Dynamic intracellular localization of Dazl protein during Xenopus germline development.

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.

Intracellular localizations of the Dead End protein in Xenopus primordial germ cells.

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.

Restriction of the Xenopus DEADSouth mRNA to the primordial germ cells is ensured by multiple mechanisms.

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.

Germes is involved in translocation of germ plasm during development of Xenopus primordial germ cells.

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).

DEADSouth protein localizes to germ plasm and is required for the development of primordial germ cells in Xenopus laevis.

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).

Developmental regulation of locomotive activity in Xenopus primordial germ cells.

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.

Single-step cloning of a hybridoma producing a monoclonal antibody against a target protein.

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.

Analysis of localization and reorganization of germ plasm in Xenopus transgenic line with fluorescence-labeled mitochondria.

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).

Ectopic formation of primordial germ cells by transplantation of the germ plasm: direct evidence for germ cell determinant in Xenopus.

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.

Germ plasm in Eleutherodactylus coqui, a direct developing frog with large eggs.

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.

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

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.

S4 protein Sll1252 is necessary for energy balancing in photosynthetic electron transport in Synechocystis sp. PCC 6803.

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.

Germ-line mitochondria exhibit suppressed respiratory activity to support their accurate transmission to the next generation.

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.

An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells.

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.

VegT, eFGF and Xbra cause overall posteriorization while Xwnt8 causes eye-level restricted posteriorization in synergy with chordin in early Xenopus development.

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.

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

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].

Bone morphogenetic protein is required for dorso-ventral patterning in the planarian Dugesia japonica.

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.

Identification and origin of the germline stem cells as revealed by the expression of nanos-related gene in planarians.

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.

Visualization of the Xenopus primordial germ cells using a green fluorescent protein controlled by cis elements of the 3' untranslated region of the DEADSouth gene.

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.

Structure of ocellus photoreceptors in the ascidian Ciona intestinalis larva as revealed by an anti-arrestin antibody.

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.

Planarian peptidylglycine-hydroxylating monooxygenase, a neuropeptide processing enzyme, colocalizes with cytochrome b561 along the central nervous system.

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.

Distribution of the stem cells (neoblasts) in the planarian Dugesia japonica.

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.

Plant 115-kDa actin-filament bundling protein, P-115-ABP, is a homologue of plant villin and is widely distributed in cells.

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.

A simple "soaking method" for RNA interference in the planarian Dugesia japonica.

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.