GEMC 1 is a critical regulator of multiciliated cell differentiation
2016; Springer Nature; Volume: 35; Issue: 9 Linguagem: Inglês
10.15252/embj.201592821
ISSN1460-2075
AutoresBerta Terré, Gabriele Piergiovanni, Sandra Segura‐Bayona, Gabriel Gil‐Gómez, Sameh A. Youssef, Camille Stephan‐Otto Attolini, Michaela Wilsch‐Bräuninger, Carole Jung, Ana M. Rojas, Marko Marjanović, Philip A. Knobel, Lluís Palenzuela, Teresa López‐Rovira, Stephen Forrow, Wieland Β. Huttner, Miguel A. Valverde, Alain de Bruin, Vincenzo Costanzo, Travis H. Stracker,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle1 March 2016free access Source DataTransparent process GEMC1 is a critical regulator of multiciliated cell differentiation Berta Terré Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Gabriele Piergiovanni FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Sandra Segura-Bayona Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Gabriel Gil-Gómez IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Barcelona, Spain Search for more papers by this author Sameh A Youssef Dutch Molecular Pathology Center, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Camille Stephan-Otto Attolini Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Michaela Wilsch-Bräuninger Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Carole Jung Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Ana M Rojas Computational Biology and Bioinformatics Group, Institute of Biomedicine of Seville, Campus Hospital Universitario Virgen del Rocio, Seville, Spain Search for more papers by this author Marko Marjanović Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Philip A Knobel Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Lluís Palenzuela Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Teresa López-Rovira Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Stephen Forrow Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Wieland B Huttner Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Miguel A Valverde Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Alain de Bruin Dutch Molecular Pathology Center, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Vincenzo Costanzo Corresponding Author FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Travis H Stracker Corresponding Author Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Berta Terré Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Gabriele Piergiovanni FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Sandra Segura-Bayona Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Gabriel Gil-Gómez IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Barcelona, Spain Search for more papers by this author Sameh A Youssef Dutch Molecular Pathology Center, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Camille Stephan-Otto Attolini Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Michaela Wilsch-Bräuninger Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Carole Jung Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Ana M Rojas Computational Biology and Bioinformatics Group, Institute of Biomedicine of Seville, Campus Hospital Universitario Virgen del Rocio, Seville, Spain Search for more papers by this author Marko Marjanović Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia Search for more papers by this author Philip A Knobel Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Lluís Palenzuela Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Teresa López-Rovira Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Stephen Forrow Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Wieland B Huttner Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Miguel A Valverde Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain Search for more papers by this author Alain de Bruin Dutch Molecular Pathology Center, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Vincenzo Costanzo Corresponding Author FIRC Institute of Molecular Oncology, Milan, Italy Search for more papers by this author Travis H Stracker Corresponding Author Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Author Information Berta Terré1,‡, Gabriele Piergiovanni2,‡, Sandra Segura-Bayona1, Gabriel Gil-Gómez3, Sameh A Youssef4, Camille Stephan-Otto Attolini1, Michaela Wilsch-Bräuninger5, Carole Jung6, Ana M Rojas7, Marko Marjanović1,8, Philip A Knobel1, Lluís Palenzuela1, Teresa López-Rovira1, Stephen Forrow1, Wieland B Huttner5, Miguel A Valverde6, Alain Bruin4,9, Vincenzo Costanzo 2 and Travis H Stracker 1 1Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain 2FIRC Institute of Molecular Oncology, Milan, Italy 3IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Barcelona, Spain 4Dutch Molecular Pathology Center, Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 5Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 6Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain 7Computational Biology and Bioinformatics Group, Institute of Biomedicine of Seville, Campus Hospital Universitario Virgen del Rocio, Seville, Spain 8Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia 9Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands ‡These authors contributed equally to this study *Corresponding author. Tel: +39 2 574303875; E-mail: [email protected] *Corresponding author. Tel: +34 93 4031183; E-mail: [email protected] EMBO J (2016)35:942-960https://doi.org/10.15252/embj.201592821 See also: EK Vladar & BJ Mitchell (May 2016) 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 The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1-deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders. Synopsis In addition to a role in DNA replicaion, Geminin-like coiled-coil containing protein 1 (GEMC1) interacts with E2F4/5-DP1 and Multicilin to control transcriptional programs required for multiciliated cell (MCC) differentiation in mammals. GEMC1 is required for normal growth and fertility in mice. Multiciliated cells and sperm require GEMC1. GEMC1 interacts with E2F4/5-DP1 and Multicilin. Multiciliated cells transcriptional programs are activated by GEMC1. GEMC1 is a candidate gene for human ciliopathies. Introduction The generation of diverse types of cilia, microtubule-based organelles that project from the cell surface, is required for many aspects of development and organismal function (Fliegauf et al, 2007; Choksi et al, 2014b). Most cells can form solitary, immotile cilia involved in sensory functions, while particular cell types exhibit motile cilia that are involved in sensory functions, motility or feeding. Multiciliated cells (MCCs), located in the brain, respiratory system, and reproductive tracts, contain dozens of motile cilia that act in a coordinated beating pattern to promote fluid flow or ovum transport along epithelia (Fliegauf et al, 2007; Brooks & Wallingford, 2014). The events that trigger MCC differentiation are not completely understood and likely vary to some extent depending on the organism and tissue (Brooks & Wallingford, 2014; Choksi et al, 2014b). In many tissues, the inhibition of Notch/Delta signaling promotes the differentiation of progenitors into MCCs (Deblandre et al, 1999; Liu et al, 2007; Stubbs et al, 2008; Guseh et al, 2009; Tsao et al, 2009; Marcet et al, 2011; Wang et al, 2013; Song et al, 2014; Lafkas et al, 2015). Notch inhibition is mediated in part by the Mir34 and Mir449 microRNA (miRNA) families that directly control Notch1 and Delta-like 1 (DLL1) (Marcet et al, 2011). Additionally, Notch inhibition leads to the activation of Multicilin (MCIDAS), considered one of the most upstream transcriptional activators of the MCC differentiation program. Multicilin promotes the transcription of key genes required for multiciliogenesis (Stubbs et al, 2012), including FOXJ1, MYB, RFX2, and RFX3 (Brooks & Wallingford, 2014; Choksi et al, 2014b) through interactions with the E2F4 and E2F5 transcription factors and their cofactor, DP1 (referred to collectively as the EDM complex) (Ma et al, 2014). This interaction occurs via a C-terminal domain of Multicilin, dubbed the TIRT domain based on its amino acid sequence (Stubbs et al, 2012). Consistent with Multicilin being linked to the E2F4/5 transcription factors, loss of E2F4 in mice impairs the development of MCCs in the airway epithelium (Danielian et al, 2007) and E2F5-deficient mice develop hydrocephaly, potentially due to defects in the formation of MCCs in the brain ependyma, although this has not been conclusively demonstrated (Davy & Robinson, 2003; Danielian et al, 2007). As each cilium in MCCs is formed from a specialized type of centriole, called the basal body, the development of MCCs requires the massive expansion of centrioles to allow the generation of dozens of motile cilia per cell (Brooks & Wallingford, 2014). Centriole duplication is normally coupled to the cell cycle through the canonical mother-centriole-dependent pathway, but as MCCs are post-mitotic, they rely on the de novo basal body formation pathway. This system of basal body expansion is dependent on deuterosomes, poorly described, electron dense ring structures that produce multiple centrioles simultaneously, enabling rapid formation of large number of centrioles (Sorokin, 1968). The genes required for deuterosome pathway centriole amplification in MCCs are transcriptionally activated by the EDM complex and include DEUP1, CCNO, and CCDC78 (Klos Dehring et al, 2013; Zhao et al, 2013; Boon et al, 2014; Ma et al, 2014; Funk et al, 2015). Dysfunction of motile cilia causes mucociliary clearance disorders, such as primary ciliary dyskinesia (PCD), characterized by defects in the upper and lower respiratory system, heart malformations, polysplenia, hydrocephaly, subfertility, or infertility, and in some cases by defects in asymmetric organ patterning (known as situs inversus) (Zariwala et al, 2011). A distinct class of mucociliary clearance disorders, referred to as congenital mucociliary clearance disorder with reduced generation of multiple motile cilia (RGMC), was shown to result from mutations in the CCNO or MCIDAS genes that are required for the generation of a fully functional multiciliated epithelium (Boon et al, 2014; Wallmeier et al, 2014). Due to reduced numbers or function of MCCs, RGMC patients develop recurring respiratory infections and in some cases have exhibited hydrocephaly and reduced fertility (Boon et al, 2014). The MCIDAS gene (encoding Multicilin) is located on chromosome 5q11.2 in humans (13 D2.2 in mice), in a locus that harbors other key regulators of MCC formation, including CCNO, CDC20B, and Mir-449a/b/c (Marcet et al, 2011; Boon et al, 2014; Song et al, 2014; Wu et al, 2014). Multicilin is a member of the Geminin superfamily, sharing a similar central coiled-coil (CC) domain with Geminin and Geminin-like coiled coil containing 1 (GEMC1, encoded by GMNC, also referred to as Lynkeas) (Balestrini et al, 2010; Pefani et al, 2011). Multicilin can interact with Geminin through the CC domain and inhibit its function as a negative regulator of DNA replication (Caillat et al, 2013). Conversely, Geminin impairs the ability of Multicilin to activate genes required for centriole biogenesis in MCCs, suggesting opposing roles that favor proliferation or differentiation, respectively (Ma et al, 2014). Mutations identified in human RGMC patients are located in the TIRT domain of Multicilin (MCIDAS) and compromise interactions with E2Fs that are required for its transcriptional functions, but are unlikely to affect interactions with Geminin (Boon et al, 2014). GEMC1 was originally identified due to its homology with the Geminin CC domain and was shown to function in DNA replication initiation through interactions with Cdc45 and CDK2/cyclin E that are important for replication initiation, and TOPBP1, a replication fork component critical for activating the ATR kinase that is mutated in Seckel syndrome (O'Driscoll et al, 2003; Mordes et al, 2008; Balestrini et al, 2010). Moreover, depletion of GEMC1 impaired DNA replication in both Xenopus extracts and mammalian cells (Balestrini et al, 2010). Like Multicilin, GEMC1 interacts with Geminin, but the functional relevance of this interaction has not yet been clearly established (Caillat et al, 2013, 2015). GEMC1 is structurally similar to Multicilin, and phylogenetic analysis strongly suggests that they are out-paralogues (Fig EV1A). In addition to the central Geminin-like CC domain, GEMC1 contains a C-terminal TIRT domain with a high degree of homology to that of Multicilin (Fig EV1B). Click here to expand this figure. Figure EV1. Evolutionary relationship and sequence homology of GEMC1 and Multicilin Phylogenetic analysis strongly suggests that GEMC1 and MCIDAS are out-paralogues that arose from primordial GEMININ, likely at the time of the two round genome duplication that shaped vertebrate genomes. This is supported by (i) the species distribution of proteins containing this domain, which resembles the species tree, (ii) the absence of identifiable similar proteins in the databases when excluding the common coiled-coil sequence, (iii) the presence of a common additional domain at the C-terminal region of both proteins, and (iv) by the bootstrap values separating the GEMININ-MCIDAS group. A possible evolutionary scenario is that after duplication, the C-terminal region was acquired by a domain-shuffling event and GEMC1 began to diverge faster. A plausible explanation for this would be a relaxation of purifying selection in this region (the CC domain), which would yield a large divergence, although the acquisition of a novel function (positive selection) could also have played a role in such a divergence. Along these lines, we can observe large branches separating the GEMC1 group, while the branches separating the GEMC1 sequences are very short. This is indicative of strong purifying selection, although this should be explored in more depth in future analyses (see Appendix Supplementary Materials and Methods for further information). The amino acid sequences of GEMC1 and Multicilin (MCIDAS) from human, mouse, and Xenopus were aligned using T-Coffee (Notredame et al, 2000) and alignments exported using ESPript (Robert & Gouet, 2014) and further annotated in Adobe Illustrator. The Geminin-like coiled-coil domain is indicated in blue and the TIRT domain in green. Phosphorylated residues reported in PhosphositePlus (Bateman et al, 2002; Li et al, 2002; Obenauer et al, 2003) are notated in blue and patient mutations in MCIDAS or corresponding mutations in GEMC1 analyzed in this study are indicated (Boon et al, 2014). Download figure Download PowerPoint Here, we report that mice lacking GEMC1 are growth impaired, develop bilateral hydrocephaly, and are infertile. These defects are accompanied by a dramatic impairment in MCC development in the brain, respiratory system, and reproductive tract of embryonic and adult animals, as well as severe defects in spermatogenesis. The trachea and oviduct that normally have large numbers of MCCs showed highly reduced expression levels of many genes linked to deuterosome-mediated centriole expansion and cilia development, including Mcidas, FoxJ1, Ccno, Ccdc78, and Deup1. We find that GEMC1 interacts with E2F4/5-DP1 and Multicilin to control the transcriptional program of MCC cells and that these interactions are required for the activation of key regulators of MCC fate. Our results demonstrate that a primary function of GEMC1 in vivo is to promote the proper differentiation of progenitor cells into the MCC lineage in multiple tissues and suggest that GEMC1 should be considered as a candidate gene for human RGMC disorders. Results GEMC1-deficient mice are runted and develop hydrocephaly As previous work linked GEMC1 to the control of DNA replication, we sought to examine its functions in vivo (Balestrini et al, 2010). For this purpose, we generated mice with a targeted deletion of the Gemc1 gene (Fig 1A and Appendix Fig S1). Gemc1−/− mice were notably smaller at birth, while no obvious phenotypes were observed in Gemc1+/− animals (Fig 1B and Appendix Fig S2). Gemc1−/− mice presented with symmetrical hydrocephaly with almost complete penetrance (Fig 1C and Appendix Fig S2), and for this reason, we were obligated to cull a number of animals in the first months of life (Fig 1D). However, those less severely affected lived up to 16 months (at which time the study was terminated) without incident. Postmortem analysis revealed hydrocephaly in all but a single Gemc1−/− animal. Given the strong effect on brain development and growth, we examined the expression of Gemc1 in wild-type animals. Although there was considerable variability between animals, Gemc1 was expressed at low levels in the kidney, spleen, heart, muscle, liver, and intestine, and at the highest levels in the brain, respiratory system and some reproductive tissues (Fig 1E and F). No Gemc1 mRNA expression was detected in any tissues examined from Gemc1−/− mice (Fig 1F and Appendix Fig S1). These results established that Gemc1 was required for normal development and that its expression was variable between tissues. Figure 1. Gemc1-deficient animals are growth impaired and develop hydrocephaly Example of littermate males of the indicated genotypes 26 days postpartum. Additional examples are provided in Appendix Fig S2. Weights of mice of the indicated genotypes from 18 to 120 days postpartum. Box plots indicate 5th–95th percentile. A minimum of eight animals are plotted at each age. Asterisks denote statistical significance (n.s., not significant, *P-value < 0.05 and ***P-value < 0.001) determined by the unpaired two-way Wilcoxon rank-sum test. Example of dissected brains (top panel) and coronal sections (bottom left and right panels) stained with H&E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1−/− mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) are also evident in the Gemc1−/− sample. Scale bars = 500 μm. Kaplan–Meier survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months, and mice were examined for histology. About 97% (31/32) of the Gemc1−/− mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2). RT–qPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a TaqMan probe. Data are compiled from duplicate samples of three male mice per tissue and mean and standard deviation are plotted. RT–qPCR analysis of Gemc1 in trachea, oviduct, and ovary tissues from Gemc1+/+ and Gemc1−/− mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from two female mice are plotted. Additional examples from other Gemc1−/− tissues are shown in Appendix Fig S1. Download figure Download PowerPoint GEMC1 is required for fertility and multiciliated cell generation Despite numerous breedings and copulation with wild-type mice, we did not obtain any offspring from Gemc1−/− males or females. As Gemc1 was more highly expressed in the germline of wild-type mice (Fig 1E and F), we histologically analyzed the reproductive tissues of Gemc1−/− mice. Testes size was similar to that of wild type but cellularity was highly reduced in tubule sections and some cell death was evident (Fig 2C). Sperm counts from the cauda epididymis of Gemc1−/− mice revealed no sperm, although some morphologically normal elongated spermatids were detected in seminiferous tubule sections (Fig 2C–E). Histological examination of the caput epididymis was consistent with sperm counts as no sperm were identified (Fig 2F). However, the epididymus appeared to be formed normally and the stereocilia lining its epithelium appeared to be intact (Fig 2G). Figure 2. GEMC1 is required for spermatogenesis A. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age. B. Testes from Gemc1−/− mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/− animals, as measured by disaggregation of the tissue and cell counting with a Neubauer chamber (n = 3). Mean and standard deviation are indicated. C. Histological analysis of tubules from 6-week-old Gemc1−/− mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 μm (left panels) and 50 μm (right panels). D. Sperm counts from the cauda epididymis of wild-type and Gemc1−/− mice revealed no sperm in mice lacking GEMC1 (n = 3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2-month-old mice. Scale bars = 10 μm. F, G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2- and 4-month-old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1−/− mice compared to wild type. Scale bars = 100 μm (F, left panels) and 50 μm (F, right panels and G). Download figure Download PowerPoint Similar to male mice, Gemc1−/− females were infertile and presented with severe defects in germline development. The ovaries of the Gemc1−/− mice examined were smaller, contained few primordial and secondary follicles, and were primarily composed of degenerated antral follicles (Fig 3A). Additionally, the oviduct was smaller in Gemc1−/− mice and was completely devoid of MCCs, in comparison with the wild-type epithelia where they were clearly abundant (Fig 3B). To address this phenotype in more detail, we performed immunofluorescence (IF) analysis with markers of cilia (γ-tubulin (γ-tub) for basal bodies and acetylated α-tubulin (Ac-tub) for cilia), as well as key transcription factors required for MCC differentiation, FOXJ1, and RFX3 (Choksi et al, 2014b). In wild-type oviducts, an organized layer of basal bodies with cilia extending from them was clearly visible (Fig 3C, left panels). However, in Gemc1−/− animals, only sporadic, punctate γ-tubulin staining was observed, likely representing individual centrosomes, and there appeared to be a complete absence of detectable cilia, as no Ac-tub staining was detected (Fig 3C, right panels). Consistent with this, staining for either FOXJ1 or RFX3 was clearly positive in wild-type animals, but no signal was detected in the Gemc1−/− oviducts. These results demonstrated that GEMC1 was required for fertility in both sexes and that in females, this was potentially due to its role in the differentiation of MCCs in the oviduct. Figure 3. GEMC1 is required for female fertility and multiciliogenesis A. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1−/− mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1−/− mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1−/− ovaries (bottom right, red arrows). Scale bars = 200 μm (left panels) and 100 μm (right top panels and right bottom panel, respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1−/− mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1−/− oviducts is observed by H&E staining. Scale bars = 50 μm (top panels). C. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild-type oviduct (left) while basal bodies and cilia are not detectable in Gemc1−/− oviducts (right). Scale bars = 50 μm. D, E. FOXJ1 (D) and RFX3 (E) staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1−/− mice. Scale bars = 50 μm. Download figure Download PowerPoint GEMC1 is required for MCCs in the brain and respiratory system Given the homology of GEMC1 to the Multicillin protein (Fig EV1) and the phenotypes we observed in the oviduct, we considered that GEMC1 could be important for the development of MCCs in other tissues, such as the brain and respiratory tract, where it was expressed (Fig 1E). To address this, we first examined cilia formation in the nasal and tracheal mucosa of wild-type and Gemc1−/− embryos at embryonic days 17.5 and 18.5 (E17.5 and E18.5). At this stage, wild-type respiratory epithelia exhibit a ciliated, pseudostratified columnar morphology with large numbers of MCCs interspersed with secretory goblet cells (Figs 4A and EV2). In sharp contrast, Gemc1−/− embryos presented with a flat columnar to squamous, non-ciliated epithelium in both the nasal and tracheal mucosa (Figs 4A and EV2) and no cilia were identifiable by staining with Ac-tubulin (Fig EV2). We next used periodic acid–Schiff (PAS) staining to detect secretory cells, as previous work demonstrated that the loss of E2F4 disrupted the development of the respiratory epithelium, leading to the loss of MCCs and increased numbers of PAS-positive cells (Danielian et al, 2007). Gemc1−/− mutants exhibited a similar phenotype as that reported for E2F4, as most of the epithelium in Gemc1−/− mice had abundant PAS-positive material (Figs 4B, right panels and EV2), in contrast to the normal epithelium where the distinct goblet cells were normally interspersed between columnar MCCs (Figs 4B, left panels and EV2). Figure 4. GEMC1 is required for the generation of multiciliated tissues H&E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ci
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