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

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.

Decapitated Hydra regenerate their heads via morphallaxis, i.e., without significant contributions made by cell proliferation or interstitial stem cells. Indeed, Hydra depleted of interstitial stem cells regenerate robustly, and Wnt3 from epithelial cells triggers head regeneration. However, we find a different mechanism controlling regeneration after midgastric bisection in animals equipped with both epithelial and interstitial cell lineages. In this context, we see rapid induction of apoptosis and Wnt3 secretion among interstitial cells at the head- (but not foot-) regenerating site. Apoptosis is both necessary and sufficient to induce Wnt3 production and head regeneration, even at ectopic sites. Further, we identify a zone of proliferation beneath the apoptotic zone, reminiscent of proliferative blastemas in regenerating limbs and of compensatory proliferation induced by dying cells in Drosophila imaginal discs. We propose that different types of injuries induce distinct cellular modes of Hydra head regeneration, which nonetheless converge on a central effector, Wnt3.

Polyps of the cnidarian Hydra maintain their adult anatomy through two developmental organizers, the head organizer located apically and the foot organizer basally. The head organizer is made of two antagonistic cross-reacting components, an activator, driving apical differentiation and an inhibitor, preventing ectopic head formation. Here we characterize the head inhibitor by comparing planarian genes down-regulated when β-catenin is silenced to Hydra genes displaying a graded apical-to-basal expression and an up-regulation during head regeneration. We identify Sp5 as a transcription factor that fulfills the head inhibitor properties: leading to a robust multiheaded phenotype when knocked-down in Hydra, acting as a transcriptional repressor of Wnt3 and positively regulated by Wnt/β-catenin signaling. Hydra and zebrafish Sp5 repress Wnt3 promoter activity while Hydra Sp5 also activates its own expression, likely via β-catenin/TCF interaction. This work identifies Sp5 as a potent feedback loop inhibitor of Wnt/β-catenin signaling, a function conserved across eumetazoan evolution.

The genes belonging to the Paired class exert primary developmental functions. They are characterized by six invariant amino acid residues in the homeodomain, while the residue at position 50 can be a serine, glutamine or lysine as in the Pax-type, Q50 Paired-like or the K50 Paired-like homeodomains respectively. Genes in this class emerged early in animal evolution: three distinct Pax genes and two Q50 Paired-like genes have recently been characterised from cnidarians. Phylogenetic molecular reconstructions taking into account homeodomain and paired-domain sequences provide some new perspectives on the evolution of the Paired-class genes. Analysis of 146 Paired-class homeodomains from a wide range of metazoan taxa allowed us to identify 18 families among the three sub-classes from which the aristaless family displays the least diverged position. Both Pax-type and K50 families branch within the Q50 Paired-like sequences implying that these are the most ancestral. Consequently, most Pax genes arose from a Paired-like ancestor, via fusion of a Paired-like homebox gene with a gene encoding only a paired domain; the Cnidaria appear to contain genes representing the 'before' and 'after' fusion events.

The conservation of developmental functions exerted by Antpclass homeoproteins in protostomes and deuterostomes suggested that homologs with related functions are present in diploblastic animals. Our phylogenetic analyses showed that Antp-class homeodomains belong either to non-Hox or to Hox͞paraHox families. Among the 13 non-Hox families, 9 have diploblastic homologs, Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh͞Hex, Not, and Dlx, reported here. Among the Hox͞paraHox, poriferan sequences were not found, and the cnidarian sequences formed at least five distinct cnox families. Two are significantly related to the paraHox Gsx (cnox-2) and the mox (cnox-5) sequences, whereas three display some relatedness to the Hox paralog groups 1 (cnox-1), 9͞10 (cnox-3) and the paraHox cdx (cnox-4). Intermediate Hox͞paraHox genes (PG 3 to 8 and lox) did not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 were not found chromosomally linked within a 150-kb range and displayed specific expression patterns in the adult head. During regeneration, cnox-1 was expressed as an early gene whatever the polarity, whereas cnox-2 was up-regulated later during head but not foot regeneration. Finally, cnox-3 expression was reestablished in the adult head once it was fully formed. These results suggest that the Hydra genes related to anterior Hox͞paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral͞aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods. T he discovery of structural and functional homologies between regulatory genes used by Drosophila and vertebrates during their development led to the hypothesis that animals would share a common set of genes for defining the head, trunk, and posterior regions at early developmental stages (1-6). The proposed genes were homeobox genes belonging either to the Antp class, like empty-spiracle (emx), even-skipped (evx), Hox genes, or to the Prd class, like orthodenticle (Otx), goosecoid. Phylogenetic analyses performed on a vast amount of Hox homeodomain (HD) sequences, including representatives from all classes of homeobox genes from animals, protozoa, fungi, and plants, confirmed the monophyly of the Antp class as well as its position as a sister group to the Paired class (7). Within the Antp class, the Hox gene organization is distinctive and enigmatic: the genes map in clusters, and the order of individual genes within a cluster correlates with their temporospatial expression pattern along the anterior-posterior body axis during development (8). Recently, it was proposed that the common bilaterian ancestor of protostomes and deuterostomes had at least seven Hox genes (9). However, the question of the composition of the ancestral HOX cluster remains open. Analysis of Hox homeobox sequences (10) suggested that the conserved HOX cluster emerged early in the evolution of metazoans from an original cluster harboring three ancestral g...

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 (mechanosensory 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 neuromuscular transmission to arise and be maintained across evolution.

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. KEY WORDS: historical perspective, transplantation, modeling, developmental reactivation, Hydra regeneration, multipotency, stemness, symbiosis, environmentThe heuristic value of the Hydra model systemIn the early 18th century the word biology was not yet in use but the nature of living organisms and their evolutionary relationships was the focus of interest for philosophers and naturalists, as evidenced by their pioneering efforts to develop new tools such as microscopes that would allow finer observation (Palm, 1996). The microscopic observation of organisms taken in the field undoubtedly helped develop morphological keys to sort between the animal and vegetal kingdoms and to group them into phyla (Linnaeus, 1758). Among the ambiguous species that could not be easily classified were the seawater corals that looked like flowers (Watson, 1753;McConnell, 1990) and the freshwater Hydra polyp that was considered to exhibit both animal and vegetal features. For example, Hydra easily reproduces asexually through budding, a trait frequently assigned to plants or fungi.Having observed some Hydra polyps in a pond, Abraham Trembley (1710-1784) (who had received a PhD in mathematics from the University of Geneva (Switzerland) and was now educating the children of the Count of Bentick in the Netherlands) decided Int. J. Dev. Biol. 56: 411-423 (2012) to solve that problem by testing their capacity to regenerate, assuming that if Hydra regenerates, then it should be considered a plant, but if it does...