bluuebunny/crossref_metadata_2025 huggingface dataset:
Two major mechanisms regulating cell‐fate decisions in the developing nervous system
Authors: Okano Hideyuki
Published: October 2003

Two types of determinants appear to be responsible for the generation of neural cell diversity: non‐cell‐autonomous and cell‐autonomous cues. We have identified both types of determinants through the intensive screening of P‐element induced Drosophila mutants affected in neural development. As a member of the first category argos (also referred to as strawberry or giant lens), which regulates cell–cell interaction in the developing nervous system, needs to be mentioned. On the basis of the phenotype on loss of function of argos. its expression pattern and the predicted structure of its product (a secreted protein with a putative epidermal growth factor (EGF) motif) we propose that argos encodes a diffusible protein with pleiotropic functions that acts as a signal involved in lateral inhibition within the developing nervous system and also as a factor involved in axonal guidance. As a member of the second category, I refer to the Drosophila musashi gene that is required for lineage formation and asymmetric division of precursor cells in the developing nervous system. The musashi gene encodes a neural RNA‐biding protein and is thus likely to regulate the asymmetric cell division of neural precursor cells by controlling the expression of target genes at the post‐transcriptional level. We have identified its mammalian homologue, mouse‐musashi‐1 (m‐Msi‐1). In the developing central nervous system (CNS), m‐Msi‐1 expression was highly enriched to neural precursor cells as is the expression of nestin. Based on the results of a single cell culture experiment, m‐Msi‐1 expression appears to be associated with multipotent cells that are capable of self‐renewal and with the generation of committed precursor cells of both neurons and glia. However, fully differentiated neuronal and glial cells lost their m‐Msi‐1 expression. The expression of m‐Msi protein showed a complementary pattern to that of another mammalian RNA‐binding protein Hu, which is localized in differentiated neurons in the CNS. Based on such differential expression patterns and its similarity to the Drosophila musashi, we propose that a combination of neural RNA‐binding proteins are required for the asymmetric distribution of intrinsic determinants in the developing mammalian nervous system. The Drosophila glial‐specific homeobox protein, Repo, can also be classified as a cell‐autonomous cue regulating cell‐fate decision during neural development. Repo expression is required for terminal differentiation and for the survival of glial cells.
Relationship between ectopic germinal vesicle breakdown in Xenopus oocytes and dorsal development of the embryo
Authors: Rivera‐Bennetts Alma Karina , Elinson Richard P.
Published: October 2003

The early development of several species involves the segregation of cytoplasmic components into different regions of the egg. In Xenopus zygotes, a 30° rotation displaces the central animal cytoplasm to the future dorsal side of the embryo. To elucidate the role of the central animal cytoplasm in dorsal determination, we induced germinal vesicle breakdown (GVBD) closer to the equator by cold/centrifugation treatment of oocytes. Centrifugation moved the germinal vesicle to the centripetal side; eggs with such displaced GVBD fertilized and began to develop normally. Dorsal embryonic structures tended to develop on the GVBD side of the egg, but displacement of the GVBD was insufficient to rescue dorsal structures in axis‐deficient embryos. The labeling of yolk platelets of oocytes with Trypan Blue revealed similar cytoplasmic patterns in control and treated eggs. Furthermore, 67% of treated eggs had Danilchik's swirl, indicative of the dorsal side, on the GVBD side. In conclusion, both the swirl and dorsal development tend to occur on the GVBD side of cold/centrifuged eggs; however, displaced GVBD cannot by itself determine dorsality.
Vertical versus planar induction in amphibian early development
Authors: Nieuwkoop P. D. , Koster K.
Published: October 2003

In the Urodeles, the archenteron roof invaginates as a single continuous sheet of cells, vertically inducing the neural anlage in the overlying ectoderm during invagination. The induction comprises first the activation process, leading, to forebrain differentiation tendencies, and then the superimposed transformation process, which changes presumptive forebrain development into that of hindbrain and spinal cord acting with a caudally increasing intensity. The activating action, being maximal anteriorly, decreases caudally to nearly zero. In the double‐layered Xenopus embryo, the internal mesodermal marginal zone shows much more independent and earlier regional segregation and involution than the external marginal zone in the Urodeles; its prechordal mesoderm already initiating vertical neural induction in overlying ectoderm at stages 10 to 10+ before any visible archenteron invagination. In Xenopus incomplete exogastrulae the prechordal mesoderm involutes normally prior to evagination of the endoderm and mesodem. Artificially produced Xenopus total exogastrulae, made at stage 9 before mesoderm involution, behave just like axolotl total exogastrulae, showing no neural differentiation. The notion of planar neural induction in Xenopus can only be applied in exogastrulae and Keller explants for the transforming action, which is maximal in the caudal archenteron roof. In normal Xenopus development, the formation of the entire nervous system is essentially due to vertical induction by the successively involuting prechordal and notochordal mesoderm. The different behavior of Xenopus embryos in comparison with Urodele embryos can essentially be explained by the double‐layered character of the animal moiety of the Xenopus embryo.
Two‐step regulation of ecdysone‐inducible late puffs in salivary glands of Drosophila melanogaster
Authors: Asaoka Miho , Myohara Maroko , Okada Masukichi
Published: October 2003

Our previous study showed that some ecdysone‐inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two‐step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin‐responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.
Role of calcium influx during the latent period in sea urchin fertilization
Authors: Créton Robbert , Jaffe Lionel F.
Published: October 2003

After ∼7–40 s following gamete fusion, a steadily increasing fraction of a sea urchin's zygotes initiate an activating calcium wave. The fertilization membrane then rises, the cell cycle resumes and development begins. This study focuses on the so‐called latent period that occurs between the time that gamete fusion occurs and the initiation of the activating calcium wave. We inhibited calcium influx during this period by adding lanthanum or by reducing external calcium with a buffer at various time points after insemination. Both of these treatments blocked the activation of eggs that had not yet started a wave at the time of treatment. This indicates that an influx of calcium is needed during the latent period to induce egg activation. These results support the sperm conduit model of egg activation in the sea urchin, where calcium flows from the sea through the fused sperms' acrosomal process into a cortical region of the eggs' endoplasmic reticulum.
Putative morphogen, DIF, of Dictyostelium discoideum induces apoptosis in rat pancreatic AR42J cells
Authors: Kubohara Yuzuru , Kimura Chiharu , Tatemoto Kazuhiko
Published: October 2003

We have recently shown that differentiation‐inducing factor‐1 (DIF‐1) of Dictyostelium discoideum is capable of raising intracellular calcium concentration ([Ca2+]i) and suppressing cell proliferation of rat pancreatic AR42J cells in a dose‐dependent manner, and that DIF‐1 at a concentration of 40 μmol/L is toxic to the cells. In this study, we have further characterized the cytotoxic effect of DIF‐1 on AR42J cells and have analyzed the effect of DIF‐1 on [Ca2+]i. In the presence of 40 μmol/L DIF‐1, cells began to bleb after approximately 6 h, and most had died within 48 h. Biochemical analysis revealed that DNA fragmentation was accompanied by cell death. Monitoring the changes in [Ca2+]i induced by DIF‐1, it was found that cells were able to adapt to stimulation with DIF‐1 so that they did not respond to subsequent stimulation by DIF‐1. These results indicate that DIF‐1 induced apoptosis in AR42J cells probably via a cell signaling system.
Oviduct histochemistry and site of synthesis of a 29.7 kDa jelly coat glycoprotein in the anuran Lepidobatrachus laevis
Authors: Perchez Michele A. , Carroll Edward J.
Published: October 2003

The oviduct of the anuran Lepidobatrachus laevis contains three morphological regions, each of which contains a histochemically distinct luminal mucosa. In the pars recta, the most anterior portion of the oviduct, there are periodic acid‐Schiff base (PAS)‐positive simple glands and epithelia. In the pars convoluta, there are alcian blue‐positive, combined alcian blue‐ and PAS‐positive and PAS‐positive gland types. The most posterior region, the pars uterina, contains alcian blue‐positive and alcian blue‐negative epithelial cells. Previous work has shown that solubilized egg jelly contains a major 29.7 kDa glycoprotein subunit that was detected in oviduct tissue extracts from the pars convoluta in the present study. Rabbit antisera to the 29.7 kDa egg jelly glycoprotein of L. laevis reacted with the major pars convoluta glycoprotein and there were no immunoreactive components in the pars uterina. The slight immunoreactivity detected at 29.0–37.0 kDa in pars recta extracts is not believed to be the jelly molecule, based on low immunoreactivity and subunit molecular weight measurements. We conclude that the synthesis of the 29.7 kDa egg jelly glycoprotein is restricted to the pars convoluta region of the oviduct.
The ter mutation responsible for germ cell deficiency but not testicular nor ovarian teratocarcinogenesis in ter / ter congenic mice
Authors: Noguchi Motoko , Watanabe Chiaki , Kobayashi Takashi , Kuwashima Miho , Sakurai Takayuki , Katoh Hideki , Moriwaki Kazuo
Published: October 2003

The ter (teratoma) gene causes germ cell deficiency and a high incidence of congenital testicular teratomas derived from primordial germ cells in 129/Sv‐ter strain mice. Ovarian teratomas in LTXBJ mice originate from ovarian parthenotes. In order to study the function of the ter gene in germ cell development and teratocarcinogenesis, we examined the influence of a foreign genetic background on the ter action by introducing the ter gene of 129/Sv‐ter strain mice into C57BL/6J, LTXBJ and C3H/HeJ genetic backgrounds by the backcross method and by thus establishing B6‐ter, LTXBJ‐ter and C3H‐ter ter congenic strains, respectively. Histological analysis showed that germ cell deficiency occurred in both sexes of the ter mutants, through the fetal stages to adulthood, but that congenital testicular teratocarcinogenesis did not occur after the fifth backcross generation. The ter/ter gonads were smaller than normal (+/+ or +/ter). Experimental testicular teratomas never developed from intratesticular grafts of B6‐ter genital ridges. LTXBJ‐ter/ter females had no ovarian teratomas. It is concluded that the ter gene is solely responsible for germ cell deficiency, but not testicular teratocarcinogenesis, in ter congenic strains having background genes other than 129/Sv‐ter and that the ter gene is not involved in ovarian teratocarcinogenesis.
Ultrastructural study on pronuclear development in fertilized eggs from goldfish, Carassius auratus
Authors: Wu Yiqing , Chen Yonglong , Liang Guixia , Mao Mingting
Published: October 2003

The formation of male and female pronuclei in physiologically monospermic fertilized eggs of the goldfish, Carassius auratus, has been investigated with transmission electron microscopy. Ultrastructural observations show that at 26°C the transformation of the sperm nucleus takes place very quickly. The sperm nuclear envelope degenerates and is replaced by a large number of smooth surface vesicles 1 min post‐insemination. Concomitantly, most of the condensed sperm chromatin is dispersed and is surrounded by vesicles. Dispersion of the chromatin is followed by the fusion of vesicles and the formation of a new bilaminar pronuclear envelope. Within 5–10 min post‐insemination, a spheroid male pronucleus with intranuclear annulate lamellae is produced. The formation of a female pronucleus is slightly different to that of the male pronucleus. The dispersing chromatin of the egg is divided into many groups, most of which are surrounded by multilaminar envelopes 5 min post‐insemination. An ellipsoid female pronucleus with a continuous bilaminar pronuclear envelope and intranuclear annulate lamellae is formed 15 min post‐insemination. Subsequently, the two pronuclei migrate towards one another. When the fully developed male and female pronuclei are located in the center of the blastodisc, each changes itself into a saccular complex 25 min post‐insemination.
Fate of the sea urchin egg receptor for sperm following fertilization
Authors: Partin Jacqueline S. , Ohlendieck Kay , Lennarz William J.
Published: October 2003

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH‐ terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi‐associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane‐bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane‐bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.
Characterization and isolation of melanocyte progenitors from mouse embryos
Authors: Kunisada Takahiro , Yoshida Hisahiro , Ogawa Minetaro , Shultz Leonard D. , Nishikawa Shin‐ichi
Published: October 2003

Whole mount immunohistochemistry and flow cytometry have been used to determine the morphological and molecular features that distinguish melanoblasts from surrounding cells. Whole mount immunohistochemistry of mouse embryos using anti‐c‐Kit monoclonal antibody revealed two distinct types of c‐Kit+ cells; one dendritic and the other round in shape. The distribution of c‐Kit+ dendritic cells in 12.5 days postcoitem embryos correlated well with that of tyrosinase‐related protein‐2 expression, while the distribution of c‐Kit+ round cells overlaps that of CD45+ cells. This observation suggests that melanoblasts are distinguishable from other c‐Kit+ cells by their dendritic shape. Mice homozygous for the steel‐Dickie mutation (Sld/Sld) were analyzed to further distinguish melanoblasts from hematopoietic progenitor cells. Sld/Sld mice are unpigmented but contain hematopoietic cells, although reduced in number. Although no c‐Kit+ dendritic cells were detectable in the Sld/Sld embryos, a significant number of c‐Kit+ round cells were present in the same embryos. To further analyze characteristic features of melanoblasts, c‐Kit+CD45− and c‐Kit+CD45+ cells were isolated from dissociated embryonic skin by fluorescent activated cell sorter and the expression of TRP2 melanogenic enzyme was analyzed. Consistent with histological analysis, most c‐Kit+CD45− cells were TRP2+.c‐Kit+CD45+ cells failed to express TRP2. These results show that most of the melanoblasts are c‐Kit+TRP2+CD45− dendritic cells and can be discriminated from other cells by flow cytometry or by their morphology.
Rhombomere formation and hind‐brain crest cell migration from prorhombomeric origins in mouse embryos
Authors: Osumi‐Yamashita Noriko , Ninomiya Youichirou , Doi Hirofumi , Eto Kazuhiro
Published: October 2003

Prior to rhombomere development, structures called prorhombomeres appear in the mammalian hindbrain. This study clarifies the developmental relationship between prorhombomeres and their descendent rhombomeres and hindbrain crest cells in mouse embryos by focal dye injections at various levels of prorhombomere A (proRhA), proRhB, and proRhC, as well as at their boundaries. ProRhA gives rise to two rhombomeres, rhombomeres 1 and 2 (r1 and r2), as well as to crest cells that migrate into the first pharyngeal arch, including the trigeminal ganglion. ProRhB develops into r3 and r4 and produces crest cells populating the second arch and acousticofacial ganglion. The anterior portion of proRhC gives rise to r5 and r6 and to crest cells migrating into the third pharyngeal arch and the IXth ganglion; its posterior portion develops into r7 and releases crest cells into the fourth pharyngeal arch region as well as the Xth ganglion. These results suggest that the boundaries between prorhombomeres serve as lineage restrictions for both hind‐brain neuroepithelial cells and for segmental origins of crest cell populations in mouse embryos. The Hox code of the mouse head can be schematized in a much simpler way based on this prorhombomeric organization of the hind‐brain, suggesting that prorhombomeres primarily underlie mammalian hind‐brain segmentation.
Relative amounts of basic nuclear proteins SP4 and SP5 in Xenopus laevis sperm correlate with gene copy number
Authors: Teshima Kaori , Abe Shin‐Ichi , Katagiri Chiaki , Takamune Kazufumi
Published: October 2003

Mature sperm of an anuran amphibian Xenopus laevis contain six sperm‐specific basic nuclear proteins (SP 1–6). In an attempt to understand how the amounts of SP produced are regulated during transcription and translation, the relative amounts of proteins and messenger RNA (mRNA), and the gene number in nuclei were compared between SP4 and SP5. Measurements of the peak areas of proteins separated by reversed‐phase high performance liquid chromatography (HPLC) indicated that SP4 is present in about a five times greater amount than SP5. Nuclease S1 protection assays showed the existence of SP4 mRNA in about a five times greater amount than SP5 mRNA. Southern hybridization analyses of restriction enzyme‐digested genomic DNA indicated a single copy of SP5 gene in the haploid genome, as contrasted with the five SP4 genes found in our previous study. Thus the deposition of different amounts of SP4 and SP5 in sperm nuclei reflects the relative number of these genes in the genome.
Centrifugal bisection of starfish oocytes
Authors: Yoshikawa Shun‐Ichi
Published: October 2003

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital‐staining experiments showed that the oocytes were elongated along the animal‐vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment‐derived embryos failed gastrulation and developed into Dauerblastulae. Two‐dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl‐prolyl cis‐trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.
Mucus‐associated antigen in epithelial cells of the chicken digestive tract: Developmental change in expression and implications for morphogenesis‐function relationships
Authors: Yoshida Naomi , Urase Koko , Takahashi Jun , Ishii Yasuo , Yasugi Sadao
Published: October 2003

The embryonic chicken digestive tract consists of endodermal epithelium and mesenchyme derived from splanchnic mesoderm. Interactions between these two tissues are important for the establishment of regionality and the subsequent differentiation of digestive organs. In the present study we obtained a monoclonal antibody that reacted with mucus‐associated antigen and named it the MA antibody. From 6 days of incubation, this antibody reacted with the esophageal, proventricular and gizzard epithelia. In the proventriculus, the MA antigen was expressed in luminal epithelial cells, while pepsinogen‐producing gland cells became MA antigen‐negative. The intestinal goblet cells, which secrete mucus, became positive to the antibody from day 13 of incubation. When the esophageal, proventricular or gizzard epithelium of a 6 day embryo was associated and cultivated with the proventricular mesenchyme, the luminal epithelial cells remained reactive to the MA antibody while gland cells were negative or only weakly positive. If the small‐intestinal epithelium was cultivated with the proventricular or gizzard mesenchyme, the antigen was detected on the apical surface of the epithelium, suggesting that the expression of the MA antigen was induced by mesenchymal influences in the small‐intestinal epithelium. These results suggest that spatio‐temporally regulated expression of the MA antigen is controlled by the epithelial‐mesenchymal interactions.
Determination, induction and pattern formation in early amphibian embryos
Authors: Tiedemann Heinz , Asashima Makoto , Born Jochen , Grunz Horst , Knöchel Walter , Tiedemann Hildegard
Published: October 2003

Determination (inducing) factors, the extracellular matrix, signaling pathways, transcription factors and genes interact in pattern formation and neural induction. Genes can either be activated or repressed. The animalvegetal and dorso–ventral polarities are determined in very early developmental stages. Factors of the TGF‐β superfamily in a graded distribution are involved in the determination of endoderm, mesoderm and ectoderm. The differentiation of mesoderm also depends on the animal ectoderm. Neural inducing factors have been partially purified.
Hormonal induction of all stages of spermatogenesis in germ‐somatic cell coculture from immature Japanese eel testis
Authors: Miura Chiemi , Miura Takeshi , Yamashita Masakane , Yamauchi Kohei , Nagahama Yoshitaka
Published: October 2003

In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11‐ketotestosterone (11‐KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. for detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11‐KT‐induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11‐KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11‐KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11‐KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11‐KT.
Morphological evaluation of microvascular networks and angiogenic factors in the selective growth of oocytes and follicles in the ovaries of mouse fetuses and newborns
Authors: Miyamoto Yohei , Nakayama Taisuke , Haraguchi Seiki , Miyamoto Hajime , Sato Eimei
Published: October 2003

In the mammalian ovary, there is a striking difference in the distribution of blood vessels to individual follicles, suggesting that a microvascular network affects the selective growth of oocytes and follicles. In the present study the role of microvascular networks and angiogenic factors on the selective growth of oocytes and follicles was evaluated histologically in fetuses and newborns of ICR strain mice. Apparent selective growth of oocytes and follicles was observed in the ovaries of 1 day old newborns and, at this time, microvascular networks were recognized electronmicroscopically around the follicle that had completed the formation of its follicular structure and contained oocytes more than about 20 μm in diameter. In 3 day old newborns, oocytes more than 30 μm in diameter were detected where blood capillaries were well vascularized. Immunoreactivity to epidermal growth factor (EGF) and a strongly negative charged (colloidal iron‐positive) substance (glycosaminoglycans; GAG), which have angiogenic activity, were detected in the ovaries of 3 day or older newborns and were identified more often around growing follicles containing oocytes more than 30 (GAG) and 40 (EGF) μm in diameter. Ovaries removed from 20 day old fetuses and cultured for 4 and 6 days in vitro showed a different distribution of growing follicles. A proportion of oocytes 20.0–24.9 μm in diameter increased during 4 and 6 days of incubation. However, the majority of oocytes did not grow further. These findings indicate that microvascular networks and angiogenic factors are deeply involved in selective oocyte growth beyond approximately 20–30 μm in diameter in mouse ovaries.
Analysis of chorion hardening of eggs of rainbow trout, Oncorhynchus mykiss
Authors: Iuchi Ichiro , Ha Chang‐Rak , Sugiyama Hitoshi , Nomura Kohji
Published: October 2003

We estimated changes of chorion hardness of rainbow trout (Oncorhynchus mykiss) egg by the use of three parameters, namely increase of resistance of an egg to rupture by extraneously applied pressure, decrease of solubility of chorion proteins in 8 mol/L urea and a change in the content of γ‐glutamyl‐ε‐lysine crosslink. Unfertilized egg chorions became hardened after egg activation. During chorion hardening, 49, 56 and 65 kDa protein components of the chorion gradually disappeared, high molecular weight intermediates (113,160–170 and higher than 250 kDa) were newly formed and, finally, all components became undetectable by sodium dodecylsulfate‐polyacrylamide gel electrophoresis. The content of γ‐glutamyl‐ε‐lysine (γ‐Glu‐ε‐Lys) crosslink in the chorion increased after hardening. Chorion hardening was inhibited by the incorporation of monodansyl‐cadaverine, a competitive inhibitor for transglutaminase (TGase), into the chorions. TGase activity was detected in unfertilized eggs and localized in the chorion fraction rather than in the ooplasmic fraction. The findings suggest that chorion hardening depends upon polymerization of the chorion components by TGase‐dependent γ‐Glu‐ε‐Lys crosslink formation.
Localization of constitutive heat shock proteins in developing ascidians
Authors: Bates William R. , Bishop Cory
Published: October 2003

Heat shock proteins (HSP) are a group of highly conserved proteins that regulate protein folding and ameliorate the effects of environmental stress. In the present study, the question of whether or not ascidian oocytes, embryos and larvae constitutively synthesize HSP was studied using HSP 60 and HSP 70 antibodies. Developmental stages obtained from Boltenia villosa, Cnemidocarpa finmarkiensis, Styela montereyensis and Corella willmeriana were examined for HSP using indirect immunocytochemistry. Myoplasm in oocytes and unfertilized eggs reacted with HSP 60 and 70 antibodies. HSP signals dramatically moved into the vegetal egg cytoplasm during ooplasmic segregation and colocalized with the myoplasm. In cleavage‐stage embryos, HSP signals were partitioned with the myoplasm into muscle progenitor blastomeres and HSP signals were evident in the tail muscle cells of larvae. Immunoblots of proteins extracted from oocytes, eggs, embryos and larvae indicate that anti‐HSP 60 recognizes a single band having an estimated molecular weight of 60 kDa. Egg centrifugation experiments suggest that most of the ascidian myoplasmic HSP are mitochondrial proteins. These results raise an intriguing possibility that mitochondria associated with the myoplasm perform biochemical functions that are unique to the embryonic muscle cell lineage.
Effect of Ca2+ on deformation, polar body extrusion and pronucleus formation in the egg of the ascidian, Ciona savignyi
Authors: Sensui Noburu , Morisawa Masaaki
Published: October 2003

Cortical deformation and polar body extrusion are the principal events that occur at fertilization in the ascidian egg. We demonstrated that the intracellular Ca2+ concentration ([Ca2+]i) in the fertilized egg of Ciona savignyi increased at egg deformation (main peak) and then several small Ca2+ spikes (1st spikes) appeared before the first polar body extrusion. Brief Ca2+ spikes (2nd spikes), then appeared in the period between the first and second polar body extrusion. When eggs were fertilized in Ca2+‐free artificial seawater, the main peak and 1st spikes appeared, but the 2nd spikes did not, suggesting that the Ca2+ required for the main peak and 1st spikes is released from the intracellular store in this species and that extracellular Ca2+ is required for the 2nd spikes. When [Ca2+]i was clamped at a low level (0.03–0.13 μmol/L) by injecting the egg with low‐Ca2+ buffers and the egg was then inseminated, deformation, polar body extrusion and pronucleus formation were suppressed. In contrast, egg deformation and first polar body extrusion were induced without insemination when [Ca2+]i was 0.9 μmol/L. A higher Ca2+ concentration of 1.2–10.1 μmol/L was required for extrusion of the second polar body and pronucleus formation. These data suggest that sequential Ca2+ increases (i.e. main peak and 1st and 2nd spikes) are prerequisite for the deformation and polar body extrusion of the egg. Furthermore, in eggs arrested at the second meiotic metaphase after first polar body extrusion by the injection of Ca2+ buffer, subsequent injection of excess Ca2+ caused formation of an irregular second polar body‐like protrusion, suggesting latent arrest at the second meiotic metaphase in the ascidian egg.
Regional expression of the transcript encoding sterol carrier protein x‐related thiolase and its regulation by homeotic genes in the midgut of Drosophila embryos
Authors: Kitamura Tomiichiro , Kobayashi Satoru , Okada Masukichi
Published: October 2003

We have isolated a gene designated terminal rings in the midgut (trim), which encodes a transcript expressed in the embryonic midgut in a region‐specific manner. The 1.4 kb trim transcript is expressed in two discrete regions of the midgut epithelium during late embryogenesis. The anterior domain of trim expression is found between the proventriculus and the first midgut constriction. The posterior domain locates in the region posterior to the third midgut constriction. Furthermore, we show that normal function of two homeotic genes, Antp and abd‐A, which express in the visceral mesoderm covering the midgut epithelium, is required for the regional expression of trim. The trim gene is thus reasoned to be activated through inductive interaction between two midgut layers. The 1.4kb mRNA encodes a sterol carrier protein x‐related thiolase. As thiolase activity is required for lipid metabolism, the expression of the 1.4 kb trim transcript can represent a state of differentiation and can be used as an appropriate marker for analyzing regional differentiation of the midgut in terms of physiologically distinct domains.
Essential role of the yolk syncytial layer for the development of isolated blastoderms from medaka embryos
Authors: Hyodo Masao , Aoki Atushi , Ando Chikara , Katsumata Masakazu , Nyui Seiichiro , Motegi Naoya , Morozumi Takeya , Matsuhashi Michio
Published: October 2003

To investigate a possible role of the yolk syncytial layer (YSL) in the development of the medaka embryo, blastoderms were isolated at different stages of embryogenesis either with or without the layer and were incubated in a culture medium. The blastoderms from cleavage stage embryos (stage 8–9), in which the YSL had not yet formed, developed into an irregular mass of cells. But some of the blastoderms isolated with the YSL from the blastula embryos (stage 10) developed into embryo‐like structures with apparent body axes and contained differentiated organs, such as the eye, ear, contractile heart, yolk sac‐like sphere and posterior body trunk with notochord. The proportion of such explants increased as the developmental stage proceeded. However, the proportion was much smaller when blastoderms were isolated at the blastula stage without the YSL. These results suggest that the YSL is essential for the development of embryonic structures. At stage 12 (early gastrula), the frequency of formation of such structures was the same among blastoderms with or without the YSL, so that these embryos are apparently committed for pattern formation.
A novel antigen Apsi is expressed in a half population of endoderm cells in normal and LiCI‐treated starfish embryos
Authors: Yoshikawa Shun‐Ichi
Published: October 2003

A novel antigen, Apsi, revealed a tissue specific expression in the starfish embryo. Apsi was detected in the stomach and intestine of the bipinnaria larva by immunofluorescence microscopy, but was not detected in the esophagus or ectoderm. The expression of Apsi was zygotic and first detected at day 3 after fertilization. Using this antigen as a molecular marker, the effect of LiCI treatment on development was examined by counting the cell number of each germ layer and endoderm tissues on serial paraffin sections. At day 5 larva stage, the ratio of the cell number of ectoderm, esophagus, Apsi‐expressing tissue (stomach and intestine) and mesoderm was 75:10:10:5. The corresponding ratio in LiCI‐treated embryo was 68:14:14:4. LiCI treatment increased the cell number of endoderm by 40%, at the expense of a 10% decrease in the cell number of ectoderm. In intact embryos, approximately half the endoderm cells expressed Apsi antigen, while the other half did not. LiCI treatment did not change this ratio of Apsi expression in endoderm tissues. These observations indicate that LiCI treatment of early blastulae affects the commitment of ectoderm/endoderm but does not affect the differentiation of the esophagus/stomach and intestine.
Change in the levels of adenine‐related compounds in starfish ovarian follicle cells following treatment with gonad‐stimulating substance
Authors: Mita Masatoshi , Yasumasu Ikuo , Nagahama Yoshitaka , Saneyoshi Mineo
Published: October 2003

The resumption of meiosis in starfish oocytes is induced by 1‐methyladenine (1‐MeA), which is produced by ovarian foilicle cells under the influence of a gonad‐stimulating substance (GSS). It has been reported that the 1‐MeA produced is newly synthesized via a process of methylation, rather than being pre‐stored within follicle cells or a breakdown product of some 1‐MeA‐containing substance. The present study examined a possible substrate for 1‐MeA biosynthesis stored in follicle cells of the starfish Asterina pectinifera. Analyses using high‐performance liquid chromatography indicated a large source of ATP among the adenine‐related compounds in these follicle cells. When follicle cells were incubated in seawater in the presence of GSS, 1‐MeA production was stimulated significantly. GSS also caused a reduction in intracellular levels of ATP. There was no change in the levels of either ADP or AMP. The amount of ATP consumed under the influence of GSS was similar to the amount of 1‐MeA produced. Methionine and selenomethionine enhanced both 1‐MeA production and ATP consumption by GSS in follicle cells. In contrast, ethionine and selenoethionine, competitive inhibitors of methionine, inhibited these processes. These results suggest that ATP is a possible substrate in the biosynthesis of 1‐MeA by starfish ovarian follicle cells.
Role of integrins in differentiation of chick retinal pigmented epithelial cellsin vitro
Authors: Mazaki Yuichi , Mochii Makoto , Kodama Ryuji , Eguchi Goro
Published: October 2003

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I‐ or type IV‐coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti‐β1 integrin antibody was added to the PEC culture medium, a decrease of cell‐substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell‐substrate adhesion.
Isolation of the Xenopus nrp‐1 gene and analysis of its expression following the midblastula transition in injected Xenopus embryos
Authors: O'Connell Marcia L. , Rebbert Martha L. , Dawid Igor B.
Published: October 2003

During embryogenesis the differentiation of distinct tissues is marked by the expression of tissue‐specific genes. In Xenopus, the neural‐specific gene nrp‐1 is activated following the midblastula transition (MBT), and is then expressed exclusively in developing neural tissue. To pursue an investigation of the transcriptional regulation of nrp‐1, the genomic DNA encoding nrp‐1 was isolated and its structure analyzed. Nrp‐1 lacks a canonical TATA box proximal to the start site of transcription, but the 5′ flanking sequence is highly GC‐rich. Injection studies with nrp‐1/chloramphenicol acetyltransferase and luciferase constructs demonstrate that in Xenopus embryos, 200 nt of the 5′ flanking sequence of nrp‐1 are sufficient to drive the expression of reporter genes soon after the MBT, albeit not in a tissue‐specific manner. This expression is enhanced by the inclusion of larger regions of the nrp‐1 gene.
The cell lineages of two types of embryo and a hermaphroditic gonad in dicyemid mesozoans
Authors: Furuya Hidetaka , Tsuneki Kazuhiko , Koshida Yutaka
Published: October 2003

The present review summarizes the patterns of cell division and the cell lineages of the two types of embryo, namely, the vermiform embryo and infusoriform embryo, of the dicyemid mesozoan Dicyema japonicum. The infusoriform embryo develops from a fertilized egg. The early cleavages are holoblastic and spiral. At around the 20‐ to 24‐cell stage, cleavages become asynchronous and the cleavage pattern changes from spiral to bilateral. The fully formed infusoriform embryo consists of 37 cells and exhibits bilateral symmetry. These 37 cells are produced after only four to eight rounds of cell division. The vermiform embryo develops asexually from an agamete. Cell division proceeds spirally in the early stages, becoming bilateral from the fifth cell division onward. The fully formed vermiform embryo consists of 23 cells and exhibits bilateral symmetry. The 23 cells are produced after only four to six rounds of cell division. During the development of both types of embryo, a few cells consistently undergo extremely unequal divisions; the much smaller daughter cells eventually degenerate and ultimately disappear during embryogenesis. This disappearance may be an example of programmed cell death. The cell lineages appear to be invariant among embryos, apart from the derivation of the lateral cells of infusoriform embryos. In addition to a description of embryogenesis, the present review includes details of development of the functionally hermaphroditic gonad, the infusorigen, and gametogenesis. The infusorigen is formed from an agamete. The first spermatogonium is produced by the second division and the first oogonium is produced by the third division. Oogonia occupy the outer surface of the axial cell of the infusorigen, while spermatogonia are incorporated into the cytoplasm of the axial cell of the infusorigen. The infusorigen consists finally of approximately 20 cells.
Cultivation of chicken proventricular epithelial cells and their potential for differentiation
Authors: Tabata Hidenori , Yasugi Sadao
Published: October 2003

Epithelial cells of chicken proventriculus (glandular stomach) differentiate into two types; luminal and glandular epithelial cells. The molecules regulating the differentiation of proventricular epithelial cells are not well understood. As the first step in screening the molecular determinants involved in the cell differentiation process, we tried to establish an in vitro culture system for isolated proventricular epithelial cells. Various basal media, growth factors and sera were tested. The medium that supports well the proliferation of epithelial cells was composed of Ham's F12 as the basal medium with epidermal growth factor (10 μg/mL), insulin (10 μg/mL), cholera toxin (1 μg/mL) and bovine pituitary extract (100 μg/mL). Fetal calf serum stimulated cell proliferation 1.7‐fold, while horse serum was rather toxic. Proventricular epithelial cells proliferated for 3 days, but began to die out within 1 week of culture. Cultured epithelial cells never expressed embryonic chicken pepsinogen (ECPg), a marker gene of glandular epithelial cells, or maintained ECPg expression. The capacity for ECPg expression in cultured epithelial cells was analyzed by recombination with the proventricular mesenchyme and ECPg was detected in epithelial cells cultured up to 3 days. We concluded therefore, that epithelial cells keep the capacity for ECPg expression for 3 days of cultivation and proventricular mesenchymal cells are required for the actual expression of the ECPg gene.
Bovine embryo cloning: Characterization of the recipient cytoplasts by phosphorylation patterns and kinase activities
Authors: Gall Laurence , Dedieu Thierry , Chesne Patrick , Ruffini Sylvie , Sevellec Claude , Peynot Nathalie , Renard Jean Paul , Heyman Yvan
Published: October 2003

When in vitro‐matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro‐matured bovine oocytes (aged oocytes, aged‐cooled oocytes, enucleated‐aged oocytes and enucleated‐aged‐cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated‐aged oocytes and in aged‐cooled oocytes, dropped to the basal level in enucleated‐aged‐cooled oocytes (recipient cytoplasts), while mitogen‐activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase‐like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.
Production of hyperdorsal larvae by exposing uncleaved Xenopus eggs to a centrifugal force directed from the animal pole to the vegetal pole
Authors: Takano Kazuhiro , Kikkawa Mika , Shinagawa Atsunori
Published: October 2003

Exposure of uncleaved Xenopus eggs to a centrifugal force directed from the animal pole to the vegetal pole produces larvae with enhanced dorsal structures, which resemble ‘hyperdorso‐anterior’ larvae produced by D2O‐treatment at 0.3 normalized time (NT). Optimal conditions are 70 g for 6 min at 20% of the first cell cycle (0.2 NT). Exposure before removal of vegetal pole cortical cytoplasm, which we find has an effect of eliminating dorsal structures, protects eggs from losing their ability to form dorsal axial structures upon removal. In contrast, exposure after a slight ultraviolet (UV)‐irradiation, which has virtually no effect on dorsal development, produces larvae with heavily reduced dorsal structures, which resemble ‘ventralized’ larvae produced by heavy UV‐irradiation. Interestingly, none of these treatments prevents cortical rotation. Morphological and histological examinations reveal that exposure to the force causes displacement of both cortical and deep egg components from around the vegetal pole to subequatorial regions. We conclude that exposure to the centrifugal force enhances dorsal structures by displacing dorsal determinants from around the vegetal pole to subequatorial regions broader than normal. This is the first experiment in which displacement of egg components, by methods independent of the rotation, are shown to perturb larval body pattern.