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A gonad is formed from germ cells and somatic mesodermal cells through their interactions. Its development is coupled with the determination and differentiation of the sex and sex-associated traits. We carried out a large-scale screening of Medaka mutants in which gonadal development is affected. Screening was performed on larvae at 8 days posthatching for abnormal abundance and/or distribution of germ cells detected by the in situ hybridization for olvas (Medaka vasa). We describe here 16 mutants of 13 genes, which are classified into four groups. Group 1, consisting of four mutants of three genes kon, tot) characterised by an increase in germ cell number. An adult tot homozygote fish has the characteristic feature of possessing hypertrophic gonads filled with immature oocytes. Group 2, represented by a single gene (zen) mutant characterized by a gradual loss of germ cells. Group 3, consisting of four mutants of distinct genes (eko, eki, sht, ano) showing irregular clustering of germ cells. Group 4, consisting of seven mutants of five genes (arr, hyo, mzr, hdr, fbk) showing fragmented clusters of germ cells. In some mutants belonging to Groups 1, 3 and 4, the expression level of ftz-f1 (sf-1/Ad4BP) in gonadal somatic cells significantly decreased, suggesting that interaction between somatic and germ cells is affected.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

We screened for mutations affecting retinotectal axonal projection in Medaka, Oryzias latipes. In wild-type Medaka embryos, all the axons of retinal ganglion cells (RGCs) project to the contralateral tectum, such that the topological relationship of the retinal field is maintained. We labeled RGC axons using DiI/DiO at the nasodorsal and temporoventral positions of the retina, and screened for mutations affecting the pattern of stereotypic projections to the tectum. By screening 184 mutagenized haploid genomes, seven mutations in five genes causing defects in axonal pathfinding were identified, whereas mutations affecting the topographic projection of RGC axons were not found. The mutants were grouped into two classes according to their phenotypes. In mutants of Class I, a subpopulation of the RGC axons branched out either immediately after leaving the eye or after reaching the midline, and this axonal subpopulation projected to the ipsilateral tectum. In mutants of Class II, subpopulations of RGC axons branched out after crossing the midline and projected aberrantly. These mutants will provide clues to understanding the functions of genes essential for axonal pathfinding, which may be conserved or partly divergent among vertebrates.

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.

We have challenged to reduce an accelerator beam power for an accelerator-based BNCT facility. The required neutron source strength at the target has been estimated so as to make the epithermal neutron flux in the patient irradiation field exceed 1.7 x 10 9 n/cm 2 s. The energy of the incident proton and the arrangement of the moderator assemblies are optimized. The beam current and the accelerating voltage are determined so that the accelerator power becomes minimum. The beam power required for the treatment in one hour is 62.5 kW. The proposed facility is equipped with a 2.5 MeV proton accelerator of 25 mA, a lithium target, and a heavy water moderator contained in an aluminum tank.