Isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening

New secondary mesenchyme specific genes, expressed exclusively in pigment cells, were isolated from sea urchin embryos using a differential screening of a macroarray cDNA library. The comparison was performed between mRNA populations of embryos having an expansion of the endo-mesodermal territory and embryos blocked in secondary mesenchyme specification. To be able to isolate transcripts with a prevalence down to five copies per cell, a subtractive hybridization procedure was employed. About 400 putative positive clones were identified and sequenced from the 5' end. Gene expression analysis was carried out on a subset of 66 clones with real time quantitative PCR and 40 clones were positive. This group of clones contained sequences highly similar to: the transcription factor glial cells missing (gcm); the polyketide synthase gene cluster (pks-gc); three different members of the flavin-containing monooxygenase gene family (fmo); and a sulfotransferase gene (sult). Using whole mount in situ hybridization, it was shown that these genes are specifically expressed in pigment cells. A functional analysis of the S. purpuratus pks and of one S. purpuratus fmo was carried out using antisense technology and it was shown that their expression is necessary for the biosynthesis of the sea urchin pigment echinochrome. The results suggest that S. purpuratus pks, fmo and sult could belong to a differentiation gene battery of pigment cells

Identification of Three Required Positive Cis-Regulated Inputs of the Sea Urchin Pigment Cell Gene Polyketide Synthase 1

Sea urchin pigment cells are single cells of mesodermal origin embedded in the aboral ectoderm. Strongylocentrotus purpuratus polyketide synthase 1 (Sp-PKS1) is required for the biosynthesis of the echinochrome pigment. Evidence suggests that pigment cells are immune cells. In order to reconstruct the gene regulatory network of pigment cells a bottom-up approach combined with comparative genomics has been used in this study. We compared the cis-regulatory regions of five pigment cell genes, Sp-Pks1, flavin monooxygenase 1, 2, and 3 (Sp-Fmo) and sulfotransferase (Sp-Sult), across three different species, Strongylocentrotus purpuratus, Mesocentrotus franciscanus, and Strongylocentrotus fragilis. The computational tool used was multiple expectation maximization motif elicitation analysis. Thirty cis-regulatory motif candidates were identified, three of which were considered for further analysis. The functionality of these motifs was tested by injecting embryos with a -2KbPks-Gfp DNA construct having one of the three motifs mutagenized. All three motifs resulted to be functional cis-regulatory sequences. Specifically, they contained DNA-binding sites for transcriptional activators of Sp-Pks1

Genome-wide assessment of differential effector gene use in embryogenesis

Six different populations of cells were isolated by FACS from disaggregated late blastula and gastrula stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered BACs producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells, and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicates the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1 to 2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic, and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus at the effector gene level a dramatic, cell type specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred

Constitutive expression of the barley dehydrin gene aba2 enhances Arabidopsis germination in response to salt stress

Dehydrins (DHNs) are a sub-family of the late embryogenesis abundant proteins generally induced during development of desiccation tolerance in seeds and water deficit or salinity stress in plants. Nevertheless, a detailed understanding of the DHNs function is still lacking. In this work we investigated the possible protective role during salt stress of a Dhn from Hordeum vulgare (L.), aba2. The coding sequence of the aba2 gene was constitutively expressed in transgenic lines of Arabidopsis thaliana (L.). During salt stress conditions germination rate, cotyledon expansion and greening were greatly improved in the transgenic lines as compared to the wild type. Between 98 and 100% of the transgenic seeds germinated after two weeks in media containing up to 250 mM NaCl, and 90% after 22 days at 300 mM NaCl. In conditions of 200 mM NaCl 93% of the transgenic cotyledons had greened after two weeks, outperforming the wild type by 45%. Our study provides further evidence that DHNs have an important role in salt stress tolerance. The production of plants constitutively expressing DHNs could be an effective strategy to improve plant breeding programs

Growth of ZnO tetrapods for gas sensor application

Zinc Oxide (ZnO) nanostructures have been obtained by vapour phase growth. Tetrapods have been grown in a reproducible way and separated from the other possible ZnO nano-morphologies, by the optimization of growth parameters. The further deposition of these nanostructures on an alumina substrate with contacts and heater, allowed us to test the gas sensing properties of the obtained ZnO tetrapods with different gases

Growth of ZnO tetrapods for gas sensor application

Zinc Oxide (ZnO) nanostructures have been obtained by vapour phase growth. Tetrapods have been grown on a large scale, in a reproducible way and separated from the other possible ZnO nano-morphologies, by the optimization of growth parameters. The deposition of these nanostructures on an alumina substrate with contacts and heater, allowed us to realize a low-cost oriented gas sensor. The obtained ZnO tetrapods-based gas sensor prototypes have been tested with different gases

Expression Pattern Of Polyketide Synthase-2 During Sea Urchin Development

Polyketide synthases (PKSs) are a large group of proteins responsible for the biosynthesis of polyketide compounds, which are mainly found in bacteria, fungi, and plants. Polyketides have a wide array of biological functions, including antibiotic, antifungal, predator defense, and light responses. In this study, we describe the developmental expression pattern of pks2, one of two pks found in the sea urchin genome. Throughout development, pks2 expression was restricted to skeletogenic cells and their precursors. Pks2 was first detected during the blastula stage. The transcript level peaked at hatched blastula, when all skeletogenic cell precursors expressed pks2. This was followed by a steady decline in expression in the skeletogenic cells on the aboral side of the embryo. By the prism stage, pks2 expression was limited to only 3-4 skeletogenic cells localized on the oral side. © 2011 Elsevier B.V. All rights reserved

Cis-Regulatory Analysis Of The Sea Urchin Pigment Cell Gene Polyketide Synthase

The Strongylocentrotus purpuratus polyketide synthase gene (SpPks) encodes an enzyme required for the biosynthesis of the larval pigment echinochrome. SpPks is expressed exclusively in pigment cells and their precursors starting at blastula stage. The 7th-9th cleavage Delta-Notch signaling, required for pigment cell development, positively regulates SpPks. In previous studies, the transcription factors glial cell missing (SpGcm), SpGatae and kruppel-like (SpKrl/z13) have been shown to positively regulate SpPks. To uncover the structure of the Gene Regulatory Network (GRN) regulating the specification and differentiation processes of pigment cells, we experimentally analyzed the putative SpPks cis-regulatory region. We established that the - 1.5. kb region is sufficient to recapitulate the correct spatial and temporal expression of SpPks. Predicted DNA-binding sites for SpGcm, SpGataE and SpKrl are located within this region. The mutagenesis of these DNA-binding sites indicated that SpGcm, SpGataE and SpKrl are direct positive regulators of SpPks. These results demonstrate that the sea urchin GRN for pigment cell development is quite shallow, which is typical of type I embryo development. © 2010 Elsevier Inc

A Novel Group Of Type I Polyketide Synthases (Pks) In Animals And The Complex Phylogenomics Of Pkss

Type I polyketide synthases (PKSs), and related fatty acid synthases (FASs), represent a large group of proteins encoded by a diverse gene family that occurs in eubacteria and eukaryotes (mainly in fungi). Collectively, enzymes encoded by this gene family produce a wide array of polyketide compounds that encompass a broad spectrum of biological activity including antibiotic, antitumor, antifungal, immunosuppressive, and predator defense functional roles. We employed a phylogenomics approach to estimate relationships among members of this gene family from eubacterial and eukaryotic genomes. Our results suggest that some animal genomes (sea urchins, birds, and fish) possess a previously unidentified group of pks genes, in addition to possessing fas genes used in fatty acid metabolism. These pks genes in the chicken, fish, and sea urchin genomes do not appear to be closely related to any other animal or fungal genes, and instead are closely related to pks genes from the slime mold Dictyostelium and eubacteria. Continued accumulation of genome sequence data from diverse animal lineages is required to clarify whether the presence of these (non-fas) pks genes in animal genomes owes their origins to horizontal gene transfer (from eubacterial or Dictostelium genomes) or to more conventional patterns of vertical inheritance coupled with massive gene loss in several animal lineages. Additionally, results of our broad-scale phylogenetic analyses bolster the support for previous hypotheses of horizontal gene transfer of pks genes from bacterial to fungal and protozoan lineages. © 2006 Elsevier B.V. All rights reserved