Embryonic and foetal expression patterns of the ciliopathy gene CEP164.

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of inherited genetic disorders that share a defect in the formation, maintenance or functioning of the primary cilium complex, causing progressive cystic kidney disease and other clinical manifestations. Mutations in centrosomal protein 164 kDa (CEP164), also known as NPHP15, have been identified as a cause of NPHP-RC. Here we have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) to perform immunohistochemistry studies on human embryonic and foetal tissues to determine the expression patterns of CEP164 during development. Notably expression is widespread, yet defined, in multiple organs including the kidney, retina and cerebellum. Murine studies demonstrated an almost identical Cep164 expression pattern. Taken together, these data support a conserved role for CEP164 throughout the development of numerous organs, which, we suggest, accounts for the multi-system disease phenotype of CEP164-mediated NPHP-RC.

Introduction

Nephronophthisis-related ciliopathies (NPHP-RC) are a collection of inherited genetic disorders, grouped together by a core defect in the formation, maintenance or functioning of the primary cilium complex [1, 2]. NPHP-RC patients typically present with nephronophthisis, a fibrotic cortico-medullary cystic kidney phenotype, which frequently leads to end stage-renal disease (ESRD) [3, 4]. In some NPHP-RC cases, including Senior-Løken syndrome (SLSN), Alström syndrome (AS), Bardet Biedl syndrome (BBS) and Joubert syndrome (JBTS), patients have retinal dysplasia and degeneration phenotypes, such as Leber congenital amaurosis, which can deteriorate to blindness [J Child Neurol. 1997 ">5-7]. Neurological abnormalities are often present; JBTS patients have midbrain cerebellar vermis hypoplasia, characterised by the “molar tooth” sign on MRI analysis [5, 8], leading to numerous problems including ataxia, hypotonia and breathing abnormalities. Intellectual disability and developmental delay, are also demonstrated throughout the spectrum of NPHP-RC. Additionally, BBS patients are often diagnosed with hypogonadism and/or obesity [9]. Severe NPHP-RC phenotypes, including Jeune syndrome and Meckel Gruber syndrome display skeletal dysplasia, and lethal occipital encephalocele. Consistent with ciliopathy syndromes, polydactyly, liver fibrosis, facial dysmorphism, cardiac abnormalities, hearing loss and type 2 diabetes can present as secondary symptoms [9-11]. Although some disease management therapies are available, notably, there is no cure for NPHP-RC.

There are at least 25 NPHP-RC causative genes currently identified [12], accounting for a molecular genetic diagnosis in around 60% of NPHP-RC patients. Mutations in CEP164, also known as NPHP15, have been identified as a cause of NPHP-RC [1, 9]. These patients have a largely heterogenous clinical presentation. The majority suffer with NPHP and a retinal phenotype, which in some patients causes blindness prior to two years of age. One patient was diagnosed with JBTS, and two others have BBS-like manifestations with an absence of renal problems [1, 9]. It has been proposed that homozygous truncating mutations of CEP164 cause a severe JBTS phenotype, whereas hypomorphic mutations may cause a milder syndromic presentation [1]. Patients with identical CEP164 mutations can display different clinical manifestations, which makes it difficult to understand the pathology and progression of the disease.

CEP164 was first identified and cloned from an adult foetal brain cDNA library [13] and is located on chromosome 11, q23.3. Its largest, most commonly cited isoform is 5,629 bp, encoding the 1,460 amino acid protein (CEP164) (NM_014956) [1, 14]. There is an alternative isoform of CEP164 (NM_001271933), with a 1,455 amino acid product [1, 13, 14]. CEP164 is a centrosomal protein that localises to each of the nine distal appendages of the primary cilia mature centriole, in a microtubule-independent manner [1, 2, 15–22]. This has been demonstrated by super-resolution microscopy techniques in multiple human and murine cell lines. Cell-cycle dependent recruitment of CEP164 to the distal appendages is hierarchical, within a network of other distal appendage proteins. These include CEP83 (mutations in CEP83 cause NPHP18), CEP89, SCLT1 (variants in SCLT1 may be associated with Orofaciodigital syndrome 9), and FBF1. CEP164 is recruited last, defining the proximal end of the transition zone [2, 15, 18, 22–26].

Protein domain analysis has identified that CEP164 has an N-terminal tryptophan-tryptophan (WW) domain, an area of lysine-rich repeats (LR), and multiple serine-glutamine/threonine-glutamine (SQ/TQ) potential phosphorylation sites [2, 14]. There are at least 3 predicted coiled-coil domains [1, 2, 14, 15] and the C-terminal domain is currently undefined (). Multiple fusion protein studies have demonstrated that the CEP164 C-terminal domain is required for localisation of CEP164 to the distal appendages [1, 15]. In some studies, CEP164 has been found within the nucleus [1, 14, 17, 27].

Numerous in vitro CEP164 knockdown studies (siRNA/CRISPR) have demonstrated that CEP164 loss causes aberration of ciliogenesis, with disruption of primary cilia production prior to transition zone formation; notably centriolar structure is not disrupted [15, 23, 25, 28, 29]. Upon initiation of ciliogenesis Rabin 11 imports the GTPase Rabin 8 to the basal body. CEP164 interacts with Rabin 8, facilitated by Chibby 1, to recruit Rab8 positive vesicles to the centrosome [15, 17, 19, 24, 30]. Vesicle docking is required for subsequent basal body anchoring to the plasma membrane and primary cilia development. Additionally, CEP164 forms a complex with tau tubulin kinase 2 (TTBK2) via its N-terminal WW domain. CEP164 recruits TTBK2 to the primary cilia basal body, which allows removal of the centriolar capping protein, CP110, potentially via phosphorylation, and then initiation of intraflagellar transport recruitment with subsequent axonemal extension [1, 15, 29, 31, 32]. The CEP164/Rabin 8 and CEP164/TTBK2 pathways work independently of each other [29]. There are other predicted interactors of CEP164 including dishevelled, NPHP3, NPHP4 and ARL13B, indicating that CEP164 is likely to have other ciliary roles [1, 15, 24, 32].

Several studies have indicated a potential role for CEP164 in ATM/ATR-mediated DNA damage response (DDR) and UV-induced nucleotide excision repair pathways, however results are conflicting [14, 17, 33]. Likewise, data supporting a role for CEP164 in cell-cycle regulation is inconsistent [1, 2, 14, 15, 25]; both of these CEP164 functions however require further validation.

CEP164 has numerous orthologs, including M.musculus, D.melanogaster, C.reinhardtii and D.rerio (). The murine ortholog of CEP164 is a 30-exon gene, located on chromosome 9, qA5.2, which shares 77% identity to the full-length human CEP164. It encodes a 1333 amino acid protein, which shares 58% identity to the 1,460 amino acid human CEP164 protein. [34, 35]. Like human CEP164, murine Cep164 may have multiple isoforms.

Given CEP164 mutations cause NPHP-RC, we sought to determine the expression of CEP164 throughout human and murine development, particularly focusing on the cerebellar-retinal-renal phenotype. Collaboration with the Medical Research Council (MRC) Human Developmental Biology Resource (HDBR) allowed the procurement of human embryonic and foetal samples, which were compared to expression data from the murine 129/OlaHsd-Cep164tm1a(EUCOMM)Wtsi/+ gene trap model.

Materials and methods

Study approval

The study was conducted with full ethical approval and consent. Ethical approval was obtained from the National Research Ethics Service Committee North East–Newcastle & North Tyneside 1 (08/H0906/21+5). Human embryonic and foetal tissue samples were collected with appropriate consent and ethical approval, via the Medical Research Council (MRC) Wellcome trust-funded Human Developmental Biology Resource (HDBR). Informed and written consent was gained prior to the collection of control urine sample for isolation of human urine derived renal epithelial cells (hURECs). All methods were performed in accordance with the relevant ethical guidelines and regulations. Animal experiments were performed under Home Office Licences (United Kingdom) in accordance with the guidelines and regulations for the care and use of laboratory animals outlined by the Animals (Scientific Procedures) Act 1986. Protocols conducted were approved by the Animal Ethics Committee of Newcastle University and the Home Office, United Kingdom.

Mouse genetics

C57BL/6NTac-Cep164 mice, generated for the International Mouse Phenotyping Consortium Initiative, were obtained from MRC Harwell [36]. These were backcrossed onto a 129/Ola-Hsd background, forming mice heterozygous for the gene trap, 129/OlaHsd-Cep164. 129/OlaHsd-Cep164 heterozygous (HET) mice were mated with wild type (WT) 129/OlaHsd-Cep164 mice, gaining pups HET:WT 1:1. Upon pre-mRNA processing of the Cep164 allele, exon 3 of Cep164 splices into the LacZ/Neomycin cassette, within intron 3, which introduces a premature termination codon and subsequent Cep164 null allele (). The LacZ cassette contains an internal ribosomal entry site (IRES), allowing translation of a beta-galactosidase fusion gene upon activation of the native Cep164 promoter, thus acting as a Cep164 reporter gene. Upon addition of X-Gal substrate, beta galactosidase hydrolyses X-Gal forming 5-bromo-4-chloro-3-hydroxyindole-1, which oxidises to a blue precipitate, 5,5’-dibromo-4,4’-dichloro-indigo-2. HET 129/OlaHsd-Cep164 mice were used for X-Gal Cep164 expression studies, as they are phenotypically normal, WT mice were used as littermate controls.

Cep164 genotyping

Ear or tail biopsies (embryos) were lysed for 1 h, at 95°C, in 50 μl alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 12) and then neutralised in 50 μl neutralising buffer (40 mM trizma hydrochloride, pH 5–5.5). A PCR reaction using GoTaq G2 DNA Polymerase (Promega) for Cep164, was completed using the following primers flanking the second loxp site; F1 5' CTC CCA CAG TGA CAA ATG CC 3', R1 5' GGT AGT TGT TAC TTC TGT CAG 3' (Eurofins Genomics). Expected amplicon sizes are as follows; WT (141 bp), homozygous (Hom) (163 bp), HET (141 bp and 163 bp). PCR products were run on a 1.5% agarose gel, with GelRed Nucleic Acid GelStain (1:10,000) (Biotium) at 150V for 45 min. To confirm correct genotyping, representative samples were Sanger-sequenced (GATC-BIOTECH).

Murine tissue collection, fixation, sectioning and staining

Murine tissue (kidney, brain, eye, heart, lung, liver, testes) was collected at P0.5/P1.5, P15.5 and P29.5/P30.5, using a standard dissection procedure. Tissues were fixed in 0.2% glutaraldehyde fix (0.2% glutaraldehyde, 2mM MgCl2, 5mM EGTA in 1 x phosphate buffered saline PBS) for 90 min at 4°C, washed in PBS and then stored in 15% sucrose in PBS overnight at 4°C. Tissues were transferred to 30% sucrose in PBS and incubated at 4°C overnight or until samples sunk. Tissues were frozen in OCT compound, and stored at -80°C.

Sections of tissues were cut (10–12 μm) using a cryostat and mounted on charged glass slides. These were incubated in 0.2% glutaraldehyde fix (0.2% glutaraldehyde, 2mM MgCl2, 5mM EGTA in PBS) for 5 min, washed in PBS for 10 min, and then washed in X-Gal wash (2 mM MgCl2, 0.01% Sodium Deoxycholate, 0.02% NP-40 in PBS) for 10 min. Slides were incubated with X-Gal stain (1mg/ml of X-Gal DMSO in 2 mM MgCl2, 0.01% Sodium Deoxycholate, 0.02% NP-40, 5mM Potassium Ferricyanide, 5mM Potassium Ferrocyanide in PBS) at 37°C in the dark, until the blue precipitate stain intensity did not further increase or WT littermate controls showed endogenous beta galactosidase staining. Slides were washed in PBS and then dehydrated to 100% ethanol before clearing in Histoclear II (National Diagnostics) and mounting in DPX mounting medium (Sigma-Aldrich). Slides were imaged using the SCN400 Side Scanner (Leica).

Murine embryo collection, wholemount fixation and staining

Murine embryos were collected at E9.5, E10.5 and E12.5 using a standard dissection procedure. Embryos were fixed in 0.2% glutaraldehyde solution (0.2% glutaraldehyde, 2mM MgCl2, 5mM EGTA in PBS) for 1 hour on ice and washed with X-Gal wash (2 mM MgCl2, 0.01% Sodium Deoxycholate, 0.02% NP-40 in PBS) prior to overnight storage at 4°C. Embryos were incubated in X-Gal stain, consisting of 25mg/ml of X-Gal/DMSO solution in a 1 in 25 dilution of X-Gal staining buffer (2 mM MgCl2, 0.01% Sodium Deoxycholate, 0.02% NP-40, 5mM Potassium Ferricyanide, 5mM Potassium Ferrocyanide in PBS) at 37°C in the dark; incubation was completed once the blue precipitate stain intensity did not further increase or WT littermate controls started to show endogenous beta galactosidase staining. Embryos were washed in 1x PBS and dehydrated in 70% ethanol, prior to imaging using a Leica Man Stereomicroscope on the Axiovision software.

Human tissue collection, fixation and processing

Human embryonic and foetal tissues were obtained from the MRC Wellcome Trust-funded Human Developmental Biology Resource (HDBR). CS23 whole embryos were fixed in 4% paraformaldehyde (PFA), for 72 hours, with an incision in the skull down the sagittal plane and a slit in the abdomen at the umbilicus. Tissues were stored in methacarn prior to further processing. This protocol was used for processing of foetal eye, brain and kidney; the embryonic and foetal kidney were cut sagittally, to allow the fixative to penetrate. Human embryonic and foetal tissues were washed in increasing concentrations of ethanol, and then incubated in 3 changes of xylene before embedding in paraffin wax. Sections, on positively charged glass slides, were de-waxed in two washes of xylene, and then rinsed in two changes of absolute ethanol. Slides were incubated in methanol peroxide solution (0.5% H2O2) for 10 min to block endogenous peroxidase. Slides were rinsed in tap water and then antigen retrieval was completed using citrate buffer. After rinsing in Tris-buffered saline (TBS), slides were incubated with 10% goat serum (Vector) for 10 min at room temperature and then incubated with the primary rabbit anti-CEP164 (Human Protein Atlas, HPA37606) or rabbit anti-PAX6 (Covance, PRB-278-P-100) in goat serum with TBS overnight at 4°C (). After washing with TBS, slides were incubated with the goat anti rabbit BA-1000 biotinylated secondary antibody (Vector Laboratories) diluted in goat serum with TBS (1/500), for 30 min at room temperature. The secondary was washed with TBS and then slides were incubated with VECTASTAIN Elite ABC kit PK6100 tertiary complex (Vector Laboratories), for 30 min at room temperature. After TBS washes, the stain was developed for 10 min at room temperature using ImmPACT DAB peroxidase substrate (SK-6100) solution (Vector Laboratories). The slides were washed thoroughly in water, and counterstained with haematoxylin, dehydrated, cleared and mounted.

Due to the presence of endogenous biotin in kidney, an alternative two-step method was used for foetal kidney samples. Samples were incubated with a HRP goat anti-rabbit IgG peroxidase secondary (Vector) (1/500). After TBS washes, the stain was developed using ImmPACT DAB peroxidase substrate (SK-6100), as described above. No primary control sections were utilised as negative controls, anti-PAX6 was utilised as a positive control for the brain and cerebellum (). Slides were imaged using the SCN400 Side Scanner (Leica).

Immunofluorescence of human urine-derived renal epithelial cell

Human urine derived epithelial cells (hURECs) were isolated and cultured as described in [37]. Immunofluorescence staining with rabbit anti-CEP164 (Human protein atlas, HPA37606) and mouse anti-ARL3B (Proteintech, 66739-1-1g), were completed as per the protocol shown in [38, 39], however primary antibodies were incubated overnight at 4°C. Cells were then mounted in Vectashield (Vector Laboratories Ltd, H-1200). Slides were imaged using the Zeiss axioimager.

Image analysis

Images of human and murine tissues were analysed using the SCN400 Slide scanner software (Leica). Immunofluorescence staining of hURECs were imaged using the Zeiss axioimager, and processed using Zen Pro 2.3. Figs 1–4, and S3–S7 Figs were generated using Adobe Photoshop CS3 Extended.

Fig 1

Expression of CEP164 throughout human and murine renal development.

Human renal development (A-D), 8 PCW (A), 14 PCW (B), 18 PCW (C) respectively and 18 PCW no primary control (D). In the developing human kidney, CEP164 expression is seen in the apical membrane of the metanephric renal vesicles (A.II, B.II), s-shaped bodies (B.II, C.I, C.III) and developing renal tubules (A.IV.VII, B.IV.VII). From 14 PCW CEP164 expression is seen at the apical membrane of all developing tubular segments including the distal and proximal tubules (B.IV, C.IV) and loop of Henle segments (B.VII, C.VII). CEP164 expression is seen in the cells of the ureteric bud (A.III, B.III, C.II), and the subsequent collecting duct at both the apical and basal membrane (A.VII, B.VII, C.VII). CEP164 expression is seen in the glomerulus of the developing immature renal corpuscle (A.V, B.V, C.V), and weakly in the matured renal corpuscle (A.VI, B.VI, C.VI). No primary controls demonstrate no background DAB staining, as represented by 18 PCW time-point (D.I-VII). Murine postnatal renal development (E-G), P1.5 (E), P30.5 (F) and P30.5 WT control (G). In the murine kidney Cep164 expression is present in the developing renal vesicles (E.I.II) and ubiquitously throughout subsequent nephron renal tubules (E.IV, F.I.III.IV). Cep164 expression is seen in the glomerulus of the developing renal corpuscle (E.V), but expression seems to be lost with maturity (E.VI, F.II). No endogenous beta galactosidase staining is present in the murine kidney, as shown by the representative image WT control at P30.5 (G). All scale bars represent 100 μm. Cap mesenchyme (CM), collecting duct (CD), distal tubule (DT), loop of Henle (LH), postnatal day (P), post conception weeks (PCW), proximal tubule (PT), renal corpuscle (RC), renal tubule (RT), renal vesicle (RV), s-shaped body (SSB), ureteric bud (UB).

Fig 4

CEP164 expression in secondary organs throughout human and murine development.

Human 8 PCW (A). Murine (B-D), P0.5 (B), P15.5 (C), P30.5 (D). In the developing human, lung CEP164 expression is seen in the respiratory epithelial lining of the bronchi and bronchioles, with weaker expression in the smooth muscle and alveoli (AI.II.III). In the developing murine lung, Cep164 is expressed in the respiratory epithelial lining of the bronchi and bronchioles (C.I.IV, D.I.IV), with additional Cep164 expression seen in the respiratory epithelial cells lining the trachea and the tertiary bronchioles (C.I.II.III.V, D.I.II.III.V.VI). Cep164 expression is seen in murine alveoli at P15.5 (C.VI), which seems to be lost with maturity (P30.5) (D.VII). Cep164 expression is seen within the cartilage of the trachea (C.II.III, D.II.III). In both the human (A.IV.V) and murine hearts (C.VII.VIII, D.VIII.IX), expression is seen the developing cardiomyocytes. In the human gastrointestinal tract CEP164 expression is seen in the inner mucosae squamous epithelial cell layer, muscularis mucosae cell layer and the external muscularis cell layer (A.VI). In the developing human gonads, CEP164 expression is seen in the germline epithelium and seminiferous cord (A.VII.VIII). In the developing murine testes, at P15.5, Cep164 expression is seen in the seminiferous tubules, specifically the smooth muscle cells spermatogonia, spermatocytes and most strongly in spermatids (C.IX.X.XI); expression in leydig cells and connective tissue can also be seen (C.X). At P30.5, Cep164 expression is defined to the spermatogonia, spermatocytes and spermatids (D.X.XI.XII). CEP164 expression is seen in the dorsal root ganglia of the human spinal cord, (A.I.IX) with weaker expression also present in the vertebrae primordia (A.I.IX) and bone primordia (A.I.X). Cep164 expression is seen in the murine developing costal cartilage (B.I.II), with weaker expression in intercostal muscle (B.I.III). The human foetal liver demonstrates some evidence of CEP164 expression (A.XI), however this is undetermined due to high endogenous peroxidase activity in the liver. The developing murine liver shows Cep164 expression in epithelial cells lining the hepatic portal veins at P15.5 (C.XII.XIII), this is not seen by P30.5. There is no expression in hepatocytes (C.XIV, D.XIV). Aveoli (Av), alveoli primordia (Av Pri), bile duct (BD), bone primordia (BP), bronchiole (Br), cardiomyocyte (CM), cartilage (Ca), connective tissue (CT), costal cartilage (CC), dorsal root ganglia (DRG), epithelial (Ep), gastrointestinal tract (GI), germline epithelium (GE), gonad (Go), heart (Ht), hepatocytes (Hep), intercostal muscle (IC), kidney (Ki), lamina propria (LM), liver (Li), lung (Lu), muscularis externa (ME), muscularis mucosae (MM), respiratory bronchiole (R), seminiferous cord (SeC), seminiferous tubule (ST), smooth muscle (SM), spermatids (Sp), spermatocytes (SC), spermatogonia (Spg), squamous cell mucosae (Mc-sq), submucosae (SB), trachea (Tr), terminal bronchi (TB), vertebrae (Vt).

CEP164 expression in the developing human and murine retina.

Human Retina (A), 11 PCW (A.I), 14 PCW (A.II), 19 PCW (A.III) and 19 PCW no primary control (A.IV). In the developing human retina, weak CEP164 expression is seen in the nerve fibre layer (NFL) and ganglion cell layer/inner plexiform layer (GCL/IPL) (A.I) and strong expression in outer neuroblastic (ONBL) photoreceptor precursors (black arrow) (A.I). By 14 PCW, CEP164 expression is seen in the developed inner plexiform layer (IPL) and the developing photoreceptor layers (A.II). At 19 PCW, CEP164 expression is seen in the nerve fibre layer (NFL), inner plexiform layer (IPL), outer plexiform layer (OPL) and photoreceptor layer, with enhancement in the inner photoreceptor segments (IS) (A.III). No background staining is present in the no primary controls (A.IV). Murine Retina (B), P1.5 (B.I), P15.5 (B.II), P29.5 (B.III) and P29.5 WT control (B.IV). In the developing murine retina, Cep164 expression is seen in the inner plexiform layer (IPL), ganglion cell layer (GCL) and outer neuroblastic layer (ONBL) (B.I). At P15.5, Cep164 expression is seen in the ganglion cell layer (GCL), the outer plexiform layer (OPL) and inner segment (IS) of the photoreceptor layer (B.II). There is also punctate expression in the inner plexiform layer (IPL) and edges of the nuclear cell layers (B.II). Retinal pigment epithelium (RPE) also shows Cep164 expression (B.I, B.II). This murine retinal expression patterning is maintained once retinal layers have been formed (B.III). Controls demonstrate no endogenous beta galactosidase activity in the murine retina (B.IV). Ganglion cell layer (GCL), inner nuclear layer (INL), inner plexiform layer (IPL), inner segment (IS), nerve fibre layer (NFL), outer neuroblastic cell layer (ONBL), outer nuclear layer (ONL), outer plexiform layer (OPL), outer segment (OS), photoreceptor segment layer (PS), retinal pigment epithelium (RPE).

CEP164 expression in the developing human and murine brain, focusing on the hindbrain.

Developing human brain (A-F), 8 PCW (A-B), 8 PCW no primary control (C), 16 PCW hindbrain (D),18 PCW hindbrain (E) and 16 PCW hindbrain no primary control (F). In the developing human brain at 8 PCW, CEP164 expression is seen in the neuroepithelium surrounding the telencephalon (A.I.II.VI, B.I.VI), diencephalon (A.I), mesencephalon (A.I), metencephalon (A.I.V) and myencephalon (A.I.VIII, B.I.IV.V), with defined expression in apical neuroepithelium cells lining the brain ventricles (A.I.VI.VIII, B.I.VI.VII.VIII) demonstrated by black arrows. Defined expression is present in the layers of the telencephalon (A.VI, B.VI). There is defined expression of CEP164 in the human brain midline (B.IV.V) and the nasal epithelium (B.IX). At 8PCW, the choroid plexus shows strong CEP164 expression in the ependymal cells (A.II.III.IV, B.II.III), with seemingly weaker expression in the choroid plexus pia matter (A.IV, B.II.III). The ependymal expression is maintained at 16 PCW and 18 PCW (D.I.V, E.I.V). CEP164 expression is seen in the migrating molecular cell layer of the developing cerebellum (D.I.II), which seems to be lost with molecular cell layer maturation (E.I.II). In the human hindbrain the apical membrane of the pons and the cerebellum demonstrate defined CEP164 expression (D.III,IV), which appears to be lost with maturation (E.III.IV). Weak CEP164 expression is seen in the human medulla oblongata (D.VI, E.VI) but not in the white matter (D.VII, E.VII). Developing murine brain (G-K), P0.5 brain (G) P0.5 hindbrain (H), P15.5 (I),P30.5 (J) and P30.5 WT control (K).Cep164 expression is widespread throughout the developing murine brain (P0.5-P30.5) (G.I, I.IV, J.IV) with expression in the cortex, striatum and cerebrum of the maturing cerebral cortex (G.I.VI, I.IV.V, J.IV.V) expression in the midbrain (G.I, I.IV.VII, J.IV.VI) and thalamus (I.IV.VI). Cep164 expression is strong and defined in the ventricular neuroepithelium (I.VII.VIII). The murine choroid plexus demonstrates strong Cep164 expression, however it is not defined to a specific cell type (G.III, H.IV). In the murine hindbrain, at P0.5, Cep164 is expressed in the migrating molecular layer of the cerebellum (G.II.IV, HI.II), this is maintained with expression also in the molecular layer, ganglion cell layer and Purkinje cell layer at P15.5 and P30.5(I.I.II,J.I.II). Cep164 expression is seen in the in the murine pons (G.VII, H.V, J. VII) and medulla oblongata (G.VIII), and weakly in the cerebellar white matter (G.IV, H.VI, I.III), which seems to be lost with maturation (J.III). The representative P30.5 control demonstrates no endogenous beta galactosidase staining (K). Aquaduct (Aq), axon tract (AT), cerebellum (Ce), cerebral cortex (CC), cerebral hemisphere (CH), cerebrum (Cr), choroid plexus (CP), cortex (Cor), cortical plate (CorP), diencephalon (Di), ependymal cells (Ep), fourth ventricle (FV), ganglion cell layer (GCL), inner plexiform layer (IPL), intermediate zone (IL), lateral ventricle (LV), marginal layer (MaL), medulla oblongata (MO), mesencephalon (Mes), metencephalon (Met), midbrain (MB), midline (Mi), molecular cell layer (MCL), myencephalon (Mye), nasal epithelium (Na), outer neuroblastic layer (ONBL), pia (P), pons (PO), purkinje cell layer (PCL), retinal pigment epithelium (RPE), striatum (St), sub-ventricular layer (SVL), telencephalon (Tel), thalamus (Th), third ventricle (TV), ventricular layer (VL), ventricular surface (VS), white matter (WM).

Results

CEP164 expression during human and murine renal development

In the human embryonic and foetal kidney, immunohistochemical staining demonstrates that CEP164 is present in the metanephric epithelial-derived renal vesicles (), comma-shaped bodies () and subsequent s-shaped bodies (), during all time points analysed (8PCW-18PCW). This expression pattern is maintained throughout nephrogenesis, with CEP164 present in the primitive nephron tubule (8PCW) (), but also in more defined proximal and distal nephron segments, as well as cells of the loop of Henle, as seen from 14 PCW (). Specifically, CEP164 expression is enriched at the apical membrane of epithelial cells lining the developing nephron lumen, during all developmental time points analysed (8PCW- 18PCW) (). CEP164 does not appear to be expressed, or is expressed very weakly, in the metanephric cap mesenchyme ().

In the developing human kidney, CEP164 is expressed in the glomerulus of the renal corpuscle at 8 PCW-18 PCW, indicating podocyte CEP164 expression (). The expression data indicates that CEP164 expression is reduced as glomeruli mature; this is most clearly seen at 18 PCW (C.V.VI).

The human embryonic/foetal ureteric bud, which forms the collecting duct, is derived from the metanephrogenic diverticulum. Both the ureteric bud and collecting duct also show CEP164 expression in the apical membrane of the epithelial cells lining the tubular lumen (8PCW-18PCW) (. CEP164 is also expressed at the basolateral membrane of the collecting duct tubule (8PCW-18PCW). CEP164 expression is not present in the human renal interstitium (). Lack of background DAB staining in no primary controls indicate that CEP164 expression in the human kidney was specific ( and ).

In the murine kidney (129/OlaHsd-Cep164), the LacZ reporter assay demonstrates that Cep164 expression correlates with human kidney CEP164 expression. Correspondingly, Cep164 is expressed in the developing murine renal vesicles, s-shaped bodies (P1.5) () and the subsequent renal tubules, including nephron segments in both the cortex and medulla (P30.5) (). The staining indicates that there is potentially cell-specific expression within each of the tubular segments. Cep164 is also expressed in the glomeruli of the renal corpuscle at P1.5, which is not present in mature glomeruli at P30.5 (). Likewise, Cep164 is expressed in the developing mesonephric ureteric bud (). Wildtype controls demonstrate that there is very low endogenous beta galactosidase expression in the kidney ( and ).

CEP164 expression during human and murine retinal development

In the developing human retina, CEP164 expression is widespread yet defined (). In early retinal development (11 PCW) CEP164 is expressed weakly in the developing nerve fibre layer (NFL) (fibrous extensions from the optic nerve) and nerve fibres in the differentiating ganglion cell layer (GCL)/inner plexiform layer (IPL) (). The basally located layer of cone precursors, which has differentiated from the outer neuroblastic cell layer (ONBL), also demonstrates strong CEP164 expression (). By 14 PCW, CEP164 expression is defined to the NFL, the IPL and the photoreceptor layer which now has both developing rods and cones (). Later in development, 19 PCW, once all of the primitive retinal layers have formed, CEP164 expression is maintained in the nerve fibres of the NFL, the ganglionic and inner nuclear nerve fibril synapses of the IPL, and the inner segments (IS) and outer segments (OS) of the photoreceptor cell layer (). At 19 PCW, CEP164 expression is also present in the developing outer plexiform layer (OPL) which contains nerve fibril synapses between the inner nuclear layer (INL) and the outer nuclear layer (ONL). CEP164 expression in the retinal pigment epithelial layer (RPE) cannot be defined due to its natural dark colouring, although no endogenous background staining is present in the other retinal layers ( and ).

Murine Cep164 retinal expression in 129/OlaHsd-Cep164 largely corresponds to the human CEP164 expression patterns, with expression maintained in the developing IPL, OPL and PS layer (P1.5-P29.5) (). However, there are some clear differences. Cep164 is expressed in retinal precursor cells throughout the murine ONBL, which in the developing human retina is restricted to the cone precursor cells (). Additionally, strong Cep164 expression is present and maintained throughout the GCL (P1.5-P29.5) (. Cep164 is also expressed sparsely in some cells of the INL and ONL, however these are at the boundaries of the plexiform layers (P15.5-P29.5) (. Notably, at later stages of development, Cep164 expression is clearly defined to the IS of the photoreceptor cell layer (P15.5-P29.5) (. Additionally, the RPE demonstrates strong, cell-specific Cep164 expression throughout development (P1.5-P29.5) (. WT controls demonstrate no endogenous X-Gal staining ( and ).

CEP164 expression during human and murine neuronal and cerebellar development

CEP164 is expressed widely throughout human brain development (8PCW-18PCW) (). At 8PCW CEP164 is strongly expressed in the neuroepithelium of the developing telencephalon (Tel) (), which is defined in cells lining the lateral ventricle (LV), including the ventricular layer (VL), subventricular layer (SVL), cortical plate (CorP) and marginal layer (MaL), (). At 8 PCW, CEP164 is also present in the developing neuroepithelium of the diencephalon (Di) (mesencephalon (Mes) (), metencephalon (Met) () and myencephalon (Mye) (), with expression strongest in the apical epithelium lining the brain ventricles as highlighted by a black arrow (). At 8PCW there is strong, well-defined CEP164 expression in the midline of the myencephalon (), as well as cells of the nasal epithelium (). Throughout human brain development (8PCW-18PCW) the choroid plexus demonstrates strong CEP164 expression, specifically in the ependymal cells, and more weakly in the choroid plexus pia matter (. The developing human cerebellar folds show defined CEP164 expression in the migrating molecular cell layer at 16 PCW (), but this seems to be reduced by 18PCW (). CEP164 is expressed at the apical membrane of the ventricular surface of the pons and cerebellum () as well as the myencephalon, specifically in the axon tracts of the medulla oblongata ().This expression pattern can be seen from 8PCW to 18 PCW, however CEP164 expression levels seem much weaker by 18 PCW. At 8PCW CEP164 is expressed in the white matter (), but this appears to be lost with maturation of the cerebellum (). Notably the human foetal brain negative controls demonstrate very low background staining, specifically in the choroid plexus region (). A PAX6 antibody was used as a positive control ().

Corresponding with the developing human brain, Cep164 is expressed in the neuroepithelium of the murine telencephalon (129/OlaHsd-Cep164) (cerebral hemisphere) (), diencephalon (), mesencephalon (midbrain) (), metencephalon (pons) () and myencephalon (medulla oblongata) at P0.5 (). At later stages of development (P15.5-P30.5), clear Cep164 expression can be seen throughout the brain including the cerebrum and striatum of the cerebral cortex (), the thalamus of the diencephalon (), the midbrain () and hindbrain pons and medulla oblongata (). Additionally, correlating with human foetal expression, Cep164 is expressed strongly in neuroepithelium lining the brain ventricles, including the third ventricle, fourth ventricle and cerebral aquaduct ().

At P0.5 to P15.5 Cep164 is expressed in the migrating molecular cell layer and Purkinje layer of the cerebellum ( Later in development, Cep164 is also expressed in the ganglion cell layer (P30.5) (). Cep164 is also expressed at the apical membrane of the ventricular surface of the cerebellum at P0.5 and P15.5 (). Unlike the human cerebellar expression, Cep164 is expressed in the white matter of the cerebellum which is present, although seems to be reduced by P30.5 (). Cep164 is also expressed in the ependymal cells of the choroid plexus, which is demonstrated in P0.5 sections (). Murine controls demonstrate that there is no endogenous beta galactosidase expression in the murine brain, apart from low endogenous staining in the choroid plexus at P0.5 ( and ).

CEP164 expression during human and murine development in other tissues

CEP164 is expressed widely throughout the developing human embryo at 8PCW (.). In the 8PCW lung, CEP164 is expressed most strongly in the epithelial cells lining the lumen of the bronchi and bronchioles (). Weaker CEP164 expression is also present in the bronchiole smooth muscle, and the alveoli primordia (). In the 8PCW developing heart, CEP164 is expressed in cardiomyocytes (). The gastrointestinal tract also shows strong defined CEP164 expression in the inner mucosa squamous epithelium cell layer, muscularis mucosae cell layer and the external muscularis externa layer at 8PCW (). At 8PCW in the developing gonads, CEP164 is expressed weakly in the germline epithelium, but strongly in the seminiferous cord tubules (). Additionally, CEP164 is expressed strongly in the dorsal root ganglia of the spinal cord, with weak expression in the developing vertebrae (). CEP164 is also expressed in developing bone primordia of the limbs at 8 PCW (). Due to endogenous beta galactosidase activity the liver () it cannot be determined whether CEP164 is expressed in human embryonic hepatocytes. The other tissues show no endogenous beta galactosidase staining ().

Murine postnatal tissues also demonstrate widespread Cep164 expression. At P0.5 Cep164 is expressed in the developing costal cartilage primordia and intercostal muscles (). Correlating with the human lung, in the developing murine P15.5 and P30.5 lung, Cep164 is present in the epithelial cells lining the trachea, bronchioles and terminal/respiratory bronchioles (), as well as the smooth muscle and cartilage of the trachea and bronchi () Weak Cep164 expression is seen in the alveoli at P15.5 (), which seems to be reduced by P29.5 (). Like human embryonic tissues, Cep164 is present in cardiomyocytes at P15.5 and P30.5 (). In postnatal P15.5 murine testes, Cep164 is expressed in the connecting tubules and Leydig cells ( In the seminiferous tubules, Cep164 is expressed in the smooth muscle cells and weakly in developing spermatogonia and spermatocytes (). Strong Cep164 expression is present in the spermatid tails (). By P30.5 Cep164 expression is defined to developing spermatogonia, spermatocytes, and spermatids (). At P15.5 Cep164 is expressed strongly in cells lining the ciliated cholangiocyte cells of the bile duct, however hepatocytes do not show any expression (). There is no Cep164 expression present in the murine liver at P30.5. X-gal staining of WT tissues, indicate that there is no endogenous beta galactosidase activity in the costal cartilage and intercostal muscles, cardiomyocytes, testes) and hepatocytes ( There is low endogenous beta galactosidase activity in the bronchioles at P15.5, but not at P30.5 ().

Wholemount Cep164 expression studies (E9.5, E10.5, E12.5), demonstrate that Cep164 expression is widespread throughout murine embryonic development, including the bronchial arches and organ primordia, as well as the developing CNS, heart and limbs (). Taken together this indicates that CEP164 expression is widespread throughout human and murine development in tissues beyond the cerebello-retinal-renal structures associated with typical disease phenotypes.

Discussion

In this study we have described the expression of CEP164 in the developing human embryo and foetus, utilising immunohistochemistry, focusing upon the kidney, retina and cerebellum. Conservation of CEP164 expression was explored using a LacZ gene trap assay to characterise Cep164 expression in corresponding murine tissues (129/OlaHsd-Cep164). Notably, during murine development, the kidney, retina and cerebellum continue to develop postnatally. This reflects the chosen murine postnatal timepoints in this study, corresponding to estimated human embryonic and foetal developmental timepoints () [40-57].

Our results demonstrate that during human and murine development CEP164 is expressed widely, in multiple organs. Notably, CEP164 expression is clearly defined within tissues. In the developing human kidney, CEP164 is expressed in the apical epithelium membrane of metanephric-derived renal vesicles and subsequent nephron tubules (8PCW-18PCW), correlating with the presence of primary cilia [58]. Primary cilia are vital in the kidney for mechanosensation and chemical signal transduction, which is required for orientated cellular divisions during development and kidney maintenance. As CEP164 expression seems to be low/not present in the cap mesenchyme, it could be speculated that CEP164 expression is switched on during mesenchymal-epithelial transition, which coincides with the formation of the primary cilium during establishment of apical-basal polarity, cell junctions and lumen formation [58]. CEP164 is expressed within the glomerulus of the renal corpuscle, and there appears to be a reduction in CEP164 expression with maturity. Interestingly, this correlates with the loss of primary cilia in podocytes seen with glomeruli maturity in rats [59]. It could be postulated that in the developing human kidney, CEP164 expression is lost in maturing glomeruli due to a similar mechanism [59]. This could protect against overstimulation of calcium-mediated signalling due to an increase in glomerular filtration rate with development [59]. CEP164 is also expressed in the ureteric bud, which develops into the primary ciliated collecting duct network (8PCW-18PCW), consistent with numerous IMCD3 expression studies [1, 19, 20, 25, 32]. The human CEP164 expression pattern is conserved in the murine kidney (P0.5-P29.5); there may be cell-specific Cep164 expression within nephron tubular segments, however this needs to be further studied.

Although our data is focused upon development, the human protein atlas indicates that CEP164 expression is maintained in the adult kidney [60]. A combination of P30.5 expression data and previous wholemount Cep164 expression studies also indicate that Cep164 expression is maintained in the adult mouse kidney [36]. Together this suggests that CEP164 may have roles in both normal renal development and maintenance of kidney function in the human and mouse. Aberrant CEP164 activity in the kidney could contribute to abnormal primary ciliary function, causing dysregulation of cell division and cell signalling, leading to cystogenesis, which has been suggested in a recent study using murine Cep164 collecting duct cell knockdown [61]. This is consistent with the NPHP-RC phenotype present in most patients with CEP164 mutations [1].

Retinal photoreceptor cells have a specialised connecting primary cilium between the inner and outer segment of the photoreceptors. It therefore seems reasonable that CEP164, a distal appendage centrosomal protein, is expressed in photoreceptor segment in the developing human and murine retina. CEP164 expression has been previously identified in the murine connecting cilium, further confirming our results [1, 62]. In the murine retina, Cep164 expression is defined to the inner segment of the photoreceptor layer by P15.5. This correlates with previous preliminary Cep164 In Situ hybridisation studies [63]. Combining human and murine data, it can be suggested that the Cep164 is transcribed and translated in the inner segment but then the CEP164 protein is transported to the outer segment of the photoreceptor cells. It can be hypothesised that with abnormal functioning/loss of CEP164, there could be atypical outer segment formation, which could lead to the accumulation of phototransduction proteins that could trigger cell loss, leading to a retinal degeneration phenotype, as seen in some CEP164 NPHP-RC patients [1, 9, 47].

Multiple studies have suggested that primary cilia are present and functional in neurones [64]. This could explain why CEP164, a cilial protein, is expressed in other retinal layers. It is also well established that CEP164 is present at the basal body of RPE cells, clarifying the RPE expression results from our study [1, 2, 15, 17, 23].

Primary cilia are vital in the developing brain for sonic hedgehog (SHH) signalling, which is needed for proliferation of neuronal granular cell precursors in vertebrates, being a major driver of cerebellar precursors [45, 46]. SHH is also required for the cell-specific expansion of postnatal progenitors [64]. Wnt signalling, transduced by the primary cilia, is also required for neuronal patterning, cell proliferation and neuronal migration [46-48]. In the adult nervous system, primary cilia are thought to be required in stem cell regulation and tissue regeneration, however the full extent of primary cilia function in the adult central nervous system is still to be determined [64, 65]. With widespread expression of the cilia protein CEP164 in the developing human and murine brain, it can be suggested that CEP164 has a functional role in human and mouse brain development. Potentially, aberrant function of CEP164, may lead to brain dysplasia via abnormal primary cilia functioning in neuronal precursors. Some CEP164 NPHP-RC patients show neurological phenotypes, including abnormal developmental delay, intellectual disability and in one patient, cerebellar vermis aplasia, an archetypal feature of Joubert syndrome. Another patient also experiences seizures [1, 9].

Leptin receptors are situated in or near the primary cilia of the choroid plexus, and in the hypothalamus [66]. Leptin signalling is vital in the hypothalamic satiety pathway. Patients with mutations in centrosomal BBS proteins demonstrate hyperleptinemia due to leptin resistance, which causes hyperphagia and subsequent weight gain. It could be hypothesised that CEP164, which is expressed in the developing choroid plexus of the human (8 PCW, 16 PCW, 18PCW) and murine brain (P0.5), may localise to the choroid plexus primary cilium, and be indirectly involved in leptin receptor localisation or functioning. This could contribute to the obesity phenotype seen in some CEP164 NPHP-RC patients [1, 9].

Ependymal cells of the choroid plexus, which contain motile cilia, are required for the production and regulation of cerebral spinal fluid (CSF). Additionally, the lateral ventricle epithelium contains motile cilia which is important for the movement of CSF throughout the brain ventricles. Previous studies have established a ciliogenesis role for CEP164 in the motile cilium and demonstrated CSF defects including hydrocephalus in zebrafish and murine models lacking functional CEP164 [24]. This further validates the strong CEP164 expression in ependymal cells and the neuroepithelium lining the brain ventricles.

CEP164 is also expressed in tissues secondary to the cerebellar-retinal-renal phenotype, some of which have motile cilium. Taking together results from a previous murine CEP164 motile cilia study [24], it is plausible that CEP164 is expressed in ciliated epithelial cells, including those of the trachea, bronchi and bronchioles in the developing human (8 PCW) and murine (P15.5-P29.5) lung. Previous studies indicate that this is likely to be maintained into adulthood [36, 60]. Cep164 is also expressed in murine spermatids, which have a flagellum, effectively a modified motile cilium [67]. Studies have demonstrated that CEP164 is expressed in the capitulum and striated columns of the human sperm neck [68]. Using information from a recent FOXJ1 mediated knockout of Cep164 and this expression data, it could be hypothesised that CEP164 is required for the formation, maintenance and functioning of motile cilia within the respiratory epithelium, reproductive tissues and the sperm flagella [24]. Mutations and subsequent aberrant functioning or loss of CEP164 may contribute to lung phenotypes such as pulmonary bronchiectasis, as seen in some patients with CEP164 mutations [1]. This may also contribute to aberrant spermatid function, which may cause infertility problems as seen typically in BBS models [9].

CEP164 is also expressed in other developing tissues that contain a primary cilium (human 8 PCW, mouse P15.5–30.5), including cardiomyocytes, cholangiocytes, tracheal and costal cartilage, smooth muscle cells of the trachea, bronchi, bronchioles and seminiferous tubules, spermatogonia, spermatocytes, bone primordia and the epithelium lining and smooth muscles of the GI tract [69-76]. Notably, CEP164 is not present in hepatocytes, which do not contain primary cilium [77].

CEP164 is expressed in tissues involved in the CEP164 NPHP-RC phenotype, but is also expressed in tissues not associated with this phenotype. It could be that CEP164 has cell specific functions, or that not all tissues require primary cilia for development or that cells have other pathways that can compensate for the abnormal functioning or loss of CEP164.

Interestingly, the patterns of human CEP164 expression described here correlate with those of other JBTS genes AHI1 and CEP290 [78]. This could indicate a universal mechanism that underlies NPHP-RC.

In summary, CEP164 demonstrates widespread yet defined expression throughout human and murine development, which is predominantly maintained into adult life. Human and murine data largely correlate and CEP164 function is likely to be conserved between the two species. CEP164 is expressed in tissues affected in CEP164-NPHP-RC patients, however clinical heterogeneity, commonly seen in ciliopathies, requires further investigation.

Working dilutions of primary antibodies used for immunohistochemistry of human tissues.

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Comparison of human and murine kidney developmental timeline.

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Comparison of human and murine retina developmental timeline.

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Comparison of human and murine cerebellar development timeline.

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Conservation and protein domains of human CEP164.

Predicted human CEP164 protein domains of the common 1460bp isoform; tryptophan-tryptophan (WW) domain conserved with two Tryptophan (W) residues, Lysine-rich repeat (LR) and predicted coiled-coil (CC) domains. Values marked are amino acid number (A). Sequence alignment of human CEP164 and its orthologs in M.musculus, D.melanogaster, C. reinhardtii and D rerio (B). Unrooted phylogenetic tree of CEP164 orthologs (C).

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Diagram of the 129/OlaHsd- Cep164 allele.

Upon pre-mRNA splicing of Cep164, exon 3 splices into the splice acceptor (SA) of the LacZ cassette, causing a frameshift and subsequent formation of a premature termination codon, this forms the tm1b allele. The LacZ has an internal ribosomal entry site (IRES), and thus the LacZ fusion gene acts as a reporter gene for Cep164. Figure adapted from MRC Harwell International Mouse Phenotyping Consortium.

Internal ribosomal entry site (IRES), splice acceptor (SA), polyadenylation site (pA).

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Digital magnification of human kidney CEP164 expression.

Human kidney 14 PCW (A), 18 PCW (B). CEP164 is expressed strongly at the apical membrane of the ureteric bud (A.I, B.I) which is maintained in the collecting duct, with CEP164 expression also at the basolateral membrane (A.II,B.II). CEP164 is also expressed strongly at the renal tubule apical membrane (A.III, B.III).

Cap mesenchyme (CM), collecting duct (CD), loop of Henle (LH), renal tubule (RT), ureteric bud (UB).

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Human CEP164 expression controls.

No primary antibody controls in the human 8 PCW kidney (A) and human 18 PCW kidney (B). Renal vesicles, comma-shaped vesicles, s-shaped body, ureteric bud and cap mesenchyme demonstrate no endogenous peroxidase staining (A.I.II.III, B.I.II.III). Renal tubule and the renal corpuscle (A.IV.V.VII, B.IV.V.VI), loop of Henle and collecting ducts (B.VII) also demonstrate no endogenous peroxidase staining. No primary antibody controls 8 PCW embryo (C). Lung, including the bronchioles and alveoli (C.I.II.III) and cardiomyocytes (C.I.IV,V) show no endogenous peroxidase staining. There is very weak endogenous staining in the Muscularis Externa (ME) of the gastrointestinal tract (C.VI) and the seminiferous cord of the gonads (C.VII.VIII). Dorsal root ganglia (C.IX) and bone primordia (C.VX) demonstrate no endogenous staining. However, the hepatocytes of the liver have endogenous peroxidase staining (C.IX), which means it cannot be determined if CEP164 is expressed in hepatocytes. The developing brain no primary controls, 8PCW (D, E) and 16 PCW (F). The developing brain (D.I, E.I) including the telencephalon (D.VI), metencephalon (D.V), midline (E.II) and myencephalon (D.VIII) do not demonstrate endogenous peroxidase staining. The neuroepithelium surrounding the brain ventricles (D.VIII, E.III.IV, F.I.III.IV) alongside the choroid plexus ependymal cells (D.II.III.IV, F.I.V) and choroid plexus pia matter (D.III.IV, F.I.V) do show some weak endogenous peroxidase staining. The molecular cell layer, purkinje and ganglion cell layers of cerebellum (F.I.II) and cerebellar white matter (F.VII) do not show endogenous staining. The ventricular surface (F.III.IV), medulla oblongata (F.VI) and choroid plexus at 16 PCW (F.V) show weak CEP164 staining. PAX6 positive control antibody (E.V, F.IX). The developing retina no primary controls, 11 PCW (G), and 19 PCW (H), both demonstrate no endogenous staining.

Alveoli primordia (Av Pri), bone primordia (BP), bronchiole (Br), cap mesenchyme (CM), cardiomyocyte (CM), cerebellum (Ce), choroid plexus (CP), collecting duct (CD), cortical plate (CorP), diencephalon (Di), dorsal root ganglia (DRG), ependymal cells (Ep), ganglion cell layer (GCL), gastrointestinal tract (GI), germline epithelium (GE), gonad (Go), intermediate Zone (IL), heart (Ht), hepatocytes (Hep), inner plexiform layer (IPL), inner nuclear layer (INL), fourth ventricle (FV), kidney (Ki), lateral ventricle (LV), lamina propria (LM), liver (Li), loop of Henle (LH), lung (Lu), marginal Layer (MaL), medulla oblongata (MO), mesencephalon (Mes), metencephalon (Met), midline (Mi), molecular Cell Layer (MCL), muscularis externa (ME), muscularis mucosae (MM), myencephalon (Mye), nasal epithelium (Na) nerve fibre layer (NFL), outer nuclear layer (ONL), outer neuroblastic cell layer (ONBL), outer plexiform layer (OPL), pia (P), photoreceptor cells (PS), post conception weeks (PCW), purkinje cell layer (PCL), renal corpuscle (RC), renal tubules (RT), renal vesicle (RV), retinal pigment epithelium (RPE), seminiferous cord (SC), s-shaped body (SSB), submucosae (SB), sub-ventricular layer (SVL), squamous epithelium mucosae (Mc-sq), telencephalon (Tel), ureteric bud (UB), ventricular layer (VL), ventricular surface (VS), white matter (WM).

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Littermate wildtype controls for murine Cep164 expression.

Murine retina wildtype controls (A). Murine kidney wildtype controls (B-C), murine secondary tissues wildtype controls (D-F), and murine cerebellar tissues (G-I). Retina at P1.5 (A.I), P15.5 (A.II), and P29.5 (A.III) demonstrate no endogenous beta galactosidase staining. Renal vesicles (B.I.II.III), ureteric bud (B.III), renal tubules (B.IV) and renal corpuscle (B.V.VI) at P1.5 do not show any endogenous staining. Renal tissue at P30.5 have low endogenous beta galactosidase expression in the renal tubules (C.I.III.IV), but not in the renal corpuscle (C.II). Developing brain (D.I), costal cartilage (D.II) and intercostal muscle (D.III) at P0.5 do not show endogenous beta galactosidase staining. The P0.5 lung bronchioles (E.I.II.III.IV) demonstrate weak endogenous beta galactosidase staining, but not the alveoli (E.V). The P30.5 murine lung trachea, bronchioles and alveoli (F.I.II.III.IV.V.VI) do not show endogenous beta galactosidase staining. Murine cardiomyocytes (E.VI.VII, F.VI.VII), developing testes including the spermatogonia, spermatocytes, spermatids and smooth muscle cells (E.VIII.IX.X, F.VIII.IX.X) do not have endogenous staining. There is also no endogenous staining in the hepatocytes of the liver, hepatic portal vein (E.XI.XII.XIII, F.XI.XII.XIII). Murine brain cerebral hemisphere (G.I.V, H.I.V, I.I.), the midbrain (G.VI,H.VII,I.V), the striatum (H.V,) the thalamus (H.VI,I.VI) the pons and the medulla oblongata (G.I.VII, H.I.VII, I.I.VII) all demonstrate no endogenous staining. The choroid plexus shows some weak endogenous beta galactosidase expression (G.I.II.IV) at P0.5. In the developing the cerebellum, the ganglion cell layer, molecular cell layer, purkinje cell layer and white matter of the cerebellum (G.II.III, H.II.III.IV, I.II.III.IV) demonstrate no endogenous beta galactosidase staining.

Alveoli (Av), bronchiole (Br), cardiomyocyte (CM), cartilage (Ca), cerebellum (Ce), cerebral hemisphere (Ch), cerebrum (Cr), choroid plexus (CP), connective tissue (CT), costal cartilage (CC), epithelial (Ep), ganglion cell layer (GCL), heart (Ht), hepatocytes (Hep), inner nuclear layer (INL), inner plexiform layer (IPL), intercostal muscle (IC), liver (Li), lung (Lu), marginal layer (MaL), medulla oblongata (MO), midbrain (MB), molecular cell layer (MCL), outer neuroblastic layer (ONBL), outer nuclear layer (ONL), outer plexiform layer (OPL), photoreceptor segment layer (PS), pons (PO), postnatal day (P), purkinje cell layer (PCL), renal corpuscle (RC), renal tubule (RT), renal vesicle (RV), respiratory bronchiole (R), retinal pigment epithelium (RPE), seminiferous tubule (ST), smooth muscle (SM), spermatids (Sp), spermatocytes (SC), spermatogonia (Spg), striatum (St), terminal bronchi (TB), thalamus (Th), trachea (Tr), ureteric bud (UB), white matter (WM).

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CEP164 wholemount expression throughout murine embryonic development and corresponding WT littermate controls.

At E9.5 (A), Cep164 widespread expression is seen, including the branchial arches, developing forebrain, midbrain and hindbrain (A.I). There is also Cep164 expression seen in the developing neural tube, including the neuroepithelium surrounding the anterior neuropore (A.II). There is Cep164 expression seen in the developing heart including the central ventricle, bulbous cordis and outflow tract (A.III), as well as the developing limb buds (A.IV). This Cep164 expression pattern is maintained at E10.5, with Cep164 expression also seen in the optic cup and olfactory pit (B). At E12.5 Cep164 expression is widespread but is defined in the spinal cord, vertebrae, cerebral cortex, midbrain, hindbrain, pons and medulla oblongata, otocyst and heart (C.I.II.III). Cep164 expression is seen in the retina (C.IV) and at the tips of the developing digits, where the apical ectodermal ridge is present (C.V.VI.VII.VIII). WT littermates do not show endogenous beta galactosidase expression at E9.5 (D.I), E10.5 (D.II), and E12.5 (D.III).

Anterior nucleopore (ANP), apical ectodermal ridge (AER), branchial arch (BA), bulbous cordis (BC), cerebral cortex (CC), common atria (CA), common ventricle (CV), forebrain (FB), forelimb bud (FL), heart (Ht), hindbrain (HB), hindlimb bud (HL), medulla oblongata (MO), midbrain (MB), neural tube (NT), olfactory pit (OF), optic cup (OC), organ primordia (Pri), otocyst (OT), outflow tract (OFT), pons (PO), somites (SM), spinal cord (SC), vertebral column (VC).

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Digital magnification of murine Cep164 testes expression.

P15.5 murine testes (A) P30.5 murine testes (B). Cep164 is expressed in the connecting tubules and leydig cells (A.I.II). In the seminiferous tubule, Cep164 is expressed in the smooth muscle cells, spermatogonia and spermatocytes (A.B). Cep164 is expressed most strongly in the spermatid, tails (A.III).

Connecting tubule (CT), Leydig cells (LD), seminiferous tubule (ST), smooth muscle cells (SM), spermatocytes (SC), spermatogonia (Spg), sperm (Sp).

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Validation of CEP164 antibody using hURECs.

Representative image of human urine derived renal epithelial cells (hURECs) with rabbit anti- CEP164 staining (Human Protein Atlas, HPA37606) (A). CEP164 (green) can be seen at the base of the primary cilium, stained with mouse anti-ARL13B (Proteintech, 66739-1-1g) (red), correlating with CEP164’s mature centriole localisation (A.I.II). Vectashield with DAPI (blue) stained cell nuclei. No primary antibody controls show no staining for CEP164 (B).

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17 Sep 2019

PONE-D-19-22989

Embryonic and foetal expression patterns of the ciliopathy gene CEP164

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. Summary of the research

In this manuscript, Devlin et al. examine the expression patterns of the centrosomal protein CEP164 during development in human and mouse tissues. They find that the expression is widespread in different organs and tissue regions, and that the expression in mouse matches that in human to a large extent, with some differences.

CEP164 has been shown to play a role in Nephronophthisis-related ciliopathies (NPHP-RC), a group of genetic disorders with has multiple clinical manifestations. The expression of CEP164 in multiple organs throughout development supports its role in the multiple phenotypes seen in NPHP-RC.

The immunohistochemistry stainings are convincing, in a range of tissues and developmental stages. The data is very informative, but qualitative.

It would help to compare respective levels of expression if the authors quantified the intensity of staining in the different regions and sub-regions investigated, especially when looking at decreasing expression with time. This might be informative when looking at specific defects associated with particular areas within tissues.

Overall this is a really nice piece of work examining CEP164 expression in human and mouse embryonic and foetal tissues. I recommend publication

2. Examples and evidence

Major issues

Please could you quantify expression or adjust text to say that ‘expression seems to be lost with maturity’, for example.

Minor issues

Please amend the following:

• Line 74: ‘in some patients’, instead of ‘is some patients’

• Line 92: what is OFD IX? Maybe replace by Orofaciodigital syndrome 9?

• Fig S1, D: not sure how this panel shows absence of CEP164 in nematoda taxa? Confusing diagram. Is there a way of labelling directly on the graph maybe?

• Line 275: should it be C.III instead of C.II for S-shape body?

• Fig1, panel E: inset for E.II is rotated 90 degree to the right, could you put it in the correct orientation please?

• Inset for E.III does not correspond to the black box on panel E.I, could you adjust the black box please?

• Black box for panel E.IV is missing, could you add it please?

• Line 283-284: Fig1: there doesn't seem to be a panel F. Please amend text, or amend Fig 1 to add panel F.

• Line 327: text mentions Inner Segment and Outer Segment of photoreceptor layer. Fig 2A.III does not mention these, please amend.

• Line 525: there is no panel D in Fig 4. Please amend text or add panel D. This may be a confusion in panel lettering, as 15.5 is panel B and 30.5 is panel C. So the correct notation could be

(Figure 4B.XV.XVI.XVII, C.XIII.XIV), instead of (Figure 4C.XV.XVI.XVII, D.XIII.XIV).

3. Other points (optional)

I will be available to look at a revised version.

Reviewer #2: Devlin et al report embryonic and fetal expression patterns of the ciliary gene CEP164 in human and mouse specimens. CEP164 is an important distal appendage protein that localizes to the distal area of centrioles/basal bodies and its function is essential for ciliogenesis. Mutations in this gene cause NPHP-RC.

The authors performed immunohistochemistry for human embryonic sections and X-gal staining for mouse embryonic sections to conduct a thorough analysis of CEP164 expression. Similar expression patterns were observed between human and mouse tissues. CEP164 is widely expressed yet show some defined expression in multiple organs.

The study is thoroughly done and detailed expression results are provided. However, errors and mislabeling in figures make is extremely hard to read through. Although it provides new expression sites of CEP164 in embryos, it is descriptive in nature and would be suitable for a more specialized journal. Some specific points are listed below.

1) Human embryonic expression studies rely on only one antibody (Sigma). I know this has been used in several studies mostly for cultured cells, it does not necessarily mean that it is specific on tissues. I recommend to use another antibody for validation. Related to this, it would be helpful to show CEP164 localization at the base of cilia for antibody specificity as high mag images of trachea or airway for example.

2) Fig1: several rectangles for high magnification are missing. For example, A.VII, B.VII, C.VII, D.II, and E.IV. In some cases, rectangles are not accurately placed for high mag images. For example, A.V and AVI. Please also check Fig4 A.VI.

3) Fig1: it is described that CEP164 expression is seen in the cells of the uteric bud at both the apical and basal membranes as well as at the basolateral membrane of the collecting duct tubule. It is novel and interesting. However, these are very difficult to see. High mag images would be helpful. There are other places where high mag images might be beneficial such as CEP164 expression in sperm tail in Fig4.

4) In some cases where CEP164 expression is weak, for example, in NFL (Fig2A), it is hard to distinguish weak expression vs background. Perhaps, side by side comparison with negative controls may help clarify this.

Here are some errors or suggestions in the text. There are many more errors especially figure numbers so please carefully check:

P4 line 89: “basal body” to “centriole”

line 95: “Proteomics analysis” to “Protein domain analysis”

line 96: “serine/glutamine” to “serine-glutamine/threonine-glutamine”

P7 line 160: “1:1” to “1:3”

P12 line 274: remove “is”

line 275: “C.II” to “C.III”

line 283: “F.IV” to “E.IV”

line 284: “D.V” to “D.V, D.Vl” (CEP164 seems to be expressed in D.Vl)

line 284: “F.IV” to “E.II”

P14 lines 309 and 312: “P29.5” to “P30.5”

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Reviewer #2: No

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Submitted filename: Devlin et al_PLoS ONE Review_2019.docx

Click here for additional data file.

20 Nov 2019

PONE-D-19-22989

Embryonic and foetal expression patterns of the ciliopathy gene CEP164

PLOS ONE

Dear Dr Lewin,

Thank you for your comments regarding our manuscript “Embryonic and foetal expression patterns of the ciliopathy gene CEP164”. We have made amendments to the manuscript, to address the points generated in the review. Please see the revised copy of the manuscript with tracked changes, and the revised manuscript.

1) In response to “Please validate your findings by demonstrating that the primary antibody for CEP164 is specific” and “Human embryonic expression studies rely on only one antibody (Sigma). I know this has been used in several studies mostly for cultured cells, it does not necessarily mean that it is specific on tissues. I recommend to use another antibody for validation. Related to this, it would be helpful to show CEP164 localization at the base of cilia for antibody specificity as high mag images of trachea or airway for example”.

We have added a supplementary figure (S8), demonstrating that the rabbit anti-CEP164 is defined to the base of the primary cilium of human urine derived renal epithelial cells (hURECs), which are phenotypically relevant to this study and the CEP164-NPHP patients. Additionally we would like to emphasise that the alternative non-immunohistochemical LacZ reporter assay used in the murine Cep164 expression studies further validates the human embryonic and foetal CEP164 antibody staining, due to correlating expression patterns. We therefore feel that CEP164 expression in the human embryo and foetal tissues is validated.

2) Reviewer 1s comment “Please could you quantify expression or adjust text to say that ‘expression seems to be lost with maturity” was thoroughly considered.

It was decided that quantification of CEP164 staining to compare against different time-points and tissue sub-regions could not be completed accurately enough due to the semi-quantitative nature of DAB immunohistochemistry and LacZ assays. Hence, the manuscript was altered so that quantification statements were not made.

3) In response to reviewer 2s comments “In some cases where CEP164 expression is weak, for example, in NFL (Fig2A), it is hard to distinguish weak expression vs background. Perhaps, side by side comparison with negative controls may help clarify this”.

Representative controls have been added to Fig 1, Fig 2 and Fig 3, to offer side-by-side comparisons. Supplementary figures S4 and S5, provide all of the controls for further analysis. Additionally, human retinal no-primary controls have been completed, to examine for endogenous peroxidase activity, and have been added to Fig 2 and S4.

4) As highlighted by reviewer 1 “Fig S1, D: not sure how this panel shows absence of CEP164 in nematoda taxa? Confusing diagram. Is there a way of labelling directly on the graph maybe?”

We acknowledge that the S1 Fig D diagram was confusing. It has been decided that it does not add sufficient information to the manuscript, and has therefore been removed.

5) All grammatical, spelling and formatting errors highlighted by the reviewers, and found within the manuscript have been resolved to the best of our knowledge. All of the Figure insets have been adjusted accordingly so that they are in the correct orientation, figures are correctly labelled and the figure panels match up with the text.

6) Reviewer 1 stated that “Black box for panel E.IV is missing, could you add it please?”, in regards to Figure 1. Additionally, reviewer 2 stated that “Fig1: several rectangles for high magnification are missing. For example…….B.VII, C.VII….and E.IV.”

There has been a misunderstanding here. The rectangles are not required in these locations due the images not being a high magnification, they are from separate medullary region of the developing kidney. This has now been clearly labelled in the figure, and figure legends.

7) Reviewer 1 stated that “Line 327: text mentions Inner Segment and Outer Segment of photoreceptor layer. Fig 2A.III does not mention these, please amend.”

Fig 2A.III has now been labelled appropriately.

8) To address the following point from reviewer 2 “Fig1: it is described that CEP164 expression is seen in the cells of the uteric bud at both the apical and basal membranes as well as at the basolateral membrane of the collecting duct tubule. It is novel and interesting. However, these are very difficult to see. High mag images would be helpful. There are other places where high mag images might be beneficial such as CEP164 expression in sperm tail in Fig4.”

Supplementary figures S3 and S7 have been generated which show digital magnification images of CEP164 expression at the nephron tubule membranes and seminiferous tubules containing sperm, respectively.

9) Reviewer 3 stated that “P7 line 160: “1:1” to “1:3””.

I feel there has been misunderstanding here. The methods are describing the mating of a heterozygous CEP164tm1a mouse with a wild type mouse, therefore the ratio of HET:WT will indeed by 1:1, as we are trying to maintain the HET line and not generate HOM mice. Please see the punnet square below.

Additionally, the discussion has been modified to be more clear and concise; amendments can be seen in the tracked changes file.

The manuscript, and associated files, have been edited to meet PLOS ONE’s style requirements. All data is provided in the manuscript, and the data availability statement has been updated accordingly.

Thank you for the helpful reviews.

Submitted filename: CEP164 rebuttalSR_JAS.docx

Click here for additional data file.

17 Dec 2019

PONE-D-19-22989R1

Embryonic and foetal expression patterns of the ciliopathy gene CEP164

PLOS ONE

Dear Prof Sayer,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Reviewer 2 recommends some minor edits to the text and to Figs. 1 and 3. Please address these suggestions or make the changes requested.

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We look forward to receiving your revised manuscript.

Kind regards,

Alfred S Lewin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have satisfcatorily addressed all the reviewer's comments, thank you very much to them. The paper is clearer and the addition of controls provides confidence in the stainings. I recommened publication.

Reviewer #2: The manuscript has been improved significantly. I have a few more edits to suggest:

line 100: remove "."

line 126: musculus

line 171: addition

line 231: glass

line 250: please add a reference here

line 265: was

Fig 1B V and VII are not properly cropped so please check

Letters in some figures are very hard to see for example Fig 3A VI and B VI.

The following paper can be cited:

Airik et al., Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys, Kidney Int, 2019

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Jan 2020

The minor comments have all been addressed in a revised version.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

6 Jan 2020

Embryonic and foetal expression patterns of the ciliopathy gene CEP164

PONE-D-19-22989R2

Dear Dr. Sayer,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Alfred S Lewin, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

16 Jan 2020

PONE-D-19-22989R2

Embryonic and foetal expression patterns of the ciliopathy gene CEP164

Dear Dr. Sayer:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE