1,721,097 research outputs found
Noninvasive Intravascular Microtransfusion in Colonial Tunicates
No full textTunicates are a diverse group of worldwide marine filter-feeders that are vertebrates' closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied
Re-engineering development to instruct tissue regeneration
No full textWith few exceptions, tissue regeneration strategies based on the conventional combination of cells, scaffolding materials, and soluble factors (tissue engineering) have introduced a rather limited clinical impact. While it is being recognized that the non-convincing benefits of engineered grafts require more fundamental knowledge on mechanisms of action and potency factors, the attempt to mimic and recapitulate developmental events has inspired an evolution of the paradigm. In the context of skeletal regeneration, a "developmental engineering" approach has been advocated to generate intermediate grafts (i.e., hypertrophic cartilage templates) which, as suggested by limb developmental biology, are capable of autonomous spatial and temporal evolution into fully functional bone organs. However, limited consideration has been given to the fact that the recipient site within adult organisms may not be compatible with well-established developmental processes. This can be due to the possibly restricted function of resident progenitors, to the critical mechanical and physical boundary conditions of mature organs, or to the strong role of inflammatory signals and immune cells at repair sites. We thus propose that predictable, orderly, and durable tissue regeneration should be based on a "developmental RE-engineering" paradigm, with the challenge to instruct the execution of developmental programs in the context of an adult system
Whole-Body Regeneration
Get PDFThis Open Access volume provides a comprehensive overview of the latest tools available to scientists to study the many facets of whole-body regeneration (WBR). The chapters in this book are organized into six parts. Part One provides a historical overview on the study of the WBR phenomena focusing on the primary challenges of this research. Parts Two and Three explore a series of non-vertebrate zoological contexts that provide experimental models for WBR, showing how they can be approached with cellular tools. Parts Four, Five, and Six discuss the future advancements of WBR, reporting about the cutting-edge techniques in genetics and omics used to dissect the underlying mechanisms of WBR, and systems biology approaches to reach a synthetic view of WBR. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and thorough, Whole-Body Regeneration: Methods and Protocols is a valuable resource for scientists and researchers who want to learn more about this important and developing field
Comparative Analysis of RNA-seq transcriptomes in aging and non-aging Hydra
No full textThe cnidarian Hydra exhibits extremely low senescence when maintained in homeostatic conditions. However H. oligactis animals that undergo sexual differentiation upon transfer from 18°C to 10°C, exhibit a visible aging. Our laboratory characterized two H. oligactis male strains that exhibit different phenotypes once they enter gametogenesis: the cold-sensitive strain (Ho_CS) that exhibits a typical cold-induced degenerative phenotype, and the cold-resistant strain (Ho_CR) that returns to homeostasis. In this study, we performed a large-scale comparative transcriptomic analysis to identify structural as well as regulatory genetic differences between these two strains. We identified a TNF receptor-associated factor (TRAF) that shows a series of deletions in Ho_CS when compared to Ho_CR. We also characterized genes differentially expressed over time between the two strains, such as CIRBP, IMPDH1, TRPA1 and TRPM3, which are orthologs to human proteins involved in response to cold or sensory perception of temperature stimulus, as well as sequences that encode uncharacterized proteins. Moreover, we characterized genes reported in the Human Ageing Genomic Resources (HAGR) GenAge database revealed supplementary interesting candidates such as orthologs for AIFM1, EIF5A2, ERCC4, GCLC, GSK3A, HDAC, HSPD1, MLH1, MAPK3, SOCS2, TRAP1, UCHL1 and ZMPSTE24, which are temporarily differentially expressed between the two strains. TRAP1 and HSPD1 are heat shock proteins, the latter being also involved in the response to cold. We thus retrieved interesting genes candidates that may influence aging phenotype following exposure to cold in Hydra
Biochemical and Molecular Bases of Lipid Metabolism in Hydra vulgaris
No full textHere we have studied some biochemical and molecular characteristics of lipid metabolism in the Hydra cnidarian polyp. We have characterized variations in lipid metabolism in response to diet by establishing lipid profiles in starved and fed Hydra, and quantifying with the Red Nile technique the accumulation of lipid droplets in the digestive cells upon starvation. As expected, Hydra uses lipid reserves as fuel for survival, suggesting that fatty acids and cholesterol in the diet are esterified and stored when abundant and then released when fasting. During regeneration, specific classes of lipids appear to be needed, possibly for cell membrane synthesis and/or specific signalling. We finally identified five orthologs of the bilaterian genes encoding the dihydrolipoamide branched-chain transacylase (DBT), hydroxyacyl-CoA dehydrogenase (HCD), choline phosphotransefrase (CHPT), phosphatidylcholine Sterol-O acyltransferase (PCSAT) and lipophorin receptor /LDL (LpR/LDL). Phylogenetic analyses confirm that these enzymes and receptor essential for lipid metabolism are conserved in eumtazoans
Random Integration Transgenesis in a Free-Living Regenerative Flatworm Macrostomum lignano
Get PDFRegeneration-capable flatworms are highly informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. Transgenesis is a powerful research tool for investigating gene function, but until recently, a transgenesis method was missing in flatworms, hampering their wider adoption in biomedical research. Here we describe a detailed protocol to create stable transgenic lines of the flatworm M. lignano using random integration of DNA constructs through microinjection into single-cell stage embryos.</p
Analyse de la régulation du gène CBP lors des processus morphogénétiques de l'hydre
No full textIn Hydra, the transcription factor CREB is rapidly phosphorylated after amputation in the endodermal cells of the head-regenerating tips, a regulation that plays an important role in the head regeneration process. In bilaterians, the phosphorylated form of CREB binds to the CREB binding protein (CBP), which acts as a general co-activator of transcription. To find out if this regulatory cascade also occurs in Hydra, we analyzed the Hydra CBP protein which we found to be highly conserved during evolution. We detected a high level of CBP transcripts in the endodermal cells of the head-forming regions, either head-regeneration tips or buds growing from intact animals. We also noted hyper-acetylation of histone-4 in head-regenerating tips, and a marked negative effect of Scriptaid, a deacetylase inhibitory drug, on the efficacy of head regeneration. These preliminary results suggest that Hydra CBP could, via chromatin modifications, participate in the reactivation of the head re-development program
The Role and Regulation of the Transcription Factor SP5 in <i>Hydra</i> Patterning
No full textThe Wnt/b-catenin signaling pathway is highly conserved in all metazoans, involved in numerous developmental processes and regulating stem cell proliferation in adulthood. Disruption of the Wnt/b-catenin signaling pathway induces a range of abnormalities in embryonic development as well as important adult pathologies such as cancer. In Hydra, the Wnt/b-catenin signaling pathway plays a key role in the apical organizer that maintains apical patterning and allows the development of new heads. The objective of this PhD project is to better understand the regulation of the Wnt/b-catenin pathway during Hydra regeneration and maintenance of the apical organizer. We designed strategies to understand the dialogue between Wnt/b-catenin signaling and Sp5 in the maintenance of the apical organizer in adult animals and in the formation of a new organizer during developmental processes such as budding and regeneration.
We first identified the transcription factor Sp5 as an inhibitor of the apical organizing center in Hydra, and show that this gene fulfills the five conditions necessary for an apical inhibitor. Briefly, this Sp5 gene is (1) predominantly expressed in the head, with a graded expression profile from apical to basal; (2) up-regulated during apical regeneration; (3) its expression is induced by the Wnt/ b-catenin signaling pathway; (4) Sp5 inhibits the expression of Wnt3 and b-catenin, thereby inhibiting the activity of this signaling pathway; and (5) Sp5 restricts apical development.
In the following chapter, we focused on the study of Sp5 regulation in vivo. We generated two transgenic lines, which constitutively express either in the epidermal layer or in the gastrodermal layer a unique construct, whose tandem structure allows the expression of the mCherry reporter gene under the control of the Hydra actin gene promoter on the one hand, and the eGFP reporter gene under the control of the Hydra Sp5 promoter on the other. This approach revealed that in the intact animal, especially in the apical organizer region, Sp5 has a distinct spatial regulation in the epidermis and in the gastrodermis.
In conclusion, this study highlights the fine regulation of Sp5, a target gene of the Wnt/b-catenin signaling pathway, as well as the putative interactions between Wnt3 and Sp5 in the different cell layers of Hydra, either in intact or developing animals, or in animals subjected to pharmacological or genetic perturbations. It also highlights in the model organism of Hydraexpression patterns of Sp5 and Wnt3 that are different in the two modes of functioning of the apical organizer, homeostatic in adult animals on the one hand, dynamic because in the process of formation or newly formed on the other hand. </p
Injury-induced signaling promoting the reactivation of developmental programs in regenerating Hydra
No full textThe purpose of my PhD thesis is to contribute to the characterization of the immediate signaling events that trigger cellular remodeling processes in an organism undergoing regeneration. The laboratory of Prof. Brigitte Galliot uses the cnidarian Hydra, one of the ‘oldest' known model system, to study regeneration. Indeed, advances in the field of Hydra regeneration contribute to a comprehensive knowledge of regenerative mechanisms across the animal kingdom. We present three questions addressed in this thesis, aimed at deepening our understanding of the amazing regenerative ability in Hydra: 1) Which immediate injury-induced signal promotes regeneration in Hydra? 2) More specifically, what are the injury-induced signals that promote MAPK activation and regenerative cell death? 3) How do injury-induced signals contribute to the reactivation of developmental programs
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