Blue-light-receptive cryptochrome is expressed in a sponge eye lacking neurons and opsin

Many larval sponges possess pigment ring eyes that apparently mediate phototactic swimming. Yet sponges are not known to possess nervous systems or opsin genes, so the unknown molecular components of sponge phototaxis must differ fundamentally from those in other animals, inspiring questions about how this sensory system functions. Here we present molecular and biochemical data on cryptochrome, a candidate gene for functional involvement in sponge pigment ring eyes. We report that Amphimedon queenslandica, a demosponge, possesses two cryptochrome/photolyase genes, Aq-Cry1 and Aq-Cry2. The mRNA of one gene (Aq-Cry2) is expressed in situ at the pigment ring eye. Additionally, we report that Aq-Cry2 lacks photolyase activity and contains a flavin-based co-factor that is responsive to wavelengths of light that also mediate larval photic behavior. These results suggest that Aq-Cry2 may act in the aneural, opsin-less phototaxic behavior of a sponge

Origin of animal epithelia: insights from the genome of the demosponge Amphimedon queenslandica

Metazoan body plans are diverse but make use of a basic set of cell and tissue types conserved across the majority of animal phyla. Epithelial tissues represent one such tissue type. They provide a cohesive unit for the construction of body walls and organs, and allow for sealing between distinct bodily compartments and between the organism and the external environment. Traditionally, an epithelial grade of organization has been considered a synapomorphy for the Eumetazoa (Ctenophora, Cnidaria and Bilateria), with tissues found in the phyla, Porifera and Placozoa, observed to lack key structural characteristics of epithelia. A scenario in which the poriferan and placozoan lineages diverged from other metazoans prior to the acquisition of true epithelia in the eumetazoan stem is consistent with recent molecular phylogenomic analyses that place Porifera as the earliest-branching metazoan phyla with Placozoa basal amongst the Eumetazoa. The evolution of the epithelial tissue phenotype involved the innovation and integration of several cellular and extracellular features. As with the evolution of any complex trait, it is possible that some of these features preceded others, with the full epithelial phenotype appearing in the lineage leading to the Eumetazoa but making use of cellular and molecular characteristics acquired earlier in metazoan evolution. In particular, the observation that epithelia-like tissues of poriferans display some of the structural characteristics of eumetazoan epithelia suggests the possibility that the evolution of this tissue type proceeded step-wise. This study applies a comparative molecular approach to address this question. Studies in model bilaterians have shown that a conserved set of genes are necessary for the development and maintenance of key epithelial characteristics. These latter include aligned cellular apical-basal polarity, belt-form junctions of the stabilizing and occluding types and an underlying basal lamina. A survey of the genome of the demosponge, Amphimedon queenslandica, reveals that this sponge encodes a near-complete complement of proteins necessary for establishing apical-basal cell polarity and forming adherens junctions. By contrast, no direct orthologues of bilaterian tight junction, septate junction or basal lamina genes were found. To further explore the timing of origin of these genes, I extend genome surveys to the completed genomes of multiple species of fungi, the choanoflagellate, Monosiga brevicollis, the placozoan, Trichoplax adhaerens, and the cnidarian, Nematostella vectensis. These analyses reveal that the majority of polarity-determining and adherens junction genes were metazoan innovations, with only Par-1 and Discs large clearly antedating the choanoflagellate-metazoan split. By contrast, the majority of septate junction and basal lamina genes are present in placozoan and cnidarian genomes, suggesting that they originated in the eumetazoan or eumetazoan sensu stricto lineages. The phylogenetic distribution of domains and domain combinations for all surveyed genes shows that most were assembled from pre-existing domains, with the occasional incorporation of novel domains or motifs. Searches for basal lamina genes reveal that Amphimedon encodes a unique set of laminin-related proteins but none that could be considered directly orthologous to bilaterian laminins. The finding of laminins with novel domain architectures prompted a more thorough comparative genomic analysis, along with domain-specific phylogenies aimed at exploring the relationships between genes with distinct architectures. Sequence analysis of the Amphimedon laminin-related proteins suggests that they may be capable of forming heterotrimers similar to those that polymerize in the basal lamina of bilaterians. However, the differences in domain structure between Amphimedon and bilaterian laminins make it impossible to fully predict the properties of the sponge proteins. The comparative genomic analysis uncovers considerable diversity in the laminin gene complements of surveyed metazoans and indicates that laminin-like genes were a holozoan innovation with typical bilaterian chain types first appearing in the eumetazoan stem. The ciliated outer layer of the Amphimedon larva resembles an epithelial tissue, particularly in the display of aligned apical-basal polarity between constituent cells. In situ hybridisation of genes identified in the genome surveys described above reveals that the polarity-related genes, AmqPar-6, AmqPar-1, AmqMPP5/7 and AmqLgl1, and the adherens junction gene, Amqp120Catenin, are expressed in epithelial-like cells of the larvae from early in embryogenesis. AmqPar-3 and AmqaPKC are also expressed during development but it is unclear whether transcripts are present in outer layer cells. Fluorescent phallacidin staining of larvae indicates that actin microfilaments are arranged in a belt around the apical circumference of epithelial-like cells, possibly in conjunction with zonula adherens junctions, which have not previously been observed in Amphimedon by microscopy. In conclusion, the results presented here suggest that the molecular bases for key features of the epithelial tissue phenotype, namely apical-basal cell polarity and zonula adherens junctions, evolved in the metazoan lineage and are likely to be functional in poriferan tissues. The origin of genes coding for components of occluding junctions and basal lamina in eumetazoan and eumetazoan sensu stricto lineages allowed for the emergence of the full epithelial phenotype subsequent to the divergence of the Porifera

Does the high gene density in the sponge NK homeobox gene cluster reflect limited regulatory capacity?

A huge discrepancy in morphological diversity exists between poriferans and eumetazoans. The disparate evolutionary outcomes of these two ancient metazoan lineages may be reflected in the composition, architecture, and regulation of genomes of modern representatives. As a case study, we compare the sizes of upstream intergenic regions of genes found within the NK homeobox cluster of the demosponge Amphimedon queenslandica with eumetazoan orthologs. This analysis includes NK genes as well as five structural genes interspersed in the cluster. The upstream intergenic regions of the homeobox genes are significantly smaller in Amphimedon than in eumetazoan orthologs, suggesting that the sponge genes house less cis-regulatory information. In contrast, the upstream intergenic regions of the structural genes are not significantly different. The simple developmental expression patterns of representative NK genes in Amphimedon lends support to the proposition that their regulatory apparatuses, unlike those of bilaterians, do not encode the information for dynamic, pleiotropic gene expression. On the basis of this example, we suggest that the size of the intergenic regions upstream of the transcription start site may act as a proxy for estimating regulatory complexity and reflect the limitations of the sponge genome to direct complex and varied morphogenetic processes

Remarkable consistency of larval release in the spermcast-mating demosponge Amphimedon queenslandica (Hooper and van Soest)

Many marine invertebrates, including many sponge species, reproduce by spermcast spawning, in which sperm released externally disperse in the water column to fertilize eggs retained internally by the maternal adult. The population consequences of a sexual reproduction mode that depends upon uptake of free spermatozoa from dilute suspension in the water column are not yet well understood. In the spermcast-spawning tropical demosponge Amphimedon queenslandica, we observed continuous fertilization and development in healthy maternal brood chambers. This results in a constant release of larvae into the water column. On average in our study population on Heron Island reef, a hermaphroditic adult will have 45 potential sperm donors available within a 4 m radius to fertilize the eggs retained within its brood chambers. A single adult may brood more than 300 embryos at one time, and all stages of development are always represented. These data can be explained by adult sponges releasing a steady trickle supply of sperm into the water column, perhaps in combination with the existence of a mechanism for sperm storage or post-fertilization developmental stasis in this species

Whole-mount in situ hybridization in Amphimedon

Developmental gene expression is analyzed predominantly via whole-mount in situ hybridization using digoxigenin-labeled RNA probes. This protocol describes how to perform this procedure in Amphimedon queenslandica, including fixation, hybridization, and sectioning of embryonic, larval, and post-larval juvenile stages

Whole-Mount In Situ Hybridization in Amphimedon

INTRODUCTIONDevelopmental gene expression is analyzed predominantly via whole-mount in situ hybridization using digoxigenin-labeled RNA probes. This protocol describes how to perform this procedure in Amphimedon queenslandica, including fixation, hybridization, and sectioning of embryonic, larval, and post-larval juvenile stages

Isolation of Amphimedon Developmental Material

INTRODUCTIONFertilization occurs internally in Amphimedon and embryos are brooded in multiple chambers throughout the adult. Each chamber contains a mixture of developmental stages, from egg to late ring stages (i.e., prehatch late embryos). At the end of embryogenesis, swimming parenchymella larvae emerge from the adult. After several hours in the water column, the larvae settle and metamorphose into juvenile sponges. This protocol details how to obtain Amphimedon larvae and post-larvae/juveniles as well as embryos. Once isolated, these biological stages can be used for a variety of molecular and cellular analyses

The Amphimedon queenslandica genome and the evolution of animal complexity

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 ĝ€̃ toolkit'trade; 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

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.</p