bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomic DNA fragmentation in red blood cells of the bullfrog during metamorphosis
Authors: Hasebe Takashi , Kawamura Kosuke , Kikuyama Sakae
Published: October 2003

Red blood cells (RBC) of the bullfrog (Rana catesbeiana) contain larval‐type hemoglobin (Hb) during the larval period. At the beginning of metamorphosis, RBC containing adult‐type Hb appear and two types of RBC coexist in the systemic circulation. During the metamorphic climax, RBC with larval‐type Hb disappear from the circulation and, simultaneously, RBC containing adult‐type Hb begin to circulate. These two types of RBC were separated by Percoll density gradient centrifugation to examine the molecular size of the genomic DNA of each population. DNA fragmentation was detected only in new RBC with adult‐type Hb that appeared in the systemic circulation and remained throughout post‐metamorphic life. Semiquantification of DNA on agarose gel showed that the degree of DNA fragmentation was highest at the metamorphic climax. As the existence of DNA fragments suggested endonucleolytic cleavage, nuclease activity was examined by an activity gel system and in vitro circular plasmid DNA digestion assays. The latter revealed that both types of RBC possess endonucleolytic activity throughout the pre‐ and post‐metamorphic periods. Assays of endogenous endonucleolytic activities under different divalent ionic conditions suggested that mobilization of intracellular Ca2+‐Mg2+ induces genomic DNA fragmentation in adult‐type RBC.
A Drosophila receptor‐type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos
Authors: Yoshioka Hidefumi , Ohuchi Hideyo , Ishimaru Yoshiyasu , Ishikawa Tetsuya , Nohno Tsutomu , Saigo Kaoru , Noji Sumihare
Published: October 2003

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila, the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca2+ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca2+ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF‐regulated genes such as Xbra, Xnot, and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF‐regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila.
Sequential gene expression during pronephric tubule formation in vitro in Xenopus ectoderm
Authors: Uochi Takaaki , Asashima Makoto
Published: October 2003

The kidney has been used as a model organ to analyze organogenesis. In in vitro experiments using Xenopus blastula ectoderm, the development of pronephric tubules (the prototype of the kidney) may be induced by treatment with activin A and retinoic acid (RA). The present study examined whether pronephric tubules induced in ectodermal explants exhibited similar characteristics to those of normal embryos at the molecular level. The experimental conditions required for high frequency induction (100%) of pronephric tubule formation from presumptive ectoderm without the development of muscle and notochord were determined. The developmental expression of the pronephros marker genes Xlim‐1 and Xlcaax‐1 was examined in induced pronephric tubules. After treatment with 10 ng/mL activin A and 10−4 mol/L RA, only pronephric tubules were induced at a high frequency. Induced pronephric tubules showed the same timing and patterns of expression for the marker genes Xlim‐1 and Xlcaax‐1 as normal embryos. These results suggest that the in vitro development of pronephric tubules induced in the presumptive ectoderm by activin A and RA parallels normal development at the molecular level.
Localization of cortical granule lectin ligand in Xenopus laevis egg jelly
Authors: Mozingo Nancy M. , Hedrick Jerry L.
Published: October 2003

The block to polyspermy in Xenopus laevis involves an interaction between a cortical granule lectin, released at fertilization, and a ligand located in the egg extracellular matrix. The egg extracellular matrix in X. laevis consists of a vitelline envelope and three distinct jelly layers, designated J1, J2 and J3. To localize cortical granule lectin ligand in the egg extracellular matrix, we used enzyme‐linked lectin assays that showed that cortical granule lectin ligands were absent in J2, J3 and the vitelline envelope. Cortical granule lectin bound to a ligand(s) in J1 in a galactose‐dependent fashion. In addition, we separated egg jelly macromolecules electrophoretically and, in conjunction with western blotting, have shown that J1 contains two major, high molecular weight ligands for cortical granule ligand. Finally, using confocal microscopy, we demonstrated that the ligand(s) for cortical granule lectin occupies a 20–30 μm thick band in a region of J1 just proximal to the vitelline envelope.
Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm
Authors: Omata Setsuko , Katagiri Chiaki
Published: October 2003

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus, and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm2) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome‐reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca2+ and Mg2+. The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate‐oxidation of VC or the pretreatment of VC with Fab fragments of anti‐VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm‐VC binding assays in combination with gel‐filtrated VC components revealed that the fractions containing 36–39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36–39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.
Expression of integrins during axolotl limb regeneration
Authors: Tsonis Panagiotis A. , Doane Kathleen , Del Rio‐Tsonis Katia
Published: October 2003

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that α1 and β1 integrins were down‐regulated as blastema formation proceeded. In contrast, the expression of α3, α6 and αv integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.
Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes
Authors: Iwamatsu Takashi , Yoshizaki Norio , Shibata Yasushi
Published: October 2003

Specific antibodies against the major chorionic glycoproteins (ZI1 ‐2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozo, play an important role in sperm guidance into the micropyle.
Cranial anomaly of homozygous rSey rat is associated with a defect in the migration pathway of midbrain crest cells
Authors: Osumi‐Yamashita Noriko , Kuratani Shigeru , Ninomiya Youichirou , Aoki Kaori , Iseki Sachiko , Chareonvit Suconta , Doi Hirofumi , Fujiwara Michio , Watanabe Tadashi , Eto Kazuhiro
Published: October 2003

Craniofacial development of vertebrates depends largely on neural crest contribution and each subdomain of the crest‐derived ectomesenchyme follows its specific genetic control. The rat small eye (rSey) involves a mutation in the Pax‐6 gene and the external feature of rSey homozygous embryos exhibits craniofacial defects in ocular and frontonasal regions. In order to identify the mechanism of craniofacial development, we examined the cranial morphology and migration of cephalic crest cells in rSey embryos. The chondrocranial defects of homozygous rSey embryos primarily consisted of spheno‐orbital and ethmoidal anomalies. The former defects appeared to be brought about by the lack of the eye. In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pair of cartilaginous rods in place of the nasal capsule. This suggests that the primary cranial defect is restricted to the lateral nasal prominence derivatives. Dil labeling revealed the abnormal migration of crest cells specifically from the anterior midbrain to the lateral nasal prominence in homozygous rSey embryos. Pax‐6 was not expressed in the crest cells but was strongly expressed in the frontonasal ectoderm. To determine whether or not this migratory defect actually resides in environmental cues, normal midbrain crest cells from wild‐type embryos were labeled with Dil and were orthotopically injected into host rSey embryos. Migration of the donor crest cells into the lateral nasal prominence was abnormal in homozygous host embryos, while they migrated normally in wild‐type or heterozygous embryos. Therefore, the cranial defects in rSey homozygous embryos are due to inappropriate substrate for crest cell migration towards the lateral nasal prominence, which consistently explains the cranial morphology of homozygous rSey embryos.
Parthenogenesis in Urechis caupo (Echiura) II. Role of intracellular pH in parthenogenesis induction
Authors: Stephano José Luis , Gould Meredith C.
Published: October 2003

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo. We have shown previously that eggs exposed to P23 for ≥3min complete meiosis but fail to cleave. However, a brief (1.5–2 min) exposure to P23 at pH 8, followed by either acidification of the seawater to pH 7 or dilution of P23 at pH 8 causes germinal vesicle breakdown (GVBD), but eggs fail to complete meiosis and many then later advance to mitosis. In the present study we investigated the hypothesis that partial activation leading to parthenogenesis occurs when there is a partial intracellular alkalinization. Measurements with the fluorescent pH indicator bis(carboxyethyl)‐carboxyfluorescein (BCECF) showed that P23 induces a pH, increase similar to that occurring during fertilization and that parthenogenesis‐inducing treatments interupt this rise in pH1 In eggs exposed to P23 for >3 min the pH1 increase was 0.31–0.49 units, slightly higher than in fertilized eggs. In partially activated eggs exposed to P23 for 1.5–2 min at pH 8, pH1 began to rise but then returned to control values or remained only partially elevated (< 0.2 pH units average increase). Electrophysiological measurements revealed that removal of P23 during the first few minutes of exposure caused the activation potential to terminate and experiments with [14C]‐P23 confirmed that dilution results in a rapid unbinding of P23 from eggs. If proton export is driven by membrane potential as well as the pH gradient, these results explain why dilution of P23 at pH 8 also interrupts the pHi increase.
Test cell migration and tunic formation during posthatching development of the larva of the ascidian, Ciona intestinalis
Authors: Sato Yoko , Terakado Kiyoshi , Morisawa Masaaki
Published: October 2003

Morphological changes in the tunic layers and migration of the test cells during swimming period in the larva of the ascidian, Ciona intestinalis, were observed by light and electron microscopy. The swimming period was divided into three stages. In stage 1, further formation of juvenile tunic layer started only in the larval trunk and neck region. In stage 2, the layer became swollen in the ventral and dorsal sides of the neck region and in stage 3, the swelling expanded backward. Concomitantly with these changes, the outermost larval tunic layer (outer cuticular layer), which had been formed before hatching, also swelled in the neck region in stage 2 and formed two humps in stage 3, although the layer did not change in the tail region during the swimming period. Test cells that were present over the entire larval tunic layer in stage 1 began to move from the surface of the fin toward that of the side of the body in stage 2, and finally gathered to form six bands running radially from the anterior end to the posterior end of the trunk region and aligned along the lateral sides of body in the tail region in stage 3. In electron microscopic observations, pseudopodia protruding from the test cells invaded the larval tunic, following which they extended proximate to the juvenile tunic in the trunk region. In the tail region, which had no juvenile tunic layer as that described, the pseudopodia invaded and remained adjacent to the surface of the epidermis or the sensory cilia protruded from the epidermis. Metamorphosis of the larvae, further tunic formation, degradation of adhesive papilla, attachment of larva to the substratum and tail resorption commenced after these morphological changes occurred. The possible role of the test cells in metamorphosis is discussed.
A gene trap approach to identify genes that control development
Authors: Takeuchi Takashi
Published: October 2003

One methodology called gene trap represents a versatile strategy by which murine genes that control developmental events can be captured and identified with corresponding mutants produced at the same time. Gene trap methodology has been developed and several genes and their mutants have been analyzed, but almost all of the genes reported are those already known or murine homologs of other species. In this study, the efficiency of the gene trap methodology was improved and a novel mutant mouse strain named jumonji established which displayed an intriguing defect. Homozygous fetal mice died in utero and a significant proportion of the homozygotes showed abnormal groove formation on the neural plate and a defect in neural tube closure with a mixed genetic background of 129/Ola and BALB/c. The trapped gene believed to be responsible for these phenotypes encodes a novel nuclear protein. The results reveal that the gene trap approach can identify unknown interesting genes in murine development. The gene trap strategy, however, has several problems, the greatest of which is the difficulty in prescreening embryonic stem (ES) cells for interesting trapped genes. Recent studies are solving this problem and show that the prescreening of ES cells for genes with several characteristics is possible.
Requirement of fibroblast growth factor signaling for regeneration of epiphyseal morphology in rabbit full‐thickness defects of articular cartilage
Authors: Otsuka Yutaka , Mizuta Hiroshi , Takagi Katsumasa , Iyama Ken‐ichi , Yoshitake Yoshino , Nishikawa Katsuzo , Suzuki Fujio , Hiraki Yuji
Published: October 2003

The involvement of fibroblast growth factor‐2 (FGF‐2) during the repair process in rabbit full‐thickness defects of articular cartilage was studied. Fibroblast growth factor‐2 (50 pg/h) was administered for 2 weeks in a 5mm defect of articular cartilage, which is large enough not to repair spontaneously. The administration of FGF‐2 resulted in the regeneration of the articular cartilage and the subchondral bone within 8 weeks. In these defects, undifferentiated mesenchymal cells initiated chondrogenic differentiation coupled with replacement by subchondral bone, resulting in the resurfacing of the defects with hyaline cartilage and the recovery of subchondral bone up to the original bone–articular cartilage junction. In rabbits, full‐thickness defects are capable of regenerating articular cartilage as long as the defect size is limited to ≤3 mm in diameter. In the defects, strong immunoreactivity for FGF‐2 was observed in the granulation tissue filling the defects in the early stage of repair, in association with the expression of FGF‐2 mRNA shown by in situ hybridization. Once the undifferentiated mesenchymal cells had differentiated into chondrocytes, both the immunoreactivity and the in situ hybridization signal declined significantly. Upon the local administration of a monoclonal antibody against FGF‐2 (bFM‐1, 50ng/h), the defects were filled with fibrous tissue and no resurfacing hyaline cartilage was formed. Compared to the non‐treated defects, there were marked increases in FGF‐2 immunoreactivity and the overexpression of FGF‐2 mRNA in the reparative tissue in the bFM‐1 ‐treated defects. This rebound phenomenon indicates that the autocrine FGF‐2 signaling is critically important for the regeneration of articular cartilage.
Histological distribution of FR‐1, a cyclic RGDS‐peptide, binding sites during early embryogenesis, and isolation and initial characterization of FR‐1 receptor in the sand dollar embryo
Authors: Katow Hideki , Yamamoto Yasuo , Sofuku Shosuke
Published: October 2003

A fibronectin‐related synthetic cyclic H‐Cys‐Arg‐Gly‐Asp‐Ser‐Pro‐Ala‐Ser‐Ser‐Cys‐OH (RGDSPASS) peptide (FR‐1) binding site in the embryo of the sand dollar Clypeaster japonicus was specified using dansyl‐labeled FR‐1 (Dns‐FR‐1) and horseradish peroxidase‐labeled FR‐1, and an FR‐1 receptor was isolated using FR‐1‐affinity column chromatography. The FR‐1 introduced to the blastocoel of blastulae inhibited primary mesenchyme cell (PMC) migration in mesenchyme blastulae, and complete gastrulation and spicule differentiation in gastrulae. The Dns‐FR‐1 bound to the entire basal side of the ectoderm in mesenchyme blastulae, and then restricted to the basal side of the ectoderm at the apical tuft region and the vegetal hemisphere in early gastrulae. The cytoplasm of the archenteron also bound to Dns‐FR‐1. In PMC, Dns‐FR‐1 bound to the nucleus and cytoplasmic reticular features. In unfertilized eggs, Dns‐FR‐1 bound to the entire cytoplasm, particularly to the oval‐shaped granules and the nuclear envelope, but only to the cytoplasm after fertilization. Relative molecular mass (Mr) of the FR‐1 ‐binding protein was 240 kDa under non‐reducing conditions and 57 kDa under reducing conditions. The FR‐1 receptor protein bound anti‐sea urchin integrin (Spl) βL subunit antibodies raised against the embryos of Strongylocentrotus purpuratus. Immunohistochemistry showed that the antibody binding site was similar to the histochemical distribution of Dns‐FR‐1. However, Mr of the FR‐1 receptor is distinctively larger than that of the Spl βL subunit.
A mutation in the cAMP signaling pathway affects sexual development of Dictyostelium discoideum
Authors: Shimizu Hajime , Morio Takahiro , Shimizu Hideki D. , Urushihara Hideko
Published: October 2003

Amoebae of cellular slime molds have two developmental modes, asexual fruiting body formation and sexual macrocyst formation. How developmental choice is made is an interesting subject of wide importance. Light exposure and dry conditions are favorable for asexual development, while conditions of darkness and high humidity are so for sexual development. In Dictyostelium discoideum, the latter conditions enhance zygote formation, which determines the fate of surrounding cells for sexual development. Here, a mutant (TMC1) defective in the post‐fusion aggregation of cells during sexual development is described. This mutant is also aggregationless in asexual development, and the level of cyclic adenosine monophosphate (cAMP) receptor is reduced. Correspondingly, a series of existing mutants with defects in cAMP signaling pathways showed the same sexual phenotype as TMC1. These results suggest that molecular mechanisms of development are shared by the two alternative developmental modes.
Decreased in vitro fertilization efficiencies in the presence of specific cyritestin peptides
Authors: Linder Britta , Heinlein Uwe A. O.
Published: October 2003

Synthetic peptides corresponding to specific regions of the mouse acrosomal transmembrane protein cyritestin have been used as competitors in in vitro fertilization experiments. One peptide representing a putative egg‐receptor binding site within the disintegrin domain of cyritestin lowered the fertilization rate to 30% of the normal value.
A 900 bp genomic region from the mouse dystrophin promoter directs lacZ reporter expression only to the right heart of transgenic mice
Authors: Kimura Shigemi , Abe Kuniya , Suzuki Misao , Ogawa Masakatsu , Yoshioka Kowashi , Kaname Tadasi , Miike Teruhisa , Yamamura Ken‐ichi
Published: October 2003

In order to study the regulatory mechanism of developmental and tissue‐specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZgene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum, and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.
An immunocytochemical analysis of the expression of thyroid hormone receptor α and β proteins during natural and thyroid hormone‐induced metamorphosis in Xenopus
Authors: Fairclough Lynne , Tata Jamshed R.
Published: October 2003

Amphibian metamorphosis is characterized by the upregulation of thyroid hormone receptor (TR) mRNA in all tissues of tadpole during both the natural and thyroid hormone (TH)‐induced development. The two TR genes, termed α and β, are members of a large multigene family of nuclear receptors related to the cellular homolog of the oncogene c‐erbA. The phenomenon of upregulation is more marked for the β than the α isoform. To determine whether or not the auto‐induction of the transcripts is paralleled by that of TR proteins, non‐cross‐reacting monoclonal antibodies were prepared against Xenopus laevis TRα and β (xTRα, β) in order to analyze immunocytochemically their expression and localization. Three tadpole tissues that exemplify three major consequences of gene re‐programing during natural and TH‐induced metamorphosis were studied: (i) Liver that undergoes extensive functional switching; (ii) small intestinal epithelium that exhibits substantial cell death prior to major structural and biochemical modifications; and (iii) hind limb‐bud as an example of de novo morphogenesis. It was shown that xTRα protein is generally more abundant in these tissues, and its expression is developmentally and hormonally less regulated, than is xTRβ. The auto‐induction of xTRβ was particularly intense at 5 days after administration of triiodo‐thyronine (T3) to both pre‐metamorphic (stage 52) tadpoles and at the onset of natural metamorphosis (stage 55). In the developing hind limb‐bud at both stages the upregulation of TRβ is topologically restricted, being particularly intense in dense pockets of cells, presumably rich in chondrocytes. It was concluded that the distribution and expression of xTRα and β proteins match partially, but not fully, those of their transcripts during natural and hormone‐induced metamorphosis.
Spfkh1 encodes a transcription factor implicated in gut formation during sea urchin development
Authors: Luke Natalie H. , Killian Christopher E. , Livingston Brian T.
Published: October 2003

A member of the forkhead class of transcription factors from sea urchins (Spfkh1) that is expressed specifically in the endoderm of developing embryos has been identified. Spfkh1 was expressed transiently in the embryo, with peak levels of messenger ribonucleic acid (mRNA) accumulating at the time endoderm invaginated into the interior of the embryo. Expression was limited to the invaginating endoderm in the early gastrula, then became further restricted to the base of the invaginating gut at the mid‐gastrula stage. Expression diminished by the end of gastrulation. This expression pattern indicates that Spfkh1 mRNA accumulates in endodermal cells as they invaginate, but disappears rapidly in endodermal cells that undergo convergent extension. Treatment of embryos during cleavage stages with lithium or phorbol esters caused an increase in Spfkh1 mRNA accumulation and expanded the domain of expression of Spfkh1, suggesting that signaling through the inos‐itol‐tris‐phosphate protein kinase C (IP3‐PKC) signaling pathway is upstream of Spfkh1 expression. The expression pattern of Spfkh1 suggests that it is centrally involved in specification and/or differentiation of the gut. Disruption of the extracellular matrix (ECM) prevents formation of the gut, but does not inhibit initiation of Spfkh1 expression. Embryos arrested prior to gastrulation continued to express Spfkh1 well past the time it was down‐regulated in normal embryos, suggesting the ECM or cell movement is required for the decrease in Spfkh1 mRNA during gastrulation.
Quantitative and spatial information on the composition of chimaeric fetal mouse eyes from single histological sections
Authors: West John D. , Hodson Benjamin A. , Keighren Margaret A.
Published: October 2003

The spatial distribution of cells in chimaeric tissues, composed of two genotypes, provides insights into the extent of cell mixing during development and growth. However, direct measurement of patch sizes is not usually meaningful because, when the proportion of one genotype is high, a single patch may encompass several adjacent coherent clones of like genotype (clone aggregation). Two previously used methods of comparing patch lengths were evaluated to overcome this problem. The corrected mean patch length (corrected for the predicted effects of random clone aggregation) is a more useful summary statistic than the median patch length of the minor genotype, because its use is not restricted to grossly unbalanced chimaeras, but its validity has been questioned. The two methods gave almost identical numerical summaries of patch sizes in the retinal pigment epithelium of fetal chimaeras, thereby validating the use of the corrected mean patch length for this tissue. The present study also showed that the corrected patch length was unaffected by the presence of cells hemizygous for the TgN(Hbb‐b1)83Clo transgene and that the proportion of pigmented cells in a single histological section was representative of the overall composition of the chimaeric fetus.
Oral/aboral ectoderm differentiation of the sea urchin embryo depends on a planar or secretory signal from the vegetal hemisphere
Authors: Yoshikawa Shun‐Ichi
Published: October 2003

A monoclonal antibody that recognizes oral ectoderm and esophagus of sea urchin larvae was newly produced. Distribution of the antigen, named Hpoe, was examined by indirect immunofluorescence microscopy. Hpoe did not exist in eggs and appeared during the cleavage stage. In hatched blastulae, Hpoe was detected on the apical surface of all cells. As embryogenesis progressed, Hpoe disappeared from the primary mesenchyme, archenteron and aboral ectoderm. Hpoe reappeared in foregut at the prism stage and was restricted to the oral ectoderm and esophagus at the pluteus stage. Using this antigen as a molecular marker of oral/aboral ectoderm differentiation, the role of the vegetal hemisphere in ectoderm differentiation was examined. All animal hemispheres isolated from 16‐cell stage embryos, mesenchyme blastulae, early gastrulae and mid gastrulae developed into epithelial balls and every cell expressed Hpoe. These epithelial balls failed in oral/aboral ectoderm differentiation. Twenty millimolar LiCI‐treated whole embryos developed into exo‐gastrulae but Hpoe restriction in ectoderm occurred in these exo‐gastrulae. These results show that oral/aboral ectoderm differentiation requires an inductive interaction from the vegetal hemisphere and indicate that the inductive interaction depends on a planar or secretory signal, rather than the contact of the esophagus and ectoderm.
A soluble extract from human spermatozoa activates ascidian oocytes
Authors: Wilding Martin , Kyozuka Keiichiro , Russo Gian Luigi , Tosti Elisabetta , Dale Brian
Published: October 2003

A soluble extract from human spermatozoa induced calcium oscillations and extrusion of the first polar body when injected into oocytes of the ascidian Ciona intestinalis. The properties of calcium oscillations and time of polar body extrusion precisely mimic oocyte activation induced by C. intestinalis sperm or sperm extracts. The data suggest that human sperm extracts can activate oocytes of different phyla by the same mechanism as homologous spermatozoa. Injection of inositol 1,4,5‐trisphosphate (IP3) into C. intestinalis oocytes mimicked to some extent the initial stages of oocyte activation, but the results demonstrate that ascidian oocyte activation by human sperm extract cannot be explained solely in terms of IP3‐induced calcium release. Injection of other calcium releasing second messengers, cyclic adenosine diphosphate ribose, or calcium ions, does not lead to oocyte activation or release intracellular calcium in ascidian oocytes. It was concluded that human spermatozoa contain one or more molecules that can trigger intracellular calcium release in oocytes from different phyla.
Oral—aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore
Authors: Akasaka Koji , Uemoto Hiroko , Wilt Fred , Mitsunaga‐Nakatsubo Keiko , Shimada Hiraku
Published: October 2003

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue‐specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral–aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral–aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oralaboral axis during sea urchin development.
A major glycoprotein of Xenopus egg vitelline envelope, gp41, is a frog homolog of mammalian ZP3
Authors: Kubo Hideo , Kawano Takehiro , Tsubuki Satoshi , Kawashima Seiichi , Katagiri Chiaki , Suzuki Akemi
Published: October 2003

A predominant glycoprotein in the vitelline envelope (VE) of the anuran Xenopus laevis is gp41, known to be proteolytically converted from gp43 of the coelomic egg envelope concomitant with the acquisition of egg fertilizability. To characterize the protein core of gp41, purified gp41 from VE was digested with lysyl endopeptidase, and peptides isolated from the digests were sequenced for amino acids to design degenerate primers for polymerase chain reaction. By reverse transcription‐polymerase chain reaction with a poly(A)+ RNA from the ovary of an ovulated female Xenopus, a specifically amplified band was obtained and sequenced. The upstream and downstream sequences of the sequenced region were completed by 5′‐ and 3′‐rapid amplification of cDNA ends, respectively. The cDNA, referred to as gp43 cDNA, comprises 1423 base pairs and contains one open reading frame with a sequence for 460 amino acids. The predicted amino acid sequence of gp43 cDNA has a close similarity with that of mammalian ZP3. Northern blot and in situ hybridization studies indicated that gp43 mRNA is expressed in oocytes, particularly in the previtellogenic oocytes. A comparison of the N‐terminal sequences of gp41 and gp43 strongly suggested that gp41 is generated at least by processing of the N‐terminal portion of gp43 with oviductin.
N‐cadherin is crucial for heart formation in the chick embryo
Authors: Nakagawa Shinichi , Takeichi Masatoshi
Published: October 2003

The developing heart primordium strongly expresses N‐cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured at stages 5–12, and injected with anti‐N‐cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody‐treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N‐cadherin‐negative but also N‐cadherin‐positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti‐N‐cadherin antibodies. These results suggest that N‐cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N‐cadherin activity.
cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43
Authors: Yang Joy C. , Hedrick Jerry L.
Published: October 2003

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N‐linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin‐like (convertase) site and a C‐terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure‐fertilization function predictions for this phylogenetically conserved family of glycoproteins.
A cis‐regulatory element within the 5′ flanking region of arylsulfatase gene of sea urchin, Hemicentrotus pulcherrimus
Authors: Morokuma Junji , Akasaka Koji , Mitsunaga‐Nakatsubo Keiko , Shimada Hiraku
Published: October 2003

The 5′ flanking region of the sea urchin Hemicentrotus pulcherrimus arylsulfatase (Ars) gene was scanned to define cis‐regulatory elements required for proper expression congruent to that of the endogenous gene. The region between −100bp and +38 bp from the transcription start site contains minimum information required for temporal initiation of transcription of the Ars gene. Progressive deletion analysis of Ars‐luciferase reporter constructs containing the Ars sequence from −3484 bp to +38bp suggests the existence of several cis‐regulatory elements within this region. Results from luciferase assays of internal deletion mutants show strong enhancer activity detected within the sequence from −194 bp to −144 bp. By gel mobility shift assay, we have identified a nuclear factor that interacts sequence‐specifically with this 50 bp region, and appears in a developmental stage‐specific manner. Further deletion analysis determined that the enhancer activity lies within a 22 bp sequence between −186 bp and −164 bp.
Murine forebrain and midbrain crest cells generate different characteristic derivatives in vitro
Authors: Chareonvit Suconta , Osumi‐Yamashita Noriko , Ikeda Masa‐aki , Eto Kazuhiro
Published: October 2003

Neural crest (NC) is a transient structure that gives rise to various types of tissues. Many NC cells are pluripotent in the sense that their progeny can generate more than one derivative. However, the potentiality to differentiate into certain derivatives, such as cartilage and bone, seems to be specified with respect to the neuraxial levels at which the NC generates. In order to compare the differentiation potentiality of different regions of head NC, the derivatives of forebrain and midbrain mouse NC have been investigated in vitro using explant cultures of neuroepithelial fragments. From morphology and expression of specific markers, the midbrain crest cultures obviously generated earlier and were greater in number of neuronal cells than were the forebrain ones. Moreover, collagen type II positive cells were detected in the midbrain but not in the forebrain crest cultures. Finally, pigment cells were only observed in the forebrain cultures. The results suggest that the forebrain and midbrain crest cells have a different potentiality to differentiate.
Adrenergic neurotransmitters and calcium ionophore‐induced situs inversus viscerum in Xenopus laevis embryos
Authors: Toyoizumi Ryuji , Kobayashi Takuhiro , Kikukawa Akihiro , Oba Junji , Takeuchi Shigeo
Published: October 2003

Xenopus laevis embryos at the blastula–early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left–right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine‐induced situs inversus was blocked by the α‐1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late‐neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut‐only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.
Double‐segment defining role of even‐skipped homologs along the evolution of insect pattern formation
Authors: Xu Xin , Xu Pin‐Xian , Amanai Kazuhito , Suzuki Yoshiaki
Published: October 2003

Recent studies on insect patterning suggest that the genetic hierarchy may be roughly conserved in phylogenetically divergent species, but pair‐rule genes may not function identically in all insects. In order to understand potential evolutionary changes in the role of the pair‐rule genes, a Bombyx even‐skipped homolog was cloned and its expression pattern during early embryogenesis studied. Eight stripes of Bombyx even‐skipped were progressively expressed in an antero–posterior order. Later, these stripes disappeared anteriorly. Under this detection system, Bombyx even‐skipped stripes clearly do not resolve into the corre sponding secondary stripes, an obvious difference from Drosophila and Tribolium. These results suggest that Bombyx even‐skipped may serve a double‐segment defining role and may determine the odd‐numbered engrailed stripes.
Separation of microvilli from Tubifex eggs upon activation: Its inhibition by concanavalin A hinders ooplasmic segregation and cleavage
Authors: Shimizu Takashi
Published: October 2003

The surface of mature eggs of the freshwater oligochaete Tubifex exhibits numerous microvilli. Upon activation, microvilli become narrower at their base and separated from the ooplasmic surface. Here it is shown that concanavalin A (Con A) reversibly inhibits the separation of microvilli from activated Tubifex eggs. The Con A‐treated eggs undergo meioses and mitoses at a normal rate. Microvilli on these eggs change their length in a meiotic cycle‐dependent manner; their core bundles of microfilaments elongate significantly during the second meiosis. The Con A‐treated eggs fail to complete polar body formation, ooplasmic segregation and cleavages. Treatment with Con A of eggs that have accomplished microvillar separation does not exert any inhibitory effect on their development. Succinyl‐Con A, a dimeric derivative of Con A, does not prevent microvillar separation, suggesting that the tetravalent form of Con A is essential for Con A to exert its inhibitory effect on microvillar separation.
The Na+, K+‐ATPase α subunit requires gastrulation in the Xenopus embryo
Authors: Uochi Takaaki , Takahashi Shuji , Ninomiya Hiromasa , Fukui Akimasa , Asashima Makoto
Published: October 2003

Na+, K+‐ATPase participates in reabsorption of ions and water and produces an electrochemical gradient between the intra‐ and extracellular spaces across the cell membrane. It also plays an important role in many developmental phenomena such as a blastocoele formation and neural formation. To elucidate the expression pattern of Na+, K+‐ATPase in the Xenopus embryo, the spatial expression patterns of the Na+, K+‐ATPase α subunit were studied in a normal embryo by whole‐mount in situ hybridization. These transcripts were localized around the dorsal blastopore at the gastrula stage, in the neural tube at the neurula stage, and then in the pronephros and cloaca at the tail‐bud stage. To study the function of Na+, K+‐ATPase in embryogenesis after mid‐blastula transition, the expression of the Na+, K+‐ATPase α subunit was inhibited by the injection of specific antisense RNA. Embryos injected with Na+, K+‐ATPase antisense RNA showed inhibition of gastrulation. When antisense RNA was injected into the dorsal blastomeres, head differentiation was markedly inhibited. These results suggest that this transcript plays an important role during gastrulation and head differentiation.
Formation of the branching pattern of blood vessels in the wall of the avian yolk sac studied by a computer simulation
Authors: Honda Hisao , Yoshizato Katsutoshi
Published: October 2003

The present study was performed to provide data to support the notion previously believed but not proved experimentally or theoretically, that blood vessels are formed by the selection of capillaries in the network. In an attempt to understand the mechanism of formation of blood vessel branching structures, the transformation of a capillary network to a branching system in the wall of quail yolk sac was successively recorded by a series of photographs, and a computer simulation was carried out for the process of in vivo vascularization based on the photographs. The simulation demonstrated that a positive feedback system participated in the formation of a branching structure. That is, vessels which had been much used were enlarged, whereas less used vessels were reduced in their size and finally extinguished. The enlarged vessels became major components of the branching system. As the body of an embryo grew, it was observed that polygonal capillary networks enlarged, which led each polygon of the network to divide into a few finer polygons. Then, some of the capillary vessels were again selected and formed a branching system. This process repeated during the body growth, indicating that the vascular system developed adaptively to the body growth. A region where the growth was fast, received much blood flow and produced finer networks of capillaries. Thus, it was experimentally demonstrated for the first time that capillaries in the network are successively selected by a positive feedback mechanism and form blood vessels.
gp49B1, an inhibitory signaling receptor gene of hematopoietic cells, is induced by leukemia inhibitory factor in the uterine endometrium just before implantation
Authors: Matsumoto Yukie , Handa Shizuo , Taki Takao
Published: October 2003

Leukemia inhibitory factor (LIF) is a cytokine involved in hematopoiesis, neuropoiesis, and embryogenesis. Transcriptional activation of various genes occurs subsequent to LIF signal transduction in its target cells. Using the mRNA differential display method, a LIF‐inducible gene was isolated from LIF‐stimulated M1 murine myeloid leukemia cells. By DNA sequencing, this gene turned out to be gp49B1, which has been reported as an inhibitory signaling receptor to attenuate mast cell activation. Because gp49B1 expression was limited to the uterus of a pregnant mouse, its uterine expression was examined especially in relation to LIF expression during pregnancy. gp49B1 was expressed specifically on day 4.0 of pregnancy, as was LIF, and the site of the most abundant expression of LIF and gp49B1 mRNA was the luminal epithelium of the uterine endometrium. These findings suggest that the gp49B1 expression in the uterine endometrium is induced just before implantation by paracrine and/or autocrine effects of LIF. Considering its function as an inhibitory signaling receptor on mast cells, a possible role for gp49B1 on the surface of the uterine endometrium as an immunoreceptor that allows blastocyst attachment is proposed.