Koichiro Hashimoto

Formation of the Primitive Streak and Mesoderm Cells in Mouse Embryos—Detailed Scanning Electron Microscopical Study

We describe morphological events of the mammalian gastrulation in pre‐ to middle‐primitive‐streakstage mouse embryos by using scanning electron microscopy. The first sign of the ingression of the mesodermal cells was disruption of the epithelial structure of ectoderm and the underlying basal lamina, thus forming a semicircular area of the presumptive primitive streak. Then, cells at periphery of the semicircular region spread on the basal lamina by extending many filopodia to it. The majority of the migrating cells formed a loosely arranged cell sheet. We found solitary cells and isolated small groups of cells migrating away from the periphery of the cell sheet. These cells were well spread on the basal lamina, and had large cell processes and many filopodia in the direction of cell migration. Filopodia of these cells were attached to the basal lamina or a meshwork of the extracellular fibrils. These observations suggest that the extracellular matrix serves as the substratum for cell adhesion and migration, and plays an important role in the mammalian gastrulation.

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.

Mouse offspring derived from fetal ovaries or reaggregates which were cultured and transplanted into adult females

Primordial germ cells (PGCs) and gonia could be promising novel targets and vehicles for manipulation of the mammalian germ line. To make such manipulation a practical possibility, PGCs or gonia must be allowed to produce gametes and offspring after they were isolated from embryos and manipulated in culture. As the first step to develop such research strategy, we obtained offspring from mouse oogonia which were isolated from embryonic ovaries and cultured as dispersed cells before transplantation into female mice as reaggregates.

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro. Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo, suggesting the presenge of other growth factor(s) in vivo. We previously reported that tumor necrosis factor‐α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4‐m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo. Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo; however, it did stop afterwards. Such proliferating PGC continue to express c‐kit and Oct‐3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES‐like EG cells.

Culture of Embryonic Cells for Analysis of Amphibian and Mammalian Early Embryogenesis

Culture of embryonic cells isolated from early embryos allows detailed analysis of the cell motility, interactions between cells, and between cells and the extracellular matrix (ECM). There is always the risk that cell behavior in culture might be artifacts not related to the behavior inside embryos. In many situations, one must use cells immediately after isolation from embryos, and try to prepare culture conditions similar to those inside the embryo.