織井 秀文

Xenopus dazl encoding an RNA-binding protein has been identified as a component of the germ plasm and is involved in the migration and differentiation of the primordial germ cells (PGCs). Here, we investigated the intracellular localization of Dazl in germline cells throughout the lifetime of Xenopus. In early embryogenesis, Dazl was detected initially in the germ plasm and then translocated to a perinuclear region. Then, it was detected within the nucleus in PGCs. Dazl was observed only in the cytoplasm in PGCs when sex differentiation began in the gonads. Dazl was distributed in both the nucleus and cytoplasm of the primary oogonium and spermatogonium, but only in the cytoplasm of the secondary oogonium and spermatogonium. In spermatocytes, Dazl was distributed throughout cytoplasm and localized at the spindles and cytoplasm during meiosis. Then, it was detected as speckles in the nucleus in the round spermatid. The dynamic intracellular localization suggests that Dazl is a multifunctional protein regulating RNA metabolism required for Xenopus germline development.

The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.

DEADSouth mRNA encoding the RNA helicase DDX25 is a component of the germ plasm in Xenopus laevis. We investigated the mechanisms underlying its specific mRNA expression in primordial germ cells (PGCs). Based on our previous findings of several microRNA miR-427 recognition elements (MREs) in the 3' untranslated region of the mRNA, we first examined whether DEADSouth mRNA was degraded by miR-427 targeting in somatic cells. Injection of antisense miR-427 oligomer and reporter mRNA for mutated MREs revealed that DEADSouth mRNA was potentially degraded in somatic cells via miR-427 targeting, but not in PGCs after the mid-blastula transition (MBT). The expression level of miR-427 was very low in PGCs, which probably resulted in the lack of miR-427-mediated degradation. In addition, the DEADSouth gene was expressed zygotically after MBT. Thus, the predominant expression of DEADSouth mRNA in the PGCs is ensured by multiple mechanisms including zygotic expression and prohibition from miR-427-mediated degradation.

We investigated the intracellular localization of Xenopus Dead end protein (Dnd1) in primordial germ cells during early development by expressing the tagged protein in transgenic Xenopus embryos, with the germ plasm visualized. Dnd1 initially localized to the germ plasm in the cortex, moved to the perinuclear region together with the germ plasm after the midblastula transition, and then entered the nucleus. Using Dnd1 deletion mutants, we identified two distinct but overlapping regions of Dnd1 that were responsible for localization to either the germ plasm or nucleus. These Dnd1 regions appeared to function in primordial germ cell- and stage-specific manners.

We have developed a simple and time-saving method to identify hybridoma clones producing an antibody against a target protein among a large number of hybridomas in a single step. This method is very useful as the primary screening tool for hybridomas producing monoclonal antibodies if several micrograms of the target protein is available.

DEADSouth mRNA is a component of germ plasm in Xenopus laevis and encodes a DDX25 DEAD-box RNA helicase. To determine the intracellular localization of DEADSouth protein, we injected mRNA encoding DEADSouth tagged with mCherry fluorescent protein into fertilized eggs from transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal. The DEADSouth-mCherry fusion protein was localized to the germ plasm, a mitochondria-rich region in primordial germ cells (PGCs). DEADSouth overexpression resulted in a reduction of PGC numbers after stage 20. Conversely, DEADSouth knockdown using an antisense locked nucleic acid gapmer inhibited movement of the germ plasm from the cortex to the perinuclear region, resulting in inhibition of PGC division at stage 12 and a decrease in PGC numbers at later stages. The knockdown phenotype was rescued by intact DEADSouth mRNA, but not mutant mRNA encoding inactive DEADSouth helicase. Surprisingly, it was also rescued by mouse vasa homolog and Xenopus vasa-like gene 1 mRNAs that encode DDX4 RNA helicases. The rescue was dependent on the 3' untranslated region (3'UTR) of DEADSouth mRNA, which was used for PGC-specific expression. The 3'UTR contributed to localization of the injected mRNA to the germ plasm, resulting in effective localization of DEADSouth protein. These results demonstrate that localization of DEADSouth helicase to the germ plasm is required for proper PGC development in Xenopus laevis.

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species-specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re-examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz-like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re-aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re-aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F-actin, myosin II activity and RhoA/Rho-associated protein kinase (ROCK) signaling.

Germes mRNA and protein are components of the germ plasm in Xenopus laevis. Previously, based on phenotypic observations of tailbud embryos expressing intact and mutant Germes, it was suggested that Germes is involved in the organization of germ plasm (Berekelya et al., 2007). Recently, to observe the germ plasm in a living embryo, we generated transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal, because germ plasm is enriched with mitochondria (Taguchi et al., 2012). Using this transgenic Xenopus, we demonstrate that Germes plays an essential role in the translocation of germ plasm from the cortex to the perinuclear region in primordial germ cells during early gastrulation.

In many animals, the germ line is specified by a distinct cytoplasmic structure called germ plasm (GP). GP is necessary for primordial germ cell (PGC) formation in anuran amphibians including Xenopus. However, it is unclear whether GP is a direct germ cell determinant in vertebrates. Here we demonstrate that GP acts autonomously for germ cell formation in Xenopus. EGFP-labeled GP from the vegetal pole was transplanted into animal hemisphere of recipient embryos. Cells carrying transplanted GP (T-GP) at the ectopic position showed characteristics similar to the endogenous normal PGCs in subcellular distribution of GP and presence of germ plasm specific molecules. However, T-GP-carrying-cells in the ectopic tissue did not migrate towards the genital ridge. T-GP-carrying cells from gastrula or tailbud embryos were transferred into the endoderm of wild-type hosts. From there, they migrated into the developing gonad. To clarify whether ectopic T-GP-carrying cells can produce functional germ cells, they were identified by changing the recipients, from the wild-type Xenopus to transgenic Xenopus expressing DsRed2. After transferring T-GP carrying cells labeled genetically with DsRed2 into wild-type hosts, we could find chimeric gonads in mature hosts. Furthermore, the spermatozoa and eggs derived from T-GP-carrying cells were fertile. Thus, we have demonstrated that Xenopus germ plasm is sufficient for germ cell determination.

Germ plasm is found in germ-line cells of Xenopus and thought to include the determinant of primordial germ cells (PGCs). As mitochondria is abundant in germ plasm, vital staining of mitochondria was used to analyze the movement and function of germ plasm; however, its application was limited in early cleavage embryos. We made transgenic Xenopus, harboring enhanced green fluorescent protein (EGFP) fused to the mitochondria transport signal (Dria-line). Germ plasm with EGFP-labeled mitochondria was clearly distinguishable from the other cytoplasm, and retained mostly during one generation of germ-line cells in Dria-line females. Using the Dria-line, we show that germ plasm is reorganized from near the cell membrane to the perinuclear space at St. 9, dependent on the microtubule system.

BACKGROUND: RNAs for embryo patterning and for germ cell specification are localized to the vegetal cortex of the oocyte of Xenopus laevis. In oocytes of the direct developing frog Eleutherodactylus coqui, orthologous RNAs for patterning are not localized, raising the question as to whether RNAs and other components of germ plasm are localized in this species. METHODS: To identify germ plasm, E. coqui embryos were stained with DiOC6(3) or examined by in situ hybridization for dazl and DEADSouth RNAs. The cDNAs for the E. coqui orthologues were cloned by RT-PCR using degenerate primers. To examine activity of the E. coqui orthologues, RNAs, made from constructs of their 3'UTRs with mCherry, were injected into X. laevis embryos. RESULTS: Both DiOC6(3) and dazl and DEADSouth in situs identified many small islands at the vegetal surface of cleaving E. coqui embryos, indicative of germ plasm. Dazl was also expressed in primordial germ cells in the genital ridge. The 3'UTRs of E. coqui dazl and DEADSouth directed primordial germ cell specific protein synthesis in X. laevis. CONCLUSIONS: E. coqui utilizes germ plasm with RNAs localized to the vegetal cortex to specify primordial germ cells. The large number of germ plasm islands suggests that an increase in the amount of germ plasm was important in the evolution of the large E. coqui egg.

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

Sll1252 was identified as a novel protein in photosystem II complexes from Synechocystis sp. PCC 6803. To investigate the function of Sll1252, the corresponding gene, sll1252, was deleted in Synechocystis 6803. Despite the homology of Sll1252 to YlmH, which functions in the cell division machinery in Streptococcus, the growth rate and cell morphology of the mutant were not affected in normal growth medium. Instead, it seems that cells lacking this polypeptide have increased sensitivity to Cl(-) depletion. The growth and oxygen evolving activity of the mutant cells was highly suppressed compared with those of wild-type cells when Cl(-) and/or Ca(2+) was depleted from the medium. Recovery of photosystem II from photoinhibition was suppressed in the mutant. Despite the defects in photosystem II, in the light, the acceptor side of photosystem II was more reduced and the donor side of photosystem I was more oxidized compared with wild-type cells, suggesting that functional impairments were also present in cytochrome b(6)/f complexes. The amounts of cytochrome c(550) and cytochrome f were smaller in the mutant in the Ca(2+)- and Cl(-)-depleted medium. Furthermore, the amount of IsiA protein was increased in the mutant, especially in the Cl(-)-depleted medium, indicating that the mutant cells perceive environmental stress to be greater than it is. The amount of accompanying cytochrome c(550) in purified photosystem II complexes was also smaller in the mutant. Overall, the Sll1252 protein appears to be closely related to redox sensing of the plastoquinone pool to balance the photosynthetic electron flow and the ability to cope with global environmental stresses.

Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3'-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation.

The involvement of myosin XI in generating the motive force for cytoplasmic streaming in plant cells is becoming evident. For a comprehensive understanding of the physiological roles of myosin XI isoforms, it is necessary to elucidate the properties and functions of each isoform individually. In tobacco cultured BY-2 cells, two types of myosins, one composed of 175 kDa heavy chain (175 kDa myosin) and the other of 170 kDa heavy chain (170 kDa myosin), have been identified biochemically and immunocytochemically. From sequence analyses of cDNA clones encoding heavy chains of 175 kDa and 170 kDa myosin, both myosins have been classified as myosin XI. Immunocytochemical studies using a polyclonal antibody against purified 175 kDa myosin heavy chain showed that the 175 kDa myosin is distributed throughout the cytoplasm as fine dots in interphase BY-2 cells. During mitosis, some parts of 175 kDa myosin were found to accumulate in the pre-prophase band (PPB), spindle, the equatorial plane of a phragmoplast and on the circumference of daughter nuclei. In transgenic BY-2 cells, in which an endoplasmic reticulum (ER)-specific retention signal, HDEL, tagged with green fluorescent protein (GFP) was stably expressed, ER showed a similar behaviour to that of 175 kDa myosin. Furthermore, this myosin was co-fractionated with GFP-ER by sucrose density gradient centrifugation. From these findings, it was suggested that the 175 kDa myosin is a molecular motor responsible for translocating ER in BY-2 cells.

BACKGROUND INFORMATION: The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90]. RESULTS: In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. CONCLUSIONS: Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.

We examined several candidate posterior/mesodermal inducing molecules using permanent blastula-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer factor chordin mRNA. Injection of chordin alone resulted in a white hemispherical neural tissue surrounded by a large circular cement gland, together with anterior neural gene expression and thus the development of the anterior-most parts of the embryo, without mesodermal tissues. When VegT, eFGF or Xbra mRNAs were injected into a different blastomere of the chordin-injected PBEs, the embryos elongated and formed eye, muscle and pigment cells, and expressed mesodermal and posterior neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8 DNA into PBEs injected with chordin resulted in eye formation and expression of En2, a midbrain/hindbrain marker, and Xnot, a notochord marker, but neither elongation, muscle formation nor more posterior gene expression. Injection of chordin and posteriorizing molecules into the same cell did not result in elongation of the embryo. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm (muscle) formation, together with embryo elongation can occur through the synergistic effect(s) of the organizer molecule chordin, and each of the 'verall posteriorizing molecules'eFGF, VegT and Xbra; (ii) Xwnt8-mediated posteriorization is restricted to the eye level and is independent of mesoderm formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.

In order to clarify the function of the Djbmp (Dugesia japonica bone morphogenetic protein) gene in planarian body patterning, we carried out knockdown of this gene by RNA interference. When the planarians were treated with double-stranded RNA of Djbmp, a bulge formed on the dorsal side, with a dent in the middle of the bulge, and the body surface inside the dent was smoothened and less pigmented. In situ hybridization of the DjIFb gene, which is expressed in the body margin, revealed that the additional body margin was formed ectopically at the region surrounding the dent. The Djbmp-knockdown planarians often had a pair of incomplete nerve cords in the dorsal side, in addition to the original pair of ventral nerve cords. Taken together, we concluded that the Djbmp-knockdown induced formation of an ectopic ventral side, suggesting that Djbmp is required for the dorso-ventral body patterning in the planarian.

We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2 cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis, clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle and phragmoplast, as well as in endocytosis.

The planarian's remarkable regenerative ability is thought to be supported by the stem cells (neoblasts) found throughout its body. Here we report the identification of a subpopulation of neoblasts, which was revealed by the expression of the nanos-related gene of the planarian Dugesia japonica, termed Djnos. Djnos-expressing cells in the asexual planarian were distributed to the prospective ovary or testes forming region in the sexual planarian. During sexualization, Djnos-expressing cells produce germ cells, suggesting that in the asexual state these cells were kept as germline stem cells for the oogonia and spermatogonia. Interestingly, the germline stem cells were indistinguishable from the neoblasts by morphology and X-ray sensitivity and did not seem to contribute to the regeneration at all. Germline stem cells initially appear in the growing infant planarian, suggesting that germline stem cells are separated from somatic stem cells in the planarian. Thus, planarian neoblasts can be classified into two groups; somatic stem cells for regeneration and tissue renewal, and germline stem cells for production of germ cells during sexualization. However, Djnos-positive cells appeared in the newly formed trunk region from the head piece, suggesting that somatic stem cells can convert to germline stem cells.

We succeeded in visualization of the primordial germ cells (PGCs) in a living Xenopus embryo. The mRNA of the reporter Venus protein, fused to the 3' untranslated region (UTR) of DEADSouth, which is a component of the germ plasm in Xenopus eggs, was microinjected into the vegetal pole of fertilized eggs and then the cells with Venus fluorescence were monitored during development. The behavior of the cells was identical to that previously described for PGCs. Almost all Venus-expressing cells were Xdazl-positive in the stage 48 tadpoles, indicating that they were PGCs. In addition, we found three sub-regions (A, B and C) in the 3' UTR, which were involved in the PGC-specific expression of the reporter protein. Sub-region A, which was identified previously as a localization signal for the germ plasm during oogenesis, participated in anchoring of the mRNA at the germ plasm and the degradation of the mRNA in the somatic cells. Sub-regions B and C were also involved in anchoring of the mRNA at the germ plasm. Sub-region B participated in the enhancement of translation.

Although there have been several studies on the structure of the ocellus photoreceptors in ascidian tadpole larvae using electron microscopy, the overall structure of these photoreceptor cells, especially the projection sites of the axons, has not been revealed completely. The number of photoreceptor cells is also controversial. Here, the whole structure of the ocellus photoreceptors in the larvae of the ascidian Ciona intestinalis was revealed by using an anti-arrestin (anti-Ci-Arr) antibody. The cell bodies of 30 photoreceptor cells covered the right side of the ocellus pigment cell and their outer segments extended through the pigment cell into the pigment cup. The axons of the photoreceptor cells were bundled together ventro-posteriorly in a single tract extending towards the midline. The nerve terminals diverged antero-posteriorly at the midline of the posterior sensory vesicle (SV). The Ci-arr gene was expressed throughout the SV at the embryonic mid-tailbud stage and it became restricted to the neighborhood of the ocellus pigment when ocellus pigmentation occurred. At the same time, the Ci-Arr protein was first detected, suggesting that the photoreceptor cells began to differentiate. The development of photoreceptor cells after hatching was also investigated using the anti-Ci-Arr antibody. Three hours after hatching, the photoreceptor terminals began to ramify and then expanded. Previous behavioral analysis showed that the larvae did not respond to the step-down of light until 2 h after hatching and then the photoresponse became robust. Accordingly, our results suggest that growth of the photoreceptor terminal is critical for the larvae to become photoresponsive.

It has been postulated that the high regeneration ability of planarians is supported by totipotent stem cells, called neoblasts. There have been a few reports showing the distribution of neoblasts in planarians. However, the findings were not completely consistent. To determine the distribution of neoblasts, we focused on proliferating cell nuclear antigen (PCNA), which is present in proliferative cells. We cloned and sequenced the cDNA of PCNA from the planarian Dugesia japonica and produced an antiserum recognizing the gene product. X-ray irradiation caused rapid loss of all PCNA-positive cells and loss of the neoblasts (which were morphologically defined by the presence of the chromatoid body), strongly suggesting that all PCNA-positive cells were true neoblasts. Using the antiserum, we were successful in identifying the neoblasts more clearly than any previous work. In addition to their dispersed distribution in the dorsal and ventral mesenchyme, the neoblasts were distributed as clusters along the midline and bilateral lines in the dorsal mesenchyme. We also examined the behavior of the neoblasts after decapitation. Decapitation did not seem to affect the migration of neoblasts far from the wound. We demonstrated here that DjPCNA is a powerful tool for identifying planarian neoblasts.

Planarians are one of the simplest animal groups with a central nervous system. Their primitive central nervous system produces large quantities of a variety of neuropeptides, of which many are amidated at their C terminus. In vertebrates, peptide amidation is catalyzed by two enzymes [peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxylglycine alpha-amidating lyase] acting sequentially. In mammals, both enzymatic activities are contained within a single protein that is encoded by a single gene. By utilizing PCR with degenerate oligonucleotides derived from conserved regions of PHM, we succeeded in cloning a full-length cDNA encoding planarian PHM. The deduced amino acid sequence showed full conservation of five His residues and one Met residue, which bind two Cu atoms that are essential for the activity of PHM. Northern blot analysis confirmed the expression of a PHM mRNA of the expected size. Distribution of the mRNA was analyzed by in situ hybridization, showing specific expression in neurons with two morphologically distinct structures, a pair of the ventral nerve cords and the brain. The distribution of PHM was very similar to that of cytochrome b561. This indicates that the ascorbate-related electron transfer system operates in the planarian central nervous system to support the PHM activity and that it predates the emergence of Plathelminthes in the evolutionary history.

In many cases, actin filaments are arranged into bundles and serve as tracks for cytoplasmic streaming in plant cells. We have isolated an actin-filament bundling protein, which is composed of 115-kDa polypeptide (P-115-ABP), from the germinating pollen of lily, Lilium longiflorum [Nakayasu et al. (1998) BIOCHEM: Biophys. Res. Commun. 249: 61]. P-115-ABP shared similar antigenicity with a plant 135-kDa actin-filament bundling protein (P-135-ABP), a plant homologue of villin. A full-length cDNA clone (ABP115; accession no. AB097407) was isolated from an expression cDNA library of lily pollen by immuno-screening using antisera against P-115-ABP and P-135-ABP. The amino acid sequence of P-115-ABP deduced from this clone showed high homology with those of P-135-ABP and four villin isoforms of Arabidopsis thaliana (AtVLN1, AtVLN2, AtVLN3 and AtVLN4), especially AtVLN4, indicating that P-115-ABP can also be classified as a plant villin. The P-115-ABP isolated biochemically from the germinating lily pollen was able to arrange F-actin filaments with uniform polarity into bundles and this bundling activity was suppressed by Ca2+-calmodulin (CaM), similar to the actin-filament bundling properties of P-135-ABP. The P-115-ABP type of plant villin was widely distributed in plant cells, from algae to land plants. In root hair cells of Hydrocharis dubia, this type of plant villin was co-localized with actin-filament bundles in the transvacuolar strands and the sub-cortical regions. Microinjection of the antiserum against P-115-ABP into living root hair cells caused the disappearance of transvaculor strands and alteration of the route of cytoplasmic streaming. In internodal cells of Chara corallina in which the P-135-ABP type of plant villin is lacking, the P-115-ABP type showed co-localization with actin-filament cables anchored on the intracellular surface of chloroplasts. These results indicated that plant villins are widely distributed and involved in the organization of actin filaments into bundles throughout the plant kingdom.

It has been known that rotenone and 1-methyl-4-phenylpyridinium ion (MPP(+), a metabolite of MPTP), which inhibit mitochondrial complex I, are useful tools for parkinsonian models in vertebrates such as primates and rodents. Planarian, an invertebrate flatworm, has a high potential for regeneration, and dopamine plays a key role in its behavior. In the present study, we examined a cloned planarian, the GI strain from Dugesia japonica. Planarians that were treated with rotenone or MPTP underwent autolysis and individual death in a concentration- and time-dependent manner. In addition, these effects induced by rotenone or MPTP were inhibited by several antiparkinsonian drugs and caspase inhibitors. These results suggest that the degeneration of planarian dopaminergic system induced by rotenone or MPTP may be mediated through caspase-like activation.

A simple method was developed for RNA interference (RNAi) in the planarian Dugesia japonica. The DjIFb ( Dugesia japonica intermediate filament b) gene was used to evaluate the effect of RNAi because both the cDNA and an antiserum against the gene product were available. After transverse cutting at the pre- and post-pharyngeal regions, the middle part of the body fragment was soaked in water containing double-stranded RNA (dsRNA) for about 5 h and then allowed to regenerate in water. On the 5th day of regeneration, little DjIFb protein was detected in the new tissues. When the worms were cut after soaking in dsRNA water, no RNAi effect was observed, suggesting that the dsRNA was introduced through the cut surface. A high concentration of dsRNA or repeated "cutting and soaking" resulted in more effective RNAi. This simple soaking method in combination with expressed sequence tag analysis should be very useful for high-throughput analyses of gene functions in planarian regeneration.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.

Arrestin is one of the key proteins for the termination of G protein signaling. Activated G protein-coupled receptors (GPCRs) are specifically phosphorylated by G protein-coupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Vertebrates possess two types of arrestin; visual arrestin expressed exclusively in photoreceptor cells in retinae and pineal organs, and beta-arrestin, which is expressed ubiquitously. Unlike visual arrestin, beta-arrestin contains the clathrin-binding domain at the C-terminus, responsible for the agonist-induced internalization of GPCRs. Here, we isolated a novel arrestin gene (Ci-arr) from the primitive chordate, the ascidian Ciona intestinalis larvae. The deduced amino acid sequence suggests that Ci-Arr be closely related to vertebrate arrestins. Interestingly, this arrestin has the feature of both visual and beta-arrestin. Whereas the expression of Ci-arr was restricted to the photoreceptors in the larvae similarly to visual arrestin, the gene product, containing the clathrin-binding domain, promoted the GPCR internalization in HEK293tsA201 cells similarly to beta-arrestin. The phylogenetic tree shows that Ci-Arr is branched from a common root of visual and beta-arrestins. Southern analysis suggests that the Ciona genome contains only one gene for the arrestin family. These results suggest that the visual and beta-arrestin genes were generated by the duplication of the prototypical arrestin gene like Ci-arr in the early evolution of vertebrates.

The planarian Dugesia japonica has two genes encoding myosin heavy chain, DjMHC-A and B (Kobayashi et al., 1998). We produced antibodies specifically recognizing each myosin heavy chain protein using their carboxyl terminal regions expressed in E. coli as antigens. Immunohistochemical analyses of sections and whole-mount specimens revealed the detailed structure and distribution of each type of muscle fiber in the planarian. In general, the MHC-A muscle fibers were distributed beneath the epithelial layers, namely, they were observable in the pharynx, the mouth, the intestine, the eyes and the body wall. In the pharynx, only MHC-A muscle fibers were present. In contrast, the MHC-B muscle fibers were distributed in the mesenchyme as dorso-ventral and transverse muscles, and in the body wall. The body-wall muscles were composed of an outer layer of circular MHC-A muscles and inner longitudinal and intermediate diagonal MHC-B muscle layers. Thus, two types of muscle fibers were distinguished by their distribution in the planarian.

We have cloned and sequenced a cDNA encoding an intermediate filament protein (IF) from the planarian Dugesia japonica named DjIFb. The deduced amino acid sequence of DjIFb has similarity to those of protostomic IFs and lamins, supporting a previous hypothesis that the protostomic IFs, including DjIFb, are evolutionarily closer to lamins than to vertebrate cytoplasmic IFs. In addition, analysis of the exon/intron organization revealed that 8 out of 10 introns of DjIFb were coincident in their position, even in the codon phase, with those of the non-neuronal IF of the snail Helix aspersa. This suggests that the Platyhelminthes are not the most primitive Bilateria but instead are evolutionarily close to the Mollusca. The DjIFb gene was expressed in particular cells, probably a kind of adhesive gland cell, which were present in the marginal region encircling the planarian body. The localization of DjIFb protein suggests that it plays an important role in the secretion of an adhesive substance. The specific expression pattern of the DjIFb gene enabled us to monitor how the body margin forms during planarian regeneration.

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.

The pharynx is a distinctive organ in the center of the body of planarians. Although the process of pharynx regeneration has been studied previously, the details and mechanism of the process remain controversial. We examined the process of regeneration of the pharynx in the planarian Dugesia japonica in detail by in situ hybridization and immunohistochemistry for myosin heavy chain-A (DjMHC-A), which is mainly expressed in the pharynx muscles and pharynx-anchoring muscles. We also monitored the behavior of the neoblasts in this process. In the regenerating posterior body fragment, the pharyngeal rudiment was formed by accumulation of cells that were probably undifferentiated cells derived from the neoblasts. The pharynx muscles appeared to differentiate in the rudiment in a manner that was coordinated with the differentiation of the pharynx-anchoring muscles in the region surrounding the rudiment. During this process, all cells containing mRNA for DjMHC-A also contained the DjMHC-A protein. These results argue against a previously proposed hypothesis that in the mesenchyme, 'pharynx-forming cells', which are committed to differentiate into the pharyngeal cells but have not yet differentiated, gather in the rudiment to form the pharynx (Agata and Watanabe, 1999). Rather, the present observations suggest that regeneration of the planarian pharynx proceeds by accumulation of cells that are probably undifferentiated cells derived from neoblasts in the rudiment, followed by their differentiation into the pharyngeal cells there.

Cytochrome b(561) is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles in the central and peripheral nervous systems of higher animals. We succeeded in cloning a full-length cDNA encoding planarian cytochrome b(561). The deduced amino acid sequence shows a very similar six transmembrane topology to those of cytochromes b(561) of higher vertebrates and contains both putative ascorbate- and monodehydro ascorbate-binding sites. Among the six totally-conserved His residues of cytochrome b(561) in higher vertebrates, one is substituted with an Asn residue, indicating that His88 and His161 of bovine cytochrome b(561) play roles as heme b ligands at the extravesicular side. Northern- and Western-blot analyses confirmed the expression of the mRNA and protein with the expected sizes in planarians. The distributions of the mRNA and apoprotein were analyzed by in situ hybridization and immunohistochemical staining, respectively, showing two morphologically distinct structures, a pair of ventral nerve cords and the cephalic ganglion cluster in the head region. The present results suggest that the usage of ascorbate to supply electron equivalents to neuroendocrine-specific copper-containing monooxygenases is likely to have originated in organisms with a very simple nervous system.

We previously showed by grafting experiments that the dorsoventral (DV) interaction evokes morphogenetic events similar to those that occur in regeneration. However, it is not yet understood whether the stem cells themselves or differentiated cells have the ability to induce regeneration. Here we demonstrated by a combination of X-ray irradiation and grafting experiments that the dorsal and ventral positional cues inducing morphogenetic events are retained in X-ray-irradiated tissues, suggesting that the differentiated cells may be responsible for the positional cues. We grafted a small piece of irradiated worm, in which the stem cells were certainly eliminated, to an intact one in DV-reversed orientation. We observed that projections were developed from the host-donor boundary, as in the previous experiments. Whole-mount in situ hybridization with several markers demonstrated that the projections had a newly established DV axis and also had anterior or posterior characteristics. Furthermore, chimeric analysis with a strain-specific marker showed that the projections consisted of nonirradiated cells and that IFb-expressing cells, which normally belonged to the ventral tissue, could be generated even from the stem cells located on the dorsal side. Taken together, the findings suggest that the stem cells may simply differentiate depending on the surroundings and that differentiated cells may present positional cues that induce morphogenesis.

The totipotent stem cells called neoblasts seem to be concerned with the remarkable regeneration ability of planarians. However, the pharynx is able to regenerate after the amputation of its distal part, in spite of a lack of neoblasts in the pharynx. The process of regeneration has been referred to as morphallaxis, based on conventional histochemical observations. We examined it again immuno-histochemically using anti-Dugesia japonica proliferating cell nuclear antigen (DjPCNA) antibody for neoblasts and anti-D. japonica myosin heavy chain-A (DjMHC-A) antibody for pharynx muscle fibers. This immuno-histochemical study, together with observations of the regeneration process of planarians irradiated with X-rays in particular regions, revealed that after the amputation, neoblasts from outside the pharynx entered that organ, moved through the mesenchyme of the pharynx to the wounded area, and differentiated into the cells that had been lost there. We show here that the regeneration after amputation of the distal part of the pharynx is an 'epimorphic' process.

Planarians can propagate asexually by fission and successive regeneration. During head regeneration, they again form a new pair of eyes, and sometimes supernumerary eyes. The positions of normal and supernumerary eyes and their regeneration abilities are expected to be highly relevant to the question of where and how the field to regenerate eyes is determined. In this study, spontaneously generated supernumerary eyes were classified into various types. In all cases, they were formed in the anterior part of the head. Enucleation of a normal eye elicited regeneration of a new eye; however, enucleation of a supernumerary eye did not. The supernumerary eyes were morphologically and functionally indistinguishable from the normal eyes, revealed by the studies of immunohistology and photophobic response, respectively. From the obtained results, we proposed a model of the eye regeneration field that changes its distribution spatiotemporally during regeneration. Immunohistological studies also showed that the optic nerves from the normal and supernumerary eyes ran independently, which might have implication about the nature of guidance cues for the optic nerves.

In the freshwater planarian Dugesia japonica, five cDNAs for HOM/HOX homeobox genes were cloned and sequenced. Together with sequence data on HOM/HOX homeobox genes of platyhelminthes deposited in databases, comparison of the deduced amino acid sequences revealed that planarians have at least seven HOM/HOX homeobox genes, Plox1 to Plox7 (planarian HOM/HOX homeobox genes). Whole-mount in situ hybridization and RT-PCR revealed that Plox4 and Plox5 were increasingly expressed along a spatial gradient in the posterior region of intact animals. During regeneration, Plox5 was expressed only in the posterior region of regenerating body pieces, suggesting that the gene is involved in the anteroposterior patterning in planarians. Plox5 was not found to be expressed in a blastema-specific manner, which contradicts a previous report (J. R. Bayascas, E. Castillo, A. M. Muños-Mármol, and E. Saló. Development 124, 141-148, 1997). X-ray irradiation experiments showed that Plox5 was expressed at least in some cells other than neoblasts, but that the induction of Plox5 expression during regeneration might require neoblasts.

Planarians are known for their strong regenerative ability. This ability has been considered to reside in the totipotent somatic stem cell called the "neoblast." Neoblasts contain a unique cytoplasmic structure called the "chromatoid body," which has similar characteristics to the germline granules of germline cells of other animals. The chromatoid bodies decrease in number and size during cytodifferentiation and disappear in completely differentiated cells during regeneration. However, germ cells maintain the chromatoid body during their differentiation from neoblasts. These observations suggest that the chromatoid body is concerned with the totipotency of cells. To understand the molecular nature of the chromatoid body in the neoblast, we focused on vasa (vas)-related genes, since VAS and VAS-related proteins are known to be components of the germline granules in Drosophila and Caenorhabditis elegans. By PCR, two vas-related genes (Dugesia japonica vasa-like gene, DjvlgA and DjvlgB) were isolated, and they were shown to be expressed in germ cells. Interestingly, DjvlgA was also expressed in a number of somatic cells in the mesenchymal space. In regenerating planarians, accumulation of DjvlgA-expressing cells was observed in both the blastema and the blastema-proximal region. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjvlgA-expressing cells decreased drastically. These results suggest that the product of DjvlgA may be a component of the chromatoid body and may be involved in the totipotency of the neoblast.

During planarian regeneration, dorsal and ventral tissues adhere to each other as a result of wound closure. To investigate the role of dorsoventral (DV) interaction in regeneration, dorsal and ventral portions were ectopically reoriented relative to each other by grafting. We sucked a small piece of planarian body into a Pasteur pipette, and then grafted the piece to the original position in DV-reversed orientation. 4 days after grafting, a blastema-like white region was formed on the boundary between the host and the graft. The region grew and formed a cup-shaped projection on both the dorsal and ventral sides of the worm after about 1 week. Histological analysis and in situ hybridization using dorsal and ventral markers revealed that the grafted piece maintained its original DV polarity. Analysis using DV boundary markers clearly indicated that a DV axis was newly established within the projection. Chimeric analysis using a strain-specific marker demonstrated that the projection grew on the DV boundary. Further analysis, using a marker specific for neoblasts and differentiating cells, showed that the projection was composed of neoblasts and/or neoblast-derived cells. Thus, ectopic DV interaction can cause blastema formation, outgrowth and establishment of a DV axis. In addition, staining with tissue- and region-specific markers showed that the projection appears to be identical to the structure from the grafted position to the most anterior or posterior tip. These results suggest that DV interaction evoked by wound closure has a important role in the onset of regeneration, especially for blastema formation and establishment of the DV axis during planarian regeneration.

Ultrastructural and physiological studies have shown that planarian muscles have some characteristics of smooth and some characteristics of striated muscles. To characterize planarian muscles, we isolated two myosin heavy chain genes (DjMHC-A and DjMHC-B) from a planarian, Dugesia japonica, by immunological screening, and analyzed their structures and spatial expression patterns. Structural analysis indicated that both MHC genes are striated-muscle-type myosin genes, although planarian muscles do not have any striation. In situ RNA hybridization showed that expression of the two myosin genes is spatially strictly segregated. DjMHC-A was expressed in pharynx muscles, pharynx cavity muscles, muscles surrounding the intestinal ducts, a subpopulation of body-wall muscles and several muscle cells in the mesenchymal region around the base of the pharynx. DjMHC-B was expressed in body-wall muscles (including circular, diagonal and longitudinal muscles), vertical muscles and horizontally oriented muscles. Double staining with DjMHC-A and -B probes clearly demonstrated that expression of the DjMHC-A and -B genes do not occur in the same cell. During regeneration, the number of cells positive for expression of each gene increased in the blastema region, suggesting that both types of muscle may be involved in blastema formation. DjMHC-B-positive cells disappeared from the body-wall muscle layer in the pharynx-cavity-forming region, whereas DjMHC-A-positive cells were markedly accumulated there, suggesting that the two types of muscle in the body wall layer may have distinct functions. These results indicate that planarians have at least two types of muscle that express striated-muscle-type MHC genes, but do not form striation.

BMP (Bone Morphogenetic Protein) acts as a morphogen for dorso-ventral patterning and organogenesis in both vertebrate and invertebrate development. A cDNA encoding BMP (named Djbmp) has been cloned and sequenced from the planarian Dugesia japonica. The mature form of DjBMP which was deduced from the cDNA sequence was composed of 114 amino acid residues. The position of seven cysteine residues of the mature DjBMP was highly conserved among the TGF-beta superfamily. DjBMP had high similarity to human BMP-2A (50% amino acid identity), BMP-4 (49%) and Drosophila decapentaplegic protein (48%), indicating that DjBMP belongs to DVR (decapentaplegic-Vg1-related) group. The expression pattern in intact and regenerating planarians revealed by whole mount in situ hybridization suggested that the DjBMP plays a role not only in dorso-ventral but also in mid-lateral body patterning.

To investigate external signals involved in germ cell differentiation from somatic stem cells, we have tried to identify protein kinases whose expression is regulated during the process of sexualization of asexual-state planarians. It is known that in planarians germ cells differentiate from totipotent somatic stem cells called "neoblasts" during sexualization. As a first step, we have isolated twelve protein kinase genes from cDNAs of sexual-state planarians, including three non-receptor tyrosine kinases, three receptor-tyrosine kinases and three non-receptor serine/threonine kinases, and then analyzed their expression patterns during sexualization. One of them, the DjPTK1 gene, is specifically expressed in germ cells of sexual-state planarians. DjPTK1-positive cells were also detected in the mesenchymal space during the process of sexualization, and it appears that these cells migrate to the dorsal side and then differentiate into spermatogonia/spermatocytes in testis. Sequence analysis indicated that the DjPTK1 gene encodes a receptor protein tyrosine kinase belonging to the FGFR/PDGF family. These results suggest that a receptor tyrosine kinase system may be involved both at an early stage of germ cell differentiation and in a step of germ cell maturation in planarians.

Pineal cells of the 8-day embryonic quail are multipotent cells which differentiate in vitro into skeletal muscle fibers, pigmented epithelial cells (PECs), lens cells and neurons. However, it was not yet clear whether precursor cells which gave such a wide repertoire of differentiation were single type or not. The present culture studies revealed that pineal cells were exclusively directed to ocular differentiation pathways by dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), suggesting a single type of precursor cell in the pineal body. DMSO directed pineal cells to differentiate into PECs. Co-administration of basic fibroblast growth factor (bFGF) with DMSO partially inhibited PEC differentiation and promoted lens cell differentiation. Northern blot analysis using cDNAs specific to PEC and lens cell confirmed this morphological observation. HMBA completely inhibited pigmentation of cultured pineal cells and markedly promoted lens cell differentiation. Ocular differentiation of pineal cells was accompanied with the loss of myogenicity. We discuss three possible pathways of lens cell differentiation from pineal cells. The agents which affect pineal cell differentiation seemed to modulate the cell-substrate interaction. And the interaction was suggested to be one of the environmental cues in the differentiation.

Pineal cells of the embryonic quail are multipotent stem cells which are able to differentiate in vitro into pigmented epithelial cells, lens cells and skeletal muscle fibers. Neuronal expression was added in this study in the repertory of differentiating potency of pineal cells. We used immunohistochemical methods to characterize neuronal properties with antibodies against serotonin, GABA, tyrosine hydroxylase and neuron-specific antigen (HPC-1) in addition to the enzyme histochemistry for acetylcholinesterase activity. Cells in the culture were found to be positively stained with these methods, suggesting that embryonic pineal cells are neuropotent to differentiate various types of neuronal cells. We have studied the culture conditions which favor increment of neuronal cells with extension of neuritic processes, and we have found that neuronal cells are maintained for quite a long period under suppressive conditions of DNA synthesis and under the effect of basic fibroblast growth factor (FGF). Suppression of DNA synthesis was achieved by the addition of aphidicolin, an inhibitor of DNA polymerase alpha, in the medium. Time lapse videograph revealed two different cell types participated in neurogenesis; a minor population of small round cells and a major one of flat epithelial cells. Since embryonic quail pineal cells have been shown to differentiate into two types of photoreceptors, the present results show wider retinal potency of cell differentiation by embryonic pineal cells. The cessation of DNA synthesis as well as growth factor(s) may be positively involved in the mechanisms of determination and differentiation of pineal neurons.

A partial cDNA sequence for chick lens beaded-filament protein CP 49 showed the greatest similarity to the sequence of acidic cytokeratins, especially human cytokeratin 18. The predicted amino acid sequence of chick CP 49 corresponded to the entire coil 1a region of the rod domain of human cytokeratin 18, spacer 1, coil 1b, spacer 2 and about half of coil 2. For this sequence of 242 amino acids, there was an overall 38% identity and 76.8% similarity between the chick CP 49 and human cytokeratin 18. This is further evidence that CP 49 belongs to the family of intermediate filament proteins.