Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex

Astrocytes are one of the most abundant cell types in the mammalian brain. They play essential roles in synapse formation, maturation, and elimination. However, how astrocytes migrate into the gray matter to accomplish these processes is poorly understood. Here, we show that, by combinational analyses of in vitro and in vivo time-lapse observations and lineage traces, astrocyte progenitors move rapidly and irregularly within the developing cortex, which we call erratic migration. Astrocyte progenitors also adopt blood vessel-guided migration. These highly motile progenitors are generated in the restricted prenatal stages and differentiate into protoplasmic astrocytes in the gray matter, whereas postnatally generated progenitors do not move extensively and differentiate into fibrous astrocytes in the white matter. We found Cxcr4/7, and integrin β1 regulate the blood vessel-guided migration, and their functional blocking disrupts their positioning. This study provides insight into astrocyte development and may contribute to understanding the pathogenesis caused by their defects.

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Software and code
Policy information about availability of computer code Data collection Time-lapse observations of living cells in brain slices or cultured cells on Matrigel were performed using Olympus FV1000.

Data analysis
Time-lapse image files (XYZT) were combined into movie files using 4D-Viewer software version 8.0.0 (Ratoc System Engineering). Confocal images (Olympus oib files, or Zeiss czi files) and Western blotting data (gel files) were processed with ImageJ software version 4.0.93 (NIH). For manual tracking of moving cells (Fig. 1a, 1b, 2a, 6d, Supplementary Fig. 1a, 1b), MTrackJ plug-in version 1.5.1 for ImageJ software was used. For automatic tracing of moving cells (Fig. 1g, 1h, 3e, 6c, Supplementary Fig.3f), TrackMate plug-in version 7.9.2 for ImageJ was used. Two photon time-lapse Images were analyzed with MATLAB software version R2014a and ImageJ software version 4.0.93. Single-cell RNA-seq data were analyzed using Cell Ranger version 1.3.0 protocol. Seurat package (version 3.2.2) in R was used to analyze Single-cell RNA-seq data. Astrocytes or astrocyte progenitors were automatically identified as GFP positive and Aldh1l1 positive (Fig. 6b, 7d, 8c) or RFP positive and Aldh1l1 positive (Fig. 8f), using Image Calculator function and 3D maxima finder plug-in version 4.0.93 for ImageJ software . The 3 dimensional distances from glial progenitor cells to blood vessels were analyzed with 3D Object Counter and 3D RoiManager plug-ins version 4.0.93 for ImageJ (Fig. 6b, 7d, Supplementary Fig. 4d). The relative positions of GFP or RFP positive/Aldh1l1 positive astrocytes (detected as mentioned above) to the upper and lower border of the CP (the borders were manually determined) were automatically measured using 3D RoiManager plug-in version 4.0.93 (Fig. 8c, 8f,

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Supplementary Fig. c). Statistical analyses were performed with Prism version 7 software (GraphPad) or R statistical package version 4.0.0. Violin and box plots were drawn by ggplot2 R package version 3.3.5. Flow cytometry data were analyzed with FlowJo software version 10.8.1 (BD Biosciences).
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Data
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Human research participants
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Life sciences study design
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Sample size
No statistical methods were applied to predetermine sample size. Three slices /brain x at least 7 brains/ (condition or group) were analyzed for statistical analyses of the distances from astrocyte progenitors to blood vessels and the distributions of astrocytes. We usually electroporated one experimental vector to 5~7 embryos, and the control vector to 3~4 embryos in the same litter. Typically, 4~6 experimental and 2~3 control brains are obtained, and they are enough for us to know the tendency. We repeated the same injection at least 2 times and confirm the reproducibility. In all other experiments (Western blots and in vitro culture), we conducted at least two biologically independent experiments to confirm the same tendency. With regards to the two-photon in vivo imaging, we provide images form one E17 embryo and one P0 pup that survived until the end of observations (monitored by circulation of blood). In these observations, we just confirmed the behaviors of astrocyte progenitors in the living mouse embryo/pup, and did not conduct statistical analysis.
Data exclusions For statistic analyses of the distances to blood vessels and the position in the CP, the brains with low transfection efficiency (less than 20 cells/ slice) were excluded from the analyses. As to two-photon imaging, the data from embryos or pups died during the observations were excluded.

Replication
All statistic analyses were done from at least two independent experiments, and we confirmed the reproducibility.
Randomization Embryos to be introduced with control vectors were randomly selected in the same litter in each in utero electroporation. Other than in utero electroporation experiments, cell lines and primary cultures are thought to be homogeneous and the bias among experimental groups can be ignored.

Blinding
We carried out the computer assisted measurements of the distances from the astrocyte progenitors to blood vessels (Fig.6b, 7d, Supplementary Fig. 4d), the relative positions of astrocytes in the CP (Fig. 8c, 8f, Supplementary Fig.3c), and the migration trajectories (Fig.1g,

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1h, 3e, 6c, Supplementary Fig.3g). When automated counting or tracing was not applicable, the investigators blinded to sample IDs performed evaluations.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. After multiple injections, eggs were collected from the hens, and IgY fractions were prepared from the yolks and then affinity-purified antibodies were prepared using GFP conjugated to an agarose matrix. The final product is a filter-sterilized mixture of both affinity-purified antibodies (30 µg/mL) and purified IgY (10 mg/mL)./ Buffer: Sodium phosphate (10 mM, pH 7.2) buffered isotonic saline (0.9%, w/v), glycerol (50%, v/v), with sodium azide (0.02%, w/v) as an anti-microbial agent./ Dilution Ranges, WB: 1:5000-1:10000, IHC: 1:2000-1:5000, ICC: 1:2000-1:5000/ Quality Control: Antibodies were analyzed by western blot analysis (1:5000 dilution) and immunohistochemistry (1:500 dilution) using transgenic mice expressing the GFP gene product. Western blots were performed using BlokHen® (Aves Labs) as the blocking reagent, and HRP-labeled goat anti-chicken antibodies (Aves Labs, Cat. #H-1004) as the detection reagent. Immunohistochemistry used tetramethyl rhodamine-labeled anti-chicken IgY.

Wild animals
No wild animals were used in the study.

Reporting on sex
We have not experienced any sexual difference in migration and positioning of astrocytes. We mixed the data from male and female mice.
Field-collected samples No filed-collected samples was used.

Ethics oversight
All protocols for animal handling and treatments conducted in Institute for Developmental Research were approved by the Animal Care and Use Committee of Institute for Developmental Research, Aichi Developmental Disability Center (approval number: 2019-013). Those in Keio University were approved by the Keio University Institutional Animal Care and Use Committee in accordance with the Institutional Guidelines on Animal Experimentation at Keio University (approval number: A2021-030).