Regeneration in the sponge Sycon ciliatum partly mimics postlarval development.

Somatic cells dissociated from an adult sponge can reorganize and develop into a juvenile-like sponge, a remarkable phenomenon of regeneration. However, the extent to which regeneration recapitulates embryonic developmental pathways has remained enigmatic. We have standardized and established a sponge Sycon ciliatum regeneration protocol from dissociated cells. Morphological analysis demonstrated that dissociated sponge cells follow a series of morphological events resembling postembryonic development. We performed high-throughput sequencing on regenerating samples and compared the data with that from regular postlarval development. Our comparative transcriptomic analysis revealed that sponge regeneration is as equally dynamic as embryogenesis. We found that sponge regeneration is orchestrated by recruiting pathways similar to those utilized in embryonic development. We also demonstrated that sponge regeneration is accompanied by cell death at early stages, revealing the importance of apoptosis in remodelling the primmorphs to initiate re-development. Because sponges are likely to be the first branch of extant multicellular animals, we suggest that this system can be explored to study the genetic features underlying the evolution of multicellularity and regeneration.

Co-expression of synaptic genes in the sponge Amphimedon queenslandica uncovers ancient neural submodules.

The synapse is a complex cellular module crucial to the functioning of neurons. It evolved largely through the exaptation of pre-existing smaller submodules, each of which are comprised of ancient sets of proteins that are conserved in modern animals and other eukaryotes. Although these ancient submodules themselves have non-neural roles, it has been hypothesized that they may mediate environmental sensing behaviors in aneural animals, such as sponges. Here we identify orthologues in the sponge Amphimedon queenslandica of genes encoding synaptic submodules in neural animals, and analyse their cell-type specific and developmental expression to determine their potential to be co-regulated. We find that genes comprising certain synaptic submodules, including those involved in vesicle trafficking, calcium-regulation and scaffolding of postsynaptic receptor clusters, are co-expressed in adult choanocytes and during metamorphosis. Although these submodules may contribute to sensory roles in this cell type and this life cycle stage, total synaptic gene co-expression profiles do not support the existence of a functional synapse in A. queenslandica. The lack of evidence for the co-regulation of genes necessary for pre- and post-synaptic functioning in A. queenslandica suggests that sponges, and perhaps the last common ancestor of sponges and other extant animals, had the ability to promulgate sensory inputs without complete synapse-like functionalities. The differential co-expression of multiple synaptic submodule genes in sponge choanocytes, which have sensory and feeding roles, however, is consistent with the metazoan ancestor minimally being able to undergo exo- and endocytosis in a controlled and localized manner.

From traveler to homebody: Which signaling mechanisms sponge larvae use to become adult sponges?

Cell-to-cell signaling is responsible for regulation of many developmental processes such as proliferation, cell migration, survival, cell fate specification and axis patterning. In this article we discussed the role of signaling in the metamorphosis of sponges with a focus on epithelial-mesenchymal transition (EMT) accompanying this event. Sponges (Porifera) are an ancient lineage of morphologically simple animals occupying a basal position on the tree of life. The study of these animals is necessary for understanding the origin of multicellularity and the evolution of developmental processes. Development of sponges is quite diverse. It finishes with the metamorphosis of a free-swimming larva into a young settled sponge. The outer surface of sponge larvae consists of a ciliated epithelial sheath, which ensures locomotion, while their internal structure varies from genus to genus. The fate of larval ciliated cells is the most intriguing aspect of metamorphosis. In this review we discuss the fate of larval ciliated cells, the processes going on in cells during metamorphosis at the molecular level and the regulation of this process. The review is based on information about several sponge species with a focus on Halisarca dujardini, Sycon ciliatum and Amphimedon queenslandica. In our model sponge, H. dujardini, ciliated cells leave the larval epithelium during metamorphosis and migrate to the internal cell mass as amoeboid cells to be differentiated into choanocytes of the juvenile sponge. Ciliated cells undergo EMT and internalize within minutes. As EMT involves the disappearance of adherens junctions and as cadherin, the main adherens junction protein, was identified in the transcriptome of several sponges, we suppose that EMT is regulated through cadherin-containing adherens junctions between ciliated cells. We failed to identify the master genes of EMT in the H. dujardini transcriptome, possibly because transcription was absent in the sequenced stages. They may be revealed by a search in the genome. The master genes themselves are controlled by various signaling pathways. Sponges have all the six signaling pathways conserved in Metazoa: Wnt, TGF-beta, Hedgehog, Notch, FGF and NO-dependent pathways. Summarizing the new data about intercellular communication in sponges, we can put forward two main questions regarding metamorphosis: (1) Which of the signaling pathways and in what hierarchical order are involved in metamorphosis? (2) How is the organization of a young sponge related to that of the larva or, in other words, is there a heredity of axes between the larva and the adult sponge?

Environmental factors related to the production of a complex set of spicules in a tropical freshwater sponge.

Adverse natural conditions will, generally, induce gemmulation in freshwater sponges. Because of this environmental dependence, gemmoscleres are given exceptional value in taxonomic, ecological and paleoenvironmental studies. Other spicules categories such as microscleres and beta megascleres have received little attention with regard to their occurrence and function during the sponge biological cycle. Metania spinata, a South American species common to bog waters in the Cerrado biome, produces alpha and beta megascleres, microscleres and gemmoscleres. To detect the environmental factors triggering the production of all these kinds of spicules, the species annual seasonal cycle was studied. Artificial substrates were devised, supplied with gemmules and placed in Lagoa Verde pond which contained a natural population of M. spinata. Field monitoring was conducted for eight months in order to observe the growth of sponges and spicules formation. Samples of water were taken monthly for physical and chemical parameters determination. The appearance of the alpha megascleres was sequentially followed by that of microscleres, gemmoscleres and beta megascleres. The first ones built the new sponge skeleton, the last three were involved in keeping inner moisture in the sponge body or its gemmules. The water level, temperature and the silicon (Si) concentration in the pond were the most important factors related to this sequential production of spicules, confirming environmental reconstructions based on the presence or absence of alpha megascleres and gemmoscleres in past sediments.

Regulatory RNA at the root of animals: dynamic expression of developmental lincRNAs in the calcisponge Sycon ciliatum.

Long non-coding RNAs (lncRNAs) play important regulatory roles during animal development, and it has been hypothesized that an RNA-based gene regulation was important for the evolution of developmental complexity in animals. However, most studies of lncRNA gene regulation have been performed using model animal species, and very little is known about this type of gene regulation in non-bilaterians. We have therefore analysed RNA-Seq data derived from a comprehensive set of embryogenesis stages in the calcareous sponge Sycon ciliatum and identified hundreds of developmentally expressed intergenic lncRNAs (lincRNAs) in this species. In situ hybridization of selected lincRNAs revealed dynamic spatial and temporal expression during embryonic development. More than 600 lincRNAs constitute integral parts of differentially expressed gene modules, which also contain known developmental regulatory genes, e.g. transcription factors and signalling molecules. This study provides insights into the non-coding gene repertoire of one of the earliest evolved animal lineages, and suggests that RNA-based gene regulation was probably present in the last common ancestor of animals.

Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer.

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.

Evolutionary origin of gastrulation: insights from sponge development.

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.

The genomic and cellular foundations of animal origins.

The first animals arose more than six hundred million years ago, yet they left little impression in the fossil record. Nonetheless, the cell biology and genome composition of the first animal, the Urmetazoan, can be reconstructed through the study of phylogenetically relevant living organisms. Comparisons among animals and their unicellular and colonial relatives reveal that the Urmetazoan likely possessed a layer of epithelium-like collar cells, preyed on bacteria, reproduced by sperm and egg, and developed through cell division, cell differentiation, and invagination. Although many genes involved in development, body patterning, immunity, and cell-type specification evolved in the animal stem lineage or after animal origins, several gene families critical for cell adhesion, signaling, and gene regulation predate the origin of animals. The ancestral functions of these and other genes may eventually be revealed through studies of gene and genome function in early-branching animals and their closest non-animal relatives.

[Concerning one obsolete tradition: does gastrulation in sponges exist?].

The analysis of comparative-embryological and molecular-biological data leads to the conclusion that universal basic mechanisms of morphogenesis occurred first in the evolution of animals in the ancestors of modern sponges and eumetazoans, which served as a basis of different evolution of individual development in Parazoa and Eumetazoa lines. In the former, morphogenesis in early embryogenesis led to formation of the water-current system as a means for capturing and delivery of food particles to different parts of the animal. In the latter, morphogenetic movements manifested themselves as gastrulation, during which the germ layers and the digestive system formed. The morphogenetic movements of cells in Metazoa emerged independently of cell specification. They are primary relative to cell differentiation. Theunity of all Metazoa is based on the similarity of mechanisms of morphogenesis rather than on the presence of germ layers.

Cellular and molecular processes leading to embryo formation in sponges: evidences for high conservation of processes throughout animal evolution.

The emergence of multicellularity is regarded as one of the major evolutionary events of life. This transition unicellularity/pluricellularity was acquired independently several times (King 2004). The acquisition of multicellularity implies the emergence of cellular cohesion and means of communication, as well as molecular mechanisms enabling the control of morphogenesis and body plan patterning. Some of these molecular tools seem to have predated the acquisition of multicellularity while others are regarded as the acquisition of specific lineages. Morphogenesis consists in the spatial migration of cells or cell layers during embryonic development, metamorphosis, asexual reproduction, growth, and regeneration, resulting in the formation and patterning of a body. In this paper, our aim is to review what is currently known concerning basal metazoans--sponges' morphogenesis from the tissular, cellular, and molecular points of view--and what remains to elucidate. Our review attempts to show that morphogenetic processes found in sponges are as diverse and complex as those found in other animals. In true epithelial sponges (Homoscleromorpha), as well as in others, we find similar cell/layer movements, cellular shape changes involved in major morphogenetic processes such as embryogenesis or larval metamorphosis. Thus, sponges can provide information enabling us to better understand early animal evolution at the molecular level but also at the cell/cell layer level. Indeed, comparison of molecular tools will only be of value if accompanied by functional data and expression studies during morphogenetic processes.

Structure and expression of conserved Wnt pathway components in the demosponge Amphimedon queenslandica.

Wnt-signalling plays a critical role in animal development, and its misregulation results in serious human diseases, including cancer. While the Wnt pathway is well studied in eumetazoan models, little is known about the evolutionary origin of its components and their functions. Here, we have identified key machinery of the Wnt-ß-catenin (canonical)-signalling pathway that is encoded in the Amphimedon queenslandica (Demospongiae; Porifera) genome, namely Wnt, Fzd, SFRP, Lrp5/6, Dvl, Axin, APC, GSK3, ß-catenin, Tcf, and Groucho. Most of these genes are not detected in the choanoflagellate and other nonmetazoan eukaryotic genomes. In contrast, orthologues of some of key components of bilaterian Wnt-planar cell polarity and Wnt/Ca(2+) are absent from the Amphimedon genome, suggesting these pathways evolved after demosponge and eumetazoan lineages diverged. Sequence analysis of the identified proteins of the Wnt-ß-catenin pathway has revealed the presence of most of the conserved motifs and domains responsible for protein-protein and protein-DNA interactions in vertebrates and insects. However, several protein-protein interaction domains appear to be absent from the Amphimedon Axin and APC proteins. These are also missing from their orthologues in the cnidarian Nematostella vectensis, suggesting that they are bilaterian novelties. All of the analyzed Wnt pathway genes are expressed in specific patterns during Amphimedon embryogenesis. Most are expressed in especially striking and highly dynamic patterns during formation of a simple organ-like larval structure, the pigment ring. Overall, our results indicate that the Wnt-ß-catenin pathway was used in embryonic patterning in the last common ancestor of living metazoans. Subsequently, gene duplications and a possible increase in complexity of protein interactions have resulted in the precisely regulated Wnt pathway observed in extant bilaterian animals.

The genome of the sponge Amphimedon queenslandica provides new perspectives into the origin of Toll-like and interleukin 1 receptor pathways.

Members of the Toll-like receptor (TLR) and the interleukin 1 receptor (IL1R) superfamilies activate various signaling cascades that are evolutionarily conserved in eumetazoans. In this study, we have searched the genome and expressed sequence tags of the demosponge Amphimedon queenslandica for molecules involved in TLR and IL1R signaling. Although we did not identify a conventional TLR or ILR, the Amphimedon genome encodes two related receptors, AmqIgTIRs, which are comprised of at least three extracellular IL1R-like immunoglobulins (Ig) and an intracellular TLR-like Toll/interleukin1 receptor/resistance (TIR) domain. The remainder of the TLR/IL1R pathway is mostly conserved in Amphimedon and includes genes known to interact with TLRs and IL1Rs in bilaterians, such as Toll-interacting protein (Tollip) and myeloid differentiation factor 88 (MyD88). By comparing the sponge genome to that of nonmetazoan eukaryotes and other basal animal phyla (i.e., placozoan and cnidarian representatives) we can infer that most components of the signaling cascade, including the receptors, evolved after the divergence of metazoan, and choanoflagellate lineages. In most cases, these proteins are composed of metazoan-specific domains (e.g., Pellino) or architectures (e.g., the association of a death domain with a TIR domain in the MyD88). The dynamic expression of the two AmqIgTIRs, AmqMyD88, AmqTollip, and AmqPellino during Amphimedon embryogenesis and larval development is consistent with the TLR/IL1R pathway having a role in both development and immunity in the last common metazoan ancestor.

Does the survivorship of activated resting stages in toxic environments provide cues for ballast water treatment?

The toxic effects of three inorganic metals (Cu, Cr, Hg), three organic (phenol, formalin, ammonium) chemicals, ozone-enriched water and peroxides (H2O2) on embryonic development were tested in 8 species from the Porifera, Bryozoa and Crustacea. Toxicants with lower molecular weight showed stronger negative impacts on post-diapause embryos than chemicals with higher molecular weight if related to the toxicity of the chemicals to active adult stages. Only few embryos of the cladoceran Moina macrocopa and none of the cladoceran Wlassicsia pannonica treated with peroxides at concentration 0.3% developed further. Ozone-enriched water had no significant effect on post-diapause embryonic development in cladocerans. Ammonium (the product of NH4OH dissociation) in concentration 100 mg/l and higher killed all embryos of M. macrocopa inside protective membranes. Peroxides and ammonium are suggested for the purification of ship ballast waters as effective, non-expensive and non-persistent toxic chemicals. Resting stages of invertebrates including at least Crustaceans, Porifera and Bryozoa seem to allow not only dispersal among toxic industrial environments such as ship ballast compartments, but may also endure serious pollution events common in seaports and estuaries. Artemia cysts due to their strong protection against different toxic substances are recommended as a model for studies of toxic effects in diapausing stages in polluted estuaries and marine environments.

The stem cell system in demosponges: insights into the origin of somatic stem cells.

The stem cell system is one of the unique systems that have evolved only in multicellular organisms. Major questions about this system include what type(s) of stem cells are involved (pluri-, multi- or uni-potent stem cells), and how the self-renewal and differentiation of stem cells are regulated. To understand the origin of the stem cell system in metazoans and to get insights into the ancestral stem cell itself, it is important to discover the molecular and cellular mechanisms of the stem cell system in sponges (Porifera), the evolutionarily oldest extant metazoans. Histological studies here provided a body of evidence that archeocytes are the stem cells in sponges, and recent molecular studies of sponges, especially the finding of the expression of Piwi homologues in archeocytes and choanocytes in a freshwater sponge, Ephydatia fluviatilis, have provided critical insights into the stem cell system in demosponges. Here I introduce archeocytes and discuss (i) modes of archeocyte differentiation, (ii) our current model of the stem cell system in sponges composed of both archeocytes and choanocytes based on our molecular analysis and previous microscopic studies suggesting the maintenance of pluripotency in choanocytes, (iii) the inference that the Piwi and piRNA function in maintaining stem cells (which also give rise to gametes) may have already been achieved in the ancestral metazoan, and (iv) possible hypotheses about how the migrating stem cells arose in the urmetazoan (protometazoan) and about the evolutionary origin of germline cells in the urbilaterian (protobilaterian).

Six major steps in animal evolution: are we derived sponge larvae?

A review of the old and new literature on animal morphology/embryology and molecular studies has led me to the following scenario for the early evolution of the metazoans. The metazoan ancestor, "choanoblastaea," was a pelagic sphere consisting of choanocytes. The evolution of multicellularity enabled division of labor between cells, and an "advanced choanoblastaea" consisted of choanocytes and nonfeeding cells. Polarity became established, and an adult, sessile stage developed. Choanocytes of the upper side became arranged in a groove with the cilia pumping water along the groove. Cells overarched the groove so that a choanocyte chamber was formed, establishing the body plan of an adult sponge; the pelagic larval stage was retained but became lecithotrophic. The sponges radiated into monophyletic Silicea, Calcarea, and Homoscleromorpha. Homoscleromorph larvae show cell layers resembling true, sealed epithelia. A homoscleromorph-like larva developed an archenteron, and the sealed epithelium made extracellular digestion possible in this isolated space. This larva became sexually mature, and the adult sponge-stage was abandoned in an extreme progenesis. This eumetazoan ancestor, "gastraea," corresponds to Haeckel's gastraea. Trichoplax represents this stage, but with the blastopore spread out so that the endoderm has become the underside of the creeping animal. Another lineage developed a nervous system; this "neurogastraea" is the ancestor of the Neuralia. Cnidarians have retained this organization, whereas the Triploblastica (Ctenophora+Bilateria), have developed the mesoderm. The bilaterians developed bilaterality in a primitive form in the Acoelomorpha and in an advanced form with tubular gut and long Hox cluster in the Eubilateria (Protostomia+Deuterostomia). It is indicated that the major evolutionary steps are the result of suites of existing genes becoming co-opted into new networks that specify new structures. The evolution of the eumetazoan ancestor from a progenetic homoscleromorph larva implies that we, as well as all the other eumetazoans, are derived sponge larvae.

Reproduction in a carnivorous sponge: the significance of the absence of an aquiferous system to the sponge body plan.

Sponges usually produce, release, and capture gametes via the aquiferous system, and so the absence of both choanocytes and an aquiferous system in the carnivorous sponge Asbestopluma occidentalis has led to unusual characteristics of development for this Phylum. Sperm are highly specialized elongate cells tightly packed into spermatic cysts in the peripheral tissue of the sponge. Mature spermatozoa have proacrosomal vesicles at the anterior end and a ciliary pit surrounding the flagellum. Clusters of four to five oocytes are in synchronous stages of cleavage, suggesting that fertilization is synchronous. All stages of embryos occur in the same individual. Early cleavage was holoblastic and equal; blastomeres in two-, four- and eight-cell embryos were compact and 16-cell stage embryos were bi-layered. Late-stage embryos show three cellular regions along the anterior-posterior axis: the anterior hemisphere with heterogeneous cells, a mid-region with cells lying perpendicular to the A-P axis in a collagenous matrix, and small cells at the posterior pole. Unusually for Porifera, multiciliated cells cover all but the posterior pole. It is inferred that fertilization occurs by capture of intact spermatic cysts whose surrounding forceps spicules become trapped in the anisochelae of neighboring sponges. The elongate shape of sperm may be designed to penetrate the loose collagenous mesohyl, such that the arrival of a packet of sperm would lead to simultaneous fertilization of oocytes in a cluster. Loss of the water canal system in carnivorous sponges has allowed the evolution of features that are highly specialized for the habitat of this animal, but such modifications were not necessarily a prerequisite for the subsequent evolution of metazoans. Given the extremely versatile mechanisms of gametogenesis, embryogenesis, and tissue/body structure in sponges, generalizations regarding basal metazoan reproduction, development, and structure must be approached with caution.

Wnt and TGF-beta expression in the sponge Amphimedon queenslandica and the origin of metazoan embryonic patterning.

BACKGROUND: The origin of metazoan development and differentiation was contingent upon the evolution of cell adhesion, communication and cooperation mechanisms. While components of many of the major cell signalling pathways have been identified in a range of sponges (phylum Porifera), their roles in development have not been investigated and remain largely unknown. Here, we take the first steps toward reconstructing the developmental signalling systems used in the last common ancestor to living sponges and eumetazoans by studying the expression of genes encoding Wnt and TGF-beta signalling ligands during the embryonic development of a sponge. METHODOLOGY/PRINCIPAL FINDINGS: Using resources generated in the recent sponge Amphimedon queenslandica (Demospongiae) genome project, we have recovered genes encoding Wnt and TGF-beta signalling ligands that are critical in patterning metazoan embryos. Both genes are expressed from the earliest stages of Amphimedon embryonic development in highly dynamic patterns. At the time when the Amphimedon embryos begin to display anterior-posterior polarity, Wnt expression becomes localised to the posterior pole and this expression continues until the swimming larva stage. In contrast, TGF-beta expression is highest at the anterior pole. As in complex animals, sponge Wnt and TGF-beta expression patterns intersect later in development during the patterning of a sub-community of cells that form a simple tissue-like structure, the pigment ring. Throughout development, Wnt and TGF-beta are expressed radially along the anterior-posterior axis. CONCLUSIONS/SIGNIFICANCE: We infer from the expression of Wnt and TGF-beta in Amphimedon that the ancestor that gave rise to sponges, cnidarians and bilaterians had already evolved the capacity to direct the formation of relatively sophisticated body plans, with axes and tissues. The radially symmetrical expression patterns of Wnt and TGF-beta along the anterior-posterior axis of sponge embryos and larvae suggest that these signalling pathways contributed to establishing axial polarity in the very first metazoans.

Vertical transmission of diverse microbes in the tropical sponge Corticium sp.

Sponges are host to extremely diverse bacterial communities, some of which appear to be spatiotemporally stable, though how these consistent associations are assembled and maintained from one sponge generation to the next is not well understood. Here we report that a diverse group of microbes, including both bacteria and archaea, is consistently present in aggregates within embryos of the tropical sponge Corticium sp. The major taxonomic groups represented in bacterial 16S rRNA sequences amplified from the embryos are similar to those previously described in a variety of marine sponges. Three selected bacterial taxa, representing proteobacteria, actinobacteria, and a clade including recently described sponge-associated bacteria, were tested and found to be present in all adult samples tested over a 3-year period and in the embryos throughout development. Specific probes were used in fluorescence in situ hybridization to localize cells of the three types in the embryos and mesohyl. This study confirms the vertical transmission of multiple, phylogenetically diverse microorganisms in a marine sponge, and our findings lay the foundation for future work on exploring vertical transmission of specific, yet diverse, microbial assemblages in marine sponges.

Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity.

Demosponges are considered part of the most basal evolutionary lineage in the animal kingdom. Although the sponge body plan fundamentally differs from that of other metazoans, their development includes many of the hallmarks of bilaterian and eumetazoan embryogenesis, namely fertilization followed by a period of cell division yielding distinct cell populations, which through a gastrulation-like process become allocated into different cell layers and patterned within these layers. These observations suggest that the last common ancestor (LCA) to all living animals was developmentally more sophisticated than is widely appreciated and used asymmetric cell division and morphogen gradients to establish localized populations of specified cells within the embryo. Here we demonstrate that members of a range of transcription factor gene classes, many of which appear to be metazoan-specific, are expressed during the development of the demosponge Reniera, including ANTP, Pax, POU, LIM-HD, Sox, nuclear receptor, Fox (forkhead), T-box, Mef2, and Ets genes. Phylogenetic analysis of these genes suggests that not only the origin but the diversification of some of the major developmental metazoan transcription factor classes took place before sponges diverged from the rest of the Metazoa. Their expression during demosponge development suggests that, as in today's sophisticated metazoans, these genes may have functioned in the regulatory network of the metazoan LCA to control cell specification and regionalized gene expression during embryogenesis.