bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronological changes in the sucrase antigen‐inducing activity and development of the regional identity in the intestinal mesoderm of the chick embryo
Authors: Matsushita Susumu
Published: October 2003

Developmental changes in mesodermal activity to induce intestine‐like differentiation expressing sucrase antigen in the endoderm and changes in endodermal reactivity to such an activity in the digestive tract of the chick embryo were analyzed. Digestive‐tract endoderms of embryos at 3 days of incubation were highly responsive to the inductive effect of the 5 day duodenal mesenchyme, with the stomach endoderm lying nearest to the intestine having the highest reactivity. Endodermal reactivity decreased with increasing age. It was almost absent in the endoderm of the esophagus or proventriculus of 6 day embryos and in the endoderm of the gizzard of 7 day embryos. The activity of the mesoderm to induce intestine‐like differentiation in 5 day gizzard endoderm was high in the 5–10 day duodenal mesenchyme, but was rarely found in 14 day duodenal mesenchyme. This activity was specific to intestinal mesenchymes, among which the duodenal mesenchyme had the highest activity in 5 day embryos. The 3 day intestinal mesenchyme may already have the inductive activity. The presumptive intestinal mesoderm of 1.5 day embryos seemed to have a slight or no activity, but it may have intestinal identity and may manifest a high inductive activity later.
Effects of 5‐azacytidine on the development of parthenogenetic mouse embryos
Authors: Penkov Leonid I. , Platonov Evgeni S. , Mironova Oksana V. , Konyukhov Boris V.
Published: October 2003

This study describes the effects of 5‐azacytidine (5‐azaC) on the development of diploid parthenogenetic embryos (PE) of CBA, C57BL/6 and (CBA × C57BL/6)F1 mice in vitro at the 1‐cell or the blastocyst stage or in vivo after implantation. Our findings indicate that genomic imprinting is modulated by genetic background. Non‐fertilized C57BL/6 eggs form diploid parthenogenetic blastocysts at a much higher frequency than CBA eggs. Eggs from F1 hybrid females form parthenogenetic blastocysts at an approximately intermediate level between these inbred strains of mice. C57BL/6 PE do not develop to the somite stages. In contrast, CBA PE and F1 PE develop to various somite stages. Following administration of 5–azaC at 1.0 μmol/L in vitro at the 1‐ ‐cell stage, the number of implantations of C57BL/6 PE transferred to pseudopregnant females increased. In contrast, the number of implantations and somite F1 PE did not significantly change following exposure to 5–azaC. However, administration of 5‐azaC at the 1‐cell stage stimulates development of somite F1 PE. Administration of 5‐azaC at 0.2 and 1.0 μmol/L in vitro at the blastocyst stage did not change the number of implantations of C57BL/6 PE. However, the number of implantations and somite CBA PE decreased. After injection of 5azaC at 0.24mg/kg in vivo at day 8 of gestation, some F1 PE developed to 26–35 somites compared with a maximum of 25 somites in controls. The different effects of 5‐azaC on the development of PE depend upon the mouse strain used and the stage of development.
Targeted disruption of genes for gp138, a cell‐fusion‐related protein in Dictyostelium discoideum, revealed the existence of a third gene
Authors: Yamaguchi Nobuyuki , Higa Mikihiko , Aiba Kazuhiro , Fang Hui , Tanaka Yoshimasa , Urushihara Hideko
Published: October 2003

The sexual cycle of the cellular slime mold, Dictyostelium discoideum, offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP138A and GP138B, have been identified so far as encoding this protein. Expression of antisense RNA for GP138B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP138A and GP138B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock‐out mutants suggested the presence of a third gene for gp138.
Does phosphatidylinositol 3‐kinase play a role in insulin‐induced outgrowth of pseudopodial cables in cultured cells derived from micromeres of sea urchin embryos?
Authors: Kuno Shin‐ichi , Yasumasu Ikuo
Published: October 2003

Cultured cells derived from micromeres isolated from sea urchin embryos at the 16‐cell stage, which have insulin receptors, undergo pseudopodial cable growth and spicule rod formation in culture with horse serum and only cable growth in culture with insulin. Genistein, an inhibitor of protein tyrosine kinase, and wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PI3kinase), inhibited pseudopodial cable growth in micromere‐derived cells cultured with insulin and also growth accompanied by spicule rod formation in horse serum‐treated cells. The PI3kinase activity in the immunoprecipitates obtained by anti‐phosphotyrosine antibody from the cells cultured with insulin was higher than that in cells cultured without insulin or with insulin and genistein. Following immunoblotting with antibody of SH‐2, Src homology 2 domains in PI3kinase regulatory subunit, a band appeared at 85 kDa in SDS‐PAGE of the immunoprecipitate, obtained from the micromere‐derived cells by anti‐phosphotyrosine antibody. This SDS‐PAGE also showed protein bands at molecular weights similar to IRS‐1 and the insulin receptor β subunit. These indicate that the insulin signal transduction pathway in micromere‐derived cells is somewhat similar to the pathway, in which PI3kinase is involved, in mammalian cells.
A combination of Buffalo rat liver cell‐conditioned medium, forskolin and membrane‐bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo
Authors: Kawase Eihachiro , Shirayoshi Yasuaki , Hashimoto Koichiro , Nakatsuji Norio
Published: October 2003

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro. Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo, suggesting the presenge of other growth factor(s) in vivo. We previously reported that tumor necrosis factor‐α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4‐m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo. Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo; however, it did stop afterwards. Such proliferating PGC continue to express c‐kit and Oct‐3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES‐like EG cells.
Developmental regulation and primary structure of murine cellular nucleic acid‐binding protein, a zinc finger‐containing protein whose general structure is present in evolutionarily diverse eukaryotes
Authors: Kingsley Paul D. , Zinkin Adam S. , Silver Louise S. , Pendola Frank L. , Palis James
Published: October 2003

Murine cellular nucleic acid binding protein (CNBP) cDNA was cloned during a differential screen of messages expressed in early postimplantation extraembryonic tissues. Sequence analysis indicates that CNBP belongs to a family of proteins that contains retroviral‐type zinc fingers (CCHC) and glycine‐rich domains and whose general structure is similar inLeishmania, the yeastSchizosaccharomyces pombeand in mammals. Transcripts representing four different CNBP proteins were detected during murine embryogenesis. Spatial analysis indicated that CNBP messages accumulated in a regulated pattern during postimplantation development. Most tissues expressed the CNBP message at E9.5 of embryonic development, with the exception of neural epithelia, developing gut and otic placode. By E12.5, the spatial pattern had become more restricted, but now included ependymal and mantal layers of the developing nervous system and differentiating lamina propria of the intestine. By E14.5, CNBP levels had decreased throughout all regions of the embryo. While the overall structure of CNBP‐like proteins is conserved in eukaryotes, the CNBP expression pattern is not consistent with a simple housekeeping role.
The ascidian embryo: An experimental system for studying genetic circuitry for embryonic cell specification and morphogenesis
Authors: Satoh Noriyuki , Makabe Kazuhiro W. , Katsuyama You , Wada Shuichi , Saiga Hidetoshi
Published: October 2003

Ascidians have served as an appropriate experimental system in developmental biology for more than a century. The fertilized egg develops quickly into a tadpole larva, which consists of a small number of tissues including the epidermis, central nervous system with two sensory organs, nerve cord, endoderm, mesenchyme, notochord and muscle. Lineage of these embryonic cells is completely described up to the gastrula stage. These features of the ascidian embryo provide an opportunity to study the mechanisms underlying the differentiation of tissues in development. To understand the molecular basis of ascidian embryogenesis, cloning of various genes has been performed, including those that exhibit a lineage‐associated expression pattern and those encoding transcription factors, which are expected to be involved in differentiation of tissues, lineage specification, axis formation and regionalization in developmental fields. Here, we present recent advances in the isolation and characterization of these genes. We emphasize the advantages of the ascidian embryo as an experimental system to study genetic circuitries that are required for cellular differentiation and morphogenesis.
Involvement of c‐Fos proto‐oncogene during palatal fusion and interdigital space formation in the rat
Authors: Yano Hiroki , Ohtsuru Akira , Ito Masahiro , Fujii Tohru , Yamashita Shunichi
Published: October 2003

c‐Fos is an indispensable proto‐oncogene product in the developmental process and a key factor in the proliferation of normal and neoplastic cells. It is also implicated in triggering epithelial–fibroblastoid cell conversion and the induction of apoptosis. To clarify the role of c‐Fos in the life span of rat embryonic cells, we examined the disappearance of the medial edge epithelium (MEE) of the palatal shelf on palatal fusion and formation of the interdigital web. Using immunohistochemical techniques with anti‐c‐Fos antibody and a TdT‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) method, we compared the pattern of c‐Fos‐positive cells and DNA fragmentation. To investigate the epithelial–mesenchymal transformation, transforming growth factor (TGF)‐β3 was examined in both regions. During palatal fusion, c‐Fos was detected in the nuclei of MEE cells just before the elevation of the palatal shelf and strongly stained at the MEE remaining in the center of the palate. c‐Fos became undetectable in accordance with the disruption of the medial edge epithelium. We also immunohistochemically recognized the colocalization of c‐Fos and TGF‐β3 in the MEE. However, DNA fragmentation was not observed at the center of fusion. Considered together, cell disappearance at the fusion site was suggested to reflect epithelial–mesenchymal transformation. In contrast, mesenchymal cells of the interdigital web and the chondrocytes of the digit expressing c‐Fos appeared to be the hallmark of programmed cell death and TGF‐β3 could not be found in the interdigital mesenchyme. c‐Fos in the interdigital space was detected more proximal than DNA fragmentation detection, suggesting that c‐Fos acted at the upper stream of apoptosis. Our results support the involvement of c‐Fos in the physiological process of cell transformation during palatogenesis and apoptosis during the interdigital formation. c‐Fos may trigger a cell specific signal during organogenesis, especially transformation of epithelial cells and apoptosis of mesenchymal cells.
Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light‐induced release of CO‐mediated inhibition
Authors: Yasumasu Ikuo , Tazawa Eigoro , Asami Kouichi , Fujiwara Akiko
Published: October 2003

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl‐p‐phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO‐mediated inhibition. The action spectra for photoreactivation of CO‐inhibited cytochrome c oxidase in isolated mitochondria and CO‐blocked respiration in TMPD‐treated eggs were found to be similar to the absorption spectrum of CO‐bound cytochrome aa3. In PMS‐treated eggs and fertilized eggs, the maximum photoreactivation of CO‐inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO‐inhibited respiration in TMPD‐treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b, which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.
Predominant expression of a cytoskeletal actin gene in mesenchyme cells during embryogenesis of the ascidian Halocynthia roretzi
Authors: Araki Isato , Tagawa Kunifumi , Kusakabe Takehiro , Satoh Noriyuki
Published: October 2003

During ascidian embryogenesis mesenchymal cells proliferate and have no known function until after metamorphosis, when they give rise to various mesodermal tissues of the adult. Despite their simple lineage, specification mechanisms of the embryonic mesenchyme cells remain to be investigated; this is mainly due to lack of specific molecular markers for this cell type. Here we report that, in Halocynthia roretzi, zygotic expression of a cytoskeletal actin gene (HrCA1) begins at the late gastrula stage and that transcripts were predominantly distributed in embryonic mesenchyme cells, although some expression was observed in special cells of the central nervous system as well as in notochord cells. When HrCA1 expression was examined in cleavage‐arrested embryos, it was found only in mesenchyme‐lineage blastomeres of arrested 8‐cell and later stages, consistent with the predominant expression of HrCA1 being in mesenchyme cells. To examine whether cell–cell contact until the 8‐cell stage is required for mesenchyme cell specification, blastomeres were continuously dissociated during the period of 2–8‐cell stages and division of the blastomeres was then arrested. Results showed that two of eight dissociated and division‐arrested blastomeres from a single fertilized egg (presumably those corresponding to B4.1 cells) expressed HrCA1, suggesting that specification of embryonic mesenchyme cells does not require cell–cell contact until the 8‐cell stage.
Effects of digit tissue on cell death and ectopic cartilage formation in the interdigital zone in chick leg buds
Authors: Omi Minoru , Ide Hiroyuki
Published: October 2003

In developing chick leg buds, large‐scale cell death occurs in the interdigital zone, which is responsible for the separation of digits from each other. Ectopic cartilage formation is known to occur upon removal of the chondrogenic digit tissue of the leg bud. To examine the mechanisms of ectopic cartilage formation in the interdigital cell death region, we performed the following operations on stage 28–29 leg buds: (i) removal of the digit‐forming area; (ii) incision between the interdigital zone and digit region; (iii) insertion of an aluminum barrier into the interdigital zone; and (iv) insertion of a permeable Nuclepore filter into the interdigital zone. In all cases, the inhibition of cell death and/or the formation of ectopic cartilage in the interdigital zone were observed, although the frequency of the inhibition of cell death and the formation of ectopic cartilage varied, depending upon the position where the operations were performed. These results suggest that cell death and cell differentiation in the interdigital zone may be controlled by some factor(s) from digit cartilage.
Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres
Authors: Amemiya Shonan
Published: October 2003

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four‐armed plutei. After feeding, the larvae developed into eight‐armed plutei. The left‐right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal‐vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five‐fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.
A developmentally regulated α2,8‐polysialyltransferase in embryos of the sea urchin Lytechinus pictus
Authors: Cho Jin Won , Troy Frederic A. , Inoue Sadako , Inoue Yasuo , Lennarz William J.
Published: October 2003

The recent chemical identification of polysialylated glycoproteins in the jelly coat and on the cell surface of the sea urchin egg raises important questions about their biosynthesis and possible function. Using CMP‐[14C]‐Neu5Ac as substrate and cell free preparations from eggs and embryos of the sea urchin Lytechinus pictus, we have identified a membrane associated CMP‐Neu5Ac:poly‐α2,8 sialosyl sialyltransferase (polyST) that transferred Neu5Ac from CMP‐Neu5Ac to an endogenous acceptor membrane protein of approximately 38kDa. An average of five to six [14C]‐Neu5Ac residues were transferred to the glycan moiety of this protein. The membrane‐associated polyST also catalyzed the polysialylation of several exogenous mammalian ganglioside acceptors, including GD3. Given that no structurally similar naturally occurring polysialylated gangliosides have been described, nor were observed in the present study, we conclude that a single polyST activity catalyzes sialylation of the endogenous acceptor protein and the gangliosides. Using an excess of GD3 as an exogenous acceptor, it was established that the expression of the polyST in L. pictus embryos increased rapidly at the mesenchyme blastula stage and reached a maximum at the gastrula stage. The finding that this polyST in the sea urchin embryo is developmentally regulated raises the possibility that it may play a role in the changing cell and tissue interactions that occur during gastrulation and the early stages of spicule formation.
Changes in subcellular localization of mtlrRNA outside mitochondria in oogenesis and early embryogenesis of Drosophila melanogaster
Authors: Amikura Reiko , Kobayashi Satoru , Saito Hiromitsu , Okada Masukichi
Published: October 2003

Mitochondrial large ribosomal RNA (mtlrRNA) has been identified as a cytoplasmic factor inducing pole cells in ultraviolet (UV)‐sterilized Drosophila embryos. In situ hybridization studies have revealed that mtlrRNA is present outside mitochondria localized on the surface of polar granules during the cleavage stage. In the present study, we describe the developmental changes in extramitochondrial mtlrRNA distribution through early embryogenesis using in situ hybridization at the light and electron microscopic level. No mtlrRNA signal was discernible on polar granules in the mature oocyte, unless the oocyte was activated for development. mtlrRNA was localized on the surface of polar granules during a limited period of stages from oocyte activation to pole bud formation and disappeared as soon as being detached from polar granules without entering pole cells. These changes in the temporal and spatial distribution of mtlrRNA outside mitochondria are compatible with the idea that mtlrRNA is required for pole cell formation but not for the differentiation of pole cells as functional germ cells.
Participation of a metalloprotease in the fertilization‐associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes
Authors: Ha Chang‐Rak , Iuchi Ichiro
Published: October 2003

The unfertilized egg envelope of medaka (Oryzias latipes) consists of two major groups of subunits, ZI‐1,2 (74–76 kDa) and ZI‐3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57–65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate‐polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57–65 kDa intermediates originated from ZI‐1,2 by limited hydrolysis. ZI‐1,2 comprises at least three distinct proteins of quite similar structure with their N‐termini undetectable by Edman degradation, while the 57–65 kDa intermediates also consist of at least three proteins with the same N‐terminal amino acid sequence: DGKPSNPQQPQVPQYPSK‐. This fact strongly suggests a participation of a protease in the conversion of ZI‐1,2 into 57–65 kDa proteins. EDTA and 1,10‐phenanthrolinium inhibited the conversion and both Ca2+ and Zn2+ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.
Spatio‐temporal distribution of the protein of Xenopus vasa homologue (Xenopus vasa‐like gene 1, XVLG1) in embryos
Authors: Ikenishi Kohji , Tanaka Tetsuya S. , Komiya Tohru
Published: October 2003

In order to know when the protein of Xenopus vasa homolog (Xenopus vasa‐like gene 1, XVLG1) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila, the spatio‐temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole‐mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta‐nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10–42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole‐mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.
Salt concentration‐dependency of vitellogenin processing by cathepsin D in Xenopus laevis
Authors: Yoshizaki Norio , Yonezawa Satoshi
Published: October 2003

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.
Suppressors of the vestigial mutant phenotype increase the level of expression of the gene
Authors: Flagiello Domenico , Fellini Nadia , Cibert Christian , Zider Alain , Cavicchi Sandro , Silber Joel
Published: October 2003

Suppressor genes of the vestigial phenotype have been isolated in a wild‐type population. These suppressors have an effect on different wing mutants and are allele‐specific in the case of vestigial. In a vgBG background they produced overgrowth of the imaginal wing disc. They also induce cell death in the wild‐type strain and alter the distribution of cell death in the mutant strain. Expression of vestigial is increased in the wing disc only. Hypotheses formed to determine the nature of these suppressors are in favor of a direct interaction between these genes and vestigial.
Multiple roles of the mitogen‐activated protein kinase kinase/mitogen‐activated protein kinase cascade in Xenopus laevis
Authors: Kosako Hidetaka , Gotoh Yukiko , Nishida Eisuke
Published: October 2003

Mitogen‐activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis. Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos.
The path leading to the discovery of cadherin: In retrospect
Authors: Okada Tokindo S.
Published: October 2003

Problems of adhesion between cells are undoubtedly one of the key major issues in developmental biology and its related field. It is little doubt that cell adhesion is one of the most fundamental mechanisms underlying the morphogenesis in multicellular animals and that it is intrinsically related to the metastasis of cancer cells as well. The historical source of adhesion studies can be traced to Wilson's work using sponges published in 1907. The present article starts to outline briefly the conceptual history up to a rise of cell adhesion in the 1950s as a problem for understanding morphogenesis in general. A crucial landmark to a recent burst of the interest to adhesion mechanisms in terms of adhesion molecules is the discovery of a group of major molecules functioning cell‐to‐cell adhesion, cadherins, from a research group at Kyoto University, Japan, which was initiated by myself and very successfully continued by Takeichi. A main part of the present review is to record the path leading to this important discovery based on my own personal experience, including some retrospective anecdotes. The path was initiated with a series of very simple experiments of a naïve question; that is, to examine whether or not different divalent cations were required for cell adhesion in qualitatively different manners.
Serum antigens detected in pars recta luminal fluid and coelomic envelope surrounding Bufo arenarum eggs
Authors: Whitacre Cecilia M. , Miceli Dora C.
Published: October 2003

In anurans, protease activity from the pars recta portion of the oviduct (under regulation by 17β‐estradiol), is known to cause ultrastructural alterations on the oocyte surface rendering fertilizability. In mammals, the presence of serum proteins in oviductal fluid via transudation is also well known. In the present study we determined the plasma proteins of the anuran Bufo arenarum that are present in pars recta fluid and oocyte extracellular matrix and characterized the 17β‐estradiol‐induced proteins synthesized de novo and secreted into the pars recta lumen. Rabbit polyclonal antibodies against the soluble proteins in pars recta fluid cross‐reacted with anuran plasma proteins and with the extracellular matrix of coelomic eggs based on immunoelectrophoresis and immunohistochemistry, respectively. Using radiolabeled leucine in the absence and presence of 17β‐estradiol, we show that a polypeptide of 66 kDa molecular mass is the principal protein synthesized and secreted into the pars recta lumen.
Feather buds exert a polarizing activity when transplanted to chick limb buds
Authors: Uchiyama Kohji , Yanazawa Masako , Kuroiwa Atsushi , Kitamura Kunio
Published: October 2003

Homeoproteins have been shown to be expressed in a position‐specific manner along the anterior‐posterior axis in the developing chick feather bud, as seen also in the developing limb bud. These facts raise the possibility that there may be common mechanistic features in the establishment of the anterior‐posterior polarity between both organs. In order to investigate this possibility, feather bud tissues were transplanted into the anterior region of limb buds to determine whether feather bud tissues possess properties such as the zone of polarizing activity of the limb bud. The manipulated limb bud formed a mirror image duplication of the skeletal elements, mainly (2)2234 digit pattern or sometimes 3(2)234. Both the anterior and posterior halves of feather bud tissue exhibited almost equal activity in inducing ectopic skeletal elements. Hoxd‐12 and Hoxa‐13 were expressed coordinately around the transplanted site of the operated limb bud. This secondary axis‐inducing activity of the feather bud was enhanced when grafts were pretreated with trypsin. In contrast, the presumptive feather bud tissue and inter‐feather bud tissue did not induce a secondary axis of the limb bud. These results suggest that the feather bud contains a region that exerts polarizing activity and that this region may play key roles in the formation of the anterior‐posterior and, if it exists, proximal‐distal axis of the feather bud, possibly via the regulation of region specific expression of Hox genes.
Embryonic cell commitment by a simultaneous alteration in nucleo/cytoplasmic ratio in sibling cells between subsequent cell cycles
Authors: Sennerstam Roland , Strömberg Jan‐Olof
Published: October 2003

Studies on murine embryonal carcinoma (EC) cell lines have revealed a mechanism for commitment of early embryonic cells. By means of a particular intraclonal cell surface glycoprotein and intermitotic time heterogeneity found in the EC lines used, we have devised a cell cycle model and written a computer program for cell cycle stimulation. In the present investigation experimental tissue culture data obtained from the EC lines were inserted into the computer program and the simulations represent a good fit to experimental data. It is shown that the dynamics of the driving forces in the ‘cell growth cycle’ and the ‘DNA‐division cycle’, when assumed to be loosely coupled and analyzed in subsequent cell cycles, reveal a mechanism that can commit the mother cell to impel her daughter cells into the next stage in embryonic development by altering the relationship between those two uncoupled subcycles. Thereby the nucleo/cytoplasmic ratio (DNA/mass) is altered in a similar way in the two daughter cells. The simulations increase the reliability of the model and open up possibilities to test other embryonic cell systems.
Integrin VLA‐6 (α6β1) is transiently expressed during the development of mouse bone marrow‐derived mast cells
Authors: Fehlner‐Gardiner Christine C. , Uniyal Shashi , Chan Bosco M. C.
Published: October 2003

In the present study the involvement of VLA‐6 (α6β1) integrin, a laminin receptor, was characterized during the course of mouse bone marrow‐derived mast cell (BMMC) development. Flow cytometry and immunoprecipitation revealed increases in α6 integrin expression during the first 3 weeks, followed by a decline, such that α6β1 was no longer detectable by week 13. Using RT‐PCR, transcripts for α6A but not the α6B isoform were detected. Results from immunoprecipitation and costaining with β1‐or β4‐specific mAb showed the expression of VLA‐6 (α6β1) and not α6β4 heterodimers. Moreover, the ability of BMMC to interact with laminin correlated with the time period of VLA‐6 expression. However, only 40% of adhesion to laminin was inhibited by blocking mAb for α6, indicating the involvement of additional laminin receptor(s). This is supported by the immunoprecipitation of VLA‐2 integrin, also known to have laminin binding properties. Heterogeneity of VLA‐6 expression was also found in connective tissue‐type mast cells; thus, VLA‐6hi and VLA‐6lo subpopulations of peritoneal mast cells were observed. The heterogeneity of VLA‐6 integrin expression in BMMC and CTMC may be relevant to the concept of mast cell heterogeneity as well as to the ability of mast cell precursors to migrate and complete their course of maturation within tissues.
Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus†
Authors: Kitajima Takashi , Tomita Masahiro , Killian Christopher E. , Akasaka Koji , Wilt Fred H.
Published: October 2003

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.
Epithelial morphogenesis in mouse embryonic submandibular gland: Its relationships to the tissue organization of epithelium and mesenchyme
Authors: Hieda Yohki , Nakanishi Yasuo
Published: October 2003

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force‐generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell‐cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.
Nerve–blastema interactions induce fibroblast growth factor‐1 release during limb regeneration in Pleurodeles waltl
Authors: Zenjari Chafika , Boilly Bénoni , Hondermarck Hubert , Boilly‐Marer Yolande
Published: October 2003

Previous studies have shown that both fibroblast growth factor (FGF)‐1 and nerves play an important function during limb regeneration, but no correlation between these two regeneration factors has yet been demonstrated. In the present study we first establish that exogenous FGF‐2, a member of the FGF family that binds to the same high‐affinity receptors as FGF‐1, is able to stimulate both [3H]‐thymidine incorporation and the mitotic index in the mesenchyme and the epidermal cells of denervated blastemas. We then use cocultures of spinal cord and blastema on heparin‐coated dishes, an in vitro system mimicking the in vivo interactions during limb regeneration, to show that interactions between nerve fibers from the spinal cord and the blastema enhance the release of bioactive FGF‐1. Release of this growth factor seemed to correlate with nerve fiber regeneration, as it decreased in the presence of the dipeptide Leu‐Ala, known to inhibit neurite outgrowth, while the inverse dipeptide Ala‐Leu was inactive. Therefore, these results support our hypothesis that the interaction between nervous tissue and blastema is permissive for the release of FGF‐1, which in turn stimulates blastema cell proliferation.
Color pattern formation on the wing of the butterfly Pieris rapae. 1. Cautery induced alteration of scale color and delay of arrangement formation
Authors: Takayama Eriko , Yoshida Akihiro
Published: October 2003

Experimental approaches to color pattern formation of lepidopteran insects have been made exclusively by analyzing pattern alterations in adult wings induced by operations. We microcauterized the presumptive black region of the dorsal forewing of the butterfly Pieris rapae and analyzed not only the resultant color pattern in the adult wing but also the cell behavior in the pupal wing epidermis around the injury. Cautery induced color alterations were as follows: (i) cautery up to 49.5 h after pupation resulted in white regions appearing within the black region while later cauteries induced larger white regions; (ii) cautery between 50 and 59.5 h resulted in the white regions induced by the cauteries being dramatically decreased; (iii) cautery after 60 h resulted in white regions that had almost disappeared. The examination of the cell behavior in the pupal wing epidermis after cauteries showed that the row formation of scale precursor cells was delayed. This delayed area varied with the time of cautery, in the same manner as that in the induced white area in the adult wing ((i) – (iii) above). The relationship between scale color alteration and the developmental delay of the scale row formation is discussed.
XBMP‐1B (Xtld), a Xenopus homolog of dorso–ventral polarity gene in Drosophila, modifies tissue phenotypes of ventral explants†
Authors: Lin Jih‐Jing , Maeda Ryu , Ong Rosa C. , Kim Jaebong , Lee Leo M. , Kung Hsiang‐fu , Maéno Mitsugu
Published: October 2003

Previously we have isolated a Xenopus cDNA homolog of bone morphogenetic protein‐1 (XBMP‐1A). In the present report we describe a new cDNA clone called XBMP‐1B (or Xtld) from a Xenopus embryonic library. Sequence analysis indicates that these two clones share an identical N‐terminal sequence, including a region of metalloprotease domain, three copies of a repeat first found in complement proteins C1r/s and an epidermal growth factor (EGF)‐like sequence. XBMP‐1B protein has an additional copy of an EGF‐like sequence followed by two copies of complement 1 r/s repeat in the C‐terminus. The overall protein structure predicted from the XBMP‐1B sequence reveals that it encodes a protein homologous to Drosophila tolloid. Three XBMP‐1 transcripts (2.9, 5.2 and 6.6 kb) were detected by northern blot analysis. However, the 2.9 kb transcript hybridized specifically with XBMP‐1A and the 5.2 and 6.6 kb transcripts hybridized with XBMP‐1B. In Drosophila, a major function of tolloid is to augment the activity of the decapentaplegic gene product, a close relative of tumor growth factor (TGF)‐β superfamily members, BMP‐2/4. Although XBMP‐1 and XBMP‐4 are detected in various adult tissues of Xenopus, the expression pattern of these two genes was not tightly correlated. In the embryo, the expression of XBMP‐1 increased gradually from the morula to the swimming tadpole stages. Injection of XBMP‐1B RNA into the ventral blastomeres at the 4‐cell stage caused an elongation of the ventral marginal zone explants and converted globin‐positive blood cells to mesenchymal and muscle tissues at later stages. It was shown that XBMP‐1A was less active and a 1A mutant lacking the signal sequence was inactive. Further studies revealed that injection of XBMP‐1B RNA into the ventral marginal zone induced up‐regulation of dorsal marginal zone markers, such as goosecoid and chordin, at the gastrulation stage. These data indicate that XBMP‐1 may have a role in determining dorso–ventral patterning in Xenopus, but in a different way from the dpp/tolloid system demonstrated in Drosophila.
Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus
Authors: Correa Liane M. , Carroll Edward J.
Published: October 2003

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol‐sea water solutions and have characterized the macromolecules by SDS‐PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N‐acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.
The developing Xenopus oocyte specifies the type of gonadotropin‐stimulated steroidogenesis performed by its associated follicle cells
Authors: Sretarugsa Prapee , Wallace Robin A.
Published: October 2003

As a response to gonadotropin, amphibian ovarian follicles primarily synthesize and secrete estradiol‐17 β (E2) during vitellogenesis and progesterone (P) when fully grown. Stage IV (vitellogenic) and stage VI (full‐grown) ovarian follicles from Xenopus laevis, as well as intermediate sizes, were used to explore this change in steroidogenesis. Optimum steroidogenesis occurred in both stage IV and stage VI follicles exposed for 6 h to 20 IU human chorionic gonadotropin/mL. Although the total amounts of steroid found were about the same, the E2/P ratios ranged from 26 to 35 for intact stage IV follicles, but only 0.02–0.03 for intact stage VI follicles. Steroid‐producing follicle cells were isolated from stage IV and stage VI follicles by non‐enzymatic procedures, were washed and were tested for steroidogenic activity in the absence of oocytes. In both cases, P was the predominant steroid produced (E2/P = 0.004–0.04), so the presence of stage IV, but not stage VI, oocytes appears to be necessary for E2 production as a response to gonadotropin. Octanol had no significant effect on the E2/P ratio of intact stage IV follicles. Dissected oocyte/follicle cell preparations from stage IV follicles were also periodically challenged with gonadotropin over 72 h, during which time most follicle cells detached from the oocyte and formed a monolayer over the bottom of the culture dish. The relatively high E2/P ratios for such preparations showed no significant change when stimulated with gonadotropin at various times over the 72 h, as long as the medium was not replaced. We conclude that the estrogenic effect of stage IV oocytes is most likely mediated by a secretory product rather than by gap junctions or by cell contact. Because the X. laevis oocyte has been shown to be a self‐differentiating cell, the steroidogenic shift that occurs in developing ovarian follicles appears to be fundamentally regulated by the growing oocyte as it undergoes a physiological change rather than by different gonadotropins.