Cell Locomotion and Contact Guidance in Amphibian Gastrulation

Presumptive mesodermal cells in amphibian gastrulae migratefrom the blastopore toward the animal pole by using the innersurface of the ectodermal layer as their substratum. Duringmigration, the mesodermal cells form lamellipodia and filopodiapredominantly in a direction toward the animal pole. There isa network of the extracellular fibrils on the inner surfaceof the ectodermal layer. The fibrils seem to serve as an adequatesubstratum for attachment of the filopodia and locomotion ofthe mesodermal cells. A significant alignment of the fibrilnetwork along the blastopore—animal pole axis suggestsa hypothesis that it directs morphogenetic cell movements bycontact guidance in combination with contact inhibition of movement.New culture conditions allow the gastrula mesodermal cells tomove actively in vitro with a similar cell shape and at a similarrate as in vivo. Such culture conditions enabled an in vitroexperiment to test the hypothesis of contact guidance. Explantedectodermal layers deposit the fibril network on the surfaceof a cover slip. Dissociated gastrula mesodermal cells seededon such a conditioned surface attach to the surface and moveabout actively. A computer analysis of the time—lapsefilms shows that the cell trails are significantly aligned alongthe blastopore—animal pole axis of the ectodermal layerthat conditioned the surface. The deposited fibril network showsthe alignment along the same axis. There is also a tendencyof the mesodermal cells to move in a polarized fashion preferentiallytoward the animal pole. These results support the hypothesisof contact guidance of mesodermal cell migration in vivo byoriented extracellular fibrils     

Establishment of the Embryo-derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer.

The ES ceii lines are embryo-derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16-day-old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. Karyotype was, however, nearly tetraploid. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G-banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines.     

Quantitative Analysis of Striped Coat-Color Patterns in Large White→Duroc Chimeric Pigs With Special Reference to the Genetic Control Mechanisms of the Dominant Black-Eyed White Phenotype

Coat colors of four chimeric pigs produced by the microinjection of dissociated blastomeres of (Landrace × Large White) blastocysts to the blastocyst cavity of (Duroc × Duroc) blastocysts (Kashiwazaki et al., 1992) exhibited characteristic horizontal stripe-patterns. We carried out quantitative analysis of those patterns in order to derive information concerning the genetic regulatory mechanisms of the dominant black-eyed white phenotypes in the pig. In the four chimeras, the theoretical mean widths of the single-clone stripe calculated from the estimated widths of minimal recognizable stripe (MRS) (Tachi, 1988) were 2.1 ± 0.1, 2.23 ± 0.15,1.89 ± 0.06, and 1.93 ± 0.28 cm respectively. The estimated number of single-clone stripes in the thoracico-lumbar region of those animals were 42.3, 40.7, 46.3, 44.2, and about twice the mean number of vertebrae in the same region (Duroc, 20 or 21; Large White, 21 or 22). Furthermore, the mean length of thoracico-lumbar vertebrate in two of the chimeric pigs, as measured on X-ray radiographs, was approximately twice the mean single-clone stripe width. It was concluded that the stripe-patterns of the chimeric pigs probably represented the dermatome patterns of epidermis; and in the pig, a single somite was likely to be derived from the clones of two primordial cells, as originally proposed by Gearhart & Mintz (1972) in the mouse. It was suggested, furthermore, that in the Large White→Duroc chimeric pigs, melanocytes that migrated into the region of skin formed by a Large White dermatome could not survive, thus creating a clearly demarcated white stripe. Possible involvement of KL or c-kit in the dominant black-eyed white phenotype of the pig is discussed.     

Laminin Fibrils in Newt Gastrulae Visualized by the Immunofluorescent Staining

An anastomosing network of extracellular fibrils on the inner surface of the ectoderm layer of amphibian gastrulae has been shown to provide an adequate substratum for attachment and migration by the mesodermal cells. These fibrils contain fibronectin as shown by immunostaining at the light and electron microscope levels. Now we report the presence of laminin, another cell adhesion glycoprotein, as a fibrillar network on the inner surface of the ectoderm layer in gastrulae of the Japanese newt ( Cynops pyrrhogaster ), but its absence on the blastula ectoderm layer, by the immunofluorescent staining using an antiserum specific for mouse laminin. The same antiserum was shown to stain basement membranes of adult newt organs as expected.     

STUDIES ON THE GASTRULATION OF AMPHIBIAN EMBRYOS: NUMERICAL CRITERIA FOR STAGE DETERMINATION DURING BLASTULATION AND GASTRULATION OF XENOPUS LAEVIS

The changing external features of Xenopus laevis embryos were examined from the first cleavage through the late gastrula as a means to develop quantitative criteria for the determination of stages for this period. During blastulation, the cell number along arcs centered on the animal or vegetal pole and representing one-sixth of a meridian - called the MCNo. - can be used as such a criterion. During gastrulation, the width of the blastopore divided by the diameter of the whole embryo can be used as such a criterion.