Unravelling Mechanisms Underlying Termination of Neuronal Migration

2017 Fiscal Year Research-status Report

• Project/Area Number: 16K07010
• Research Institution: National Institute of Genetics
• Principal Investigator: ZHU YAN 国立遺伝学研究所, 総合遺伝研究系, 助教 (50464235)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: neuronal migration / termination of migration / RNA sequencing / Tag-1

Outline of Annual Research Achievements

Neurons migrate from birthplaces to various final destinations to be incorporated into neural circuits. This research aims to unravel the mechanisms underlying termination of neuronal migration using two complementary approaches: (1) RNA-seq based transcriptome profiling; (2) down-regulation of cell surface molecules during termination of migration. During this fiscal year, we obtained and analyzed the RNA-seq data for approach (1), and for approach (2), we discovered a role of Tag-1 in neuronal migration and are testing hypotheses on mechanisms that down-regulate Tag-1 protein.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

(1)During this fiscal year, we have obtained the transcriptome data of 3 neuronal populations: neurons undergoing migration, neurons having terminated migration, and neurons destined to terminate at different sites. Following bioinformatic analyses and literature search, we are now performing functional screening of 17 candidate genes.
(2)Tag-1, an Ig superfamily adhesion molecule, is highly expressed in migrating neurons but its protein becomes drastically down-regulated in neurons undergoing termination. During this fiscal year, we performed detailed phenotypic analysis of Tag-1 knockout mice and found that neuronal migration was compromised. We are now in the process of identifying extrinsic cues and proteases that mediate Tag-1 down-regulation.

Strategy for Future Research Activity

Following gain-of-function screening of the candidate list, those that show effect on migration termination will be subjected to loss-of-function analyses by shRNA-mediated knockdown, or CRISPR/CAS9 mediated gene knockout. Molecules identified from this set of experiment will serve as keys for us to further unravel the regulatory network that contributes to the spatial and temporal precision of the termination of neuronal migration.
The role of Tag-1 and its regulation during migration termination will be further studied in terms of identifying the proteases that down-regulate Tag-1 as well as searching for the extrinsic cues that trigger the Tag-1 down-regulation.

Causes of Carryover

The third fiscal year budget will be used for shRNA and/or CRISPR/Cas9 knock-down experiments, molecular cloning, experimental animals for in vivo functional experiments, in vitro dissociated neuronal culture and explants culture, protease inhibitors, histology and electron microscopy analysis.
The shRNA and/or CRISPR/Cas9 loss of function experiments will be performed on mouse embryos. The in vitro neuronal and explants culture systems will be used to screen for the protease and extrinsic cues that control Tag-1 down-regulation.