Detrimental effects of glucocorticoids on neuronal migration during brain development.

Glucocorticoids, the most downstream effectors of the hypothalamus-pituitary-adrenal axis, are one of main mediators of the stress reaction. Indeed, exposure to high levels of stress-triggered glucocorticoids is detrimental to brain development associated with abnormal behaviors in experimental animals and the risk of psychiatric disorders in humans. Despite the wealth of this knowledge, the cellular and molecular mechanisms underlying the detrimental effects of glucocorticoids on brain development remain unclear. Here, we show that excess glucocorticoids retard the radial migration of post-mitotic neurons during the development of the cerebral cortex, and identify an actin regulatory protein, caldesmon, as the glucocorticoids' main target. The upregulation of caldesmon expression is mediated by glucocorticoid receptor-dependent transcription of the CALD1 gene encoding caldesmon. This upregulated caldesmon negatively controls the function of myosin II, leading to changes in cell shape and migration. The depletion of caldesmon in vivo impairs radial migration. The overexpression of caldesmon also causes delayed radial migration during cortical development, mimicking the excessive glucocorticoid-induced retardation of radial migration. We conclude that an appropriate range of caldesmon expression is critical for radial migration, and that its overexpression induced by excess glucocorticoid retards radial migration during cortical development. Thus, this study provides a novel insight into the underlying mechanism of glucocorticoid-related neurodevelopmental disorders.

DNG1, a Dictyostelium homologue of tumor suppressor ING1 regulates differentiation of Dictyostelium cells.

dng1 is a Dictyostelium homologue of the mammalian tumor suppressor ING gene. DNG1 protein localizes in the nucleus, and has a highly conserved PHD finger domain found in chromatin-remodeling proteins. Both dng1 disruption and overexpression impaired cell proliferation. In dng1-null cells, the progression of differentiation was delayed in a cell-density-dependent manner, and many tiny aggregates were formed. Exogenously applied cAMP pulses reversed the inhibitory effect caused by dng1 disruption on the aggregation during early development, but formation of tiny aggregates was not restored. dng1-overexpressing cells acquired the ability to undergo chemotaxis to cAMP earlier and exhibited enhanced differentiation. These phenotypes were found to be coupled with altered expressions of early genes such as cAMP receptor 1 (car1) and contact site A (csA). Furthermore, disordered histone modifications were demonstrated in dng1-null cells. These results suggest a regulatory role of dng1 in the transition of cells from growth to differentiation.

Cloning, sequencing, and expression of the genomic DNA encoding the protein phosphatase cdc25 in Dictyostelium discoideum.

A genomic DNA ( Dd-cdc25) encoding the protein phosphatase cdc25 was isolated from the cellular slime mold Dictyostelium discoideum. The Dd-cdc25 DNA sequence, with a length of 2,958 bp, encodes a protein consisting of 986 amino acid (aa) residues. The sequence shares significant identities with cdc25 from human, mouse, Xenopus, Drosophila, and Shizosaccharomyces pombe, particularly at the C-terminal region including the catalytic site for phosphatase activity. The deduced Dictyostelium cdc25 protein (Dd-cdc25) has the highest molecular mass (109.9 kDa) in several cdc25 species so far reported and contains four regions consisting of unusually long asparagine repeats (22-31) in the sequence. Unexpectedly, however, Western blot analysis using a specific antibody raised against the C terminus (aa 892-986) of Dd-cdc25 demonstrated that the protein exists as a short form (56 kDa), which has the C-terminal active site of phosphatase, during the course of Dictyostelium development. The Western blot analysis also revealed marked changes in the phosphorylated state of the Dd-cdc25, coupling with cellular development.

Association of culture of mouse urogenital complexes in media containing rodent sera with the appearance of primordial germ cell-like cells.

Primordial germ cells differentiate into germ cells and have the ability to reacquire totipotency. Mouse primordial germ cells are identified by alkaline phosphatase staining of the extraembryonic mesoderm, and they proliferate and migrate to reach the genital ridges. Mouse primordial germ cells have never been maintained in culture exclusively for longer than a week without differentiation or dedifferentiation. Moreover, primordial germ cells have not been proliferated with urogenital complexes in vitro, because gonad culture has never been successful. It was thought that primordial germ cells could proliferate in a culture of urogenital complex under modified medium conditions resembling those in vivo; however, organ culture of mouse gonad has been performed with fetal calf serum or equine serum, and those sera produce conditions different from those in vivo. Therefore, mouse urogenital complexes were cultured in media containing rodent sera. As a result, it was possible to proliferate primordial germ cell-like cells outside gonads, and these cells very closely resembled primordial germ cells. In addition, motile primordial germ cell-like cells could be obtained. The ability to maintain primordial germ cell-like cells in culture by this intra-species culture method is important in the study of gametogenesis. Furthermore, this method is useful as a source of stem cells such as embryonic germ cells.

Purification of mouse primordial germ cells by Nycodenz.

Primordial germ cells are important cells for the study of germ cell lineage. It has proved difficult to obtain highly purified primordial germ cells for preparation of a specific antibody. In the present study, a new method for purifying mouse primordial germ cells was developed using a Nycodenz gradient. Furthermore, the polyclonal anti-mouse primordial germ cells IgG derived from mouse primordial germ cells was prepared. As this IgG reacted only with primordial germ cells obtained at day 12.5 after mating, this antibody appeared to recognize the stage-specific antigen of primordial germ cells. One reason that a continuous primordial germ cell marker has not been obtained is because the purity of the primordial germ cells used has been too low to prepare the antibody. This new method represents a significant improvement in the purification of primordial germ cells; it is simpler than previous methods, and produced mouse primordial germ cells with a purity of more than 95%. In addition, the separation reagent Nycodenz is non-toxic and achieved separation of primordial germ cells without attachment of antibodies against the primordial germ cell membrane surface. This new purification method and stage-specific antibody will be useful for the analysis of the mechanisms of primordial germ cell migration.