Morphological Changes of the Inner Surface of the Blastocoelic Wall before and during Gastrulation in the Newt, Cynops pyrrhogaster

Ultrastructural observations were made of morphological changes in the inner surface of the blastocoelic wall (BW) in newt embryos during the period from morula to gastrula. A scanning electron microscopic investigation distinguished three phases of process formation on the inner surface of the BW. First, small blebs and filopodia were found in the morula. These then changed to large blebs in the blastula and early gastrula. Finally, they acquired lamella-like structures (lamellipodia) in the middle and late gastrula. In addition to the changes in the cell surface, cytoplasmic changes such as of the organization of the cortical microfilamentous layer (CML) were found by transmission electron microscopy (TEM). The morphological changes on the inner surface of the BW and those in the cytoplasmic ultrastructures are considered to be closely related, playing some important roles in the mechanisms of morphogenetic cell movements.     

Movement of an Epithelial Layer Isolated from Early Embryos of the Newt, Cynops pyrrhogaster I. Development of Folding Movement of the Blastocoelic Wall Isolated from Embryos before and during Gastrulation

The blastocoelic wall (BW) was isolated from embryos of the newt, Cynops pyrrhogaster , before and during gastrulation. The mechanism responsible for the folding movement of the isolated BW toward the basal side was investigated. The BW isolated from the early gastrula was induced to fold toward the basal side by treatment with serum. The folding movement of the isolated BW took place from the late blastula to early gastrula stage. Electron microscopy, rhodamine-phalloidin staining and experiments with inhibitors show that the development of the folding movement was correlated with the appearance of a submembranous microfilaments layer (SML) which was formed beneath the cell membrane on the basal side of the BW and suggest that the contraction of actin filaments in the SML is involved in the folding movement of the isolated BW toward the basal side.     

Accumulation and Distribution of Extracellular Matrix as Revealed by Scanning Electron Microscopy in Freeze-Dried Newt Embryos Before and During Gastrulation

A scanning electron-microscopic study was carried out on the extracellular matrices (ECMs) in freeze-dried newt embryos from the cleavage to the gastrula stage. The results revealed the appearance, accumulation and distribution of two types of ECMs, a fibrillar ECM in the blastocoel and an amorphous ECM on the inner surface of the blastocoelic wall (BW). The fibrillar ECM first appeared in the blastocoel at the cleavage stage and increased notably in quantity at the blastula and gastrula stages. On the other hand, the amorphous ECM was initially detected on the inner surface of the BW at the beginning of gastrulation and it increased in quantity during gastrulation. With the progress of archenteron invagination, the amorphous ECM was found to be deposited in the space between the BW and migrating cells.     

An Ultrastructural and Carotenoid Analysis of the Red Ventrum of the Japanese Newt,Cynops pyrrhogaster

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)‐reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab‐reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, β‐carotene and five other kinds of carotenoids were detected by thin‐layer chromatography (TLC). In the ventral yellow skin of lab‐reared newts, however, only β‐carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab‐reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.     

Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X-Ray Microanalysis

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster , was examined by cytochemical methods and X-ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron-dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron-dense organelles and yolk granules.     

Changes of Chromosome Length and Constitutive Heterochromatin in Association with Cell Division during Early Development of Cynops pyrrhogaster embryo

The length of chromosomes in the presumptive ectoderms of Cynops embryos was measured at nine successive cell divisions from the 6th (cleavage stage) to the 14th (gastrula stage). Up to the 10th cell division (cleavage stage) the chromosome length remained constant. At the 11th cell division the chromosomes began to shorten and continued to shorten at every cell division up to the 14th cell division. The degree of shortening and the mode of variation in length corresponded to the respective developmental stages of cleavage, blastula and gastrula. During those periods when chromosomes became shortened, some fine C-bands of the paracentromeric region found in earlier stages fused together. The chromatin of interphase nuclei also showed considerable changes during chromosome shortening. Besides the size reduction of interphase nuclei, the number of C-band granules in an interphase nucleus decreased in parallel with chromosome shortening and fusion of C-bands in mitotic chromosome.     

Persistence of Disorganized Pathways and Tortuous Trajectories of Regenerating Retinal Fibers in the Adult Newt Cynops pyrrhogaster

The anatomical pathways and trajectories of regenerating retinal fibers within the optic tract and tectum of adult newts were examined, 7 months after transection of the optic nerve. In spite of restoration of retinotopic ordered central connection within the tectum, the pathways of individual regenerating retinal fibers within the optic tract were greatly disorganized; the misrouted retinal fibers exhibited tortuous trajectories on the tectal surface in approach to their sites of normal innervation. These results suggest that the regenerating retinal fibers with abnormal pathways and tortuous trajectories can be maintained provided they are in contact with appropriate targets.     

An Ultrastructural Study on the Sertoli Cell in the Newt (Trituroides hongkongensis)

An electron microscopical study was made on the Sertoli cell in testicular lobules carrying mature spermatozoa in the newt, Trituroides hongkongensis. The ultrastructural features suggested that the Sertoli cells in newt might be capable of performing three possible functions: one, as a support for spermatozoa; two, carrying out phagocytosis; and thirdly, facilitate the liberation of spermatozoa.     

Meiosis of Primary Spermatocytes and Early Spermiogenesis in the Resultant Spermatids in Newt, Cynops pyrrhogaster in vitro

Dissociated spermatogenic cells were cultivated within the collagen matrix at low cell density. The largest cell type in the culture was identified as the primary spermatocytes by their size and the morphological characteristics revealed by ultra-thin sections. Chromosome analysis showed that about 90% of the cells examined were either in first or second meiosis. Within the collagen matrix, the fates of 282 single primary spermatocytes at meiotic stage in diakinesis or metaphase were followed. In a few days, most of them gave rise to four spermatids, passing through first and second meiotic divisions. About 80% of the spermatids formed motile flagella. They grew about 20–60 μm a day. The final state of the differentiation attained in our culture conditions was the spermatids with localized spherical nuclei and motile flagella, about 500 μm in length after 1-month's culture. Ultra-thin sections of the spermatids show that the rings, neck-pieces, and acrosomes developed in the cells.     

Epibolic extension of the presumptive ectodermal layer of embryos of the newt Cynops pyrrhogaster before and during gastrulation.

Epibolic extension of the presumptive ectodermal layer (PEL) was investigated in embryos of the newt Cynops pyrrhogaster before and during gastrulation. The PEL was composed of only one layer of columnar cells at all stages examined. The cells of the PEL became elongated from the blastula to the early gastrula stage. They were most elongated at the early gastrula stage and then shortened during gastrulation. Present observations suggest that changes in cell shape of the PEL play an important role in the control of the epibolic extension of the newt embryos. The morphology and movement of the isolated cells from the PEL were examined in an attempt to elucidate the role of cell movement in epibolic extension of the PEL. Blebbing and vermiform cells which showed active cell movement appeared at the early blastula stage. The blebbing cells, which formed large hyaline blebs that moved around the circumference of each cell, appeared in large numbers at the early blastula stage. The frequency of the blebbing cells decreased from the early blastula to the early gastrula stage and increased again during gastrulation. The vermiform cells, which had an elongated cell body and moved in a worm-like manner, increased in frequency from the early blastula to the early gastrula stage. The relative number of such vermiform cells was maximal at the early gastrula stage and decreased abruptly during gastrulation. These results suggest that the elongation of the cells of the PEL is controlled by the active cell movement which resembles that of a worm.     

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis

We have previously shown that mammalian follicle-stimulating hormone (FSH) promotes the proliferation of spermatogonia and their differentiation into primary spermatocytes in organ culture of newt testis. In the current study, we performed microarray analysis to isolate local factors secreted from somatic cells upon FSH treatment and acting on the germ cells. We identified neuregulin 1 (NRG1) as a novel FSH-upregulated clone homologous to mouse NRG1 known to control cell proliferation, differentiation and survival in various tissues. We further isolated cDNAs encoding two different clones. Amino acid sequences of the two clones were 75% and 94% identical to Xenopus leavis immunoglobulin (Ig)-type and cysteine-rich domain (CRD)-type NRG1, respectively, which had distinct sequences in their N-terminal region but identical in their epidermal growth factor (EGF)-like domain. Semi-quantitative and quantitative PCR analyses indicated that both clones were highly expressed at spermatogonial stage than at spermatocyte stage. In vitro FSH treatment increased newt Ig-NRG1 (nIg-NRG1) mRNA expression markedly in somatic cells, whereas newt CRD-NRG1 (nCRD-NRG1) mRNA was only slightly increased by FSH. To elucidate the function of newt NRG1 (nNRG1) in spermatogenesis, recombinant EGF domain of nNRG1 (nNRG1-EGF) was added to organ and reaggregated cultures with or without somatic cells: it promoted spermatogonial proliferation in all cases. Treatment of the cultures with the antibody against nNRG1-EGF caused remarkable suppression of spermatogonial proliferation activated by FSH. These results indicated that nNRG1 plays a pivotal role in promoting spermatogonial proliferation by both direct effect on spermatogonia and indirect effect via somatic cells in newt testes.     

Live Imaging of Whole Mouse Embryos during Gastrulation: Migration Analyses of Epiblast and Mesodermal Cells

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.     

Effects of Extracellular Matrix Molecules on Subepidermal Neural Crest Cell Migration in Wild Type and White Mutant (dd) Axolotl Embryos

Migration of neural crest (NC) derived pigment cells is restricted in the white mutant (dd) axolotl embryo (Ambystoma mexicanum). Transplantations between mutant and wild type embryos show that the extracellular matrix (ECM) of the white mutant is unable to support the migration of prospective pigment cells in wild type embryos (Löfberg et al., 1989, Dev. Biol. 131:168–181). In the present study, we test the effects of various purified ECM molecules on NC cell migration in the subepidermal migratory pathway of wild type (D/-) and white mutant (dd) axolotl embryos. We adsorbed the ECM molecules onto membrane microcarriers, which were then implanted under the epidermis. Fibronectin (FN), tenascin (TN), collagens I and VI, and a chick aggrecan stimulated migration in both types of embryos. Laminin-nidogen, rat chondrosarcoma aggrecan, and shark aggrecan stimulated migration in dd embryos but did not affect migration in D/- embryos. Collagen III, fibromodulin and bovine aggrecan had no effect on migration in either type of embryo. NC cells did not migrate on control micro-carriers, which lacked ECM molecules. Some cells observed contacting, and presumably migrating on, coated microcarriers could be identified as pigment cells by their ultra-structure. Enzymatic digestion in vivo with chondroitinase ABC had no effect on NC cell migration. The neutral or stimulatory effect of the aggrecans is surprising; when tested in vitro they inhibited NC cell migration. The effect of three-dimensionality and other molecules present either in the embryonic ECM or in solution may overcome the inhibitory effect of aggrecans.     

Movement of an Epithelial Layer Isolated from Early Embryos of the Newt, Cynops pyrrhogaster II. Formation of a Blastoporal Groove and Archenteron in a Superficial Epithelial Layer Isolated from Initial Gastrula

The roles of folding movement of epithelial layer in amphibian gastrulation were investigated. A superficial epithelial layer was isolated from the vegetal hemisphere of the initial gastrula (stage 11) of the newt, Cynops pyrrhogaster . The isolated epithelial layers were cultured and morphogenetic movements of the epithelial layers were analysed. Two types of folding movement, folding toward the apical side in the blastopore-forming region and folding toward the basal side in the dorsal marginal zone, arose autonomously in the cultured epithelial layers. These movements caused morphogenesis similar to the formation of the blastoporal groove and archenteron in the control embryo. Treatment with chemical reagents that affect the morphogenetic movement of cells and electron microscopy of the submembranous microfilaments layer (SML) suggested that contraction of actin filaments in the SML was involved in both types of folding movement but that they are controlled, respectively, by different mechanisms in terms of involvement of Ca²⁺ ions. The present results suggest that two types of folding movement arise in the superficial epithelial layer of the embryo and play important roles in the formation of the blastoporal groove and archenteron during early steps of amphibian gastrulation.     

Regional specification of the head and trunk-tail organizers of a urodele (Cynops pyrrhogaster) embryo is patterned during gastrulation

The dorsal marginal zone (DMZ) of an amphibian early gastrula is thought to consist of at least two distinct domains: the future head and trunk-tail organizers. We studied the mechanism by which the organizing activities of the lower half of the DMZ (LDMZ) of the urodelean (Cynops pyrrhogaster) embryo are changed. The uninvoluted LDMZ induces the notochord and then organizes the trunk-tail structures, whereas after cultivation in vitro or suramin treatment, the same LDMZ loses the notochord-inducing ability and organizes the head structures. A cell-lineage experiment indicated that the change in the organizing activity of the LDMZ was reflected in the transformation of the inductive ability: from notochord-inducing to neural-inducing activity. Using RT-PCR, we showed that the LDMZ expressed gsc, lim-1, chordin, and noggin, but not the mesoderm marker bra. In the sandwich assay, the LDMZ induced bra expression in the animal cap ectoderm, but the inductive activity was inhibited by cultivation or suramin treatment. The present study indicates that the change in the organizing activity of the LDMZ from trunk-tail to head is coupled with the loss of notochord-inducing activity. Based on these results, we suggest that this change is essential for the specification of the head and trunk-tail organizers during gastrulation.     

Changes in Surface Morphology of Myogenic Cells during the Cell Cycle, Fusion and Myotube Formation

The surface morphology of chick myogenic cells during development in cell culture was examined by scanning electron microscopy. Myoblasts at the G₁ and S phases of the cell cycle had a relatively smooth surface. In late G₂ and mitosis, they had many microvilli and some blebs on their surfaces. Ca²⁺-deficient fusion-arrested myoblasts had a relatively smooth surface. When the cells underwent cell fusion, many microvilli, small spherical protrusions, and some blebs appeared on their surface. In newly formed myotubes, the surface over the nucleus was smooth whereas that over perinuclear regions had many flat excrescences and other surface protrusions. This mosaic appearance of the surface was less prominent in striated myotubes. Scanning electron microscopy combined with fluorescence microscopy using rhodamine-labeled erabutoxin b revealed that sites of accumulation of acetylcholine receptor had a smooth surface. These results suggest that changes in surface structure occur in association with the cell cycle, fusion and subsequent development of myotubes.     

Seasonal Changes in the Newt,Trituroides hongkongensis,Testis. II. An Ultrastructural Study on the Lobule Boundary Cell

An electron microscopical study was made on the lobule boundary cells. They were found to show characteristic ultrastructures of a typical steroid-producing cell. Changes in these cells were divided into five stages according to the development within the lobules. The possible significance of the changes and their correlation with the degree of steroid production at different stages are discussed.     

Presence of D-aspartate oxidase and free D-aspartate in amphibian (Xenopus laevis, Cynops pyrrhogaster) tissues.

1. This paper is the first report on the presence of D-aspartate oxidase activity and free D-aspartate in the amphibian tissues. 2. The presence of D-aspartate oxidase activity in tissues of clawed toad (Xenopus laevis) and Japanese newt (Cynops pyrrhogaster) was demonstrated by requirements for enzyme activity, selective inhibition with meso-tartrate and substrate specificity. 3. In each animal, the highest activity was found in kidney, followed by liver and brain, and no gender difference in the specific activity was observed in each tissue. 4. A small but significant amount of D-aspartate was detected in liver and kidney, irrespective of species. 5. In the newt, there was a gender difference in the hepatic and renal content of D-aspartate and not in the D-/D+L-aspartate ratio.     

周期性机械应力对髓核细胞增殖和细胞外基质表达的影响

目的:探讨周期性机械应力对髓核细胞增殖和细胞外基质表达的影响。方法:对兔髓核细胞进行体外细胞培养,对细胞施加周期性机械应力(0.25Mpa,0.1Hz)。实验分为2组,不加压组和加压组,不加压组置于单纯旋转式生物反应器内,加压组每天置于周期性机械应力场内2小时。分别于3天,7天检测细胞数目以及聚集蛋白聚糖(aggrecan)和Ⅱ型胶原的基因表达。结果:髓核细胞的增殖和聚集蛋白聚糖、Ⅱ型胶原基因的表达水平与周期性压力密切相关,在周期性机械应力刺激下髓核细胞增殖明显,细胞外基质的分泌增加,组织工程髓核细胞的活性显著提高。结论:周期性机械应力能够显著促进髓核细胞增殖,同时上调聚集蛋白聚糖、Ⅱ型胶原的基因的表达。