For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore–microtubule attachments are disrupted (error correction) by Aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to allow fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to Aurora B regulation, but attachment to the microtubule plus-end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how Aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable – experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and (4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

Embryonic stem cells (ESCs) express heterogeneous levels of pluripotency and developmental transcription factors (TFs) and their cell cycle is unsynchronised when grown in the presence of serum. Here, we asked whether the cell cycle and developmental heterogeneities of ESCs are coordinated by determining the state identities of G1-and G2M-enriched mouse ESCs (mESCs) at single cell resolution. We found that G2M cells were not all the same and demonstrate their split into the naïve and formative (intermediate) pluripotency states marked by high or low Esrrb expression, respectively. The naïve G2M sub-state resembles 'ground' state pluripotency of the LIF/2i cultured mESCs. The naïve and formative G2M sub-states exist in the pre-and post-implantation stages of the mouse embryo, respectively, verifying developmental distinction. Moreover, the G2M sub-states partially match between the mouse and human ESCs, suggesting higher similarity of transcriptional control between these species in G2M. Our findings propose a model whereby G2M separates mESCs into naïve and formative pluripotency states. This concept of G2M-diverted pluripotency states provides new framework for understanding the mechanisms of pluripotency maintenance and lineage specification in vitro and in vivo, and the development of more efficient and clinically relevant reprogramming strategies.

In the version of this Article originally published online, the lines connecting the data points were missing from the chart on the left of Fig. 8c; the correct graph is shown below. This has been corrected in all versions of the Article.

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563that only a subset of ZII − Purkinje cells are ectopic in cdf and taken together this indicates that there are at least two classes of ZII − Purkinje cells.Interestingly, recent experiments examining the origins of ZII − cells during development also suggest that at least two immunologically distinct populations of Purkinje cells contribute to the ZII − population in the adult cerebellum -one that expresses neurogranin alone (NG+) and a second that expresses a combination of heat shock protein25, calbindin and neurogranin (HSP25+). Our data reveals that both Purkinje cell phenotypes are present in the cdf cerebellum. However, we observed that the overwhelming majority of ectopic Purkinje cells in the perinatal cdf cerebellum are represented by the HSP25+ subset. This observation supports the adult data because a population of cells predicted to contribute to the adult ZII − population is ectopic in the perinatal cerebellum. Finally, despite the ectopia present in the cdf cerebellum, overall, parasagittal patterning is very similar to wild type. Thus, our evidence indicates that mediolateral patterning is established prior to Purkinje cell dispersal and that this dispersal process is differentially regulated across Purkinje cell subclasses.The basic helix-loop-helix (bHLH) genes encode for a family of transcription factors involved in developmental processes such as neurogenesis, myogenesis, hematopoeisis and sex determination. NSCL1 is a bHLH transcription factor expressed in various populations of postmitotic neurons of the CNS and the PNS. In situ hybridization demonstrated expression of NSCL1 in the subependymal layer of the neuroepithelium throughout the CNS, in the dorsal root ganglia, trigeminal and other cranial ganglia, sensory nasal epithelium, sensory layer of the developing optic cup, in the forebrain, hippocampus, septum, tectum, hypothalamic nuclei, hindbrain and spinal cord both in mouse embryos, postnatal and adult animals.NSCL1 behaves as an initiator factor of cerebellar granule cell growth and differentiation. Expression in the cerebellum, spinal cord and dorsal root ganglia corresponds to the expression of the adhesion molecule TAG-1/axonin-1. NSCL1 is detected in rhombomere boundaries both in mouse and chick. NSCL1 expression in these structures is coincident with their formation and is maintained until these structures are gone at later stages of development. cNSCL1 expression is also observed during rhombomere boundary regeneration in chick embryos, after their microsurgical ablation. In addition, a stronger NSCL1 signal is observed in r4, corresponding to facial branchiomotor (fbm) and visceromotor neurons. The expression pattern of mNSCL1 in Hoxb1 −/− mice is strongly affected in r4 where migrating fbm neurons are located.To further investigate the role of NSCL-1 as a transcriptional activator or repressor, we performed luciferase assays using different truncated forms of cNSCL-1 fused to GAL-4, and a UAS luciferase reporter. In a complementary system, ME1a, a binding partner of NSCL-1 ...