The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts

Genome-wide analysis of the repertoire of TRIM genes in sea urchins

The eukaryotic TRIM (TRIpartite Motif) super-family represents one of the largest classes of putative E3 ubiquitin ligases involved in several processes, including epigenetic control of development and disease. In the post-genomic era, new approaches allow genome-wide studies of gene family. In particular, we performed a comprehensive analysis of the TRIM repertoire in selected sea urchin species. By combining iterations of ab initio predictions and pairwise comparative methods, we first retrieved the full complement of TRIM genes in Strongylocentrotus purpuratus, whose full genome sequence was available. Interestingly, such a DNA sequence set includes not previously classified, echinoderm-specific, TRIM genes as well as multiple copies of known ones. We also retrieved a landscape of cDNA sequences from staged EST libraries, indicating that most of these genes are actively transcribed during development. Phylogenetic analysis of the deduced proteins, using set of TRIMs from various species, revealed a degree of genetic variation between species. Worth of mention, we predicted the occurrence of transposition events involving some of these genes, according with the documented rapid evolution of this family. Next, we adopted heuristic algorithms and post-processing steps to investigate the evolutionarily distant Paracentrotus lividus, Allocentrotus fragilis and Lytechinus variegatus genomes, whose sequencing projects are actually in progress. We assembled partial pools of TRIM genes and specifically associated them to EST-derived cDNA sequences. Such a collection of data should provide a framework for unravel gene regulatory networks involving TRIM genes from an evolutionary perspective. Indeed, in the Pl species, we have previously isolated and functionally characterized the cDNA sequence encoding the first echinoderm TRIM factor, Strim1. Here, we identified five strim1 genes, all sharing a intronless organization, and roughly located their cis-regulatory apparatus

EFFECT OF Γ-AMINOBUTYRRIC ACID (GABA) EXPOSURE ON EMBRYOGENESIS OF PARACENTROTUS LIVIDUS AND IDENTIFICATION OF GABA-RECEPTOR GENES IN SEA URCHINS

Developmental processes are controlled by regulatory genes encoding for transcription factors and signaling molecules. Functional relationships between these genes are described by gene regulatory networks (GRN), models which allow integration of various levels of information. The sea urchin embryo is an experimental model system which offers many advantages for the analysis of GRN. Recently, the GRN that governs the biomineralization of the sea urchin embryonic skeleton has begun to be deciphered. Preliminary evidence suggest that the γ- aminobutyric acid (GABA) signaling pathway is involved in skeletal morphogenesis during development of the sea urchin. GABA is a molecule synthesized by nearly all organism, from bacteria to humans, and it acts through ionotropic and metabotropic receptors (GABAA-Rs and GABAB-Rs, respectively). We report that Paracentrotus lividus embryos exposed to GABA at concentrations ranging from 0.01 to 1.0 mM showed aberrations in axial patterning, with a dose dependent effect. Washout experiments allowed to determine that the period of sensitivity is restricted from the blastula to the gastrula stage. In order to identify GABA-R genes we performed a comprehensive in silico analysis in selected sea urchin species (P. lividus, Strongylocentrotus purpuratus, and Lytechinus variegatus), and in phylogenetically related organisms, such as the hemichordate Saccoglossus kowalevskii, the chordate Ciona intestinalis, and the nematode Caenorhabditis elegans. By combining iteration of ab initio predictions and pairwise comparative methods, we identified the orthologous genes encoding for GABAB1 and GABAB2, the two subunits which assemble GABAB-R, and we confirmed that all of these organisms possess a unique α/β GABAA-R gene pair clustered in the genome. Furthermore, we have observed that the reciprocal disposition of GABAA-R genes is also evolutionarily conserved. Interestingly, in adjacent position to these genes, we have identified an additional gene, which shows significant sequence similarity to a invertebrate-specific GABAA-R gene. Indeed, such a gene has been only identified in C. elegans, Drosophila melanogaster, and Nematostella vectensis. We also retrieved several cDNA sequences from staged EST databases of the three sea urchin species inspected, indicating that these genes are actively transcribed during development. Some selected cDNA plasmids were also isolated from P. lividus total RNA samples and fully sequenced. Hypothetical proteins were deduced and used for phylogenetic analysis, including a selection of vertebrate and invertebrate GABAA-R subunit sequences. The resulting phylogenetic tree strongly support the hypothesis that the sea urchins contain genes encoding for both canonical and invertebrate-specific GABAA-R subunits. Such a collection of data should provide a support to better understand the involvement of GABA-signalling pathway in the skeletal GR

Specific expression of a TRIM-containing factor in ectoderm cells affects the skeletal morphogenetic program of the sea urchin embryo

In the indirect developing sea urchin embryo, the primary mesenchyme cells (PMCs) acquire most of the positional and temporal information from the overlying ectoderm for skeletal initiation and growth. In this study, we characterize the function of the novel gene strim1, which encodes a tripartite motif-containing (TRIM) protein, that adds to the list of genes constituting the epithelial-mesenchymal signaling network. We report that strim1 is expressed in ectoderm regions adjacent to the bilateral clusters of PMCs and that its misexpression leads to severe skeletal abnormalities. Reciprocally, knock down of strim1 function abrogates PMC positioning and blocks skeletogenesis. Blastomere transplantation experiments establish that the defects in PMC patterning, number and skeletal growth depend upon strim1 misexpression in ectoderm cells. Furthermore, clonal expression of strim1 into knocked down embryos locally restores skeletogenesis. We also provide evidence that the Otp and Pax2/5/8 regulators, as well as FGFA, but not VEGF, ligand act downstream to strim1 in ectoderm cells, and that strim1 triggers the expression of the PMC marker sm30, an ectoderm-signaling dependent gene. We conclude that the strim1 function elicits specific gene expression both in ectoderm cells and PMCs to guide the skeletal biomineralization during morphogenesis

Suppression of nodal expression in prospective dorsal cells of the early sea urchin embryo by the hbox12 homeodomain regulator

Dorsal/Ventral (DV) axis formation in the sea urchin embryo depends upon the expression of nodal on the ventral side, which behaves as a DV organizing centre. However, only fuzzy clues are known as to the early symmetry-breaking steps that lead to the positioning of such an organizer. An extremely interesting candidate for this role is the hbox12 homeobox-containing gene. In Paracentrotus lividus, hbox12 expression is antecedent and complementary with respect to that of nodal, being confined in prospective dorsal cells. We show that ectopic expression of Hbox12 provokes DV abnormalities and attenuates nodal as well as nodal-dependent gene transcription. By blastomere transplantation, we also establish that DV defects arise from hbox12 misexpression in the animal hemisphere. To impair Hbox12 function we expressed ubiquitously a truncated form of the protein, encoding for the homeodomain. Such a perturbation disrupts DV axis formation by allowing ectopic expression of nodal across the embryo. Moreover, clonal loss-of-function imposed by either blastomere transplantation or gene transfer assays highlights that Hbox12 action in prospective dorsal cells is necessary for DV polarization. Remarkably, the localized knock-down of nodal restores DV polarity of embryos lacking hbox12 function. Finally, we show that hbox12 is involved in the dorsal-specific inactivation of the p38 MAPK, which is known to be required for nodal expression. Altogether, our results indicate that Hbox12 prevents the ectopic activation of nodal transcription within the future dorsal side of the early sea urchin embryo