Characteristics of extracted ion beam from a cesium-free negative ion source using sheet plasma.

We report the characteristics of extracted beam current in the test plasma produced by direct current for sheet plasma upgrade producing negative ions by volume production without cesium (Cs) seeding. The negative hydrogen ion beam is extracted by a two-grid extraction system, which is located at the periphery of the sheet plasma. Experimental observations show that (i) negative hydrogen ions are successfully extracted from the sheet plasma by single/multi-aperture grids and (ii) the ratio of the extracted electron current IEG(e) and the hydrogen negative ion current IEG(H-), IEG(e)/Ic(H-), decreases from 8.0 to 2.0 with an increase in the height of the electron fence (HEF), which is a filter that prevents electron diffusion from the extraction region.

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs.

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.

Efficient targeting of gene expression in chick embryos by microelectroporation.

During vertebrate embryonic development, a key to unraveling specific functions of gene products is the capability to manipulate expression of the gene of interest at the desired time and place. For this, we developed a 'microelectroporation' technique by which DNA can be locally introduced into a targeted site of avian embryos, restricting spatial expression of the protein products during development. This technique involved injection of DNA solution in ovo around the target tissue and pinpoint application of an electric field by tungsten electrodes, allowing efficient and reproducible targeted gene transfer, for example, into an optic vesicle, somites, cranial mesoderm and limb mesenchyme. Because of the locality of gene introduction and its expression, survival rates of the embryos were high: approximately 90% of the embryos injected in optic vesicles were alive for at least 1 day after microelectroporation. The instantaneous gene transfer into embryonic cells allowed rapid expression of protein products such as green fluorescence protein within 2.5 h with fluorescence maintained for 3 days of incubation. This improved technique provides a convenient and efficient way to express transgenes in a spatially and temporally restricted manner in chicken embryos.

Somitogenesis controlled by Noggin.

In vertebrates, the segmented somites, which are the medial-most component in the paraxial mesoderm, are the entity giving rise to the axial bones and skeletal muscles. We previously demonstrated that the mechanism that distinguishes the somite from the more lateral mesoderm (lateral plate) involves different levels of BMP-4 activity which is highest in the lateral plate. We report that Noggin, an antagonist of BMP-4, is expressed in the presumptive somite and appears to control effective levels of BMP-4 to differentiate somitic mesoderm from the lateral plate. When Noggin-producing cells were implanted into the presumptive lateral plate, they produced ectopic somites that were respecified from the lateral plate precursors. These somites exhibited no mediolateral (M-L) polarity, but acquired it when implanted Noggin was eliminated. Thus, in normal embryogenesis no or low BMP-4 activity realized by Noggin specifies the somites in the medial-most portion of the paraxial mesoderm, and then BMP-4 emanating from the lateral plate subsequently establishes the M-L polarity in the somites.

Mesodermal subdivision along the mediolateral axis in chicken controlled by different concentrations of BMP-4.

Molecular mechanisms by which the mesoderm is subdivided along the mediolateral axis in early chicken embryos have been studied. When the presomitic mesoderm (medial mesoderm) was transplanted into the lateral plate, the graft was transformed into lateral plate tissue, indicating that the primitive somite was not fully committed and that the lateral plate has a cue for mesodermal lateralization. Since the lateral plate expresses a high level of BMP-4 mRNA, a member of the TGF-beta family, we hypothesized that it is the molecule responsible for the lateralization of the somite. To test this, we transplanted COS cells producing BMP-4 into the presomitic region. Those cells locally prevented the presomitic cells from differentiating into somites, converting them instead into lateral plate mesoderm, which was revealed by expression of cytokeratin mRNA, a marker for the lateral plate. The effect was dependent on the level of effective BMP-4: with a high level of BMP-4, the somite was transformed completely to lateral plate; with a low level, the somite formed but was occupied by the lateral somitic component expressing cSim 1, a marker for the lateral somite. These results suggest that different thresholds of effective BMP-4 determine distinct subtypes of the mesoderm as a lateralizer during early development.

BMP-4 mediates interacting signals between the neural tube and skin along the dorsal midline.

BACKGROUND: The neural tube and its overlying tissues (skin and mesenchyme) interact along the dorsal midline during early development. This has been previously demonstrated experimentally in chicken embryos by the fact that the dorsal neural tube transplanted ectopically induced expression of Msx 2 in the adjacent tissues. It is important to identify the molecules responsible for these interactions. RESULTS: We observed that BMP-4, a member of the TGFbeta-family, is expressed in the dorsal neural tube in a pattern closely correlated with that of Msx 2. In order to investigate whether BMP-4 mediates the signal between the neural tube and the skin/mesenchyme, BMP-4 was ectopically administered in ovo either by implantation of the recombinant protein or transplantation of COS cells producing BMP-4. Both manipulations resulted in ectopic induction of Msx 2 expression in the adjacent skin/mesenchyme. In addition, the activity of BMP-4 in inducing Msx 2 was counteracted by the floor plate. CONCLUSION: These data suggest that BMP-4 positively mediates the signals from the neural tube to the adjacent tissues and that this signal may be an essential step for the establishment of the dorsal midline structures.

TAB1: an activator of the TAK1 MAPKKK in TGF-beta signal transduction.

Transforming growth factor-beta (TGF-beta) regulates many aspects of cellular function. A member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, TAK1, was previously identified as a mediator in the signaling pathway of TGF-beta superfamily members. The yeast two-hybrid system has now revealed two human proteins, termed TAB1 and TAB2 (for TAK1 binding protein), that interact with TAK1. TAB1 and TAK1 were co-immunoprecipitated from mammalian cells. Overproduction of TAB1 enhanced activity of the plasminogen activator inhibitor 1 gene promoter, which is regulated by TGF-beta, and increased the kinase activity of TAK1. TAB1 may function as an activator of the TAK1 MAPKKK in TGF-beta signal transduction.