2016 Fiscal Year Research-status Report
Unravelling Mechanisms Underlying Termination of Neuronal Migration
| Project/Area Number |
16K07010
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| Research Institution | National Institute of Genetics |
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Principal Investigator |
ZHU YAN 国立遺伝学研究所, 総合遺伝研究系, 助教 (50464235)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | RNA-seq / transcriptome profile / degradation / neuronal migration / termination |
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| Outline of Annual Research Achievements |
This research takes two complementary approaches to unravel mechanisms underlying termination of neuronal migration: (1) RNA-seq based transcriptome profiling; (2) regulation and the functional significance of the dynamic changes of cell surface molecules during migration termination.
(1) During the first fiscal year, we established methods by which we could successfully obtain three neuronal populations with high purity: neurons during migration, neurons undergoing migration termination, and neurons that termination at a different site. We extracted total RNA and thereafter prepared cDNA libraries for next generation sequencing from these populations. The 15 cDNA libraries are presently undergoing deep sequencing.
(2) A number of cell surface molecules are being dynamically regulated during the termination of neuronal migration. In the first fiscal year, using both in vitro and in vivo approaches, we found that the guidance receptor DCC and adhesion molecule Tag-1 are down-regulated in neurons undergoing termination. Furthermore, we found that these down-regulations take place at the post-transcriptional level. We are currently dissecting sub-domains of DCC that mediate its degradation.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The RNA-seq experiment has been progressing in accordance with our original plan.
For the investigation of the down-regulation of cell surface molecules during migration termination, my plan in the grant application was to investigate the extrinsic cues that cause this down-regulation first. However, the fact that we found DCC and Tag-1 are down-regulated at the protein levels provided us an opportunity to dissect out protein sub-domains that mediate their down-regulation. This information will provide useful clues to our investigation of the extrinsic cues in the next step.
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| Strategy for Future Research Activity |
Following the completion of deep sequencing, we will perform bioinformatics analysis of the RNA-seq data to identify genes or molecules that are differentially expressed between the three neuronal populations. Candidate molecules will be selected from this in silico list and their true differential expression will be validated by other methods such as in situ hybridization. Molecules of potential interest will then be investigated for their involvement in mediating various aspects of termination of neuronal migration.
We are expecting to identify sub-domains of DCC /Tag-1 that are involved in their degradation. Following the clues deduced from the nature of these degradation domains, we will then investigate the extrinsic cues that trigger DCC and Tag-1 degradation. We will also make degradation resistant variants of these molecules and investigate the functional significance of the dynamic regulation of these molecules at the migration termination sites.
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| Causes of Carryover |
The second fiscal year funding will be budgeted for reagents/kits for performing in situ hybridization and immunohistochemistry; reagents for FACS sorting, and next generation sequencing.; reagents and lab wares for in vitro neuronal culture and imaging; reagents/kits for molecular biology, and reagents and experimental animals for in vivo functional experiments.
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| Expenditure Plan for Carryover Budget |
In situ hybridization and immunohistochemistry will be used to identify the true differential expressed molecules between the three neuronal populations. FACS sorting and next generation sequencing reagents are to prepare for possible addition of the fourth neuronal populations to the RNA-seq experiment. In vitro neuronal culture and imaging are to understand the function and protein regulation of DCC/Tag-1 in in vitro systems. Finally, reagents/kits for molecular biology will be used to construct variants of DCC/Tag-1 as well as cloning the expression constructs of identified molecules from RNA-seq experiments. Experimental animals and other in vivo experiment reagents are for analysing the function of these molecules in vivo.
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