KIF20A/MKLP2 regulates the division modes of neural progenitor cells during cortical development

ARTICLE DOI: 10.1038/s41467-018-05152-1 OPEN KIF20A/MKLP2 regulates the division modes of neural progenitor cells during cortical development 1234567890():,; Anqi Geng1, Runxiang Qiu1, Kiyohito Murai1,6, Jiancheng Liu1, Xiwei Wu2, Heying Zhang1, Henry Farhoodi1, Nam Duong1, Meisheng Jiang3, Jiing-kuan Yee4, Walter Tsark5 & Qiang Lu 1 Balanced symmetric and asymmetric divisions of neural progenitor cells (NPCs) are crucial for brain development, but the underlying mechanisms are not fully understood. Here we report that mitotic kinesin KIF20A/MKLP2 interacts with RGS3 and plays a crucial role in controlling the division modes of NPCs during cortical neurogenesis. Knockdown of KIF20A in NPCs causes dislocation of RGS3 from the intercellular bridge (ICB), impairs the function of Ephrin-B–RGS cell fate signaling complex, and leads to a transition from proliferative to differentiative divisions. Germline and inducible knockout of KIF20A causes a loss of progenitor cells and neurons and results in thinner cortex and ventriculomegaly. Interestingly, loss of function of KIF20A induces early cell cycle exit and precocious neuronal differentiation without causing substantial cytokinesis defect or apoptosis. Our results identify a RGS–KIF20A axis in the regulation of cell division and suggest a potential link of the ICB to regulation of cell fate determination. 1 Department of Developmental and Stem Cell Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. 2 Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. 3 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. 4 Department of Virology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. 5 Transgenic/Knockout Mice Facility, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. 6 Present address: Department of Anatomy and Neurobiology, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. These authors contributed equally: Anqi Geng, Runxiang Qiu. Correspondence and requests for materials should be addressed to Q.L. (email: ) NATURE COMMUNICATIONS | (2018)9:2707 | DOI: 10.1038/s41467-018-05152-1 | www.nature.com/naturecommunications 1 ARTICLE D NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05152-1 uring brain development, neural progenitor cells (NPCs) have to maintain a tight control on the balance between proliferation and differentiation, so that desired neural cell types (including neurons, glia, and other cells) can be produced in an appropriate order and with the correct numbers. The regulation of such a fate decision in NPCs manifests in the form of symmetric (self-renewal) versus asymmetric (differentiation) cell divisions. Symmetric cell division expands the NPC pool, whereas asymmetric cell division allows NPCs to simultaneously maintain the progenitor pool and generate cellular diversity. The mechanisms that govern the mode of cell divisions (symmetric versus asymmetric) have been studied extensively in the nervous systems of Drosophila and Caenorhabditis elegans1–4 and the knowledge gained from invertebrate studies has provided a framework to understand how symmetric and asymmetric cell divisions might be regulated in the mammalian systems. Consequently, homologs of many of the invertebrate genes implicated in regulation of symmetric versus asymmetric cell divisions were tested for a similar role as cell fate regulators in the mammalian brains. However, the mammalian studies have so far yielded contradicting results. For examples, knockout of Numb and Numbl was initially reported to compromise the maintenance of NPCs in the cortex5,6, but it was later found to induce hyperproliferation of NPCs as well as impaired neuronal differentiation7, or to cause delamination and displacement of apical radial glial cells (RGCs) into basal regions of the ventricular zone (VZ), but the progenitor fate was maintained8. Knockout of LGN/ GPSM2, a modulator of G protein signaling, caused randomization of mitotic spindle orientation and ectopic distribution of the apical NPCs in the cortex, but did not have an obvious impact on proliferative versus neurogenic divisions9,10. Protein phosphatase PP4c was found to regulate spindle orientation and inhibit neurogenesis in one study11 and was shown to promote neurogenesis and suppress NPC proliferation in another study12. These divergent results indicated that the actual process by which symmetric versus asymmetric cell division occurs in mammalian brains and the regulatory protein network of the process remain to be further explored13,14. The complexity of mammalian cells highlights the importance of identifying additional cell fate determinants crucial for regulating proliferative versus differentiative cell divisions, particularly those factors that work in close association with the cell division machinery. We have previously shown that a regulator of G protein signaling (RGS) motif-mediated Ephrin-B reverse signaling pathway15 and the Gα signaling pathway work together to regulate self-renewal and differentiation of NPCs during cortical neurogenesis16–18. RGS domain functions as a GTPase activating protein and links transmembrane receptor Ephrin-B to inhibition of Gαi and Gαo subunits. In the developing cortex, the Ephrin-B/ RGS reverse signaling is required for the maintenance of NPCs16,17, while Gα signaling functions to activate neurogenesis18, and the balance between the two signaling pathways regulates the decision of NPCs to stay as a progenitor or to become a neuron18. In an earlier experiment, we found that in utero electroporation (IUE)-mediated knockdown of Ephrin-B1 or its cytoplasmic binding protein PDZ-RGS3 (RGS3 isoform 1) in the mouse cortex could induce early neuronal differentiation within 24 h of cell transfection16. This fast onset of knockdown effect prompted us to reason that the Ephrin-B/RGS signaling may be directly linked to cell division machinery, rather than modulating the outcome of NPC division via an indirect route, for example, through transcriptional regulation of downstream cell fate genes. We therefore searched for additional molecules that may interact with RGS3. In this study, we identify the interaction between KIF20A/MKLP2 and RGS3 within the intercellular bridge (ICB) of dividing NPCs. We further present functional data implicating 2 a key role of the RGS–KIF20A axis of interaction in cell fate determination during NPC divisions. Results KIF20A binds to the RGS domain of RGS3. We screened an embryonic mouse yeast two-hybrid complementary DNA (cDNA) library using both the full-length RGS3 and a C-terminal fragment containing the RGS domain alone as bait. Full-length RGS3 identified a number of preys including α-tubulin and KIF20A (Supplementary Fig. 1) and the RGS domain pulled out KI (...truncated)