Diego R Revinski

Laure-Emmanuelle Zaragosi

Camille Boutin

Marie Deprez

Virginie Thomé

Olivier Rosnet

Anne-Sophie Gay

Agnès Paquet

Nicolas Pons

Gilles Ponzio

Sandra Ruiz-Garcia

Olivier Mercey

Brice Marcet
email: marcet@ipmc.cnrs.fr

Laurent Kodjabachian
email: laurent.kodjabachian@univ-amu.fr

Pascal Barbry
email: barbry@ipmc.cnrs.fr

CDC20B is required for deuterosome-mediated centriole production in 1 multiciliated cells

Keywords: multiciliated cell, centriole, deuterosome, motile cilia, CDC20B, Separase, PLK1, cell cycle, Xenopus, mouse, human

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Introduction

Multiciliated cells (MCCs) are present throughout metazoan evolution and serve functions ranging from locomotion of marine larvae and flatworms, to brain homeostasis, mucociliary clearance of pathogens and transportation of oocytes in vertebrates [START_REF] Meunier | Multiciliated Cells in Animals[END_REF][START_REF] Spassky | The development and functions of multiciliated epithelia[END_REF][START_REF] Brooks | Multiciliated cells[END_REF] . The formation of MCCs requires the production of numerous motile cilia through a complex process called multiciliogenesis [START_REF] Spassky | The development and functions of multiciliated epithelia[END_REF][START_REF] Brooks | Multiciliated cells[END_REF] . The transcriptional control of multiciliogenesis has been decrypted to a large extent, through studies in Xenopus and mouse [START_REF] Spassky | The development and functions of multiciliated epithelia[END_REF] . Seating at the top of the cascade, the Geminin-related factors GemC1 [START_REF] Kyrousi | Mcidas and GemC1 are key regulators for the generation of multiciliated ependymal cells in the adult neurogenic niche[END_REF][START_REF] Arbi | GemC1 controls multiciliogenesis in the airway epithelium[END_REF][START_REF] Terre | GEMC1 is a critical regulator of multiciliated cell differentiation[END_REF][START_REF] Zhou | Gmnc Is a Master Regulator of the Multiciliated Cell Differentiation Program[END_REF] and Multicilin [START_REF] Stubbs | Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation[END_REF][START_REF] Ma | Multicilin drives centriole biogenesis via E2f proteins[END_REF] (MCIDAS in mammals) are both necessary and sufficient to initiate MCC differentiation. GemC1 and Multicilin in complex with E2F transcription factors have been reported to activate the expression of Myb, FoxJ1, Rfx2 and Rfx3, which collectively regulate the expression of a large body of effectors required for the formation of multiple motile cilia [START_REF] Kyrousi | Mcidas and GemC1 are key regulators for the generation of multiciliated ependymal cells in the adult neurogenic niche[END_REF][START_REF] Arbi | GemC1 controls multiciliogenesis in the airway epithelium[END_REF][START_REF] Stubbs | Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation[END_REF][START_REF] Ma | Multicilin drives centriole biogenesis via E2f proteins[END_REF][START_REF] Quigley | Rfx2 Stabilizes Foxj1 Binding at Chromatin Loops to Enable Multiciliated Cell Gene Expression[END_REF][START_REF] Chung | RFX2 is broadly required for ciliogenesis during vertebrate development[END_REF] . Recently, defective multiciliogenesis caused by mutations in MCIDAS and Cyclin O (CCNO) has been associated with congenital respiratory and fertility syndromes in human [START_REF] Boon | MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia[END_REF][START_REF] Wallmeier | Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia[END_REF] .

Each cilium sits atop a modified centriole, called a basal body (BB). After they exit from the cell cycle, maturing MCCs face the challenge of producing dozens to hundreds of centrioles in a limited time window. In vertebrate MCCs, bulk centriole biogenesis is mostly achieved through an acentriolar structure named the deuterosome, although canonical amplification from parental centrioles also occurs [START_REF] Meunier | Multiciliated Cells in Animals[END_REF][START_REF] Spassky | The development and functions of multiciliated epithelia[END_REF][START_REF] Brooks | Multiciliated cells[END_REF] . The deuterosome was first described in early electron microscopy studies of various multiciliated tissues including the mammalian lung [START_REF] Sorokin | Reconstructions of centriole formation and ciliogenesis in mammalian lungs[END_REF] and oviduct [START_REF] Dirksen | Centriole morphogenesis in developing ciliated epithelium of the mouse oviduct[END_REF][START_REF] Anderson | The formation of basal bodies (centrioles) in the Rhesus monkey oviduct[END_REF] , the avian trachea [START_REF] Kalnins | Centriole replication during ciliogenesis in the chick tracheal epithelium[END_REF] , and the Xenopus tadpole epidermis and trachea [START_REF] Steinman | An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis[END_REF] . In mammalian MCCs, the deuterosome was described as a spherical mass of fibers organized into an inner dense region and an outer, more delicate, corona [START_REF] Anderson | The formation of basal bodies (centrioles) in the Rhesus monkey oviduct[END_REF] . In Xenopus, deuterosomes were initially named procentriole organizers and were reported as dense amorphous masses [START_REF] Steinman | An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis[END_REF] .

Recent studies have revealed that deuterosome-mediated centriole synthesis mobilizes key components of the centriole-dependent duplication pathway of the cell cycle, including CEP152, PLK4 and SAS6 [START_REF] Jord | Centriole amplification by mother and daughter centrioles differs in multiciliated cells[END_REF][START_REF] Klos Dehring | Deuterosome-mediated centriole biogenesis[END_REF][START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] . However, the deuterosome itself differs from the centriole and may contain specific components. The identification of one such component, called DEUP1

for Deuterosome assembly protein 1, opened the possibility to investigate the deuterosome at the molecular level [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] . In mouse tracheal ependymal cells, DEUP1 was detected in the core of the deuterosome [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] . DEUP1, also known as CCDC67, is a conserved vertebrate paralogue of CEP63, itself known for its importance in initiation of centriole duplication during the cell cycle [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF][START_REF] Sir | A primary microcephaly protein complex forms a ring around parental centrioles[END_REF] . Consistently, DEUP1 was shown to be essential for centriole multiplication in mouse and Xenopus MCCs [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] . Both CEP63 and DEUP1 interact with CEP152, an essential event for centriole duplication and multiplication in cycling cells and MCCs, respectively [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF][START_REF] Sir | A primary microcephaly protein complex forms a ring around parental centrioles[END_REF] .

Once centriole multiplication is over, neo-synthesized centrioles must disengage from deuterosomes and parental centrioles, convert into BBs and migrate apically to dock at the plasma membrane to initiate cilium elongation.

In this study, we aimed at better understanding deuterosome biology. We found that the gene CDC20B was specifically expressed in maturing MCCs during the phase of centriole multiplication. We established the corresponding CDC20B protein as an essential regulator of centriole-deuterosome disengagement. This work illustrates well the strong functional relationships that exist between centriole release from deuterosomes and centriole disengagement in mitotic cells. It also posits CDC20B as a new component of a "multiciliary locus" that contains several gene products, either proteins, such as MCIDAS, CCNO or CDC20B itself, or microRNAs, such as miR-449abc, which are all actively involved into multiciliogenesis.

Results

MCC single-cell transcriptome at deuterosome-stage

To identify new regulators of centriole multiplication, we analyzed the transcriptome of human airway epithelial cells (HAECs) at the differentiation stage corresponding to active centriole multiplication [START_REF] Marcet | Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway[END_REF] at the single-cell level (Fig. 1a). Gene expression data from 1663 cells was projected on a 2D space by t-distributed Stochastic Neighbor Embedding (tSNE) (Fig. 1b). We identified a small group of 37 cells corresponding to maturing MCCs engaged in deuterosome-mediated centriole amplification, as revealed by the specific expression of MCIDAS [START_REF] Stubbs | Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation[END_REF] , MYB [START_REF] Tan | Myb promotes centriole amplification and later steps of the multiciliogenesis program[END_REF] , and DEUP1 [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] (Fig. 1c,d and Supplementary Figure 1). This subpopulation was characterized by the expression of known effectors of centriole synthesis, such as PLK4, STIL, CEP152, SASS6, but also of cell cycle regulators, such as CDK1, CCNB1, CDC20, SGOL2 and NEK2 (Fig. 1d, Supplementary Figure 1 and Supplementary Table 1). We reasoned that uncharacterized cell cycle-related genes that are specific to this subpopulation could encode new components of the deuterosome-dependent centriole amplification pathway. A particularly interesting candidate in this category was CDC20B (Fig. 1d), which is related to the cell cycle regulators CDC20 and FZR1 [START_REF] Yu | Cdc20: a WD40 activator for a cell cycle degradation machine[END_REF] (Supplementary Figure 2a). First, the CDC20B gene is present in the vertebrate genomic locus that also contains the key MCC regulators MCIDAS [START_REF] Stubbs | Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation[END_REF] and CCNO [START_REF] Wallmeier | Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia[END_REF] . Co-expression of CDC20B, MCIDAS and CCNO throughout HAEC differentiation was indeed observed in an independent RNA sequencing study, performed on a bulk population of HAECs (Supplementary Figure 2b). These results fit well with the observation that the promoter of human CDC20B was strongly activated by the MCIDAS partners E2F1 and E2F4 (Supplementary Figure 2c), as also shown in Xenopus by others [START_REF] Ma | Multicilin drives centriole biogenesis via E2f proteins[END_REF] (Supplementary Figure 2d). Second, the CDC20B gene bears in its second intron the miR-449 microRNAs, which were shown to contribute to MCC differentiation [START_REF] Marcet | Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway[END_REF][START_REF] Song | miR-34/449 miRNAs are required for motile ciliogenesis by repressing cp110[END_REF][START_REF] Otto | Cell cycle-targeting microRNAs promote differentiation by enforcing cell-cycle exit[END_REF][START_REF] Wu | Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis[END_REF][START_REF] Chevalier | miR-34/449 control apical actin network formation during multiciliogenesis through small GTPase pathways[END_REF][START_REF] Mercey | Characterizing isomiR variants within the microRNA-34/449 family[END_REF] . Finally, in Xenopus epidermal MCCs, cdc20b transcripts were specifically detected during the phase of centriole amplification (Supplementary Figure 2e-m). This first set of data pointed out the specific and conserved expression pattern of CDC20B in immature MCCs. In the rest of this study, we analyzed the putative role of CDC20B in deuterosome-mediated centriole multiplication.

Composition and organization of vertebrate deuterosomes

We first conducted a series of immunofluorescence analyses to gain a better understanding of deuterosome organization in mouse ependymal and Xenopus epidermal MCCs as models. In whole-mounts of mouse ependymal walls, mature deuterosomes revealed by DEUP1 staining appeared as circular structures around a lumen (Fig. 2a). We noticed that DEUP1 also stained fibers emanating from the core into the corona. Nascent centrioles revealed by the marker FOP were organized around the DEUP1-positive core ring. STED super-resolution microscopy helped to better appreciate the regular organization of individual FOP-positive procentrioles (Fig. 2b). Proximity labeling assays have revealed that when ectopically expressed in centrosomes CCDC67/DEUP1 is found close to Pericentrin (PCNT) and γtubulin, two main components of the pericentriolar material (PCM) [START_REF] Firat-Karalar | Proximity interactions among centrosome components identify regulators of centriole duplication[END_REF] . Interestingly, we found that PCNT was present in the deuterosome corona (Fig. 2a), and STED microscopy further revealed that PCNT formed fibers around growing procentrioles (Fig. 2b). γ-tubulin staining was detected in the DEUP1-positive deuterosome core, as well as in the corona (Fig. 2a).

STED microscopy indicated that PCNT and γ-tubulin stained distinct interwoven fibers in the deuterosome corona. Next, we stained immature Xenopus epidermal MCCs with γ-Tubulin and Centrin to reveal centriole amplification platforms. These platforms displayed irregular shapes and sizes (Fig. 2c), in agreement with early electron microscopy studies [START_REF] Steinman | An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis[END_REF] . Expression of low amounts of GFP-Deup1 in MCCs induced by Multicilin confirmed that active deuterosomes are embedded in γ-Tubulin-positive masses (Fig. 2d). Overall, this analysis is consistent with early ultrastructural studies, as the deuterosome core and corona can be distinguished by the presence of DEUP1 and PCNT, respectively. Moreover, γ-tubulin is a conserved marker of centriole amplification platforms in vertebrate MCCs. By analogy to the organization of the centrosome, we propose to coin the term perideuterosomal material (PDM) to describe the corona, as this region may prove important for deuterosome function.

CDC20B associates to vertebrate deuterosomes

We then analyzed the subcellular localization of CDC20B protein in deuterosome-stage mouse and Xenopus MCCs. In immature mouse tracheal MCCs, double immunofluorescence revealed the association of CDC20B to DEUP1-positive deuterosomes (Fig. 3a). We noticed that CDC20B tended to associate primarily to large DEUP1 foci. As deuterosomes grow as they mature [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] , this suggests that CDC20B may penetrate into the deuterosomal environment at a late stage of the centriole multiplication process. The same observation was made when comparing CDC20B staining in the region of immature and mature deuterosomes of mouse ependymal MCCs (Fig. 3b). As double DEUP1/CDC20B staining could not be performed on these cells, we analyzed CDC20B distribution relative to FOP-positive procentrioles. In early deuterosome-stage MCCs, CDC20B was expressed at low levels and FOP staining was mostly concentrated in a large amorphous cloud (Fig. 3b). In such cells, no CDC20B staining was detected in association to FOP-positive procentrioles growing around deuterosomes. In contrast, in mature deuterosome-stage MCCs, CDC20B was enriched in the innermost part of the PDM, probably very close to the deuterosome core (Fig. 3b). Further evidence was provided with a custom-made polyclonal antibody (Supplementary Figure 3b,c) used to analyze Cdc20b protein distribution in Xenopus epidermal MCCs. Here also, Cdc20b was found associated to Deup1-positive deuterosomes actively engaged in centriole synthesis (Fig. 3c). We finally analyzed the distribution of CDC20B in mature MCCs. As previously reported, the CDC20B protein was detected near BBs [START_REF] Marcet | Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway[END_REF] , but also in cilia of fully differentiated human airway MCCs (Supplementary Figure 4a-c). This was confirmed by proximity ligation assays that revealed a tight association of CDC20B with Centrin2 and acetylated α-Tubulin, in BBs and cilia, respectively (Supplementary Figure 4d-f). Fluorescent immunostaining also revealed the presence of Cdc20b in the vicinity of BBs in Xenopus epidermal MCCs (Supplementary Figure 4g-i). In contrast, no cilia staining was observed in these cells.

Altogether, our analyses revealed that in three distinct types of MCCs in two distant vertebrate species, CDC20B is tightly associated to mature deuterosomes. We next investigated whether it may control their function.

CDC20B is required for multiciliogenesis in vertebrates

For that purpose, Cdc20b was knocked down in mouse ependymal MCCs, through post-natal brain electroporation of three distinct shRNAs. One of them, sh274, which targets the junction between exons 3 and 4, and can therefore only interact with mature mRNA, was useful to rule out possible interference with the production of miR-449 molecules from the Cdc20b pre-mRNA (Supplementary Figure 5a). Five days after electroporation, all three shRNAs significantly reduced the expression of CDC20B in deuterosome-stage MCCs (Fig. 4c), but did not alter MCC identity as revealed by FOXJ1 expression (Fig. 4a,b,d). Centriole production by deuterosomes was analyzed by FOP/DEUP1 double staining nine days after electroporation. At this stage, control MCCs had nearly all released their centrioles and disassembled their deuterosomes (Fig. 4e,g). In sharp contrast, Cdc20b shRNAs caused a significant increase in the number of defective MCCs that displayed centrioles still engaged on deuterosomes (Fig. 4f,g). Fifteen days after electroporation, a majority of CDC20Bdeficient MCCs still showed a severely reduced number of released centrioles, and consequently lacked cilia (Fig. 4h-k).

Cdc20b was also knocked down in Xenopus epidermal MCCs, through injection of two independent morpholino antisense oligonucleotides targeting either the ATG (Mo ATG), or the exon 1/intron 1 junction (Mo Spl) (Supplementary Figure 5b). The efficiency of Mo ATG was verified through fluorescence extinction of co-injected Cdc20b-Venus (Supplementary Figure 5c). RT-PCR confirmed that Mo Spl caused intron 1 retention (Supplementary Figure 5d), which was expected to introduce a premature stop codon, and to produce a Cdc20b protein lacking 96% of its amino-acids, likely to undergo unfolded protein response-mediated degradation. Thus, both morpholinos were expected to generate severe loss of Cdc20b function. Consistent with this interpretation, both morpholinos strongly reduced Cdc20b immunostaining in deuterosome-stage MCCs (Supplementary Figure 5e). We verified that neither morpholinos caused p53 transcript up-regulation (Supplementary Figure 5f), a non-specific response to morpholinos that is sometimes detected in zebrafish embryos [START_REF] Robu | p53 activation by knockdown technologies[END_REF] .

Importantly, whole-mount in situ hybridization indicated that miR-449 expression was not perturbed in the presence of either morpholino (Supplementary Figure 5g). We found that cdc20b knockdown did not interfere with acquisition of the MCC fate (Supplementary Figure 6a-e), but severely impaired multiciliogenesis, as revealed by immunofluorescence and electron microscopy (Fig. 5a-i). This defect stemmed from a dramatic reduction in the number of centrioles, and poor docking at the plasma membrane (Fig. 5g-o and Supplementary Figure 6f-k). Importantly, centrioles and cilia were rescued in Mo Spl MCCs by co-injection of cdc20b, venus-cdc20b or cdc20b-venus mRNAs (Fig. 5j-o and Supplementary Figure 6f-k). In normal condition, Xenopus epidermal MCCs arise in the inner mesenchymal layer and intercalate into the outer epithelial layer, while the process of centriole amplification is underway [START_REF] Werner | Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1[END_REF] . To rule out secondary defects due to poor radial intercalation, we assessed the consequences of cdc20b knockdown in MCCs induced in the outer layer by Multicilin overexpression [START_REF] Stubbs | Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation[END_REF] . Like in natural MCCs, Cdc20b proved to be essential for the production of centrioles and cilia in response to Multicilin activity (Supplementary Figure 7a-g). We also noted that the apical actin network that normally surrounds BBs was disrupted in absence of Cdc20b, although this defect could be secondary to the absence of centrioles (Supplementary Figure 7d-g). Centrioles in Cdc20b morphant cells often formed clusters, suggesting that disengagement from deuterosomes could have failed (Fig. 5l,m). To better assess this process, we injected GFP-Deup1 in Multicilin-induced MCCs and stained centrioles with Centrin. In mature control MCCs, deuterosomes were disassembled, centrioles were converted into BBs, had docked and initiated cilium growth (Fig. 5p,s). In contrast, both morpholinos caused a dramatic increase in the number of defective MCCs, which were devoid of cilia and displayed centrioles still engaged on deuterosomes (Fig. 5q-u). Altogether our functional assays in mouse and Xenopus indicate that CDC20B is required for centriole disengagement from deuterosomes and subsequent ciliogenesis in MCCs. We next investigated the molecular mechanism of action of CDC20B underlying its role in centriole release.

Partners and effectors of CDC20B reveal its mechanism of action

In mitotic cells, centriole disengagement is necessary to license centriole duplication in the following cell cycle [START_REF] Firat-Karalar | The centriole duplication cycle[END_REF] . This process is known to depend on the coordinated activities of the mitotic kinase PLK1 and the protease Separase [START_REF] Tsou | Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells[END_REF] . One proposed mechanism involves the phosphorylation of PCNT by PLK1, which induces its cleavage by Separase, thereby allowing centriole disengagement through disassembly of the PCM [START_REF] Kim | PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit[END_REF][START_REF] Matsuo | Kendrin is a novel substrate for separase involved in the licensing of centriole duplication[END_REF] . Separase is known to be activated by the degradation of its inhibitor Securin, which is triggered by the Anaphase Promoting Complex (APC/C) upon binding to CDC20 [START_REF] Yu | Cdc20: a WD40 activator for a cell cycle degradation machine[END_REF] . PLK1, Separase (ESPL1), Securin (PTTG1), CDC20 and PCNT were all found to be expressed in human deuterosome-stage MCCs (Fig. 1d and Supplementary Figure 1). We have shown above that PCNT is present in the PDM and a recent study revealed the presence of CDC20 and the APC/C component APC3 in mouse ependymal MCCs at the stage of centriole disengagement [START_REF] Jord | Calibrated mitotic oscillator drives motile ciliogenesis[END_REF] . Based on this large body of information, we hypothesized that centriole-deuterosome disengagement involves the coordinated activities of PLK1 and Separase, and that CDC20B would be involved in this scenario. CDC20B encodes a protein of about 519 amino-acids largely distributed across the vertebrate phylum [START_REF] Marcet | Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway[END_REF] . In its C-terminal half, CDC20B contains seven well conserved WD40 repeats, predicted to form a β-propeller, showing 49% and 37% identity to CDC20 and FZR1 repeats, respectively (Supplementary Figure 2a). However, CDC20B lacks canonical APC/C binding domains (Supplementary Figure 2a). Using mass spectrometry on immunoprecipitated protein complexes from transfected HEK cells, we could identify multiple APC/C components interacting with CDC20 but not with CDC20B (Supplementary Table 2). We conclude that CDC20B is probably incapable of activating APC/C. Interestingly, an unbiased interactome study reported association of CDC20B with PLK1 [START_REF] Huttlin | The BioPlex Network: A Systematic Exploration of the Human Interactome[END_REF] . Using reciprocal co-immunoprecipitation assays in HEK transfected cells, we confirmed that CDC20B and PLK1 could be found in the same complex (Fig. 6a and Supplementary Figure 8). This suggested that CDC20B could cooperate with PLK1 to trigger centriole disengagement. Consistent with this hypothesis, we found that PLK1 was enriched in the PDM of mature deuterosomes in mouse ependymal MCCs (Fig. 6b), in agreement with a recent report [START_REF] Jord | Calibrated mitotic oscillator drives motile ciliogenesis[END_REF] . Another interesting partner of CDC20B identified in a second unbiased interactome study [START_REF] Rual | Towards a proteome-scale map of the human protein-protein interaction network[END_REF] was SPAG5 (Astrin), which was reported to control timely activation of Separase during the cell cycle [START_REF] Thein | Astrin is required for the maintenance of sister chromatid cohesion and centrosome integrity[END_REF][START_REF] Chiu | The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation[END_REF] . Using the same strategy as above, we could detect CDC20B and SPAG5 in the same complex (Fig. 6c and Supplementary Figure 8). As SPAG5 was found associated to DEUP1 in a proximity labeling assay [START_REF] Firat-Karalar | Proximity interactions among centrosome components identify regulators of centriole duplication[END_REF] , we assessed its localization in deuterosomes. Strikingly, SPAG5 was detectable in mature deuterosomes of mouse ependymal MCCs, with a clear enrichment in the deuterosome core (Fig. 6d). Finally, reciprocal co-immunoprecipitations revealed that CDC20B and DEUP1 were detected in the same complex when co-expressed in HEK cells (Fig. 6e and Supplementary Figure 8).

Consistent with this result, we observed that RFP-Cdc20b was recruited around spherical Deup1-GFP structures positive for γ-Tubulin and Centrin in Xenopus epidermal MCCs (Supplementary Figure 7h-m). This series of experiments suggested that CDC20B could participate in the assembly of a protein complex in mature deuterosomes, required to coordinate the activities of PLK1 and Separase for centriole disengagement. As Separase is the last effector in this scenario, we tested whether over-expressing human Separase in Xenopus cdc20b morphant MCCs could rescue centriole disengagement. In support to our hypothesis, over-expression of wild-type, but not protease-dead Separase, efficiently rescued centriole disengagement and cilia formation in cdc20b morphant MCCs (Fig. 7a-g and Supplementary Figure 7n-s). Separase could also rescue multiciliogenesis in Multicilin-induced MCCs injected with cdc20b Mos (Supplementary Figure 7t-z). We conclude that CDC20B is involved in Separase-mediated release of mature centrioles from deuterosomes in vertebrate MCCs (Fig. 7h).

Discussion

In this study, we report for the first time the essential and conserved role of CDC20B in vertebrate multiciliogenesis. Our data suggest that the presence of CDC20B in the perideuterosomal region is necessary to allow centriole disengagement. We note, however, that our data, which are based on partial knockdowns, remain compatible with an earlier function of CDC20B in promoting deuterosome assembly and/or activity. A total genetic knockout of Cdc20b should help to assess this possibility in mouse tracheal and ependymal MCCs. By analogy to mitosis, we propose that CDC20B is involved in Separase-dependent proteolysis at deuterosomes, allowing the release of mature centrioles and subsequent ciliogenesis. This view is consistent with a recent report showing that centriole disengagement in murine ependymal MCCs involves the activities of PLK1, a partner of CDC20B, and APC/C, the activator of Separase [START_REF] Jord | Calibrated mitotic oscillator drives motile ciliogenesis[END_REF] . The central question arising from our work then becomes: how are CDC20B and Separase activities integrated? The simple scenario of a CDC20-like function of CDC20B is very unlikely as it does not appear to bind APC/C (Supplementary Table 2). CDC20 was detected in cultured murine ependymal MCCs during the phase of centriole disengagement [START_REF] Jord | Calibrated mitotic oscillator drives motile ciliogenesis[END_REF] , and FZR1 genetic ablation was reported to cause reduced production of centrioles and cilia in the same cells [START_REF] Eguren | The APC/C cofactor Cdh1 prevents replicative stress and p53dependent cell death in neural progenitors[END_REF] . APC/C is therefore likely activated in maturing MCCs by its classical activators, CDC20 and/or FZR1, leading to Separase activation through degradation of its inhibitor Securin. In that context, we propose that additional factors linked directly or indirectly to CDC20B may contribute to activation of Separase. It was shown that SPAG5 inhibits or activates Separase depending on its status of phosphorylation [START_REF] Thein | Astrin is required for the maintenance of sister chromatid cohesion and centrosome integrity[END_REF][START_REF] Chiu | The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation[END_REF] . As the phosphorylation status of SPAG5 was shown to be controlled by PLK1 [START_REF] Chung | Phosphorylation of Astrin Regulates Its Kinetochore Function[END_REF] , our data suggest that the CDC20B/PLK1/SPAG5 complex could control the timing of Separase activation locally in deuterosomes. It is therefore possible that multiple modes of activation of Separase may act in parallel to trigger the release of neo-synthesized centrioles in maturing MCCs. Alternatively, different pathways may be used in distinct species, or in distinct types of MCCs. An important question for future studies regards the identity of PLK1 and Separase substrates involved in centriole disengagement. Work on mitotic cells [START_REF] Kim | PLK1 regulation of PCNT cleavage ensures fidelity of centriole separation during mitotic exit[END_REF][START_REF] Matsuo | Kendrin is a novel substrate for separase involved in the licensing of centriole duplication[END_REF] and our own analysis suggest that PCNT may represent a prime target. Another potentially relevant candidate could be DEUP1 itself as it is clear that deuterosomes are disassembled after the release of centrioles. In that respect, it is interesting to note the presence of multiple PLK1 consensus phosphorylation sites in human, mouse and Xenopus DEUP1.

In this study, we have introduced the notion of perideuterosomal material, in analogy to the pericentriolar material. It is striking that the two main components of the PCM, PCNT and γ-Tubulin, are also present in the PDM, which begs the question whether additional PCM proteins may be present in the PDM. The PDM may constitute a platform to sustain procentriole growth, through the concentration and delivery of elementary parts. It could also play a mechanical role to hold in place the growing procentrioles. Future work should evaluate deuterosome-mediated centriole synthesis in absence of major PDM components.

We found that beyond its association to deuterosomes during the phase of centriole amplification, CDC20B was also associated to BBs and cilia in fully differentiated mammalian MCCs. This dual localization is consistent with failed ciliogenesis upon CDC20B knockdown in mouse ependymal MCCs. However, while we could detect Cdc20b near BBs of mature MCCs in Xenopus, we found no evidence of its presence in cilia. Furthermore, cilia were rescued by Separase overexpression in Cdc20b morphant MCCs. This suggests that Cdc20b is not required for ciliogenesis in this species, although it could potentially contribute to cilium structure and/or function. Thus, refined temporal and spatial control of CDC20B inhibition will be needed to study its function beyond centriole synthesis. This and previous studies [START_REF] Marcet | Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway[END_REF][START_REF] Song | miR-34/449 miRNAs are required for motile ciliogenesis by repressing cp110[END_REF][START_REF] Otto | Cell cycle-targeting microRNAs promote differentiation by enforcing cell-cycle exit[END_REF][START_REF] Wu | Two miRNA clusters, miR-34b/c and miR-449, are essential for normal brain development, motile ciliogenesis, and spermatogenesis[END_REF] establish that the miR-449 cluster and its host gene CDC20B are commonly involved in multiciliogenesis. Consistent with its early expression, it was suggested that miR-449 controls cell cycle exit and entry into differentiation of MCCs [START_REF] Marcet | Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway[END_REF][START_REF] Otto | Cell cycle-targeting microRNAs promote differentiation by enforcing cell-cycle exit[END_REF][START_REF] Mercey | Characterizing isomiR variants within the microRNA-34/449 family[END_REF] .

This study reveals that CDC20B itself is involved in the production of centrioles, the first key step of the multiciliogenesis process. From that perspective, the nested organization of miR-449 and CDC20B in vertebrate genomes, which allows their coordinated expression, appears crucial for successful multiciliogenesis.

It is also noteworthy to point out the location of this gene in a genomic locus where congenital mutations in MCIDAS and CCNO were recently shown to cause a newlyrecognized MCC-specific disease, called Reduced Generation of Multiple motile Cilia (RGMC). RGMC is characterized by severe chronic lung infections and increased risk of infertility [START_REF] Boon | MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia[END_REF][START_REF] Wallmeier | Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia[END_REF] . Its location in the same genetic locus as MCIDAS and CCNO makes CDC20B a putative candidate for RGMC. By extension, new deuterosome-stage specific genes uncovered by scRNA-seq in this study also represent potential candidates for additional RGMC mutations.

Previous works have established the involvement of the centriole duplication machinery active in S-phase of the cell cycle, during centriole multiplication of vertebrate post-mitotic

MCCs [START_REF] Jord | Centriole amplification by mother and daughter centrioles differs in multiciliated cells[END_REF][START_REF] Klos Dehring | Deuterosome-mediated centriole biogenesis[END_REF][START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] . Our study further reveals a striking analogy between centriole disengagement from deuterosomes in MCCs, and centriole disengagement that occurs during the M/G1 transition of the cell cycle (Fig. 7g). Thus, it appears that centriole production in MCCs recapitulates the key steps of the centriole duplication cycle [START_REF] Firat-Karalar | The centriole duplication cycle[END_REF] . However, the cell cycle machinery must adapt to the acentriolar deuterosome to massively produce centrioles. Such adaptation appears to involve physical and functional interactions between canonical cell cycle molecules, such as CEP152 and PLK1, and recently evolved cell cycle-related deuterosomal molecules, such as DEUP1 [START_REF] Zhao | The Cep63 paralogue Deup1 enables massive de novo centriole biogenesis for vertebrate multiciliogenesis[END_REF] and CDC20B. It remains to examine whether additional deuterosomal cell cycle-related molecules have emerged in the vertebrate phylum to sustain massive centriole production.

In conclusion, this work illustrates how coordination between ancestral and recently evolved cell cycle-related molecules can give rise to a new differentiation mechanism in vertebrates.

Methods

Subjects/human samples

Inferior turbinates were from patients who underwent surgical intervention for nasal obstruction or septoplasty (provided by L. Castillo, Nice University Hospital, France). The use of human tissues was authorized by the bioethical law 94-654 of the French Public Health Code and written consent from the patients.

Single-cell RNA sequencing of Human Airway Epithelial Cells (HAECs)

HAECs cultures were derived from nasal mucosa of inferior turbinates. After excision, nasal inferior turbinates were immediately immersed in Ca2+/Mg2+-free HBSS supplemented with 25 mM HEPES, 200 U/mL penicillin, 200 µg/mL streptomycin, 50 µg/mL gentamicin sulfate, and 2.5 µg/ml amphotericin B (all reagents from Gibco). After repeated washes with cold supplemented HBSS, tissues were digested with 0.1% Protease XIV from Streptomyces Griseus (Sigma) overnight at 4°C. After incubation, fetal calf serum (FCS) was added to a final concentration of 10%, and nasal epithelial cells were detached from the stroma by gentle agitation. Cell suspensions were further dissociated by trituration through a 21G-needle and then centrifuged at 150g for 5 min. The pellet was resuspended in supplemented HBSS containing 10% FCS and centrifuged again. The second cell pellet was then suspended in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% FCS and cells were plated (20 000 cells per cm²) on 75 cm²-flasks coated with rat-tail collagen I (Sigma-Aldrich).

Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Culture medium was replaced with Bronchial Epithelium Basal Medium (BEBM, Lonza) supplemented with BEGM SingleQuot Kit Supplements (Lonza) on the day after and was then changed every other day. After 4 to 5 days of culture, after reaching about 70% confluence, cells were detached with trypsin-EDTA 0.05% (Gibco) for 5 min and seeded on Transwell® permeable supports (6.5 mm diameter; 0.4 µm pore size; Corning), in BEGM medium, with a density of 30 000 cells per Transwell®. Once the cells have reached confluence (typically after 5 days), they were induced to differentiate at the air-liquid interface by removing medium at the apical side of the Transwell®, and by replacing medium at the basal side with DMEM:BEBM (1:1) supplemented with BEGM SingleQuot Kit Supplements. Culture medium was changed every other day. Single-cell analysis was performed after 14 days of culture at the air-liquid interface, which corresponds to the maximum centriole multiplication stage. To obtain a single-cell suspension, cells were incubated with 0.1% protease type XIV from Streptomyces griseus in supplemented HBSS for 4 hours at 4°C degrees. Cells were gently detached from Transwells® by pipetting and then transferred to a microtube. 50 units of DNase I (EN0523 Thermo Fisher Scientific) per 250 µL were directly added and cells were further incubated at room temperature for 10 min. Cells were centrifuged (150g for 5 min) and resuspended in 500 µL supplemented HBSS containing 10% FCS, centrifuged again (150g for 5 min) and resuspended in 500 µL HBSS before being mechanically dissociated through a 26G syringe (4 times). Finally, cell suspensions were filtered through a Scienceware® Flowmi™ Cell Strainer (40µm porosity), centrifuged (150g for 5 min) and resuspended in 500 µL of cold HBSS. Cell concentration measurements were performed with Scepter™ 2.0 Cell Counter (Millipore) and Countess™ automated cell counter (ThermoFisher Scientific). Cell viability was checked with Countess™ automated cell counter (ThermoFisher Scientific). All steps except the DNAse I incubation were performed on ice. For the cell capture by the 10X genomics device, the cell concentration was adjusted to 300 cells/µl in HBSS aiming to capture 1500 cells. We then followed the manufacturer's protocol (Chromium™ Single Cell 3' Reagent Kit, v2 Chemistry) to obtain single cell 3' libraries for Illumina sequencing.

Libraries were sequenced with a NextSeq 500/550 High Output v2 kit (75 cycles) that allows up to 91 cycles of paired-end sequencing: the forward read had a length of 26 bases that included the cell barcode and the UMI; the reverse read had a length of 57 bases that contained the cDNA insert. CellRanger Single-Cell Software Suite v1.3 was used to perform sample demultiplexing, barcode processing and single-cell 3′ gene counting using standards default parameters and human build hg19. Additional analyses were performed using R.

Pseudotemporal ordering of single cells was performed with the last release of the Monocle package [START_REF] Qiu | Single-cell mRNA quantification and differential analysis with Census[END_REF] . Cell cycle scores were calculated by summing the normalized intensities of genes belonging to phase-specific gene sets then centered and scaled by phase. Gene sets for each phase were curated from previously described sets of genes [START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF] (Table S2). Data was submitted to the GEO portal under series reference GSE103518. Data shown in Figure 1 is representative of 4 independent experiments performed on distinct primary cultures.

RNA sequencing of HAECs

For Figure S3A, three independent HAEC cultures (HAEC1, HAEC2, HAEC3) were triggered to differentiate in air-liquid interface (ALI) cultures for 2 days (ALI day 2, undifferentiated), ALI day 14 (first cilia) or ALI day 28 (well ciliated). RNA was extracted with the miRNeasy mini kit (Qiagen) following manufacturer's instructions. mRNA-seq was performed from 2 µg of RNA that was first subjected to mRNA selection with Dynabeads® mRNA Purification Kit (Invitrogen). mRNA was fragmented 10 min at 95°C in RNAseIII intersection-nonempty" option. Normalization and statistical analysis were performed using Bioconductor package DESeq2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF] . Differential expression analysis was done between Multicilin-hGR alone versus Multicilin-hGR in the presence of E2f4ΔCT. Reads from ChIPseq were mapped to the Xenopus laevis genome release 7.1 using Bowtie2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] . Peaks were called and annotated according to their positions on known exons with HOMER [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities[END_REF] . Peak enrichments of E2F4 binding site in the promoters of centriole genes and cell cycle genes [START_REF] Ma | Multicilin drives centriole biogenesis via E2f proteins[END_REF] were estimated in presence or absence of Multicilin and a ratio of E2F4 binding (Multicilin vs no Multicilin) was calculated.

Promoter reporter studies

The human CDC20B promoter was cloned into the pGL3 Firefly Luciferase reporter vector (Promega) with SacI and NheI cloning sites. The promoter sequenced ranged from -1073 to +104 relative to the transcription start site. 37.5 ng of pGL3 plasmid were applied per well. pCMV6-Neg, pCMV6-E2F1 (NM_005225) and pCMV6-E2F4 (NM_001950) constructs were from Origene. 37.5 ng of each plasmid was applied per well. 25 ng per well of pRL-CMV (Promega) was applied in the transfection mix for transfection normalization (Renilla luciferase). HEK 293T cells were seeded at 20 000 cells per well on 96-well plates. The following day, cells were transfected with the indicated plasmids (100 ng of total DNA) with lipofectamine 3000 (Invitrogen). After 24 hours, cells were processed with the DualGlo kit (Promega) and luciferase activity was recorded on a plate reader.

Proximity ligation Assays

Fully differentiated HAECs were dissociated by incubation with 0.1% protease type XIV from Streptomyces griseus (Sigma-Aldrich) in HBSS (Hanks' balanced salts) for 4 hours at 4°C. Cells were gently detached from the Transwells® by pipetting and then transferred to a microtube. Cells were then cytocentrifuged at 300 rpm for 8 min onto SuperFrostPlus slides using a Shandon Cytospin 3 cytocentrifuge. Slides were fixed for 10 min in methanol at -20°C for Centrin2 and ZO1 assays, and for 10 min in 4% paraformaldehyde at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min for Acetylated-α-tubulin assays. Cells were blocked with 3% BSA in PBS for 30 min. The incubation with primary antibodies was carried out at room temperature for 2 h. Then, mouse and rabbit secondary antibodies from the Duolink® Red kit (Sigma-Aldrich) were applied and slides were processed according to manufacturer's instructions. Images were acquired using the Olympus Fv10i confocal imaging systems with 60X oil immersion objective and Alexa

Immunostaining on mouse ependyma

Dissected brains were subjected to 12 min fixation in 4% paraformaldehyde, 0.1% Triton X-100, blocked 1 hour in PBS, 3% BSA, incubated overnight with primary antibodies diluted in PBS, 3% BSA, and incubated 1 h with secondary antibodies at room temperature. Ependyma were dissected further and mounted with Mowiol before imaging using an SP8 confocal microscope (Leica microsystems) equipped with a 63x oil objective. The same protocol was used to prepare samples for super-resolution acquisition. Pictures were acquired with a TCS SP8 STED 3X microscope equipped with an HC PL APO 93X/1.30 GLYC motCORR TM objective (Leica microsystems). Pericentrin was revealed using Alexa 514 (detection 535-564nm, depletion 660nm), γ-tubulin was revealed using Alexa 568 (detection 582-667nm, depletion 775), and FOP was revealed using Alexa 488 (detection 498-531nm, depletion 592nm). Pictures were deconvoluted using Huygens software. Maximum intensity projection of 3 deconvoluted pictures is presented in Figure 4G. Primary antibodies: rabbit anti-CDC20B (1:500; Proteintech, 133376-1-AP), mouse IgG anti-PLK1 (1:500; Thermo Fisher, 33-1700), rabbit anti-Pericentrin (1:500, Abcam, ab4448), mouse IgG1 anti-FoxJ1 (1:1000; eBioscience,

Postnatal mouse brain electroporation

The detailed protocol for postnatal mouse brain electroporation established by Boutin and colleagues [START_REF] Boutin | Efficient in vivo electroporation of the postnatal rodent forebrain[END_REF] was used with minor modifications. Briefly, P1 pups were anesthetized by hypothermia. A glass micropipette was inserted into the lateral ventricle, and 2 μl of plasmid solution (concentration 3μg/μl) was injected by expiratory pressure using an aspirator tube assembly (Drummond). Successfully injected animals were subjected to five 95V electrical pulses (50 ms, separated by 950 ms intervals) using the CUY21 edit device (Nepagene, Chiba, Japan), and 10 mm tweezer electrodes (CUY650P10, Nepagene) coated with conductive gel (Signagel, Parker laboratories). Electroporated animals were reanimated in a 37°C incubator before returning to the mother.

Statistical analyses of mouse experiments

Analysis of CDC20B signal intensity in deuterosomes (graph in Fig. 3b). For each category, >25 cells from two different animals were analyzed. Deuterosome regions were delineated based on FOP staining and the intensity of CDC20B fluorescent immunostaining was recorded using ImageJ software, and expressed as arbitrary units. Unpaired t test vs immature: p=0,0005 (intermediate,***); p<0,0001 (Mature, ****).

Analysis of Cdc20b shRNAs efficiency (Fig. 4c): For each cell at the deuterosomal stage, the intensity of CDC20B fluorescent immunostaining was recorded using ImageJ software and expressed as arbitrary units. Data are mean ± sem. Two independent experiments were analyzed. A minimum of 35 cells per condition was analyzed. n= 3, 4, 5 and 5 animals for sh control, sh273, sh274 and sh277, respectively. Unpaired t test vs sh control: p<0.0001 (sh273, sh274 and sh277 ****).

Analysis of the number of FOXJ1 positive cells at 5dpe (Fig. 4d): Unpaired t test vs sh control: 0.3961 (sh273, ns), 0.1265 (sh274, ns), 0.3250 (sh277, ns).

Analysis of the number of cells with non-disengaged centrioles at 9dpe (Fig. 4g): 15-20 fields were analyzed per condition. n= 4, 4, 3, and 4 animals for sh control, sh273, sh274 and sh277, respectively, from 2 independent experiments. Unpaired t test vs sh control: p<0.0001 (sh273, sh274, sh277 ****).

Analysis of the number of centrioles per cell at 15dpe (Fig. 4j): >100 cells were analyzed per condition. n= 3, 3, 3, and 3 animals for sh control, sh273, sh274 and sh277, respectively, from 2 independent experiments. Unpaired t test vs sh control: p<0.0001 (sh273, sh274, sh277 ****).

Analysis of ependymal cell categories at 15dpe (Fig. 4k): Data are mean ± sem from three independent experiments. More than 500 cells were analyzed for each condition. n= 4, 4, 3, and 3 animals for sh control, sh273, sh274 and sh277, respectively. Unpaired t test vs sh control: p= 0.0004 (sh273, ***), 0.0001 (sh274, ****), 0.0038 (sh277, **).

Mouse tracheal epithelial cells (MTECs)

MTECs cell cultures were established from the tracheas of 12 weeks-old mice. After dissection, tracheas were placed in cold DMEM:F-12 medium (1:1) supplemented with 15 mM HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL gentamicin sulfate, and 2.5 µg/ml amphotericin B. Each trachea was processed under a binocular microscope to remove as much conjunctive tissue as possible with small forceps and was opened longitudinally with small dissecting scissors. Tracheas were then placed in supplemented DMEM:F-12 containing 0.15% protease XIV from Streptomyces Griseus. After overnight incubation at 4°C, FCS was added to a final concentration of 10%, and tracheal epithelial cells were detached by gentle agitation. Cells were centrifuged at 400g for 10 min and resuspended in supplemented DMEM:F-12 containing 10% FCS. Cells were plated on regular cell culture plates and maintained in a humidified atmosphere of 5% CO2 at 37°C for 4 hours to allow attachment of putative contaminating fibroblast. Medium containing cells in suspension was further centrifuged at 400g for 5 min and cells were resuspended in supplemented DMEM:F-12 containing BEGM Singlequots kit supplements and 5% FCS.

Immunostaining on HAECs and MTECs

Three days after setting-up the air-liquid interface, MTECs on Transwell membranes were pre-extracted with 0.5% Triton X-100 in PBS for 3 min, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. HAECs were treated 21 days after setting-up the air-liquid interface. They were fixed directly on Transwells® with 100% cold methanol for 10 min at -20°C (for CDC20B and Centrin2 co-staining, Supplementary Figure 4a,b) or with 4% paraformaldehyde in PBS for 15 min at room temperature (for CDC20B single staining, Supplementary Figure 4c). All cells were then permeabilized with 0.5% Triton X-100 in PBS for 5 min and blocked with 3% BSA in PBS for 30 min. The incubation with primary and secondary antibodies was carried out at room temperature for 2 h and 1 h, respectively. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Transwell® membranes were cut with a razor blade and mounted with ProLong Gold medium (Thermofisher). Primary antibodies: rabbit anti-CDC20B (1:500; Proteintech, 133376-1-AP), rabbit anti-DEUP1 (1:500; Proteintech, 24579-1-AP), anti-Centrin2 (Clone 20H5, 1:500; Millipore, 04-1624). Secondary antibodies: Alexa Fluor 488 goat anti-rabbit (1:1000; Thermo Fisher Scientific, A-11034), Alexa Fluor 647 goat anti-mouse (1:1000; Thermo Fisher Scientific, A-21235). For co-staining of CDC20B and DEUP1, CDC20B primary antibody was directly coupled to CF TM 633 with the Mix-n-Stain TM kit (Sigma-Aldrich) according to the manufacturer's instruction. Coupled primary antibody was applied after secondary antibodies had been extensively washed and after a 30 min blocking stage in 3% normal rabbit serum in PBS.

Western blot and immunofluorescence on transfected cells

Co-immunoprecipitation studies

Asynchronous HEK cells were rinsed on ice with chilled Ca++ and Mg++ free Dulbecco's PBS (DPBS, Invitrogen), harvested using a cell scraper and lysed on ice for 5 min in lysis buffer (0.025M Tris, 0.15M NaCl, 0.001M EDTA, 1% NP-40, 5% glycerol; pH 7.4) supplemented with EDTA and Halt™ Protease and Phosphatase Inhibitor Cocktail (Pierce, Thermofisher). Lysates were clarified (12000g, 4°C, 10 min) and the protein concentrations were determined using the Bradford assay (Bio-Rad). Immunoprecipitations were performed with the Pierce co-immunoprecipitation kit (Pierce, Thermofisher) according to the manufacturer's instructions. For each immunoprecipitation, 1-1.5 mg of total lysate was precleared on a control column, then incubated on columns coupled with 20 µg of anti-GFP or anti-c-myc-antibody (clone 9E10). Incubation was performed overnight at 4°C. Columns were washed and eluted with 50 µL elution buffer. Samples were denatured at 70°C for 10 min with Bolt™ LDS Sample Buffer and Bolt reducing agent, then separated on 4-12% gradient Bolt precast gels (Thermofisher), transferred onto nitrocellulose (Millipore), and subjected to immunoblot analysis using either anti-CDC20B (ProteinTech, 133376-1-AP) or anti-c-myc antibody (clone 9E10). In Figure 6, note that the high level of expression of myc-PLK1 (a) and myc-SPAG5 (b) drained out locally the ECL reagent at the peak of the protein.

The resulting double bands correspond in fact to unique ones. Human SPAG5, subcloned into pCMV6-MT, was from OriGene. Human DEUP1 and PLK1 were cloned into pCS2-MT vector (Addgene). Human CDC20B was cloned into pEGFP-C1, pEGFP-N1 (Clontech) for the GFP fusion protein and pIRES-EYFP (Addgene) for the untagged protein.

In-Gel digestion, NanoHPLC, and Q-exactiveplus analysis For mass spectrometry analysis, protein spots were manually excised from the gel and destained with 100 µL of H2O/ACN (1/1). After 10 min vortexing, liquid was discarded, and the procedure was repeated 2 times. They were rinsed with acetonitrile and dried under vacuum. Extracts were reduced with 50 μL of 10 mM dithiothreitol for 30 min at 56 °C, then alkylated with 15 μL of 55 mM iodoacetamide for 15 min at room temperature in the dark.

They were washed successively by: i) 100 µL of H2O/ACN (1/1) (2 times) and ii) 100 µL of acetonitrile. Gel pieces were rehydrated in 60 µL of 50 mM NH 4 HCO 3 containing 10 ng/µL of trypsin (modified porcine trypsin, sequence grade, Promega) incubated for one hour at 4°C. After the removal of trypsin, samples were incubated overnight at 37°C. Tryptic peptides were extracted with: i) 60 µL of 1% FA (formic acid) in water (10 min at RT), ii) 60 µL acetonitrile (10 min at RT). Extracts were pooled, concentrated under vacuum, resuspended in 15 µL of aqueous 0.1% formic acid for NanoHPLC separation.

Separation was carried out using a nanoHPLC (Ultimate 3000, Thermo Fisher Scientific).

After concentration on a µ-Precolumn Cartridge Acclaim PepMap 100 C 18 (i.d. 5 mm, 5 µm, 100 Å, Thermo Fisher Scientific) at a flow rate of 10 µL/min, using a solution of H 2 O/ACN/FA 98%/2%/0.1%, a second peptide separation was performed on a 75 µm i.d. x 250 mm (3 µm, 100 Å) Acclaim PepMap 100 C 18 column (Thermo Fisher Scientific) at a flow rate of 300 nL/min. Solvent systems were: (A) 100% water, 0.1% FA, (B) 100% acetonitrile, 0.08% FA. The following gradient was used t = 0min 6% B; t = 3 min 6%B; t = 119min, 45% B; t = 120 min, 90% B; t = 130 min 90% B (temperature at 35°C).

NanoHPLC was coupled via a nanoelectrospray ionization source to the Hybrid Quadrupole-Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific). MS spectra were acquired at a resolution of 70 000 (200 m/z) in a mass range of 300-2000 m/z with an AGC target 3e6 value of and a maximum injection time of 100ms. The 10 most intense precursor ions were selected and isolated with a window of 2m/z and fragmented by HCD (Higher energy C-Trap Dissociation) with normalized collision energy (NCE) of 27. MS/MS spectra were acquired in the ion trap with an AGC target 2e5 value, the resolution was set at 17 500 at 200 m/z combined with an injection time of 100 ms. Data were reprocessed using Proteome Discoverer 2.1 equipped with Sequest HT. Files were searched against the Swissprot Homo sapiens FASTA database (update of February 2016). A mass accuracy of ± 10 ppm was used to precursor ions and 0.02 Da for product ions. Enzyme specificity was fixed to trypsin, allowing at most two miscleavages. Because of the previous chemical modifications, carbamidomethylation of cysteines was set as a fixed modification and only oxydation of methionine was considered as a dynamic modification. Reverse decoy databases were included for all searches to estimate false discovery rates, and filtered using the Percolator algorithm at a 1% FDR.

Xenopus embryo injections, plasmids, RNAs, and morpholinos Eggs obtained from NASCO females were fertilized in vitro, dejellied and cultured using standard protocols [START_REF] Marchal | BMP inhibition initiates neural induction via FGF signaling and Zic genes[END_REF] . All injections were done at the 8-cell stage in one animal-ventral blastomere (presumptive epidermis), except for electron microscopy analysis for which both sides of the embryo was injected, and for RT-PCR analysis for which 2-cell embryos were injected.

cdc20b riboprobe was generated from Xenopus laevis cDNA. Full-length sequence was subcloned in pGEM™-T Easy Vector Systems (Promega). For sense probe it was linearized by SpeI and transcribed by T7. For antisense probe it was linearized by ApaI and transcribed by Sp6 RNA polymerase. Synthetic capped mRNAs were produced with the Ambion mMESSAGE mMACHINE Kit. pCS105/GFP-CAAX was linearized with AseI and mRNA was synthesized with Sp6 polymerase. pCS2-mRFP and pCS2-GFP-gpi were linearized with NotI and mRNA was synthesized with Sp6 polymerase. pCS-Centrin4-YFP (a gift from Reinhard Köster, Technische Universität Braunschweig, Germany) was linearized with Notl and mRNA was synthesized with Sp6 polymerase. pCS2-GFP-Deup1 and pCS2-Multicilin(MCI)-hGR were kindly provided by Chris Kintner; both plasmids were linearized with ApaI, and mRNAs were synthesized with Sp6 polymerase. Embryos injected with MCI-hGR mRNA were cultured in Dexamethasone 20μM in MBS 0,1X from st11 until fixation. pCS2-Separase wild-type and phosphomutant 2/4 (protease dead, PD) were provided by Marc Kirchner and Olaf Stemann, respectively; plasmids were linearized with NotI and mRNAs were synthesized with Sp6 polymerase. Venus-cdc20b, cdc20b-Venus and cdc20b were generated by GATEWAY™ Cloning Technology (GIBCO BRL) from Xenopus laevis cdc20b cDNA. cdc20b was also subcloned in pCS2-RFP to make RFP-cdc20b and cdc20b-RFP fusions. All cdc20b constructs were linearized with NotI and mRNAs were synthesized with Sp6 polymerase. Quantities of mRNA injected: 500pg for GFP-CAAX, RFP, GFP-gpi, Separase and Separase(PD); 25 to 500pg for GFP-Deup1; 40 to 500pg for MCI-hGR; 1ng for Venus-cdc20b, cdc20b-Venus, cdc20b, and cdc20b-RFP; 500pg to 1ng for RFP-cdc20b.

Two independent morpholino antisense oligonucleotides were designed against cdc20b (GeneTools, LLC). cdc20b ATG Mo: 5'-aaatcttctctaacttccagtccat-3', cdc20b Spl Mo 5'acacatggcacaacgtacccacatc-3'. 20ng of MOs was injected per blastomere or 10ng of each Mo for co-injection.

PCR and Quantitative RT-qPCR

Xenopus embryos were snap frozen at different stages and stored at -80°C. Total RNAs were purified with a Qiagen RNeasy kit (Qiagen). Primers were designed using Primer-BLAST Software. PCR reactions were carried out using GoTaq® G2 Flexi DNA Polymerase (Promega). RT reactions were carried out using iScript™ Reverse Transcription Supermix for RT-qPCR (BIO-RAD). qPCR reactions were carried out using SYBRGreen on a CFX Biorad qPCR cycler. To check cdc20b temporal expression by qPCR we directed primers to exons 9/10 junction (Forward: 5'-ggctatgaattggtgcccg-3') and exons 10/11 junction (Reverse: 5'gcagggagcagatctggg-3') to avoid amplification from genomic DNA. The relative expression of cdc20b was normalized to the expression of the housekeeping gene ornithine decarboxylase (ODC) for which primers were as follows: forward: 5'gccattgtgaagactctctccattc-3': reverse: 5'-ttcgggtgattccttgccac-3'.

To check the efficiency of Mo SPL, expected to cause retention of intron1 in the mature mRNA of cdc20b we directed forward (5'-cctcccgagagttagagga-3') and reverse (5'gcatgttgtactttctgctcca-3') primers in exon1 and exon2, respectively.

To check the expression of p53 in morphants by qPCR, primers were as follows: forward: 5'cgcagccgctatgagatgatt-3'; reverse: 5'-cacttgcggcacttaatggt-3'. The relative expression of p53 was normalized to Histone4 expression (H4) for which primers were as follows: forward: 5'ggtgatgccctggatgttgt-3'; reverse: 5'-ggcaaaggaggaaaaggactg-3'.

Immunostainining on Xenopus embryos

Embryos were fixed in 4% paraformaldehyde (PFA) overnight at 4°C and stored in 100% methanol at -20°C. Embryos were rehydrated in PBT and washed in MABX (Maleic Acid Buffer + Triton X100 0,1% v/v). Next, embryos were incubated in Blocking Reagent (Roche) 2% BR + 15% Serum + MABX with respective primary and secondary antibodies. The anti-Xenopus laevis CDC20B antibody was obtained by rabbit immunization with the peptide SPDQRRIFSAAANGT (amino acids 495-509) conjugated to keyhole limpet hemocyanin, followed by affinity purification (Eurogentec). For immunofluorescence, embryos were fixed at RT in PFA 4% in PBS, and incubated in the CDC20B antibody diluted 1/150 in BSA 3% in PBS. For all experiments secondary antibodies conjugated with Alexa were used. GFP-CAAX in Supplementary Figure 5g was revealed using a rabbit anti-GFP antibody together with a secondary antibody coupled to Alkaline Phosphatase (AP), which was revealed as follows: embryos incubated with the AP-conjugated antibody were washed twice in alkaline phosphatase buffer (PAB) (NaCl 0.1M, Tris HCl pH 9.5 0.1M, MgCl2 0.05M, Tween 0.1%),

In situ hybridization on Xenopus embryos

Whole-mount chromogenic in situ hybridization and whole-mount fluorescent in situ hybridization (FISH) was performed as detailed by Marchal and colleagues [START_REF] Marchal | BMP inhibition initiates neural induction via FGF signaling and Zic genes[END_REF] , and Castillo-Briceno and Kodjabachian [START_REF] Castillo-Briceno | Xenopus embryonic epidermis as a mucociliary cellular ecosystem to assess the effect of sex hormones in a non-reproductive context[END_REF] , respectively. For single staining, all RNA probes were labeled with digoxigenin. For FISH on section, embryos were fixed in 4% paraformaldehyde (PFA), stored in methanol for at least 4 h at -20°C, then rehydrated in PBT (PBS + Tween 0.1% v/v), treated with triethanolamine and acetic anhydride, incubated in increasing sucrose concentrations and finally embedded with OCT (VWR Chemicals). 12μm-thick cryosections were made. Double FISH on sections was an adaptation of the whole-mount FISH method. 80ng of cdc20b digoxigenin-labeled sense and antisense riboprobes and 40ng of antisense αtubulin fluorescein-labeled riboprobe [START_REF] Deblandre | A two-step mechanism generates the spacing pattern of the ciliated cells in the skin of Xenopus embryos[END_REF] were used for hybridization. All probes were generated from linearized plasmids using RNA-labeling mix (Roche). FISH was carried out using Tyramide Signal Amplification -TSA TM Plus Cyanine 3/Fluorescein System (PerkinElmer). Antibodies: Anti-DigAP (Roche, 11266026, 1:5000), Anti-DigPOD (Roche, 11207733910, 1:500), Anti-FluoPOD (Roche, 11426346910, 1:500).

Microscopy

Confocal: Flat-mounted epidermal explants were examined with a Zeiss LSM 780 confocal microscope. Four-colors confocal z-series images were acquired using sequential laser excitation, converted into single plane projection and analyzed using ImageJ software.

Scanning Electron Microscopy (SEM): stage 37 Xenopus embryos were fixed in 3% glutaraldehyde in 0.1 M phosphatase buffer pH 7.4 (19 ml monosodium phosphate 0.2 M and 81 ml disodium phosphate 0.2 M) made with filtered (0.22 μm) bi-distilled water, during 4 h with vigorous agitation, then washed with phosphatase buffer and filtered bi-distilled water, to be successively dehydrated in ethanol at 25, 50 and 70% for 30 minutes each; then, embryos were stored in fresh ethanol 70% at 4°C for 1-2 days before further processing.

Embryos in 70% ethanol were further dehydrated with vigorous agitation in ethanol once at 90% and twice at 100% for 30 minutes each; they were subsequently subjected to CO2 critical point drying (CPD030, Balzers) at 31°C and 73 atm. Finally, samples were sputter-coated with gold (vacuum 1×10-12 Torr, beam energy 3-4 keV) for immediate SEM digital imaging (FEI TENEO) of the skin epidermis. Transmission Electron Microscopy (TEM): stage 25 Xenopus embryos were fixed overnight at 4°C in 2.5% glutaraldehyde, 2% paraformaldehyde, 0.1% tannic acid in a sodium cacodylate buffer 0.05 M pH7.3. Next, embryos were washed 3x15 min in cacodylate 0.05 M at 4°C. Post-fixation was done in 1% osmium buffer for 2 h. Next, embryos were washed in buffer for 15 min. Then, embryos were washed in water and dehydrated conventionally with alcohol, followed by a step in 70% alcohol containing 2% uranyl during 1 to 2 h at RT, or overnight at 4°C. Following 3 incubations in 100% alcohol, completed with 3 washes of acetone, embryos were included in classical epon resin, which was polymerized in oven at 60°C for 48 h. Sections of 80 nm were made and analyzed into an FMI TECNAI microscope with acceleration of 200kV.

Statistical analysis of Xenopus experiments

To quantify the effect of our different experiments, we applied One-way ANOVA analysis and Bonferroni's multiple comparisons test (t test). ***P<0.05; ns = not significant. Statistical analyses were done using GraphPad Prism 6.

Figures and legends

  647 detection parameters. Animals All experiments were performed following the Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes. Experiments on Xenopus laevis and mouse were approved by the 'Direction départementale de la Protection des Populations, Pôle Alimentation, Santé Animale, Environnement, des Bouches du Rhône' (agreement number F 13 055 21). Mouse experiments were approved by the French ethical committee n°14 (permission number: 62-12112012). Timed pregnant CD1 mice were used (Charles Rivers, Lyon, France).

  14-9965), rabbit anti-Deup1 (1:1000; kindly provided by Dr Xueliang Zhu), rabbit anti-Deup1 (1:250; Proteintech, 24579-1-AP), mIgG1 anti-γ-Tubulin (clone GTU88) (1:250; Abcam, Ab 11316), rabbit anti-ZO1 (1:600; Thermo Fisher Scientific, 61-7300), rabbit anti-Spag5 (1:500; Proteintech, 14726-1-AP), mouse IgG1 anti-ZO1 (1:600; Invitrogen, 33-9100), mouse IgG2b anti-FGFR1OP (FOP) (1:2000; Abnova, H00011116-M01), mouse IgG1 anti-α-tubulin (1:500; Sigma-Aldrich, T9026). Secondary antibodies: Alexa Fluor 488 goat anti-rabbit (1:800; Thermo Fisher Scientific, A-11034), Alexa Fluor 647 goat anti-rabbit (1:800; Thermo Fisher Scientific, A-21244), Alexa Fluor 514 goat anti-rabbit (1:800; Thermo Fisher Scientific, A-31558), Alexa Fluor 488 goat anti-mouse IgG2b (1:800; Thermo Fisher Scientific, A-21141), Alexa Fluor 568 goat anti-mouse IgG2b (1:800; Thermo Fisher Scientific, A-21144), Alexa Fluor 488 goat anti-mouse IgG2a (1:800; Thermo Fisher Scientific, A-21131), Alexa Fluor 568 goat anti-mouse IgG1 (1:800; Thermo Fisher Scientific, A-21134), Alexa Fluor 647 goat anti-mouse IgG1 (1:800; Thermo Fisher Scientific, A-21240). Mouse constructs Expression constructs containing shRNA targeting specific sequences in the CDC20B coding sequence under the control of the U6 promoter were obtained from Sigma-Aldrich (ref. TRCN0000088273 (sh273), TRCN0000088274 (sh274), TRCN0000088277 (sh277)). PCX-mcs2-GFP vector (Control GFP) kindly provided by Xavier Morin (ENS, Paris, France), and U6 vector containing a validated shRNA targeting a specific sequence in the NeuroD1 coding sequence 52 (Control sh, ref. TRCN0000081777, Sigma-Aldrich) were used as controls for electroporation experiments.

  Cells were plated on rat tail collagen I-coated Transwell®. Typically, 5 tracheas resulted in 12 Transwells®. Medium was changed every other day. Air-liquid interface culture was conducted once transepithelial electrical resistance had reached a minimum of 1000 ohm/cm2 (measured with EVOM2, World Precision Instruments). Air-liquid interface culture was obtained by removing medium at the apical side of the Transwell®, and by replacing medium at the basal side with supplemented DMEM:F-12 containing 2% Ultroser-G TM (Pall Corporation). 10 µM DAPT (N-[N-(3,5difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) (Sigma) was added one day after setting-up the air-liquid interface.

  Cos1 or Hela cells cells were grown in DMEM supplemented with 10% heat inactivated FCS and transfected with Fugene HD (Roche Applied Science) according to manufacturer's protocol. Transfected or control cells were washed in PBS and lysed in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1mM EDTA, containing 1% NP-40 and 0.25% sodium deoxycholate (modified RIPA) plus a Complete Protease Inhibitor Cocktail (Roche Applied Science) on ice. Cell extracts separated on polyacrylamide gels were transfered onto Optitran membrane (Whatman) followed by incubation with rabbit anti-mouse CDC20B (1:500, Proteintech, 24579-1-AP) or homemade rabbit anti-Xenopus Cdc20b (1:300) antibody and horseradish peroxidase conjugated secondary antibody (Jackson Immunoresearch Laboratories, 711-035-152 and 715-035-150). Signal obtained from enhanced chemiluminescence (Western Lightning ECL Pro, Perkin Elmer) was detected with MyECL Imager (Thermo Fisher Scientific). For immunofluorescence staining, transfected cells were grown on glass coverslips and fixed for 6 min in methanol at -20°C. Cells were washed in PBS, blocked in PBS, 3% BSA and stained with rabbit anti-Xenopus Cdc20b (1:300) or rabbit anti-CFTR (1:200, Santa-Cruz Biotechnology, 10747) as a negative control, in blocking buffer. After washings in PBS 0.1% Tween-20, cells were incubated with Alexa fluor 488 donkey anti-rabbit antibody (Thermo Fisher Scientific, R37118), washed, and DNA was stained with 250 ng/ml DAPI. Coverslip were then rinsed and mounted in Prolong Gold antifade reagent (ThermoFisher Scientific) and confocal images were acquired by capturing Z-series with 0.3 μm step size on a Zeiss LSM 510 laser scanning confocal microscope.

  10 minutes each. Next, embryos were incubated in PAB with INT/BCIP substrate (Roche, REF:11681460001) until appropriate staining. Finally embryos were washed twice in MABX and fixed in MEMFA 30min at RT. To mark cortical actin in MCCs, embryos were fixed in 4% paraformaldehyde (PFA) in PBT (PBS + 0,1% Tween v/v) for 1h at room temperature (RT), washed 3x10 min in PBT at RT, then stained with phalloidin-Alexa Fluor 555 (Invitrogen, 1:40 in PBT) for 4 h at RT, and washed 3x10 min in PBT at RT. Primary antibodies: mouse anti-Acetylated-α-Tubulin (Clone 6-11B-1, Sigma-Aldrich, T7451, 1:1000), rabbit anti-γ-Tubulin (Abcam, Ab 16504, 1:500), mouse anti-γ-Tubulin (Clone GTU88, Ab 11316, Abcam, 1:500), Chicken anti-GFP (AVES, GFP-1020, 1:1000), rabbit anti-GFP (Torrey Pines Biolabs, TP401, 1:500), mouse anti-Centrin (Clone 20H5, EMD Millipore, 04-1624, 1:500). Secondary antibodies: donkey anti-rabbit-AP (Jackson ImmunoResearch, 711055152, 1:1000), Alexa Fluor 647 goat anti-mouse IgG2a (1:500; Thermo Fisher Scientific, A-21241), Alexa Fluor 488 goat anti-chicken (1:500; Thermo Fisher Scientific, A-11039), Alexa Fluor 568 goat anti-rabbit (1:500; Thermo Fisher Scientific, A-11011).

Fig. 5o

  Fig. 5o and Fig. S6k: 10 cells per condition were analyzed and the total number of Centrin-YFP or γ-tubulin positive spots per injected cell was counted.

Fig.

  Fig. 7g: 5 fields (20x zoom) per condition were analyzed, and the total number of properly ciliated MCCs based on acetylated α-tubulin staining among GFP positive cells per field was counted. Each field corresponded to a different embryo.

Fig. 5s :

 5s Fig. 5s : 160-200 cells per condition were analyzed. n= 6, 8, and 10 embryos from 3 independent experiments for control, Mo ATG and Mo Spl, respectively. Unpaired t test vs control: p=0,0037 (Mo ATG **) and 0,0004 (Mo Spl ***).

Figure 1 :

 1 Figure 1: Single-cell RNA-seq analysis reveals MCC transcriptome at deuterosome-

Figure 2 :

 2 Figure 2: Composition and organization of vertebrate deuterosomes (a-b) Maturing mouse ependymal MCCs were immunostained as indicated, pictures were taken with confocal (a) or STED (b) microscope. (a) DEUP1 stains the deuterosome core (ring) and a close fibrous area that defines the perideuterosomal region. The centriolar marker FOP reveals procentrioles arranged in a circle around the deuterosome. Pericentrin (PCNT) is enriched in the perideuterosomal region. γ-Tubulin (γ-TUB) stains the core as well as the periphery of the deuterosome. (b) STED pictures showing the organization of FOP, PCNT and γ-TUB around deuterosomes. Individual centrioles identified by FOP staining are pointed out with arrowheads. The diagram was drawn from the adjacent FOP photograph to help reveal the regular concentric organization of nascent centrioles in a typical deuterosomal figure. (c) Xenopus embryos were immunostained for γ-Tubulin (γ-Tub) and Centrin and high-magnification pictures of immature epidermal MCCs were taken. In these cells, Centrinpositive procentrioles grow around γ-Tubulin positive structures. (d) Xenopus embryos were

Figure 3 :

 3 Figure 3: CDC20B associates to vertebrate deuterosomes. (a) Double immunofluorescence was performed on mouse tracheal MCCs after 3 days of culture in air-liquid interface. Low magnification confocal panels show coincident CDC20B and DEUP1 staining in several individual MCCs. High magnification on a single MCC reveals the prominent association of CDC20B to large deuterosomes marked by DEUP1 (arrowheads). Note that some smaller deuterosomes do not contain CDC20B (arrows). (b) Mouse ependymal MCCs were immunostained as indicated, and high magnification confocal pictures of cells with immature and mature deuterosomal figures were taken. In these cells, centrioles revealed by FOP form a ring around deuterosomes. CDC20B staining forms a ring inside the ring of FOP-positive procentrioles indicating that CDC20B is tightly associated to deuterosomes. Note that the CDC20B signal associated to deuterosome increased with their maturation (high-magnification pictures of >25 cells per category from two different animals were quantified in the graph; horizontal bars are mean values and vertical lines are standard deviations). Unpaired t test vs immature: p=0,0005 (intermediate, ***); p<0,0001 (mature, ****). (c) Xenopus embryos were injected with GFP-Deup1 mRNA and immunostained at neurula st18 as indicated. Scale bars: 5µm (a, b, large view), 1.5µm (a, high magnification), 500nm (b, high magnification), 10µm (c).

Figure 4 :

 4 Figure 4: CDC20B knockdown impairs multiciliogenesis in mouse ependymal MCCs.

Figure 5 :

 5 Figure 5: cdc20b knockdown impairs multiciliogenesis in Xenopus epidermal MCCs. (a-c) 8-cell embryos were injected in presumptive epidermis with GFP-CAAX mRNA and cdc20b morpholinos, as indicated. Embryos at tailbud st25 were processed for fluorescent staining against GFP (injection tracer, green) and Acetylated-α-Tubulin (Ac-α-Tub, cilia, white). White dotted lines indicate the position of orthogonal projections shown in bottom panels. Note that cdc20b morphant MCCs display cytoplasmic filaments but do not grow cilia (white arrowheads). (d-f) Scanning Electron Microscopy (SEM) of control (d) and cdc20b morphant (e,f) embryos at tadpole st31. Yellow arrowheads point at normal (d) and defective MCCs (e,f). (g-i) Transmission Electron Microscopy (TEM) of control (g) and cdc20b morphant (h,i) embryos at tailbud st25. Yellow arrowheads point at normally docked basal bodies supporting cilia (g) and undocked centrioles unable to support cilia (h,i). (j-n) 8-cell embryos were injected in presumptive epidermis with centrin-YFP mRNA, cdc20b morpholinos, and cdc20b mRNA, as indicated. Centrin-YFP fluorescence was observed directly to reveal centrioles (yellow). Nuclei were revealed by DAPI staining in blue. White dotted lines indicate the position of orthogonal projections shown in bottom panels. Yellow arrowheads point at undocked centrioles. (o) Bar graph showing the number of BBs per MCC, and standard error mean, as counted by Centrin-YFP dots. One way ANOVA and Bonferroni's Multiple Comparisons Test on two experiments, *** = p<0.0001. cdc20b knockdown significantly reduced the number of BBs per cell, and this defect could be corrected by cdc20b co-injection with Mo Spl. (p-u) Embryos were injected with Multicilin-hGR and GFP-Deup1 mRNAs, treated with dexamethasone at gastrula st11 to induce Multicilin activity, and immunostained at neurula st23 against Acetylated-α-tubulin (cilia,

Figure 6 :

 6 Figure 6: CDC20B interacts with PLK1, SPAG5 and DEUP1 (a, c, e) Co-immunoprecipitations of PLK1, SPAG5 and DEUP1 with CDC20B were tested after transfections of different constructs in HEK cells, indicated at the top of each panel. Proteins (left legend) were revealed by immunoblotting. (b, d) Maturing mouse ependyma were immunostained for the indicated proteins, and pictures were taken with a confocal microscope. PLK1 and SPAG5 are expressed in maturing MCCs. High magnifications show that PLK1 is enriched in the perideuterosomal region, while SPAG5 is enriched in the deuterosome core. Scale bars: 5µm (d, e, large view), 1µm (d, e, high magnification).

Figure 7 :

 7 Figure 7: Separase overexpression rescues multiciliogenesis in absence of Cdc20b. (a-f) 8-cell Xenopus embryos were injected in the presumptive epidermis with GFP-gpi mRNA, cdc20b morpholinos, and human Separase mRNA, as indicated. Embryos were fixed at tailbud st25 and immunostained against GFP (injection tracer, green), Acetylated-α-Tubulin (cilia, white) and ɣ-Tubulin (BBs, red). White dotted lines indicate the position of orthogonal projections shown in bottom panels. Red arrowheads point undocked BBs. Left inset in (e) shows zoom on clustered centrioles. (g) Bar graph showing the number of properly ciliated MCCs among injected cells, per field of observation. Bars are standard error

Table 2 : CDC20, but not CDC20B, interacts with APC/C.

 2

		G1/S		S		G2/M		M		M/G1
	Symbol	Ensembl	Symbol	Ensembl	Symbol	Ensembl	Symbol	Ensembl	Symbol	Ensembl
	ORC1	ENSG00000085840	ANKRD18A	ENSG00000273170	IQGAP3	ENSG00000183856	CKS1B	ENSG00000268942	TROAP	ENSG00000135451
	ZNF367	ENSG00000165244	REEP1	ENSG00000068615	TRAIP	ENSG00000183763	DEPDC1B	ENSG00000035499	CDKN3	ENSG00000100526
	ADAMTS1	ENSG00000154734	DEPDC7	ENSG00000121690	CDC25C	ENSG00000158402	SHCBP1	ENSG00000171241	PRC1	ENSG00000198901
	CCNE2	ENSG00000175305	CDC7	ENSG00000097046	NEIL3	ENSG00000109674	FAM64A	ENSG00000129195	HSD17B11	ENSG00000198189
	CDC25A	ENSG00000164045	DNA2	ENSG00000138346	PIF1	ENSG00000140451	FYN	ENSG00000010810	BTBD3	ENSG00000132640
	RECQL4	ENSG00000160957	EXO1	ENSG00000174371	KIFC1	ENSG00000237649	KIF2C	ENSG00000142945	SLC39A10	ENSG00000196950
	DTL	ENSG00000143476	KDELC1	ENSG00000134901	HJURP	ENSG00000123485	SPAG5	ENSG00000076382	GTF3C4	ENSG00000125484
	CDC6	ENSG00000094804	ANKRD18A	ENSG00000180071	NCAPH	ENSG00000121152	CIT	ENSG00000122966	WWC1	ENSG00000113645
	CCNE1	ENSG00000105173	BRIP1	ENSG00000136492	KIF23	ENSG00000137807	CENPA	ENSG00000115163	ELP3	ENSG00000134014
	MCM2	ENSG00000073111	PKMYT1	ENSG00000127564	SKA3	ENSG00000165480	DIAPH3	ENSG00000139734	FOXK2	ENSG00000141568
	GINS3	ENSG00000181938	CDC45	ENSG00000093009	KIAA1524	ENSG00000163507	CADM1	ENSG00000182985	OPN3	ENSG00000054277
	CHAF1B	ENSG00000159259	C11orf82	ENSG00000165490	NDC80	ENSG00000080986	KIF14	ENSG00000118193	KIAA0586	ENSG00000100578
	WDR76	ENSG00000092470	BLM	ENSG00000197299	CCNF	ENSG00000162063	PLK1	ENSG00000166851	ANTXR1	ENSG00000169604
	MCM6	ENSG00000076003	RAD51	ENSG00000051180	CDCA8	ENSG00000134690	MDC1	ENSG00000137337	CEP70	ENSG00000114107
	CLSPN	ENSG00000092853	CCDC150	ENSG00000144395	PSRC1	ENSG00000134222	DEPDC1	ENSG00000024526	HMGCR	ENSG00000113161
	CDCA7	ENSG00000144354	CDCA5	ENSG00000146670	FANCD2	ENSG00000144554	BUB1	ENSG00000169679	TULP4	ENSG00000130338
	OSBPL6	ENSG00000079156	CPNE8	ENSG00000139117	ESPL1	ENSG00000135476	DLGAP5	ENSG00000126787	ZNF281	ENSG00000162702
	RAB23	ENSG00000112210	MCM8	ENSG00000125885	CDR2	ENSG00000140743	NUF2	ENSG00000143228	CDK7	ENSG00000134058
	PLCXD1	ENSG00000182378	ESCO2	ENSG00000171320	AURKB	ENSG00000178999	CEP55	ENSG00000138180	LYAR	ENSG00000145220
	SKP2	ENSG00000145604	GOLGA8B	ENSG00000215252	BORA	ENSG00000136122	GTSE1	ENSG00000075218	PPP6R3	ENSG00000110075
	MDM1	ENSG00000111554	ASF1B	ENSG00000105011	LMNB1	ENSG00000113368	HMMR	ENSG00000072571	DCP1A	ENSG00000162290
	GINS2	ENSG00000131153	FANCA	ENSG00000187741	TRIM59	ENSG00000213186	FOXM1	ENSG00000111206	FAM189B	ENSG00000160767
	E2F1	ENSG00000101412	INTS7	ENSG00000143493	CHEK2	ENSG00000183765	E2F5	ENSG00000133740	AGPAT3	ENSG00000160216
	MCM5	ENSG00000100297	POLA1	ENSG00000101868	MND1	ENSG00000121211	PRR11	ENSG00000068489	PSEN1	ENSG00000080815
	SNHG10	ENSG00000247092	FANCI	ENSG00000140525	CDCA2	ENSG00000184661	NEK2	ENSG00000117650	NUP37	ENSG00000075188
	HSF2	ENSG00000025156	RRM2	ENSG00000171848	CKAP2L	ENSG00000169607	TACC3	ENSG00000013810	MSL1	ENSG00000188895
	UBR7	ENSG00000012963	TTLL7	ENSG00000137941	STIL	ENSG00000123473	CENPE	ENSG00000138778	AGFG1	ENSG00000173744
	RMI2	ENSG00000175643	RAD51AP1	ENSG00000111247	POLQ	ENSG00000051341	CCNB2	ENSG00000157456	SNUPN	ENSG00000169371
	NUP43	ENSG00000120253	KAT2B	ENSG00000114166	MELK	ENSG00000165304	CDC20	ENSG00000117399	STAG1	ENSG00000118007
	ACD	ENSG00000102977	CHML	ENSG00000203668	CENPL	ENSG00000120334	BIRC5	ENSG00000089685	LRIF1	ENSG00000121931
	ZMYND19	ENSG00000165724	BRCA1	ENSG00000012048	LIX1L	ENSG00000152022	CCDC88A	ENSG00000115355	PAK1IP1	ENSG00000111845
	MSH2	ENSG00000095002	ABHD10	ENSG00000144827	KIF11	ENSG00000138160	POC1A	ENSG00000164087	NCOA3	ENSG00000124151
	CDCA7L	ENSG00000164649	TYMS	ENSG00000176890	C14orf80	ENSG00000185347	MKI67	ENSG00000148773	PTTG1	ENSG00000164611
	KIAA1586	ENSG00000168116	PRIM1	ENSG00000198056	UBE2C	ENSG00000175063	MKI67	ENSG00000148773	CTR9	ENSG00000198730
	PMS1	ENSG00000064933	TTC31	ENSG00000115282	NCAPD3	ENSG00000151503	HSPA13	ENSG00000155304	DKC1	ENSG00000130826
	UNG	ENSG00000076248	E2F8	ENSG00000129173	HAUS8	ENSG00000131351	CDC25B	ENSG00000101224	FOPNL	ENSG00000133393
	KIAA1147	ENSG00000257093	CENPQ	ENSG00000031691	FAM83D	ENSG00000101447	TPX2	ENSG00000088325	VCL	ENSG00000035403
	POLD3	ENSG00000077514	PHTF1	ENSG00000116793	CDK1	ENSG00000170312	AURKA	ENSG00000087586	MRPS2	ENSG00000122140
	ANKRD10	ENSG00000088448	MASTL	ENSG00000120539	MAD2L1	ENSG00000164109	ANKRD40	ENSG00000154945	WIPF2	ENSG00000171475
	CHAF1A	ENSG00000167670	OSGIN2	ENSG00000164823	GABPB1	ENSG00000104064	CENPF	ENSG00000117724
	BARD1	ENSG00000138376	GOLGA8A	ENSG00000175265	SAP30	ENSG00000164105	CNTROB	ENSG00000170037
	INTS8	ENSG00000164941	PHTF2	ENSG00000006576	CFD	ENSG00000197766	NCAPD2	ENSG00000010292
	APEX2	ENSG00000169188	BBS2	ENSG00000125124	TTF2	ENSG00000116830	SGOL2	ENSG00000163535
	ACYP1	ENSG00000119640	BMI1	ENSG00000168283	MID1	ENSG00000101871	SRF	ENSG00000112658
	MRI1	ENSG00000037757	FEN1	ENSG00000168496	GAS1	ENSG00000180447	DZIP3	ENSG00000198919
	INSR	ENSG00000171105	RMI1	ENSG00000178966	TUBA1A	ENSG00000167552	ECT2	ENSG00000114346
	TOPBP1	ENSG00000163781	NSUN3	ENSG00000178694	ZNF587	ENSG00000198466	ORAOV1	ENSG00000149716
	FAM105B	ENSG00000154124	KAT2A	ENSG00000108773	TUBD1	ENSG00000108423	NUP35	ENSG00000163002
	NPAT	ENSG00000149308	CENPM	ENSG00000100162	FAN1	ENSG00000198690	PTPN9	ENSG00000169410
	PCDH7	ENSG00000169851	ZWINT	ENSG00000122952	CDKN2C	ENSG00000123080	HS2ST1	ENSG00000153936
	GMNN	ENSG00000112312	ORC3	ENSG00000135336	TUBB2A	ENSG00000137267	RCAN1	ENSG00000159200
	RNPC3	ENSG00000185946	KIAA1598	ENSG00000187164	TNPO2	ENSG00000105576	SS18	ENSG00000141380
	RNF113A	ENSG00000125352	BIVM	ENSG00000134897	ZNHIT2	ENSG00000174276	HCFC1	ENSG00000172534
	FAM122A	ENSG00000187866	DNAJB4	ENSG00000162616	TRMT2A	ENSG00000099899	NUP98	ENSG00000110713
	CAPN7	ENSG00000131375	CCDC84	ENSG00000186166	PKNOX1	ENSG00000160199	POM121	ENSG00000196313
	TIPIN	ENSG00000075131	DCAF16	ENSG00000163257	ENTPD5	ENSG00000187097	TOMM34	ENSG00000025772
	C14orf142	ENSG00000170270	NUP160	ENSG00000030066	KDM4A	ENSG00000066135	CKAP5	ENSG00000175216
	LNPEP	ENSG00000113441	RFC2	ENSG00000049541	STK17B	ENSG00000081320	GRK6	ENSG00000198055
	USP53	ENSG00000145390	CDKN2AIP	ENSG00000168564	KLF6	ENSG00000067082	SEPHS1	ENSG00000086475
	PANK2	ENSG00000125779	UBE2T	ENSG00000077152	KATNA1	ENSG00000186625	QRICH1	ENSG00000198218
	VPS72	ENSG00000163159	DHFR	ENSG00000228716	H2AFX	ENSG00000188486	AHI1	ENSG00000135541
	DIS3	ENSG00000083520	PTAR1	ENSG00000188647	BRD8	ENSG00000112983	CNOT10	ENSG00000182973
			RAD18	ENSG00000070950	RCCD1	ENSG00000166965	KLF9	ENSG00000119138
			OGT	ENSG00000147162	CDKN1B	ENSG00000111276	SETD8	ENSG00000183955
			EZH2	ENSG00000106462	UACA	ENSG00000137831	ATF7IP	ENSG00000171681
			C5orf42	ENSG00000197603	KCTD9	ENSG00000104756	RAD51C	ENSG00000108384
			LYRM7	ENSG00000186687	ATL2	ENSG00000119787	CDC42EP1	ENSG00000128283
			CCDC14	ENSG00000175455	KPNA2	ENSG00000182481	HPS4	ENSG00000100099
			NAB1	ENSG00000138386	HRSP12	ENSG00000132541	GOT1	ENSG00000120053
			SP1	ENSG00000185591	VTA1	ENSG00000009844	MZT1	ENSG00000204899
			RPA2	ENSG00000117748	HMGB2	ENSG00000164104	RRP1	ENSG00000160214
			RBBP8	ENSG00000101773	C2orf69	ENSG00000178074	AKIRIN2	ENSG00000135334
			RRM1	ENSG00000167325	FADD	ENSG00000168040	CDC27	ENSG00000004897
			FAM178A	ENSG00000119906	HIPK2	ENSG00000064393	SMARCD1	ENSG00000066117
			SAP30BP	ENSG00000161526	KIF22	ENSG00000079616	BIRC2	ENSG00000110330
			NT5DC1	ENSG00000178425	MGAT2	ENSG00000168282
			CERS6	ENSG00000172292	NR3C1	ENSG00000113580
			ZBED5	ENSG00000236287	DHX8	ENSG00000067596
			MAP3K2	ENSG00000169967	NMB	ENSG00000197696
					TFAP2A	ENSG00000137203
					HINT3	ENSG00000111911
					CDC16	ENSG00000130177
					NUMA1	ENSG00000137497
					ARMC1	ENSG00000104442
					STAT1	ENSG00000115415
					CCDC107	ENSG00000159884
					TMPO	ENSG00000120802

Protein ranking from highest to lowest Sum PEP score CDC20, but not CDC20B, interacts with APC/C CDC20 IP -no overexpression CDC20 IP + CDC20 overexpression CDC20B IP -no overexpression CDC20B IP + CDC20B overexpression

Acknowledgements

We are grateful to Chris Kintner, Marc Kirschner, Olaf Stemmann, Reinhard Köster, Xavier Morin and Xueliang Zhu for reagents. Imaging in IBDM was performed on PiCSL-FBI core facility, supported by the French National Research Agency through the program "Investments for the Future" (France-BioImaging, ANR-10-INBS-04). Sequencing at UCAGenomiX (IPMC), a partner of the National Infrastructure France Génomique, was supported by Commissariat aux Grands Investissements (ANR-10-INBS-09-03, ANR-10-INBS-09-02) and Canceropôle PACA. The authors thank Florian Roguet for Xenopus care, and Nathalie Garin from Leica Microsystems GmbH for technical advice on STED microscopy. We are grateful to Rainer Waldmann, Kévin Lebrigand, Virginie Magnone and Nicolas Nottet for fruitful discussions on single cell RNA sequencing, and Delphine Debayle for help with mass spectrometry experiments. We thank Julien Royet and Harold Cremer for insightful comments on the manuscript. This project was funded by grants from ANR (ANR-11-BSV2-021-02, ANR-13-BSV4-0013, ANR-15-CE13-0003), FRM (DEQ20141231765, DEQ20130326464, DEQ20180339158), Fondation ARC (PJA 20161204865, PJA 20161204542), the labex Signalife (ANR-11-LABX-0028-01), the association Vaincre la Mucoviscidose (RF20140501158, RF20120600738, RF20150501288), and the Chan Zuckerberg Initiative (Silicon Valley Fundation, 2017-175159 -5022). OM, CB and DRR were supported by fellowships from Ligue Nationale contre le Cancer (OM and CB), and Fondation ARC (DRR).

Data availability statement scRNAseq data was submitted to the GEO portal under series reference GSE103518. All other relevant data are available from the authors.

buffer (Invitrogen) then adapter-ligated, reverse transcribed and amplified (6 cycles) with the reagents from the NEBNext Small RNA Library Prep Set for SOLiD. Small RNA-seq was performed from 500 ng RNA with the NEBNext Small RNA Library Prep Set for SOLiD (12 PCR cycles) according to manufacturer's instructions. Both types of amplified libraries were purified on Purelink PCR micro kit (Invitrogen), then subjected to additional PCR rounds (8 cycles for RNA-seq and 4 cycles for small RNA-seq) with primers from the 5500 W Conversion Primers Kit (Life Technologies). After Agencourt® AMPure® XP beads purification (Beckman Coulter), libraries were size-selected from 150 nt to 250 nt (for RNAseq) and 105 nt to 130 nt (for small RNA-seq) with the LabChip XT DNA 300 Assay Kit (Caliper Lifesciences), and finally quantified with the Bioanalyzer High Sensitivity DNA Kit (Agilent). Libraries were sequenced on SOLiD 5500XL (Life Technologies) with single-end 50b reads. SOLiD data were analyzed with lifescope v2.5.1, using the small RNA pipeline for miRNA libraries and whole transcriptome pipeline for RNA-seq libraries with default parameters. Annotation files used for production of raw count tables correspond to Refseq Gene model v20130707 for mRNAs and miRBase v18 for small RNAs. Data generated from RNA sequencing were then analyzed with Bioconductor (http://www.bioconductor.org) package DESeq and size-factor normalization was applied to the count tables. Heatmaps were generated with GenePattern using the "Hierarchical Clustering" Module, applying median row centering and Euclidian distance.

Re-analysis of Xenopus E2F4 Chip-seq and RNA-seq RNA-seq (samples GSM1434783 to GSM1434788) and ChIP-seq (samples GSM1434789 to GSM1434792) data were downloaded from GSE59309. Reads from RNA-seq were aligned to the Xenopus laevis genome release 7.1 using TopHat2 47 with default parameters. Quantification of genes was then performed using HTSeq-count [START_REF] Anders | HTSeq--a Python framework to work with highthroughput sequencing data[END_REF]

Competing financial interests

The authors declare no competing interests.

Supplementary information

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

Revinski et al.

Supplementary Figure 1: Single cell RNA-seq analysis of HAECs. tSNE plots for a selection of genes expressed at the single-cell level, in deuterosomal-stage differentiating HAECs. Genes were grouped into functional categories. Immunofluorescence against GFP (injection tracer, green), and Acetylated-a-Tubulin (cilia, white) was performed at tailbud st25. Cells in dotted squares were blown up for better visualization. Note that multiciliogenesis was rescued in cdc20b morphant MCCs by wild-type (p,q) but not protease-dead Separase (r,s). (t-z) 4-cell embryos were injected in one ventral blastomere (presumptive epidermis) with MCI-hGR and GFP-GPI mRNAs, in the presence or not of cdc20b morpholinos, as indicated. Next, at 16-cell stage, half of those embryos were injected with human Separase and RFP mRNAs in one ventral-animal blastomere. This setup was designed to avoid co-injection of cdc20b morpholinos with Separase mRNA, ruling out non-specific interference in vitro between these reagents. MCI-hGR-injected embryos were induced with dexamethasone at st11. All embryos were fixed at tailbud st25 and stained for GFP (cdc20b Mo tracer, green), RFP (Separase tracer, red) and Acetylated-a-Tubulin (cilia, white). Note that multiciliogenesis failed in MCI-induced cdc20b morphant MCCs (w,y). The presence of Separase rescued multiciliogenesis in MCI-induced cdc20b morphant MCCs (x,z).

Scale bars: 5µm (a, h, o, t), 20µm (n).

Supplementary Figure 8: Uncropped Western blots.

Red boxes mark the parts of the Western blot images that are shown in the indicated parts of Figure 6.

Supplementary Table 1: Gene sets used to assess enrichment of cell cycle phases related transcripts in individual cells analyzed by scRNA-seq.