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Sponges (Porifera) are a large phylum that includes an enormous number of species. They are classified into four classes. Among these four classes, class Demospongiae is the largest and contains more than 90% of sponge species. In the last decade, methodologies for molecular studies and sequencing resources in sponge biology have dramatically advanced and made it possible to clearly define particular types of cells based on the genes they are expressing. Here we describe in detail the method of high-resolution WISH (whole mount in situ hybridization) and dual color fluorescent detection of in situ hybridization (dual color FISH) that we have established to detect particular types of cells, especially their stem cells known as archeocytes, in juveniles of freshwater demosponge, E. fluviatilis.

Animal morphogenesis can be summarized as a reconfiguration of a mass of cells. Although extracellular matrices that include rigid skeletal elements, such as cartilage/bones and exoskeletons, have important roles in morphogenesis, they are also secreted in situ by accumulated cells or epithelial cells. In contrast, recent studies of the skeleton construction of sponges (Porifera) illuminate a conceptually different mechanism of morphogenesis in which cells manipulate rather fine rigid materials (spicules) to form larger structures. Here, two different types of sponge skeleton formation using calcareous spicules or siliceous spicules are compared with regard to the concept of the production of rigid materials and their use in skeletons. The comparison highlights the advantages of their different strategies of forming sponge skeletons.

The evolution of multicellular organisms is generally thought (and seems likely) to have been accompanied by the evolution of a stem cell system. Sponges, some of the early-evolved metazoans, have totipotent/pluripotent stem cells. Thus, uncovering the cellular and molecular bases of the sponge stem cells will not only be crucial for understanding the ancestral gene repertoire of animal stem cells, but will also give us clues to understanding the evolution of molecular mechanisms for maintaining multipotency (pluripotency) and differentiation ability during animal evolution. Sponges (Porifera) are a large phylum that includes an enormous number of species, whose cellular compositions and life cycles show striking variations. In the last decade, methodologies for molecular studies and sequencing resources have dramatically advanced and made it possible to clearly define stem cells in sponges in cellular and molecular terms. In this review, together with recent studies of sponges in various classes, the following issues will be discussed: i) recent findings that revealed that the previously proposed model that “archeocytes and choanocytes are the two types of stem cells” originally based on work in demosponges can be applied as a unified view of the stem cell system in sponges that have various cellular organizations, ii) the fact that sponge cells are more plastic than previously thought, as shown by recent studies of sponge regeneration both from dissociated cells and upon injury, and iii) the importance of transdifferentiation in sponge stem cell systems and regeneration.

Animal bodies are shaped by skeletons, which arebuilt inside the body by biomineralization of condensed mesenchymal cells in vertebrates [1, 2] and echinoderms [3, 4], or outside the body by apical secretion of extracellular matrices by epidermal cell layers in arthropods [5]. In each case, the skeletons' shapes are a direct reflection of the pattern of skeleton-producing cells [6]. Here we report a newly discovered mode of skeleton formation: assembly of sponges' mineralized skeletal elements (spicules) in locations distant from where they were produced. Although it was known that internal skeletons of sponges consist of spicules assembledinto large pole-and-beam structures with a variety of morphologies [7-10], the spicule assembly process (i.e., how spicules become held up and connected basically in staggered tandem) and what types of cells act in this process remained unexplored. Here we found that mature spicules are dynamically transported from where they were produced and then pierce through outer epithelia, and their basal ends become fixed to substrate orconnected with such fixed spicules. Newly discovered "transport cells" mediate spicule movement and the "pierce" step, and collagen-secreting basal-epithelial cells fix spicules to the substratum, suggesting that the processes of spiculous skeleton construction are mediated separately by specialized cells. Division of labor by manufacturer, transporter, and cementer cells, and iteration of thesequential mechanical reactions of "transport, " "pierce, " "raise up, " and "cementation, " allows construction of the spiculous skeleton spicule by spicule as a self-organized biological structure, with the great plasticity in size and shape required for indeterminate growth, and generating the great morphological diversity of individual sponges., カイメン体内で微細な建築資材（ガラス質の骨片）を細胞が運び、立て、組み上げる全く新しい骨格形成機構を発見. 京都大学プレスリリース. 2015-09-24.

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

We previously revealed that the mechanism of demosponge skeleton construction is self-organization by multiple rounds of sequential mechanical reactions of player cells. In these reactions, "transport cells" dynamically carry fine skeletal elements (spicules) on epithelia surrounding the inner body space of sponges (basal epithelium (basopinacoderm) and the endodermal epithelium (ENCM)). Once spicules pierce ENCM and apical pinacoderm, subsequently they are cemented to the substratum under the sponge body, or connected to other skeleton-constructing spicules. Thus, the "pierce" step is the key to holding up spicules in the temporary periphery of growing sponges' bodies. Since sponges can regress as well as grow, here we asked how skeleton construction occurs during local regression of the body. We found that prior to local basopinacoderm retraction (and thus body regression), the body became thinner. Some spicules that were originally carried outward stagnated for a while, and were then carried inwards either on ENCM or basopinacoderm. Spicules that were carried inwards on ENCM pierced epithelia after a short transport, and thus became held up at relatively inward positions compared to spicules carried on outwardly extending basopinacoderm. The switch of epithelia on which transport cells migrate efficiently occurred in thinner body spaces where basopinacoderm and ENCM became close to each other. Thus, the mechanisms underlying this phenomenon are rather mechanical: the combination of sequential reactions of skeleton construction and the narrowed body space upon local retraction of basopinacoderm cause spicules to be held up at more-inward positions, which might strengthen the basopinacoderm's attachment to substratum.

Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans.

Various organisms have color patterns on their body surfaces, and these color patterns are thought to contribute to physiological regulation, communication with conspecifics, and signaling with the environment. An adult fly of Drosophila guttifera (Insecta: Diptera: Drosophilidae) has melanin pigmentation patterns on its body and wings. Though D. guttifera has been used for research into color pattern formation, how its pupal development proceeds and when the pigmentation starts have not been well studied. In this study, we defined the pupal stages of D. guttifera and measured the pigment content of wing spots from the pupal period to the period after eclosion. Using a transgenic line which carries eGFP connected with an enhancer of yellow, a gene necessary for melanin synthesis, we analyzed the timing at which the yellow enhancer starts to drive eGFP. We also analyzed the distribution of Yellow-producing cells, as indicated by the expression of eGFP during pupal and young adult periods. The results suggested that Yellow-producing cells were removed from wings within 3 h after eclosion, and wing pigmentation continued without epithelial cells. Furthermore, the results of vein cutting experiments showed that the transport of melanin precursors through veins was necessary for wing pigmentation. These results showed the importance of melanin precursors transported through veins and of extracellular factors which were secreted from epithelial cells and left in the cuticle.

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.

Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA-expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU-incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell-lineage-specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell-lineage-specific markers during archeocyte differentiation into other cell types. Choanocytes are food-entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell-like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.

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).

Siliceous spicules of sponges are morphologically diverse and provide good models for understanding the morphogenesis of biomineralized products. The silica deposition enzyme silicatein is a component of siliceous spicules of sponges and is thought to be the key molecule determining the morphology of spicules. Here, we focused on the silicateins of the freshwater sponge Ephydatia fluviatilis, which has two types of morphologically and functionally different spicules, called megascleres and gemmoscleres. We isolated six isoforms of silicateins and examined their mRNA expression in the cells producing megascleres and gemmoscleres. The spicule-type-specific mRNA expression of these isoforms and differential expression during spicule development suggest that the characteristic morphology of spicules is due to the specific properties and combinatory functions of silicatein isoforms.

International audience; 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.

The caspases, a family of cysteine proteases, play multiple roles in apoptosis, inflammation, and cellular differentiation. Caspase-8 (Casp8), which was first identified in humans, functions as an initiator caspase in the apoptotic signaling mediated by cell-surface death receptors. To understand the evolution of function in the Casp8 protein family, casp8 orthologs were identified from a comprehensive range of vertebrates and invertebrates, including sponges and cnidarians, and characterized at both the gene and protein levels. Some introns have been conserved from cnidarians to mammals, but both losses and gains have also occurred; a new intron arose during teleost evolution, whereas in the ascidian Ciona intestinalis, the casp8 gene is intronless and is organized in an operon with a neighboring gene. Casp8 activities are near ubiquitous throughout the animal kingdom. Exogenous expression of a representative range of nonmammalian Casp8 proteins in cultured mammalian cells induced cell death, implying that these proteins possess proapoptotic activity. The cnidarian Casp8 proteins differ considerably from their bilaterian counterparts in terms of amino acid residues in the catalytic pocket, but display the same substrate specificity as human CASP8, highlighting the complexity of spatial structural interactions involved in enzymatic activity. Finally, it was confirmed that the interaction with an adaptor molecule, Fas-associated death domain protein, is also evolutionarily ancient. Thus, despite structural diversity and cooption to a variety of new functions, the ancient origins and near ubiquitous distribution of this activity across the animal kingdom emphasize the importance and utility of Casp8 as a central component of the metazoan molecular toolkit.

Ecological developmental biology (eco-devo) explores the mechanistic relationships between the processes of individual development and environmental factors. Recent studies imply that some of these relationships have deep evolutionary origins, and may even pre-date the divergences of the simplest extant animals, including cnidarians and sponges. Development of these early diverging metazoans is often sensitive to environmental factors, and these interactions occur in the context of conserved signaling pathways and mechanisms of tissue homeostasis whose detailed molecular logic remain elusive. Efficient methods for transgenesis in cnidarians together with the ease of experimental manipulation in cnidarians and sponges make them ideal models for understanding causal relationships between environmental factors and developmental mechanisms. Here, we identify major questions at the interface between animal evolution and development and outline a road map for research aimed at identifying the mechanisms that link environmental factors to developmental mechanisms in early diverging metazoans. Also watch the Video Abstract.

The shufflon, a multiple DNA inversion system in the plasmid R64, consists of four DNA segments flanked and separated by seven 19-bp repeat sequences. Site-specific recombinations mediated by the rci product occur between each inverted repeat sequence, resulting in inversions of the four segments independently or in groups. The seven 19-bp repeat sequences are classified into four types (repeat-a, -b, -c, and -d), according to their 3-bp variable sequences. We individually cloned A, B, and C segments of the R64 shufflon and determined the in vivo inversion frequency of each segment. The inversion frequencies of three segments differed greatly. The inversion frequency declined in the following order: segments A, B, and C. Synthetic 19-mer oligonucleotides corresponding to both strands of repeat-a, -b, -c, and -d sequences were inserted into appropriate sites of pBR322. The rci-mediated DNA inversion occurred between two synthetic inverted repeats, indicating that the 19-bp inverted repeat sequences are the sole elements required in cis for the shufflon system. The inversion frequencies of DNA segments flanked by various sequences indicate that the four types of repeat sequences determine the inversion frequency of the four DNA segments of the R64 shufflon. Deletion of a DNA segment flanked by direct repeat sequences could not be detected.

beta-Catenin, a cytoplasmic protein known for its association with cadherin cell adhesion molecules, is also part of a signaling cascade involved in embryonic patterning processes such as the determination of the dorsoventral axis in Xenopus and determination of segment polarity in Drosophila. Previous studies suggest that increased cytoplasmic levels of beta-catenin correlate with signaling, raising questions about the need for in- teraction with cadherins in this process. We have tested the role of the beta-catenin-cadherin interaction in axis formation. Using beta-catenin deletion mutants, we demonstrate that significant binding to cadherins can be eliminated without affecting the signaling activity. Also, depletion of the soluble, cytosolic pool of beta-catenin by binding to overexpressed C-cadherin completely inhibited beta-catenin-inducing activity. We conclude that binding to cadherins is not required for beta-catenin signaling, and therefore the signaling function of beta-catenin is independent of its role in cell adhesion. Moreover, because beta-catenin signaling is antagonized by binding to cadherins, we suggest that cadherins can act as regulators of the intracellular beta-catenin signaling pathway.

beta-catenin was identified as a cytoplasmic cadherin-associated protein required for cadherin adhesive function (Nagafuchi, A., and M. Takeichi. 1989. Cell Regul. 1:37-44; Ozawa, M., H. Baribault, and R. Kemler. 1989. EMBO [Eur. Mol. Biol. Organ.] J. 8:1711-1717). Subsequently, it was found to be the vertebrate homologue of the Drosophila segment polarity gene product Armadillo (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science [Wash. DC]. 254:1359-1361; Peifer, M., and E. Wieschaus. 1990. Cell. 63:1167-1178). Also, antibody perturbation experiments implicated beta-catenin in axial patterning of the early Xenopus embryo (McCrea, P. D., W. M. Brieher, and B. M. Gumbiner. 1993. J. Cell Biol. 123:477-484). Here we report that overexpression of beta-catenin in the ventral side of the early Xenopus embryo, by injection of synthetic beta-catenin mRNA, induces the formation of a complete secondary body axis. Furthermore, an analysis of beta-catenin deletion constructs demonstrates that the internal armadillo repeat region is both necessary and sufficient to induce axis duplication. This region interacts with C-cadherin and with the APC tumor suppressor protein, but not with alpha-catenin, that requires the amino-terminal region of beta-catenin to bind to the complex. Since alpha-catenin is required for cadherin-mediated adhesion, the armadillo repeat region alone probably cannot promote cell adhesion, making it unlikely that beta-catenin induces axis duplication by increasing cell adhesion. We propose, rather, that beta-catenin acts in this circumstance as an intracellular signaling molecule. Subcellular fractionation demonstrated that all of the beta-catenin constructs that contain the armadillo repeat domain were present in both the soluble cytosolic and the membrane fraction. Immunofluorescence staining confirmed the plasma membrane and cytoplasmic localization of the constructs containing the armadillo repeat region, but revealed that they also accumulate in the nucleus, especially the construct containing only the armadillo repeat domain. These findings and the beta-catenin protein interaction data offer several intriguing possibilities for the site of action or the protein targets of beta-catenin signaling activity.

Major questions about stem cell systems include what type(s) of stem cells are involved (unipotent/totipotent/pluripotent/multipotent stem cells) and how the self-renewal and differentiation of stem cells are regulated. Sponges, the sister group of all other animals and probably the earliest branching multicellular lineage of extant animals, are thought to possess totipotent stem cells. This review introduces what is known about the stem cells in sponges based on histological studies and also on recent molecular biological studies that have started to reveal the molecular and cellular mechanisms of the stem cell system in sponges (mainly in demosponges). The currently proposed model of the stem cell system in demosponges is described, and the possible applicability of this model to other classes of sponges is discussed. Finally, a possible scenario of the evolution of stem cells, including how migrating stem cells arose in the urmetazoan (the last common ancestor of metazoans) and the evolutionary origin of germ line cells in the urbilaterian (the last common ancestor of bilaterians), are discussed.

Little is known about the stem cells of organisms early in metazoan evolution. To characterize the stem cell system in demosponges, we identified Piwi homologs of a freshwater sponge, Ephydatia fluviatilis, as candidate stem cell (archeocyte) markers. EfPiwiA mRNA was expressed in cells with archeocyte cell morphological features. We demonstrated that these EfPiwiA-expressing cells were indeed stem cells by showing their ability to proliferate, as indicated by BrdU-incorporation, and to differentiate, as indicated by the coexpression of EfPiwiA with cell-lineage-specific genes in presumptive committed archeocytes. EfPiwiA mRNA expression was maintained in mature choanocytes forming chambers, in contrast to the transition of gene expression from EfPiwiA to cell-lineage-specific markers during archeocyte differentiation into other cell types. Choanocytes are food-entrapping cells with morphological features similar to those of choanoflagellates (microvillus collar and a flagellum). Their known abilities to transform into archeocytes under specific circumstances and to give rise to gametes (mostly sperm) indicate that even when they are fully differentiated, choanocytes maintain pluripotent stem cell-like potential. Based on the specific expression of EfPiwiA in archeocytes and choanocytes, combined with previous studies, we propose that both archeocytes and choanocytes are components of the demosponge stem cell system. We discuss the possibility that choanocytes might represent the ancestral stem cells, whereas archeocytes might represent stem cells that further evolved in ancestral multicellular organisms.

A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map. It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.

We propose two major evolutionary origins of stem cell systems in the animal kingdom. Adult pluripotent stem cell systems are found in many invertebrates and probably evolved as components of asexual reproduction. Lineage-specific stem cell systems probably evolved later and include neural and hematopoietic stem cell types. We propose that these two types of stem cell systems evolved independently. The vasa-like genes regulate reproductive stem cells, but not lineage-specific stem cells, which may be regulated by gcm genes. Here, we review the evidence for the molecular basis for the evolutionary origin of these two different stem cell systems.

Sponges (phylum Porifera) have remarkable regenerative and reconstitutive abilities and represent evolutionarily the oldest metazoans. To investigate sponge stem cell differentiation, we have focused on the asexual reproductive system in the freshwater sponge Ephydatia fluviatilis. During germination, thousands of stem cells proliferate and differentiate to form a fully functional sponge. As an initial step of our investigation of stem cell (archeocyte) differentiation, we isolated molecular markers for two differentiated cell types: spicule-making sclerocyte cells, and cells involved in innate immunity. Sclerocyte lineage-specific Ef silicatein shares 45% to 62% identity with other sponge silicateins. As in situ hybridization of Ef silicatein specifically detects archeocytes possibly committed to sclerocytes, as well as sclerocytes with an immature or mature spicule, therefore covering all the developmental stages, we conclude that Ef silicatein is a suitable sclerocyte lineage marker. Ef lectin, a marker for the cell type involved in innate immunity, shares 59% to 65% identity with the marine sponge Suberites domuncula galactose-binding protein (Sd GBP) and horseshoe crab Tachypleus tridentatus tachylectin1/lectinL6. Since Sd GBP and tachylectin1 are known to bind to bacterial lipopolysaccharides and inhibit the growth of bacteria, Ef lectin may have a similar function and be expressed in a specialized type of cell involved in defense against invading bacteria. Ef lectin mRNA and protein are not expressed in early stages of development, but are detected in late stages. Therefore, Ef lectin may be specifically expressed in differentiating and/or differentiated cells. We suggest Ef lectin as a marker for cells that assume innate immunity in freshwater sponges.

Most triploblastic animals including vertebrates have a coelomic cavity that separates the outer and inner components of the body. The coelom is lined by two different tissue components, somatopleure and splanchnopleure, which are derived from the lateral plate region. Thus, the coelom is constructed as a result of a binary decision during early specification of the lateral plate. In this report we studied the molecular mechanisms of this binary decision. We first demonstrate that the splitting of the lateral plate into the two cell sheets progresses in an anteroposterior order and this progression is not coordinated with that of the somitic segmentation. By a series of embryological manipulations we found that young splanchnic mesoderm is still competent to be respecified as somatic mesoderm, and the ectoderm overlying the lateral plate is sufficient for this redirection. The lateral ectoderm is also required for maintenance of the somatic character of the mesoderm. Thus, the ectoderm plays at least two roles in the early subdivision of the lateral plate: specification and maintenance of the somatic mesoderm. We also show that the latter interactions are mediated by BMP molecules that are localized in the lateral ectoderm. Evolutionary aspects of the coelom formation are also considered.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.

Analysis of genomic resources available for cnidarians revealed that several key components of the vertebrate innate immune repertoire are present in representatives of the basal cnidarian class Anthozoa, but are missing in Hydra, a member of the class Hydrozoa, indicating ancient origins for many components of the innate immune system., Background Characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest - it not only provides insights into the basic immunological 'tool kit' of the common ancestor of all animals, but is also likely to be important in understanding the global decline of coral reefs that is presently occurring. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag (EST) datasets are available for these and for the coral Acropora millepora. Results To better understand the basis of innate immunity in cnidarians, we scanned the available EST and genomic resources for some of the key components of the vertebrate innate immune repertoire, focusing on the Toll/Toll-like receptor (TLR) and complement pathways. A canonical Toll/TLR pathway is present in representatives of the basal cnidarian class Anthozoa, but neither a classic Toll/TLR receptor nor a conventional nuclear factor (NF)-κB could be identified in the anthozoan Hydra. Moreover, the detection of complement C3 and several membrane attack complex/perforin domain (MAC/PF) proteins suggests that a prototypic complement effector pathway may exist in anthozoans, but not in hydrozoans. Together with data for several other gene families, this implies that Hydra may have undergone substantial secondary gene loss during evolution. Such losses are not confined to Hydra, however, and at least one MAC/PF gene appears to have been lost from Nematostella. Conclusion Consideration of these patterns of gene distribution underscores the likely significance of gene loss during animal evolution whilst indicating ancient origins for many components of the vertebrate innate immune system.

Molecular mechanisms by which the mesoderm is subdivided along the mediolateral axis in early chicken embryos have been studied. When the presomitic mesoderm (medial mesoderm) was transplanted into the lateral plate, the graft was transformed into lateral plate tissue, indicating that the primitive somite was not fully committed and that the lateral plate has a cue for mesodermal lateralization. Since the lateral plate expresses a high level of BMP-4 mRNA, a member of the TGF-β family, we hypothesized that it is the molecule responsible for the lateralization of the somite. To test this, we transplanted COS cells producing BMP-4 into the presomitic region. Those cells locally prevented the presomitic cells from differentiating into somites, converting them instead into lateral plate mesoderm, which was revealed by expression of cytokeratin mRNA, a marker for the lateral plate. The effect was dependent on the level of effective BMP-4: with a high level of BMP-4, the somite was transformed completely to lateral plate; with a low level, the somite formed but was occupied by the lateral somitic component expressing cSim 1, a marker for the lateral somite. These results suggest that different thresholds of effective BMP-4 determine distinct subtypes of the mesoderm as a lateralizer during early development.

In order to investigate the cellular system of the freshwater sponge, Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mount in situ hybridization shows Ef annexin expression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude that Ef annexin can be used as a choanocyte marker. In the early development stage, Ef annexin expression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate that Ef annexin is initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte.