bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathogenesis in Transgenic Mice Expressing Bovine Cellular Retinoic Acid‐Binding Protein
Authors: Wei Li‐Na , Lee Chih‐Hao , Chang Shu‐Ling , Chu Ya‐Shu
Published: January 2005

Transgenic mice with ectopic expression of bovine CRABP under the control of the human metallotheionein IIA promoter have shown a variety of pathological consequences. Expression of the transgene has been detected in most of the tissues examined, including heart, lung, liver, spleen, kidney, intestine, testis, and ovary, except pancreas. Two independent lines have been able to produce normal non‐transgenic F1 animals of both sexes but only female transgenic progenies. All of these F1 female transgenic animals derived from both lines are sterile, and the ovaries from these animals appear to be significantly smaller as compared to their non‐transgenic littermates. Histopathological examinations have shown no maturing follicles in these transgenic ovaries in which abnormal cells have been observed. Another independent line has generated transgenic F1 animals which have been growing retardly. These animals all have small spleen and liver and have become very sick at the age of 4 to 5 weeks. Histopathological examinations on these transgenic progenies have shown hepatocytes to be reduced in the cytoplasmic portion in which glycogen is highly depleted. The spleen is poorly developed as no well organized germinal centers can be observed in the spleen sections of these transgenic animals.
Elevation of Cyclic AMP‐Dependent Protein Kinase Activity during Migration of Primary Mesenchyme Cell in Sand Dollar Blastulae
Authors: Katow Hideki , Ishida Katsumi
Published: January 2005

Concetration of intracellular cyclic AMP (cAMP), and activities of adenylate cyclase and cAMP‐dependent protein kinase were examined in swimming and mesenchyme blastulae and primary mesenchyme cells (PMCs) of the sand dollar, Clypeaster japonicus, respectively. In mesenchyme blastulae, the concentration of cAMP increased 45% from that in swimming blastulae. PMCs contained a concentration of cAMP 40% higher than that in whole embryos at the mesenchyme blastula stage. The activity of adenylate cyclase in mesenchyme blastulae was 100% higher than that in swimming blastulae. The activites of cAMP‐dependent protein kinase in whole embryos at the above two developmental stages, on the other hand, were quite similar to each other. However, in PMCs the activity of the enzyme was conspicuously higher than that in these embryos, and it reached 190% higher than that in these embryos. Inhibition of cAMP‐dependent protein kinase activity by a synthetic inhibitor, H8, caused severe inhibition of PMC migration but it did not exert any effect on PMC ingression. These results suggest that the cAMP‐dependent protein kinase activity is involved in PMC migration, but not in PMC ingression.
Positive and Negative Regulation of the Differentiation of Ventral Mesoderm for Erythrocytes in Xenopus laevis
Authors: Maéno Mitsugu , Ong Rosa Chua , Kung Hsiang‐fu
Published: January 2005

To elucidate the mechanism of determination and regulation of hemopoiesis in the early Xenopus embryo, explants of dorsal and ventral mesoderm from various stage embryos were cultured alone or combined with various tissues derived from the same stage embryo. Western blot analysis of larvae‐specific globin expression using monoclonal antibody L5.41 revealed that extensive erythropoiesis occurred in the explants of ventral mesoderm from st. 22 tailbud embryo, but not in those of dorsal mesoderm. Experiments using combined explants at this stage demonstrated that the in vitro differentiation of erythrocytes in the ventral mesoderm could be completely inhibited by the dorsal tissue, including neural tube, notochord, and somite mesoderm, but not by other mesoderms, gut endoderm, or forebrain. Subsequent explant studies showed that the notochord alone is sufficient for this inhibition. Furthermore, the ventral mesoderm explant from the st. 10+ early gastrula embryo was not able to differentiate into erythroid cells. However, small amounts of globin were expressed if ventral mesoderm of this stage was combined with animal pole cells which were mainly differentiated to epidermis. This stimulation was enhanced when both tissues were excised together without separation, while none of the other parts of st. 10+ embryo had this stimulatory effect. These observations found in the combined explants suggest that in vivo interactions between the ventral mesoderm and adjacent tissues are important for normal development of erythroid precursor cells.
The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos
Authors: Grunz Horst
Published: January 2005

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions (“beating hearts”). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.
Developmentally Regulated Splicing of the Third Intron of P Element in Somatic Tissues in Drosophila Embryos
Authors: Kitamura Tomiichiro , Kobayashi Satoru , Okada Masukichi
Published: January 2005

In Drosophila, it has been suggested that the third intron of the P element is spliced out only in germ‐line cells. However, we found that the splicing took place in some somatic tissues during embryogenesis, as well as in pole cells, progenitor cells of the germ line. To detect the splicing activity in vivo, we designed an assay system to visualize the cells where the third intron was spliced out by the histochemical staining for β‐galactosidase activity. In this paper, we describe the somatic tissues where the third intron is spliced out during embryogenesis, with reference to the stages when the splicing occurs. Based on these findings we propose a modification of the previous documentation that the complete processing of transcripts from P element takes place only in germ‐line cells.
Role of Germ Nuclei in Conjugation of Paramecium caudatum
Authors: Yanagi Akira
Published: January 2005

Ciliated protozoa have two distinct nuclei, germinal micronuclei and somatic macronuclei. Macronuclei are transcriptionally active, but micronuclei are almost inert. To know whether micronuclei have any function in conjugation of Paramecium caudatum, expression of the conjugation‐specific nuclear antigen, CSN‐1, in amicronucleate cells during conjugation was examined. Micro‐ (mic) and amicronucleate (amic) cells were conjugated in three combinations (mic‐mic, mic‐amic and amic‐amic). In conjugation of two amicronucleate cells, the cells were negative against the CSN‐1 antibody by indirect immunofluorescence staining and underwent abnormal macronuclear fragmentation. These were not observed in conjugation of micronucleate cells. In conjugation between micro‐ and amicronucleate cells, the amicronucleate mates were negative against the antibody in the beginning, but after receiving a pronucleus from the micronucleate mates, they became positive. This indicates that the CSN‐1 antigen or factors involved in the expression of the antigen can hardly pass across the fused cell membrane between conjugating cells before pronuclear exchange. The present results suggest that micronuclei have some function in expression of the CSN‐1 antigen and the macronuclear fragmentation, both of which are specific events in conjugation.
Induction of Pronephric Tubules by Activin and Retinoic Acid in Presumptive Ectoderm of Xenopus laevis
Authors: Moriya Naomi , Uchiyama Hideho , Asashima Makoto
Published: January 2005

Correlation between activin and retinoic acid (RA), both of which affect early amphibian development, was studied using Xenopus laevis embryos. In the first set of experiments, two isomers of RA, all‐trans RA and 13‐cis RA, were compared in terms of stability of biological activity against light. Xenopus blastulae were dipped in RA solutions which had either been kept away from light, or had been exposed to light for a few hours. At doses ranging from 10–4to 10–6M, RA elicited head deformity. All‐trans RA, under both dark and light conditions, had similarly potent effects. On the other hand, 13‐cis RA under dark conditions had much weaker effects than it did under light conditions.

In the second set of experiments, activin was mixed with all‐trans RA, and the inducing effects on the animal cap explants were investigated. Activin at a concentration of 10 ng/ml induced notochord. In combination with 10–6M RA, muscle was well induced instead of notochord. In combination with 10–5M RA, pronephric tubules were markedly induced. Pronephric tubules were never induced by activin alone, at any of the various concentrations employed. This is the first report on the very high frequency of induction of pronephric tubules by the combination of activin A and all‐trans RA in the Xenopus ectoderm.
A Factor That Interrupts the Cell Cycle at G2 or Prophase is Present in Full‐Grown Frog Oocytes
Authors: Sadakane Yutaka , Iwao Yasuhiro
Published: January 2005

Full‐grown amphibian oocytes that had been arrested at meiotic prophase I contained an activity that prevented the cell cycle from progressing beyond a G2‐like stage. Injection of the contents of germinal vesicles (GV‐content) or cytoplasm obtained from oocytes of the frog Rana rugosa prevented fertilized eggs of Cynops pyrrhogaster or Bufo japonicus from cleaving. The nuclei in the arrested eggs consisted of thin chromosomes and nucleolus‐like particles enclosed within clear nuclear membrane and their volume increased as a function of time after injection. Cycling of maturation‐promoting factor (MPF) did not occur in the injected eggs, but DNA synthesis was not disturbed. The injection of exogenous MPF into the eggs induced the reinitiation of the cell cycle with progression to the M phase and subsequent cleavage. Furthermore, the injection into the full‐grown oocytes of Bufo inhibited induction of the maturation of oocytes by progesterone. These results demonstrate that a factor that arrests the cell cycle either at a G2‐like stage of mitosis or at prophase in meiosis is present both in the GV and cytoplasm of frog oocytes. We refer to this factor as a G2‐specific cytostatic factor (G2‐CSF). G2‐CSF may play an important role not only in the physiological arrest at prophase I in meiosis, but also in regulation of the G2/M transition in the cell cycle of early embryonic cells.
Immunochemical Analysis of Arylsulfatase Accumulation in Sea Urchin Embryos
Authors: Yang Qing , Kingsley Paul D. , Kozlowski David J. , Angerer Robert C. , Angerer Lynne M.
Published: January 2005

We have determined the expression pattern of arylsulfatase in embryos of the sea urchin Strongylocentrotus purpuratus. Polyclonal antibodies raised against a fusion protein containing sequences encoded by SpARSI (Yang et al., 1989, Dev. Biol. 135: 53–61, 1989) detect several peptides of 65–70 kD on immunoblots. Treatment with glycopeptidase F shows that at least one of these peptides is modified by N‐linked glycosylation, which accounts for some of the peptide diversity. We have also identified a second arylsulfatase gene (SpARSII) whose sequence is highly similar to ARS, a gene expressed in the Hemicentrotus pulcherrimus embryo. Arylsulfatase activity is detectable in unfertilized eggs, in which only SpARSII mRNA can be detected. Both SpARSI and SpARSII mRNAs increase greatly in abundance during embryogenesis accompanied by parallel changes in arylsulfatase activity and immunoreactivity. Immunohistochemistry with the anti‐SpARSI antibody shows that arylsulfatase accumulates primarily along the apical surface of the aboral ectoderm of pluteus larvae, and to a lesser extent along portions of oral ectoderm. At earlier stages, the protein is more uniformly distributed along all presumptive ectoderm, reflecting a more uniform mRNA distribution. Treatment of embryos with glycine‐EDTA, which dissociates but does not lyse cells of the embryo, releases virtually all enzymatic activity and all immunoreactive protein. Embryos cultured in sulfate‐free sea water, which arrest at gastrula stage, show normal accumulation and secretion of peptide detected with the SpARSI antibody.
Engrailed Expression during Development of a Lamprey, Lampetra japonica: A Possible Clue to Homologies between Agnathan and Gnathostome Muscles of the Mandibular Arch
Authors: Holland Nicholas D. , Holland Linda Z. , Honma Yoshiharu , Fujii Tamotsu
Published: January 2005

Developing lampreys were fixed at frequent intervals between the gastrula stage (6 days) and the earliest ammocoete larva (31 days). Expression of lamprey engrailed (en) gene was studied by labeling with a polyclonal antiserum (αEnhb‐1) raised against mouse en protein. Western blotting of proteins from developing lampreys reveals a major band (40±103Mr), which is probably lamprey en protein. Expression domains of en were demonstrated in developing lampreys by immunohistochemistry of whole mounts and histological sections. Expression of en first becomes detectable at the head protrusion stage (11/12 days) in neural tube cells at the mid/hindbrain boundary and soon thereafter in some mesodermal cells of the mandibular arch. These en‐expressing cells of the mandibular arch are located in the walls of vesicles of paraxial mesoderm that originate by enterocoely on either side of the pharynx. At the tailbud stage (15 days), en expression is also detectable in mesodermal cells of the anterior lip and in some mesodermal and epidermal cells in the region of the tailbud. By the eye spot stage (18 days), detectable en expression in the mandibular arch becomes limited to cells of the velothyroideus muscles, which drive the power stroke of the recently formed velum. At later stages, while the preceding expression domains fade, en expression begins in some epidermal cells associated with the lip papillae, gill slits, and nostril. We suggest that the velothyroideus muscles of lampreys may be homologous to certain jaw muscles of teleosts–namely, the levator arcus palatini and the dilator operculi, which express en continuously while differentiating from the myogenic mesoderm into identifiable muscle types.
The Timing of Appearance and Pathway of Migration of Cranial Neural Crest‐derived Precursors of Melanocytes in Chick Embryos
Authors: Takiguchi‐Hayashi Keiko , Kitamura Kunio
Published: January 2005

The timing of appearance and pathway of migration of precursors of melanocytes in cranial regions of chick embryos were examined by the monoclonal antibody MEBL‐1, which can identify precursors of melanocytes soon after their emigration from the neural tube (7). Precursors of melanocytes were first detected on the dorsal side of the mesencephalic neural tube at stage 16, when other neural crest cells had already left the dorsal side of the neural tube. Then precursors of melanocytes at more rostral and caudal levels appeared. After the first appearance on the neural tube, precursors of melanocytes migrated along a dorsolateral pathway under the superficial ectoderm, which followed other neural crest cells. These results indicate that precursors of melanocytes migrate along spatially the same pathway as other neural crest cells, but temporally the different time as considered previously.
Cooperative Activation of HoxD Homeobox Genes by Factors from the Polarizing Region and the Apical Ridge in Chick Limb Morphogenesis
Authors: Koyama Eiki , Noji Sumihare , Nohno Tsutomu , Myokai Fumio , Ono Katsuhiko , Nishijima Katsumi , Kuroiwa Atsushi , Ide Hiroyuki , Taniguchi Shigehiko , Saito Taiichi
Published: January 2005

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.
Anti‐peptide Antibody Identifies a 57 kDa Protein Tyrosine Kinase in the Sea Urchin Egg Cortex
Authors: Peaucellier Gerard , Shartzer Kirsten , Jiang Wanping , Maggio Katia , Kinsey William H.
Published: January 2005

The egg plasma membrane and cortical structures are highly enriched in protein tyrosine kinase activity which is thought to play an important role in the fertilization process. In order to identify the tyrosine protein kinases in the egg cortex, a site directed polyclonal antibody was produced against a peptide duplicating a conserved region of the catalytic domain of the sea urchin c‐abl gene product. The region chosen as an antigen had a high degree of homology (57%) to other protein tyrosine kinases. The antibody was found to bind with a high degree of specificity to a 57 kDa protein tyrosine kinase in S. purpuratus eggs. The antibody was capable of immunoprecipitating the enzyme as a 57 kDa phosphoprotein from purified egg cortex fractions solubilized in NP‐40. Immunoprecipitation was completely inhibited by prior incubation of the antibody with the synthetic peptide used as an antigen. Binding of the antibody completely inhibited kinase activity. However, the immunoprecipitated kinase activity could be eluted from the Sepharose‐coupled antibody and was shown to have catalytic activity towards a tyrosine containing peptide substrate. The enzyme also underwent autophosphorylation on tyrosine in vitro. Ultrastructural localization of the kinase by immuno‐electron microscopy revealed that the enzyme was primarily restricted to the egg plasma membrane.
Remodeling of the Nauplius Eye into the Adult Ocelli during Metamorphosis of the Barnacle, Balanus amphitrite hawaiiensis
Authors: Takenaka Makoto , Suzuki Akiko , Yamamoto Takashi , Yamamoto Masamichi , Yoshida M.
Published: January 2005

The planktonic barnacle larva has a single median ocellus (nauplius eye), while the adult possesses two distinct sets of photoreceptors; a pair of lateral ocelli and a single median ocellus. The nauplius eye of the cypris larva of Balanus amphitrite hawaiiensis is composed of 14 visual cells grouped into three components (a pair of lateral components and a single ventral component) surrounding two centrally located pigment cells; each lateral component consists of 5 visual cells and the ventral component, 4 visual cells. In each component, the rhabdom is made up of apposing microvilli arising directly from the neighboring visual cell bodies.

During metamorphosis into the adult form, the three components of the median ocellus become separated. Each lateral component migrates laterally on the mantle and is remodeled into the adult lateral ocellus, losing two visual cells but gaining new pigment and tapetum cells in the process. The ventral component remains in the mid portion and becomes the adult median ocellus without fundamental modification in composition. The visual cells in both ocelli undergo a marked increase in volume and form many finger‐like dendrites. Rhabdomes are made up of interdigitating microvilli arising from the the dendrite tips.
Proteins ADP‐ribosylated in Nuclei and Plasma Membrane Vesicles Isolated from Sea Urchin Embryos at Various Stages of Early Development
Authors: Kamata Yasuyuki , Furuya Shigehisa , Yasumasu Ikuo
Published: January 2005

The ADP‐ribosylations of proteins in nuclei, plasma membrane vesicles, mitochondria, microsome vesicles and the soluble fraction of sea urchin embryos isolated at various stages of development were examined by measuring the radioactivities of proteins after exposure of these subcellular fractions to [adenosine‐14C]NAD or [adenylate‐32P]NAD. ADP‐ribosylation of proteins was detected only in the nuclear and plasma membrane fractions. In the nuclear fraction, the rate of ADP‐ribosylation of the histone fraction did not change appreciably during early development. In the TCA‐insoluble protein fraction of the nuclei, the rate of ADP‐ribosylation increased from fertilization to the morula stage, then decreased and again increased from the mesenchyme blastula to the late gastrula stage. After exposure of the nuclear fraction to [adenylate‐32P]NAD, a protein band with a molecular weight of 90 kDa was detected by SDS‐polyacrylamide gel electrophoresis and radioautography at all stages examined. Its labeling intensity indicated that its ADP‐ribosylation is higher at the morula and late gastrula stages than at other stages. In the plasma membrane fraction, proteins with molecular weights of 22 and 68 kDa were ADP‐ribosylated and their rates of ADP‐ribosylation hardly changed during early development.
Role of the Core Promoter for the Preferential Transcription of Fibroin Gene in the Posterior Silk Gland Extract
Authors: Takiya Shigeharu , Suzuki Yoshiaki
Published: January 2005

Preferential transcription of the fibroin gene in homologous cell‐free transcription systems prepared from the posterior silk gland (psg) of Bombyx mori is supported by the modulators upstream and downstream of the core promoter. However, we found that the cognate fibroin core promoter composed of the TATA box and transcription initiation region was indispensable for this preferential transcription in the psg extract. Studies on the transcriptional efficiencies of reconstituted fibroin promoters indicated that the fibroin core promoter functions synergistically with the modulators. The core promoter of adenovirus major late promoter (AdMLP) which carries the same TATA box sequence as the fibroin promoter could not substitute for the fibroin core promoter for the preferential transcription. In DNase I footprint experiments a characteristic protection pattern was obtained on the fibroin core promoter specifically in the psg extract but not in the HeLa cell extract, and the pattern on the fibroin core promoter was distinguished from that on AdMLP. Appearance of the characteristic footprint pattern on the core promoter and promoter activity of the fibroin gene were correlated in the psg extract. These results suggest that specific interaction of the transcription machinery with fibroin core promoter in the psg extract is a basis of psg specific transcription of the fibroin gene.
Purification and Heterogeneous Localization of Sarcophaga Lectin Receptor on the Surface of Imaginal Discs of Sarcophaga peregrina (Flesh Fly)
Authors: Nagasawa Yasuo , Kurata Shoichiro , Komano Hiroto , Natori Shunji
Published: January 2005

Previously, we reported autocrine involvement of Sarcophaga lectin in the development of Sarcophaga imaginal discs (Kawaguchi et al., Dev. Biol. 144, 86–93 (1991)). In this study, we purified Sarcophaga lectin binding protein from the membrane fraction of cultured embryonic cells of Sarcophaga to near homogeneity and raised a monoclonal antibody against it. Histochemical analysis using the monoclonal antibody revealed that this binding protein is distributed heterogeneously on the surface of leg imaginal discs. This binding protein was especially clearly localized in the central region of the basal side of leg discs which forms the junction between the leg and body, suggesting the participation of Sarcophaga lectin in morphogenesis of the basal region of the developing leg.
Removal of Heparan Sulfate Chains Halted Epithelial Branching Morphogenesis of the Developing Mouse Submandibular Gland in vitro
Authors: Nakanishi Yasuo , Uematsu Jun , Takamatsu Hiroshi , Fukuda Yuh , Yoshida Keiichi
Published: January 2005

The physiological function of heparan sulfate chains in the mouse embryonic submandibular gland was studied by the use of heparitinases purified from Flavobacteriu heparinum. Heparitinase I, which catalyzes the cleavage of specific glycosaminidic linkages adjacent to non‐or monosulfated disaccharides of heparan sulfate chains, in the culture medium of the mid and late 12‐day gland inhibited the branch‐initiation and changed their round epithelial shape to elongated one, together with a concommitant reduction in lobular growth. [3H]Thymidine incorporation experiments indicated that heparitinase I treatment blocked 24% of the DNA synthesis compared with controls. Analysis of 35S‐inorganic sulfate labeled glycosaminoglycans extracted from cultured rudiments revealed that the glands with heparitinase I contained no heparan sulfate, while in the glands without the enzyme more than 20% of total glycosaminoglycans was heparan sulfate.

The heparitinase effect on morphogenesis was mimicked by the addition of heparan sulfate (1 mg ml−1) or heparin (75 μg ml−1), but not by chondroitin sulfate (1 mg ml−1) in the culture medium. Transmission electron microscopic study indicated that at the epithelial‐mesenchymal interface close contacts between the fibroblast and epithelial cells were much fewer in heparitinase‐treated glands than in controls. Immunohistochemical analysis demonstrated that the core protein of basement membrane heparan sulfate proteoglycan and type IV collagen accumulated abnormally inside the epithelial lobules of glands cultured with heparitinase I. These results strongly suggested that glycosaminoglycan chains of heparan sulfate or heparin is involved in the epithelial morphogenesis of the mouse embryonic submandibular gland.
Temperature Sensitivity of a Hybrid between Two Species of Sea Urchin Differing in Thermotolerance
Authors: Fujisawa Hirosuke
Published: January 2005

Eggs of Dendraster excentricus can be fertilized with sperm of Strongylocentrotus purpuratus at a high success rate according to Whiteley and Baltzer's method. The resulting hybrids are well suited for investigations of the factors determining embryonic thermosensitivity, because the embryos of both the above species differ significantly with regard to egg size, blastula shape, and particularly thermotolerance and the temperature dependence of their developmental rate. Morphologically, the hybrids show some paternally inherited traits, but their thermosensitivity is strictly determined by the egg cytoplasm.
Furrow Formation in the Vegetal Hemisphere of Xenopus Eggs
Authors: Kubota Tomoyuki , Sakamoto Masahiko
Published: January 2005

The mechanism of furrow formation in the vegetal hemisphere of amphibian eggs was studied using Xenopus eggs. Injection of colchicine into the eggs after the furrow tip had entered the vegetal hemisphere arrested the subsequent cleavage. The effect of impairing the continuity between the animal and vegetal hemispheres was examined by squeezing the equator of uncleaved eggs from both sides with the edges of coverslips. On gentle squeezing a shallow vegetal furrow was formed at the first cleavage, whereas on strong squeezing furrowing was arrested at the equator. The mechanism of furrow formation in the vegetal hemisphere of amphibian eggs is discussed on the basis of these findings.
Xenopus Heterochronic Presumptive Primordial Germ Cells (pPGCs) Implanted in the Correct Position in Host Neurula Embryos can Differentiate into PGCs
Authors: Ikenishi Kohji , Tanaka Tetsuya S.
Published: January 2005

Presumptive primordial germ cells (pPGCs) in explants, derived from single germ plasm‐bearing cells of Xenopus 32‐cell embryos, at the equivalent of neurula stage (stage 20) in control embryos (designated as ‘stage‐20′ explants) were demonstrated to be able to differentiate into PGCs, when implanted into a prospective place of pPGCs in host embryos (stage 20) (Ikenishi & Tsuzaki, 1988). According to a recent proposal that individual early embryonic cells in Xenopus, at both in vivo and in vitro, are able to measure elapsed time since fertilization (Cooke and Smith, 1990), the result means that the implanted pPGCs having the same elapsed time as the host embryos (isochronic pPGCs) could differentiate into PGCs. In the present study, in order to know whether the compatibility in elapsed times of implanted pPGCs and host embryos is necessary for the differentiation of PGCs, labelled, heterochronic pPGCs in ‘stages 12–33/34′ explants were implanted into unlabelled, host neurulae (stage 19).

Those heterochronic pPGCs could differentiate into PGCs like isochronic pPGCs in ‘stage‐19′ explants as the control. By comparing the average diameters and yolk contents of labelled PGCs with those of unlabelled, host ones in experimental tadpoles, the possibility that a certain mechanism modulating the elapsed time of heterochronic pPGCs to that of host pPGCs is present in host embryos was also suggested.
Movement of an Epithelial Layer Isolated from Early Embryos of the Newt, Cynops pyrrhogaster I. Development of Folding Movement of the Blastocoelic Wall Isolated from Embryos before and during Gastrulation
Authors: Komazaki Shinji
Published: January 2005

The blastocoelic wall (BW) was isolated from embryos of the newt, Cynops pyrrhogaster, before and during gastrulation. The mechanism responsible for the folding movement of the isolated BW toward the basal side was investigated. The BW isolated from the early gastrula was induced to fold toward the basal side by treatment with serum. The folding movement of the isolated BW took place from the late blastula to early gastrula stage. Electron microscopy, rhodamine‐phalloidin staining and experiments with inhibitors show that the development of the folding movement was correlated with the appearance of a submembranous microfilaments layer (SML) which was formed beneath the cell membrane on the basal side of the BW and suggest that the contraction of actin filaments in the SML is involved in the folding movement of the isolated BW toward the basal side.
Movement of an Epithelial Layer Isolated from Early Embryos of the Newt, Cynops pyrrhogaster II. Formation of a Blastoporal Groove and Archenteron in a Superficial Epithelial Layer Isolated from Initial Gastrula
Authors: Komazaki Shinji
Published: January 2005

The roles of folding movement of epithelial layer in amphibian gastrulation were investigated. A superficial epithelial layer was isolated from the vegetal hemisphere of the initial gastrula (stage 11) of the newt, Cynops pyrrhogaster. The isolated epithelial layers were cultured and morphogenetic movements of the epithelial layers were analysed. Two types of folding movement, folding toward the apical side in the blastopore‐forming region and folding toward the basal side in the dorsal marginal zone, arose autonomously in the cultured epithelial layers. These movements caused morphogenesis similar to the formation of the blastoporal groove and archenteron in the control embryo. Treatment with chemical reagents that affect the morphogenetic movement of cells and electron microscopy of the submembranous microfilaments layer (SML) suggested that contraction of actin filaments in the SML was involved in both types of folding movement but that they are controlled, respectively, by different mechanisms in terms of involvement of Ca2+ ions. The present results suggest that two types of folding movement arise in the superficial epithelial layer of the embryo and play important roles in the formation of the blastoporal groove and archenteron during early steps of amphibian gastrulation.
Low‐Na+Seawater Induces the Acrosome Reaction and Histone Degradation of Starfish Sperm in the Absence of Egg Jelly
Authors: Amano Tosikazu , Okita Yoshihito , Hoshi Motonori
Published: January 2005

Egg jelly induces the degradation of histones as well as the acrosome reaction in the spermatozoa of Asterina pectinifera. Much similar degradation of histones without any apparent morphological changes such as the acrosome reaction was induced in the spermatozoa by merely dispersing them into Na+‐free seawater. It required external Ca2+ much less than the jelly‐induced one in normal seawater, and was not susceptible to Ca2+‐channel antagonists, verapamil and diltiazem. Once spermatozoa were incubated with egg jelly in Ca2+‐free seawater, they did not undergo the histone degradation even after subsequent addition of Ca2+, but Na+‐free seawater rescued such blockage. Spontaneous acrosome reaction occurred in seawater containing 10–30 mM Na+ in a Ca2+‐dependent manner. This reaction was accompanied by a rapid increase in intracellular pH (pHi) followed by a large pHi decrease. Diltiazem blocked a large decrease in pHi but scarcely inhibited the acrosome reaction induced by low‐Na+ seawater. Increasing K+ inhibited both pHi changes and the acrosome reaction induced by low‐Na+ seawater. Decreasing pH of seawater also inhibited the pHi changes but did not affect the acrosome reaction. Strontium was also effective to induce a rapid increase, followed by a gradual decrease, in pHi and the acrosome reaction.
Localization of an Exogastrula‐Inducing Peptide (EGIP) in Embryos of the Sea Urchin Anthocidaris crassispina
Authors: Mizuno Nobuhiko , Uemura Isao , Yamasu Kyo , Suyemitsu Takashi , Ishihara Katsutoshi
Published: January 2005

Exogastrula‐inducing peptides (EGIPs) are present in the unfertilized eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. The localization of EGIP‐D during embryogenesis has been explored using polyclonal antibodies against EGIP‐D. Immunofluorescent staining revealed that EGIP‐D is stored in the cytoplasm of immature oocytes and is concentrated into vesicles in unfertilized eggs. At fertilization, the vesicles containing EGIP‐D (EGIP‐vesicles) migrate to the cortical surface of the zygotes and are distributed in a ring‐like pattern at the apical surface of blastomeres, disappearing from basal surfaces and those adjacent to neighboring cells, during development from cleavage stages to larval stages. Mesenchyme cells also contain the vesicles but no such polarized distribution of vesicles is apparent. Acidic vesicles with a similar polarized distribution were examined by staining with acridine orange, which revealed that acidic vesicles were in close proximity to the surface of eggs at fertilization and were then distributed in a ring‐like pattern at the apical surface of blastomeres as are the EGIP‐vesicles. Furthermore, immunoelectron microscopy revealed that EGIP‐D is present in vesicles that are located at the apical surface of blastomeres. The significance of the localized distribution of EGIP‐D is discussed in relation to its function.
A Novel Mesoderm‐Specific cDNA Isolated from a Mouse Embryonal Carcinoma Cell Line
Authors: Sado Takashi , Nakajima Noboru , Tada Masako , Takagi Nobuo
Published: January 2005

A novel mesoderm‐specific cDNA clone has been isolated by differential screening of cDNA library from an embryonal carcinoma (EC) cell line MC12. The cDNA clone 121a is about 2.5 kb in length and apparently encodes a putative polypeptide of 335 amino acids which may be secreted or membrane anchored glycoprotein since it has a possible signal sequence and a potential N‐linked glycosylation site. In situ hybridization using mouse embryos revealed that 121a expression was confined to mesoderm and its derivatives such as allantois, the mesodermal layer of amnion, chorion and yolk sac, somites, heart, etc. These findings suggest that 121 a may be essential for mesodermal differentiation or function, although nothing definite is known. Conservation of 121a homolog in mammals and even in Drosophila seems to support this presumption. Fluorescence in situ hybridization successfully localized 121a to B1 band of mouse chromosome 6.
Laterally Asymmetric Development of Duck Syrinx: Inhibition of Growth and Chondrogenesis of the Right Syringeal Half by the Left Half
Authors: Takahashi Mayumi M. , Noumura Tetsuo
Published: January 2005

For determination of whether position‐related tissue interaction is involved in the laterally asymmetric development of the male duck syrinx, a cartilaginous vocal organ, the syrinxes from 10‐day embryos were cut into right and left halves and cultured organotypically. The lateral halves developed equally when cultured separately, but their developments were unequal when they were cultured together in the same culture medium: that is, the right half developed less than the left half in terms of increase in size and chondrogenesis. When the right half from a normal or estrogen‐treated embryo was cultured with the left half from a normal embryo, it accumulated less sulfated proteoglycans than when it was cultured with a left half from an estrogen‐treated embryo. These results suggest that a soluble inhibitor that is susceptible to estrogen in the left half of the male syrinx inhibits the intrinsic increase in size and chondrogenesis of the right half and plays an important role in the laterally asymmetric development of the male duck syrinx.
Distribution of Retinoids Applied Exogenously to Chick Limb Buds: an Autoradiographic Analysis
Authors: Tamura Koji , Hashimoto Yuichi , Shudo Koichi , Ide Hiroyuki
Published: January 2005

An autoradiographic analysis was undertaken to examine the localization of retinoids applied exogenously to chick limb buds. Ion‐exchange beads (AGI‐X2) containing a tritium‐labeled synthetic retinoid, Am80, were implanted to various regions of chick wing buds. This synthetic retinoid is known to induce a duplicated limb pattern as retinoic acid (RA) does. One to 24 hours after the application, wing buds were fixed, sectioned, and prepared for autoradiography. Heavy labeling was observed in the peripheral region of the wing mesoderm, but no gradient along the antero‐posterior axis was found.

These results suggest that the peripheral region of the limb bud may be important for the morphogenetic function of RA. Tissue‐bound retinoids may not form an antero‐posterior concentration gradient when retinoids are added to the anterior margin of the chick limb bud.
Effect of 5‐Hydroxytryptamine on Steroidogenesis and Oocyte Maturation in Pre‐ovulatory Follicles of the Medaka Oryzias latipes
Authors: Iwamatsu Takashi , Toya Yoshiaki , Sakai Noriyoshi , Terada Yasutaka , Nagata Rieko , Nagahama Yoshitaka
Published: January 2005

The effect of 5‐hydroxytryptamine (5‐HT) on steroidogenesis and oocyte maturation in pre‐ovulatory follicles of the medaka Oryzias lalipes was examined using in vitro culture system. The earliest breakdown of the germinal vesicle of intrafollicular oocytes occurred about 17 hr after the beginning of incubation in the presence of 5‐HT at concentration of 10 ng/ml or more. 5‐HT induced oocyte maturation in a dose‐dependent manner. Cyanoketone inhibited this stimulation. The concentration of 5‐HT required to induce oocyte maturation corresponded to that required to enhance the production (secretion) of estradiol‐17β and 17α,20β‐dihydroxy‐4‐pregnen‐3‐one by pre‐ovulatory follicle cells. At a concentration of 1 μg/ml, the follicle had to be exposed to 5‐HT for at least 4 hr for oocyte maturation accompanied by ovulation to occur. These results indicate that 5‐HT induces in vitro maturation of medaka oocytes by stimulating 17α,20β‐dihydroxy‐4‐pregnen‐3‐one production by pre‐ovulatory follicular cells.
A Tripolar Spindle Formed at Meiosis I Assures the Retention of the Original Ploidy in the Gynogenetic Triploid Crucian Carp, Ginbuna Carassius auratus langsdorfii
Authors: Yamashita Masakane , Jiang Jianqiao , Onozato Hiroshi , Nakanishi Teruyuki , Nagahama Yoshitaka
Published: January 2005

A triploid crucian carp, ginbuna (Carassius auratus langsdorfii), reproduces by gynogenesis, in which sperm of diploid ginbuna or of other species triggers the development of the triploid eggs, but a male genome makes no contribution to the zygotic genome. Gynogenesis is maintained by two mechanisms: exclusion of male genome during fertilization and retention of somatic ploidy levels during oogenesis. We examined the mechanisms responsible for producing unreduced eggs. Microfluorometry with a DNA staining dye showed that DNA content in the ginbuna oocytes was not reduced in half during meiosis I. Cytological observations revealed that a tripolar spindle was formed at meiosis I and the first polar body was not extruded, whereas an ordinary bipolar spindle was formed and the second polar body was extruded at meiosis II. Activity of histone H1 kinase (as an indicator of maturation‐promoting factor) decreased transiently between meiosis I and II, strongly suggesting a “normal” meiotic cycle progression in the ginbuna oocytes. These results have indicated that in the gynogenetic ginbuna the somatic ploidy levels are maintained by inhibiting the first polar body extrusion via the formation of the tripolar spindle at meiosis I.
A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes
Authors: Akiyama Takahiro , Okada Masukichi
Published: January 2005

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee. We named the allele chickadeebin, in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadeewc57 and WF57 (2). In addition, the egg chamber of chickadeebin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.
Prostaglandin Metabolism during Cell Aggregation in the Regenerating Vertebrate Appendage
Authors: Jayadeep Appukuttan , Sailesh Surapureddi , Reddanna Pallu , Menon Venugopal P.
Published: January 2005

Prostaglandin metabolism during cell aggregation period was studied in the regenerating tail of the house lizard. On the basis of scanning electron microscopy it was observed that similar kinds of cells in the blastema aggregate to form promuscie aggregate and procartilage aggregate on the 13th day of tail regeneration. In order to understand the prostaglandin metabolism the following parameters were analysed. Fatty acid composition of phospholipids and free fatty acids analysed by gas chromatography. The activity of two rate limiting enzymes‐phosholipase A and C, and the activity of the enzymes which are responsible for the oxygenation of polyunsaturated fatty acids‐lipoxygenase and cycloxygenase were also estimated. The characterization of the endogenous prostaglandins were carried out by high performance liquid chromatography. On the basis of the above investigations, we observed an increase in phospholipase C activity and resultant increase in free arachidonic acid level. High activity of cycloxygenase and presence of prostaglandin E2 (PGE2) were also observed PGE2 was reported to stimulate cAMP production and resultant cell differentiation. These observations suggest the involvement of prostaglandin metabolism during cell aggregation period in the regenerating blastema and resultant cytodifferentiation of blastemal cells.
Growth and Morphogenesis of Embryonic Mouse Organs on Non‐Coated and Extracellular Matrix‐Coated Biopore Membrane
Authors: Hardman Patricia , Klement Brenda J. , Spooner Brian S.
Published: January 2005

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non‐coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM‐coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.
Localization of Erythropoietin and Erythropoietin‐Receptor in Postimplantation Mouse Embryos
Authors: Yasuda Yoshiko , Nagao Masaya , Okano Masaki , Masuda Seiji , Sasaki Ryuzo , Konishi Hiroyoshi , Tanimura Takashi
Published: January 2005

We used RT‐PCR (reverse transcription‐polymerase chain reaction) and immunocytochemical methods to demonstrate the presence of erythropoietin (Ep) and its receptor (EpR) in postimplantation mouse embryos from the egg‐cylinder to the unturned stage. Expression of mRNA for EpR was detected in total RNA from embryos and decidua in all these stages, but Ep mRNA was confined to embryos in the primitive streak stage and beyond and was not detected in the decidua. Staining of Ep and EpR was seen in all tissues, embryo proper and extra‐embryonic. Moreover, regions of marked staining of Ep and EpR were detected in the extra‐embryonic endoderm, embryonic ectoderm, neural folds and yolk sac, chronologically. No conspicuous differences were present in the staining patterns between Ep and EpR until primitive streak stage; however, after this stage, Ep was predominantly present in the nucleus and EpR on the surface of almost all cells; in the visceral yolk sac endoderm EpR was also detected in adsorption vacuoles and lipid droplets. These studies suggest that Ep first of exogenous and then endogenous origin and EpR of endogenous origin are involved not only in embryogenesis but also in neurogenesis and hematopoiesis in early postimplantation mouse embryos.
Oocyte Maturation and Furrow Formation in an Unfertilized Egg by Fusion with a Fertilized Egg or Blastomeres in the Ascidian, Ascidia sydneiensis divisa
Authors: Sensui Noburu , Ishikawa Masaru , Morisawa Masaaki
Published: January 2005

A technique for fusing an ascidian egg with blastomeres using a chemical fusiogen was established and then used to identify cytoplasmic factors that regulate the process of oocyte maturation in ascidian eggs. Unfertilized eggs fused with fertilized eggs or blastomeres in hypotonic artificial sea water containing 20% polyvinyl alcohol within 10 min. After fusion polar bodies were extruded from the unfertilized portion of the fused eggs. Furrows were formed not only in the fertilized portion but also in the unfertilized portion in the fused eggs. No polar body extrusion and furrow formation occur in either portion of fused unfertilized eggs. These results suggest that fertilized eggs and blastomeres contain a factor that induces oocyte maturation. Polar body extrusion and furrow formation were not suppressed in the fertilized portion of fused eggs, suggesting that unfertilized eggs do not contain a factor that inhibits oocyte maturation.