Masato Kiyomoto

The 5-HT receptor cell is a new member of secondary mesenchyme cell descendants and forms a major blastocoelar network in sea urchin larvae

A gene encoding the serotonin (5-hydroxytryptamine, 5-HT) receptor (5-HT-hpr) was identified from the sea urchin, Hemicentrotus pulcherrimus. Partial amino acid sequence deduced from the cDNA showed strong similarity to Aplysia californica 5-HT2 receptor. Immunoblotting analysis of this 5-HT-hpr protein (5-HT-hpr) with an antibody raised against a deduced peptide showed two bands. Their relative molecular masses were 69 and 53 kDa, respectively. The larger band alone disappeared after N-glycopeptidase F digestion, indicating the protein was N-glycosylated. Immunolocalization analysis showed that cells expressing the 5-HT-hpr (SRC) first appeared near the tip of the archenteron in 33-h post-fertilization (33 hpf) prism larvae. Their cell number doubled in 2 h, and 5-HT-hpr protein expression increased further without cell proliferation. SRC spread ventrally on the basal surface of the oral ectoderm in 36 hpf prism larvae, and then clockwise on the ventral ectoderm to the posterior region to complete formation of the SRC network in 48 hpf early plutei. The SRC network was comprised of 7 main tracts: 4 spicule system-associated tracts and 3 spicule system-independent tracts. The network extended short fibers to the larval body surface through the ectoderm, implicating a signal transmission system that receives exogenous signal. Double-stain immunohistochemistry with antibodies to primary mesenchyme cells showed that SRC were not stained by the antibody. In embryos deprived of secondary mesenchyme cell (SMC) by microsurgery, the number of SRC decreased considerably. These two data indicate that SRC are SMC descendants, adding a new member to the SMC lineage.

Heterochronic activation of VEGF signaling and the evolution of the skeleton in echinoderm pluteus larvae.

The evolution of the echinoderm larval skeleton was examined from the aspect of interactions between skeletogenic mesenchyme cells and surrounding epithelium. We focused on vascular endothelial growth factor (VEGF) signaling, which was reported to be essential for skeletogenesis in sea urchin larvae. Here, we examined the expression patterns of vegf and vegfr in starfish and brittle stars. During starfish embryogenesis, no expression of either vegfr or vegf was detected, which contrast with previous reports on the expression of starfish homologs of sea urchin skeletogenic genes, including Ets, Tbr, and Dri. In later stages, when adult skeletogenesis commenced, vegfr and vegf expression were upregulated in skeletogenic cells and in the adjacent epidermis, respectively. These expression patterns suggest that heterochronic activation of VEGF signaling is one of the key molecular evolutionary steps in the evolution of the larval skeleton. The absence of vegf or vegfr expression during early embryogenesis in starfish suggests that the evolution of the larval skeleton requires distinct evolutionary changes, both in mesoderm cells (activation of vegfr expression) and in epidermal cells (activation of vegf expression). In brittle stars, which have well‐organized skeletons like the sea urchin, vegfr and vegf were expressed in the skeletogenic mesenchyme and the overlying epidermis, respectively, in the same manner as in sea urchins. Therefore, the distinct activation of vegfr and vegf may have occurred in two lineages, sea urchins and brittle stars.

Abnormal Gametogenesis, Male Dominant Sex Ratio, and Sertoli Cell Morphology in Induced Triploid Mussels, Mytilus galloprowincialis

Abstract Gametogenesis of one year-old induced triploid mussels, Mytilus galloprovincialis, was examined histologically and compared to sibling diploid mussels. Histological analysis revealed that triploid mussels developed a number of primary spermatocytes that were arrested at prophase I. Late in the reproductive season, triploid mussels produced an extremely small number of spermatozoa (9/10000 &mgr;m2 gonadal section) compared to diploid mussels (1072/10000 &mgr;m2 gonadal section). All triploid mussels were identified as males, whereas the sex ratio of diploid mussels was almost equal (1.12:1.0, male:female), indicating that sex determination for this species may follow a Z:W model. Sertoli cells in triploid mussels were prominent, had an enlarged cytoplasm, and were easily seen using light microscopy. In comparison, Sertoli cells in diploids were thin and could only be seen by electron microscopy. Sertoli cell hypertrophy in triploid mussels probably reflects their role in eliminating abnormal and degenerating germ cells.

Study of Larval and Adult Skeletogenic Cells in Developing Sea Urchin Larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.

All meiotic chromosomes and both centrosomes at spindle pole in the zygotes discarded as two polar bodies in clam Corbicula leana: unusual polar body formation observed by antitubulin immunofluorescence

Abstract To understand the unusual polar body formation in the androgenetic clam, Corbicula leana, whole-mount eggs stained with monoclonal antibodies against α-tubulin, γ-tubulin, and 4’-6’-diamidino-2-phenylindole were examined. The meiotic spindle was located at the peripheral region of the egg at metaphase I, and its axis was parallel to the egg surface. After segregation of chromosomes at anaphase I, cytoplasmic bulges formed at both meiotic spindle pole sites. Centrosomes were located at the apical portion of the each bulge. From the apical portion of the bulge a bundle of astral microtubules radiated toward the bulge base in late anaphase resembling a half spindle. Maternal chromosomes and both centrosomes were all distributed in two ”first polar bodies” and were eventually discarded. After the polar body formation only one male pronucleus existed in the egg cytoplasm. The present study showed that the anaphase microtubules originating from a single aster can induce the polar body formation without overlapping of microtubules from the opposing aster.

Functional evolution of Ets in echinoderms with focus on the evolution of echinoderm larval skeletons

Convergent evolution of echinoderm pluteus larva was examined from the standpoint of functional evolution of a transcription factor Ets1/2. In sea urchins, Ets1/2 plays a central role in the differentiation of larval skeletogenic mesenchyme cells. In addition, Ets1/2 is suggested to be involved in adult skeletogenesis. Conversely, in starfish, although no skeletogenic cells differentiate during larval development, Ets1/2 is also expressed in the larval mesoderm. Here, we confirmed that the starfish Ets1/2 is indispensable for the differentiation of the larval mesoderm. This result led us to assume that, in the common ancestors of echinoderms, Ets1/2 activates the transcription of distinct gene sets, one for the differentiation of the larval mesoderm and the other for the development of the adult skeleton. Thus, the acquisition of the larval skeleton involved target switching of Ets1/2. Specifically, in the sea urchin lineage, Ets1/2 activated a downstream target gene set for skeletogenesis during larval development in addition to a mesoderm target set. We examined whether this heterochronic activation of the skeletogenic target set was achieved by the molecular evolution of the Ets1/2 transcription factor itself. We tested whether starfish Ets1/2 induced skeletogenesis when injected into sea urchin eggs. We found that, in addition to ectopic induction of mesenchyme cells, starfish Ets1/2 can activate some parts of the skeletogenic pathway in these mesenchyme cells. Thus, we suggest that the nature of the transcription factor Ets1/2 did not change, but rather that some unidentified co-factor(s) for Ets1/2 may distinguish between targets for the larval mesoderm and for skeletogenesis. Identification of the co-factor(s) will be key to understanding the molecular evolution underlying the evolution of the pluteus larvae.

The Major Yolk Protein is Synthesized in the Digestive Tract and Secreted Into the Body Cavities in Sea Urchin Larvae

Major yolk protein (MYP), a transferrin superfamily protein contained in yolk granules of sea urchin eggs, also occurs in the coelomic fluid of male and female adult sea urchins regardless of their reproductive cycle. MYP in the coelomic fluid (CFMYP; 180 kDa) has a zinc‐binding capacity and has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). CFMYP is thought to be synthesized in the digestive tract and secreted into the coelomic fluid where it is involved in the transport of zinc derived from food. To clarify when and where MYP synthesis starts, we investigated the expression of MYP during larval development and growth in Pseudocentrotus depressus. MYP mRNA was detected using RT‐PCR in the early 8‐arm pluteus stage and its expression persisted until after metamorphosis. Real‐time RT‐PCR revealed that MYP mRNA increased exponentially from the early 8‐arm stage to metamorphosis. Western blotting showed that maternal EGMYP disappeared by the 4‐arm stage and that newly synthesized CFMYP was present at and after the mid 8‐arm stage. In the late 8‐arm larvae, MYP mRNA was detected in the digestive tract using in situ hybridization, and the protein was found in the somatocoel and the blastocoel‐derived space between the somatocoel and epidermis using immunohistochemistry. These results suggest that CFMYP is synthesized in the digestive tract and secreted into the body cavities at and after the early 8‐arm stage. We assume that in larvae, CFMYP transports zinc derived from food via the body cavities to various tissues, as suggested for adults. Mol. Reprod. Dev. 76: 142–150, 2009.

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm–extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)‐beta growth factor, Pl‐univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton‐defective embryos rescues skeleton development. In addition, PMC from skeleton‐defective embryos transplanted into normal or PMC‐less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.

Skeletogenesis by transfated secondary mesenchyme cells is dependent on extracellular matrix–ectoderm interactions in Paracentrotus lividus sea urchin embryos

In the sea urchin embryo, primary mesenchyme cells (PMCs) are committed early in development to direct skeletogenesis, provided that a permissive signal is conveyed from adjacent ectoderm cells. We showed that inhibition of extracellular matrix (ECM)–ectoderm cells interaction, by monoclonal antibodies (mAb) to Pl‐nectin, causes an impairment of skeletogenesis and reduced expression of Pl‐SM30, a spicule‐specific matrix protein. When PMCs are experimentally removed, some secondary mesenchyme cells (SMCs) switch to skeletogenic fate. Here, for the first time we studied SMC transfating in PMC‐less embryos of Paracentrotus lividus. We observed the appearance of skeletogenic cells within 10 h of PMCs removal, as shown by binding of wheat germ agglutinin (WGA) to cell surface molecules unique to PMCs. Interestingly, the number of WGA‐positive cells, expressing also msp130, another PMC‐specific marker, doubled with respect to that of PMCs present in normal embryos, though the number of SM30‐expressing cells remained constant. In addition, we investigated the ability of SMCs to direct skeletogenesis in embryos exposed to mAbs to Pl‐nectin after removal of PMCs. We found that, although phenotypic SMC transfating occurred, spicule development, as well as Pl‐SM30‐expression was strongly inhibited. These results demonstrate that ectoderm inductive signals are necessary for transfated SMCs to express genes needed for skeletogenesis.

Ars insulator protects transgenes from long-term silencing in sea urchin larva

Reporter genes have been used as a powerful tool to analyze cis-regulatory elements responsible for temporal and spatial expression in the early development of sea urchin. However, here we show that the transgenes introduced into the sea urchin embryos undergo suppression in larval stage. The transgene silencing could be one of major obstacle in the analysis of regulatory regions in the late stages of development. We previously demonstrated that a DNA fragment (ArsI) located in the upstream region of sea urchin (Hemicentrotus pulcherrimus) arylsulfatase gene has the property of an insulator. We show that tandem ArsI prevents silencing of a transgene in sea urchin larvae when the ArsI is fused to the 5′ end of the reporter gene. Furthermore, we demonstrate that DNA of the reporter gene introduced into the sea urchin eggs is methylated during development and that the ArsI protects the transgene from the DNA methylation.