Project description:Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offer protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e. mediating the population´s response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype. Moreover, positive and negative regulators of the Wnt/β-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to interfere with both head and foot regeneration, suggesting an active role in setting up tissue gradients and oro-aboral polarity through delivery of short and long-distance signals. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems
Project description:Hydra has long been studied for its remarkable ability to regenerate its head. Previous studies focusing on molecular mechanisms of axial patterning and head regeneration using a candidate gene approach have revealed a central role for the canonical Wnt pathway. We performed a global gene expression analysis during Hydra magnipapillata head regeneration using RNA-seq to identify additional genes that are transcriptionally regulated during the regeneration of the head organizer in hydra. Differential expression analysis revealed a set of 4,978 genes with significant changes during a 48-hour head regeneration time-course that includes many key genes in the Wnt, TGF-M-NM-2/BMP and MAP kinase pathways. We observed the differential regulation of several genes that are part of the epithelial-to-mesenchymal transition in bilaterians such as Snail. We assembled 806 novel putative lincRNAs with 176 of these are differentially expressed during the time course. We observed the coordinated transcriptional regulation of several factors that regulate the effective pool of free M-NM-2-catenin that together synergize to increase the amount of M-NM-2-catenin available for transcriptional regulation of downstream genes. The differential expression of Snail and some of its interacting regulators and downstream targets suggests that a partial-EMT-like response is involved in hydra head regeneration. This time-course is a valuable resource for the study of the transcriptional dynamics of head regeneration in hydra. mRNA profiling of regenerating head from 6 time points post bisection of Hydra head (H. magnipapillata), generated by deep sequencing, in duplicates, using Illumina HiSeq2500.
Project description:In Hydra, Notch inhibition causes defects in head patterning and prevents differentiation of proliferating nematocyte progenitor cells into mature nematocytes. To understand the molecular mechanisms by which the Notch pathway regulates these processes, we performed RNA-seq and identified genes that are differentially regulated in response to 48 h of treating the animals with the Notch inhibitor DAPT. To identify candidate direct regulators of Notch signalling, we profiled gene expression changes that occur during subsequent restoration of Notch activity and performed promoter analyses to identify RBPJ transcription factor-binding sites in the regulatory regions of Notch-responsive genes. Interrogating the available single-cell sequencing data set revealed the gene expression patterns of Notch-regulated Hydra genes. Through these analyses, a comprehensive picture of the molecular pathways regulated by Notch signalling in head patterning and in interstitial cell differentiation in Hydra emerged. As prime candidates for direct Notch target genes, in addition to Hydra (Hy)Hes, we suggest Sp5 and HyAlx. They rapidly recovered their expression levels after DAPT removal and possess Notch-responsive RBPJ transcription factorbinding sites in their regulatory regions
Project description:Recent body of evidence demonstrates that extracellular vesicles (EVs) represent the first language of cell-cell communication emerged during evolution. In aquatic environments, transferring signals between cells by EVs offer protection against degradation, allowing delivering of chemical information in high local concentrations to the target cells. The packaging of multiple signals, including those of hydrophobic nature, ensures target cells to receive the same EV-conveyed messages, and the coordination of a variety of physiological processes across cells of a single organisms, or at the population level, i.e. mediating the population´s response to changing environmental conditions. Here, we purified EVs from the medium of the freshwater invertebrate Hydra vulgaris, and the molecular profiling by proteomic and transcriptomic analyses revealed multiple markers of the exosome EV subtype. Moreover, positive and negative regulators of the Wnt/β-catenin signaling pathway, the major developmental pathway acting in body axial patterning, were identified. Functional analysis on amputated polyps revealed EV ability to interfere with both head and foot regeneration, suggesting an active role in setting up tissue gradients and oro-aboral polarity through delivery of short and long-distance signals. Our results open the path to unravel EV biogenesis and function in all cnidarian species, tracing back the origin of the cell-cell, cross-species or cross-kingdom communication in aquatic ecosystems
Project description:Hydra has long been studied for its remarkable ability to regenerate its head. Previous studies focusing on molecular mechanisms of axial patterning and head regeneration using a candidate gene approach have revealed a central role for the canonical Wnt pathway. We performed a global gene expression analysis during Hydra magnipapillata head regeneration using RNA-seq to identify additional genes that are transcriptionally regulated during the regeneration of the head organizer in hydra. Differential expression analysis revealed a set of 4,978 genes with significant changes during a 48-hour head regeneration time-course that includes many key genes in the Wnt, TGF-β/BMP and MAP kinase pathways. We observed the differential regulation of several genes that are part of the epithelial-to-mesenchymal transition in bilaterians such as Snail. We assembled 806 novel putative lincRNAs with 176 of these are differentially expressed during the time course. We observed the coordinated transcriptional regulation of several factors that regulate the effective pool of free β-catenin that together synergize to increase the amount of β-catenin available for transcriptional regulation of downstream genes. The differential expression of Snail and some of its interacting regulators and downstream targets suggests that a partial-EMT-like response is involved in hydra head regeneration. This time-course is a valuable resource for the study of the transcriptional dynamics of head regeneration in hydra.
Project description:After section, Hydra regenerate each missing part within 2 to 3 days. This study was designed to allow tracking of gene expression levels during apical and basal regeneration and to compare these different regenerative contexts systematically. We determine that transient early genetic events are generic, i.e. that modulations in gene expression have analogous directions, magnitudes and durations whatever the level along the central body column and orientation of the amputation plane. In turn, early modulated transcripts with sustained expression patterns during regeneration are generally distinct during apical and basal regeneration. Genes that were previously shown to be instrumental in defining the apical organizer (Wnt signalling pathway) are among the first genes to be expressed in the condition where Hydra regenerates its apical part, Wnt3 is actually the only detected transcript encoding a signalling protein already upregulated by 2h. Regarding basal regeneration, we identify a number of transcripts with sustained expression patterns already established by 4h, some of them encoding evolutionary conserved signalling proteins, which are almost exclusively consisting of agonists and antagonists of the BMP signalling pathway. The processed data deposited here are also accessible in a graphical manner from a blast-based web interface available at https://hydratlas.unige.ch
Project description:Primary cilia are microtubule based sensory organelles that protrude from almost every cell type. Their membrane contains highly specialized receptors important for receiving and processing extracellular signals, which enables them to regulate several signalling pathways. A recently discovered characteristic is that cilia are also able to release extracellular vesicles (EVs) from the ciliary membrane. Since EVs have been shown to exert numerous functions in physiology and pathology, these findings have the potential to dramatically alter our understanding of how the primary cilium is able to regulate various signalling pathways in development and disease. In our study we focused on the release of EVs from a ciliated kidney cell line. Using control and mutant cell lines, in which ciliary trafficking was disrupted, we observed that loss of primary cilia function leads to altered EV secretion and composition in NTA (Nanoparticle Tracking Analysis) and Western blot. Cilia mutant cells released more small EVs compared to control, and their composition was also changed between mutant and control. Protein identification via mass spectrometry identified both cilia- as well as WNT signalling-associated proteins and miRNA sequencing determined WNT-related miRNAs, which were differentially expressed in small EVs isolated from the cilia mutant cell line. Because of the presence of differentially expressed WNT related molecules in these small EVs, we tested whether this would have an effect on the WNT activity of recipient cells. We observed that small EVs secreted from cilia mutant cells differentially modulated the WNT response in recipient cells compared to control. Our results highlight a possible new small EV-dependent ciliary signalling mechanism, since deficient primary cilia lead to a change in EV secretion, resulting in an altered signal transduction. These results provide us with new insights into ciliopathy disease pathogenesis.
Project description:The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps.
Project description:The cnidarian freshwater polyp Hydra sp. exhibits an unparalleled regeneration capacity in the animal kingdom. Using an integrative transcriptomic and stable isotope labeling by amino acids in cell culture proteomic/phosphoproteomic approach, we studied stem cell-based regeneration in Hydra polyps.