Cyclin O ( Ccno ) functions during deuterosome‐mediated centriole amplification of multiciliated cells
2015; Springer Nature; Volume: 34; Issue: 8 Linguagem: Inglês
10.15252/embj.201490805
ISSN1460-2075
AutoresMaja C. Funk, Agata Bera, Tabea Menchen, Georg Kuales, Kerstin Thriene, Soeren S. Lienkamp, Jörn Dengjel, Heymut Omran, Marcus Frank, Sebastian J. Arnold,
Tópico(s)Microtubule and mitosis dynamics
ResumoArticle23 February 2015free access Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells Maja C Funk Maja C Funk University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany Spemann Graduate School of Biology and Medicine, Freiburg, Germany Search for more papers by this author Agata N Bera Agata N Bera University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Tabea Menchen Tabea Menchen Department of Pediatrics, University Hospital Muenster, Muenster, Germany Search for more papers by this author Georg Kuales Georg Kuales University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany Search for more papers by this author Kerstin Thriene Kerstin Thriene Department of Dermatology, ZBSA Centre for Biological Systems Analysis, Medical Centre, University of Freiburg, Freiburg, Germany Search for more papers by this author Soeren S Lienkamp Soeren S Lienkamp University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Jörn Dengjel Jörn Dengjel BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Department of Dermatology, ZBSA Centre for Biological Systems Analysis, Medical Centre, University of Freiburg, Freiburg, Germany Search for more papers by this author Heymut Omran Heymut Omran Department of Pediatrics, University Hospital Muenster, Muenster, Germany Search for more papers by this author Marcus Frank Marcus Frank Medical Biology and Electron Microscopy Centre, University Medicine Rostock, Rostock, Germany Search for more papers by this author Sebastian J Arnold Corresponding Author Sebastian J Arnold University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Maja C Funk Maja C Funk University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany Spemann Graduate School of Biology and Medicine, Freiburg, Germany Search for more papers by this author Agata N Bera Agata N Bera University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Tabea Menchen Tabea Menchen Department of Pediatrics, University Hospital Muenster, Muenster, Germany Search for more papers by this author Georg Kuales Georg Kuales University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany Search for more papers by this author Kerstin Thriene Kerstin Thriene Department of Dermatology, ZBSA Centre for Biological Systems Analysis, Medical Centre, University of Freiburg, Freiburg, Germany Search for more papers by this author Soeren S Lienkamp Soeren S Lienkamp University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Jörn Dengjel Jörn Dengjel BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Department of Dermatology, ZBSA Centre for Biological Systems Analysis, Medical Centre, University of Freiburg, Freiburg, Germany Search for more papers by this author Heymut Omran Heymut Omran Department of Pediatrics, University Hospital Muenster, Muenster, Germany Search for more papers by this author Marcus Frank Marcus Frank Medical Biology and Electron Microscopy Centre, University Medicine Rostock, Rostock, Germany Search for more papers by this author Sebastian J Arnold Corresponding Author Sebastian J Arnold University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Author Information Maja C Funk1,2, Agata N Bera1,3, Tabea Menchen4, Georg Kuales1, Kerstin Thriene5, Soeren S Lienkamp1,3, Jörn Dengjel3,5, Heymut Omran4, Marcus Frank6 and Sebastian J Arnold 1,3 1University Medical Centre, Renal Department, Centre for Clinical Research, Freiburg, Germany 2Spemann Graduate School of Biology and Medicine, Freiburg, Germany 3BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany 4Department of Pediatrics, University Hospital Muenster, Muenster, Germany 5Department of Dermatology, ZBSA Centre for Biological Systems Analysis, Medical Centre, University of Freiburg, Freiburg, Germany 6Medical Biology and Electron Microscopy Centre, University Medicine Rostock, Rostock, Germany *Corresponding author. Tel: +49 761 270 63120; E-mail: [email protected] The EMBO Journal (2015)34:1078-1089https://doi.org/10.15252/embj.201490805 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs. Synopsis Cyclin O mutations cause congenital mucociliary clearance disorder linked to a reduced number of motile cilia in the airway epithelium. This is caused by malfunctioning deuterosomes that lead to fewer and partially defective centrioles. Ccno is expressed early in the embryonic node, and in respiratory and ependymal multiciliated cells (MCCs) later on. MCCs lacking Ccno generate fewer centrioles that often fail to dock to the plasma membrane, resulting in fewer motile cilia. Deuterosomes of Ccno-deficient MCCs are enlarged in size, reduced in number and functionally compromised. These deuterosomes generate fewer and partially non-functional centrioles. Introduction Cilia are microtubule-based, hairlike organelles that protrude from the surfaces of cells and are present in the majority of animal phyla (Carvalho-Santos et al, 2011). While most cells possess one cilium, then referred to as primary cilium, specialized epithelial cells can generate up to several hundred cilia on their surface, then referred to as multiciliated cells (MCCs). Cilia of MCCs exhibit coordinated beating motility that creates a directional flow enabling fluid transport along epithelial surfaces (Fliegauf et al, 2007). Fluid flow directs the distribution of signalling molecules, or generates mechanical forces for fluid transportation, as found in the ventricular system of the brain. Additionally, fluid flow is essential for the clearance of environment-exposed epithelia by mucus transport, such as in the respiratory tract epithelium. Human individuals with congenital ciliary motility disorders present with clinical features of recurrent airway infections, infertility, situs ambiguous and increased incidence of hydrocephalus, collectively referred to as primary ciliary dyskinesia (PCD) (reviewed by Knowles et al, 2013). Recently, human cases of congenital mucociliary clearance disorders with predominant respiratory symptoms have been reported that are primarily caused by the reduced generation of multiple motile cilia (RGMC) (Wallmeier et al, 2014). Mutations in the CCNO gene lead to the compromised generation of sufficient cilia numbers on MCCs, while the beating motility of remaining cilia is not generally disrupted (Wallmeier et al, 2014). This study suggested requirements of CCNO function for docking of centrioles to the apical cell membrane. However, a more detailed analysis of CCNO function during the generation of multiple motile cilia may reveal alternative roles of CCNO for cilia biogenesis in MCCs. During ciliogenesis, centrioles dock to the plasma membrane, thereby forming basal bodies, which serve as platform for the outgrowth of ciliary axonemes (reviewed by Nigg & Raff, 2009). While in monociliated cells the cilium extends from the former mother centriole, MCCs massively amplify centrioles as initiating event of multiciliogenesis (Sorokin, 1968). Two alternative modes of centriole amplification have been described based on ultrastructural studies (Sorokin, 1968). In the canonical pathway of centriole amplification, several procentrioles form in association with the mother centriole, denoted as the mother-centriole-dependent pathway (MCD). Additionally, the massive amplification of centrioles is achieved in a mother-centriole-independent, acentriolar fashion at so-called deuterosomes (deuterosome-dependent pathway, DD). These serve as additional procentriole nucleation centres and are specific for MCCs (Sorokin, 1968). A recent study has demonstrated that deuterosomes are generated in a consecutive manner strictly from daughter centrioles, implicating that predominantly daughter centrioles contribute to centriole amplification in MCCs (Al Jord et al, 2014). Previously, deuterosomes were solely described by their characteristic strongly electron-dense, ring-like appearance in transmission electron microscopy and until recently their composition was unknown (Sorokin, 1968). Two studies have elucidated some molecular components of deuterosomes by the identification of DEUP1 and CCDC78 as proteins that specifically localize to deuterosomes, but not to the foci of mother centrioles during procentriole formation (Klos Dehring et al, 2013; Zhao et al, 2013). Forced expression of Deup1 leads to amplification of centrioles, and bacterially expressed, recombinant DEUP1 forms spherical structures reminiscent of deuterosomes in MCCs (Zhao et al, 2013). To date, the knowledge about regulation of deuterosome formation, number and control of their function during centriole amplification remains limited. However, two recent studies have identified the transcription factor MULTICILIN (Mcidas) as direct and broad regulator of the transcriptional programme responsible for DD-ciliogenesis in MCCs, including the positive regulation of Ccno (Ma et al, 2014). Additionally, it is assumed that centriole formation via deuterosomes is at least partially controlled by the same set of known factors for centriole biogenesis of cycling cells, such as PLK4 (Kleylein-Sohn et al, 2007), SAS-6 (Strnad et al, 2007) and CEP152 (Blachon et al, 2008). However, the precise roles of these players have yet to be identified in MCCs (Tang, 2013). In this study, we describe highly MCC-specific expression of Ccno from early phases of centriole amplification and ciliogenesis during embryonic development and in differentiating MCCs in culture. Using targeted genetics, we analysed Ccno-deficient mice, which show characteristics of PCD-like phenotypes, as a result of significantly reduced cilia numbers of MCCs. Impaired ciliogenesis of Ccno-deficient MCCs is caused by functional defects of deuterosome-mediated centriole amplification. Reduced numbers of deuterosomes are formed and newly generated centrioles exhibit defects of maturation and localization, resulting in reduced numbers of properly docked and fully functional ciliary basal bodies. However, Ccno does not seem to impact on the MCD mode of centriole amplification. In summary, we demonstrate that Ccno plays critical roles in the regulation of deuterosome formation and function, required for amplification of functional centrioles and thus the generation of multiple motile cilia in MCCs. Results Ccno is specifically expressed in MCCs and the embryonic node In an expression screen that aimed for identifying transcripts with regional specific expression during cell lineage formation in the embryonic day 7.5 (E7.5) mouse embryo, we discovered highly regional specific transcription of the Ccno gene in cells of the most anterior tip of the primitive streak and in the embryonic node (Fig 1A). The node is the transient organizing structure that establishes the left-right body axis in a cilia-dependent manner. Pit cells in the centre of the node carry motile monocilia that generate a leftward fluid flow across the ventral node surface, required for establishing left-right asymmetry of the body axis (Shiratori & Hamada, 2006). Ccno expression was exclusively found in pit cells, but was excluded from the crown cells that constitute the margins of the node and carry non-motile cilia (Fig 1A). Figure 1. Ccno is specifically expressed in multiciliated cells and the embryonic node A. Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane. B. Schematic of the CcnoTA targeted LacZ knock-in allele used in (A) and (C–G). C. Whole embryo X-Gal staining of E16 CcnoTA/+ and wild-type control embryos and indicated section planes of (D–F'). D–F'. Histological sections of LacZ-stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus. G. Co-expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X-Gal and immunohistochemistry using a FOXJ1-specific antibody. Data information: Scale bars: 100 μm in (A), 1 mm in (C), 500 μm in (D-F') and 100 μm in (G). Download figure Download PowerPoint For the robust detection of Ccno gene expression, we established a reporter mouse line by blastocyst injection of ES cells generated by the EUCOMM consortium (Skarnes et al, 2011), which carry a LacZ reporter allele in intron 1 of the Ccno gene (Fig 1B and Supplementary Fig S1A). LacZ expression from this CcnoLacZ reporter allele (referred to as CcnoTA hereafter) recapitulates endogenous mRNA expression (Fig 1A and Supplementary Fig S1C) and was used for successive analyses of Ccno expression. At later stages of development, Ccno expression was restricted to epithelial tissues that share as common feature the presence of multiciliated cells (MCCs) (Fig 1C). Ccno-expressing epithelia include the ependymal epithelium lining the ventricular surfaces of the brain (Fig 1C), the plexus choroideus within the ventricular lumen (Fig 1D), and the epithelium of the upper (snout epithelium; Fig 1E) and lower respiratory tract (trachea and bronchi, Fig 1F). Expression analysis in tracheal epithelium revealed co-expression of Ccno and Foxj1, a key transcriptional regulator for the generation of motile cilia (Fig 1G) (Chen et al, 1998; Brody et al, 2000). Collectively, these expression patterns suggest specific functions of Ccno in MCCs. Ccno deletion in mouse causes functional defects of MCCs due to reduced cilia number To study Ccno gene function, we generated mice with a targeted deletion at the Ccno gene locus by crossing CcnoTA/+ animals to the transgenic Sox2::Cre germline deleter strain (Hayashi et al, 2002) (Supplementary Fig S1A). The deletion of coding exons 2 and 3 resulted in a Ccno null allele configuration (referred to as CcnoRA hereafter). To generate homozygous deletions of the Ccno gene locus, we intercrossed CcnoRA/+ heterozygous animals. CcnoRA/RA mice were born, however were not represented at Mendelian ratio (25%) at weaning age (postnatal day 21; P21). Instead, only 14% of CcnoRA/RA homozygously deleted mutants could be observed (35/246; Fig 2E), while CcnoRA/RA embryos were found at expected frequency of 25% until E17 (Fig 2E), indicating perinatal lethality until P21 in Ccno-deficient offspring. From the remaining CcnoRA/RA mice, ~57% (20/35) developed a prominent hydrocephalus, leading to abnormal head morphology (Fig 2A and B) and growth retardation. MRI analysis and dissected brains revealed massively enlarged ventricles and a substantial thinning of the cerebral cortex (Fig 2B and C). All CcnoRA/RA animals that developed severe hydrocephalus died within the first 6 weeks of life. Remaining CcnoRA/RA homozygous animals (15/35, 43%) appeared grossly normal and did not suffer from untimely lethality (Fig 2E). Importantly, no obvious signs of laterality defects in Ccno-deficient embryos or adult animals, and no sneezing or coughing were observed. Male and female animals that were not severely affected by hydrocephalus were fertile at maturity age when used for breeding. Figure 2. Genetic deletion of Ccno leads to severe hydrocephalus, mucociliary clearance deficits and reduced cilia number of MCCs A. Ccno-deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow). B, C. Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation. D, D'. Alcian-blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows). E. Survival table of different genotypes at embryonic stages (E13–E17) and at P21 from offspring of heterozygous CcnoRA/+ intercrosses. 11% of CcnoRA/RA homozygous animals die between E17 and P21. Of CcnoRA/RA homozygous animals at P21, roughly 60% develop severe hydrocephalus (20/35 animals), and 40% (14/35) appear grossly unaffected. F, F'. Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno-deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows). G. Transmission electron microscopy (TEM) of wild-type and CcnoRA/RA MCCs from adult trachea. Ccno-deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron-dense material is found within the cytoplasm of Ccno-deficient MCCs (arrowheads). Data information: Scale bars: 500 μm in (D), 100 μm in (D'), 7 μm in (F), 2 μm in (F') and 1 μm in (G). Download figure Download PowerPoint Hydrocephalus in mice is frequently caused by defective ciliary function of ependymal MCCs leading to the reduced intraventricular transport of cerebrospinal fluid (reviewed by Lee, 2013). To investigate additional manifestations associated with disturbed MCC function, we examined paranasal cavities by mucus staining of CcnoRA/RA animals. Obvious mucosal congestion was found in paranasal cavities of all examined CcnoRA/RA animals (n = 3), but not in control littermates from the same cage (n = 3) (Fig 2D and D'). The predominant manifestation of MCC dysfunction in human individuals is insufficient airway clearance leading to recurrent airway infections. Histologicalanalysis of adult Ccno-deficient lung tissue did not reveal signs of inflammation or mucus congestions of CcnoRA/RA mice that were maintained in a specific pathogen-free environment (Supplementary Fig S2A and B). However, scanning electron microscopy (SEM) of tracheal epithelium from CcnoRA/RA adult mice showed a pronounced reduction of cilia number on the surface of tracheal MCCs (Fig 2F and F'). In comparison to wild-types, cilia of Ccno-deficient MCCs appeared less grouped and extended mostly from the central regions of the apical cell surface, leaving the margins of MCCs undecorated (Fig 2F and F'). Otherwise, single cilia did not show gross morphological abnormalities and the overall cell number of MCCs appeared unchanged (Fig 2F). Immunofluorescence (IF) staining of adult CcnoRA/RA tracheal epithelium for acetylated α-tubulin, which marks ciliary axonemes, similarly showed reduced staining in accordance with overall reduced number of cilia (Supplementary Fig S2D). The presence of the motor protein dynein outer arm heavy chain 5 (DNAH5) suggested that remaining cilia are motile (Supplementary Fig S2D') (Fliegauf et al, 2005). This assumption was confirmed by video microscopy of tracheal epithelium that revealed ciliary motility of remaining cilia on Ccno-deficient MCCs (Supplementary Movies S1 and S2). To uncover ultrastructural details of MCCs, we employed transmission electron microscopy (TEM) of adult tracheal epithelium. Confirming the results from SEM and IF analyses, TEM also revealed considerably reduced numbers of cilia in Ccno-deficient MCCs (Fig 2G). Instead of properly docked basal bodies, we observed multiple mislocalized and misshaped basal bodies that failed to correctly dock to the apical plasma membrane and to extend ciliary axonemes (Supplementary Fig S3B–E). Additionally, the cytoplasm of Ccno-deficient cells frequently contained electron-dense structures that most likely represent remnants of microtubular-based organelles such as centrioles and/or basal bodies (Fig 2G and Supplementary Fig S3B–E). Ultrastructural analysis of the few cilia that extend from correctly docked basal bodies showed pleiotropic axonemal defects at increased frequencies (Supplementary Fig S3H–J). These included microtubular alterations, such as singlet (Supplementary Fig S3H), or triplet microtubules (Supplementary Fig S3I), abnormal microtubule doublet number and lack of the central tubule pair (Supplementary Fig S3J). However, also axonemes with normal morphology were observed (Supplementary Fig S3G), likely explaining conservation of some ciliary beating motility that could be seen in Ccno-deficient tracheal epithelium (Supplementary Movies S1 and S2). Dynamic expression of Ccno suggests roles during early stages of ciliogenesis To examine if the observed ciliary defects are caused by disturbed ciliary assembly, or result from altered ciliary maintenance or disassembly, we focused our analyses on tracheal and bronchial epithelium during early stages of MCC development. First, we monitored expression onset of Ccno during lung development (Fig 3A–H). Using the CcnoTA reporter allele, we found first expression in tracheal epithelium at E13, coinciding with the early initiation of MCCs (Fig 3E) (Rawlins et al, 2007). During the following developmental stages, Ccno expression extended to more distal bronchi. By E16, expression could be found from the trachea to the terminal bronchioles (Fig 3G and H). Thus, Ccno expression reflects the spatio-temporal order of MCC development in the respiratory tract (Rawlins et al, 2007; Vladar & Stearns, 2007). Figure 3. Ccno is dynamically expressed from early phases of ciliogenesis and Ccno deficiency impacts on the transcriptional programme for ciliogenesis A–H. X-Gal-staining of (A–C) control and (D–H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli. I. X-Gal-staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation-onset by switching to air–liquid interface (ALI) conditions (ALI d5). J. Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT–PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side. K. Relative mRNA expression levels for indicated genes were measured by qRT–PCR in wild-type and Ccno-deficient mTEC cultures at indicated days after differentiation-onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures. Data information: Scale bars: 500 μm in (A–H) and 250 μm in (I). Download figure Download PowerPoint To examine stage-dependent Ccno expression during ciliogenesis in more detail, we applied mouse tracheal epithelium cell culture (mTEC) techniques (Fig 3I) and compared Ccno mRNA expression with transcription of previously described key components for MCC ciliogenesis in wild-type mTECs (Zhao et al, 2013). mRNA levels were quantified by qRT–PCR after onset of air–liquid interface culture (ALI) conditions that induces MCC differentiation and ciliogenesis in a semi-synchronized fashion (Fig 3J). Expression profiles of Deup1, Cep152, Cep63 and Ift57 recapitulated published data sets (Zhao et al, 2013). Ccno mRNA is strongly induced during the first day after onset of ALI conditions and peaked around day 3 (ALI day 3) of differentiation with a 90-fold induction of mRNA levels compared to day 0, thereby paralleling the expression of Deup1 (Fig 3J). DEUP1 is one of the core components of deuterosomes and becomes upregulated during the early phase of deuterosome formation in mTECs (Zhao et al, 2013). The immediate and strong induction of Ccno expression at early stages of tracheal epithelium and mTEC differentiation thus suggests that Ccno, similar to Deup1, may function during the early steps of centriole amplification. To test if loss of Ccno function impacts on the previously described transcriptional programme for deuterosome-dependent (DD) centriole amplification (Ma et al, 2014), we performed qRT–PCRs in control and Ccno-deficient mTECs (Fig 3K). In wild-type mTECs, key components of DD centriole amplification are strongly induced during the first 3 days of differentiation and expression declines shortly afterwards. Recently, it was demonstrated that MULTICILIN is the key transcriptional regulator for the expression of multiple factors for DD centriole amplification, including Deup1, Ccno, Cep152 and Cp110 (Ma et al, 2014). In Ccno-deficient mTECs, Multicilin and all tested downstream targets were strongly induced (Fig 3K) during the first 3 days of differentiation similar to the wild-type cultures. However, unexpectedly Ccno-deficient mTECs failed to downregulate Multicilin and other components of the DD pathway after the first induction phase and showed further increasing expression levels until day 7. Interestingly, no increase of Cep63 expression, a key component for mother-centriole-dependent (MCD) centriole formation, was observed. This could indicate that loss of Ccno function specifically impacts on deuterosome-mediated centriole amplification, but not on the MCD pathway. Ccno-deficient MCCs show ultrastructural and functional defects of deuterosomes and forming centrioles To analyse functional consequences of Ccno deletion at early stages of MCC ciliogenesis in more detail, we performed SEM of tracheal tissue at E17. In contrast to SEM analysis of adult trachea (Fig 2F), we observed a complete absence of multiple cilia on the epithelial surface of Ccno-deficient trachea at E17 (Fig 4A–B'). However, ciliogenesis was not generally ablated at this stage in CcnoRA/RA MCCs, since cells with mono- or few cilia with normal morphology could be detected (Fig 4B'). The absence of multiple cilia in E17 tracheal MCCs was also reflected by a significant reduction of acetylated α-tubulin, marking ciliary axonemes (Fig 4C–E). To investigate the underlying cause for the failure to generate multiple cilia, we performed TEM analysis of E17 tracheal epithelium. As expected from SEM, we observed only very few cilia and similarly only few basal bodies that had properly docked to the apical cell membrane in Ccno-deficient MCCs (Supplementary Fig S4A and B). At E17, wild-type MCCs frequently exhibited deuterosomes with forming procentrioles that arrange in a symmetrical fashion surrounding the deuterosome cortex (Fig 4F, F' and H). In striking contrast, deuterosomes of Ccno-deficient MCCs only rarely displayed clearly discernable procentrioles (Fig 4G, G' and I), and the few emerging procentrioles were significantly shorter in comparison to controls (Fig 4J). Additionally, the usual symmetrical orientation of centrioles was lost, showing an uneven distribution around the deuterosomes (Fig 4H and I, and Supplementary Fig S4E and E'). As most obvious morphological alteration, the great majority of deuterosomes of Ccno-deficient MCCs showed an irregular shape and were significantly enlarged by 42% (Fig 4K) when compared to deuterosomes of wild-type cells (Fig 4F–I and Supplementary Fig S4). Enlarged deuterosomes often showed more than one electron-transparent central region, surrounded by the electron-dense cortex (Fig 4G and G'). 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