Nek2, a mammalian structural homologue of Aspergillus protein kinase NIMA, is predominantly known as a centrosomal kinase that controls centriole-centriole linkage during the cell cycle. However, its dynamic subcellular localization during mitosis suggested that Nek2 might be involved in diverse cell cycle events in addition to the centrosomal cycle. In order to determine the importance of Nek2 during mammalian development, we investigated the expression and function of Nek2 in mouse early embryos. Our results show that both Nek2A and Nek2B were expressed throughout early embryogenesis. Unlike cultured human cells, however, embryonic Nek2A appeared not to be destroyed upon entry into mitosis, suggesting that the Nek2A protein level is controlled in a unique manner during mouse early embryogenesis. Suppression of Nek2 expression by RNAi resulted in developmental defects at the second mitosis. Many of the blastomeres in Nek2-suppressed embryos showed abnormality in nuclear morphology, including dumbbell-like nuclei, nuclear bridges and micronuclei. These results indicate the importance of Nek2 for proper chromosome segregation in embryonic mitoses.

A rapid induction of mouse period1 (mPer1) gene expression is supposed to be critical in the clock gene regulation, especially in the phase resetting of the clock, but its molecular mechanism is poorly understood. Based on the previous finding that the process does not involve de novo synthesis of proteins, we postulated the involvement of CLOCK:BMAL1 heterodimer, a positive regulator of circadian oscillator, in the rapid induction of mPer1 transcription. To test this hypothesis, we utilized CLOCKdelta19, a dominant-negative mutant, to suppress the function of CLOCK:BMAL1 in vitro. Serum-evoked rapid increases of mPer1 mRNA expression and promoter activity were significantly blunted when CLOCK:BMAL1 function was interfered with. Furthermore, DNA binding activity of CLOCK:BMAL1 heterodimer to five E-boxes of mPer1 promoter markedly increased shortly after serum shock. Taken together, these results suggest that CLOCK:BMAL1 heterodimer is not only a core component of negative feedback loop driving circadian oscillator, but also involved in the rapid induction of mPer1during phase resetting of the clock.

Heat shock proteins (HSPs) are known to play an important role not only in various stress conditions such as exposure to heat shock, but also in normal development and/or differentiation. The role of small heat shock proteins such as HSP25 in early embryo development remains largely unknown. In the present study, we examined temporal and spatial expression patterns of HSP25 during mouse preimplantation embryo development. Reverse transcription-polymerase chain reaction (RT-PCR) showed that hsp25 mRNA was detected in unfertilized eggs. Hsp25 mRNA was induced by zygotic gene activation at 2-cell stage, decreased slightly at 4-cell, and re-increased at morula, with the highest level at blastocyst stage. Interestingly, another form of hsp25 variant of which 156 bp (52 a.a.) was truncated within the exon1 region was observed in all stages of preimplantation embryos. We also investigated the sub-cellular localization of HSP25 by fluorescence immunocytochemistry. HSP25 was detected in the cytoplasm under normal developmental condition. While acute heat shock (at 43 degrees C for 30 min) caused no significant changes in the sub-cellular localization of HSP25 in the developing mouse embryos, chronic heat shock (at 43 degrees C for 3 hr) resulted in a denser immunostaining of HSP25 in the nucleus than in the cytoplasm, indicating a nuclear translocation of HSP25 by heat shock. As hsp25 mRNA was detected in the unfertilized egg as a maternal transcript, we examined the expression of hsp25 mRNA with RT-PCR during oocyte maturation under normal and heat shock conditions. Hsp25 mRNA was detected at GV (germinal vesicle)-, GVBD (germinal vesicle breakdown)-, and MII (metaphase II)-oocytes. The expression of hsp25 mRNA was increased markedly by both acute (for 30 min and 1 hr) and chronic (for 4 hr) heat shock, but returned to the basal level during recovery from heat shock in a time-dependent manner, suggesting a thermo-protective role of HSP25. In contrast to preimplantation embryos, HSP25 was detected both in the cytoplasm and the nucleus except for the nucleolus, and the cellular localization was not altered by heat shock. Finally, we investigated the effect of heat shock on oocyte maturation. When GV-oocytes were exposed to acute heat shock (at 43 degrees C for 15 min to 1 hr), they underwent the GVBD and the PB (polar body) emission successfully. However, under more stringent heat shock conditions (at 43 degrees C for 2-4 hr), most oocytes were arrested at the GV-stage, and the first PB was not developed, indicating that chronic heat shock might be inhibitory to the mouse oocyte maturation. Taken together, these findings suggest that HSP25 is important for mouse preimplantation embryo development and oocyte maturation.

The preimplantation development of mammalian embryo after fertilization encompasses a series of events including cleavage, compaction, and differentiation into blastocyst. These events are likely to be associated with substantial changes in embryonic gene expression. In the present study, we explored the expression patterns and function of epithin, a mouse type II transmembrane serine protease, during preimplantation embryo development. RT-PCR analysis showed that epithin mRNAs were detectable during the cleavage stages from a 1-cell zygote to the blastocyst. Immunocytochemical studies revealed that epithin protein was expressed at blastomere contacts of the compacted 8-cell and later embryonic stages. Epithin colocalized with E-cadherin at the membrane contacts of the compacted morula-stage embryo as revealed by double-staining immunocytochemistry and confocal microscopy, respectively. Post-transcriptional epithin gene silencing by RNA interference (RNAi) resulted in the blockade of 8-cell in vitro-stage embryo compaction and subsequent embryonic deaths after several rounds of cell division. These results strongly suggest that epithin plays an important role in the compaction processes that elicit the signal for the differentiation into trophectoderm and inner cell mass.

Recently, we demonstrated that activation of the protein kinase C (PKC) signalling pathway promoted morphological differentiation of GT1 hypothalamic neurones via an increase in b-catenin, a cell-cell adhesion molecule, indicating a possible involvement of PKC in cellular motility. In this study, we explored the differential roles of PKC isoforms in GT1 cell migration. First, we transiently transfected GT1 cells with enhanced green¯uorescence protein (EGFP)-tagged actin to monitor the dynamic rearrangement of ®lamentous-actin (F-actin) in living cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, markedly promoted lamellipodia formation, while sa®ngol (a PKCa-selective inhibitor) blocked the TPA-induced lamellipodial actin structure. Both wound-healing and Boyden migration assays showed that TPA treatment promoted neuronal migration of GT1 cells; however, cotreatment of TPA with sa®ngol or rottlerin (a PKCd-selective inhibitor) clearly blocked this TPA effect, indicating that both PKCa and PKCd may be positive regulators of neuronal migration. By contrast, PKCg-EGFP-expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress ®bres, suggesting that PKCg may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130 Cas , a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130 Cas was induced in a PKCa-dependent manner. Together, these results demonstrate that PKCa promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130 Cas and FAK.