Transient microglial absence assists postmigratory cortical neurons in proper differentiation

In the developing cortex, postmigratory neurons accumulate in the cortical plate (CP) to properly differentiate consolidating subtype identities. Microglia, despite their extensive surveying activity, temporarily disappear from the midembryonic CP. However, the mechanism and significance of this absence are unknown. Here, we show that microglia bidirectionally migrate via attraction by CXCL12 released from the meninges and subventricular zone and thereby exit the midembryonic CP. Upon nonphysiological excessive exposure to microglia in vivo or in vitro, young postmigratory and in vitro-grown CP neurons showed abnormal differentiation with disturbed expression of the subtype-associated transcription factors and genes implicated in functional neuronal maturation. Notably, this effect is primarily attributed to interleukin 6 and type I interferon secreted by microglia. These results suggest that “sanctuarization” from microglia in the midembryonic CP is required for neurons to appropriately fine-tune the expression of molecules needed for proper differentiation, thus securing the establishment of functional cortical circuit.

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Yuki Hattori and Takaki Miyata
Feb 27, 2020 Image data were collected using FV10-ASW software version 4.1 on Fluoview FV1000 (Olympus) and NIS-Elements software AR Analysis version 5.01.00 on TiEA1R (Nikon) and A1Rsi (Nikon). Live-imaging data were collected using CV1000 software version 1.06.06 on CellVoyager CV1000 (Yokogawa Electric Corporation). Flow cytometry analysis was performed using FACSDiva software version 8.0 on FACS Canto II and FACS SORP Aria II (BD Biosciences). RNA samples were sequenced on the Illumina NovaSeq6000 platform using a 100 bp paired-end strategy. ELISA data were obtained using PowerScan4.
For RNA-Seq, read qualities were assessed by the FASTQC tool on Galaxy. Reads were mapped to the mouse genome assembly (mm10) using TopHat version 2.1.1 using the corresponding sample's mean inner distance between mate pairs. The mRNA read counts were quantified during transcript assembly with Cufflinks version 2.2.1.2. For the heat map, the count data were transformed using the DESeq2 algorithm. Seeking an unbiased approach to pathway analysis, we used the gene set enrichment analysis (GSEA) tool developed by Broad Institute (http://software.broadinstitute.org/gsea/index.jsp), which identifies groups of coordinately regulated genes present in gene sets annotated in the Molecular Signatures Database (MSigDB). Flow cytometry data were analyzed using FlowJo software version 7.6. Statistical analyses were performed using R software version 3.6.0.
The source data for all experiments in this study are provided as a Source Data file. The raw data have been deposited in the DNA Data Bank of Japan (DDBJ) under nature research | reporting summary

October 2018
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All studies must disclose on these points even when the disclosure is negative. the DRX accession number: DRX199371-DRX199376. These sequence data are also available at NCBI Sequence Read Archive (SRA) under the ID code: DRP005827 (https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=DRP005827).
No statistical methods were used to predetermine the sample size owing to experimental limitations. Sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.
No samples were basically excluded from the analysis. We only excluded the data obtained from failed experiments by some reasons, e.g., failure in sample preparations.
We confirmed that replicate experiments were successful by repeating at least three times for all experiments.
No randomization of mice was performed. Mice analyzed were litter mates whenever possible.
Basically, investigators were blinded during experiments.
Information on all antibodies used in the study (their supplier name, catalog number and the concentration) was described in the Method section.
Validation about all antibodies was described on the manufacturer's website. For some antibodies (such as anti-Satb2 antibody, anti-Ctip2 antibody, anti-Brn2 and anti-RORb antibody) that we used for intracellular FACS staining, we separately evaluated whether these antibodies could be used because the information about the application for FACS was missed in the manufacturer's site. The antibody concentration we determined is provided in the Method section.
NB2a mouse neuroblastoma cell line was introduced from RIKEN BRC.
Short Tandem Repeat (STR) profiling was applied.
NB2a tested negative for mycoplasma.
No use.