We isolated a novel bHLH protein gene Mesp2 (for mesoderm posterior 2) that cross-hybridizes with Mespl expressed in the early mouse mesoderm. Mesp2 is expressed in the rostral presomitic mesoderm, but down-regulated immediately after the formation of the segmented somites. To determine the function of MesP2 protein (MesP2) in somitogenesis, we generated Mesp2.deficient mice by gene targeting. The homozygous Mesp2 (-/-) mice died shortly after birth and had fused vertebral columns and dorsal root ganglia, with impaired sclerotomal polarity. The earliest defect in the homozygous embryos was a lack of segmented somites. Their disruption of the metameric features, altered expression of Mox-1, Pax-l, and Dill, and lack of expression of Notchl, Notch2, and FGFR1 suggested that MesP2 controls sclerotomal polarity by regulating the signaling systems mediated by notch-delta and FGF, which are essential for segmentation.

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The larger genomic segment, RNA1 (~3.1 kb), encodes an RNA-dependent RNA polymerase (protein A), and the smaller genomic segment RNA2 (~1.4 kb) codes for the coat protein. These viruses can be classified into four genotypes, designated striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV), and barfin flounder nervous necrosis virus (BFNNV), based on similarities in their partial RNA2 sequences. The optimal temperatures for the growth of these viruses are 20-25°C (SJNNV), 25-30°C (RGNNV), 20°C (TPNNV), and 15-20°C (BFNNV). However, little is known about the mechanisms underlying the temperature sensitivity of these viruses. We first constructed two reassortants between SJNNV and RGNNV to test their temperature sensitivity. The levels of viral growth and RNA replication of these reassortants and parental viruses in cultured fish cells were similar at 25°C. However, the levels of all of the viruses but RGNNV were markedly reduced at 30°C. These results indicate that both RNA1 and RNA2 control the temperature sensitivity of betanodaviruses by modulating RNA replication or earlier viral growth processes. We then constructed ten mutated RGNNVs, the RNA1 segments of which were chimeric between SJNNV and RGNNV, and showed that only chimeric viruses bearing the RGNNV RNA1 region, encoding amino acid residues 1-445, grew similarly to the parental RGNNV at 30°C. This portion of protein A is known to serve as a mitochondrial-targeting signal rather than functioning as an enzymatic domain.

A subtractive hybridization strategy was used to isolate putative genes involved in the development of mouse primordial germ cells (PGC). Complimentary DNA was amplified on RNA isolated from the base of the allantois where PGC are located in the 7.5 days post coitum (dpc) mouse embryo. It was then subtracted by hybridization with cDNA amplified on RNA of the anterior region where PGC are absent. A novel gene thus isolated is designated as Mesp1 and encodes a possible transcription factor MesP1 containing a basic helix-loop-helix motif. Its earliest expression was observed at the onset of gastrulation, as early as 6.5 dpc, in the nascent mesodermal cells that first ingressed at the end of the primitive streak. These expressing cells in the lateral and extraembryonic mesoderm showed a wing-shaped distribution. Its initial expression was soon down-regulated at 7.5 dpc before the completion of gastrulation, except at the proximal end of the primitive streak which included the extraembryonic mesoderm and the base of allantois. At 8 dpc, the expression at the base of the allantois moved laterally. This distribution between 7.0 and 8.0 dpc was similar to that of PGC detected by the alkaline phosphatase activity. However, the expression of Mesp1 was down-regulated thereafter, when PGC entered in the migration stage. After birth, Mesp1 expression was detected only in mature testes, but in a different isoform from that expressed in the embryo. Mesp1 was mapped to the mid region of chromosome 7, near the mesodermal deficiency gene (mesd). However, a Southern hybridization study clearly showed that Mesp1 was distinctly different from mesd. The amino acid sequence and its expression pattern suggest that MesP1 plays an important role in the development of the nascent mesoderm including PGC.

-Among the four types of betanodaviruses, redspotted grouper nervous necrosis virus (RGNNV) has the highest optimum temperature (25-30∞C) for its multiplication. We tested 16 RGNNV isolates for their temperature sensitivity in cultured cells and demonstrated that their upper temperature limits ranged from less than 30∞C to 35∞C. At the temperatures over the upper limits, viral RNA replication was inhibited similarly. These results indicate that temperatures mainly affect RNA replication or earlier virus multiplication processes. The incompetence of betanodaviruses at 37∞C suggests their avirulence in human.