bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation
Authors: Wang Weihua , Hosoe Misa , Li Rongfeng , Shioya Yasuo
Published: October 2003

Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate‐conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22h in vitro, 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro, only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.
Involvement of the protein of Xenopus vasa homolog (Xenopus vasa‐like gene 1, XVLG1) in the differentiation of primordial germ cells
Authors: Ikenishi Kohji , Tanaka Tetsuya S.
Published: October 2003

In order to understand the role of the protein of Xenopus vasa homolog (Xenopus vasa‐like gene 1, XVLG1) in germ line cells, an attempt was made to perturb the function of the protein with the anti‐vasa antibody 2L‐13. The 2L‐13 or the control antibody was microinjected with a lineage tracer (FITC‐dextran‐lysine, FDL) into single vegetal blastomeres containing the germ plasm of Xenopus 32‐cell embryos, the descendants of which were destined to differentiate into a small number of primordial germ cells (PGC) and a large number of somatic cells, mostly of endodermal tissues at the tadpole stage. No significant effect of the injection of the antibodies on FDL‐labeled, presumptive PGC (pPGC) was observed in embryos until stage 37/38. However, FDL‐labeled PGC were not observed in almost all the 2L‐13 antibody‐injected tadpoles, although a similar number of labeled somatic cells were always present. As 2L‐13 antibody specifically reacts with XVLG1 protein in the embryos by immunoblotting, the present results suggest that the antibody perturbed the function of XVLG1 protein in the pPGC, resulting in failure of PGC differentiation at the tadpole stage.
Transferrin is necessary and sufficient for the neural effect on growth in amphibian limb regeneration blastemas
Authors: Mescher Anthony L. , Connell Elise , Hsu Christine , Patel Chetna , Overton Bonnie
Published: October 2003

Cell proliferation during the early phase of growth in regenerating amphibian limbs requires a permissive influence of nerves. Based on analyses of proliferative activity in denervated blastemas, it was proposed that nerves provide factors important for cells to complete the proliferative cycle rather than for mitogenesis itself. One such factor, the iron‐transport protein transferrin (Tf), is abundant in regenerating peripheral nerves where it is axonally transported and released at growth cones. Using blastemas in organ culture, which have been widely used in previous investigations of the neural effect on growth, it was shown here that the growth‐promoting activity of neural extract was completely removed by immuno‐absorption with antiserum against Tf and restored by addition of Tf. Purified Tf or a low molecular weight ferric ionophore were as active as the neural extract in this assay, indicating that the trophic effect of Tf involves its capacity for iron delivery. Both Tf and ferric ionophore also maintained DNA synthesis in denervated blastemas in vivo. A dose‐response assay indicated that purified axolotl Tf stimulates growth of cultured blastemal cells at concentrations as low as 100 ng/mL. The Tf mRNA in axolotl nervous tissue was shown by northern analysis to be similar in size to that of liver. These results are discussed together with those from previous in vitro studies of blastemal growth and support the hypothesis that cell division in the blastema depends on axonally released Tf during the early, nerve‐dependent phase of limb regeneration.
Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)‐signal transfer in chick limb development
Authors: Hara Kenji , Ide Hiroyuki
Published: October 2003

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1, in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER‐mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero‐proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re‐expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf‐8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.
Expressions ofPDGF receptor alpha, c‐KitandFlk1genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells
Authors: Kataoka Hiroshi , Takakura Nobuyuki , Nishikawa Satomi , Tsuchida Kunihiro , Kodama Hiroaki , Kunisada Takahiro , Risau Werner , Kita Toru , Nishikawa Shin‐Ichi
Published: October 2003

In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRα), c‐Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post‐gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1+mesodermal cells could be further classified by the level of c‐Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1+mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supportedin vitrohematopoiesis from ES cells. Flk1+cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1+cells will be required forin vitroES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos orin vitrocultured ES cells.
Electric fusion of unfertilized starfish oocytes
Authors: Yoneda Mitsuki
Published: October 2003

An electric power device to fuse starfish oocytes was constructed. The outputs were supplied to a pair of parallel platinum wires in a solution of mannitol to which small amounts of salts were added. Oocytes of the starfish Asterina pectinifera, deprived of the vitelline envelope, were fused by several repetitions of a combination of 2.5 MHz AC field for a few seconds and a 50 μs DC pulse. Observation of fusing pairs of immature oocytes revealed that: (i) the oocyte placed near to the cathode forms a bulge at the surface facing the anode when subjected to DC pulses and, with subsequent DC pulses, a similar bulge is formed on another oocyte; and (ii) the pair of bulges then fuse together leading to fusion of the main body of the two oocytes. The conjugate of maturing oocytes soon became a single sphere, usually within 10 min, but this process toward spherical form paused when the oocyte was extruding its polar bodies. The conjugate of immature oocytes took 1 h to become a single sphere. The fusion did not disturb the progress of meiotic events, but electric pulses at an intensity suitable for the fusion often activated maturing oocytes and mature eggs.
Chicken keratin‐19: Cloning of cDNA and analysis of expression in the chicken embryonic gut
Authors: Sato Kazuna , Yasugi Sadao
Published: October 2003

From many recent studies, it has been argued that keratins (cytokeratins) play important roles in the morphogenesis and differentiation of organ development. To learn the role of keratin in digestive tract development, a cDNA of the chicken homolog of keratin‐19 (GK‐19) was cloned and its expression pattern was analyzed in the digestive tract of chicken embryos. TheGK‐19full‐length sequence was approximately 1.6 kb and showed more than 80% similarity to human and mouse keratin‐19. The result of in situ hybridization with the proventriculus (glandular stomach) of different developmental stages showed thatGK‐19expression disappeared specifically in the glandular epithelium from day 6 to day 9 of incubation. Furthermore,GK‐19was localized in the notochord, floor plate, anterior lobe of the pituitary gland and mesonephros. These results suggest the possibility thatGK‐19may have multiple roles in organogenesis during embryogenesis.
Identification of a new sea urchin vitelline envelope sperm binding glycoprotein
Authors: Correa Liane M. , Carroll Edward J.
Published: October 2003

The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N‐acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation‐free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE‐A, VE‐B and VE‐C, and one minor component, VE‐D, were identified with antisera against whole VE preparations and against glutaraldehyde‐fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl‐mannoside comigrated with VE‐B when analyzed by immunoblotting. Whole VE preparations and VE‐B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE‐B in gamete binding.
Crossregulation between En‐2 and Wnt‐1 in chick tectal development
Authors: Sugiyama Sayaka , Funahashi Jun‐ichi , Kitajewski Jan , Nakamura Harukazu
Published: October 2003

En‐1, En‐2 and Wnt‐1 are proposed to be essential signals for the development of the optic tectum in chick embryos. Drosophila engrailed and wingless, homologs of En (En‐1 and En‐2) and Wnt‐1, respectively, have been shown to crossregulate each other. In the present paper, it is reported that crossregulation between En‐2 and Wnt‐1 is preserved in the development of the chick optic tectum. When En‐2 is overexpressed by the replication competent retroviral vector, Wnt‐1 is expressed ectopically at the dorsal midline of the diencephalon. When Wnt‐1 is introduced extrinsically either by ectopic transplantation of mesencephalon, or by implantation of Wnt‐1 producing cells, En‐2 is induced ectopically at the dorsal midline of the tel‐diencephalic border. Thus, ectopic expression of En‐2 and Wnt‐1 leads to crossregulation of each other in the chick brain. As diencephalon transdifferentiates into the optic tectum by an appropriate signal, the crossregulation of En‐2 and Wnt‐1 in the diencephalon may mimic the relationship required for early development in the tectum.
Early embryonic interaction of retinal pigment epithelium and mesenchymal tissue induces conversion of pigment epithelium to neural retinal fate in thesilvermutation of the Japanese quail
Authors: Araki Masasuke , Yamao Mikaru , Tsudzuki Masaoki
Published: October 2003

The neural retina and retinal pigment epithelium (RPE) diverge from the optic vesicle during early embryonic development. They originate from different portions of the optic vesicle, the more distal part developing as the neural retina and the proximal part as RPE. As the distal part appears to make contact with the epidermis and the proximal part faces mesenchymal tissues, these two portions would encounter different environmental signals. In the present study, an attempt has been made to investigate the significance of interactions between the RPE and mesenchymal tissues that derive from neural crest cells, using a unique quail mutantsilver (B/B)as the experimental model. Thesilvermutation is considered to affect neural crest‐derived tissues, including the epidermal melanocytes. The homozygotes of thesilvermutation have abnormal eyes, with double neural retinal layers, as a result of aberrant differentation of RPE to form a new neural retina. Retinal pigment epithelium was removed from early embryonic eyes (before the process began) and cultured to see whether it expressed any phenotype characteristic of neural retinal cells. When RPE of the B/B mutant was cultured with surrounding mesenchymal tissue, neural retinal cells were differentiated that expressed markers of amacrine, cone or rod cells. When isolated RPE of theB/Bmutant was cultured alone, it acquired pigmentation and did not show any property characteristic of neural retinal cells. The RPE of wild type quail always differentiated to pigment epithelial cells. In the presence of either acidic fibroblast growth factor (aFGF) or basic FGF (bFGF), the RPE of theB/Bmutant differentiated to neural retinal cells in the absence of mesenchymal tissue, but the RPE of wild type embryos only did so in the presence of 10–40 times as much aFGF or bFGF. These observations indicate that genes responsible for theB/Bmutation are expressed in the RPE as well as in those cells that have a role in the differentiation of neural crest cells. They further suggest that development of the neural retina and RPE is regulated by some soluble factor(s) that is derived from or localized in the surrounding embryonic mesenchyme and other ocular tissues, and that FGF may be among possible candidates.
Blastomeres show differential fate changes in 8‐cell Xenopus laevis embryos that are rotated 90° before first cleavage
Authors: Huang Sen , Johnson Kurt E. , Wang H. Z. Pearl
Published: October 2003

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis‐respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8‐cell stage, blastomeres were injected with the lineage tracers, Texas Red‐ or FITC‐Dextran Amines. The distribution of the labeled progeny was mapped at the tail‐bud stages (stages 35–38) and compared with the fate map of an 8‐cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8‐cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.
Cloning and in situ hybridization analysis of estrogen receptor in the developing gonad of the red‐eared slider turtle, a species with temperature‐dependent sex determination
Authors: Bergeron Judith M. , Gahr Manfred , Horan Karen , Wibbels Thane , Crews David
Published: October 2003

Many reptiles exhibit temperature‐dependent sex determination where the incubation temperature of the egg determines the gonadal sex of the individual. If exogenous estrogen is administered during the temperature‐sensitive period to embryos incubating at a male‐producing temperature, the temperature effects can be over‐ridden and females will be produced. Inhibiting production of endogenous estrogens at female‐biased incubation temperatures results in embryos developing as males rather than females. Thus, estroge–nestrogen receptor‐dependent mechanisms appear to play a key role in female sex determination. The present study characterized the expression of the estrogen receptor during the critical period of temperature sensitivity in the red‐eared slider turtle, Trachemys scripta. Polymerase chain reaction was used to amplify estrogen receptor cDNA. A portion of the estrogen receptor cDNA was used to produce probes for in situ hybridization analyses to localize and quantitate levels of estrogen receptor mRNA at different stages of development in embryos from different incubation temperatures. Estrogen receptor mRNA is expressed in the gonadal tissues of both putative males and putative females even before the gonads begin to resolve as ovaries or testes. There is a greater abundance of estrogen receptor mRNA in putative females at the beginning of the temperature‐sensitive period as compared to putative males. In embryos from a female‐producing incubation temperature, levels of estrogen receptor mRNA are higher in the beginning of the temperature‐sensitive window compared to levels after the ovary is differentiated. These results support the hypothesis that estrogen–estrogen receptor dependent processes are important during sex determination and gonadal differentiation in temperature‐dependent sex determination.
Neural induction in embryos
Authors: Tiedemann Heinz , Asashima Makoto , Grunz Horst , Knöchel Walter , Tiedemann Hildegard
Published: October 2003

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP‐4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105 000 g), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.
Dorsal specification in blastoderm at the blastula stage in the goldfish, Carassius auratus
Authors: Yamaha Etsuro , Mizuno Toshiro , Hasebe Yutaka , Takeda Hiroyuki , Yamazaki Fumio
Published: October 2003

The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid‐ to late‐blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180°, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed: the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso‐anterior index. Furthermore, the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.
Effects of wheat germ agglutinin on tunicate egg activation and fertilization: Is there a plasma membrane sperm receptor system on Ascidia ceratodes eggs?
Authors: Flannery Brendan , Epel David
Published: October 2003

Little work has been carried out on the sperm recognition systems present on the egg plasma membrane. Here it is shown that wheat germ agglutinin (WGA) interferes with the sperm‐interacting system on the plasma membrane of eggs of the ascidian, Ascidia ceratodes. The WGA activates the dechorionated egg, indicating that a plasma membrane sugar residue can be directly tied to egg activation. Low concentrations of this lectin do not activate the eggs, but reduce fertilizability. This observation suggests that the WGA binding site might be part of a sperm reception–activation complex in the plasma membrane. While WGA also affects sperm binding to the chorion, the mechanisms of sperm interaction at the plasma membrane and chorion show different sensitivities to lectins, sugars and enzymes.
Calcium transients accompany ooplasmic segregation in zebrafish embryos
Authors: Leung Christina F. , Webb Sarah E. , Miller Andrew L.
Published: October 2003

Through the injection of f‐aequorin (a calcium‐specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (KD= 1.5 μmol/L) calcium buffer 5,5′‐dibromo‐BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: ≥ 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5′‐dimethyl‐BAPTA (KD= 0.15 μmol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.
Patterns of gene expression in the core of Spemann's organizer and activin‐treated ectoderm in Cynops pyrrhogaster
Authors: Yokota Chika , Ariizumi Takashi , Asashima Makoto
Published: October 2003

The presumptive pharyngeal endoderm region of the Cynops early gastrula induces head or trunk–tail structures in sandwich culture. Activin‐treated ectoderm can mimic this phenomenon at least at the histological level. The patterns of expression of organizer‐specific genes were examined to compare these two inductive materials at the molecular level. A chordin cDNA clone from Cynops pyrrhogaster (Cychd) was isolated by reverse transcription‐polymerase chain reaction (RT‐PCR). Cychd mRNA was first detected in the presumptive pharyngeal endoderm and prechordal plate regions of stage 11 embryos, and was expressed continuously until stage 20. The spatiotemporal expression pattern of Cychd was similar to that of Xenopus chordin. The patterns of expression of organizer‐related genes in the pharyngeal endoderm and activin‐treated ectoderm were compared by RT‐PCR analysis. Expression of Cychd in these two materials peaked at the time when they can induce head structures in sandwich culture. Expression of fork head and goosecoid did not change in the presumptive pharyngeal endoderm over this period. Cychd may play a key role in head formation in the Cynops embryo.
Sequence and expression of a novel mouse gene PRDC (protein related to DAN and cerberus) identified by a gene trap approach
Authors: Minabe‐Saegusa Chika , Saegusa Hironao , Tsukahara Masatoshi , Noguchi Shigeru
Published: October 2003

Gene trapping in embryonic stem (ES) cells was used to identify a novel gene involved in mouse development. In order to screen trapped ES cell lines for the presence of developmentally regulated genes, an in vitro differentiation test was used. One of the G418 resistant cell lines, in conjunction with the lacZ reporter gene, showed differential expression patterns under differentiated and undifferentiated conditions. The gene trap insertion in this cell line was germ‐line transmitted and X‐gal staining was used to assess the expression pattern of lacZ in embryos heterozygous for the trapped allele. The reporter gene's expression was detected in commissural neurons in the developing spinal cord, suggesting functions for the trapped gene in mouse neural development. Structural analysis of the cDNA revealed that this trapped gene, named PRDC (protein related to DAN and cerberus), is a novel gene that encodes a putative secretory protein consisting of 168 amino acid residues. PRDC gene product shows limited similarities to the products of DAN (differential screening‐selected gene aberrative in neuroblastoma) and cerberus. (DAN is a possible tumor‐suppressor for neuroblastoma in human. Cerberus can induce an ectopic head in Xenopus embryos when ectopically expressed.) These three gene products may form a novel family of signaling molecules.
Isolation and characterization of a gene expressed mainly in the gastric epithelium, a novel member of the ep37 family that belongs to the βγ‐crystallin superfamily
Authors: Ogawa Masanori , Takahashi Tadashi C. , Takabatake Takashi , Takeshima Kazuhito
Published: October 2003

EP37 family proteins are non‐lens members of the βγ‐crystallin superfamily, of which expression is observed in integumental tissues of the Japanese newt, Cynops pyrrhogaster. In the present study, a gene was isolated that has high homology with ep37 and is transcribed mainly in the gastric epithelial cells and hence designated gep. The predicted amino acid sequence of the gep cDNA contains four βγ‐crystallin motifs in the N‐terminal half, as is the case in the integumental EP37 proteins. Immunohistochemical analysis showed that GEP protein was mainly localized on the luminal content of the surface mucous cells of the gastric epithelium in both premetamorphic larvae and adults. In addition, GEP protein was also expressed in fundic glands after metamorphosis. Considering the fact that β‐ and γ‐crystallins are evolutionarily related to stress‐induced proteins, this localization suggests that GEP protein may have an evolutionarily conserved role in protection against physicochemical stresses, such as physical abrasion and autodigestion, during assimilation.
Juxtaposition of two heterotypic monolayer cell cultures of chick limb buds
Authors: Sato‐Maeda Mika , Ide Hiroyuki
Published: October 2003

In chick limb buds, mesenchymal cells of the progress zone (PZ‐cells) at different developmental stages segregate one from the other in mixed cell cultures, suggesting they have different cell affinity. In order to learn the possible roles of such differences in the cells, two heterotypic leg PZ‐cell populations (cells from stages 25/26 and 20/21) in vitro were juxtaposed to allow them to form the boundary. A method with double cylindrical columns was used to make adjoining monolayer cell cultures. It was shown that heterotypic juxtaposition produced two chondrogenic patterns along the boundary: aggregates of chondrocytes formed by stage 20/21 PZ‐cells and a chondrocyte‐free band formed by those at stage 25/26. Juxtaposition of PZ‐cells and proximal cells also formed these patterns, while that between cells from anterior and posterior PZ formed indistinct patterns along the boundary. Homotypic PZ‐cell juxtaposition did not produce these patterns. The results suggest that different cell affinity has a role in the segmentation of cartilage patterns at a point along the proximodistal axis, as well as a role in retaining cells in one area so as not to be recruited to other condensation areas.
Apical ectodermal ridge induction by the transplantation of En‐1‐overexpressing ectoderm in chick limb bud
Authors: Tanaka Mikiko , Shigetani Yasuyo , Sugiyama Sayaka , Tamura Koji , Nakamura Harukazu , Ide Hiroyuki
Published: October 2003

In the early chick embryo, the dorsal–ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Here it is reported that Engrailed‐1 (En‐1), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En‐1 in the dorsal side of the limb bud by transplantation of En‐1‐overexpressing ectoderm induces ectopic AER at the boundary of En‐1‐positive and ‐negative cells. The results suggest that En‐1 is involved in AER formation at the DV boundary of the limb bud.
An ascidian gene encoding an SH2‐domain protein is expressed in the notochord cells of the embryo
Authors: Takahashi Hiroki , Satoh Noriyuki
Published: October 2003

Differentiation of notochord cells in the ascidian embryo requires cell–cell interactions and signal transduction pathways. Isolation and characterization of an ascidian gene (HrSH2) from Halocynthia roretzi is reported. Sequence analysis suggests that HrSH2 encodes a polypeptide with an SH2 domain and a tyrosine kinase phosphorylation site, that are implicated in signaling pathways through tyrosine phosphorylation. Zygotic expression of HrSH2 was transient. The gene expression began at the 110‐cell stage but was downregulated by the larval stage. Whole‐mount in situ hybridization, taking advantage of well‐known lineage, revealed that the HrSH2 transcript first appeared in primordial notochord cells as well as a few endoderm cells of the 110‐cell embryo. During gastrulation and neurulation, expression in the endoderm was downregulated, and instead HrSH2 transcript became evident in notochord cells, nerve cord cells, endodermal strand cells and epidermal cells of the tail. These results suggest the possibility that HrSH2 is involved in the signal transduction pathways required for notochord formation and for differentiation of other cells in conjunction with the notochord.
Improved hatching for in vitro quail embryo culture using surrogate eggshell and artificial vessel
Authors: Kamihira Masamichi , Oguchi Satoshi , Tachibana Ayuko , Kitagawa Yuki , Iijima Shinji
Published: October 2003

The establishment of avian embryonic culture is important both for the analysis of the developmental process and the establishment of transgenic chickens that produce useful biological materials in eggs. However, the hatchability of cultured embryos has been ∼50%. We identified that the low rate of hatchability of cultured embryos was caused by limited oxygen and calcium availability. In quail embryo culture using chicken eggshell as a culture vessel,viability in the middle stage of culture was improved and 30% of embryos were hatched by oxygen enrichment. Furthermore, hatchability increased to 80% by supplementation with calcium lactate in addition to oxygen aeration. In the present study, a fully artificial vessel for quail embryo culture was designed using a gas‐permeable Teflon membrane. By the addition of fine eggshell powder and calcium lactate, quail embryos grew and developed normally, and 43% of embryos hatched. Although the hatchability was lower than that of cultures using a surrogate eggshell, we achieved in hatching an avian embryo using a fully artificial vessel.
Evidence for introduction of a variable G1 phase at the midblastula transition during early development in axolotl
Authors: Lefresne Jacques , Andéol Yannick , Signoret Jacques
Published: October 2003

After fertilization in axolotl, the synchronous cell cleavages are triphasic (S, G2 and M phases). Midblastula transition (MBT) begins at the ninth cleavage and is the consequence of lengthening of cell cycles. By spectro‐fluorometry and incorporation of 3H thymidine into the nuclear DNA followed by autoradiography on individual cells, the time at which a G1 phase appears during early development was investigated. The present results show that the G1 phase was introduced for the first time at MBT and its duration was variable from one blastomere to another. This variability could account for lengthening of cell cycles and be required for zygotic transcriptions necessary for DNA replication. From this point of view, axolotl represents an interesting alternative amphibian model to identify regulators involved in the G1–S transition at MBT during early development.
Expression of hindlimb abnormalities under rearing temperature effects during the larval development of the salamander Pleurodeles waltl (urodele amphibian)
Authors: Dournon Christian , Bautz Alain , Membre Hervé , Lauthier Michel , Collenot Alain
Published: October 2003

Anomalie m.p. is a spontaneous and heritable hindlimb abnormality described earlier. Twenty years later, Pleurodeles waltl larvae from the strain bearing anomalie m.p. and reared at room temperature or at 30°C, expressed abnormalities (ectrodactylia, hemimelia, ectromelia). A morphological study of all the hindlimbs and an analysis of the hindlimb skeleton of samples from the experimental animals confirmed that most of the skeletal malformations were identical to those previously reported and affected the disto‐proximal and prepostaxial pattern of the hindlimb. Analysis of the effects of rearing temperature on the expression of anomalie m.p. showed that the effects varied according to the developmental period at which the heat treatment was applied; the sooner the heat treatment began, the more numerous and more various were the degrees of severity of the malformations. Moreover, heat treatment induced the expression of two additional malformations not yet described: the first one, named ‘reversed knee joint’, was characterized by a reversal of the knee joint, and the second one, named ‘twisted foot’, by a downward twisting of the foot. The epigenetic effects of rearing at 30°C on hindlimb development are discussed with regard to the differentiation or patterning.
Expression pattern analysis of SGF‐3/POU‐M1 in relation to sericin‐1 gene expression in the silk gland
Authors: Matsunami Katsuyoshi , Kokubo Hiroki , Ohno Kaoru , Suzuki Yoshiaki
Published: October 2003

Embryonic and larval expression patterns of the sericin‐1 gene and its presumed transcription factor, SGF‐3/POU‐M1, in the silk gland were analyzed by in situ hybridization and immunohistochemistry. The sericin‐1 transcripts were first detected at embryonic stage 26 in an increasing gradient pattern in the middle and posterior part of the middle silk gland (MSG), while at the same stage the SGF‐3/POU‐M1 was already present in the entire anterior silk gland (ASG) and in the MSG but with a decreasing gradient pattern. The latter expression pattern was consistently maintained through all larval stages, while the sericin‐1 expression was detected during the feeding stages but disappeared at the molting stages. These observations suggest that, although the SGF‐3/POU‐M1 was proposed to be a positive transcription factor for the sericin‐1 gene, the protein might function in a negative manner on sericin‐1 gene transcription. Alternatively, it is also possible that the sericin‐1 gene might require another unidentified factor or mediator for in vivo transcription.
Cloning of cDNA for newt WT1 and the differential expression during spermatogenesis of the Japanese newt, Cynops pyrrhogaster
Authors: Nakayama Yuki , Yamamoto Takashi , Matsuda Yoshiko , Abé Shin‐Ichi
Published: October 2003

To investigate the function of Wilms' tumor 1 (WT1) during spermatogenesis, cDNA for newt WT1 homolog was cloned and the expression of WT1 in newt testes was examined. The cDNA is 2089 bp in length and encodes 426 amino acid (aa) residues. The deduced aa sequence shares 76 and 79% homology with human and Xenopus WT1, respectively. Northern blot analysis shows that WT1 mRNA, 3.2 and 4.5kb in length, are expressed in the testis and kidney. Both WT1 mRNA species are detected in various stages of spermatogenesis, but the 3.2kb mRNA is highly expressed in spermatogonia and mature sperm stages, while the amount of 4.5kb mRNA is almost constant throughout spermatogenesis. In situ hybridization reveals that WT1 mRNA is localized in Sertoli cells. Moreover, immunohistochemical analysis shows that WT1 protein is highly expressed in the nuclei of Sertoli cells in early spermatogonia and mature sperm stages, but not in pericystic cells or germ cells. These results suggest that WT1 is involved in the regulation of gene expression in Sertoli cells, depending on the spermatogenic stage.
Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera)
Authors: Yamashiki Naoko , Kawamura Naoko
Published: October 2003

The behavior of centrioles during eupyrene and apyrene meiosis was examined in the silkworm, Bombyx mori, by transmission electron microscopy and indirect immunofluorescence for tubulin. In eupyrene spermatocytes the centrioles, accompanied by axonemes, attached temporarily to the nucleus at diplotene, then detached from the nucleus in diakinesis. After the separation, a beret‐shaped structure consisting of a double membrane covered the proximal region of the pair of centrioles. The structure disappeared after breakdown of the nuclear membrane. The centriole, with the axoneme, reattached to the nucleus at telophase I. The process was repeated during meiosis II until the centrioles maintained their nuclear attachment in newly developed spermatids. In stark contrast to their eupyrene counterparts, apyrene spermatocytes were conspicuously devoid of any attachment of the centrioles to the nucleus. These eupyrene‐specific and apyrene‐specific relationships were consistently and repeatedly found between the nuclear membrane and centrioles, giving rise to suspicion that the behavioral phenomena may be related to differentiation of the dimorphic sperm types.
An alternatively spliced gene encoding a Y‐box protein showing maternal expression and tissue‐specific zygotic expression in the ascidian embryo
Authors: Wada Michiko R. , Ohtani Yoshiaki , Shibata Yumiko , Tanaka Kimio J. , Tanimoto Naomi , Nishikata Takahito
Published: October 2003

An ascidian Y‐box protein gene was cloned, designated as CiYB, which consists of a highly conserved cold shock domain and an auxiliary tail domain with alternating modules of acidic and basic amino acids. CiYB is a single copy gene in the ascidian genome. During oogenesis and early development, CiYB produces three different transcripts (CiYB1, CiYB2 and CiYB3) by alternate splicing. CiYB1 and CiYB2 were expressed during oogenesis, suggesting that they are recruited into maternal ribonucleoprotein particles. According to gel mobility shift assay, the CiYB1 protein has the ability to bind RNA. The sequence preference of RNA binding is similar to that of the Xenopus Y‐box protein (FRGY2), which is a major component of the maternal messenger ribonucleoprotein particles (mRNP) in the oocyte. These results suggest that the ascidian Y‐box protein may have an important role for masking and translational regulation of maternal mRNA. Furthermore, CiYB1, CiYB2 and CiYB3 were expressed zygotically in a tissue restricted manner. CiYB1 was expressed specifically in muscle precursor blastomeres and tail muscle cells suggesting its important role in muscle differentiation.
Developmental studies for identification of the inhibitory center of melanotropes in the toad, Bufo japonicus
Authors: Kouki Tom , Kawamura Kosuke , Kikuyama Sakae
Published: October 2003

Two series of experiments were performed to identify the inhibitory center of the melanotropes in the intermediate lobe of hypophysis of the toad, Bufo japonicus. First, developmental changes in the distribution of dopaminergic neurons were examined from hatching stage to postmetamorphosis using an antiserum against dopamine synthase (tyrosine hydroxylase, TH). In the postmetamorphic toads, TH‐positive cell bodies were localized in three clusters. One was the preoptic recess organ (PRO) in the prechiasmatic area, the other two were the paraventricular organ (PVO) and infundibular nucleus (IN) in the postchiasmatic area. Each of them exhibited different ontogenetic changes. During larval development, TH‐positive cell bodies were first detected in the PVO and IN at a premetamorphic stage. The number of immunoreactive cells increased rapidly in both loci as metamorphosis proceeded, although the two nuclei showed different growth profiles. By contrast, in the PRO, a very small number of immunoreactive cells were observed before the onset of the prometamorphic period. Although the number of immunoreactive neurons increased as metamorphosis progressed, early neurons were confined to the caudal area of the PRO (cPRO), the rostral area of the PRO (rPRO) being devoid of TH‐positive cells. Immunoreactive TH neurons appeared in the rPRO for the first time at the end of meta‐morphic climax. This timing coincided well with the development of TH‐positive nerve endings in the pars intermedia (PI) and median eminence. In the second series of experiments, the embryonic primordium of the PRO was surgically extirpated from open neurulae to examine the effects of PRO‐ectomy. In 75% of the operated animals, background adaptation was not observed, their dermal melanophores remained permanently dispersed even on the white background. Dopaminergic neurons in the rPRO and the immunoreactive nerve endings in the PI and median eminence were scarcely observed in these animals. It was concluded that the present data strongly support the hypothesis that rPRO is the center of white‐background adaptation.
Coelomic pouch formation in reconstructing embryos of the starfish Asterina pectinifera
Authors: Tamura Miwa , Dan‐Sohkawa Marina , Kaneko Hiroyuki
Published: October 2003

The morphogenetic processes of coelomic pouch (CP) formation in starfish embryos that were experimentally dissociated and induced to undergo reconstruction were studied. An analysis of these embryos randomly chosen from several cultures showed that CP always form on either side of the esophagus, even though the CP formation can differ in timing of initiation and duration, and can vary in number and size from embryo to embryo. Successive observations of CP formation in living embryos revealed two distinct sequences of CP development that were accompanied by different appearances of the blastocoele. These processes were named ‘enterocoelic‐like’ and ‘schizocoelic‐like’ CP formation. The former resembled normal development and occurred in embryos with a transparent blastocoele. The latter was characterized by the aggregation and epithelialization of mesenchyme‐like cells on either side of the esophagus and was observed in embryos possessing a cloudy blastocoele. In a few embryos, both types of CP formation were seen in the same individual (‘mosaic type’ CP formation). Thick sections of embryos possessing a cloudy blastocoele revealed that aggregates of mesenchyme‐like cells undergoing CP formation directly contact the developing esophagus. Together, these data demonstrate flexibility in the morphogenetic processes that regulate CP formation, and suggest that positional cues in the esophagus regulate the placement of CP.
Detection of phospholipase Cγ in sea urchin eggs
Authors: De Nadai Céline , Cailliau Katia , Epel David , Ciapa Brigitte
Published: October 2003

Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium‐sensitive phospholipase C activity. We have investigated whether phospholipase Cγ (PLCγ), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCγ revealed the presence of this protein in cortical regions. It was also observed that PLCγ displayed calcium‐sensitive activity. The present results suggest that PLCγ may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.
Risks for unplanned hospital readmission in a teaching hospital in southern Taiwan
Authors: Chang Su‐Hsien , Chiu Ya Hung , Liou I‐Ping
Published: October 2003

The purpose of this study was to assess and analyse hospital readmission and its risk factors for patients who were visited by a discharge planning coordinator during hospitalization in a teaching hospital located in southern Taiwan. Results found that 67 patients (5.7%) were readmitted within 14 days of discharge during the data collection period. Twenty‐eight patients (41.8%) were readmitted because of complications. Patients’ previous diagnoses and complications were two major reasons for patients to be re‐hospitalized within 14 days of discharge. In addition, significant predictors for unplanned hospital readmission within 14 days of discharge were patients who received care from home health care nurses or hospice home health care nurses.