Giovanni Spinelli

Impairing Otp homeodomain function in oral ectoderm cells affects skeletogenesis in sea urchin embryos

In the sea urchin embryo skeletogenesis is the result of a complex series of molecular and cellular events that coordinate the morphogenetic process. Past and recent evidence strongly indicate that skeletal initiation and growth are strictly dependent on signals emanating from the oral ectodermal wall. As previously suggested, Orthopedia (Otp), a homeodomain-containing transcription factor specifically expressed in a small subset of oral ectoderm cells, might be implicated in this signalling pathway. In this study, we utilize three different strategies to address the issue of whether Otp is an upstream regulator of sketelogenesis. We describe the effects of microinjection of Otp morpholino-substituted antisense oligonucleotides and dominant-negative Otp-engrailed mRNA in Paracentrotus lividus embryos. We demonstrate that inhibition of Otp expression completely abolishes skeletal synthesis. By contrast, coinjection of Otp mRNA and the morpholino antisense oligonucleotide specifically rescues the skeletogenic program. In addition, localized ectodermal expression of the Otp-GFP fusion gene construct driven by the hatching enzyme promoter, induces ectopic and abnormal spiculogenesis. We further show that an indirect target of this homeoprotein is the skeletogenic specific gene SM30, whose expression is known to be under the strict control of the oral ectoderm territory. Based on these results, we conclude that Otp triggers the ectoderm-specific signal that promotes skeletogenesis.

p53-Mediated downregulation of H ferritin promoter transcriptional efficiency via NF-Y

The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53-NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis. These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation.

Synthesis of histones during sea urchin oogenesis.

Abstract Oocytes at the stage of vitellogenesis were isolated from sea urchin gonads and incubated in vitro with labelled lysine. The synthesis of arginine-rich and some other histone fraction was detected by column chromatography and electrophoresis. An attempt to calculate the absolute rate of histone synthesis is also reported.

Early asymmetric cues triggering the dorsal/ventral gene regulatory network of the sea urchin embryo

Dorsal/ventral (DV) patterning of the sea urchin embryo relies on a ventrally-localized organizer expressing Nodal, a pivotal regulator of the DV gene regulatory network. However, the inceptive mechanisms imposing the symmetry-breaking are incompletely understood. In Paracentrotus lividus, the Hbox12 homeodomain-containing repressor is expressed by prospective dorsal cells, spatially facing and preceding the onset of nodal transcription. We report that Hbox12 misexpression provokes DV abnormalities, attenuating nodal and nodal-dependent transcription. Reciprocally, impairing hbox12 function disrupts DV polarity by allowing ectopic expression of nodal. Clonal loss-of-function, inflicted by blastomere transplantation or gene-transfer assays, highlights that DV polarization requires Hbox12 action in dorsal cells. Remarkably, the localized knock-down of nodal restores DV polarity of embryos lacking hbox12 function. Finally, we show that hbox12 is a dorsal-specific negative modulator of the p38-MAPK activity, which is required for nodal expression. Altogether, our results suggest that Hbox12 function is essential for proper positioning of the DV organizer. DOI: http://dx.doi.org/10.7554/eLife.04664.001

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.

Smouldering hepatitis b virus replication in patients with chronic liver disease and hepatitis delta virus superinfection

Hepatitis B virus deoxyribonucleic acid (HBV-DNA) was studied by Southern blot analysis in liver biopsy specimens from 75 HBsAg-positive patients with chronic liver disease living in southern Italy. Twenty-seven of the patients were hepatitis delta virus (HDV) superinfected. Intrahepatic HBV-DNA was detected in 54 (72%) patients, 32 (59%) of them with replicative forms. The presence of replicative forms was directly related to liver HBcAg and inversely related to liver HDAg, as shown by multivariate analysis. However, 14 patients with intrahepatic HBV-DNA non-replicative pattern and about half of HDV-infected patients were liver HBcAg and/or serum HBV-DNA positive, mostly in low amounts. Histological inflammatory activity was strongly related to liver HBcAg expression regardless of HDV superinfection, as confirmed by multivariate analysis. Our results confirm previous studies about the concordance between intrahepatic HBV-DNA replicative pattern and liver HBcAg expression and about inhibition by HDV of high-level HBV replication. However, they suggest that low-level HBV replication may have an important role in causing liver damage also among HDV-infected patients, in a population where the spreading of HBV and HDV is a naturally occurring event.

The Sea Urchin sns5 Insulator Protects Retroviral Vectors From Chromosomal Position Effects by Maintaining Active Chromatin Structure

Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.

cis-Regulatory sequences driving the expression of the Hbox12 homeobox-containing gene in the presumptive aboral ectoderm territory of the Paracentrotus lividus sea urchin embryo

Embryonic development is coordinated by networks of evolutionary conserved regulatory genes encoding transcription factors and components of cell signalling pathways. In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. To obtain insights on the regulation of Hbox12 expression, we have explored the cis-regulatory apparatus of the gene. In this paper, we show that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined aboral ectoderm (AE) module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for a Sox factor and the binding of the Otx activator are both required for correct Hbox12 gene expression. Spatial restriction to the aboral ectoderm is achieved by a combination of different repressive sequence elements. Negative sequence elements necessary for repression in the endomesoderm map within the most upstream 60 bp region and nearby the Sox binding site. Strikingly, a Myb-like consensus is necessary for repression in the oral ectoderm, while down-regulation at the gastrula stage depends on a GA-rich region. These results suggest a role for Hbox12 in aboral ectoderm specification and represent our first attempt in the identification of the gene regulatory circuits involved in this process.

REGULATION OF THE SEA URCHIN EARLY H2A HISTONE GENE EXPRESSION DEPENDS ON THE MODULATOR ELEMENT AND ON SEQUENCES LOCATED NEAR THE 3' END

Abstract Transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, we used promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven by the early H2A promoter. We found that morula and gastrula nuclear proteins produced indistinguishable DNase I footprint patterns on the H2A promoter. Two sites of interactions, centred on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3′ coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator is essential for the expression of early H2A gene and that sequences for down-regulation are localized near the 3′ end of the H2A gene.

The sea urchin embryo: A model to study Alzheimer’s beta amyloid induced toxicity

Alzheimers disease (AD) is the most common form of dementia. The cause of AD is closely related to the accumulation of amyloid beta peptide in the neuritic plaques. The use of animal model systems represents a good strategy to elucidate the molecular mechanism behind the development of this pathology. Here we use the Paracentrotus lividus embryo to identify molecules and pathways that can be involved in the degenerative process. As a first step, we identified the presence of an antigen related to the human APP, called PlAPP. This antigen, after gastrula stage, is processed producing a polypeptide of about 10kDa. By immunohistochemistry we localized the PlAPP antigen in some serotonin expressing cells. Similarly, after 48 or 96h incubation, a recombinant beta-amyloid peptide, rAbeta42, accumulates around the intestinal tube and oesophagus. In addition, incubation of sea urchin embryos with two different solutions rich in oligomers and fibrillar aggregates of rAbeta42 induce activation of apoptosis as detected by TUNEL assay. Moreover, we demonstrate that aggregates induce apoptosis by extrinsic pathway activation, whereas oligomers induce apoptosis both by extrinsic and intrinsic pathway activation. Utilizing an apoptotic inhibitor, caspases activation was offset and morphological damage rescued. Taken together all these observations suggest that the sea urchin may be a simple and suitable model to characterize the mechanism underlining the cytotoxicity of Abeta42.