GoKawiilLists of the antibodies and reagents used are provided in Supplementary Tables 1 and 2.
Plasmids
The pCAG-mScarlet plasmid was generated using the mScarlet sequence obtained from Addgene (85042; a gift from D. Gadella). To generate the pCAG-53BP1-mNeonGreen construct, mouse 53BP1 cDNA was amplified from a mouse brain cDNA library and inserted into the pENTR1A vector. In parallel, the Gateway cassette from pDest-eGFP-N1 was inserted into the EcoRI site of pmNeonGreen using In-Fusion Cloning to generate pDest-mNeonGreen. The 53BP1 cDNA in pENTR1A was recombined into pCAG-Dest-mNeonGreen using LR Clonase II Enzyme Mix (Invitrogen, 11791020) to produce the final pCAG-53BP1-mNeonGreen construct. The pTRIP-CMV-GFP-Flag-cGAS plasmid was obtained from Addgene (86674; a gift from N. Manel). mCherry-KASH1 was constructed as previously decribed13. The pAAV-Neurod1-GFP plasmid was constructed by replacing the CAG promoter in pAAV-CAG-EGFP51 with the Neurod1 promoter, which was cloned from the cDNA of CGNs isolated from P6 ICR mice.
Animals
All animal experiments were approved by the Animal Experiment Committee of Kyoto University (KUIAS 1-9) and conducted in accordance with the guidelines of the National Centre for the Replacement, Refinement and Reduction of Animals in Research. For some in vitro experiments, timed-pregnant C57BL/6J and ICR mice were purchased from Japan SLC. We generated conditional deletion mice by breeding Lig4flox/flox mice30 with Neurod1-cre (Tg (Neurod1-cre) RZ24Gsat/Mmucd, MMRRC_036320-UCD) or Gabra6-cre (B6.129P2-Gabra6tm2(cre)Wwis/Mmucd, MMRRC_015968-UCD) mice from MMRRC at UC Davis. The Neurod1-GFP (Tg (Neurod1-eGFP) CR99Gsat/Mmucd, MMRRC_000329-UCD) mouse strain was also obtained from MMRRC. These lines were maintained on a predominantly C57BL/6J mixed background, with mice of both sexes used for experiments. Animals were housed under a 12 h–12 h light–dark cycle at 23 ± 3 °C and 50% humidity.
Genotyping
Tail or nail clippings were digested in 50 mM NaOH at 95 °C for 10 min. After neutralizing by adding 1 M Tris-HCl (pH 8.0), samples were centrifuged at 4 °C for 15 min. The supernatant was subjected to PCR using the following primers: Lig4flox allele, 5′-GAGCTGCAACAGTTTGTGAAGTTTGTGAGGA-3′ and 5′-GTGTTGGTCAGGACCAGAAGGAAAGCA-3′; Neurod1-cre allele, 5′-TAGGATTAGGGAGAGGGAGCTGAA-3′ and 5′-CGGCAAACGGACAGAAGC-3′; Neurod1-GFP allele, 5′-TAGGATTAGGGAGAGGGAGCTGAA-3′ and 5′-Gabra6-cre allele, 5′-GATCTCCGGTATTGAAACTCCAGC-3′ and 5′-GCTAAACAT GCTTCATCGTCGG-3′.
In vivo and in utero electroporation and brain slice imaging
Organotypic cerebellar slice culture was performed as previously described52. Plasmid electroporation into P5 cerebella was conducted using an electroporator (CUY21, Nepagene). A total of six square-wave pulses (70 mV, 50 ms duration, 150 ms intervals) were applied. Cerebella were collected at P7 and embedded in 3.5% low-melting-point agarose (Nacalai, 01651-76), and coronally sliced into 300-μm-thick sections using a vibratome (NLS-AT, Dosaka EM). The slices were placed onto Millicell-CM membrane inserts (Millipore, PICM0RG50) and embedded in a collagen gel matrix (FUJIFILM Wako, 631-00651). The slices were maintained in medium composed of 60% basal medium Eagle (BME, Sigma-Aldrich, B9638), 25% Earle’s balanced salt solution (Sigma-Aldrich, E7510), 15% heat-inactivated horse serum (HS, Invitrogen, 26050070), 3 mM l-glutamine (Gibco, 35050-061), 1 mM sodium pyruvate (Sigma-Aldrich, S8636), 5.6 g l−1 glucose (Sigma-Aldrich, G7021), 1.8 g l−1 sodium bicarbonate (Sigma-Aldrich, S5761), and N-2 supplement (Invitrogen, 17502001). In utero electroporation into cerebral cortex was performed at E12. Brains were dissected at E14 and coronally sliced using a surgical knife53. Slices were mounted in a collagen gel matrix (FUJIFILM Wako, 631-00651) in DMEM/F12 (Sigma-Aldrich, D2906) supplemented with 5% HS, 5% FBS (FBS, BioWest, S1400-500), penicillin–streptomycin (Invitrogen, 15140122), and N-2 supplement. The slices were placed in a stage-top incubator at 37 °C with humidified 85% O 2 /5% CO 2 gas flow, mounted onto an upright microscope (BX61WI, Olympus)54. Time-lapse imaging was performed using a laser-scanning confocal microscope (FV1000, Olympus) equipped with a GaAsP detector and a ×40 water-immersion objective lens (NA 0.8). Images were acquired every 5 or 10 min over 6 h.
Live-cell imaging on microfabricated constriction substrates
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