Inductive Capacities for the Dorsal Mesoderm of the Dorsal Marginal Zone and Pharyngeal Endoderm in the Very Early Gastrula of the Newt,and Presumptive Pharyngeal Endoderm as an Initiator of the Organization Center*

The organization center of Cynops pyrrhogaster was divided into Parts 1, 2 and 3 of equal size (0.3×0.4 mm²) with presumptive fates as pharyngeal, pharyngeal+prechordal+trunk notochord, and trunk-tail notochord, respectively. Movements and changes in size and shape of each part were followed through gastrulation. Differentiation tendencies of each part were examined under three conditions: I, isolated; II, sandwiched with presumptive ectoderm; 111, sandwiched with presumptive ectoderm after preculture in isolation for various times. In I, Parts 2 and 3 differentiated into dorsal mesoderm. In II, each part induced dorsal mesoderm and neural tissues, the frequency being highest in Part 2 and lowest in Part 3. In III, Parts 1 and 2 realized their presumptive fates, through changes in inductive capacities from trunk-tail to head. This change progressed rapidly in Part 1, and slowly in Part 2. Part 3 required induction by neighbouring Part 2 to realize its presumptive fate. Changes of inductive capacity of Parts 1 and 2 respectively, were chronologically similar in normal development and in preculture experiments. Lastly, the primary presumptive pharyngeal zone at blastula was proposed to act as an initiator of the organization center, its programmed information being transmitted to Part 2, and then to Part 3.     

Conditions for seedling establishment and probable function of seed in the clonal sedge Carex rugulosa Kük. in riverside marshes

Carex rugulosa Kük. forms large meadows in moist zones along estuarine riversides. These meadows are usually maintained by rhizomatous ramet production (clonal growth), and the appearance of seedlings is uncommon. We studied the conditions required for seedling establishment. In areas in which clonal ramets were dense (906–1050/m²), and the relative light intensity at ground level was low (0.8–3.8%), seed germination was entirely suppressed. In contrast, many seedlings (288–513/m²) were observed in areas in which clonal ramets had been considerably reduced (13–269/m²) by continuous inundation in the previous year. In these areas, the relative light intensity had increased (20.5–79.3%). It was inferred that seeds resisted the prolonged inundation that killed the ramets, and that germination was induced under these open conditions. These results suggest that the main functions of sexual reproduction in C. rugulosa are recovery after severe damage to vegetation using seeds from the seed bank and the dispersal of seeds to areas without dense vegetation. Most seedlings died when the marshy meadow was temporarily inundated by heavy rain. However, transplanted seedlings survived better at artificially elevated sites that were not submerged. This shows that inundation during the seedling stage impedes seedling survival. Therefore, seeds can contribute to recovery after ramet decline or colonization to open area when (i) the rainfall is not heavy to cause seedling submergence or (ii) seeds are dispersed to higher area which is not inundated.     

Relationship between seed and clonal growth in the reproduction of Carex rugulosa Kük. in riverside meadows

Carex rugulosa Kük. develops riverside vegetation in brackish marshes along the lower parts of rivers. Because the rhizomatous ramets grow densely (590–950/m²) in its developed population, it is expected that seedlings cannot get enough light to grow if seeds germinate in crowded ramets. We studied the reproductive contribution of seed in a marshy meadow of the Ohashi River, Japan. The number of seeds produced was 6900–14 200/m² in 2002. The number of buried seeds in the following germination season differed among sites from 1190 to 2690/m². No seedlings were observed at plots where the ramets from rhizomes grew densely. In contrast, seedlings appeared in plots where all rhizomatous ramets were artificially cut. The number of seedlings corresponded to 17.5–39.5% of that of buried seeds. All these seedlings were submerged and died when it rained heavily. In the genotype analysis using allozyme detection, one of the phosphoglucoisomerase loci ( Pgi-2 ) indicated a pair of alleles. Among eight plots (each plot was 50 or 60-cm square), four showed an excess of heterozygotes and two showed homozygote domination in the Pgi-2 genotype. These results suggest that: (i) seed germination is suppressed in crowded ramets; (ii) seedling survival is severely reduced by inundation; and (iii) the population is usually maintained by clonal growth. As germination is induced in open areas, it is hypothesized that the main role of seed is recovery after vegetation decline caused by environmental stresses or colonization to other areas without dense vegetation.     

Biological Role of Tyrosinase Related Protein and its Biosynthesis and Transport From TGN to Stage I Melanosome,Late Endosome,Through Gene Transfection Study

Tyrosinase-related protein (TRP)-1 is one of the most abundant melanosomal glycoproteins involved in melanogenesis. This report summarizes our recent research efforts related to the biological role and biosynthesis of TRP-1 and its transport from TGN (trans-Golgi network) to the stage I melanosome. Our UV irradiation and tyrosinase and TRP-1 cDNA co-transfection studies indicated that human TRP-1 is involved in not only melanogenesis but also prevention of melanocyte death, which may occur during biosynthesis of melanin pigment in the presence of tyrosinase. Furthermore, a coordinated gene interaction was indicated between tyrosinase and TRP-1, resulting in upregulation of mRNA and protein expression of LAMP (lysosome-associated membrane protein)-1 that would directly prevent the tyrosinase-mediated programmed cell death of melanocytes. Similar to tyrosinase, however, TRP-1 appears to require a molecular chaperone, calnexin, which we have cloned recently. Our cDNA transfection study of tyrosinase with calnexin showed clearly the necessity of calnexin in order to have efficient, functional activity of melanosomal glycoprotein, especially tyrosinase. Once glycosylation is completed, TRP-1 will be transported from TGN to the stage I melanosome. At this stage, TRP-1 will have its own target signal, in particular, tyrosine-rich leucine residues in cytoplasmic tail. Our TRP-1 cDNA transfection and immunoelectron microscopy study shows that TRP-1 will be transported through small vesicles, probably non-clathrin-coated type, to large vacuoles, identical to the MPR (mannose-6-phosphate receptor)-positive, late endosomes. In this transport process, a low molecular weight G-protein, rab-7, was isolated from the purified melanosomal protein on 2D-PAGE and identified by subsequent sequencing and PCR amplification. Confocal microscopy with double immunostaining and immunoelectron microscopy confirmed the co-localization of rab-7 and TRP-1 in the melanosomes with early stages of maturation (I-III). Furthermore, this process will also be regulated by phosphatidylinositol 3-kinase (PI-3 kinase).