Distinct gene expression dynamics in developing and regenerating crustacean limbs.

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

The Hazards of Regeneration: From Morgan's Legacy to Evo-Devo.

In his prominent book Regeneration (1901), T.H. Morgan's collected and synthesized theoretical and experimental findings from a diverse array of regenerating animals and plants. Through his endeavor, he introduced a new way to study regeneration and its evolution, setting a conceptual framework that still guides today's research and that embraces the contemporary evolutionary and developmental approaches.In the first part of the chapter, we summarize Morgan's major tenets and use it as a narrative thread to advocate interpreting regenerative biology through the theoretical tools provided by evolution and developmental biology, but also to highlight potential caveats resulting from the rapid proliferation of comparative studies and from the expansion of experimental laboratory models. In the second part, we review some experimental evo-devo approaches, highlighting their power and some of their interpretative dangers. Finally, in order to further understand the evolution of regenerative abilities, we portray an adaptive perspective on the evolution of regeneration and suggest a framework for investigating the adaptive nature of regeneration.

Pattern regulation in a regenerating jellyfish.

Jellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around 'hubs' which serve as positional landmarks. Stabilization of these hubs, and associated expression of Wnt6, depends on the configuration of the adjoining muscle fiber 'spokes'. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/ß-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.

The multifaceted role of nerves in animal regeneration.

The discovery that the nervous system plays a critical role in salamander limb regeneration, in 1823, provided the first mechanistic insights into regenerative phenomena and stimulated a long quest for molecular regulators. A role for nerves in the context of regeneration has been suggested for most vertebrate and invertebrate groups, thus offering a possible shared mechanism for the regulation of regenerative processes among animals. Methodological differences and technical limitations, especially in invertebrate groups, have so far hampered broad comparisons and the search for common principles on the role of nerves. This review considers both old and recent work in this topic and provides a broad perspective on the roles of nerves during regeneration. Nerves are found consistently to have important roles in regeneration, but their mode of action varies across species. The ongoing technological developments in a broad range of invertebrate models are now paving the way for the discovery of the shared and unique roles of nerves in animal regeneration.

The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle.

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterized by distinct transcriptome signatures reflecting abrupt life-cycle transitions and all deploy a mixture of phylogenetically old and new genes. Medusa-specific transcription factors, including many with bilaterian orthologues, associate with diverse neurosensory structures. Compared to Clytia, the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content evolution and sequence evolution. Absence of expression and gene loss among Clytia orthologues of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys the ancestral cnidarian-bilaterian transcription factor gene complement.

A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol.

In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence-the hybridization step, followed by detection of annealed sequences by means of fluorescent or colorimetric reactions. Successful hybridization requires permeabilization of tissues, followed by denaturation of nucleic acids strands, which is usually carried out in a formamide-based buffer and at high temperatures. Such reaction conditions, besides posing a health hazard (both concerning manipulation and waste disposal), can be excessively harsh for the delicate tissues of some species or developmental stages. We detail here an alternative method for in situ hybridization, where the toxic formamide is replaced with a urea solution. This substitution improved both tissues preservation and signal-to-noise detection, in several animal species. The protocol described here, originally developed for the hydrozoan jellyfish Clytia hemisphaerica, provides guidelines for adapting formamide-based traditional protocols to the urea variant. Urea-based protocols have already been successfully applied to diverse invertebrate and vertebrate species, showing the ease of such a modification, and providing the scientific community with a promising, safer and versatile tool.

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species.

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has greatly advanced our understanding of biological processes, including developmental regulation. In situ protocols are today routinely followed in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acid sequence. This step is generally carried out at high temperatures and in a denaturing solution, called hybridization buffer, commonly containing 50% (v/v) formamide - a hazardous chemical. When applied to the soft-bodied hydrozoan medusa Clytia hemisphaerica, we found that this traditional hybridization approach was not fully satisfactory, causing extensive deterioration of morphology and tissue texture which compromised our observation and interpretation of results. We thus tested alternative solutions for in situ detection of gene expression and, inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8M urea solution in the hybridization buffer. Our new protocol not only yielded better morphologies and tissue consistency, but also notably improved the resolution of the signal, allowing more precise localization of gene expression and reducing aspecific staining associated with problematic areas. Given the improved results and reduced manipulation risks, we tested the urea protocol on other metazoans, two brachiopod species (Novocrania anomala and Terebratalia transversa) and the priapulid worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea during in situ hybridization offers a safer alternative, potentially of widespread use in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

Development of the aboral domain in Nematostella requires ß-catenin and the opposing activities of Six3/6 and Frizzled5/8.

The development of the oral pole in cnidarians and the posterior pole in bilaterians is regulated by canonical Wnt signaling, whereas a set of transcription factors, including Six3/6 and FoxQ2, controls aboral development in cnidarians and anterior identity in bilaterians. However, it is poorly understood how these two patterning systems are initially set up in order to generate correct patterning along the primary body axis. Investigating the early steps of aboral pole formation in the sea anemone Nematostella vectensis, we found that, at blastula stage, oral genes are expressed before aboral genes and that Nvß-catenin regulates both oral and aboral development. In the oral hemisphere, Nvß-catenin specifies all subdomains except the oral-most, NvSnailA-expressing domain, which is expanded upon Nvß-catenin knockdown. In addition, Nvß-catenin establishes the aboral patterning system by promoting the expression of NvSix3/6 at the aboral pole and suppressing the Wnt receptor NvFrizzled5/8 at the oral pole. NvFrizzled5/8 expression thereby gets restricted to the aboral domain. At gastrula stage, NvSix3/6 and NvFrizzled5/8 are both expressed in the aboral domain, but they have opposing activities, with NvSix3/6 maintaining and NvFrizzled5/8 restricting the size of the aboral domain. At planula stage, NvFrizzled5/8 is required for patterning within the aboral domain and for regulating the size of the apical organ by modulation of a previously characterized FGF feedback loop. Our findings suggest conserved roles for Six3/6 and Frizzled5/8 in aboral/anterior development and reveal key functions for Nvß-catenin in the patterning of the entire oral-aboral axis of Nematostella.

A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.

Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.

Molecular characterization of the apical organ of the anthozoan Nematostella vectensis.

Apical organs are sensory structures present in many marine invertebrate larvae where they are considered to be involved in their settlement, metamorphosis and locomotion. In bilaterians they are characterised by a tuft of long cilia and receptor cells and they are associated with groups of neurons, but their relatively low morphological complexity and dispersed phylogenetic distribution have left their evolutionary relationship unresolved. Moreover, since apical organs are not present in the standard model organisms, their development and function are not well understood. To provide a foundation for a better understanding of this structure we have characterised the molecular composition of the apical organ of the sea anemone Nematostella vectensis. In a microarray-based comparison of the gene expression profiles of planulae with either a wildtype or an experimentally expanded apical organ, we identified 78 evolutionarily conserved genes, which are predominantly or specifically expressed in the apical organ of Nematostella. This gene set comprises signalling molecules, transcription factors, structural and metabolic genes. The majority of these genes, including several conserved, but previously uncharacterized ones, are potentially involved in different aspects of the development or function of the long cilia of the apical organ. To demonstrate the utility of this gene set for comparative analyses, we further analysed the expression of a subset of previously uncharacterized putative orthologs in sea urchin larvae and detected expression for twelve out of eighteen of them in the apical domain. Our study provides a molecular characterization of the apical organ of Nematostella and represents an informative tool for future studies addressing the development, function and evolutionary history of apical organ cells.

The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian.

The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone Nematostella vectensis. We show that orthologs of the bilaterian anterior developmental genes six3/6, foxQ2, and irx have dynamic expression patterns in the aboral region of Nematostella. Functional analyses reveal that NvSix3/6 acts upstream of NvFoxQ2a as a key regulator of the development of a broad aboral territory in Nematostella. NvSix3/6 initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of NvSix3/6 and NvFoxQ2a in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by NvFGFa1 is specifically required for the development of the apical organ, whereas NvSix3/6 has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.

Muscle differentiation in a colonial ascidian: organisation, gene expression and evolutionary considerations.

BACKGROUND: Ascidians are tunicates, the taxon recently proposed as sister group to the vertebrates. They possess a chordate-like swimming larva, which metamorphoses into a sessile adult. Several ascidian species form colonies of clonal individuals by asexual reproduction. During their life cycle, ascidians present three muscle types: striated in larval tail, striated in the heart, and unstriated in the adult body-wall. RESULTS: In the colonial ascidian Botryllus schlosseri, we investigated organisation, differentiation and gene expression of muscle beginning from early buds to adults and during zooid regression. We characterised transcripts for troponin T (BsTnT-c), adult muscle-type (BsMA2) and cytoplasmic-type (BsCA1) actins, followed by in situ hybridisation (ISH) on sections to establish the spatio-temporal expression of BsTnT-c and BsMA2 during asexual reproduction and in the larva. Moreover, we characterised actin genomic sequences, which by comparison with other metazoans revealed conserved intron patterns. CONCLUSION: Integration of data from ISH, phalloidin staining and TEM allowed us to follow the phases of differentiation of the three muscle kinds, which differ in expression pattern of the two transcripts. Moreover, phylogenetic analyses provided evidence for the close relationship between tunicate and vertebrate muscle genes. The characteristics and plasticity of muscles in tunicates are discussed.