Generation of Fertile and Diploid Fish, Medaka (Oryzias latipes), from Nuclear Transplantation of Blastula and Four-Somite-Stage Embryonic Cells into Nonenucleated Unfertilized Eggs

The fine structure of lobopodia in dissociated embryonic cells of the freshwater fish, Oryzias latipes, was observed with the electron microscope in order to understand the mechanism of the circus movements which they display. Dense material (grandular or fibrillar) is present in the zone between the lobopodium and the endoplasm, as well as in the cortical layer around the cell circumference. The direction of lobopodial movement is related to the distribution of this dense material. The band between the lobopodium and the endoplasm is conspicuous and is connected to the cortical dense layer around the cell periphery at the advancing front of the lobopodium, while the dense material is usually almost absent beneath the cell membrane in the anterior region of the lobopodium. The band between lobopodium and endoplasm is blurred or disrupted near the hind end of the lobopodiu, where the peripheral dense layer is well developed. In situ localization of actin/heavy meromyosin complexes in the cell showed that the dense material has actin-like properties. Cytochalasin B(0-5 mug/ml) induced constriction of the neck of the bleb, shrinkage of the endoplasm, and herniation of the endoplasmic contents to the enlarged hemispherical bleb, and thus arrested the circus movement. On the basis of these results, an hypothesis concerning the mechanism of circus movement is proposed and discussed.

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Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (Oryzias latipes)

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.98.3.1071 ◽

2001 ◽

Vol 98 (3) ◽

pp. 1071-1076 ◽

Cited By ~ 69

Author(s):

Y. Wakamatsu ◽

B. Ju ◽

I. Pristyaznhyuk ◽

K. Niwa ◽

T. Ladygina ◽

...

Keyword(s):

Oryzias Latipes ◽

Small Laboratory ◽

Embryonic Cells

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Viability and metabolic activity of homozygous Brachyury (T) embryos

Development ◽

10.1242/dev.40.1.271 ◽

1977 ◽

Vol 40 (1) ◽

pp. 271-276

Author(s):

K. O. Yanagisawa ◽

H. Fujimoto

Keyword(s):

T Cells ◽

Cell Lines ◽

Metabolic Activity ◽

In Utero ◽

Embryonic Cells ◽

Somite Stage

Some features of metabolic activity of homozygous mutants for the Brachyury (T) mutation were studied. The embryos incorporated tritium-labelled thymidine up to about the ‘32–36 somite’ stage. The total amount of protein per embryo increased until the same stage. T/T cells proved viable in vitro over the in utero lethal period. Several cell lines were established from +/+ and T/T embryonic cells.

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147 IDENTIFICATION OF PRIMORDIAL GERM CELLS IN PORCINE EMBRYOS FROM THE PRIMITIVE STREAK STAGE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab147 ◽

2006 ◽

Vol 18 (2) ◽

pp. 181 ◽

Cited By ~ 1

Author(s):

M. Vejlsted ◽

H. Offenberg ◽

P. Maddox-Hyttel

Keyword(s):

Germ Cells ◽

Primordial Germ Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Primitive Streak ◽

Posterior Pole ◽

Embryonic Cells ◽

Somite Stage ◽

Mesodermal Origin ◽

A Site

In embryonic stem cell research, Oct-4 is one of the most widely used markers of pluripotency. Moreover, at least in the mouse, this marker is restricted to primordial germ cells (PGCs) after gastrulation. Vimentin is often used as a marker of mesoderm/mesenchyme in embryonic tissues and appears to localize to the same embryonic cells as Oct-4, at least in the bovine epiblast. The expression of neither of these markers has been completely addressed in the pig. Therefore, the purpose of the present study was to examine the expression of Oct-4 and vimentin in the porcine epiblast during differentiation and establishment of the three germ layers, i.e. the process of gastrulation. A total of 410 porcine embryos were collected at 8 to 17 days post-insemination from 29 sows of the Danish Landrace breed. Embryos were categorized based on stereo-microscopic observations into the following stages: pre-streak stages 1 and 2, primitive streak stage, neural groove stage, and somite stage. Specimens were fixed at all stages, dehydrated and embedded in paraffin wax. Selected embryos at each stage (n = 28) were completely cut into serial sections for immunohistochemical evaluation of Oct-4 and vimentin. Pre-streak stage 1 embryos were defined by lack of polarization of the embryonic disk, whereas in pre-streak stage 2 embryos a crescent shaped thickening was seen at the posterior pole of the disk. This thickening, marking the first morphological anterior-posterior polarization of the embryo proper, was shown to be a site of incipient ingression of cells from the epiblast. Immunohistochemical analyses localized Oct-4 to nuclei and vimentin to cytoplasm of both founding and ingressing epiblast cells. During formation of mesoderm and endoderm, at the primitive streak stage, solitary Oct-4 positive cells, i.e. potential PGCs, were seen scattered in the endoderm. Cells of the epiblast displayed positive labeling for Oct-4 until specification for the ectoderm cell lineage at the subsequent neural groove stage. In mesoderm, Oct-4 likewise disappeared by the time of formation of the first somites, defining the following somite stage. Thus, at this stage the only cells labeled for Oct-4, i.e. potential PGCs, were seen solitarily scattered in the endoderm. By the 15-somite stage, such cells were no longer visible in the endoderm but were seen located in the mesoderm, spreading from the stalk of the yolk sac and allantois and extending through the mid- and hindgut areas into the incipient genital ridge. Vimentin localized to the mesenchyme and most other derivatives of neural crest and mesodermal origin. In conclusion, based on Oct-4 labeling and distribution pattern, we strongly believe that we have identified the porcine PGCs from the primitive streak stage.

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Circus movement in dissociated embryonic cells of a teleost, Oryzias latipes

Journal of Cell Science ◽

10.1242/jcs.19.1.169 ◽

1975 ◽

Vol 19 (1) ◽

pp. 169-182

Author(s):

N. Fujinami ◽

T. Kageyama

Keyword(s):

Oryzias Latipes ◽

Time Lapse ◽

Cell Diameter ◽

Fresh Water Fish ◽

C Cells ◽

Embryonic Cells ◽

Salt Solutions ◽

Circus Movement ◽

Water Fish ◽

Effects Of Ph

The dissociated early embryonic cells of the fresh water fish, Oryzias latipes, protrude hyaline lobopodia, which tend to rotate around the cell circumference in a propagating wave. Cells from late blastula or gastrula continuously show this “circus movement”, while most cells up to early blastula are rounded. The linear velocity of the lobopodium was estimated by means of time-lapse cinemicrography. The velocity increases slightly as cell diameter increases. The effects of pH, temperature and osmotic pressure of the immersion media on the movement were also quantitatively investigated. Cells become rounded and do not form lobopodial blebs when immersed in media below pH 5. The velocity is reduced by decreasing temperature, but the movement continues even at 5 degrees C. Cells placed in hypertonic salt solutions become crenated and do not continuously demonstrate the circus movement.

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Cytosolic free calcium-ion concentration in cleaving embryonic cells of Oryzias latipes measured with calcium-selective microelectrodes.

The Journal of Cell Biology ◽

10.1083/jcb.100.3.947 ◽

1985 ◽

Vol 100 (3) ◽

pp. 947-954 ◽

Cited By ~ 23

Author(s):

A R Schantz

Keyword(s):

Membrane Potential ◽

Calcium Ion ◽

Oryzias Latipes ◽

Potassium Acetate ◽

Ion Concentration ◽

Free Calcium ◽

Embryonic Cells ◽

Cell Stage ◽

Partial Digestion ◽

Calcium Ion Concentration

Calcium-selective microelectrodes were used to measure the free calcium-ion concentration ([Ca2+]i) in early-cleaving embryonic cells of the golden medaka, Oryzias latipes, a fresh water teleost fish. Embryos could be dechorionated as early as the four-cell stage using a three-step technique consisting of removal of some yolk to enlarge the perivitelline space, partial digestion of the chorion with pancreatin, and removal of the weakened chorion with forceps. Dechorionated embryos underwent cleavage at a normal rate. Intracellular cytosolic [Ca2+]i was monitored by impaling blastomeres first with a microelectrode filled with 5 M potassium acetate to measure membrane potential, and a few minutes later with a calcium-selective microelectrode. During nine rounds of cytokinesis from a total of six different embryos, cytosolic [Ca2+]i remained constant (with apparently random fluctuations of less than +/- 0.1 microM). During two successive cleavages in one embryo, however, [Ca2+]i rose transiently fourfold above the original resting level to 1.32 and 1.20 microM in synchrony with each period of cytokinesis and returned after each rise to submicromolar levels. Because a calcium-selective microelectrode can detect [Ca2+]i changes only in the immediate vicinity of its 2-microns tip, we interpreted these data to suggest that, although [Ca2+]i in most areas of the cytosol remains between 0.01 and 0.40 microM (mean of 0.14 microM), there may be small regions of the cell in which [Ca2+]i undergoes a substantial increase at the time of cleavage. Evidence also is presented to suggest that the membrane potential in these blastomeres undergoes a slow net hyperpolarization during early cleavage stages.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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