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Paracentrotus lividus

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Generation of genomic resources for the Mediterranean sea urchin Paracentrotus lividus: EST libraries from embryonic stages and selected genomic sequences

Metazoans develop from a single cell, the zygote, into a complex multicellular organism comprising many differentiated cell types that express different sets of genes. The genome is at the centre of this complex process as it encodes both the final state and the developmental processes to establish it. Together with systematic description of spatial patterns of gene expression and functional analyses, genomic and transcriptomic data provide a background to dissect gene regulatory networks that have a key role during development. Integration of gene network dynamic with 4D description of embryo development will lead to a system approach appropriate to better understand the complexity of multicellular development.

Sea urchins are well-known marine animals that are found all over the world. The adult sea urchin has an unusual fivefold symmetry (fig. 1A), most evident for its close relative the sea star. Despite this derived feature, sea urchins are Bilaterians as revealed by the bilateral symmetry of their embryo (fig. 1B). Sea urchins belong to the phylum Echinodermata, which together with Hemichordata and Xenoturbella form a sister group to the Chordates (a group that includes Vertebrates).

The embryo from Paracentrotus lividus, the main edible sea urchin species on the Mediterranean and North-East Atlantic coasts, is an exceptional experimental model for developmental biology. Embryos from this species have been used by 19th century European biologists to perform classic embryological studies that led to major basic discoveries in biology. The role of gametes pronuclei and their fusion at fertilization was discovered by Hertwig and Fol, the requirement for the complete chromosome set in each blastomere for development was demonstrated by Boveri, Driesch discovered regulative development and Horstadius developed ideas on gradients and obtained evidence for inductive interactions.

Like other sea urchins, P. lividus produce large numbers of gametes (> 107 eggs per female) and fertilization can be carried out in vitro with a yield of almost 100%, providing very large populations of embryos that develop rapidly and synchronously. The P. lividus egg displays a unique feature, the sub-equatorial pigmented band, which allows orientation with respect to the primordial animal-vegetal axis (fig. 2A). Both eggs and embryos are very sturdy and can easily endure egg bisection and blastomeres recombination. Microinjections into one blastomere at the 8-cell stage are routinely performed in P. lividus, while they are considered as nearly impossible in other species. The embryo is exceptionally transparent (fig. 2B-D), allowing observation of tissues and cells, movements of germ layers and cell behaviour as well as easy readings of signals from whole-mount in situ hybridisation (WMISH), immunolocalisation, GFP-tagged proteins and reporter genes.

It is particularly well suited for all imaging methods (fig. 3).

A large-scale screen by WMISH has been performed [1], and the first 4D reconstructions of the developing embryo from confocal imaging have been carried out successfully on P. lividus embryo allowing precise description of the complete cell lineage up to 500 cells. [2]. Most important, microinjection in eggs of DNA constructs permits standard analyses of cis-regulatory activities, while microinjection of mRNAs and morpholino-oligonucleotides allows functional studies by gain and loss of function analyses. The sea urchin embryo is one of the leading models to study gene regulatory networks [3].

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