Yoshiki Masuda

Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges

SUMMARY 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.

Ribosomal ITS Sequences Allow Resolution of Freshwater Sponge Phylogeny with Alignments Guided by Secondary Structure Prediction

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatiamuelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.

Toward understanding the morphogenesis of siliceous spicules in freshwater sponge: Differential mRNA expression of spicule‐type‐specific silicatein genes in Ephydatia fluviatilis

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. Developmental Dynamics 237:3024–3039, 2008.

Monophyletic origin of freshwater sponges in ancient lakes based on partial structures of COXI gene

The phylogenetic relationships between freshwater sponges (families Lubomirskiidae, Spongillidae and Potamolepidae) are still poorly understood. Partial sequences of the cytochrome oxidase subunit I (COXI) gene were obtained from seven freshwater sponge species and one marine sponge. Phylogenetic analyses showed that all the freshwater sponge species formed one common clade and they appeared to be closely related. These results indicate that freshwater sponges are of monophyletic origin. Potamolepidae and Spongillidae are situated at the base of the tree and are assumed to be phylogenetically older than Lubomirskiidae.

The active stem cell specific expression of sponge Musashi homolog EflMsiA suggests its involvement in maintaining the stem cell state

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.

Sponge Cytogenetics — Mitotic Chromosomes of Ten Species of Freshwater Sponge

Abstract Porifera (sponges) are the most basal phylum of extant metazoans. To gain insight into sponge genome construction, cytogenetic analysis was performed for ten freshwater sponge species of six genera, using conventional Giemsa staining, chromosome banding, and fluorescence in-situ hybridization. The karyotypes were very similar among the ten species, exhibiting a diploid chromosome number of 2n=46 or 48, and usually consisted of microchromosomes with one or two pairs of large chromosomes. The 18S-28S rRNA genes were localized to a single pair of microchromosomes in two Ephydatia species. Hybridization signals of the telomere (TTAGGG)n sequences were observed at the ends of metaphase chromosomes. The genome sizes of Ephydatia fluviatilis and Ephydatia muelleri were estimated by flow cytometric analysis as about 0.7 pg per diploid complement. These freshwater sponge species appear to represent a fairly homogeneous group with respect to karyotypes.