bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of furrowing activity following regional cortical tension reduction in sand dollar blastomere and fertilized egg fragment surfaces
Authors: Rappaport R.
Published: March 2003

The purpose of the present investigation was to test experimentally the possibility that division mechanism establishment at the equator of sand dollar eggs may be a consequence of cortical tension gradients between the equator and the poles. Cytochalasin has been shown to decrease tension at the sea urchin egg surface. The concave ends of cytochalasin D‐containing agarose cylinders were held against regions of the surface of Echinarachnius parma blastomeres and enucleated fertilized egg fragments. The ability to interfere with normal furrowing activity was used as a biological indicator of the effectiveness of cytochalasin. When agarose containing 2 μg/mL cytochalasin contacted the equatorial region of the blastomeres resulting from the first cleavage, or the equatorial surfaces of nucleated fertilized egg halves, furrowing was blocked, stalled or delayed, indicating that the concentration of cytochalasin was effective. When the same concentration of cytochalasin was applied to the poles, the cells and nucleated fertilized egg fragments divided in the same way as the controls, indicating that the effectiveness of the cytochalasin did not spread from the poles to the equator and that bisection did not interfere with the division of nucleated fertilized egg fragments. When the same concentration of cytochalasin was applied to diametrically opposed surfaces of enucleated, spherical egg fragments, there was no evidence of furrowing activity between the areas that contacted the cytochalasin or in any other part of the surface. Because of the tension‐reducing effect of cytochalasin, a tension gradient existed between the regions affected and unaffected by cytochalasin. The results strongly suggest that establishment of the division mechanism by simple gradients of tension at the surface is unlikely.
Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis
Authors: Nakatani Yuki , Nishida Hiroki
Published: March 2003

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32‐cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32‐cell stage and is completed by the 64‐cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32‐cell embryos, and were heterochronously recombined at various stages ranging from the early 32‐cell stage to the 64‐cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32‐cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64‐cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.
A maternal RNA encoding smad1/5 is segregated to animal blastomeres during ascidian development
Authors: Kobayashi Ako , Sasakura Yasunori , Ogasawara Michio , Makabe Kazuhiro W.
Published: March 2003

Expressed Sequence Tag (EST) research on the ascidian Halocynthia roretzi revealed that Hrsmad1/5, a homolog of smad genes, is expressed in H. roretzi eggs. A comparison of amino acid sequences of smad family members showed that the isolated clone was a homolog of smad1 and smad5 of vertebrates. A molecular phylogenetic analysis showed that Hrsmad1/5 was separated from the common ancestor with the group containing smad1 and smad5. A northern blot analysis showed that transcript of Hrsmad1/5 was abundant in the fertilized egg. The amount of the transcript remained constant until the gastrulae and then rapidly decreased at the neurulae. The spatial expression of Hrsmad1/5 was investigated by means of whole‐mount in situ hybridization. Maternal transcripts of Hrsmad1/5 were detected in the entire fertilized egg. The signals were localized preferentially to the animal blastomeres of the 8‐, 16‐, 32‐ and 64‐cell stages. The zygotic expression of Hrsmad1/5 started in prospective epidermal blastomeres in the animal hemisphere at the 64‐cell stage but not in cells of the central nervous system, and it decreased rapidly around the neural‐plate stage. At the tail‐bud stage, signals were detected broadly all through the trunk and in a small part of the epidermis in the tail region. This is the first report of a maternal RNA that preferentially accumulates in the animal hemisphere in early ascidian embryos. Animal blastomeres of ascidian embryos differentiate mainly into epidermis in a cell‐autonomous manner and partly differentiate into neural tissues by induction. The Hrsmad1/5 gene may play a role in the signal transduction process in epidermal precursor cells of ascidian embryos.
Timing of the potential of micromere‐descendants in echinoid embryos to induce endoderm differentiation of mesomere‐descendants
Authors: Minokawa Takuya , Amemiya Shonan
Published: March 2003

It has been reported that the micromeres of echinoid embryos have the potential to induce an archenteron in animal cap mesomeres recombined at the 16‐ or 32‐cell stage. In the present study, experiments were performed to determine the exact period when the micromeres transmit their inductive signal to respecify the cell fate of mesomeres as endo‐mesoderm. An animal cap was recombined with a quartet of micromeres, or micromere‐descendants cultured in isolation, to form a recombinant embryo. The micromere‐descendants were completely removed at various developmental stages, resulting in an embryo composed only of mesomere‐descendants that had been under the inductive influence of micromeres for a limited period. The resulting embryos were cultured and examined for their potential to differentiate endoderm. The results indicated that the signal effective for inducing an archenteron in mesomere‐descendants emanated from the micromere‐descendants at the early blastula stage around hatching onward. Before this stage, the micromeres and micromere‐descendants showed this potential slightly or not at all. The inductive signal emanated from the micromere‐descendants almost on time even when the cells were cultured in isolation. The micromere‐descendants completed transmission of the signal for inducing the archenteron in the animal cap within 2 h of recombination. The animal cap at between the 28‐cell stage and 2 h after the 32‐cell stage could react with the inductive signal from the micromere‐descendants. Embryos composed of only animal cap mesomeres that had received the inductive signal from micromere‐descendants for a limited period had the potential to develop into 8‐armed plutei. Each pluteus formed an adult rudiment essentially on the left side of the larval body, and metamorphosed into a juvenile with pentaradiate symmetry.
Phosphorylation‐dependent regulation of skeletogenesis in sea urchin micromere‐derived cells and embryos
Authors: Cervello Melchiorre , Sanfilippo Rosalia , Isola Gabriella , Virruso Lucrezia , Scalia Giuliana , Cammarata Giuseppe , Gambino Roberto
Published: March 2003

Sea urchin embryo micromeres when isolated and culturedin vitrodifferentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signalling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose‐dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.
A starfish homolog of mouse T‐brain‐1 is expressed in the archenteron of Asterina pectinifera embryos: Possible involvement of two T‐box genes in starfish gastrulation
Authors: Shoguchi Eiichi , Satoh Nori , Maruyama Yoshihiko K.
Published: March 2003

A cDNA clone for a starfish T‐box gene (Ap‐Tbr) was isolated and characterized. Molecular phylogenetic analysis showed that the Ap‐Tbr gene was a member of the T‐brain subfamily, which includes mouse T‐brain‐1 and Xenopus Eomesodermin. Ap‐Tbr was expressed as early as in late blastulae, and the transcript was evident in a disc‐like region at the vegetal end or the vegetal plate. In early gastrulae, the gene was expressed in the cells of the invaginated archenteron, from which the majority of mesodermal cells as well as some endodermal cells are derived. The Ap‐Tbr expression disappeared by the end of gastrulation, and was not detected in early bipinnaria larvae. This expression pattern of Ap‐Tbr suggests its role in an early step of archenteron invagination, associated with mesoderm and endoderm formation. Furthermore, double staining of late blastulae and early gastrulae with a probe specific for Ap‐Tbr and one for ApBra (the starfish Brachyury gene) demonstrated that the regions of Ap‐Tbr and ApBra expression at the vegetal/posterior end of the embryo did not overlap, the region with Ap‐Tbr expression being encircled by a ring‐shaped region of ApBra expression. A gap without expression of the two genes was located between them, and the gap was seen around the blastoporal lip in the early gastrula. These observations suggest implication of the two T‐box genes, with different roles, in starfish gastrulation.
Cloning a novel developmental regulating gene, Xotx5: Its potential role in anterior formation in Xenopus laevis
Authors: Kuroda Hiroki , Hayata Tadayoshi , Eisaki Akira , Asashima Makoto
Published: March 2003

The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. In Xenopus, Xotx1, Xotx2, and Xotx4 have already been isolated and analyzed. Here the cloning, developmental expression and functions of the additional Otx Xenopus gene, Xotx5 are reported. This latter gene shows a greater degree of homology to Xotx2 than Xotx1 and Xotx4. Xotx5 was initially expressed in Spemann’s organizer and later in the anterior region. Ectopic expression of Xotx5 had similar effects to other Xotx genes in impairing trunk and tail development, and especially similar effects to Xotx2 in causing secondary cement glands. Taken together, these findings suggest that Xotx5 stimulates the formation of the anterior regions and represses the formation of posterior structures similar to Xotx2.
Spatio‐temporal expression of Xenopus vasa homolog, XVLG1, in oocytes and embryos: The presence of XVLG1 RNA in somatic cells as well as germline cells
Authors: Ikenishi Kohji , Tanaka Tetsuya S.
Published: March 2003

The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole‐mount in situ hybridization and reverse transcription–polymerase chain reaction (RT‐PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm‐bearing cells (GPBC) from 32‐cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I–III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life‐span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49–50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT‐PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells.
In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid
Authors: Moriya Naomi , Komazaki Shinji , Asashima Makoto
Published: March 2003

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10–4M retinoic acid for 3 h differentiated into pancreas‐like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus‐like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas‐specific molecular markers, XlHbox8 and insulin, were induced in retinoic acid‐treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas‐like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.
Gene transfer into chicken embryos as an effective system of analysis in developmental biology
Authors: Yasugi Sadao , Nakamura Harukazu
Published: March 2003

Chicken embryos have been used as a model animal in developmental biology since the time of comparative and experimental embryology. Recent application of gene transfer techniques to the chicken embryo increases their value as an experimental animal. Today, gene transfer into chicken cells is performed by three major systems, lipofection, electroporation and the virus‐mediated method. Each system has its own features and applicability. In this overview and the associated four minireviews, the methods and application of each system will be presented.
Expression of testicular fatty acid‐binding protein PERF 15 during germ cell apoptosis
Authors: Kido Tatsuo , Namiki Hideo
Published: March 2003

PERF 15 is a testicular germ cell specific fatty acid‐binding protein (FABP) isolated from rat. Indirect immunofluorescent analysis of juvenile rat testis showed that there were some strongly PERF 15‐positive spermatocytes. These cells showed unclear nuclear structure and were predicted to undergo apoptosis. Apoptosis in germ cells is an important regulatory event to limit the number of germ cells in the seminiferous epithelium, but the physiological significance and molecular mechanisms of this testicular germ cell apoptosis are poorly understood. To determine whether PERF 15 participates in germ cell apoptosis, juvenile rat testis was examined by immunohistochemical and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL) methods. Strongly PERF 15‐positive cells and TUNEL‐positive cells were co‐localized in adjacent sections. Exposure to methoxyacetic acid (MAA), known to induce apoptosis in spermatocytes, increased the number of strongly PERF 15‐positive cells in 25‐day‐old rats’ testes. Therefore, it seems that PERF 15 is involved in both spermatogenesis and testicular germ cell apoptosis.
Loss of reactivity to pan‐cadherin antibody in epidermal cells as a marker for metamorphic alteration of Xenopus skin
Authors: Izutsu Yumi , Tochinai Shin , Onoé Kazunori
Published: March 2003

Pan‐cadherin antibodies recognize the conserved C‐terminal region of the family of cell–cell adhesion molecules, cadherins, and have a broad spectrum of reactivity to the molecules. In the present study, by immunohistochemistry using an anti‐pan cadherin monoclonal antibody (mAb), expression dynamics of cadherins in epidermal tissues were analyzed during metamorphosis of Xenopus laevis. At early stages of development, the anti‐pan cadherin mAb detected signals at cell–cell boundaries and in the cytoplasm of both trunk and tail epidermal cells. During metamorphosis, the immunoreactivity decreased in the trunk skin tissue but remained in the tail. At the climax stage, immunoreactivity was observed only in the regressing tail epidermis. The signals disappeared completely from the trunk epidermis, which had already transformed into adult‐type tissue. This observation was confirmed by western blot analysis. A specific band was detected in the larval skin, but not in the adult lysate, at approximately 135 kDa in molecular size, corresponding to the molecular mass of cadherins. This different immunoreactivity in larvae and adults was observed in the epidermis of the skin, but not in any other tissues examined, that is, brain, kidney and liver. The immunoreactivity seen in larval epidermal cells was drastically downregulated by thyroid hormone treatment in vitro. These changes of immunoreactivity were specific for the C‐terminal region of cadherins, suggesting intracellular alteration of the molecules during metamorphosis, and the anti‐pan cadherin mAb can be a marker for larval‐type epidermal cells that is applicable to analysis of Xenopus metamorphosis.
Activity of the medaka translation elongation factor 1α‐A promoter examined using the GFP gene as a reporter
Authors: Kinoshita Masato , Kani Shuichi , Ozato Kenjiro , Wakamatsu Yuko
Published: March 2003

The translation elongation factor 1α (EF‐1α) is known to have several isoforms, which are expressed in a tissue‐ and stage‐specific manner. Two genes encoding EF‐1α exist per haploid genome in the medaka. In the present study, the promoter activity of the 5′‐flanking region of the medaka EF‐1α‐A gene, an isoform of EF‐1α, was characterized using transgenic techniques. First, using CAT gene as a reporter, it was revealed that about 1.8 kbp 5′‐flanking sequence from the transcription initiation site of EF‐1α‐A was sufficient for high‐level promoter activity. Second, the green fluorescent protein (GFP) gene fused to this region was introduced into medaka eggs using the microinjection method. Three germline transgenic individuals (one male and two female) were mated with non‐transgenic medaka to obtain F1 offspring. In the case of embryonic and adult F1 transgenic individuals, GFP fluorescence was observed in almost all the tissues examined (e.g. kidney, liver, heart, gill, ovary, and testis), except for the skeletal muscle. In the case of F2 transgenic embryos derived from F1 transgenic males and non‐transgenic females, the fluorescence was observed from the early gastrula stage. On the other hand, in the case of F2 transgenic embryos derived from F1 transgenic females and non‐transgenic males, the fluorescence was observed even at the 1‐cell stage, suggesting that this region is transcriptionally active during oogenesis. The usefulness of the EF‐1α‐A promoter as a tool for introducing foreign proteins into oocytes is discussed.
Direct molecular interaction of a conserved yolk granule protein in sea urchins
Authors: Wessel Gary M. , Zaydfudim Victor , Hsu Yuin‐tsee Jennifer , Laidlaw Michael , Brooks Jacqueline M.
Published: March 2003

The regulation of yolk storage in oocytes and subsequent utilization in embryos is critical for embryogenesis. In sea urchins, the major yolk protein is made in the intestines, transported to the ovaries and accumulated in developing oocytes within membrane‐bound vesicles comprising approximately 10% of the mass of an egg. Here, a non‐yolk protein that accumulates specifically in yolk granules is reported. This protein was identified by cDNA cloning and, by use of antibodies to the recombinant protein, it was shown that this molecule is stored selectively in yolk granules of oocytes and embryos. No accumulation was seen in the accessory cells, testis, or intestines. In situ ribonucleic acid (RNA) hybridizations showed that the transcript accumulated only in oocytes, and was more highly concentrated in young oocytes. However, later in oogenesis, the messenger ribonucleic acid (mRNA) levels decreased significantly so that no signal was detectable in mature haploid eggs or at any later stage in development. However, by immunofluorescence and western blot analysis, the 30 kDa band was present throughout development. The predicted sequence of this protein shows that it is a member of the bep, HLC‐32, EBP family of sea urchin proteins, but as it does not accumulate at the cell surface, nor in the hyaline layer in the two species studied here, as do other members of the family, it has been referred to as YP30 (30 kDa protein of the yolk platelet). To address its potential function, yeast two‐hybrid analysis was performed to screen for proteins that potentially interact with YP30. It was found that it binds itself, and forms strongly interacting dimers. It is hypothesized that YP30 participates in the packaging and storage of major yolk protein during oogenesis, or in the utilization of the major yolk protein in development.
Ontogeny of telomerase in chicken: Impact of downregulation on pre‐ and postnatal telomere lengthin vivo
Authors: Taylor Heather A. , Delany Mary E.
Published: March 2003

Telomeres are the termini of linear chromosomes composed of tandem repeats of a conserved DNA sequence. Telomerase provides a mechanism for proliferating cells to offset telomeric sequence erosion by synthesizing new repeats onto the end of each parental DNA strand. Reduced or absent telomerase activity can lead to telomere shortening and genome instability. Telomeres and telomerase have not previously been characterized during ontogeny of any avian species. In the present study, telomerase activity in the chicken model was examined from early differentiation embryos through to adulthood. Telomerase activity was detected in all early embryos (preblastula through neurula) and in tissues throughout organogenesis. Subsequently, telomerase was downregulated in the majority of somatic tissues, either pre‐ or postnatally. A subset of tissues, such as intestine, immune and reproductive organs, exhibited constitutive activity. The impact of telomerase downregulation on telomere length was investigated and a telomere reduction of 3.2 kb in somatic tissues compared with germ line was observed in 5‐year‐old adults. The present results suggest that the telomere clock function is a conserved feature of avians as well as mammals. Knowledge regarding the relationships among telomerase regulation, proliferation/senescence profiles and differentiation status will be useful for numerous applications of chicken cells.
Identification of the aromatic L‐amino acid decarboxylase (AADC) gene and its expression in the attachment and metamorphosis of the barnacle, Balanus amphitrite
Authors: Okazaki Yuichi , Shizuri Yoshikazu
Published: March 2003

Serotonin and dopamine are involved in the attachment and metamorphosis of cypris larvae of barnacles. Aromatic L‐amino acid decarboxylase (AADC) gene, the product of which catalyzes the synthesis of serotonin and dopamine from L‐5‐hydroxytryptophan and L‐3,4‐dihydroxyphenylalanine, respectively, was characterized. A DNA clone containing part of an AADC sequence was obtained from the genomic DNA library of the barnacle, Balanus amphitrite. This clone had four putative exons consisting of 226 amino acids with an identity of 63.2% and a similarity of 92.1% with human AADC. Northern blot analysis showed that AADC mRNA was expressed at all stages of barnacles: naupliar larvae, cypris larvae and adult barnacles. Two inducers of larval attachment and metamorphosis; that is, serotonin and extract of adult barnacles, obviously increased the expression of AADC mRNA at an early cypris larval stage. These results suggest that intracellular biosynthesis of serotonin, or dopamine, or both is at least partly involved in the control of the attachment and metamorphosis of cypris larvae.
Serotonin localization in Phallusia mammillata larvae and effects of 5‐HT antagonists during larval development
Authors: Pennati Roberta , Groppelli Silvia , Sotgia Cristina , Candiani Simona , Pestarino Mario , De Bernardi Fiorenza
Published: March 2003

The neurotransmitter 5‐hydroxytryptamine (5‐HT, serotonin) plays an important role in a wide range of non‐neural processes. Using immunofluorescence with an antiserotonin antibody, 5‐HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5‐HT on development, we treated embryos with two different 5‐HT receptor subtype antagonists. Treatment at the gastrula stage with 10 μM ondansetron, an antagonist of the 5‐HT3 receptor, induced anterior truncation and a short tail. At 10 μM, ritanserin, a 5‐HT2B receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5‐HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5‐HT is suggested.
Cloning and characterization of the Xenopus laevis p8 gene
Authors: Igarashi Toshime , Kuroda Hiroki , Takahashi Shuji , Asashima Makoto
Published: March 2003

The p8 gene encodes a transcription factor with a basic helix‐loop‐helix motif and has been cloned in rat, mouse and human. It is upregulated in acute pancreatitis. In the present study, the Xenopus laevis homolog of p8 (Xp8) was isolated by PCR. The full‐length Xp8 cDNA consists of 677 bp and encodes 82 amino acids. The basic helix‐loop‐helix region is well conserved between X. laevis and mammals. Overexpression of an Xp8‐EGFP fusion protein indicated that Xp8 was localized to nucleus, as in mammals. Analysis by RT–PCR showed that expression of Xp8 was increased after gastrulation and maintained into later developmental stages. Expression was detected in the prospective neural region at the neurula stage by whole‐mount in situ hybridization. At the larval stage, Xp8 was expressed in central nervous regions, such as ventrolateral brain, trigeminal nerve, vestibulocochlear nerve and dorsal neural tube. Expression was also detected in the basal cement gland, olfactory placode, ear vesicle, notochord and anus. This specific pattern of expression suggests that Xp8 may play a role in the development of the central nervous system in X. laevis.
Absorption, transportation and digestion of egg white in quail embryos
Authors: Yoshizaki Norio , Ito Yasushi , Hori Hideyuki , Saito Hiroshi , Iwasawa Atsushi
Published: March 2003

The present study was done to reveal how egg white is taken up by embryonic tissues, the pathway through which egg white is transported, and the location where it is digested during the development of the quail Coturnix japonica. Antiserum against quail ovalbumin was raised in rabbit and used as a probe. By immunoelectron microscopy, the uptake of ovalbumin on a small scale by receptor‐mediated endocytosis was observed in the ectodermal cells of the yolk sac on days four to seven of incubation. The uptake of egg white on a large scale by fluid‐phase endocytosis took place in the cells generally referred to collectively as the ‘albumen sac’. The ovalbumin was transported through the albumen sac into the extraembryonic cavity during days eight to 10, and then into the amniotic cavity through the amnion approximately on day 10. Ovalbumin was present in the intestinal lumen on days 11 and 14, but it was not digested in the intestinal epithelial cells. The ovalbumin was detected in the yolk of embryos after day 10. Immunoblot testing, as well as a fluoroimmunoassay, revealed that the location where the amount of ovalbumin was highest changed chronologically from the extraembryonic cavity on day 10 to the amniotic cavity on day 11, the intestinal lumen on day 12 and then to the yolk on day 13. Several low molecular proteins which cross‐reacted with the antiserum were observed in the extracts of the yolk. The reaction producing these proteins depended on low pH (approximately 3.0) and was inhibited by pepstatin A. The ovotransferrin was similarly digested. These results indicate that egg white is, for the most part, transported through the albumen sac to the yolk via the extraembryonic cavity, the amniotic cavity, and the intestinal lumen, and is digested in the yolk by aspartic proteinases.
Xenopus Brachyury regulates mesodermal expression of Zic3, a gene controlling left–right asymmetry
Authors: Kitaguchi Tetsuya , Mizugishi Kiyomi , Hatayama Minoru , Aruga Jun , Mikoshiba Katsuhiko
Published: March 2003

The Brachyury gene has a critical role in the formation of posterior mesoderm and notochord in vertebrate development. A recent study showed that Brachyury is also responsible for the formation of the left–right (L–R) axis in mouse and zebrafish. However, the role of Brachyury in L–R axis specification is still elusive. Here, it is demonstrated that Brachyury is involved in L–R specification of the Xenopus laevis embryo and regulates expression of Zic3, which controls the L–R specification process. Overexpression of Xenopus Brachyury (Xbra) and dominant‐negative type Xbra (Xbra‐EnR) altered the orientation of heart and gut looping, concomitant with disturbed laterality of nodal‐related 1 (Xnr1) and Pitx2 expression, both of which are normally expressed in the left lateral plate mesoderm. Furthermore, activation of inducible type Xbra (Xbra‐GR) induces Zic3 expression within 20 min. These results suggest that a role of Brachyury in L–R specification may be the direct regulation of Zic3 expression.
In vitro cultivation of spermatocysts to matured sperm in the silkworm Bombyx mori
Authors: Kawamura Naoko , Sahara Ken
Published: March 2003

Bombyx spermatogonia are bipotential, producing nucleate eupyrene sperm and anucleate apyrene sperm. An in vitro cultivation of spermatocysts of Bombyx mori from spermatocytes to matured sperm was established. The present experiment made clear that: (i) spermatocysts must be isolated; (ii) constant shaking at 45 r.p.m. was necessary; and (iii) the addition of Bombyx hemolymph (BH) was indispensable for successful cultivation. In the absence of BH, spermatogenesis proceeded normally for 2 or 3 days and, thereafter, spermatocytes and sperm bundles began to degenerate. The best results for normal eupyrene spermatogenesis were obtained when culture medium containing BH of the corresponding stage was used in every exchange of the medium at 72 h intervals. None or only a small number of apyrene sperm bundles was produced by this culture system when spermatocysts from larval testes were used, although eupyrene spermatogenesis proceeded normally to form matured, or squeezed, sperm bundles.
Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis
Authors: Obinata Akiko , Akimoto Yoshihiro , Omoto Yasumasa , Hirano Hiroshi
Published: March 2003

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline‐rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two‐fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.
Localization and functional role of a 41 kDa collagenase/gelatinase activity expressed in the sea urchin embryo
Authors: Mayne Janice , Robinson John J.
Published: March 2003

The egg storage compartment of the sea urchin embryo was investigated for a protein destined for export to the extracellular matrices. Using an antiserum prepared against a 41 kDa collagenase/gelatinase localized to the extraembryonic matrices (the hyaline layer and basal lamina), the egg storage compartment was mapped for this antigen. Indirect immunofluorescence analysis revealed the 41 kDa collagenase/gelatinase in the cortical granules as well as a second compartment which was dispersed throughout the egg cytoplasm. High resolution immunogold labeling defined this cytoplasmic compartment as the yolk granule organelle. Gelatin substrate gel zymography revealed the presence of a 41 kDa gelatin cleavage activity in purified yolk granules. These results suggest a role for yolk granules in regulated protein export and challenge the traditional view of this organelle as a benign storage compartment for nutrients. In additional experiments, embryos grown in the presence of the 41 kDa cleavage activity or the anti‐41 kDa antiserum had severely delayed gut formation and spicule elongation. These results demonstrate a requirement for defined levels of the 41 kDa activity in the extracellular matrices of the developing embryo.
Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique
Authors: Knobloch Jürgen , Kunz Werner , Grevelding Christoph G.
Published: March 2003

The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4′,6‐diamidino‐2‐phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5‐bromo‐2′‐deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.
Spore dispersal of Dictyophora fungi (Phallaceae) by flies
Authors: Tuno Nobuko
Published: March 2003

The composition and food habits of insects visiting fungi of two Dictyophora species, D. indusiata (Vent. & Pers.) and D. duplicata Fisch, were examined in Furano, northern Japan, and in Kyoto, central Japan. As well as field work being carried out, the quantity and the viability of spores in the recta of drosophilid and muscid flies were examined in vitro. Although the composition of insects varied locally and temporally, most of the insects were observed to feed on gleba, which contains spores. Among the insect assemblies, a few insects were specialized for mycophagy but most were secondarily or not at all mycophagous. Although Dictyophora‐feeders rarely attached the spores on their body surfaces, they contained a quantity of spores in their gut, which was estimated to be about 35 000–240 000 for drosophilids and about 1.7 million for muscids. Those spores showed high germination rates, which were not significantly different from the intact spores. Thus, spores of Dictyophora are dispersed as excrement through the gut of Dictyophora‐feeding insects but not as adherers on the insect body.
Foraging habitat shift in the narcissus flycatcher, Ficedulanarcissina, due to the response of herbivorous insects to the strengthening defenses of canopy trees
Authors: Murakami Masashi
Published: March 2003

The indirect effect of tree defense on bird foraging habitat via lepidopterous larvae was studied by focusing on phenological changes in each component. The seasonal changes in foraging habitat of the narcissus flycatcher (Ficedula narcissina), the distribution pattern of arthropod populations, and the leaf characteristics related to the defense system against insect herbivores were studied in a cool‐temperate deciduous forest in northern Japan. Narcissus flycatchers foraged in the canopy from late May to mid June, on the ground from late June, with lepidopterous larvae being the most preferred prey (about 80% of total prey items), and again in the canopy in July. The biomass of lepidopterous larvae was much more abundant within 3 weeks after budbreak in the canopy in late May, and thereafter decreased rapidly in mid June. By contrast, the larvae were abundant on the forest floor from early to late June. These were mainly caused by their downward migration in early to mid June. After budbreak, the leaf toughness and tannin content of two deciduous tree species (maple and oak) increased, while nitrogen and water content decreased. The strengthening of defense traits of canopy leaves caused changes in the abundance and distribution of the lepidopterous larvae. The foraging habitat of the flycatcher subsequently shifted in response to the changes in abundance and distribution of the lepidopterous larvae. In this study, the evidence of the indirect effects of trees on bird foraging habitat was presented by focusing on the phenological changes in relationships among each component in this three‐trophic system.
Effects of micro‐environmental factors on photosynthetic CO2 uptake and carbon fixation metabolism in a spring ephemeral, Erythronium japonicum, growing in native and open habitats
Authors: Sawada Shinichi , Harada Akira , Asari Yoshiko , Asano Sachie , Kuninaka Makoto , Kawamura Hitomi , Kasai Minobu
Published: March 2003

Microenvironmental factors and physiological parameters (such as water potential, activity of ribulose 1,5‐bisphosphate carboxylase (RuBPcase), levels of ribulose 1,5‐bisphosphate (RuBP), 3‐phosphoglyceric acid (PGA) and sucrose in leaves) affecting photosynthetic processes of the typical vernal species Erythronium japonicum Decne. were examined on the floor of a deciduous broad‐leaved Quercus mongolica forest (Q.m. stand) and on bare land left undisturbed for 7 years after forest clearing (bare stand). Daytime solar radiation and the air and leaf temperatures at the bare stand were significantly higher than those at the Q.m. stand. The relative air humidity was very low and did not differ much between the stands, whereas the leaf–air vapor pressure difference (VPD) at the bare stand was twice as high as that at the Q.m. stand. The water potential in leaves at the bare stand was lower than two times that at the Q.m. stand. Therefore, the aboveground parts of the plants at the bare stand were subjected to much more severe heat stress than those at the Q.m. stand. When these environmental factors observed at the bare stand were reproduced in an assimilation chamber, the rate of photosynthetic CO2 uptake, stomatal conductance and water potential in leaves were significantly low in comparison with those when the factors at the Q.m. stand were simulated. The internal CO2 partial pressure in leaves at the bare stand was considerably lower than that at the Q.m. stand. Consequently, the decrease in the photosynthetic rate of the plants at the bare stand was caused mainly by a decrease in stomatal conductance through a lowering of water potential due to subjection of the aboveground parts to much more severe heat stress than that at the Q.m. stand. The possibility that an inhibition of the photosynthetic carbon fixation metabolism induced by the decrease in water potential contributes to the reduction in photosynthetic CO2 uptake in the plants at the bare stand is also discussed in light of physiological characteristics such as the activity of RuBPcase and levels of PGA, RuBP and sucrose in the leaves.
Belowground carbon storage of Micronesian mangrove forests
Authors: Fujimoto Kiyoshi , Imaya Akihiro , Tabuchi Ryuichi , Kuramoto Shigeo , Utsugi Hajime , Murofushi Tamon
Published: March 2003

Belowground carbon storage was examined for mangrove forests on Pohnpei Island, Micronesia. Stored carbon in a coral reef‐type mangrove habitat consisting of a 2 m thick mangrove peat layer, which is a type of mangrove habitat in tropical Pacific islands, was estimated at 1300 t C ha−1. The carbon burial rate during the phase of gradual sea‐level rise, which was calculated at 93 g m−2 year−1 between 1800 and 1380 years
BP using the medians of the radiocarbon ages, was significantly higher than that between 1380 years
BP and present in a stable sea‐level phase.
Contribution of fern gametophytes to the growth of produced sporophytes on the basis of carbon gain
Authors: SAKAMAKI YOSHIAKI , INO YOSHIO
Published: March 2003

Sporophytes appeared on most gametophytes of Thelypteris palustris (Salisb.) Schott that reached a certain size, which is interpreted to be a critical size of gametophytes for the production of sporophytes. After sporophytes were produced, attached gametophytes ceased dry weight growth, but the gametophytes which did not produce sporophytes grew successively. It was hypothesized that matter produced by gametophytes was being supplied to young sporophytes. Photosynthesis and respiration of gametophytes with attached sporophytes were not significantly different from that of gametophytes without sporophytes. Photosynthetic activity of gametophytes dropped from 0.18 to 0.03 μmol CO2 g−1 s−1 during the growth period. The higher photosynthetic rates of gametophytes in the early growth stage were important for reaching the critical size for sporophyte production in a short time. Sporophytes in the ‘one leaf stage’ averaged 0.14 μmol CO2 g−1 s−1 of photosynthetic activity. The results show that sporophytes that had expanded the first leaf grow by their own photosynthetic production. Gametophytes allocated the photosynthate for sporophytes and it was an important aid before the one‐leaf stage. The supportive role of gametophytes ended at that stage.
Nitrogen resorption from needles of Pinus thunbergii Parl. growing along a topographic gradient of soil nutrient availability
Authors: ENOKI TSUTOMU , KAWAGUCHI HIDEYUKI
Published: March 2003

To examine the relative importance of nutrient resorption in increasing the nutrient‐use efficiency of Pinus thunbergii Parl., we investigated the nitrogen contents of green and senescent needles of P. thunbergii trees growing at five positions along a slope (LS, lower slope; TR, transitional site; MS, middle slope; US, upper slope; RG, ridge) and found that soil nitrogen availability tended to decrease upslope. Nitrogen concentration in green and senescent needles decreased upslope. Nitrogen resorption efficiencies (percentage change in nitrogen content between green and senescent needles) increased upslope from 43 to 77% with decreasing soil nitrogen availability. Nitrogen resorption efficiency was related to green needle dry mass per unit length, but there was no clear correlation between nitrogen resorption efficiency and nitrogen content in green needles. We concluded that the increase in nitrogen resorption efficiency of P. thunbergii enhanced the nitrogen‐use efficiency as a response to the low nitrogen availability.
Seed dispersal and predation by Yakushima macaques, Macaca fuscata yakui, in a warm temperate forest of Yakushima Island, southern Japan
Authors: Otani Tatsuya , Shibata Ei’ichi
Published: March 2003

The relationships between fruit morphology and the foraging behavior of the Yakushima macaque, Macaca fuscata yakui were studied during a 9‐month field study and series of laboratory experiments on Yakushima Island, southern Japan. These relationships may affect seed germination traits through seed dispersal. The macaques’ foraging behavior was observed in order to obtain data concerning the treatment of pulp and seeds. Seeds in feces and spat seeds were collected and analyzed. A linear discriminant analysis of the data led to the following results: (i) an increase in the relative flesh volume of a fruit was one of the potential factors which increased the probability of seed dispersal by the macaques; and (ii) seed size was an important parameter in relation to the type of seed dispersal (by defecation or spitting out). The germination test was conducted in a laboratory, then the germination rates and delays of dispersed seeds were compared with those of seeds collected directly from trees. The germination of dispersed seeds of Ficus thunbergii, Eurya japonica, and Vaccinium bracteatum, was significantly enhanced by passage through the macaques’ gut. The enhanced germination behavior was not seen in larger seeds, such as those of Psychotria serpens, Myrsine seguinii, Diospyros morrisiana, and Neolitsea sericea. The germination enhancement in small‐seeded plants could be due to a sorting effect from the passage through the gut, which selects seeds with a narrower range of germination traits.
No distributional association between the tick Haemaphysalis longicornis (Acari: Ixodidae) and plant surface area
Authors: Tsunoda Takashi , Mori Keiji
Published: March 2003

The questing height of the tick Haemaphysalis longicornis Neumann was studied on the plant species Solidago altissima L., Pteridium aquilinum Kuhn, Miscanthus sinensis Anderss, and Pleioblastus chino Makino in an abandoned rice field and in an ume (Japanese Apricot) orchard in a forest. On all plant species, the vertical distribution of tick adults and nymphs was not significantly correlated with the distribution of plant surface area. This result suggests that the questing height of this tick is related to the body height of its main animal host, the sika deer.