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Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors

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Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors Chuanqing Wu1,2, Mei Yang2,4, Juan Li2,5¤, Chengbing Wang2, Ting Cao2,6, Kaixiong Tao1*, Baolin Wang2,3* 1 Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China, 2 Department of Genetic Medicine, Weill Medical College of Cornell University, New York, New York, United States of America, 3 Department of Cell Biology and Development, Weill Medical College of Cornell University, New York, New York, United States of America, 4 Department of Human Anatomy, Institute of Neuroscience, Chongqing Medical University, Chongqing, China, 5 Institute of Developmental Immunology, College of Life Science, Shandong University, Jinan, Shandong, China, 6 Institute of Life Science, Nanjing University, Nanjing, Jiangsu, China Abstract Granule neuron progenitors (GNPs) are the most abundant neuronal type in the cerebellum. GNP proliferation and thus cerebellar development require Sonic hedgehog (Shh) secreted from Purkinje cells. Shh signaling occurs in primary cilia originating from the mother centriole. Centrioles replicate only once during a typical cell cycle and are responsible for mitotic spindle assembly and organization. Recent studies have linked cilia function to cerebellar morphogenesis, but the role of centriole duplication in cerebellar development is not known. Here we show that centrosomal protein Cep120 is asymmetrically localized to the daughter centriole through its interaction with Talpid3 (Ta3), another centrosomal protein. Cep120 null mutant mice die in early gestation with abnormal heart looping. Inactivation of Cep120 in the central nervous system leads to both hydrocephalus, due to the loss of cilia on ependymal cells, and severe cerebellar hypoplasia, due to the failed proliferation of GNPs. The mutant GNPs lack Hedgehog pathway activity. Cell biological studies show that the loss of Cep120 results in failed centriole duplication and consequently ciliogenesis, which together underlie Cep120 mutant cerebellar hypoplasia. Thus, our study for the first time links a centrosomal protein necessary for centriole duplication to cerebellar morphogenesis. Citation: Wu C, Yang M, Li J, Wang C, Cao T, et al. (2014) Talpid3-Binding Centrosomal Protein Cep120 Is Required for Centriole Duplication and Proliferation of Cerebellar Granule Neuron Progenitors. PLoS ONE 9(9): e107943. doi:10.1371/journal.pone.0107943 Editor: Sudipto Roy, Institute of Molecular and Cell Biology, Singapore Received June 11, 2014; Accepted August 18, 2014; Published September 24, 2014 Copyright: ß 2014 Wu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding: Funding from National Institutes of Health grants (R01CA111673 and R01GM070820) to BW and a CNSF grant 81172294 to KXT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] (KXT); [email protected] (BW) ¤ Current address: Laboratory of Molecular Oncology, Clinical Research Institute of Montre´al, Montre´al, Que´bec, Canada Introduction size and surface area. Defects in any of these stages of cerebellar development result in cerebellar hypoplasia in humans and mice. The cerebellum controls motor movement coordination, Sonic Hedgehog (Shh) is a secreted molecule that plays balance, equilibrium, and muscle tone. Granule neurons are the important roles in embryonic development. In the developing most abundant neurons in the cerebellum and in fact the entire cerebellum, Shh is expressed in Purkinje cells and controls GNP brain. Cerebellar development is a complex process requiring proliferation [4–7] in part by upregulating the protooncogene N- coordinated regulation of progenitor proliferation, neuronal myc [8]. Hedgehog (Hh) signaling occurs in the primary cilium, a differentiation and migration [1,2]. Granule neuron progenitors microtubule-based organelle that protrudes from the cell surface of (GNPs) originate in the rhombic lip (RL), a region of the hindbrain most vertebrate cells [9], including both Purkinje cells and GNPs bordering the fourth ventricle. In the moue, the GNPs leave the in the EGL [10,11]. Defects in ciliogenesis are associated with RL at around embryonic day 13 (E13) and migrate rostrally over human congenital cerebellar malformations [12]. Mutations in the surface of the cerebellar anlage to form the external germinal several ciliary genes in the mouse have also been shown to cause layer (EGL). During the first two weeks after birth, the GNPs in significantly reduced GNP proliferation and cerebellar hypoplasia the EGL undergo significant proliferation to generate the large [13,14]. pool of GNPs required for producing granule neurons. They then Primary cilia originate from the basal body, a specialized exit cell cycle and migrate internally into the cortex of the mother centriole, during the G0 phase of cell cycle [15]. Centrioles cerebellum to establish the inner granule layer (IGL) under the are cylindrical microtubule-based structures, and a typical cell Purkinje cell monolayer (PCL) [3]. During the same extended contains two centrioles, the core components of a centrosome. period, the cerebellum becomes foliated, significantly increasing its Before cells enter the cell cycle, primary cilia have to be PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e107943 Cep120 in Centriole Duplication and Cerebellar Development disassembled, and the two centrioles replicate, a process that is genotyping with the following primers: BW946F, 59-AT- strictly controlled to occur only once per cell cycle for a typical CACTGTGGAGCCTTGGGCA-39, and BW946R, 59- cell. As a result, the two centrioles are distinct in age and maturity TGTTACTCAGCAGCTGGTACC-39, for the wild type allele, as well as function. The younger of the two centrioles is known as which produced a 220 bp fragment; BW1241F, 59- daughter centriole, and the older one is called mother centriole. CCTCTGCCTCCTTAGTGGATC-39, and BW946R, for the During the G0 phase, the mother centriole matures and becomes Cep120f allele, which produced a 280 bp fragment; BW294, 59- the basal body of the primary cilium [16]. Thus, centriole ATTGGGAAGACAATAGCAGGCA-39 and BW947, 59- duplication and ciliogenesis are two closely related processes. The AGACCAGCCTCAGTTACACCG-39 for Cep120- allele, which list of known centrosomal proteins that have been found to be produced a 230 bp fragment. Ta3 knockout mice were created by essential for ciliogenesis keeps increasing, but only a small number the deletion of the first two exons of the Ta3 gene using the of them are also required for centriole duplication [17]. Whether standard targeted gene knockout approach, and the detail of the these centrosomal proteins regulate cerebellar development is still strategy will be described elsewhere. Nestin-Cre or nes-Cre mice unknown. were obtained from Dr. Tao Sun and originally purchased from In the present study, we identify the centrosomal protein Jackson Laboratories. Actin-Flpe and actin-Cre mice were Cep120 as an interacting protein of Talpid3 (Ta3), another obtained from Dr. Licia Selleri and originally purchased from centrosomal protein that is required for ciliogenesis and Hh Jackson Laboratories. Since DNA recombination from maternally signaling [18,19]. Cep120 is asymmetrically localized to the inherited nes-Cre allele is less efficient than that from paternally daughter centriole [20], while Ta3 is predominantly found in the inherited allele [21], for all crosses involving nes-Cre, only the mother centriole. The asymmetrical localization of Cep120 is males carrying the transgene were used for crosses. All animal care dependent on Ta3. Cep120 null mutant mice die at an early was done in accordance with the guidelines of the National gestation stage with defect in heart looping. Most Cep120 mutant Institutes of Health. cells either lack centrioles or only have one, confirming a recent study showing that Cep120 is required for centriole duplication Cell Lines and Cell Culture [20]. Inactivation of Cep120 in the central nervous system results Wild type, Cep120-/-, and Ta3 primary mouse embryonic in severe hydrocephalus and cerebellar hypoplasia, subsequently fibroblasts (pMEFs) were prepared from E9.5 and E10.5 mouse causing lethality. In the Cep120 mutant, cilia are also absent on embryos, respectively. The procedure was approved by Institu- ependymal cells and cerebellar GNPs (CGNPs) and centriole tional Animal Care and Use Committee at Weill Medical College. duplication fails to occur in CGNPs. As a result, the CGNPs lack The pMEFs and HEK293 cells were cultured in DMEM Hh pathway activity, thus proliferating. The mutant cerebellum supplemented with heat-inactivated 10% FBS, penicillin, and also never becomes foliated. These phenotypes are more severe streptomycin. The calcium phosphate precipitation method was than those of the known cilia mutants [13,14]. Therefore, Cep120 used to transfect HEK293
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