2017 Fiscal Year Research-status Report
Identification and characterization of PLA2 enzyme required for sensory nervous system development
| Project/Area Number |
17K14970
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
GUY ADAM・TSUDA 国立研究開発法人理化学研究所, 脳科学総合研究センター, 研究員 (30634779)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | Molecular Biology / Cell Biology / Neuroscience |
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| Outline of Annual Research Achievements |
I have imported into Riken three secreted PLA2 knockout (KO) mouse lines in Riken animal facility: pla2g5 (sPLA2 V) and pla2g10 (sPLA2 X) and pla2g3 (sPLA2 III).
I have obtained antibodies to sPLA2 isozymes.
I have carried out antibody staining of phosphatidyl glucoside (PG), the precursor to lysophosphatidyl glucoside (LPG), and found that distribution of PG is unchanged in sPLA2 V and X knockout mouse spinal cord. This is an important control.
I have carried out analyses of sPLA2 V and sPLA2 X KO mice spinal cord sensory axon connections, using DiI to label primary afferents and confocal microscopy imaging. Both V and X lines display axon mis-projection at developmental stage E14 in spinal cord. Specifically, nociceptive axons extend outside the dorsal root entry zone and into inappropriate zones of the spinal cord including dorsal grey matter and medial white matter. In general, sPLA2 X showed a stronger (ie greater abnormal projection) than sPLA2 V. These are very promising data.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Progress in analyses of in vivo phenotypes of knockout mice have been delayed because of low breeding rate of knockout mice. All three knockout lines show reduced male fertility. To overcome this, I have requested Riken animal facility to perform in vitro fertilisation (IVF) and cryostorage of zygotes, to be implanted into wildtype (WT) surrogates for developmental analyses. So far, in total >80 pla2g5 and pla2g10 homozygote KO embryos have been successfully produced and placed in cryostorage. pla2g3 line has yet to be IVF-treated (due to waiting list in animal facility).
Progress in mass spectrometry analyses of LPG concentration in spinal cord of WT and sPLA2 KO mouse embryos is slightly delayed due to laboratory amalgamation and purchase of new mass spectrometer which requires time to optimise. However, the new mass spectrometer, manufactured by Shimazu corporation, will have high sensitivity for lipid analyses and I am confident measurements of spinal cord LPG will be possible in FY2018.
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| Strategy for Future Research Activity |
I will carry out the following research plans:
1) Continue DiI and antibody labelling analyses of spinal cord projection of sensory axons in the KO mouse lines pla2g5 and pla2g10. This study is being carried out blind to strengthen its data. In particular, I will carry out sensory axon labelling mixed with WT littermates as an essential control.
2) IVF of pla2g3 KO embryos to allow analyses to begin on this KO mouse line.
3) Mass spectrometry analyses of WT, pla2g5 KO and pla2g10 KO embryonic spinal cord concentration of LPG.
4) Present data so far obtained at international conference (Molecular Mechanisms of Neuronal Connectivity, CSHL, September 2018).
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