WDR11‐mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome
2017; Springer Nature; Volume: 19; Issue: 2 Linguagem: Inglês
10.15252/embr.201744632
ISSN1469-3178
AutoresY. Kim, Daniel P. S. Osborn, Ji‐Young Lee, Masatake Araki, Kimi Araki, Timothy J. Mohun, Johanna Känsäkoski, Nina Brandstack, Hyun–Taek Kim, Francesc Miralles, Cheol‐Hee Kim, Nigel A. Brown, Hyung‐Goo Kim, Juan Pedro Martı́nez-Barberá, Paris Ataliotis, Taneli Raivio, Lawrence C. Layman, Soo‐Hyun Kim,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoArticle20 December 2017Open Access Source DataTransparent process WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome Yeon-Joo Kim Yeon-Joo Kim orcid.org/0000-0001-8896-1153 Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Daniel PS Osborn Daniel PS Osborn Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Ji-Young Lee Ji-Young Lee Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Masatake Araki Masatake Araki Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan Search for more papers by this author Kimi Araki Kimi Araki Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan Search for more papers by this author Timothy Mohun Timothy Mohun Francis Crick Institute, London, UK Search for more papers by this author Johanna Känsäkoski Johanna Känsäkoski Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Nina Brandstack Nina Brandstack Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Hyun-Taek Kim Hyun-Taek Kim Department of Biology, Chungnam National University, Daejeon, Korea Search for more papers by this author Francesc Miralles Francesc Miralles orcid.org/0000-0003-3069-2725 Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Cheol-Hee Kim Cheol-Hee Kim Department of Biology, Chungnam National University, Daejeon, Korea Search for more papers by this author Nigel A Brown Nigel A Brown Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Hyung-Goo Kim Hyung-Goo Kim Medical College of Georgia, Augusta University, Augusta, GA, USA Search for more papers by this author Juan Pedro Martinez-Barbera Juan Pedro Martinez-Barbera Developmental Biology and Cancer Programme, Birth Defects Research Centre, UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Paris Ataliotis Paris Ataliotis Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Taneli Raivio Taneli Raivio Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Lawrence C Layman Lawrence C Layman Medical College of Georgia, Augusta University, Augusta, GA, USA Search for more papers by this author Soo-Hyun Kim Corresponding Author Soo-Hyun Kim [email protected] orcid.org/0000-0002-9394-1437 Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Yeon-Joo Kim Yeon-Joo Kim orcid.org/0000-0001-8896-1153 Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Daniel PS Osborn Daniel PS Osborn Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Ji-Young Lee Ji-Young Lee Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Masatake Araki Masatake Araki Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan Search for more papers by this author Kimi Araki Kimi Araki Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan Search for more papers by this author Timothy Mohun Timothy Mohun Francis Crick Institute, London, UK Search for more papers by this author Johanna Känsäkoski Johanna Känsäkoski Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Nina Brandstack Nina Brandstack Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Hyun-Taek Kim Hyun-Taek Kim Department of Biology, Chungnam National University, Daejeon, Korea Search for more papers by this author Francesc Miralles Francesc Miralles orcid.org/0000-0003-3069-2725 Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Cheol-Hee Kim Cheol-Hee Kim Department of Biology, Chungnam National University, Daejeon, Korea Search for more papers by this author Nigel A Brown Nigel A Brown Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Hyung-Goo Kim Hyung-Goo Kim Medical College of Georgia, Augusta University, Augusta, GA, USA Search for more papers by this author Juan Pedro Martinez-Barbera Juan Pedro Martinez-Barbera Developmental Biology and Cancer Programme, Birth Defects Research Centre, UCL Great Ormond Street Institute of Child Health, London, UK Search for more papers by this author Paris Ataliotis Paris Ataliotis Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Taneli Raivio Taneli Raivio Helsinki University Central Hospital, Helsinki, Finland Search for more papers by this author Lawrence C Layman Lawrence C Layman Medical College of Georgia, Augusta University, Augusta, GA, USA Search for more papers by this author Soo-Hyun Kim Corresponding Author Soo-Hyun Kim [email protected] orcid.org/0000-0002-9394-1437 Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK Search for more papers by this author Author Information Yeon-Joo Kim1, Daniel PS Osborn1, Ji-Young Lee1, Masatake Araki2, Kimi Araki2, Timothy Mohun3, Johanna Känsäkoski4, Nina Brandstack4, Hyun-Taek Kim5,8, Francesc Miralles1, Cheol-Hee Kim5, Nigel A Brown1, Hyung-Goo Kim6, Juan Pedro Martinez-Barbera7, Paris Ataliotis1, Taneli Raivio4, Lawrence C Layman6 and Soo-Hyun Kim *,1 1Molecular and Clinical Sciences Research Institute, St. George's, University of London, London, UK 2Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan 3Francis Crick Institute, London, UK 4Helsinki University Central Hospital, Helsinki, Finland 5Department of Biology, Chungnam National University, Daejeon, Korea 6Medical College of Georgia, Augusta University, Augusta, GA, USA 7Developmental Biology and Cancer Programme, Birth Defects Research Centre, UCL Great Ormond Street Institute of Child Health, London, UK 8Present address: Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany *Corresponding author. Tel: +44 2082666198; E-mail: [email protected] EMBO Reports (2018)19:269-289https://doi.org/10.15252/embr.201744632 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 WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum. Synopsis WDR11, the causative gene for congenital hypogonadotrophic hypogonadism and Kallmann syndrome, promotes Hedgehog singaling and ciliogenesis, linking these diseases to the human ciliopathy spectrum. WDR11 functions as a novel element of the Hedgehog (Hh) signal pathway, which regulates fundamental aspects of mammalian development. WDR11 shuttles between the nucleus and cytoplasm in response to Hh-signaling. WDR11 is required for the processing of GLI3 protein, forms a tertiary complex with EMX1 and GLI3 and regulates the expression of novel target genes EMX1/2 and GNRH1. WDR11 is a cilia-associated protein suggesting that CHH/KS with WDR11 mutations are ciliopathy disorders. Introduction Motile cilia are essential for fluid transport and confer motility. Non-motile primary cilia are the organizing hub for extracellular signals. Defective ciliogenesis leads to abnormal embryonic development and disrupted tissue homeostasis, resulting in ciliopathies—a group of developmental and degenerative disorders including Bardet–Biedl syndrome (BBS), Joubert syndrome, polycystic kidney disease and Meckel syndrome 12. The severity of ciliopathy phenotypes ranges from generally mild to congenitally lethal and can include hypogonadism, anosmia, obesity, retinal degeneration, renal dysfunction, developmental delay, speech deficit, syndactyly, dental dysgenesis, ataxia, diabetes mellitus and congenital heart disease 345. The cilia are required for the proper delivery of the Hedgehog (Hh) signalling essential for growth, survival, cell fate and embryonic patterning 6. Hh initiates signalling through activation of Smoothened (SMO) which translocates to primary cilia and modulates the processing of GLI transcription factors 78, to induce downstream target genes 9. Ciliary intraflagellar transport is required for the trafficking of GLI transcription factors to the ciliary tip where SMO is concentrated, and back to the cell body and nucleus 78. Although defective Hh signalling is associated with numerous congenital disorders and cancers 10, its downstream target genes that critically regulate specific organogenesis are poorly understood. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) are classically defined by delayed puberty and infertility secondary to gonadotrophin-releasing hormone (GnRH) dysfunction 11. However, CHH/KS is a complex disease, as the patients can also exhibit developmental defects in various organs including the brain, spinal cord, heart, kidney, skeleton, ear and eye, along with intellectual disability, neurosensory and neuromotor anomalies at reduced penetrance 12. We previously identified WD repeat domain 11 (WDR11) as the underlying locus for CHH/KS based on a break point at 10q26.12 and subsequent identification of five missense mutations 13. A splice site mutation in WDR11 has also been found in a combined pituitary hormone deficiency disorder 14. WDR11 is located within the 600-kb minimal microdeletion region associated with the 10q26 deletion syndrome (MIM 609625) 1516. WDR11 contains twelve WD domains—minimally conserved motifs of approximately 40 amino acids forming two beta-propellers potentially serving as molecular scaffolds 13. We previously identified the homeobox protein EMX1 as a binding partner of WDR11 in a yeast 2-hybrid screen and CHH/KS-associated mutations of WDR11 resulted in failed binding of EMX1 13. The nature of their interactions, however, remains unclear. Due to evolutionary conservation and broad tissue expression of WDR11 13, we hypothesized that it may have roles beyond the development of the reproductive system. Here we report that WDR11 is involved in the Hh signalling pathway and is essential for normal ciliogenesis. WDR11-defective mice and zebrafish exhibit complex developmental abnormalities in multiple organs, resembling features known to be associated with Hh signalling and ciliogenesis, thus linking CHH/KS with ciliopathy spectrum disorders. WDR11 undergoes Hh-dependent intracellular trafficking and interacts with GLI3 and EMX1 to regulate the expression of GnRH. We further show that loss of WDR11 leads to obesity in both mice and men, suggesting that Hh signalling via WDR11 is one of the key links between reproduction and metabolism. WDR11 may also be an underlying locus for the holoprosencephaly (HPE) spectrum and responsible for many of the phenotypes associated with 10q26 deletion syndrome. Our study proposes a new paradigm for the diagnosis of these genetically overlapping disorders and may expand the disease spectrum of CHH/KS. Results Generation of Wdr11 knockout mouse To better comprehend the biological activities of WDR11, we generated a knockout (KO) mouse line Ayu 21-KBW205 by genetrap mutagenesis. Analysis by 5′-RACE, allele-specific PCR, sequencing and Western blot confirmed the insertion of the pU-21W vector in exon 3 of the mouse Wdr11 gene, and functional KO of endogenous Wdr11 expression (Fig 1A, Appendix Fig S1A, Appendix Table S1). There was no disruption of neighbouring genes Emx2 and Fgfr2 (Appendix Fig S1B) that are also located within the 10q26 deletion syndrome region and known to be important in forebrain and genitalia development 1516. Figure 1. Generation and characterization of Wdr11 knockout mouse Genotypes were determined by PCR analyses of genomic DNA using specific primers designed for the knockout (KO) or wild-type (WT) alleles. Western blot analyses of total protein lysates extracted from the brain and testis tissue samples demonstrate the absence of endogenous Wdr11 protein at ˜130 kDa in the KO. β-actin is a loading control. Sagittal sections of E12.5 Wdr11+/− embryos were stained with antibodies against Wdr11 and x-GAL. Zoomed images are from pituitary (a), branchial arch (b), mesonephric duct (c) and limb bud (d). Scale bars indicate 1 mm in the whole embryo images and 500 μm in the zoomed images. Wdr11-deficient mice at various stages exhibit significantly retarded development and growth. Images of Wdr11−/− forelimbs showing abnormal digit separation at E13.5 and digit fusion at P1. Skeletal defects of the mice were demonstrated by double staining with alizarin red (for ossified bones) and alcian blue (for cartilage). H/E-stained coronal sections of E18.5 embryos exhibit multiple defects in forebrain and midline craniofacial structures, and present enlarged ventricles in Wdr11−/−. Scale bars, 1 mm. Abbreviations are e, eye; t, tongue; ns, nasal septum. 3D reconstruction images of whole embryos at E12.5 by HREM displaying the details of facial features. Scale bar indicates 1 mm. Photographs of 12-week-old brain displaying the OB dysgenesis in the null mutant. Coronal sections of E18.5 Wdr11−/− brain demonstrated a rudimentary OB that failed to separate. 3D reconstruction images of HREM sections of Wdr11−/− and WT hearts. Double-outlet right ventricle (two red arrows in upper left panel) is observed in Wdr11−/− compared to the normal outlets of the right and left ventricles (red arrow in bottom panels). Ventricular septal defect (a red arrow in upper right panel) is also observed in Wdr11−/− compared to the normal intact ventricular septum in bottom panel. The septum is marked by double-headed green arrows in all images. Note that the mutant was processed as a whole embryo and the WT as an isolated heart, and thus, poorer dye penetration in the mutant explains the different appearance of the reconstructions. Source data are available online for this figure. Source Data for Figure 1 [embr201744632-sup-0004-SDataFig1.pptx] Download figure Download PowerPoint Expression profiles of Wdr11 in mouse Wdr11 is broadly expressed in various adult organs including the brain, eye, ear, lung, heart, kidney and gonads (Appendix Fig S1). In E10.5–12.5 embryos, Wdr11 signal is visible in the ventricles of the heart, branchial arches and mesonephric duct. Expression is also detected in the head mesenchyme, developing eye and forebrain (Fig 1B and Appendix Fig S1C). Detailed analysis of the brain revealed Wdr11 expression in the GnRH neuronal migratory niche including nasal cavity and cribriform plate area in E12.5 embryo as well as the median eminence in the adult brain, showing co-localization with GnRH (Appendix Fig S2 and S3A). Moreover, Wdr11 is expressed throughout the developing and adult olfactory bulb (OB) with particularly high levels in the glomerular layer (Fig EV1A). Click here to expand this figure. Figure EV1. WDR11 co-localizes with cilia in various ciliated tissues A–C. Immunofluorescence images stained for acetylated tubulin (ACT) and WDR11 demonstrate their co-localization in the sagittal sections of 10-week-old brain, especially in the olfactory bulb (A), the sperm flagellum (B) and the coronal sections of 12-week-old brain, showing the hypothalamus and median eminence (C). The zoomed images of the dotted area are shown below. Wdr11−/− brain exhibits virtually absent ACT staining in all areas. Abbreviations are GLL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; GCL, granule cell layer; ME, median eminence. Scale bars, 500 μm. Download figure Download PowerPoint Wdr11 deficiency causes retardation of growth and development Gross morphological examination of the embryos and newborn pups revealed significant developmental defects and growth retardation in the null homozygotes (Wdr11−/−), compared to the WT (Wdr11+/+) and heterozygote (Wdr11+/−) littermates (Fig 1C). There was a significant underrepresentation of homozygotes from E13 onwards with less than 6% detected beyond E17.5 (Table 1), suggesting a mid-gestation embryonic lethality. The majority of null mice were either stillborn or died within 1–2 days after birth. Rare null individuals survived through adulthood. The null mutants exhibited abnormal digit separation and syndactyly, as well as shortened limbs and hypoplastic skeletons with reduced or absent bone mineralization. The heads of Wdr11−/− mice showed a diminutive and curved nasal midline and a small lower jaw (Fig 1D). Table 1. Mouse phenotype and genotype summary Genotype Wdr11 +/+ Wdr11 +/− Wdr11 −/− Expected Mendelian ratio 25% 50% 25% Total observed pups 34% (86/251) 61% (153/251) 5%a (12/251) E12.5b 28% (12/43) 49% (21/43) 23% (10/43) E14.5 30% (18/60) 65% (39/60) 5%c (3/60) E17.5 32% (16/50) 62% (31/50) 6%c (3/50) Total observed embryos 30% (46/153) 59% (91/153) 10%c (16/153) Growth and development delay 3% (8/244) 43% (12/28) Eye defect 6% (15/244) 15% (4/28) Skeletal defect 32% (9/28) Heart defect (embryos) 31% (5/16) Infertility (pups) 75% (9/12) Hydrocephalus (pups) 33% (4/12) Ovary cyst (pups, female) 33% (2/6) a Significant deviation from the expected Mendelian ratio (P < 0.01 × 10−9) by chi-square test. b No significant deviation from the expected ratio by chi-square test. c Significant deviation from the expected ratio (P < 0.01) by chi-square test. Wdr11-null mice display features of Holoprosencephaly Wdr11-null mutants exhibited several characteristics of holoprosencephaly (HPE) associated with Hh signal deficiency. Most notable was the small head (microcephaly), with closely spaced eyes (hypotelorism) or single/absent eyes (microphthalmia/ anophthalmia), indicative of failed eye field development (Fig 1C and E). In some cases, Wdr11-null mutants exhibited exencephaly, indicating a neural tube closure defect, without any sign of spina bifida. The 3D reconstructed images of the mutant embryos by high-resolution episcopic microscopy (HREM) revealed facial features often associated with midline defects such as protruding tongue and fusion of medial nasal processes (Fig 1F). Coronal sections of E18.5 Wdr11−/− brain further demonstrated midline craniofacial defects, including a partially divided forebrain and narrow mid-facial region, along with the hypoplasia of the neuroepithelium, optic eminence, olfactory pit, nasal septum and Rathke's pouch. The developing Wdr11-null brain also displayed enlarged ventricles (Fig 1E). We observed a hypoplastic OB with incomplete separation of the telencephalon similar to that in lobar HPE (Fig 1G). Congenital heart defects have been reported in CHH/KS 17. We found a double-outlet right ventricle and ventricular septal defects in E12.5 Wdr11−/− hearts (Fig 1H). Sometimes thoracic skeletal defects and lung airway abnormalities accompanied an abnormal heart (Movie EV1). The observed frequencies of these phenotypes with respect to the genotypes are shown (Table 1). Wdr11 mutants show hypothalamic GnRH deficiency and pituitary dysgenesis The total number of GnRH-positive cells was significantly reduced in Wdr11 KO mice (Fig 2A). Interestingly, in Wdr11−/− brains, a relative accumulation of GnRH-positive cells under the cribriform plate area compared to the forebrain area was observed, potentially suggesting a delayed migration of GnRH neurons (Appendix Fig S3A). Wdr11-null embryos also exhibited dysmorphogenesis of the pituitary gland, such as a bifurcation of the anterior lobe and abnormal shaping of the lumen of Rathke's pouch (Fig 2B). Therefore, we analysed the expression of genes essential for normal hypothalamic–pituitary function using samples from 8- to 10-week-old age-matched males and dioestrous females. Wdr11 KO caused a significant reduction in the expression of Gnrh1, Gnrhr, Lhb, Fshb, Gh and Prl in females. The null males also showed a significant reduction but not in Gnrhr and Fshb (Fig 2C and Appendix Fig S3B), suggesting that female pituitary functions were more severely affected. Figure 2. Wdr11 knockout mouse shows neuroendocrine and metabolic dysfunctions Total numbers of GnRH neurons determined by counting the positive immunoreactivity in every fourth section of serially sectioned whole embryo head at E12.5 (WT, n = 5; Wdr11−/−, n = 5). Data are presented as means ± SEM. Statistical analysis by unpaired Student's t-test (****P < 0.0001). Sagittal sections of the brain at E12.5–E14.5 and coronal sections at E18.5 were imaged after H/E staining to investigate pituitary development. rp, Rathke's pouch; vd, ventral diencephalon; m, mesenchyme; inf, infundibulum; rt, rostral tip; al, anterior lobe; il, intermediate lobe; hy, hypothalamus; 3v, third ventricle. Scale bars, 100 μm. Quantitative RT–PCR analyses of genes expressed in the hypothalamus and pituitary of 8- to 10-week-old males (n = 5) and dioestrous females (n = 5). Data are presented as means ± SEM. Statistical analysis by unpaired Student's t-test (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). GnRHR, GnRH receptor; LH, luteinizing hormone; FSH, follicle-stimulating hormone; GH, growth hormone; PRL, prolactin. Numbers of pups per litter produced after mating with WT B6 females were tracked in one WT and two null male mice for a period of 7 months. One-way ANOVA with Dunnett's post hoc test indicates significantly reduced fertility in the null (***P < 0.001; ****P < 0.0001). Data presented as means ± SEM. A photograph showing the male testes. H/E-stained sections of testes show hypoplastic Leydig cells and vacuolated seminiferous tubules (asterisk) containing few spermatozoa and spermatids in Wdr11−/− (Scale bars, 100 μm). Phase-contrast images of the sperm show morphological abnormalities in the null (Scale bars, 20 μm). A photograph showing the female uteri. H/E-stained sections of the uterine wall and ovaries are shown. FL, follicle; CL, corpus luteum; HC, haemorrhagic cyst. Scale bars, 500 μm. Body length (BL) and weight (BW) were measured, and body mass index (BW/BL, g/cm2) was calculated from the measurements at 1 week (WT, n = 6; Wdr11+/−, n = 6; Wdr11−/−, n = 6) and 25 weeks (WT, n = 8; Wdr11+/−, n = 8; Wdr11−/−, n = 6). Body fat percentages of WT and Wdr11+/− mice at 25 weeks were calculated by dividing fat weight by body weight. Data are presented as means ± SEM. Statistical analysis by unpaired Student's t-test (ns, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Source data are available online for this figure. Source Data for Figure 2 [embr201744632-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint Wdr11 deficiency causes delayed puberty, reproductive dysfunctions and obesity Both male and female null mutants showed hypoplasia of the reproductive organs. Wdr11-null male mice exhibited underdeveloped external genitalia and microphallus, along with a reduced anogenital distance index (Appendix Fig S4A). The timing of balanopreputial separation was significantly (P = 0.00076; F(2,12) = 24.68) delayed in the null (34 ± 0.32 days, n = 5), compared to WT (30 ± 0.55 days, n = 5) or heterozygotes (29.4 ± 0.6 days, n = 5). Wdr11-deficient males had smaller testes with abnormal seminiferous tubules containing fewer spermatozoa and spermatids. Notably, Wdr11 was expressed throughout sperm flagella (Fig EV1B) and loss of Wdr11 caused a high frequency (> 50%) of morphologically abnormal sperm, leading to subfertility or infertility (Fig 2D and E). In females, the null mutants had smaller ovaries and uteri, with thin and poorly differentiated walls. Wdr11−/− ovaries showed arrested follicle development with absent corpora lutea and antral follicles. Widespread atresia and haemorrhagic cysts were observed (Fig 2F). Wdr11−/− females exhibited dysregulated oestrous cycles (Appendix Fig S4B). Wdr11-null mice showed substantial growth retardation in terms of body weight (Appendix Fig S4C). However, because they also had a short stature, their body mass index indicated early-onset obesity, while the heterozygotes showed late-onset obesity with signs of a fatty liver (Fig 2G, Appendix Table S3, and Appendix Fig S4D). Wdr11 is localized to the primary cilium and required for ciliogenesis Since Hh signalling requires functional primary cilia and the phenotypes of Wdr11-null mutants resemble ciliopathy spectrum disorders, we hypothesized that Wdr11 deficiency disrupts normal ciliogenesis. Mouse embryo fibroblasts (MEFs) derived from Wdr11−/− mice exhibited defective ciliogenesis with a significant reduction in the length of the ciliary axoneme and the frequency of ciliated cells, but no defects were found in the formation of the basal body itself (Fig 3A). Since both GnRH neurons and olfactory axons are known to be ciliated in vivo 181920, we examined the effects of Wdr11 KO on the OB and ME. There was significant deprivation of axoneme structures in these organs in Wdr11−/− embryos, indicating disrupted ciliogenesis (Fig EV1A and C). The abnormal choroid plexus present in Wdr11−/− brain, which led to hydrocephalus, contained much fewer epithelial cilia compared to the WT, although the overall axonemal structure appeared normal (Fig 3B). Figure 3. WDR11 is required for ciliogenesis Immunofluorescence images of MEFs derived from E12.5 WT and null embryos. Arl13b stains axoneme and gamma-tubulin (g-TUB) stains the basal bodies of cilia. The percentages of ciliated cells were assessed against the total DAPI-stained cells in random fields. The length of cilia was assessed in 150 cells per genotype by measuring the maximum projection using ImageJ. Data represent mean ± SEM from three independent experiments using two-tailed unpaired t-test (*****P < 0.00001). Scale bar, 50 μm. Choroid plexus sections of 8-week-old brain exhibited morphological alterations and lack of ciliary acetylated tubulin (ACT) staining in Wdr11−/−. Scale bars, 1 mm (main image) and 500 μm (zoomed images). TEM micrographs of longitudinal (1,000×) and horizontal (15,000×) sections of the choroid plexus epithelium show no significant difference in the 9+2 microtubule arrangement of the axoneme. Immunofluorescence analyses of NIH3T3 cells transfected with GFP-tagged WDR11 construct and treated with purmorphamine (Pur), cyclopamine (Cyc), leptomycin B (Lep) and the solvent dimethylformamide (Solv) for 10 h before staining with anti-ACT or anti-g-TUB antibodies followed by DAPI. Scale bar, 10 μm. The percentage of cells showing either nuclear or cytoplasmic localization of WDR11-GFP were quantified in HEK293 cells after each treatment as in (C). Data from four independent experiments, counting 300–400 cells in each experiment, are presented as mean ± SEM with two-way ANOVA followed by Bonferroni's post hoc test (****P < 0.0001). Download figure Download PowerPoint Wdr11 knockdown in zebrafish causes developmental defects associated with aberrant Hh signal and ciliogenesis To further investigate the function of WDR11 during vertebrate development and ciliogenesis, we employed a zebrafish model system where the endogenous wdr11 was knocked down using two non-overlapping antisense morpholinos (MOs) targeting the exon 3–intron 3 (E3I3 MO) and exon 9–intron 9 (E9I9 MO) splice sites, independently injected at the 1- to 2-cell stage. E3I3 MO caused an inclusion of intron 3, resulting in a predicted in-frame stop codon in intron 3 (Appendix Fig S5A). E9I9 MO caused an exclusion of exon 9, predicting a premature stop codon
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