Department of Genetics and Evolution, Institute of Genetics and Genomics in Geneva (iGE3), University of Geneva, Geneva, Switzerland
BACKGROUND: Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. RESULTS: To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48'909 unique sequences including splice variants, representing approximately 24'450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10'597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11'270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. CONCLUSIONS: We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events.
Department of Genetics and Evolution, University of Geneva, 30 quai Ernest Ansermet, CH-1211 Geneva-04, Switzerland. email@example.com
The freshwater Hydra polyp provides a unique model system to decipher the mechanisms underlying adult regeneration. Indeed, a single cut initiates two distinct regenerative processes, foot regeneration on one side and head regeneration on the other side, the latter relying on the rapid formation of a local head organizer. Two aspects are discussed here: the asymmetric cellular remodeling induced by mid-gastric bisection and the signaling events that trigger head organizer formation. In head-regenerating tips (but not in foot ones), a wave of cell death takes place immediately, leading the apoptotic cells to transiently release Wnt3 and activate the beta-catenin pathway in the neighboring cycling cells to push them through mitosis. This process, which mimics the apoptosis-induced compensatory proliferation process deciphered in Drosophila larvae regenerating their discs, likely corresponds to an evolutionarily conserved mechanism, also at work in Xenopus tadpoles regenerating their tail or mice regenerating their skin or liver. How is this process generated in Hydra? Several studies pointed to the necessary activation of the extracellular signal-regulated kinase (ERK) 1-2 and mitogen-activated protein kinase (MAPK) pathways during early head regeneration. Indeed inhibition of ERK 1-2 or knockdown of RSK, cAMP response element-binding protein (CREB), and CREB-binding protein (CBP) prevent injury-induced apoptosis and head regeneration. The current scenario involves an asymmetric activation of the MAPK/CREB pathway to trigger injury-induced apoptosis in the interstitial cells and in the epithelial cells a CREB/CBP-dependent transcriptional activation of early genes essential for head-organizing activity as wnt3, HyBra1, and prdl-a. The question now is how bisection in the rather uniform central region of the polyp can generate this immediately asymmetric signaling.
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA. firstname.lastname@example.org
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
Department of Genetics and Evolution, University of Geneva, Switzerland. email@example.com
The discovery of Hydra regeneration by Abraham Trembley in 1744 promoted much scientific curiosity thanks to his clever design of experimental strategies away from the natural environment. Since then, this little freshwater cnidarian polyp flourished as a potent and fruitful model system. Here, we review some general biological questions that benefitted from Hydra research, such as the nature of embryogenesis, neurogenesis, induction by organizers, sex reversal, symbiosis, aging, feeding behavior, light regulation, multipotency of somatic stem cells, temperature-induced cell death, neuronal transdifferentiation, to cite only a few. To understand how phenotypes arise, theoricists also chose Hydra to model patterning and morphogenetic events, providing helpful concepts such as reaction-diffusion, positional information, and autocatalysis combined with lateral inhibition. Indeed, throughout these past 270 years, scientists used transplantation and grafting experiments, together with tissue, cell and molecular labelings, as well as biochemical procedures, in order to establish the solid foundations of cell and developmental biology. Nowadays, thanks to transgenic, genomic and proteomic tools, Hydra remains a promising model for these fields, but also for addressing novel questions such as evolutionary mechanisms, maintenance of dynamic homeostasis, regulation of stemness, functions of autophagy, cell death, stress response, innate immunity, bioactive compounds in ecosystems, ecotoxicant sensing and science communication.
University of Geneva, Switzerland
As scientists it is our duty to fight against obscurantism and loss of rational thinking if we want politicians and citizens to freely make the most intelligent choices for the future generations. With that aim, the scientific education and training of young students is an obvious and urgent necessity. We claim here that Hydra provides a highly versatile but cheap model organism to study biology at any age. Teachers of biology have the unenviable task of motivating young people, who with many other motivations that are quite valid, nevertheless must be guided along a path congruent with a 'syllabus' or a 'curriculum'. The biology of Hydra spans the history of biology as an experimental science from Trembley's first manipulations designed to determine if the green polyp he found was plant or animal to the dissection of the molecular cascades underpinning, regeneration, wound healing, stemness, aging and cancer. It is described here in terms designed to elicit its wider use in classrooms. Simple lessons are outlined in sufficient detail for beginners to enter the world of 'Hydra biology'. Protocols start with the simplest observations to experiments that have been pretested with students in the USA and in Europe. The lessons are practical and can be used to bring 'life', but also rational thinking into the study of life for the teachers of students from elementary school through early university.
Department of Genetics and Evolution, Faculty of Science, University of Geneva, Switzerland.
In the freshwater cnidarian polyp Hydra, cell death takes place in multiple contexts. Indeed apoptosis occurs during oogenesis and spermatogenesis, during starvation, and in early head regenerating tips, promoting local compensatory proliferation at the boundary between heterografts. Apoptosis can also be induced upon exposure to pro-apoptotic agents (colchicine, wortmannin), upon heat-shock in the thermosensitive sf-1 mutant, and upon wounding. In all these contexts, the cells that undergo cell death belong predominantly to the interstitial cell lineage, whereas the epithelial cells, which are rather resistant to pro-apoptotic signals, engulf the apoptotic bodies. Beside this clear difference between the interstitial and the epithelial cell lineages, the different interstitial cell derivatives also show noticeable variations in their respective apoptotic sensitivity, with the precursor cells appearing as the most sensitive to pro-apoptotic signals. The apoptotic machinery has been well conserved across evolution. However, its specific role and regulation in each context are not known yet. Tools that help characterize apoptotic activity in Hydra have recently been developed. Among them, the aposensor Apoliner initially designed in Drosophila reliably measures wortmannin-induced apoptotic activity in a biochemical assay. Also, flow cytometry and TUNEL analyses help identify distinctive features between wortmannin-induced and heat-shock induced apoptosis in the sf-1 strain. Thanks to the live imaging tools already available, Hydra now offers a model system with which the functions of the apoptotic machinery to maintain long-term homeostasis, stem cell renewal, germ cell production, active developmental processes and non-self response can be deciphered.
Department of Genetics and Evolution, Faculty of Science, University of Geneva, Sciences III, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.
Cnidarians belong to the first phylum differentiating a nervous system, thus providing suitable model systems to trace the origins of neurogenesis. Indeed corals, sea anemones, jellyfish and hydra contract, swim and catch their food thanks to sophisticated nervous systems that share with bilaterians common neurophysiological mechanisms. However, cnidarian neuroanatomies are quite diverse, and reconstructing the urcnidarian nervous system is ambiguous. At least a series of characters recognized in all classes appear plesiomorphic: (1) the three cell types that build cnidarian nervous systems (sensory-motor cells, ganglionic neurons and mechanosensory cells called nematocytes or cnidocytes); (2) an organization of nerve nets and nerve rings [those working as annular central nervous system (CNS)]; (3) a neuronal conduction via neurotransmitters; (4) a larval anterior sensory organ required for metamorphosis; (5) a persisting neurogenesis in adulthood. By contrast, the origin of the larval and adult neural stem cells differs between hydrozoans and other cnidarians; the sensory organs (ocelli, lens-eyes, statocysts) are present in medusae but absent in anthozoans; the electrical neuroid conduction is restricted to hydrozoans. Evo-devo approaches might help reconstruct the neurogenic status of the last common cnidarian ancestor. In fact, recent genomic analyses show that if most components of the postsynaptic density predate metazoan origin, the bilaterian neurogenic gene families originated later, in basal metazoans or as eumetazoan novelties. Striking examples are the ParaHox Gsx, Pax, Six, COUP-TF and Twist-type regulators, which seemingly exert neurogenic functions in cnidarians, including eye differentiation, and support the view of a two-step process in the emergence of neurogenesis.
Department of Genetics and Evolution, University of Geneva, Sciences III, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.
After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.
The Hydra model system is well suited to decipher the mechanisms underlying adult regeneration, specifically those that were robust enough to be maintained across evolution. After mid-gastric bissection head regeneration in Hydra relies on apoptosis-induced compensatory proliferation via the release of Wnt3 by the apoptotic interstitial cells and activation of the beta-catenin pathway in the surrounding cycling interstitial cells. As apoptosis-induced compensatory proliferation is also at work in Drosophila regenerating imaginal discs, Xenopus tadpole regenerating their tail and mice regenerating their skin or their liver, this mechanism might represent an evolutionarily-conserved way to launch a regenerative response. However after decapitation, the analysis of the activation of the canonical Wnt pathway in decapitated Hydra showed that apoptosis-induced compensatory proliferation does not take place in this context. Given that the proportion of interstitial stem cells is significantly higher in the middle part than in the upper part of the body column, this suggested that the route taken to regenerate a structure as complex as the head dramatically varies according to the homeostatic status of the tissue at the time of injury. From these observations, we propose a tri-modular model for animal regeneration where the first module or "wound healing module" is followed by a transient module named "inducing module of regeneration" that allows the recruitment of the third module named "re-development module", necessary for repatterning the missing structure. We claim that among these three modules, the inducing module of regeneration is the most drastically constrained by the homeostatic conditions of any given tissue or organ at the time of injury and therefore the most variable.
Department of Zoology and Animal Biology, Faculty of Sciences, University of Geneva, 30 Quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland. firstname.lastname@example.org
The Hydra model system is well suited for the eludication of the mechanisms underlying regeneration in the adult, and an understanding of the core mechanisms is likely to cast light on pathways conserved in other species. Recent detailed analyses of the activation of the Wnt-beta-catenin pathway in bisected Hydra shows that the route taken to regenerate a structure as complex as the head varies dramatically according to the level of the amputation. When decapitation induces direct re-development due to Wnt3 signaling from epithelial cells, head regeneration after mid-gastric section relies first on Wnt3 signaling from interstitial cells, that undergo apoptosis-induced compensatory proliferation, and subsequently on activation of Wnt3 signaling in the epithelial cells. The relative distribution between stem cells and head progenitor cells is strikingly different in these two contexts, indicating that the pre-amputation homeostatic conditions define and constrain the route that bridges wound-healing to the re-development program of the missing structure.
University of Hereford, BioComputation Research Library, College Lane, Hatfield AL10 9AB, United Kingdom.
Faculty of Sciences, Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland.
Over the past decades, genetic analyses performed in vertebrate and invertebrate organisms deciphered numerous cellular and molecular mechanisms deployed during sexual development and identified genetic circuitries largely shared among bilaterians. In contrast, the functional analysis of the mechanisms that support regenerative processes in species randomly scattered among the animal kingdom, were limited by the lack of genetic tools. Consequently, unifying principles explaining how stress and injury can lead to the reactivation of a complete developmental program with restoration of original shape and function remained beyond reach of understanding. Recent data on cell plasticity suggest that beside the classical developmental approach, the analysis of homeostasis and asexual reproduction in adult organisms provides novel entry points to dissect the regenerative potential of a given species, a given organ or a given tissue. As a clue, both tissue homeostasis and regeneration dynamics rely on the availability of stem cells and/or on the plasticity of differentiated cells to replenish the missing structure. The freshwater Hydra polyp provides us with a unique model system to study the intricate relationships between the mechanisms that regulate the maintenance of homeostasis, even in extreme conditions (starvation and overfeeding) and the reactivation of developmental programs after bisection or during budding. Interestingly head regeneration in Hydra can follow several routes according to the level of amputation, suggesting that indeed the homeostatic background dramatically influences the route taken to bridge injury and regeneration. Mol. Reprod. Dev. (c) 2010 Wiley-Liss, Inc.