Battling EMF reports.

Background: The genome of the sea urchin Strongylocentrotus purpuratus has recently been sequenced because it is a major model system for the study of gene regulatory networks. Embryonic expression patterns for most genes are unknown, however. Results: Using large-scale screens on arrays carrying 50% to 70% of all genes, we identified novel territory-specific markers. Our strategy was based on computational selection of genes that are differentially expressed in lithium-treated embryos, which form excess endomesoderm, and in zinctreated embryos, in which endomesoderm specification is blocked. Whole-mount in situ hybridization (WISH) analysis of 700 genes indicates that the apical organ region is eliminated in lithium-treated embryos. Conversely, apical and specifically neural markers are expressed more broadly in zinc-treated embryos, whereas endomesoderm signaling is severely reduced. Strikingly, the number of serotonergic neurons is amplified by at least tenfold in zinc-treated embryos. WISH analysis further indicates that there is crosstalk between the Wnt (wingless int), Notch, and fibroblast growth factor signaling pathways in secondary mesoderm cell specification and differentiation, similar to signaling cascades that function during development of presomitic mesoderm in mouse embryogenesis. We provide differential expression data for more than 4,000 genes and WISH patterns of more than 250 genes, and more than 2,400 annotated WISH images. Conclusion: Our work provides tissue-specific expression patterns for a large fraction of the sea urchin genes that have not yet been included in existing regulatory networks and await functional integration. Furthermore, we noted neuron-inducing activity of zinc on embryonic development; this is the first observation of such activity in any organism. Published: 16 May 2007 Genome Biology 2007, 8:R85 (doi:10.1186/gb-2007-8-5-r85) Received: 15 January 2007 Revised: 12 April 2007 Accepted: 16 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R85 Genome Biology 2007, 8:R85 R85.2 Genome Biology 2007, Volume 8, Issue 5, Article R85 Poustka et al. http://genomebiology.com/2007/8/5/R85 Background Body plan development is controlled by large gene regulatory networks (GRNs). Such networks consist of components that accurately specify cell fate at defined times during development via their physical interaction, or in the case of transcription factors via their binding to cis-regulatory DNA elements. One of the best studied developmental GRNs is the sea urchin endomesoderm GRN, which includes almost 50 genes [1,2]. These genes were uncovered in part through three array screens: a subtractive screen, in which RNA from lithiumtreated embryos was subtracted with RNA isolated from cadherin injected embryos [3]; a Brachyury target gene screen [4]; and a screen for pigment cell-specific genes [5]. Comparison of the endoderm network between vertebrates (mouse, xenopus, and zebrafish) showed that many components have been conserved. Common key zygotic factors are the Nodalrelated transforming growth factor-β ligands, the Mixlike (paired box) family of homeodomain transcription factors, the Gata4/Gata5/Gata6 zinc-finger transcription factors and the HMG box transcription factor Sox17 [6-10]. Orthologs of some of these genes are components of the sea urchin endomesoderm GRN. Examples include SpGataE and SpGataC (orthologs of Gata4/Gata5/Gata6 and Gata1/Gata2/ Gata3, respectively), SpFoxA (ortholog of FoxA1 [HNF3b], which in Xenopus is a target of Mixer), and SpOtx (ortholog of Otx2, which in Xenopus is induced by Sox17). However, comparison of the vertebrate and sea urchin endomesoderm network also reveals that many sea urchin orthologs of vertebrate endomesoderm genes are absent from the respective sea urchin GRN. This could be due to the fact that the existing sea urchin endomesoderm GRN is built progressively, starting from genes found to be regulated in the initial screens; this raises the possibility that nodes of the endomesoderm network that are not affected by the above subtractive hybridizations have not yet been explored. In addition, some genes employed in the sea urchin endomesoderm GRN are apparently absent from vertebrate endomesoderm GRNs. The aim of this study is to identify additional genes that are associated with developmental patterning, primarily focusing on endomesoderm specific genes but also on genes that are involved in ectoderm differentiation and patterning. We then add these genes to the existing GRNs or create novel GRNs that describe sea urchin embryonic development. The early sea urchin embryo develops two primary axes: the animal-vegetal axis and the oral-aboral axis. Most of the endodermal and mesodermal cells are derived from the vegetal half, whereas the animal cells contribute to neural and non-neural ectodermal territories. During gastrulation the ectoderm is divided into an oral side, which flattens and is the site where the mouth secondarily breaks through, and a rounded aboral side, which is seperated by the ciliary band region. Activation of the sea urchin endomesoderm GRN is initiated at the molecular level as a result of nuclearization of β-catenin initially in the vegetal micromeres (at the fourth cleavage) and subsequently in the macromeres and their progenitor blastomeres veg2 and part of veg1. The nuclearization of βcatenin in the micromeres at the 16-cell stage is also the earliest molecular evidence of an animal-vegetal axis in Strongylocentrotus purpuratus [11-14]. Reagents exist for manipulation of the GRNs that specify the embryonic axis. Lithium chloride acts as a vegetalizing (posteriorizing) agent by directly binding glycogen synthase kinase-3β, thus freeing up β-catenin, which then enters the nucleus and activates target genes via a complex with Tcf/Lef [14] (Figure 1 shows a sketch of the resulting axis perturbations). As result of the vegetalization, the endomesodermal domain is expanded at the expense of ectodermal territories. A recent study suggested that lithium chloride treatment induces an increase in endoderm at the expense of the ectoderm, but without alterating the mesodermal territories, because the expression domain of Frizzled5/8 at the animal pole is eliminated whereas its expression at the secondary mesenchyme cells (SMCs) is not affected [15]. Furthermore, recent evidence based on study of Nodal suggests that lithium chloride also intervenes with the oral-aboral axis of the embryo, because the region expressing the oral marker Nodal is reduced and shifted to the animal side [16], which is consistent with the conversion of part of the ectoderm to endoderm. Oral-aboral axis is established before the sixth cleavage and is dependant on signals from the vegetal pole [16,17]. Complementary to lithium treatment, zinc treatment animalizes (anteriorizes) the embryos and leads to embryos with no or reduced endomesodermal cells [18-20]. Using these reagents we conducted separate array hybridizations of lithium chloride or zinc sulfate treated and normal embryos. Because lithium vegetalizes and zinc complementarily animalizes embryos, we would expect endomesodermspecific genes to be upregulated in embryos treated with lithium and downregulated in embryos treated with zinc sulfate, whereas ectoderm-specific genes should exhibit the opposite pattern. Hybridizations were carried out on nonredundant arrays that correspond to 50% to 70% of all sea urchin genes [21]. In our experimental design we have used repetitions of experiments in order to calculate sensitivity as a factor of reproducibility. We deliberately did not amplify or subtract any probes, because these procedures run the risk for distorting the representation of different sequences in the RNA sample. In addition, they can interfere with the identification of (for instance, they may remove) highly expressed genes, which can also be territory specific markers. Differentially expressed genes were analyzed by whole-mount in situ hybridization (WISH) from early blastula stages (10 hours) to the pluteus stage (90 hours) during normal embryonic Genome Biology 2007, 8:R85 http://genomebiology.com/2007/8/5/R85 Genome Biology 2007, Volume 8, Issue 5, Article R85 Poustka et al. R85.3