing protein gene were identified in each transcriptome, illustrating their conservation during long evolutionary periods and their presence as early as lungfish and tetrapod ancestors, 400 millions years ago.

At the end of the first part of the project we think that 15-20 jaw transcriptomes will be sequenced and available for further analyzes, in particular tracing back the origins of these mineralizing proteins. Their role in the control of skeletal tissue mineralization is really important as illustrated by genetic diseases occurring when some genes are mutated. Also, we will annotate all transcriptome using bioinformatic techniques and will give the scientific community access to this important set of data.

A this early step of our project we have no scientific production to refer to.


It is now largely admitted that the recruitment, more than 460 million years (Ma) ago, of a CaPO4-based mineralized skeleton was a crucial innovation that provided adaptive trait for vertebrates (protection, locomotion, predatory behaviour, ...). This important event gave rise to teeth, bones, and various skeletal elements. The well-mineralized skeletal tissues (enamel, dentin, bone) of these organs display an overall evolutionary stability of their structure, and thus of their underlying cellular and molecular mechanisms. This means that the specialized cells, the regulatory gene networks, the specific proteins of their organic matrix, and the type of mineral were recruited early in the vertebrate story and have not changed drastically for more than 460 Ma.
The major components of these early-mineralized tissues were recruited after the two-genome duplications that predated vertebrate differentiation. Among the new molecules that have favoured the development of a CaPO4-based mineralized integument are the SCPPs (for Secretory Calcium-binding PhosphoProteins). They are a group of phylogenetically related proteins that are involved in the regulation of matrix mineralization in mammals, and a number of them play a so important role that they are responsible for genetic diseases when mutated in humans. It is thought that the genes coding for these crucial extracellular matrix proteins arose from SPARCL1 (SPARC-like 1), which originated from SPARC (secreted protein, acidic, cystein-rich), at about the same period when fibrillar collagen genes used for the vertebrate skeletal tissues were differentiated. The SCPP gene family was subsequently enlarged in various lineages through tandem duplications. To date, 22 SCPP genes have been identified in humans. They are distributed into two groups: the proline / glutamine rich protein genes that regroups 17 genes, among which at least five enamel matrix protein genes (AMEL, AMBN, ENAM, AMTN, ODAM), and the acidic protein genes, with five dentin/bone matrix protein genes (DSPP, DMP1, IBSP, MEPE, SPP1).
Our group has acquired an internationally and nationally recognized expertise in the evolutionary analysis of these proteins (see reference list) and we recently improved our impact through a scientific collaboration with Dr K. Kawasaki (Penn State University, USA) who had discovered the SCPP family in 2003. Our studies benefitted from the large number of sequences of vertebrate genomes available in Genbank: several SCPPs were found in Xenopus tropicalis, an amphibian, and in five teleost fish species. However, SCPPs are unstructured proteins and their genes accumulate various mutations that render difficult finding them, even in the sequenced genome of phylogenetically distant species. The main objective of our four years long project is to overcome this problem using large scale transcriptome analyses and, therefore, to considerably improve our knowledge of the origin, evolution and relationships of the SCPPs.
In this project we plan to obtain the transcriptome sequences from the jaws (containing developing teeth, bones, etc.) of 15 vertebrate species representative of important, and phylogenetically distant lineages that are not currently available as sequenced genomes (salamander, lungfish, bichir, sturgeon, gar pike, bowfin, several teleosts, shark, ...). We already obtained good results in a preliminary analysis of a crocodile jaw transcriptome (30,000 contigs).
In addition, we have identified several issues in order to take advantage of the large amount of data that will be generated by these transcriptomes. They include (i) comparative studies (evo-devo) of SCPP gene expression (in situ hybridization experiments), (ii) the annotation of the transcriptome sequence data sets in order to identify new candidate genes involved in skeletal tissue development and mineralization, (iii) make available the annotated datasets to the scientific community.

Project coordinator

Monsieur Jean-Yves SIRE (Université Pierre et Marie Curie, UMR 7138) – jean-yves.sire@upmc.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

UPMC Université Pierre et Marie Curie, UMR 7138
UMR 5554 CNRS-IRD-Université Montpellier Institut des Sciences de l'Evolution

Help of the ANR 379,991 euros
Beginning and duration of the scientific project: December 2012 - 48 Months

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