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The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.
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Proteínas de Ciclo Celular , Centrossomo , Pontos de Checagem da Fase M do Ciclo Celular , Mitose , Centrossomo/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Divisão Celular , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Células HeLaRESUMO
Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.
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Axonema , Caenorhabditis elegans , Animais , Camundongos , Axonema/metabolismo , Axonema/ultraestrutura , Caenorhabditis elegans/metabolismo , Cílios/metabolismo , Mamíferos , Microtúbulos/metabolismo , Movimento , Tubulina (Proteína)/metabolismoRESUMO
Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines and has been found to have distinct anti-inflammatory and pro-inflammatory properties in various cellular and disease contexts. OSM signals through two receptor complexes, one of which includes OSMRß. To investigate OSM-OSMRß signaling in adult hematopoiesis, we utilized the readily available conditional Osmrfl/fl mouse model B6;129-Osmrtm1.1Nat/J, which is poorly characterized in the literature. This model contains loxP sites flanking exon 2 of the Osmr gene. We crossed Osmrfl/fl mice to interferon-inducible Mx1-Cre, which is robustly induced in adult hematopoietic cells. We observed complete recombination of the Osmrfl allele and loss of exon 2 in hematopoietic (bone marrow) as well as non-hematopoietic (liver, lung, kidney) tissues. Using a TaqMan assay with probes downstream of exon 2, Osmr transcript was lower in the kidney but equivalent in bone marrow, lung, and liver from Osmrfl/fl Mx1-Cre versus Mx1-Cre control mice, suggesting that transcript is being produced despite loss of this exon. Western blots show that liver cells from Osmrfl/fl Mx1-Cre mice had complete loss of OSMR protein, while bone marrow, kidney, and lung cells had reduced OSMR protein at varying levels. RNA-seq analysis of a subpopulation of bone marrow cells (hematopoietic stem cells) finds that some OSM-stimulated genes, but not all, are suppressed in Osmrfl/fl Mx1-Cre cells. Together, our data suggest that the B6;129-Osmrtm1.1Nat/J model should be utilized with caution as loss of Osmr exon 2 has variable and tissue-dependent impact on mRNA and protein expression.
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BACKGROUND: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). METHODS: SYK and FLT3 mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60 mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. CONCLUSIONS: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.
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The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium. Specifically, we describe in detail how to capture and quantify the 2D dynamics of individual transmembrane proteins PTCH1 and SMO and how to map the 3D nanoscale distributions of the inversin compartment proteins INVS, ANKS6, and NPHP3. This protocol can, with minor modifications, be adapted for studies of other proteins and cell lines to further elucidate the structure and function of the primary cilium at the molecular level.
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Cílios , Doenças Renais Císticas , Humanos , Cílios/metabolismo , Imagem Individual de Molécula , Doenças Renais Císticas/metabolismo , Transdução de Sinais , Linhagem CelularRESUMO
Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.
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Calcineurina , Cílios , Calcineurina/metabolismo , Cílios/metabolismo , Cálcio/metabolismo , Centrossomo/metabolismo , Centríolos/metabolismo , Proteínas/metabolismoRESUMO
Microtubule doublets (MTDs) are a well conserved compound microtubule structure found primarily in cilia. However, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we characterize microtubule-associated protein 9 (MAP9) as a novel MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. Loss of MAPH-9 caused ultrastructural MTD defects, dysregulated axonemal motor velocity, and perturbed cilia function. As we found that the mammalian ortholog MAP9 localized to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in supporting the structure of axonemal MTDs and regulating ciliary motors.
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The Mouse Phenome Database (MPD; https://phenome.jax.org; RRID:SCR_003212), supported by the US National Institutes of Health, is a Biomedical Data Repository listed in the Trans-NIH Biomedical Informatics Coordinating Committee registry. As an increasingly FAIR-compliant and TRUST-worthy data repository, MPD accepts phenotype and genotype data from mouse experiments and curates, organizes, integrates, archives, and distributes those data using community standards. Data are accompanied by rich metadata, including widely used ontologies and detailed protocols. Data are from all over the world and represent genetic, behavioral, morphological, and physiological disease-related characteristics in mice at baseline or those exposed to drugs or other treatments. MPD houses data from over 6000 strains and populations, representing many reproducible strain types and heterogenous populations such as the Diversity Outbred where each mouse is unique but can be genotyped throughout the genome. A suite of analysis tools is available to aggregate, visualize, and analyze these data within and across studies and populations in an increasingly traceable and reproducible manner. We have refined existing resources and developed new tools to continue to provide users with access to consistent, high-quality data that has translational relevance in a modernized infrastructure that enables interaction with a suite of bioinformatics analytic and data services.
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Bases de Dados Genéticas , Fenômica , Camundongos , Animais , Camundongos Endogâmicos , Fenótipo , GenótipoRESUMO
DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Before differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication or four centrioles after. We find that the average nuclear area increases approximately eight-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inhibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number but to a limited extent compared with DNA reduplication.
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Centríolos , Trofoblastos , Gravidez , Feminino , Humanos , Centríolos/metabolismo , Trofoblastos/metabolismo , Centrossomo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Gigantes/metabolismo , Poliploidia , Proteínas Serina-Treonina QuinasesRESUMO
In adult acute myeloid leukemia (AML), the acquisition of driver somatic mutations may be preceded by a benign state termed clonal hematopoiesis (CH). To develop therapeutic strategies to prevent leukemia development from CH, it is important to understand the mechanisms by which CH-driving and AML-driving mutations cooperate. Here, we use mice with inducible mutant alleles common in human CH (DNMT3AR882; mouse Dnmt3aR878H) and AML (NPM1c; mouse Npm1cA). We find that Dnmt3aR878H/+ hematopoietic stem cells (HSCs), but not multipotent progenitor cell (MPP) subsets, have reduced cytokine expression and proinflammatory transcriptional signatures and a functional competitive advantage over their wild-type counterparts. Dnmt3aR878H/+ HSCs are the most potent cell type transformed by Npm1cA, generating myeloid malignancies in which few additional cooperating somatic mutation events were detected. At a molecular level, Npm1cA, in cooperation with Dnmt3aR878H, acutely increased the accessibility of a distinct set of promoters in HSCs compared with MPP cells. These promoters were enriched for cell cycling, PI3K/AKT/mTOR signaling, stem cell signatures, and targets of transcription factors, including NFAT and the chromatin binding factor HMGB1, which have been implicated in human AML. These results demonstrate cooperativity between preexisting Dnmt3aR878H and Npm1cA at the chromatin level, where specific loci altered in accessibility by Npm1cA are dependent on cell context as well as Dnmt3a mutation status. These findings have implications for biological understanding and therapeutic intervention in the transformation from CH to AML.
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Leucemia Mieloide Aguda , Transtornos Mieloproliferativos , Animais , Cromatina , Hematopoiese Clonal , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Transtornos Mieloproliferativos/patologia , Nucleofosmina , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/uso terapêuticoRESUMO
Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50-100 µm from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 µm/minute. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium before other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.
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Centríolos , Neurônios Receptores Olfatórios , Animais , Centríolos/metabolismo , Cílios/metabolismo , Camundongos , Microtúbulos , Mucosa OlfatóriaRESUMO
CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.
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Ciclo Celular , Citometria de Fluxo/métodos , Edição de Genes/métodos , Sistemas CRISPR-Cas , Linhagem Celular , Química Click , Imunofluorescência , Humanos , TransfecçãoRESUMO
The mechanisms of vertebrate Hedgehog signaling are linked to the biology of the primary cilium, an antenna-like organelle that projects from the surface of most vertebrate cell types. Although the advantages of restricting signal transduction to cilia are often noted, the constraints imposed are less frequently considered, and yet they are central to how Hedgehog signaling operates in developing tissues. In this Review, we synthesize current understanding of Hedgehog signal transduction, ligand secretion and transport, and cilia dynamics to explore the temporal and spatial constraints imposed by the primary cilium on Hedgehog signaling in vivo.
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Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais/fisiologia , Animais , Ciclo Celular , Proliferação de Células , Humanos , VertebradosRESUMO
Decline in hematopoietic stem cell (HSC) function with age underlies limited health span of our blood and immune systems. In order to preserve health into older age, it is necessary to understand the nature and timing of initiating events that cause HSC aging. By performing a cross-sectional study in mice, we discover that hallmarks of aging in HSCs and hematopoiesis begin to accumulate by middle age and that the bone marrow (BM) microenvironment at middle age induces and is indispensable for hematopoietic aging. Using unbiased approaches, we find that decreased levels of the longevity-associated molecule IGF1 in the local middle-aged BM microenvironment are a factor causing HSC aging. Direct stimulation of middle-aged HSCs with IGF1 rescues molecular and functional hallmarks of aging, including restored mitochondrial activity. Thus, although decline in IGF1 supports longevity, our work indicates that this also compromises HSC function and limits hematopoietic health span.
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Medula Óssea , Nicho de Células-Tronco , Envelhecimento , Animais , Estudos Transversais , Hematopoese , Células-Tronco Hematopoéticas , CamundongosRESUMO
Multiciliated cells are considered terminally differentiated, yet tissues bearing them are remodeled during development and after injury. In this issue of Developmental Cell, Tasca et al. (2021) show that multiciliated epithelial cells are lost via two different Notch-dependent processes, apoptosis and transdifferentiation, during developmental remodeling of the Xenopus epidermis.
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Células Epidérmicas , Células Epiteliais , Animais , Diferenciação Celular , Transdiferenciação Celular , Xenopus laevisRESUMO
Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, the cellular location with the fastest intrinsic rhythm should act as a pacemaker for the process. Here we traced the waves of tubulin polymerization and depolymerization that occur at mitotic entry and exit in Xenopus egg extracts back to their origins. We found that mitosis was commonly initiated at sperm-derived nuclei and their accompanying centrosomes. The cell cycle was ~20% faster at these initiation points than in the slowest regions of the extract. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.
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Relógios Biológicos/fisiologia , Ciclo Celular/fisiologia , Núcleo Celular/fisiologia , Animais , Mitose/fisiologia , Oócitos , Xenopus laevisRESUMO
Loss of the cilium is important for cell cycle progression and certain developmental transitions. Chytrid fungi are a group of basal fungi that have retained centrioles and cilia, and they can disassemble their cilia via axoneme internalization as part of the transition from free-swimming spores to sessile sporangia. While this type of cilium disassembly has been observed in many single-celled eukaryotes, it has not been well characterized because it is not observed in common model organisms. To better characterize cilium disassembly via axoneme internalization, we focused on chytrids Rhizoclosmatium globosum and Spizellomyces punctatus to represent two lineages of chytrids with different motility characteristics. Our results show that each chytrid species can reel in its axoneme into the cell body along its cortex on the order of minutes, while S. punctatus has additional faster ciliary compartment loss and lash-around mechanisms. S. punctatus retraction can also occur away from the cell cortex and is partially actin dependent. Post-internalization, the tubulin of the axoneme is degraded in both chytrids over the course of about 2 hr. Axoneme disassembly and axonemal tubulin degradation are both partially proteasome dependent. Overall, chytrid cilium disassembly is a fast process that separates axoneme internalization and degradation.
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Axonema/metabolismo , Cílios/metabolismo , Fungos/patogenicidadeRESUMO
The centriole duplication cycle normally ensures that centriole number is maintained at two centrioles per G1 cell. However, some circumstances can result in an aberrant increase in centriole number-a phenotype that is particularly prevalent in several types of cancer. Following an artificial increase in centriole number without tetraploidization due to transient overexpression of the kinase PLK4, human cells return to a normal centriole number during the proliferation of the population. We examine the mechanisms responsible for this return to normal centriole number at the population level in human retinal pigment epithelial cells. We find that the return to normal centriole number in the population of induced cells cannot be explained by limited duplication of centrioles, instability of extra centrioles, or by grossly asymmetric segregation of extra centrioles in mitosis. However, cells with extra centrioles display heterogenous phenotypes including extended cell cycle arrest, longer interphase durations, and death, which overall results in a proliferative disadvantage relative to normal cells in the population. Although about half of cells with extra centrioles in a population were able to divide, the extent of the disadvantages conferred by other fates is sufficient to account for the observed rate of return to normal centriole number. These results suggest that only under conditions of positive selection for cells with extra centrioles, continuous generation of such centrioles, or alleviation of the disadvantageous growth phenotypes would they be maintained in a population.
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Centríolos/metabolismo , Centríolos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células/fisiologia , Centrossomo/metabolismo , Homeostase , Humanos , Interfase/fisiologia , Mitose , Proteínas Serina-Treonina Quinases/fisiologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Olfaction in most animals is mediated by neurons bearing cilia that are accessible to the environment. Olfactory sensory neurons (OSNs) in chordates usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how the epithelium generates cells with many centrioles is not yet understood. We show that centrioles are amplified via centriole rosette formation in both embryonic development and turnover of the olfactory epithelium in adult mice, and rosette-bearing cells often have free centrioles in addition. Cells with amplified centrioles can go on to divide, with centrioles clustered at each pole. Additionally, we found that centrioles are amplified in immediate neuronal precursors (INPs) concomitant with elevation of mRNA for polo-like kinase 4 (Plk4) and SCL/Tal1-interrupting locus gene (Stil), key regulators of centriole duplication. These results support a model in which centriole amplification occurs during a transient state characterized by elevated Plk4 and Stil in early INP cells. These cells then go on to divide at least once to become OSNs, demonstrating that cell division with amplified centrioles, known to be tolerated in disease states, can occur as part of a normal developmental program.