bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for cell surface and internal phospholipase activity in ascidian eggs
Authors: Goode Christina A. , Gamboa‐Pinto Antonio J. , Cruz Rowelle , Gough Lisa Lucio , Lund Caren V. , Lambert Charles C.
Published: October 2003

Upon fertilization, ascidian eggs release a cell surface glycosidase used in the block to polyspermy and undergo cortical contractions resulting from increased intracellular calcium levels. The glycosidase is released by fertilization, calcium ionophores or added phospholipase C (PLC) activity. The PLC inhibitor D609 blocks glycosidase release. Intact Ascidia ceratodes eggs cleave 4‐methylumbelliferyl‐phospho‐choline when it is added to seawater. This yields highly fluorescent 4‐methylumbelliferone. Authentic phospholipase C but not phospholipase D can cleave this substrate. Thus, the authors believe that cleavage of the substrate is specific for PLC activity. Eggs incubated in the fluorogenic substrate after having been washed and detergent extracted were not fluorescent. Therefore the substrate failed to enter intact cells. Glycosidase release and PLC activity were stimulated by ionomycin. Octylglucoside or Triton X‐100 extracts of ascidian eggs had two forms of phospholipase activity as shown by ion affinity chromatography: PL1 eluting at 0.25 mol/L NaCl and PL2 eluting at 0.6mol/L NaCl. The PL1 appeared to be isolated as a single protein. When surface proteins were labeled with non‐penetrating biotin and were subsequently reacted with streptavidin, half of the PLC activity bound. This demonstrates that half the ascidian egg PLC activity is located on the surface of either the egg or follicle cell, and half is located within the egg.
Hierarchy of inductive events
Authors: Lopashov G. V. , Zemchikhina V. N.
Published: October 2003

Examination of data on the inducing capacity of mesoderm‐inducing factors shows that none of them induces mesoderm alone, but that they also induce endoderm and neural tissues, while in the mesoderm itself, they induce tissues belonging to its main levels. Tests on the inducing capacity of living retina have shown that it does not induce mesoderm, but does induce a spectrum of tissues, including retina, pigmented epithelium, lens and a piece of brain. This situation seems to be indispensable because if it evokes only one differentiation, an increase of diversity in development would be impossible. Selectivity occurs only at the end of the sequence of inductions, as it does in the induction of lens tissue by lens epithelium. Such mechanisms, however, are insufficient for the development of different kinds of cells into homogeneous tissues. This is achieved through the aggregation or separation of initially induced cells, their own products determining (or not) their further fate and leading to homogeneity of tissues. These mechanisms of the first two levels of inductive interactions overlap with events that allow or prevent the access of inducing factors and are, therefore, also involved in the manifestation of competence of potentially reactive cells.
Distribution pattern of HNF‐3β proteins in developing embryos of two mammalian species, the house shrew and the mouse
Authors: Yasui Kinya , Sasaki Hiroshi , Arakaki Rieko , Uemura Masanori
Published: October 2003

The pattern of expression of HNF‐3β in organizing centers and axial structures during early vertebrate development suggests an important role for this protein in the establishment of the vertebrate body plan. To establish whether the pattern of expression during embryogenesis is species specific, a comparative immunohistochemical study of two mammalian species, the house shrew, insectivore, and the mouse was carried out; it is difficult to obtain accurate morphological differences from the study of remotely related animals. The earliest expression of HNF‐3β appeared in the node and hypoblast (or endoderm) in both species, where the presumptive foregut endoderm showed intense immunoreactivity prior to the formation of the axial mesoderm, suggesting a role different from that in axial formation. The anterior extension of immunopositive axial mesoderm and the median band of the neural plate varied between the two species, and was delayed in the house shrew. HNF‐3β in the anterior end of the foregut disappeared transiently in the house shrew but persisted in the mouse embryo. An asymmetric pattern of distribution in the primitive streak was also observed in the mouse but not in the house shrew. The present immunohistochemical study elucidated that the distribution of HNF‐3β is conserved initially but soon manifests species specificities in development even between mammalian species.
Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis
Authors: Miura Takeshi , Kawamura Seichi , Miura Chiemi , Yamauchi Kohei
Published: October 2003

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG‐injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well‐developed testis and the milt could be acquired by hand‐stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand‐stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 23 to 26 late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.
Formation of new plasma membrane during the first cleavage cycle in the egg of Xenopus laevis: An immunocytological study
Authors: Aimar Christian
Published: October 2003

Polyclonal antibodies (IS1) reacting specifically with plasma membrane proteins of the Xenopus oocyte were used to study the formation of new plasma membrane in cleavage furrows. Membrane precursors were detected in the inner cytoplasm, then under the plasma membrane of the animal hemisphere and finally on the furrow's edges. Cycloheximide and colchicine caused abnormal distribution of stained material. IS1 antibodies conjugated to colloidal gold, were used to examine the local insertion of membrane precursors into the furrow region by electron microscopy. Membrane precursors were only detected in intracytoplasmic vesicles that fused with the plasma membrane at the edges of the furrow walls. Arrays of microtubules may guide membrane precursors to the site of their insertion in the furrow walls.
Behaviors of nucleus, basal bodies and microtubules during eupyrene and apyrene spermiogenesis in the silkworm, Bombyx mori (Lepidoptera)
Authors: Yamashiki Naoko , Kawamura Naoko
Published: October 2003

Observations with immunostaining for tubulin and electron microscopy revealed that silkworm eupyrene spermiogenesis was characterized by an attachment of the basal body to the nucleus except in the period of movement for unidirectional arrangement. In young eupyrene sperm, a microtubule basket caught the nucleus, which thereafter was transformed elliptically. Microtubules were also observed along the elongated acrosome and mitochondrial derivatives. During apyrene spermiogenesis, however, the basal body was not attached to the nucleus and approached the head cyst cell after the completion of unidirectional arrangement, leaving the round nucleus in the middle of the cell. The presence or absence of the phenomenon in which the basal body attaches to the nucleus seems to be essential in the course of diverse spermatogenesis of the silkworm.
A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain
Authors: Umesono Yoshihiko , Watanabe Kenji , Agata Kiyokazu
Published: October 2003

To analyze the organization of planarian brain, a homolog of the homeobox‐containing gene Orthopedia (Otp) from planarian, Djotp, was isolated. The homeodomain of Djotp differs from mouse Otp by only two amino acids. This conservation extends to include a 12 amino acid motif downstream of the homeodomain. Whole mount in situ hybridization studies indicated that Djotp is specifically expressed in the branch structures of the normal planarian adult brain. During regeneration, Djotp is expressed in the presumptive branch region prior to branch formation. These observations implicate a role for Djotp in establishing and maintaining the identity of the planarian brain branch region. The results suggest that recruitment of Otp for its role in brain pattern formation occurred very early in evolution.
A metaphase pause: Hormone‐induced maturation progresses through a long pause at the first meiotic metaphase in oocytes of the starfish, Pisaster ochraceus
Authors: Yamamoto Kenya
Published: October 2003

In several species of starfish, it has been reported that the meiotic divisions in fertilized oocytes occur precociously compared to those in unfertilized oocytes. The nature of the ‘acceleration’ of meiosis was studied using Pisaster ochraceus oocytes. The extent of the acceleration of first polar body formation was found to be completely dependent on the time of fertilization (or artificial activation); fertilization at about 100 min after 1–methyladenine application accelerated meiosis I the most, while earlier or later fertilization resulted in a smaller extent of accelerations of meiosis I. Observation of isolated meiotic spindles and fluorescent visualization of meiotic spindles in whole oocytes showed that progression of meiosis I in Pisaster oocytes pauses transiently at metaphase I for more than 40min unless they are activated. The activation shortened the duration of metaphase I, which resulted in the acceleration of first polar body formation. A new term ‘metaphase pause’ is proposed to define this long duration of metaphase I in starfish oocytes.
Unequal cleavage in the early Tubifex embryo
Authors: Shimizu Takashi , Ishii Ryuichi , Takahashi Hirokazu
Published: October 2003

Unequal cleavage that produces two blastomeres of different size is a cleavage pattern that many animals in a variety of phyla, particularly in Spiralia, adopt during early development. This cleavage pattern is apparently instrumental for asymmetric segregation of developmental potential, but it is also indispensable for normal embryogenesis in many animals. Mechanically, unequal cleavage is achieved by either simple unequal cytokinesis or by forming a polar lobe at the egg's vegetal pole. In the present paper, the mechanisms for unequal cytokinesis involved in the first three cleavages in the oligochaete annelid Tubifex are reviewed. The three unequal cleavages are all brought about by an asymmetrically organized mitotic apparatus (MA). The MA of the first cleavage is monastral in that an aster is present at one pole of a bipolar spindle but not at the other. This monastral form, which arises as a result of the involvement of a single centrosome in the MA assembly, is both necessary and sufficient for unequal first cleavage. The egg cortex during the first mitosis is devoid of the ability to remodel spindle poles. In contrast to the non‐cortical mechanisms for the first cleavage, asymmetry in the MA organization at the second and third cleavages depends solely on specialized properties of the cell cortex, to which one spindle pole is physically connected. A cortical attachment site for the second cleavage spindle is generated de novo at the cleavage membrane resulting from the first cleavage; it is an actin‐based, cell contact‐dependent structure. The cortical microtubule attachment site for the third cleavage, which functions independently of contact with other cells, is not generated at the cleavage membrane resulting from the second cleavage, but is located at the animal pole; it may originate from the second polar body formation and become functional at the 4‐cell stage.
Genetic evidence for posterior specification by convergent extension in the Xenopus embryo
Authors: Yamada Tuneo , Modak Sohan P.
Published: October 2003

Genetic studies substantiate that mesodermal convergent extension expressed behind the anteroposterior borderline, in the form of a gradient with the posterior apex after gastrulation, regulates morphogenesis of the posterior zone at the dorsal and dorso‐lateral levels which is in full agreement with the model of dorsalization–caudalization. In contrast, how anteroposterior specification of mesodermal tissues occurs at the ventral and latero‐ventral levels is not yet understood.
Combinatorial expression patterns of individual TLE proteins during cell determination and differentiation suggest non‐redundant functions for mammalian homologs of Drosophila Groucho
Authors: Yao Jing , Liu Yanling , Husain Junaid , Lo Rita , Palaparti Anuradha , Henderson Janet , Stifani Stefano
Published: October 2003

The Drosophila protein Groucho is involved in the regulation of cell‐determination events during insect neurogenesis and segmentation. A group of mammalian proteins, referred to as transducin‐like Enhancer of split (TLE) 1 through 4, share with Groucho identical structures and molecular properties. The aim was to determine whether individual TLE proteins participate in the regulation of cell determination in mammals like their Drosophila counterpart. It is here reported that TLE family members are expressed in combinatorial ways during the in vitro differentiation of mouse P19 embryonic carcinoma cells (a model for neural determination) and rat CFK2 cells (a model for chondrocytic determination). TLE1 is up‐regulated and TLE2 and TLE4 are down‐regulated to different extents during early stages of differentiation. In contrast, later stages correlate with up‐regulation of TLE2 and TLE4, and decreased expression of TLE1. Individual TLE proteins are also expressed in combinatorial as well as complementary patterns during the development of the cerebral cortex and spinal cord of mouse embryos. In particular, TLE1 is robustly expressed in both neural progenitor cells and postmitotic neurons of the outer layers of the cortical plate, whereas TLE4 expression marks preferentially postmitotic neurons of the inner layers. Taken together, these results strongly suggest non‐redundant roles for individual TLE proteins during both cell‐determination and cell‐differentiation events.
Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl
Authors: Penrad‐Mobayed May , Moreau Nicole , Angelier Nicole
Published: October 2003

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop‐associated proteins (RNA/protein interactions). North‐western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.
Metamorphosis‐associated and region‐specific expression of calbindin gene in the posterior intestinal epithelium of Xenopus laevis larva
Authors: Amano Tosikazu , Noro Nobuhiro , Kawabata Hiromi , Kobayashi Yoshie , Yoshizato Katsutoshi
Published: October 2003

The present study used a molecular approach toward understanding the mechanism of hormone‐ and region‐dependent remodeling of the small intestine during metamorphosis of Xenopus laevis. A protein spot was noticed on a two‐dimensional polyacrylamide gel as a protein whose expression was metamorphic stage‐ and region‐dependent. The protein was identified as the Xenopus homolog (Xcalbindin) of chick calbindin D28k. Xcalbindin expression in the intestine was restricted to absorptive cells in the posterior part, being detectable at stages 49–61, not detectable at stages 62–63, detectable again at stages 64–66, and finally becoming undetectable in the adult. During spontaneous metamorphosis, the level of Xcalbindin mRNA was significantly increased between stages 57 and 58, dramatically reduced at stage 59, and the mRNA was undetectable from stages 60–63, after which it was weakly re‐expressed until the end of metamorphosis. Such up‐ and down‐regulation of Xcalbindin mRNA was induced precociously by exogenous thyroid hormone. These results indicated that Xcalbindin is a specific marker of the differentiated absorptive cells of the intestine. Immunohistochemistry with specific antibodies against Xcalbindin demonstrated that precursor cells of adult intestinal epithelial cells expressed Xcalbindin. Considering these results, the origin of adult intestinal epithelial cells was discussed.
Activin‐treated ectoderm has complete organizing center activity in Cynops embryos
Authors: Ninomiya Hiromasa , Ariizumi Takashi , Asashima Makoto
Published: October 2003

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae–early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk‐rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin‐treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin‐treated ectoderm. These findings show that activin‐treated ectoderm can act as the complete organizing center in Cynops.
Hypoblast cells of chick pre‐streak stage embryos invaded basement membrane analogues in vitro: Implications for hypoblast layer formation
Authors: Mogi Kazue , Toyoizumi Ryuji , Takeuchi Shigeo
Published: October 2003

In early chick blastodermal morphogenesis, the hypoblast layer is organized beneath the epiblast and induces an axial structure. However, the origin of hypoblast cells and the mechanism of hypoblast layer formation are poorly understood. We hypothesized that the hypoblast layer is formed by an invasive process across the basement membrane of the juxtaposing epiblast, and tested the idea in vitro. Primary and secondary hypoblast cells from embryos at various pre‐streak stages were dissociated into single cells and cultured on reconstituted basement membrane gel, laminin gel or fibronectin gel in the culture medium with or without serum for 24–48 h. As a result, we found that after 24 h of serum‐supplemented culture, up to 35% of the hypoblast cells dissolved the gel and made holes on it. Similarly, up to 36% of the hypoblast cells showed invasiveness after 48 h in the serum‐free culture. Furthermore, it was observed that Koller's sickle cells, which are regarded to be the progenitors of secondary hypoblast cells, penetrated those gels on which they were seeded. The posterior epiblast cells covering Koller's sickle were also invasive. These results suggest that the presumptive primary hypoblast cells that are known to mingle with epiblast cells invade through the basement membrane to form the hypoblast layer. Furthermore, the present results imply that invasion through the basement membrane may be involved in the formation of Koller's sickle, the anlage of secondary hypoblast.
Inhibition of sea urchin fertilization by jaspisin, a specific inhibitor of matrix metalloendoproteinase
Authors: Kato Koichi H. , Takemoto Kazuhisa , Kato Etsuko , Miyazaki Kaoru , Kobayashi Hiroki , Ikegami Susumu
Published: October 2003

Jaspisin, originally isolated from a marine sponge as an inhibitor of the hatching of the sea urchin (Hemicentrotus pulcherrimus) embryo, causes inhibition of sea urchin fertilization. Electron microscopic examination revealed that the acrosome reaction was induced in jaspisin‐treated sperm when they were incubated with an intact egg. The acrosome‐reacted sperm bound to the vitelline layer by the acrosomal material surrounding the acrosomal process. However, fusion of the acrosomal process and the egg plasma membrane failed to take place. Membrane potential changes were monitored using eggs preloaded with a membrane potential‐sensitive fluorochrome, di‐8‐ANEPPS. Depolarization of the membrane potential, normally observed in the fertilized egg was not observed in the egg inseminated in the presence of jaspisin, indicating the absence of electrical continuity between the jaspisin‐treated egg and sperm. Jaspisin inhibited the activities of matrix metallo‐endoproteinase members but not of other types of proteinases. These results provide strong, albeit indirect, evidence that a matrix metallo‐endoproteinase(s) is involved in the process of gamete fusion during sea urchin fertilization.
Crosstalk between the phosphatidylinositol cycle and MAP kinase signaling pathways in Xenopus mesoderm induction
Authors: Rose Linda , Busa William B.
Published: October 2003

Recent studies have established a role for the phosphoinositide (PI) cycle in the early patterning of Xenopus mesoderm. In explants, stimulation of this pathway in the absence of growth factors does not induce mesoderm, but when accompanied by growth factor treatment, simultaneous PI cycle stimulation results in profound morphological and molecular changes in the mesoderm induced by the growth factor. This suggests the possibility that the PI cycle exerts its influence via crosstalk, by modulating some primary mesoderm‐inducing pathway. Given recent identification of mitogen‐activated protein kinase (MAPK) as an intracellular mediator of some mesoderm‐inducing signals, the present study explores MAPK as a potential site of PI cycle‐mediated crosstalk .We report that MAPK activity, like PI cycle activity, increases in intact embryos during mesoderm induction. Phosphoinositide cycle stimulation during treatment of explants with basic fibroblast growth factor (bFGF) synergistically increases late‐phase MAPK activity and potentiates bFGF‐induced expression of Xbra, a MAPK‐dependent mesodermal marker.
Embigin/basigin subgroup of the immunoglobulin superfamily: Different modes of expression during mouse embryogenesis and correlated expression with carbohydrate antigenic markers
Authors: Fan Qi‐Wen , Kadomatsu Kenji , Uchimura Kenji , Muramatsu Takashi
Published: October 2003

Embigin and basigin are highly glycosylated transmembrane glycoproteins with two immunoglobulin domains and form a subgroup in the immunoglobulin superfamily. Previous studies have demonstrated the functional significance of these molecules. In the present study, in situ hybridization analysis of their expression was performed during mouse embryogenesis. Embigin was strongly expressed in the endoderm during early postimplantation embryogenesis, and in the somite stage in the gut and visceral endoderm. Embryonic ectoderm and its derivative tissues weakly to moderately expressed this molecule. From day 10 to 15 of gestation, no embigin signal was detected. Basigin was more broadly expressed. During the organogenesis period, basigin was expressed in various epithelial tissues, brain ventricles, the spinal cord and dorsal root ganglion. The modes of expression of these two proteins throughout the egg cylinder stage correlated with the expression of the carbohydrate markers that they carry; embigin with Dolichos biflorus agglutinin binding sites and basigin with Lex antigen and more closely with fucosyltransferase IV, which forms the antigenic epitope. These findings imply that proteins with specific carbohydrate epitopes play roles in early postimplantation embryogenesis.
Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy
Authors: Omoto Takatsugu , Katow Hideki
Published: October 2003

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus. A molecular image analysis of pamlin was conducted using immuno‐electron microscopy, rotary shadowing and negative staining technique‐applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α‐subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α‐subunit that attaches to one end of a 105 nm long 180 kDa β‐subunit, and a 15.6 nm diameter globular 23 kDa γ‐subunit that binds to the middle of the β‐subunit. The α‐ and β‐subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β‐subunits of the αβγ pamlin molecule, leaving the entire α‐subunit surface free. Occasionally associations between the ends of α‐subunits, or between an α‐subunit and the middle of a β‐subunit also occurred, but no aggregations of pamlin formed through the γ‐subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a ‘loop’, suggesting the presence of a calcium sensitive region in the molecule.
Induction of metamorphosis in the sand dollar Peronella japonica by thyroid hormones
Authors: Saito Minoru , Seki Masanori , Amemiya Shonan , Yamasu Kyo , Suyemitsu Takashi , Ishihara Katsutoshi
Published: October 2003

The larva of the sand dollar Peronella japonica lacks a mouth and gut, and undergoes metamorphosis into a juvenile sand dollar without feeding. In the present study, it was found that thyroid hormones accelerate the metamorphosis of P. japonica larvae. The contents of thyroid hormones in larvae increased gradually during development. Thiourea and potassium perchlorate, inhibitors of thyroid hormone synthesis, delayed larval metamorphosis and simultaneously repressed an increase in the content of thyroxine in the larval body. These results suggest that the P. japonica larva has a system for synthesis of thyroid hormones that act as factors for inducing metamorphosis.
Effects of mesenchyme on epithelial tissue architecture revealed by tissue recombination experiments between the submandibular gland and lung of embryonic mice
Authors: Iwai Kazuma , Hieda Yohki , Nakanishi Yasuo
Published: October 2003

Lung epithelium during morphogenesis maintains a sheet structure of polarized cells lining a lumen, in which E‐cadherin, β‐catenin and tight junctional proteins are localized at the cell–cell contact sites. On the other hand, the submandibular gland epithelium at early stages of development forms a non‐cavitated mass of cells where E‐cadherin/β‐catenin are present on the entire cell surfaces and tight junctional proteins are almost absent or weakly scattered. In the present study, tissue recombination experiments were performed between the two organs to explore roles of mesenchyme in the architectural development of the epithelium. Homotypic recombinants of both submandibular gland and lung showed the tissue architecture as observed in the intact organs. In contrast, 11‐day lung epithelium cultured with 13‐day submandibular mesenchyme formed multilayers of cells with the lumen being less visible. It was accompanied by redistribution of E‐cadherin/β‐catenin along the entire cell surfaces and by an irregular distribution of tight junctional proteins. A similar redistribution of these molecules was observed in 15‐day lung epithelium cultured with the submandibular mesenchyme, although the epithelial sheet structure lining the lumen was formed. On the other hand, the tissue architecture of submandibular gland epithelium was little affected by lung mesenchyme, although the epithelium was flattened and showed branching morphogenesis.
Existence of gradient in cell adhesiveness along the developing Xenopus hind limb bud, shown by a cellular sorting‐out experiment in vitro
Authors: Koibuchi Nobutaka , Tochinai Shin
Published: October 2003

To examine the possibility of a difference in cell adhesiveness along the developing Xenopus hind limb bud axes, single mesenchymal cells from developing hind limb buds were cultured, allowing them to form an aggregate in a gyratory culture system. By observing the distribution of cells within aggregates, it was found that sorting‐out occurred between cells from different positions and different stages. Cells derived from more distal positions tended to be situated interiorly in the aggregates. According to Steinberg's differential adhesion hypothesis, these results support the idea that there is a graded difference in cell adhesiveness along the proximo‐distal axis of the developing limb, with adhesiveness increasing distally. Although similar sorting‐out was observed between anterior and posterior cell populations, it could not be determined which cell populations were definitely more cohesive. These properties may be correlated with the experimentally demonstrated ‘positional value', which should be different among cells located at different positions along the axes of the developing vertebrate limb bud.
DNA replication cycle in parthenogenetically developing eggs of the starfish Asterina pectinifera
Authors: Nomura Akira , Nemoto Shin‐ichi
Published: October 2003

Starfish oocytes artificially activated by a calcium ionophore will develop normally if the formation of polar bodies is suppressed. In the present paper, schedules of the DNA replication period (S phase) of these parthenogenotes were explicitly timed using 5‐bromo‐2'‐deoxyuridine (BrdU) and anti‐BrdU monoclonal antibody. Their schedule of S phase was identical to that of fertilized eggs. Consequently, an S phase regulation system is triggered even in parthenogenotes raised by dual treatment of egg activation and polar body suppression. The S phase schedule of parthenogenotes confirms the temporal pattern of chromosome duplication, observed by other researchers, leading to tetraploid parthenogenotes. The S phase determination also provides a basis for argument concerning the number of centrioles participating in parthenogenetic development. If polar body formation of activated eggs was not suppressed, the first S phase was normal, but the second S phase did not recur on time. A rigidly regulated system of DNA replication cycle, which should be an essential prerequisite for parthenogenesis, thus requires the content of polar bodies.
Developmental regulation and tissue‐specific localization of calmodulin mRNA in the protochordate Ciona intestinalis
Authors: Gregorio Anna Di , Villani Maria Grazia , Locascio Annamaria , Ristoratore Filomena , Aniello Francesco , Branno Margherita
Published: October 2003

A full‐length cDNA encoding a highly conserved calmodulin was isolated from a cDNA library prepared from hatched larvae of the ascidian Ciona intestinalis. Sequence analysis has identified a 447 b.p. open reading frame, encoding a putative protein of 149 amino acid residues, with a predicted molecular weight of 16.8 kDa, showing 85–98% identity to known calmodulins. Northern blot analysis revealed a single transcript of about 0.8 kb in length, which was maternally expressed and progressively increased during development, until late tail‐bud stage. Whole‐mount in situ hybridizations, carried out on embryos at different stages of development, showed that starting from the neurula stage, the C. intestinalis calmodulin (Ci‐CaM) expression became restricted to the neuroectoderm and that in larvae it was specifically detected in the nervous system.
Mapping of cholecystokinin transcription in transgenic mouse brain using Escherichia coli β‐galactosidase reporter gene
Authors: Itoh Yasuhiro , Kozakai Ikuo , Toyomizu Masaaki , Ishibashi Teru , Kuwano Ryozo
Published: October 2003

Cholecystokinin (CCK), a neuro‐gut peptide, occurs not only in the nervous but also in the digestive system. As a first step in elucidating whether CCK gene expression and its physiological functions co‐operate in these separate organs, transgenic mice were produced using CCK promoter that directs bacterial β‐galactosidase as a reporter gene. A new transgenic vector was constructed, inserting the SV40 poly A signal 5' to the CCK promoter to impede any transcription upstream of the transgene. A 2.4 kb.p. region upstream to the transcription start site of the mouse CCK gene was used as the promoter. Transgene expression was detected first at embryonic 13.5 days in the central nervous system and increased after birth. The distribution of cells expressing β‐galactosidase transgene agreed fairly well with that of in situ hybridization. In addition, the upregulation of CCK gene expression was clearly demonstrated by expressing β‐galactosidase after injury to the brain. These results indicated that the 2.4 kb.p. of the CCK gene promoter region was sufficient to direct appropriate tissue‐specific gene expression in mice.
Retinoic acid affects patterning along the anterior–posterior axis of the ascidian embryo
Authors: Katsuyama You , Saiga Hidetoshi
Published: October 2003

Because retinoic acid (RA) is known to affect anterior‐posterior patterning in vertebrate embryos, it was questioned whether it shows similar effects in a more primitive chordate, the ascidian Halocynthia roretzi. Ascidian embryos treated with RA exhibited truncated phenotypes in a dose‐dependent manner similar to the anterior truncations seen in vertebrate embryos. The most severely affected larvae possessed a round trunk without the papillae characteristic of the anterior terminal epidermis. Retinoic acid also altered the expression of HrHox‐1 and Hroth in a dose‐dependent manner. Expression of HrHox‐1 increased, whereas expression of Hroth decreased with increasing levels of RA. In treated embryos, HrHox‐1 was first expressed pan‐ectodermally, then degraded in all but specific regions of the embryo. By contrast, initiation of Hroth expression was not affected, but epidermal expression was lost while expression in the neural tube narrowed toward the anterior in tail‐bud embryos. These alterations in the expression of homeobox genes appear to correlate closely to the morphological defects elicited by RA treatment, suggesting broad conservation of developmental patterning mechanisms within the Phylum Chordata.
Timing and mechanisms of mesodermal and neural determination revealed by secondary embryo formation in Cynops and Xenopus
Authors: Imoh Hiroshi , Yamamoto Yoshiyuki , Terahara Tadashi , Moody Sally A. , Suzuki Akio S.
Published: October 2003

We examined the timing and mechanisms of mesodermal and neural determination in Cynops, using the secondary embryo induced by transplantation of the prechordal endomesoderm. Two unique approaches were used: one was to observe gastrulation movements induced by the graft, and the other to measure the volumes of formed tissues. Transplanted graft pulled host animal cap cells inside to form a new notochord and other mesoderm of the secondary embryo, showing determination of mesoderm during gastrulation. The graft attained a certain width beneath the host ectoderm and moved near to the animal pole of the host by late gastrula, and a neural plate, which had a similar width to the graft, was formed covering the graft. The volume of neural tissues of the secondary embryo at tail‐bud stages was about half that of the normal embryo, while the volumes of notochord were comparable in each case. These data suggest that prechordal endomesoderm, rather than notochord, determines the limit of neural plate in the overlying ectoderm. Similar dorsal grafts were transplanted at early gastrula in Xenopus but did not form well developed secondary embryos, demonstrating that the timing and mechanisms of mesoderm formation in Xenopus are different from those in Cynops.
Shh, Bmp‐2 and Hoxd‐13 gene expression in chick limb bud cells in culture
Authors: Kimura Jun , Ide Hiroyuki
Published: October 2003

The anteroposterior axis of the vertebrate limb bud is determined by signals from the zone of polarizing activity (ZPA). Sonic hedgehog (Shh) is expressed in the posterior mesoderm, which corresponds closely to ZPA activity. Moreover, Bmp‐2 and HoxD genes are expressed in the broader posterior mesoderm, and it is thought that the ZPA signaling pathway consists of these gene products. Limb outgrowth and patterning, including expression of these genes, depend on the apical ectodermal ridge (AER). Fibroblast growth factors (FGF) have been identified as candidates for signal molecules from the AER. To further understand the ZPA signaling pathway and the participation of FGF, expressions of these genes were examined by reverse transcription‐polymerase chain reaction in chick limb bud cells cultured with FGF‐4. The present results indicate that FGF‐4 cannot maintain Shh expression but can maintain Hoxd‐13 expression in cultured posterior cells; moreover, Bmp‐2 is expressed independently of FGF‐4. These results suggest that Bmp‐2 and Hoxd‐13 expressions do not require a continuous expression of Shh. Further, it was demonstrated that posterior cells cultured with FGF‐4 recovered Shh expression when grafted to the limb bud, indicating that FGF‐4 maintains not Shh expression itself but competence of Shh expression.
Overexpression of CAF1 encoding a novel Ca2+‐binding protein stimulates the transition of Dictyostelium cells from growth to differentiation
Authors: Itoh Mikiko , Noguchi Maiko , Maeda Yasuo
Published: October 2003

Among the expressed genes associated with the switch‐over of Dictyostelium cells from cell proliferation to differentiation, the Calfumirin‐1 (CAF1) gene has been shown to be preferentially expressed at the initial step of differentiation, encoding a novel Ca2+‐binding protein (Abe & Maeda 1995). To analyze precisely the function of CAF1, transformants overexpressing the CAF1 mRNA at the vegetative growth phase and also CAF1‐null mutants were prepared, and their developmental features were compared with those of parental wild‐type cells. As a result, the CAF1‐overexpression was found to promote cell differentiation, possibly through prompt induction of the cAMP receptor 1 (CAR1) gene expression. In addition, the CAF1‐overexpressing cells were able to differentiate even under low external Ca2+ ([Ca2+]e) conditions around 10−6mol/L at which non‐transformed wild‐type cells never differentiated. Unexpectedly, however, the CAF1‐null mutant produced by homologous recombination exhibited apparently normal development to form fruiting bodies on non‐nutrient agar. These results seem to indicate that CAF1‐overexpression has a stimulatory effect on differentiation, but that the CAF1 protein is not necessarily required for the phase‐shift of cells from growth to differentiation.
Hormone‐inducible expression of secreted factors in zebrafish embryos
Authors: de Graaf Mark , Zivkovic Danica , Joore Jos
Published: October 2003

The study of gene function at later stages of embryonic development by overexpression experiments is often complicated by genes exerting different functions at multiple stages of development, which renders analysis of stage‐specific effects difficult. To address this problem an inducible expression system that supports timed expression of essentially any protein, including secreted proteins was designed. The system is based on a two step mechanism. A glucocorticoid inducible, Gal4‐site binding chimeric transcription factor is expressed ubiquitously, whereas a gene of interest is placed under the control of a Gal4‐site driven promoter. Treatment of zebrafish embryos injected with such constructs with the synthetic glucocorticoid dexamethasone results in readily detectable reporter activity within 3h. The system was tested with induced expression of XactivinβB and Xwnt, which both were shown to induce morphological abnormalities, as well as alterations in the expression patterns of goosecoid and otx2, respectively. Coinjection of an inducible lacZ reporter vector served as an indicator for expressing cells in embryos. The present results demonstratrate that this is a versatile inducible expression system for use in vertebrate embryos, that also supports expression of secreted proteins.
Studies on fertilization in the teleost. I. Dynamic responses of fertilized medaka eggs
Authors: Iwamatsu Takashi
Published: October 2003

In order to understand the mechanisms of fertilization in the teleost, the movements of the egg cortex, cytoplasmic inclusions and pronuclei were observed in detail in fertilized medaka Oryzias latipes eggs. The first cortical contraction occurred toward the animal pole region following the onset of exocytosis of cortical alveoli. The cortical contraction caused movement of oil droplets toward the animal pole where the germinal vesicle had broken down during oocyte maturation. The movement of oil droplets toward the animal pole region was frequently twisted in the right or left direction. The direction of the twisting movement has been correlated with the unilateral bending of non‐attaching filaments on the chorion. The female pronucleus, which approached the male pronucleus from the vicinity of the second polar body, took a course to the right, left or straight along the s‐p axis connecting the male pronucleus and the second polar body. The course of approach by the female pronucleus correlated with the bending direction of the non‐attaching filaments that had been determined by rotation of the oocyte around the animal–vegetal axis during oogenesis. The first cleavage furrow also very frequently coincided with the axis. These observations suggest that dynamic responses of medaka eggs from fertilization to the first cleavage reflect the architecture dynamically constructed during oogenesis.
Correlation between tectum formation and expression of two PAX family genes, PAX7 and PAX6, in avian brains
Authors: Nomura Tadashi , Kawakami Atsushi , Fujisawa Hajime
Published: October 2003

Heterotopic transplantation of brain vesicles between chick and quail were performed, and the correlation between tectum formation and the expression of two PAX family genes, PAX7 and PAX6, analyzed. Reciprocal transplantation between the prosencephalon and mesencephalon showed that formation of the tectum always coincided with induction/maintenance of PAX7 and suppression of PAX6, indicating that switch‐on or ‐off of these two PAX family genes in region specific manners are responsible for the differentiation of brain vesicles into the tectum. On the other hand, transplantation of the mesencephalic floor plate into the dorsal mesencephalon suppressed PAX7 expression in the dorsal mesencephalon and changed its fate from the tectum to the tegmentum, indicating that factors in the mesencephalic floor plate suppress PAX7 and limit tectum territory to the dorsal part of the mesencephalon.
Gravity influences the position of the dorsoventral axis in medaka fish embryos (Oryzias latipes)
Authors: Fluck Richard A. , Krok Karen L. , Bast Brian A. , Michaud Sarah E. , Kim Cecilia E.
Published: October 2003

To determine whether gravity influences the plane of bilateral symmetry in medaka embryos, zygotes were placed with their animal‐vegetal axis orientated vertically and with their vegetal pole elevated. Then, at regular intervals during the first cell cycle, the zygotes were tilted 90° for about 10 min and subsequently returned to their original orientation. In embryos tilted during the first half of the first cell cycle, the embryonic shield formed on the side that had been lowermost when the zygote was tilted. In embryos that were tilted twice, first in one direction and then in the opposite direction, the embryonic shield formed on the side that was lowermost the first time. When zygotes were centrifuged at 5 g, the embryonic shield formed on the outwardly radial (centrifugal) side of the embryo. The orientation of the array of parallel microtubules in the vegetal pole region was also influenced by tilting or centrifuging zygotes. No correlation was found between the positions of the polar body and the micropyle and the plane of bilateral symmetry. It was concluded that gravity influences both the plane of bilateral symmetry and the orientation of microtubules in the vegetal pole region of medaka embryos.
Tissue interaction regulates expression of a spasmolytic polypeptide gene in chicken stomach epithelium
Authors: Tabata Hidenori , Yasugi Sadao
Published: October 2003

The primitive epithelium of embryonic chicken proventriculus (glandular stomach) differentiates, after day 6 of incubation, into luminal epithelium, which faces the lumen and abundantly secretes mucus, and glandular epithelium, which invaginates into mesenchyme and later expresses embryonic chicken pepsinogen (ECPg). So far it is not well understood how undifferentiated epithelial cells differentiate into these two distinct cell populations. Spasmolytic polypeptide (SP) is known to be expressed in surface mucous cells of mammalian stomach. In order to obtain the differentiation marker for proventricular luminal epithelial cells, we cloned a cDNA encoding chicken SP (cSP). Sequence analysis indicated that cSP has the duplicated cysteine‐rich domain characteristic of SP. Examination of the spatial and temporal expression pattern of cSP gene revealed that, during embryogenesis, cSP was expressed in luminal epithelial cells of the proventriculus, gizzard, small intestine, and lung, but not the esophagus. In the proventriculus, cSP mRNA was first detected on day 8 of incubation and was localized to differentiated luminal epithelial cells. By using cSP as a molecular marker, the effects of mesenchyme on the differentiation of epithelium were analyzed in vitro. On the basis of these data, a model is presented concerning the differentiation of proventricular epithelium.