Abstract: New perspectives on the origin of neurogenesis emerged with the identification of genes encoding post-synaptic proteins as well as many "neurogenic" regulators as the NK, Six, Pax, bHLH proteins in the Demosponge genome, a species that might differentiate sensory cells but no neurons. However, poriferans seem to miss some key regulators of the neurogenic circuitry as the Hox/paraHox and Otx-like gene families. Moreover as a general feature, many gene families encoding evolutionarily-conserved signaling proteins and transcription factors were submitted to a wave of gene duplication in the last common eumetazoan ancestor, after Porifera divergence. In contrast gene duplications in the last common bilaterian ancestor, Urbilateria, are limited, except for the bHLH Atonal-class. Hence Cnidaria share with Bilateria a large number of genetic tools. The expression and functional analyses currently available suggest a neurogenic function for numerous orthologs in developing or adult cnidarians where neurogenesis takes place continuously. As an example, in the Hydra polyp, the Clytia medusa and the Acropora coral, the Gsx/cnox2/Anthox-2 ParaHox gene likely supports neurogenesis. Also neurons and nematocytes (mechano-sensory cells) share in hydrozoans a common stem cell and several regulatory genes indicating that they can be considered as sister cells. Performed in anthozoan and medusozoan species, these studies should tell us more about the way(s) evolution hazards achieved the transition from epithelial to neuronal cell fate, and about the robustness of the genetic circuitry that allowed neuro-muscular transmission to arise and be maintained across evolution.

Abstract: The Hydra polyp provides a powerful model system to investigate the regulation of cell survival and cell death in homeostasis and regeneration as Hydra survive weeks without feeding and regenerates any missing part after bisection. Induction of autophagy during starvation is the main surviving strategy in Hydra as autophagic vacuoles form in most myoepithelial cells after several days. When the autophagic process is inhibited, animal survival is actually rapidly jeopardized. An appropriate regulation of autophagy is also essential during regeneration as Hydra RNAi knocked-down for the serine protease inhibitor Kazal-type (SPINK) gene Kazal1, exhibit a massive autophagy after amputation that rapidly compromises cell and animal survival. This excessive autophagy phenotype actually mimics that observed in the mammalian pancreas when SPINK genes are mutated, highlighting the paradigmatic value of the Hydra model system for deciphering pathological processes. Interestingly autophagy during starvation predominantly affects ectodermal epithelial cells and lead to cell survival whereas Kazal1(RNAi)-induced autophagy is restricted to endodermal digestive cells that rapidly undergo cell death. This indicates that distinct regulations that remain to be identified, are at work in these two contexts. Cnidarian express orthologs for most components of the autophagy and TOR pathways suggesting evolutionarily-conserved roles during starvation.

Abstract: In early October 2008, researchers from diverse backgrounds gathered at an EMBO conference entitled 'The Molecular and Cellular Basis of Regeneration and Tissue Repair' to discuss the basic biology of regeneration. Topics included cell plasticity in regenerative and developmental contexts, and the link between wound healing and regeneration. The meeting also highlighted the progress made in identifying the molecular networks that underlie regeneration in a variety of model systems.

Abstract: Hox and ParaHox (H/P) genes belong to evolutionary-sister clusters that arose through duplication of a ProtoHOX cluster early in animal evolution. In contrast to bilaterians, cnidarians express, beside PG1, PG2 and Gsx orthologs, numerous Hox-related genes with unclear origin. We characterized from marine hydrozoans three novel Hox-related genes expressed at medusa and polyp stages, which include a Pdx/Xlox ParaHox ortholog induced 1 day later than Gsx during embryonic development. To reconstruct H/P genes' early evolution, we performed multiple systematic comparative phylogenetic analyses, which identified derived sequences that blur the phylogenetic picture, recorded dramatically different evolutionary rates between ParaHox and Hox in cnidarians and showed the unexpected grouping of [Gsx-Pdx/Xlox-PG2-PG3] families in a single metagroup distinct from PG1. We propose a novel more parsimonious evolutionary scenario whereby H/P genes originated from a [Gsx-Pdx/Xlox-PG2-PG3]-related ProtoHox gene, the "posterior" and "anterior" H/P genes appearing secondarily. The ProtoHOX cluster would have contained the three Gsx/PG2, Pdx/PG3, Cdx/PG9 paralogs and produced through tandem duplication the primordial HOX and ParaHOX clusters in the Cnidaria-Bilateria ancestor. The stronger constraint on cnidarian ParaHox genes suggests that the primary function of pre-bilaterian H/P genes was to drive cellular evolutionary novelties such as neurogenesis rather than axis specification.

Abstract: In hydra, the regulation of the balance between cell death and cell survival is essential to maintain homeostasis across the animal and promote animal survival during starvation. Moreover, this balance also appears to play a key role during regeneration of the apical head region. The recent finding that autophagy is a crucial component of this balance strengthens the value of the Hydra model system to analyze the implications of autophagy in starvation, stress response and regeneration. We describe here how we adapted to Hydra some established tools to monitor steady-state autophagy. The ATG8/LC3 marker used in biochemical and immunohistochemical analyses showed a significant increase in autophagosome formation in digestive cells after 11 days of starvation. Moreover, the maceration procedure that keeps intact the morphology of the various cell types allows the quantification of the autophagosomes and autolysosomes in any cell type, thanks to the detection of the MitoFluor or LysoTracker dyes combined with the anti-LC3, anti-LBPA, and/or anti-RSK (ribosomal S6 kinase) immunostaining. The classical activator (rapamycin) and inhibitors (wortmannin, bafilomycin A(1)) of autophagy also appear to be valuable tools to modulate autophagy in hydra, as daily-fed and starved hydra display slightly different responses. Finally, we show that the genetic circuitry underlying autophagy can be qualitatively and quantitatively tested through RNA interference in hydra repeatedly exposed to double-stranded RNAs.

Abstract:

Abstract: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Abstract: In eukaryotic cells, a multiplicity of extra-cellular signals can activate a unique signal transduction system that at the nuclear level will turn on a variety of target genes, eliciting thus diverse responses adapted to the initial signal. How distinct signals can converge on a unique signalling pathway that will nevertheless produce signal-specific responses provides a theoretical paradox that can be traced back early in evolution. In bilaterians, the CREB pathway connects diverse extra-cellular signals via cytoplasmic kinases to the CREB transcription factor and the CBP co-activator, regulating according to the context, cell survival, cell proliferation, cell differentiation, pro-apoptosis, long-term memory, hence achieving a "hub" function for cellular and developmental processes. In hydra, the CREB pathway is highly conserved and activated during early head regeneration through RSK-dependent CREB phosphorylation. We show here that the CREB transcription factor and the RSK kinase are co-expressed in all three hydra cell lineages including dividing interstitial stem cells, proliferating nematoblasts, proliferating spermatogonia and spermatocytes, differentiating and mature neurons as well as ectodermal and endodermal myoepithelial cells. In addition, CREB gene expression is specifically up-regulated during early regeneration and early budding. When the CREB function was chemically prevented, the early post-amputation induction of the HyBraI gene was no longer observed and head regeneration was stacked. Thus, in hydra, the CREB pathway appears already involved in multiple tasks, such as reactivation of developmental programs in an adult context, self-renewal of stem cells, proliferation of progenitors and neurogenesis. Consequently, the hub function played by the CREB pathway was established early in animal evolution and might have contributed to the formation of an efficient oral pole through the integration of the neurogenic and patterning functions.

Abstract: The recent establishment of gene silencing through RNA interference upon feeding opens avenues to decipher the genetic control of regeneration in hydra. Following that approach, we identified three main stages for head regeneration. Immediately post-amputation, the serine protease inhibitor Kazal1 gene produced by the gland cells prevents from an excessive autophagy in regenerating tips. This cytoprotective function, or self-preservation, is similar to that played by Kazal-type proteins in the mammalian exocrine pancreas, in homeostatic or post-injury conditions, likely reflecting an evolutionarily conserved mechanism linking cell survival to tissue repair. Indeed, in wild-type hydra, within the first hours following mid-gastric section, an extensive cellular remodelling is taking place, including phenotypic cellular transitions and cell proliferation. The activation of the MAPK pathway, which leads to the RSK-dependent CREB phosphorylation, is required for these early cellular events. Later, at the early-late stage, the expression of the Gsx/cnox-2 ParaHox gene in proliferating apical neuronal progenitors is required for the de novo neurogenesis that precedes the emergence of the tentacle rudiments. Hence, head regeneration in wild-type hydra relies on spatially restricted and timely orchestrated cellular modifications, which display similarities with those reported during vertebrate epimorphic regeneration. These results suggest some conservation across evolution of the mechanisms driving the post-amputation reactivation of developmental programs.

Abstract: Because head regeneration occurs in nerve-free hydra mutants, neurogenesis was regarded as dispensable for this process. Here, in wild-type hydra, we tested the function of the ParaHox gsx homolog gene, cnox-2, which is a specific marker for bipotent neuronal progenitors, expressed in cycling interstitial cells that give rise to apical neurons and gastric nematoblasts (i.e. sensory mechanoreceptor precursors). cnox-2 RNAi silencing leads to a dramatic downregulation of hyZic, prdl-a, gsc and cnASH, whereas hyCOUP-TF is upregulated. cnox-2 indeed acts as an upstream regulator of the neuronal and nematocyte differentiation pathways, as cnox-2(-) hydra display a drastic reduction in apical neurons and gastric nematoblasts, a disorganized apical nervous system and a decreased body size. During head regeneration, the locally restricted de novo neurogenesis that precedes head formation is cnox-2 dependent: cnox-2 expression is induced in neuronal precursors and differentiating neurons that appear in the regenerating tip; cnox-2 RNAi silencing reduces this de novo neurogenesis and delays head formation. Similarly, the disappearance of cnox-2(+) cells in sf-1 mutants also correlates with head regeneration blockade. Hence in wild-type hydra, head regeneration requires the cnox-2 neurogenic function. When neurogenesis is missing, an alternative, slower and less efficient, head developmental program is possibly activated.