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Sponges (phylum Porifera) exhibit surprisingly complex tissue dynamics, maintaining constant cell turnover and migration, rearranging internal structures, and regenerating after severe injuries. Such tissue plasticity relies on the activity of proliferating cells represented primarily by the food-entrapping cells, choanocytes. Although there are plenty of studies regarding the dynamics of regeneration and tissue rearrangement in sponges, cell cycle kinetics of choanocytes in intact tissues remains a controversial issue. This study is devoted to the comparative description of choanocyte cell cycle dynamics in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia corallorrhiza (class Calcarea). We have identified populations of proliferating cells and synchronized them in the S-phase to estimate the growth fraction of cycling cells. Using continuous exposure to labeled thymidine analog ethynyl deoxyuridine (EdU), we calculated choanocyte cell cycle duration and the length of the S phase. We also applied double labeling with EdU and antibodies against phosphorylated histone 3 to estimate the lengths of choanocyte M and G2 phases. Finally, flow-cytometry-based quantitative analysis of DNA content provided us with the lengths of G2 and G1 phases. We found that tissue growth and renewal in the studied sponges are generally maintained by a relatively large population of slowly cycling choanocytes with a total cell cycle duration of 40 h in H. dujardinii and 60 h in L. corallorrhiza. In both species, choanocytes are characterized by an extremely short M-phase and heterogeneity in the duration of the G2 phase., (© 2024 Federation of European Biochemical Societies.)

Microtubules are an indispensable component of all eukaryotic cells due to their role in mitotic spindle formation, yet their organization and number can vary greatly in the interphase. The last common ancestor of all eukaryotes already had microtubules and microtubule motor proteins moving along them. Sponges are traditionally regarded as the oldest animal phylum. Their body does not have a clear differentiation into tissues, but it contains several distinguishable cell types. The choanocytes stand out among them and are responsible for creating a flow of water with their flagella and increasing the filtering and feeding efficiency of the sponge. Choanocyte flagella contain microtubules, but thus far, observing a developed system of cytoplasmic microtubules in non-flagellated interphase sponge cells has been mostly unsuccessful. In this work, we combine transcriptomic analysis, immunofluorescence, and electron microscopy with time-lapse recording to demonstrate that microtubules appear in the cytoplasm of sponge cells only when transdifferentiation processes are activated. We conclude that dynamic cytoplasmic microtubules in the cells of sponges are not a persistent but rather a transient structure, associated with cellular plasticity.

Sponges (phylum Porifera) are early-branching animals, whose outwardly simple body plan is underlain by a complex genetic repertoire. The transition from a mobile larva to an attached filter-feeding organism occurs by metamorphosis, a process accompanied by a radical change of the body plan and cell transdifferentiation. The continuity between larval cells and adult tissues is still obscure. In a previous study, we have produced polyclonal antibodies against the major protein of the flagellated cells covering the larva of the sponge Halisarca dujardini , used them to trace the fate of these cells and shown that the larval flagellated cells transdifferentiate into the choanocytes. In the present work, we identified the sequence of this novel protein, which we named ilborin. A search in the open databases showed that multiple orthologues of the newly identified protein are present in sponges, cnidarians, flatworms, ctenophores and echinoderms, but none of them has been described yet. Ilborin has two conserved domains: triosephosphate isomerase-barrel, which has enzymatic activity against macroergic compounds, and canonical EF-hand, which binds calcium. mRNA of ilborin is expressed in the larval flagellated cells. We suggest that the new protein is involved in the calcium-mediated regulation of energy metabolism, whose activation precedes metamorphosis.

Glycan-to-glycan binding was shown by biochemical and biophysical measurements to mediate xenogeneic self-recognition and adhesion in sponges, stage-specific cell compaction in mice embryos, and in vitro tumor cell adhesion in mammals. This intermolecular recognition process is accepted as the new paradigm accompanying high-affinity and low valent protein-to-protein and protein-to-glycan binding in cellular interactions. Glycan structures in sponges have novel species-specific sequences. Their common features are the large size >100 kD, polyvalency >100 repeats of the specific self-binding oligosaccharide, the presence of fucose, and sulfated and/or pyruvylated hexoses. These structural and functional properties, different from glycosaminoglycans, inspired their classification under the glyconectin name. The molecular mechanism underlying homophilic glyconectin-to-glyconectin binding relies on highly polyvalent, strong, and structure-specific interactions of small oligosaccharide motifs, possessing ultra-weak self-binding strength and affinity. Glyconectin localization at the glycocalyx outermost cell surface layer suggests their role in the initial recognition and adhesion event during the complex and multistep process. In mammals, Le x -to-Le x homophilic binding is structure-specific and has ultra-weak affinity. Cell adhesion is achieved through highly polyvalent interactions, enabled by clustering of small low valent structure in plasma membranes.

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10 mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and β-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sponges (Phylum Porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis . A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

A widely held-but rarely tested-hypothesis for the origin of animals is that they evolved from a unicellular ancestor, with an apical cilium surrounded by a microvillar collar, that structurally resembled modern sponge choanocytes and choanoflagellates 1-4 . Here we test this view of animal origins by comparing the transcriptomes, fates and behaviours of the three primary sponge cell types-choanocytes, pluripotent mesenchymal archaeocytes and epithelial pinacocytes-with choanoflagellates and other unicellular holozoans. Unexpectedly, we find that the transcriptome of sponge choanocytes is the least similar to the transcriptomes of choanoflagellates and is significantly enriched in genes unique to either animals or sponges alone. By contrast, pluripotent archaeocytes upregulate genes that control cell proliferation and gene expression, as in other metazoan stem cells and in the proliferating stages of two unicellular holozoans, including a colonial choanoflagellate. Choanocytes in the sponge Amphimedon queenslandica exist in a transient metastable state and readily transdifferentiate into archaeocytes, which can differentiate into a range of other cell types. These sponge cell-type conversions are similar to the temporal cell-state changes that occur in unicellular holozoans 5 . Together, these analyses argue against homology of sponge choanocytes and choanoflagellates, and the view that the first multicellular animals were simple balls of cells with limited capacity to differentiate. Instead, our results are consistent with the first animal cell being able to transition between multiple states in a manner similar to modern transdifferentiating and stem cells.

The evolution of cell-adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges-a divergent animal lineage-indicate that their tissues are organized primarily by an alternative, sponge-specific cell-adhesion mechanism called "aggregation factor." In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei ( Op ). Our results indicate that Op vinculin localizes to both cell-cell and cell-ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Coral reefs harbor diverse assemblages of organisms yet the majority of this diversity is hidden within the three dimensional structure of the reef and neglected using standard visual surveys. This study uses Autonomous Reef Monitoring Structures (ARMS) and amplicon sequencing methodologies, targeting mitochondrial cytochrome oxidase I and 18S rRNA genes, to investigate changes in the cryptic reef biodiversity. ARMS, deployed at 11 sites across a near- to off-shore gradient in the Red Sea were dominated by Porifera (sessile fraction), Arthropoda and Annelida (mobile fractions). The two primer sets detected different taxa lists, but patterns in community composition and structure were similar. While the microhabitat of the ARMS deployment affected the community structure, a clear cross-shelf gradient was observed for all fractions investigated. The partitioning of beta-diversity revealed that replacement (i.e. the substitution of species) made the highest contribution with richness playing a smaller role. Hence, different reef habitats across the shelf are relevant to regional diversity, as they harbor different communities, a result with clear implications for the design of Marine Protected Areas. ARMS can be vital tools to assess biodiversity patterns in the generally neglected but species-rich cryptic benthos, providing invaluable information for the management and conservation of hard-bottomed habitats over local and global scales.

How animals evolved from a single-celled ancestor over 700 million years ago is poorly understood. Recent transcriptomic and chromatin analyses in the sponge Amphimedon queenslandica, a morphologically-simple representative of one of the oldest animal phyletic lineages, have shed light on what innovations in the genome and its regulation underlie the emergence of animal multicellularity. Comparisons of the regulatory genome of this sponge with those of more complex bilaterian model species and even simpler unicellular relatives have revealed that fundamental changes in genome regulatory complexity accompanied the evolution of animal multicellularity. Here, we review and discuss the results of these recent investigations by specifically focusing on the contribution of long non-coding RNAs to the evolution of the animal regulatory genome.

Acquisition of multicellularity is a central event in the evolution of Eukaryota. Strikingly, animal multicellularity coincides with the emergence of three intercellular communication pathways - Notch, TGF-β and Wnt - all considered as hallmarks of metazoan development. By investigating Oopsacas minuta and Aphrocallistes vastus, we show here that the emergence of a syncytium and plugged junctions in glass sponges coincides with the loss of essential components of the Wnt signaling (i.e. Wntless, Wnt ligands and Disheveled), whereas core components of the TGF-β and Notch modules appear unaffected. This suggests that Wnt signaling is not essential for cell differentiation, polarity and morphogenesis in glass sponges. Beyond providing a comparative study of key developmental toolkits, we define here the first case of a metazoan phylum that maintained a level of complexity similar to its relatives despite molecular degeneration of Wnt pathways.

A complex genetic repertoire underlies the apparently simple body plan of sponges. Among the genes present in poriferans are those fundamental to the sensory and nervous systems of other animals. Sponges are dynamic and sensitive animals and it is intuitive to link these genes to behaviour. The proposal that ctenophores are the earliest diverging metazoan has led to the question of whether sponges possess a 'pre-nervous' system or have undergone nervous system loss. Both lines of thought generally assume that the last common ancestor of sponges and eumetazoans possessed the genetic modules that underlie sensory abilities. By corollary extant sponges may possess a sensory cell homologous to one present in the last common ancestor, a hypothesis that has been studied by gene expression. We have performed a meta-analysis of all gene expression studies published to date to explore whether gene expression is indicative of a feature's sensory function. In sponges we find that eumetazoan sensory-neural markers are not particularly expressed in structures with known sensory functions. Instead it is common for these genes to be expressed in cells with no known or uncharacterized sensory function. Indeed, many sensory-neural markers so far studied are expressed during development, perhaps because many are transcription factors. This suggests that the genetic signal of a sponge sensory cell is dissimilar enough to be unrecognizable when compared to a bilaterian sensory or neural cell. It is possible that sensory-neural markers have as yet unknown functions in sponge cells, such as assembling an immunological synapse in the larval globular cell. Furthermore, the expression of sensory-neural markers in non-sensory cells, such as adult and larval epithelial cells, suggest that these cells may have uncharacterized sensory functions. While this does not rule out the co-option of ancestral sensory modules in later evolving groups, a distinct genetic foundation may underlie the sponge sensory system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sponges have a high capacity for regeneration and this process improves biomass production in some species, thus contributing to a solution for the biomass supply problem for biotechnological applications. The aim of this work is to characterize the dynamics of cell behavior during the initial stages of sponge regeneration, using bright-field microscopy, confocal microscopy and SEM. We focused on the first 20 h of regeneration, during which blastema formation and epithelium initialization occur. An innovative sponge organotypic culture of the regenerating internal region is described and investigated by confocal microscopy, cell transplantation and vital staining. Cell-cell interaction and cell density are shown to affect events in morphogenesis such as epithelial/mesenchymal and mesenchymal/epithelial transitions as well as distinct cell movements required for regeneration. Extracellular matrix was organized according to the morphogenetic process observed, with evidence for cell-signaling instructions and remodeling. These data and the method of organotypic culture described here provide support for the development of viable sponge biomass production.

Marine sponges are important members of coral reef ecosystems. Thus, their responses to changes in ocean chemistry and environmental conditions, particularly to higher seawater temperatures, will have potential impacts on the future of these reefs. To better understand the sponge thermal stress response, we investigated gene expression dynamics in the shallow water sponge, Haliclona tubifera (order Haplosclerida, class Demospongiae), subjected to elevated temperature. Using high-throughput transcriptome sequencing, we show that these conditions result in the activation of various processes that interact to maintain cellular homeostasis. Short-term thermal stress resulted in the induction of heat shock proteins, antioxidants, and genes involved in signal transduction and innate immunity pathways. Prolonged exposure to thermal stress affected the expression of genes involved in cellular damage repair, apoptosis, signaling and transcription. Interestingly, exposure to sublethal temperatures may improve the ability of the sponge to mitigate cellular damage under more extreme stress conditions. These insights into the potential mechanisms of adaptation and resilience of sponges contribute to a better understanding of sponge conservation status and the prediction of ecosystem trajectories under future climate conditions., Competing Interests: CC was supported by a L’Oreal-UNESCO For Women in Science National Fellowship. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Background: The Planar Cell Polarity pathway (PCP) has been described as the main feature involved in patterning cell orientation in bilaterian tissues. Recently, a similar phenomenon was revealed in cnidarians, in which the inhibition of this pathway results in the absence of cilia orientation in larvae, consequently proving the functional conservation of PCP signaling between Cnidaria and Bilateria. Nevertheless, despite the growing accumulation of databases concerning basal lineages of metazoans, very few information concerning the existence of PCP components have been gathered outside of Bilateria and Cnidaria. Thus, the origin of this module or its prevalence in early emerging metazoans has yet to be elucidated., Results: The present study addresses this question by investigating the genomes and transcriptomes from all poriferan lineages in addition to Trichoplax (Placozoa) and Mnemiopsis (Ctenophora) genomes for the presence of the core components of this pathway. Our results confirm that several PCP components are metazoan innovations. In addition, we show that all members of the PCP pathway, including a bona fide Strabismus ortholog (Van gogh), are retrieved only in one sponge lineage (Homoscleromorpha) out of four. This highly suggests that the full PCP pathway dates back at least to the emergence of homoscleromorph sponges. Consequently, several secondary gene losses would have occurred in the three other poriferan lineages including Amphimedon queenslandica (Demospongiae). Several proteins were not retrieved either in placozoans or ctenophores leading us to discuss the difficulties to predict orthologous proteins in basally branching animals. Finally, we reveal how the study of multigene families may be helpful to unravel the relationships at the base of the metazoan tree., Conclusion: The PCP pathway antedates the radiation of Porifera and may have arisen in the last common ancestor of animals. Oscarella species now appear as key organisms to understand the ancestral function of PCP signaling and its potential links with Wnt pathways.

Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the "germ-line multipotency program" and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.

Recent phylogenomic evidence suggests that ctenophores may be the sister group to the rest of animals. This phylogenetic arrangement opens the possibility that sponges and placozoans could have lost neural cell types or that the ctenophore nervous system evolved independently. We critically review evidence to date that has been put forth in support of independent evolution of neural cell types in ctenophores. We observe a reluctance in the literature to consider a lost nervous system in sponges and placozoans and suggest that this may be due to historical bias and the commonly misconstrued concept of animal complexity. In support of the idea of loss (or modification beyond recognition), we provide hypothetical scenarios to show how sponges and placozoans may have benefitted from the loss and/or modification of their neural cell types., (© 2015 The Author(s).)

The Porifera (sponges) is one of the earliest phyletic lineages to branch off the metazoan tree. Although the body-plan of sponges is among the simplest in the animal kingdom and sponges lack nervous systems that communicate environmental signals to other cells, their larvae have sensory systems that generate coordinated responses to environmental cues. In eumetazoans (Cnidaria and Bilateria), the nervous systems of larvae often regulate metamorphosis through Ca(2+)-dependent signal transduction. In sponges, neither the identity of the receptor system that detects an inductive environmental cue (hereafter "metamorphic cues") nor the signaling system that mediates settlement and metamorphosis are known. Using a combination of behavioral assays and surgical manipulations, we show here that specialized epithelial cells-referred to as flask cells-enriched in the anterior third of the Amphimedon queenslandica larva are most likely to be the sensory cells that detect the metamorphic cues. Surgical removal of the region enriched in flask cells in a larva inhibits the initiation of metamorphosis. The flask cell has an apical sensory apparatus with a cilium surrounded by an apical F-actin-rich protrusion, and numerous vesicles, hallmarks of eumetazoan sensory-neurosecretory cells. We demonstrate that these flask cells respond to metamorphic cues by elevating intracellular Ca(2+) levels, and that this elevation is necessary for the initiation of metamorphosis. Taken together, these analyses suggest that sponge larvae have sensory-secretory epithelial cells capable of converting exogenous cues into internal signals via Ca(2+)-mediated signaling, which is necessary for the initiation of metamorphosis. Similarities in the morphology, physiology, and function of the sensory flask cells in sponge larvae with the sensory/neurosecretory cells in eumetazoan larvae suggest this sensory system predates the divergence of Porifera and Eumetazoa., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Sponges are known to possess remarkable reconstitutive and regenerative abilities ranging from common wounding or body part regeneration to more impressive re-building of a functional body from dissociated cells. Among the four sponge classes, Homoscleromorpha is notably the only sponge group to possess morphologically distinct basement membrane and specialized cell-junctions, and is therefore considered to possess true epithelia. The consequence of this peculiar organization is the predominance of epithelial morphogenesis during ontogenesis of these sponges. In this work we reveal the underlying cellular mechanisms used during morphogenesis accompanying ectosome regeneration in the homoscleromorph sponge model: Oscarella lobularis. We identified three main sources of novel exopinacoderm during the processes of its regeneration and the restoration of functional peripheral parts of the aquiferous system in O. lobularis: (1) intact exopinacoderm surrounding the wound surface, (2) the endopinacoderm from peripheral exhalant and inhalant canals, and (3) the intact choanoderm found on the wound surface. The basic morphogenetic processes during regeneration are the spreading and fusion of epithelial sheets that merge into one continuous epithelium. Transdifferentiation of choanocytes into exopinacocytes is also present. Epithelial-mesenchymal transition is absent during regeneration. Moreover, we cannot reveal any other morphologically distinct pluripotent cells. In Oscarella, neither blastema formation nor local dedifferentiation and proliferation have been detected, which is probably due to the high morphogenetic plasticity of the tissue. Regeneration in O. lobularis goes through cell transdifferentiation and through the processes, when lost body parts are replaced by the remodeling of the remaining tissue. Morphogenesis during ectosome regeneration in O. lobularis is correlated with its true epithelial organization. Knowledge of the morphological basis of morphogenesis during Oscarella regeneration could have important implications for our understanding of the diversity and evolution of regeneration mechanisms in metazoans, and is a strong basis for future investigations with molecular-biological approaches.

The concept of germ layers has been one of the foremost organizing principles in developmental biology, classification, systematics and evolution for 150 years (refs 1 - 3). Of the three germ layers, the mesoderm is found in bilaterian animals but is absent in species in the phyla Cnidaria and Ctenophora, which has been taken as evidence that the mesoderm was the final germ layer to evolve. The origin of the ectoderm and endoderm germ layers, however, remains unclear, with models supporting the antecedence of each as well as a simultaneous origin. Here we determine the temporal and spatial components of gene expression spanning embryonic development for all Caenorhabditis elegans genes and use it to determine the evolutionary ages of the germ layers. The gene expression program of the mesoderm is induced after those of the ectoderm and endoderm, thus making it the last germ layer both to evolve and to develop. Strikingly, the C. elegans endoderm and ectoderm expression programs do not co-induce; rather the endoderm activates earlier, and this is also observed in the expression of endoderm orthologues during the embryology of the frog Xenopus tropicalis, the sea anemone Nematostella vectensis and the sponge Amphimedon queenslandica. Querying the phylogenetic ages of specifically expressed genes reveals that the endoderm comprises older genes. Taken together, we propose that the endoderm program dates back to the origin of multicellularity, whereas the ectoderm originated as a secondary germ layer freed from ancestral feeding functions.

Recent phylogenetic analyses resulting from collection of whole genome data suggest that ctenophores, or comb jellies, are sister to all other animals. Even before publication, this result prompted discussion among researchers. Here, I counter common criticisms raised about this result and show that assumptions placing sponges as the basal-most extant animal lineage are based on limited evidence and questionable premises. For example, the idea that sponges are simple and the reported similarity of sponge choanocytes to Choanflagellata do not provide useful characters for determining the positions of sponges within the animal tree. Intertwined with discussion of basal metazoan phylogeny is consideration of the evolution of neuronal systems. Recent data show that neural systems of ctenophores are vastly different from those of other animals and use different sets of cellular and genetic mechanisms. Thus, neural systems appear to have at least two independent origins regardless of whether ctenophores or sponges are the earliest branching extant animal lineage., (© 2015. Published by The Company of Biologists Ltd.)

Three-dimensional (3D) porous collagen sponges incorporated with single-walled carbon nanotubes (SWCNTs) were prepared and used for 3D culture of bovine articular chondrocytes (BACs). The pore structures of the sponges were controlled by using ice particulates as a porogen material. The responses of cells to SWCNTs were investigated in this 3D cell culture system by evaluation of cell functions and cellular uptake of SWCNTs. The results showed that cells adhered and spatially distributed in the porous sponges. The incorporation of SWCNTs in the porous sponges promoted cell proliferation and production of sulfated glycosaminoglycans (sGAG). Confocal Raman imaging revealed that SWCNTs could be internalized by cells. The hybrid porous sponges not only provided nanostructured pore surfaces to facilitate cell proliferation and extracellular matrix (ECM) secretion but also supplied nanomaterials for cellular uptake which may be useful for biomedical applications., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Demosponges possess a leucon-type canal system which is characterized by a highly complex network of canal segments and choanocyte chambers. As sponges are sessile filter feeders, their aquiferous system plays an essential role in various fundamental physiological processes. Due to the morphological and architectural complexity of the canal system and the strong interdependence between flow conditions and anatomy, our understanding of fluid dynamics throughout leuconoid systems is patchy. This paper provides comprehensive morphometric data on the general architecture of the canal system, flow measurements and detailed cellular anatomical information to help fill in the gaps. We focus on the functional cellular anatomy of the aquiferous system and discuss all relevant cell types in the context of hydrodynamic and evolutionary constraints. Our analysis is based on the canal system of the tropical demosponge Tethya wilhelma, which we studied using scanning electron microscopy. We found a hitherto undescribed cell type, the reticuloapopylocyte, which is involved in flow regulation in the choanocyte chambers. It has a highly fenestrated, grid-like morphology and covers the apopylar opening. The minute opening of the reticuloapopylocyte occurs in an opened, intermediate and closed state. These states permit a gradual regulation of the total apopylar opening area. In this paper the three states are included in a theoretical study into flow conditions which aims to draw a link between functional cellular anatomy, the hydrodynamic situation and the regular body contractions seen in T. wilhelma. This provides a basis for new hypotheses regarding the function of bypass elements and the role of hydrostatic pressure in body contractions. Our study provides insights into the local and global flow conditions in the sponge canal system and thus enhances current understanding of related physiological processes.

Sponges are simple animals with few cell types, but their genomes paradoxically contain a wide variety of developmental transcription factors, including homeobox genes belonging to the Antennapedia (ANTP) class, which in bilaterians encompass Hox, ParaHox and NK genes. In the genome of the demosponge Amphimedon queenslandica, no Hox or ParaHox genes are present, but NK genes are linked in a tight cluster similar to the NK clusters of bilaterians. It has been proposed that Hox and ParaHox genes originated from NK cluster genes after divergence of sponges from the lineage leading to cnidarians and bilaterians. On the other hand, synteny analysis lends support to the notion that the absence of Hox and ParaHox genes in Amphimedon is a result of secondary loss (the ghost locus hypothesis). Here we analysed complete suites of ANTP-class homeoboxes in two calcareous sponges, Sycon ciliatum and Leucosolenia complicata. Our phylogenetic analyses demonstrate that these calcisponges possess orthologues of bilaterian NK genes (Hex, Hmx and Msx), a varying number of additional NK genes and one ParaHox gene, Cdx. Despite the generation of scaffolds spanning multiple genes, we find no evidence of clustering of Sycon NK genes. All Sycon ANTP-class genes are developmentally expressed, with patterns suggesting their involvement in cell type specification in embryos and adults, metamorphosis and body plan patterning. These results demonstrate that ParaHox genes predate the origin of sponges, thus confirming the ghost locus hypothesis, and highlight the need to analyse the genomes of multiple sponge lineages to obtain a complete picture of the ancestral composition of the first animal genome.

Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and β subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on sponge cells and regulating the symbiotic relationship.

Background: The evolutionary origin of gastrulation--defined as a morphogenetic event that leads to the establishment of germ layers--remains a vexing question. Central to this debate is the evolutionary relationship between the cell layers of sponges (poriferans) and eumetazoan germ layers. Despite considerable attention, it remains unclear whether sponge cell layers undergo progressive fate determination akin to eumetazoan primary germ layer formation during gastrulation., Results: Here we show by cell-labelling experiments in the demosponge Amphimedon queenslandica that the cell layers established during embryogenesis have no relationship to the cell layers of the juvenile. In addition, juvenile epithelial cells can transdifferentiate into a range of cell types and move between cell layers. Despite the apparent lack of cell layer and fate determination and stability in this sponge, the transcription factor GATA, a highly conserved eumetazoan endomesodermal marker, is expressed consistently in the inner layer of A. queenslandica larvae and juveniles., Conclusions: Our results are compatible with sponge cell layers not undergoing progressive fate determination and thus not being homologous to eumetazoan germ layers. Nonetheless, the expression of GATA in the sponge inner cell layer suggests a shared ancestry with the eumetazoan endomesoderm, and that the ancestral role of GATA in specifying internalised cells may antedate the origin of germ layers. Together, these results support germ layers and gastrulation evolving early in eumetazoan evolution from pre-existing developmental programs used for the simple patterning of cells in the first multicellular animals.

Representatives of all major metazoan lineages form biominerals. The molecular mechanisms that underlie this widespread and evolutionarily ancient ability are gradually being revealed for some lineages. However, until a wider range of metazoan biomineralization strategies are understood, the true diversity, and therefore the evolutionary origins of this process, will remain unknown. We have previously shown that the coralline demosponge, Astrosclera willeyana, in some way employs its endobiotic bacterial community to form its highly calcified skeleton. Here, using in situ hybridization and immunohistochemistry, we show that an ortholog of ATG8 (most likely a GABARAPL2/GATE-16 ortholog) is expressed in cells that construct the individual skeletal elements of the sponge. In TEM sections sponge cells can be observed to contain extensive populations of bacteria, and frequently possesses double-membrane structures which we interpret to be autophagosomes. In combination with our previous work, these findings support the hypothesis that the host sponge actively degrades a proportion of its bacterial community using an autophagy pathway, and uses the prokaryotic organic remains as a framework upon which calcification of the sponge skeleton is initiated.

Many sponge-derived natural products with applications to human health have been discovered over the past three decades. In vitro production has been proposed as one biological alternative to ensure adequate supply of marine natural products for preclinical and clinical development of drugs. Although primary cell cultures have been established for many marine phyla, no cell lines with an extended life span have been established for marine invertebrates. Hybridoma technology has been used for production of monoclonal antibodies for application to human health. We hypothesized that a sponge cell line could be formed by fusing sponge cells of one species with those of another, or by fusing sponge cells with rapidly dividing, marine-derived, non-sponge cells. Using standard methods for formation of hybridomas, with appropriate modifications for temperature and salinity, cells from individuals of the same sponge species, as well as cells from individuals of two different sponge species were successfully fused. Research in progress is focused on optimizing fusion to produce a cell line and to stimulate expression of natural products with therapeutic relevance. Experimental hybridomas may also be used as models to test hypotheses related to naturally occurring sponge chimeras and hybridomas.

The family Oscarellidae is one of the two families in the class Homoscleromorpha (phylum Porifera) and is characterized by the absence of a skeleton and the presence of a specific mitochondrial gene, tatC. This family currently encompasses sponges in two genera: Oscarella with 17 described species and Pseudocorticium with one described species. Although sponges in this group are relatively well-studied, phylogenetic relationships among members of Oscarellidae and the validity of genus Pseudocorticium remain open questions. Here we present a phylogenetic analysis of Oscarellidae using four markers (18S rDNA, 28S rDNA, atp6, tatC), and argue that it should become a mono-generic family, with Pseudocorticium being synonymized with Oscarella, and with the transfer of Pseudocorticium jarrei to Oscarella jarrei. We show that the genus Oscarella can be subdivided into four clades, each of which is supported by either a small number of morphological characters or by molecular synapomorphies. In addition, we describe two new species of Oscarella from Norwegian fjords: O. bergenensis sp. nov. and O. nicolae sp. nov., and we compare their morphology, anatomy, and cytology with other species in this genus. Internal anatomical characters are similar in both species, but details of external morphology and particularly of cytological characters provide diagnostic features. Our study also confirms that O. lobularis and O. tuberculata are two distinct polychromic sibling species. This study highlights the difficulties of species identification in skeleton-less sponges and, more generally, in groups where morphological characters are scarce. Adopting a multi-marker approach is thus highly suitable for these groups.

The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

A hallmark of stem cells is the ability to sustainably generate stem cells themselves (self-renew) as well as differentiated cells. Although a full understanding of this ability will require clarifying underlying the primordial molecular and cellular mechanisms, how stem cells maintain their stem state and their population in the evolutionarily oldest extant multicellular organisms, sponges, is poorly understood. Here, we report the identification of the first stem cell-specific gene in demosponges, a homolog of Musashi (an evolutionarily conserved RNA binding protein that regulates the stem cell state in various organisms). EflMsiA, a Musashi paralog, is specifically expressed in stem cells (archeocytes) in the freshwater sponge Ephydatia fluviatilis. EflMsiA protein is localized predominantly in the nucleus, with a small fraction in the cytoplasm, in archeocytes. When archeocytes enter M-phase, EflMsiA protein diffuses into the cytoplasm, probably because of the breakdown of the nuclear membrane. In the present study, the existence of two types of M-phase archeocytes [(M)-archeocytes] was revealed by a precise analysis of the expression levels of EflMsiA mRNA and protein. In Type I (M)-archeocytes, presumably archeocytes undergoing self-renewal, the expression levels of EflMsiA mRNA and protein were high. In Type II (M)-archeocytes, presumably archeocytes committed to differentiate (committed archeocytes), the expression levels of EflMsiA mRNA and protein were about 60% and 30% lower than those in Type I (M)-archeocytes. From these results, archeocytes can be molecularly defined for the first time as EflMsiA-mRNA-expressing cells. Furthermore, these findings shed light on the mode of cell division of archeocytes and suggest that archeocytes divide symmetrically for both self-renewal and differentiation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Residents of the marine environment, sponges (Porifera) have the ability to produce organic compounds known as secondary metabolites, which are not directly involved in the normal growth, development, or reproduction of an organism. Because of their sessile nature, the production of these bioactive compounds has been interpreted as a functional adaptation to serve in an important survival role as a means to counter various environmental stress factors such as predation, overgrowth by fouling organisms, or competition for limited space. Regardless of the reasons for this adaptation, a variety of isolated compounds have already proven to demonstrate remarkable anticancer, fungicidal, and antibiotic properties. A major obstacle to the isolation and production of novel compounds from sponges is the lack of a large, reliable source of sponge material. Sponge collection from the sea would be environmentally detrimental to the already stressed and sparse sponge populations. Sponge production in an aquaculture setting might appear to be an ideal alternative but would also be cost-ineffective and sponge growth is extremely slow. A third approach involves the development of a sponge cell culture system capable of producing the necessary cell numbers to harvest for research purposes as well as for the eventual commercial-scale production of promising bioactive compounds. Unfortunately, little progress has been made in this direction other than the establishment of temporary cultures containing aggregates and fragments of cells. One impediment toward successful sponge cell culture might be ascribed to a lack of published knowledge of failed methodologies, and thus, time and effort is wasted on continued reinvention of the same methods and procedures. Consequently, we have undertaken here to chart some of our unsuccessful research efforts, our methodology, and results to provide the sponge research community with knowledge to assist them to better avoid taking the same failed pathways.

Despite the importance of trophic interactions between organisms, and the relationship between primary production and benthic diversity, there have been few studies that have quantified the carbon flow from pelagic to benthic environments as a result of the assemblage level activity of suspension-feeding organisms. In this study, we examine the feeding activity of seven common sponge species from the Taputeranga marine reserve on the south coast of Wellington in New Zealand. We analysed the diet composition, feeding efficiency, pumping rates, and the number of food particles (specifically picoplanktonic prokaryotic cells) retained by sponges. We used this information, combined with abundance estimates of the sponges and estimations of the total amount of food available to sponges in a known volume of water (89,821 m(3)), to estimate: (1) particulate organic carbon (POC) fluxes through sponges as a result of their suspension-feeding activities on picoplankton; and (2) the proportion of the available POC from picoplankton that sponges consume. The most POC acquired by the sponges was from non-photosynthetic bacterial cells (ranging from 0.09 to 4.69 g C d(-1) with varying sponge percentage cover from 0.5 to 5%), followed by Prochlorococcus (0.07 to 3.47 g C d(-1)) and then Synechococcus (0.05 to 2.34 g C d(-1)) cells. Depending on sponge abundance, the amount of POC that sponges consumed as a proportion of the total POC available was 0.2-12.1% for Bac, 0.4-21.3% for Prochlo, and 0.3-15.8% for Synecho. The flux of POC for the whole sponge assemblage, based on the consumption of prokaryotic picoplankton, ranged from 0.07-3.50 g C m(2) d(-1). This study is the first to estimate the contribution of a sponge assemblage (rather than focusing on individual sponge species) to POC flow from three groups of picoplankton in a temperate rocky reef through the feeding activity of sponges and demonstrates the importance of sponges to energy flow in rocky reef environments.

Background: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available., Results: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene., Conclusion: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Proliferation of sponge cells is generally measured via cell counts or viability assays. However, more insight into the proliferative state of a sponge cell population can be obtained from the distribution of the cells over the different phases of the cell cycle. Cell cycle distribution of sponge cells was measured via flow cytometry after staining the DNA with propidium iodide. The five sponges studied in this paper all showed a large fraction of cells in G1/G0 compared to G2/M and S, indicating that cells were not actively dividing. In addition, some sponges also showed a large apoptotic fraction, indicating cell death. Additional apoptosis measurements, based on caspase activity, showed that harvesting and dissociation of sponge tissue to initiate a primary cell culture was directly correlated with an increase in apoptotic cells. This indicates that for the development of cell cultures, more attention should be given to harvesting, dissociation, and quality of starting material. Finally, cultivation conditions used were ineffective for proliferation, since after 2 d of cultivating Haliclona oculata cells, most cells shifted towards the apoptotic fraction, indicating that cells were dying. For development of in vitro sponge cell cultures, flow cytometric cell cycle analysis is a useful method to assess the proliferative state of a sponge cell culture and can be used to validate improvements in harvesting and dissociation, to select sponges with good proliferative capacities and to study the influence of culture conditions for stimulating cell growth., (© The Author(s) 2011. This article is published with open access at Springerlink.com)

Sponges are an ancient group of animals that diverged from other metazoans over 600 million years ago. Here we present the draft genome sequence of Amphimedon queenslandica, a demosponge from the Great Barrier Reef, and show that it is remarkably similar to other animal genomes in content, structure and organization. Comparative analysis enabled by the sequencing of the sponge genome reveals genomic events linked to the origin and early evolution of animals, including the appearance, expansion and diversification of pan-metazoan transcription factor, signalling pathway and structural genes. This diverse 'toolkit' of genes correlates with critical aspects of all metazoan body plans, and comprises cell cycle control and growth, development, somatic- and germ-cell specification, cell adhesion, innate immunity and allorecognition. Notably, many of the genes associated with the emergence of animals are also implicated in cancer, which arises from defects in basic processes associated with metazoan multicellularity.

The application of synthetic cancellous bone has been shown to be highly successful when its architecture mimics that of the naturally interconnected trabeculae bone it aims to replace. The following investigation demonstrates the potential use of marine sponges as precursors in the production of ceramic based tissue engineered bone scaffolds. Three species of natural sponge, Dalmata Fina (Spongia officinalis Linnaeus, Adriatic Sea), Fina Silk (Spongia zimocca, Mediterranean) and Elephant Ear (Spongia agaricina, Caribbean) were selected for replication. A high solid content (80 %wt), low viscosity (126 mPas) hydroxyapatite slurry was developed, infiltrated into each sponge species and subsequently sintered, producing a scaffold structure that replicated pore architecture and interconnectivity of the precursor sponge. The most promising of the ceramic tissue engineered bone scaffolds developed, Spongia agaricina replicas, demonstrated an overall porosity of 56-61% with 83% of the pores ranging between 100 and 500 microm (average pore size 349 microm) and an interconnectivity of 99.92%.

Many marine and freshwater organisms are rocky bottom dwellers, and the mineralogical composition of the substratum is known to potentially condition their biology and ecology. In this work, we propose the use of 3D sponge cellular aggregates, called primmorphs, as suitable models for a multidisciplinary study of the mechanisms which regulate the biological responses triggered by the contact with different inorganic substrata. In our experiments, primmorphs obtained from the marine sponge Petrosia ficiformis (Poiret, 1789) were reared on calcium carbonate or on quartzitic substrata, respectively, and their morphological development was described. In parallel, the quantitative expression levels of two genes, silicatein and heat shock protein 70 (HSP70), were evaluated. The first gene is strictly correlated to spiculogenesis and sponge growth, while the second is an important indicator of stress. The results achieved with this in vitro model clearly demonstrate that quartzitic substrata determine the increase of silicatein gene expression, a lower expression of HSP70 gene, and a remarkable difference in primmorphs morphology compared to the analogous samples grown on marble.

This study reveals the peculiar in vivo cell kinetics and cell turnover of the marine sponge Halisarca caerulea under steady-state conditions. The tropical coral reef sponge shows an extremely high proliferation activity, a short cell cycle duration and massive cell shedding. Cell turnover is predominantly confined to a single cell population, i.e. the choanocytes, and in this process apoptosis only plays a minor role. To our knowledge, such fast cell kinetics under steady-state conditions, with high turnover by shedding in the absence of apoptosis, has not been observed previously in any other multicellular organism. The duration of the cell cycle in vivo resembles that of unicellular organisms in culture. Morphological and histochemical studies demonstrate compartmentalization of choanocytes in the sponge tissue, which corresponds well with its remarkable cellular kinetics. Coral reef cavity sponges, like H. caerulea, inhabit low nutrient tropical waters, forcing these organisms to filter large volumes of water and to capture the few nutrients efficiently. Under these oligotrophic conditions, a high cell turnover may be considered as a very useful strategy, preventing permanent damage to the sponge by environmental stress. Halisarca caerulea maintains its body mass and keeps its food uptake system up to date by constantly renewing its filter system. We conclude that studies on cell kinetics and functional morphology provide new and essential information on the growth characteristics and the regulation of sponge growth in vivo as well as in vitro and the role of choanocytes in tissue homeostasis.

Marine sponges (Porifera) are ancient and simple eumetazoans. They constitute key organisms in the evolution from unicellular to multicellular animals. We now demonstrated that pure sulfated polysaccharides from marine sponges are responsible for the species-specific cell-cell interaction in these invertebrates. This conclusion was based on the following observations: (1) each species of marine sponge has a single population of sulfated polysaccharide, which differ among the species in their sugar composition and sulfate content; (2) sulfated polysaccharides from sponge interact with each other in a species-specific way, as indicated by an affinity chromatography assay, and this interaction requires calcium; (3) homologous, but not heterologous, sulfated polysaccharide inhibits aggregation of dissociated sponge cells; (4) we also observed a parallel between synthesis of the sulfated polysaccharide and formation of large aggregates of sponge cells, known as primmorphs. Once aggregation reached a plateau, the demand for the de novo synthesis of sulfated polysaccharides ceased. Heparin can mimic the homologous sulfated polysaccharide on the in vitro interaction and also as an inhibitor of aggregation of the dissociated sponge cells. However, this observation is not relevant for the biology of the sponge since heparin is not found in the invertebrate. In conclusion, marine sponges display an ancestor event of cell-cell adhesion, based on the calcium-dependent carbohydrate-carbohydrate interaction.

One set of evolutionary features that has received less attention than the evolution of genes or species is the evolution of cellular machines, the self-contained structures in cells with dedicated functions. Here I suggest that domain expansion through shuffling, duplication, and changes in protein expression level are critical drivers in the evolution of cellular machines. Once established, evolutionary change in these cellular machines tends to occur by paralogy or expansion and modification of the existing core genes. A comparative genomics approach to one cellular machine--the post-synaptic complex--provided preliminary validation of these views. A comparative genomics approach to the entire cellulome may reveal the diversity of cellular machines and their inter-relationships.

We know little about the genomic events that led to the advent of a multicellular grade of organization in animals, one of the most dramatic transitions in evolution. Metazoan multicellularity is correlated with the evolution of embryogenesis, which presumably was underpinned by a gene regulatory network reliant on the differential activation of signaling pathways and transcription factors. Many transcription factor genes that play critical roles in bilaterian development largely appear to have evolved before the divergence of cnidarian and bilaterian lineages. In contrast, sponges seem to have a more limited suite of transcription factors, suggesting that the developmental regulatory gene repertoire changed markedly during early metazoan evolution. Using whole-genome information from the sponge Amphimedon queenslandica, a range of eumetazoans, and the choanoflagellate Monosiga brevicollis, we investigate the genesis and expansion of homeobox, Sox, T-box, and Fox transcription factor genes. Comparative analyses reveal that novel transcription factor domains (such as Paired, POU, and T-box) arose very early in metazoan evolution, prior to the separation of extant metazoan phyla but after the divergence of choanoflagellate and metazoan lineages. Phylogenetic analyses indicate that transcription factor classes then gradually expanded at the base of Metazoa before the bilaterian radiation, with each class following a different evolutionary trajectory. Based on the limited number of transcription factors in the Amphimedon genome, we infer that the genome of the metazoan last common ancestor included fewer gene members in each class than are present in extant eumetazoans. Transcription factor orthologues present in sponge, cnidarian, and bilaterian genomes may represent part of the core metazoan regulatory network underlying the origin of animal development and multicellularity.

Natural chimerism, the fusion between genetically distinct conspecifics, is a process known to occur in various marine benthic invertebrates. Sponges (phylum Porifera) have proven to be a useful model to study the origin and evolution of allorecognition. Like some other invertebrates, they display an ontogenetic shift in their allorecognition response: genetically different individuals can fuse during early development, but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of this allorecognition response, which prevents integration of incompatible genotypes. Here we follow the behaviours and fates of cells derived from genetically distinct larvae of the demosponge Amphimedon queenslandica that have fused together at metamorphosis. By labelling individual larvae with different fluorescent dyes, we can follow cell movement in the postlarval chimeras. We observed that cells from the two individuals readily mixed for 2 weeks after the initial fusion. After that time, differently labelled cells began to sort into different postlarval cellular territories, with one lineage giving rise to choanocytes and the other to pinacocytes and cells of the mesohyl. These results suggest that a rapid ontogenetic shift in the allogeneic response of A. queenslandica occurs about 2 weeks after the initiation of metamorphosis and that the molecular basis of this response is also involved in creating differential cell affinities that underlie the construction of the sponge body plan. Compatible with this proposition is the observation that cells from postlarvae that are allowed to develop for 2 weeks before contact do not fuse and form a distinct boundary between genotypes. The successful chimeras remained stable for the duration of the experiment (3 weeks) raising the possibility that reproductive chimeras might persist in the natural environment, with a single genotype giving rise to germ cells.

Sponges are a promising source of organic compounds of potential interest regarding industrial and medical applications. For detailed studies on such compounds, large amounts of sponge biomass are required. Obtaining that is at present extremely difficult because most sponges are relatively rare in nature and their mass cultivation in the laboratory has not yet been accomplished. In this study the possibility of culturing Aplysina aerophoba fragments in laboratory was examined. While a substantial biomass increase was not yet observed, we achieved fragmented sponge tissue to develop into a functional sponge as a first success.