Materials and methods Animals All animal manipulations were performed in accordance with the guidelines for animal experiments at the RIKEN Centre for Developmental Biology. DNA constructs and RNAi CCPM (GE Healthcare) and EB3-tagRFP vector (Evrogen) were purchased. Other mammalian expression vectors were based on pCAGGS (Niwa et al, 1991). NLS-GFP was constructed by inserting oligonucleotides for three copies of a nuclear localization sequence (NLS) into EGFP-C1 (Clontech). For GFP-TPX2, full-length mouse TPX2-coding sequence was ampliﬁed from RIKEN FANTOM cDNA (Accession No. AK090101) and ligated into pCAG-EGFP-C1. For 6myc-TPX2, the GFP sequence in the plasmid above was substituted with six copies of the myc epitope sequence. For p18Ink4c expression, mouse p18Ink4c-coding sequence was ampliﬁed from Mammalian Gene Collection cDNA (Accession No. BC027026) and ligated into pCAGGS (constructed by Dr Konno, RIKEN CDB). To knockdown TPX2 by vector-type RNAi, the BLOCK-iT Pol II miR RNAi Expression Vector Kit (Invitrogen) was used (RNAi-target sequences: for LacZ, 50-AAATCGCTGATTTGTGTAGTC-30, and for TPX2, 50-TAATGATAGTGCATCCTCTGG-30). To enhance RNAi efﬁciency, the plasmid was modiﬁed to have four copies of the target sequence in tandem, then cloned into pCAGGS. To knock- down TPX2 with double-stranded RNA, StealthRNAi (Invitrogen) (50-AAGUGUUGUCAACUGCCUUCAACGG-30) was electroporated into E12.5 mouse brain at a concentration of 50 mM in physiological saline solution. Antibodies The primary antibodies used were Tpx2 (rabbit, Santa Cruz; 1:50), BrdU (rat, Abcam; 1:300), GFP (chick, Aves Labs; 1:500), myc (mouse, Upstate; 1:100), Par-3 (rabbit, Upstate; 1:250), ZO-1 (mouse, Zymed; 1:100), b-catenin (mouse, BD; 1:1000), g-tubulin (mouse, Sigma; 1:100), TACC3 (rabbit, 1:300) (Yao et al, 2007), Ki67 (mouse, Novocastra; 1:100), p18Ink4c (rabbit, Santa Cruz; 1:50), Tuj1 (mouse, Covance; 1:600), Tuj1 (rabbit, Covance; 1:500), PCNA (mouse, BD; 1:100), mouse mAb against phospho-Histone H3 (pH 3) (mouse, Sigma; 1:100), pH 3 (rabbit, Upstate; 1:500), Pax6 (rabbit, Chemicon; 1:300), Sox2 (rabbit, Chemicon; 1:500), a-tubulin (rat, Serotec; 1:500) and Tbr2 (rabbit, Abcam; 1:100). To recognize CldU and IdU, BrdU (rat, Abcam; 1:300) and BrdU (mouse, BD; 1:50) were used, respectively. References Barr AR, Gergely F (2007) Aurora-A: the maker and breaker of spindle poles. 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Nature 185: 702–703 Go¨ tz M, Huttner WB (2005) The cell biology of neurogenesis. Nat Rev Mol Cell Biol 6: 777–788 Go¨ tz M, Stoykova A, Gruss P (1998) Pax6 controls radial glia differentiation in the cerebral cortex. Neuron 21: 1031–1044 Graham V, Khudyakov J, Ellis P, Pevny L (2003) SOX2 functions to maintain neural progenitor identity. Neuron 39: 749–765 & 2011 European Molecular Biology Organization Mechanisms of interkinetic nuclear migration Y Kosodo et al Brain slice culture and incorporation of ﬂuorescent microbeads Coronal brain tissue slices were prepared from ICR mice and cultured in collagen gels as described previously (Miyata et al, 2001) with slight modiﬁcations. For details, see the Supplementary data. Time-lapse imaging analysis Time-lapse images of the movements of NLS-GFP-containing nuclei and ﬂuorescent beads in embryonic mouse brain slice cultures are described in the Supplementary data. Computational modelling Construction of the two-dimensional model to evaluate the displacement effect for INM, and the effect of parameter settings, are described in Supplementary data. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Acknowledgements We thank Dr Shigenobu Yonemura and Kisa Kakiguchi (RIKEN CDB) for electron microscopy studies. HVEM observations at the National Institute of Physiological Sciences (NIPS) were performed under the instruction of Dr Tatsuo Arii (NIPS). We thank Drs Tetsuo Noda and Ryoji Yao (The Cancer Institute) for providing anti-TACC3 antibody. We thank Drs Raj Ladher and Guojun Sheng (RIKEN CDB) for critical reading of the manuscript. YMK was supported by Takeda Science Foundation. Author contributions: YK and FM designed the project and wrote the manuscript. YK and TS performed most of the experiments and data analysis. AK designed and performed the computational modelling. 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