Vincenzo Cavalieri

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.

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.

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.

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.

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.

Promoter activity of the sea urchin ( Paracentrotus lividus ) nucleosomal H3 and H2A and linker H1 α-histone genes is modulated by enhancer and chromatin insulator

Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) α-histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence suggests that the MBF-1 transcription factor participates also in the expression of the H3 gene and that the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer. Altogether, these results provide a clear demonstration of the enhancer-blocking function of a chromatin insulator in a natural gene context. In addition, they suggest that both the H2A enhancer and the sns5 insulator may account for the diverse accumulation of the linker H1 versus the core nucleosomal histones during early development of the sea urchin embryo.

Extracellular Hsp70 Enhances Mesoangioblast Migration via an Autocrine Signaling Pathway

Mouse mesoangioblasts are vessel‐associated progenitor stem cells endowed with the ability of multipotent mesoderm differentiation. Therefore, they represent a promising tool in the regeneration of injured tissues. Several studies have demonstrated that homing of mesoangioblasts into blood and injured tissues are mainly controlled by cytokines/chemokines and other inflammatory factors. However, little is known about the molecular mechanisms regulating their ability to traverse the extracellular matrix (ECM). Here, we demonstrate that membrane vesicles released by mesoangioblasts contain Hsp70, and that the released Hsp70 is able to interact by an autocrine mechanism with Toll‐like receptor 4 (TLR4) and CD91 to stimulate migration. We further demonstrate that Hsp70 has a positive role in regulating matrix metalloproteinase 2 (MMP2) and MMP9 expression and that MMP2 has a more pronounced effect on cell migration, as compared to MMP9. In addition, the analysis of the intracellular pathways implicated in Hsp70 regulated signal transduction showed the involvement of both PI3K/AKT and NF‐κB. Taken together, our findings present a paradigm shift in our understanding of the molecular mechanisms that regulate mesoangioblast stem cells ability to traverse the extracellular matrix (ECM). J. Cell. Physiol. 232: 1845–1861, 2017.

Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo

Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

Functional studies of regulatory genes in the sea urchin embryo.

Sea urchin embryos are characterized by an extremely simple mode of development, rapid cleavage, high transparency, and well-defined cell lineage. Although they are not suitable for genetic studies, other approaches are successfully used to unravel mechanisms and molecules involved in cell fate specification and morphogenesis. Microinjection is the elective method to study gene function in sea urchin embryos. It is used to deliver precise amounts of DNA, RNA, oligonucleotides, peptides, or antibodies into the eggs or even into blastomeres. Here we describe microinjection as it is currently applied in our laboratory and show how it has been used in gene perturbation analyses and dissection of cis-regulatory DNA elements.