Onami S et al. (2006) Bioessays "Genome-wide prediction of genetic interactions in a metazoan."

Kitano H, Onami S

[Bioessays, 2006]

Genetic interactions provide information about genes and processes with overlapping functions in biological systems. For Saccharomyces cerevisiae, computational integration of multiple types of functional genomic data is used to generate genome-wide predictions of genetic interactions. However, this methodology cannot be applied to the vastly more complex genome of metazoans, and only recently has the first metazoan genome-wide prediction of genetic interactions been reported. The prediction for Caenorhabditis elegans was generated by computationally integrating functional genomic data from S. cerevisiae, C. elegans and Drosophila melanogaster. This achievement is an important step toward system-level understanding of biological systems and human diseases. BioEssays 28: 1087-1090, 2006. (c) 2006 Wiley Periodicals, Inc.

Onami S et al. (1997) International C. elegans Meeting "CHANGES IN POLY(A) TAIL LENGTH OF MATERNAL MESSAGES."

Kohara Y, Onami S

[International C. elegans Meeting, 1997]

In Drosophila and Xenopus, lots of maternal mRNA have been reported whose translation is regulated through cytoplasmic polyadenylation; their poly(A) tails elongate when their translation is activated. Aiming to fish out the maternal mRNA of C. elegans whose translation is regulated through cytoplasmic polyadenylation, we have developed a method to assess the poly(A) tail length of given mRNA in embryos at a specific stage. First, we examined the poly(A) tail lengths of several known maternal mRNA whose translation is considered to be regulated. Maternal glp-1 mRNA exists in embryo before the first division and is detected in both blastomeres at 2cell stage, while GLP-1 protein first appears at 2cell stage and is restricted in AB blastomere(Evans et al., Cell 77:183). In order to examine the poly(A) tail length of glp-1 mRNA, total RNA was extracted from 15 embryos at the same stage. An RNA-oligonucleotide-tag was added to its 3'-end. This RNA was examined by RT-PCR using a glp-1-specific and a tag-specific primer. Results showed that glp-1 mRNA has poly(A) tail of -40 bases in matured oocyte while 2cell stage embryo contains glp-1 mRNA with longer poly(A) tail(>110bases). 2cell stage embryo also contains glp-1 mRNA with shorter poly(A) tail(-25bases). These results suggest that cytoplasmic polyadenylation is involved in the translational regulation of glp-1 mRNA. Fem-3 mRNA and skn-1 mRNA were examined and elongation of their poly(A) tails during embryogenesis was also observed. We are currently examining the exact time at which the poly(A) tails start to elongate. Also, we are examining their poly(A) tail lengths in AB and P1 blastomere.

Onami S et al. (2001) Foundations in Systems Biology "Automatic aquisition of cell lineage through 4D microscopy and analysis of early C. elegans embryogenesis."

Hamahashi S, Kitano H, Onami S, Miyano S, Nagasaki M

[Foundations in Systems Biology, 2001]

S Onami et al. (1999) International C. elegans Meeting "Protein Expression Pattern Analysis of Maternal mRNAs."

Y Kohara, S Onami, T Nagaoka

[International C. elegans Meeting, 1999]

Hatakeyama, A. et al. (2019) International Worm Meeting "Mechanical functions of eggshell in C. elegans development."

Tanaka, Y., Onami, S., Sato, A., Hatakeyama, A.

[International Worm Meeting, 2019]

A nematode embryo is surrounded by chitinous eggshell. It has been reported that, in C.elegans, about 75% of eggs without eggshell at the stage later than the 2-cell stage can develop to hatch1. Therefore, it has been believed that eggshell does not play any particular role in the development at later stages except the mechanical protection of the embryo. However, our earlier studies showed that the body length of the first-stage larva (L1) hatched from the eggs without eggshell and with the permeability barrier inside the eggshell was about 10% shorter than that from intact eggs2. This result suggests that eggshell plays some role in epidermal morphogenesis. To investigate mechanical functions of eggshell, we have developed a microdevice as artificial eggshell. We have shown that incubation of eggs without eggshell in our microdevice recovers the defects in body length3. In the present study, we further investigated the effects of eggshell on L1 morphologies. Arrangements of epidermal cells were observed by using the strain in which cell boundaries were visualized by the AJM-1::GFP. As a result, the number and the line-pattern arrangements of seam cells were unchanged but contacts between some seam cells (H1-H2 cells and V1-P1/2 cells) were altered. The variation was observed mainly at an anterior half of the L1 larva hatched from the eggs without eggshell. In contrast, cell-cell contacts of seam cells in the embryo from the comma to the 3-fold stages showed no noticeable difference between intact eggs and eggs without eggshell. The eggshell would have functions at the very last stage of embryonic development such as the stage just before hatching. To manipulate mechanical environments around embryos during development, we fabricated microwells with various sizes (20 -200 m in width) and shapes (round, oval and rectangle), which adapt to our device. When eggs without eggshell were incubated in oval-shaped microwells (well size 23 x 36 m2), which was smaller in size than normal eggs, no recovery was found in the body length, unlike the case when oval-shaped microwells (well size 43x59 m2), as small as normal embryos, were used. We speculate that mechanical environments around an embryo affect efficiencies in mechanotransduction during morphogenesis. 1. Schierenberg E and Junkersdorf B, 1992, Roux's Arch Dev Biol, 202, 10-16. 2. Hatakeyama A and Onami S, 2017, 21th International C. elegans Conference Abstracts, LA. 3. Hatakeyama A et al, 2018, EMBO Workshop: C. elegans Development, Cell Biology, and Gene Expression Abstracts, Barcelona.

Takayama J et al. (2016) Cell Rep "The Sperm TRP-3 Channel Mediates the Onset of a Ca(2+) Wave in the Fertilized C.elegans Oocyte."

Takayama J, Onami S

[Cell Rep, 2016]

Sperm induce Ca(2+) waves in the fertilized egg by introducing soluble factors or by surface interactions, which activate egg Ca(2+) channels. Involvement of sperm Ca(2+) channels is predicted by the conduit model; however, this model has not been validated. In Caenorhabditis elegans, the sperm-specific TRP family Ca(2+) channel TRP-3 mediates sperm-oocyte fusion. Here, using high-speed invivo imaging and image analyses, we show that sperm induce an immediate local Ca(2+) rise followed by a Ca(2+) wave in fertilized C.elegans oocytes. Oocytes fertilized by rare trp-3 escaper sperm showed a lack of local rise and a delay in onset of the Ca(2+) wave. Sperm Ca(2+) imaging suggests that the local rise is not due to the bolus introduction of stored Ca(2+). These results suggest that, along with its primary function in sperm-oocyte fusion, TRP-3 induces Ca(2+) waves in fertilized oocytes, consistent with the conduit model.

Takayama, J. et al. (2013) International Worm Meeting "Sperm-derived TRP-3 channel specifies the onset of the fertilization Ca2+ wave in the oocyte of C. elegans."

Onami, S., Takayama, J.

[International Worm Meeting, 2013]

Fertilization induces a Ca2+ wave in the egg to start embryogenesis. How sperm triggers the Ca2+ wave is not well understood. Here we show that sperm-derived TRP-3 channel specifies the onset of the Ca2+ wave in the oocyte upon plasma membrane fusion in C. elegans. First, by live confocal imaging, we found that sperm entry triggered a biphasic Ca2+ response: a fast local rise and a following slow global wave. The fast local rise emerged near the sperm entry point immediately after sperm entry, whereas the slow global wave traveled from that point to the opposite pole. This biphasic waveform was reproduced by simulations assuming Ca2+-induced Ca2+ release and local Ca2+ influx. Next, to understand how sperm triggers the Ca2+ wave, we examined Ca2+ responses in sperm mutants. In spe-9 mutants, whose sperm cannot enter the oocyte, no Ca2+ responses were observed. On the other hand, in spe-11 mutants, whose sperm cannot activate but can enter the oocyte, a wild type-like response was observed. In trp-3/spe-41 mutants, which lack the sperm-specific Ca2+-permeable channel TRP-3, fertilized oocytes generated no local Ca2+ rise but a slow global wave with delayed onset. Approximately half of the fertilized trp-3 embryos were arrested during embryogenesis. Finally, we investigated how the sperm plasma membrane channel mediates the local Ca2+ rise in the oocyte. By high-speed imaging, we found that Ca2+ concentration in the fused sperm cytoplasm increased from a resting level rather than decreased from a high level equivalent to the local rise. In addition, visualization of the plasma membrane of the oocyte revealed that sperm entered the oocyte by fusion at the plasma membrane, which contains the sperm channels. Altogether, these results suggest that sperm-derived plasma membrane that contains TRP-3 channels acts as a signaling membrane domain to specify the onset of the Ca2+ wave in the oocyte. TRP-3 channels might ensure successful embryogenesis by timely triggering the Ca2+ wave to coordinate with other events occurring during oocyte-to-embryo transition.

Takayama, J. et al. (2015) International Worm Meeting "A sperm-derived ion channel TRP-3 induces a Ca2+ wave in the fertilized C. elegans oocyte."

Onami, S., Takayama, J.

[International Worm Meeting, 2015]

Embryonic development is triggered by Ca2+ waves during fertilization. How these Ca2+ waves are induced is poorly understood. Here we show that a sperm-derived plasma membrane channel TRP-3 induces a Ca2+ wave in fertilized oocytes in C. elegans by using high-speed in vivo imaging and genetic analysis. We visualized the fertilization Ca2+ dynamics by using spinning disk confocal microscopy and a chemical Ca2+ indicator. Image analysis revealed that the fertilized oocyte showed a local Ca2+ rise near the sperm entry point upon sperm entry, followed by a traveling Ca2+ wave. Both the local Ca2+ rise and the traveling Ca2+ wave was absent in sperm-defective mutants such as spe-9 and spe-42, in which sperm cannot enter the oocyte. The local Ca2+ rise was dependent on a sperm ion channel, TRP-3, also known as SPE-41, because no local Ca2+ rise was observed in the fertilized oocyte in trp-3 mutants. The traveling Ca2+ wave was observed in the trp-3 mutant, but the onset was significantly delayed. These defects were sperm-dependent because wild-type oocytes fertilized by trp-3 mutant sperm showed a trp-3 mutant-type Ca2+ response, and trp-3 mutant oocytes fertilized by wild-type sperm showed the wild-type Ca2+ response. Moreover, sperm-specific expression of a trp-3 transgene partially rescued both the absence of the local Ca2+ rise and the delay in the onset of the traveling wave. Transgenic experiments revealed the sperm-specific expression of the trp-3 gene and the surface localization of TRP-3::TagRFP-T fusion protein in mature sperm. We visualized the fertilization between oocytes whose plasma membrane was labeled with GFP::PH and the TRP-3::TagRFP-T-expressing sperm, and found that sperm entered the oocyte via direct plasma membrane fusion. Moreover, during the sperm-oocyte fusion, TRP-3::TagRFP-T was transferred from the sperm plasma membrane to the oocyte plasma membrane. The Ca2+ concentration of the mature trp-3 mutant sperm before fertilization was not different from that of the wild-type sperm. The estimated Ca2+ concentration in the fused sperm cytoplasm increased from a resting level after the sperm-oocyte fusion. Furthermore, transgenic rescue experiments for the trp-3 mutant showed that the larger the local Ca2+ rise, the earlier the traveling wave was induced. This correlation was explained by a numerical simulation, which assumes Ca2+-induced Ca2+ release machinery in the oocyte. These results suggest that sperm-derived TRP-3 channel acts as a Ca2+ conduit to induce the local Ca2+ rise in the fertilized oocyte. The local Ca2+ rise then induces the traveling Ca2+ wave probably via the Ca2+-induced Ca2+ release machinery.

Fujita M et al. (2012) PLoS One "Cell-to-cell heterogeneity in cortical tension specifies curvature of contact surfaces in Caenorhabditis elegans embryos."

Onami S, Fujita M

[PLoS One, 2012]

In the two-cell stage embryos of Caenorhabditis elegans, the contact surface of the two blastomeres forms a curve that bulges from the AB blastomere to the P blastomere. This curve is a consequence of the high intracellular hydrostatic pressure of AB compared with that of P. However, the higher pressure in AB is intriguing because AB has a larger volume than P. In soap bubbles, which are a widely used model of cell shape, a larger bubble has lower pressure than a smaller bubble. Here, we reveal that the higher pressure in AB is mediated by its higher cortical tension. The cell fusion experiments confirmed that the curvature of the contact surface is related to the pressure difference between the cells. Chemical and genetic interferences showed that the pressure difference is mediated by actomyosin. Fluorescence imaging indicated that non-muscle myosin is enriched in the AB cortex. The cell killing experiments provided evidence that AB but not P is responsible for the pressure difference. Computer simulation clarified that the cell-to-cell heterogeneity of cortical tensions is indispensable for explaining the pressure difference. This study demonstrates that heterogeneity in surface tension results in significant deviations of cell behavior compared to simple soap bubble models, and thus must be taken into consideration in understanding cell shape within embryos.

Kimura A et al. (2005) Dev Cell "Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration."

Kimura A, Onami S

[Dev Cell, 2005]

A male pronucleus migrates toward the center of an egg to reach the female pronucleus for zygote formation. This migration depends on microtubules growing from two centrosomes associated with the male pronucleus. Two mechanisms were previously proposed for this migration: a "pushing mechanism," which uses the pushing force resulting from microtubule polymerization, and a "pulling mechanism," which uses the length-dependent pulling force generated by minus-end-directed motors anchored throughout the cytoplasm. We combined two computer-assisted analyses to examine the relative contribution of these mechanisms to male pronuclear migration. Computer simulation revealed an intrinsic difference in migration behavior of the male pronucleus between the pushing and pulling mechanisms. In vivo measurements using image processing showed that the actual migration behavior in Caenorhabditis elegans confirms the pulling mechanism. A male pronucleus having a single centrosome migrated toward the single aster. We propose that the pulling mechanism is the primary mechanism for male pronuclear migration.