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.

Given CEP164 mutations cause NPHP-RC, we sought to determine the expression of CEP164 throughout human and murine development, particularly focusing on the cerebellarretinal-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-Cep164 tm1a(EUCOMM)Wtsi/+ gene trap model.

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.
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 MgCl 2 , 5mM EGTA in PBS) for 5 min, washed in PBS for 10 min, and then washed in X-Gal wash (2 mM MgCl 2, 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 MgCl 2, 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 MgCl 2 , 5mM EGTA in PBS) for 1 hour on ice and washed with X-Gal wash (2 mM MgCl 2, 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 MgCl 2, 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% H 2 O 2 ) 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 (S1 Table). 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 (S1 Table). 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.

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 (Fig 1A, 1B and 1C), comma-shaped bodies (Fig 1B) and subsequent s-shaped bodies (Fig 1C), during all time points analysed (8PCW-18PCW). This expression pattern is maintained throughout nephrogenesis, with CEP164 present in the primitive nephron tubule (8PCW) (Fig 1A), but also in more defined proximal and distal nephron segments, as well as cells of the loop of Henle, as seen from 14 PCW (Fig 1B and 1C). 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) (S3 Fig). CEP164 does not appear to be expressed, or is expressed very weakly, in the metanephric cap mesenchyme (Fig 1A, 1B and 1C).
In the developing human kidney, CEP164 is expressed in the glomerulus of the renal corpuscle at 8 PCW-18 PCW, indicating podocyte CEP164 expression (Fig 1A, 1B and 1C). 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) (Fig 1A, 1B and 1C). 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 (Fig 1A, 1B and 1C). Lack of background DAB staining in no primary controls indicate that CEP164 expression in the human kidney was specific (Fig 1D and S4 Fig).
In the murine kidney (129/OlaHsd-Cep164 tm1a(EUCOMM)Wtsi/+ ), 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) (Fig 1E) and the subsequent renal tubules, including nephron segments in both the cortex and medulla (P30.5) (Fig 1E and 1F). The staining indicates that there is potentially cellspecific 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 (Fig 1E  and 1F). Likewise, Cep164 is expressed in the developing mesonephric ureteric bud (Fig 1E). Wildtype controls demonstrate that there is very low endogenous beta galactosidase expression in the kidney (Fig 1G and S5 Fig).

CEP164 expression during human and murine retinal development
In the developing human retina, CEP164 expression is widespread yet defined (Fig 2A). 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) (Fig 2A). The basally located layer of cone precursors, which has differentiated from the outer neuroblastic cell layer (ONBL), also demonstrates strong CEP164 expression (Fig 2A). By 14 PCW, CEP164 expression is defined to the NFL, the IPL and the photoreceptor layer which now has both developing rods and cones (Fig  2A). 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 (Fig 2A). 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 (Fig 2A and S4 Fig).
Cep164 is expressed in retinal precursor cells throughout the murine ONBL, which in the developing human retina is restricted to the cone precursor cells (Fig 2B). Additionally, strong Cep164 expression is present and maintained throughout the GCL (P1.5-P29.5) (Fig 2B). 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) (Fig 2B). Notably, at later stages of development, Cep164 expression is clearly defined to the IS of the photoreceptor cell layer (P15.5-P29.5) (Fig 2B). Additionally, the RPE demonstrates strong, cell-specific Cep164 expression throughout development (P1.5-P29.5) (Fig 2B). WT controls demonstrate no endogenous X-Gal staining (Fig 2B and S5 Fig).

CEP164 expression during human and murine neuronal and cerebellar development
CEP164 is expressed widely throughout human brain development (8PCW-18PCW) (Fig 3). At 8PCW CEP164 is strongly expressed in the neuroepithelium of the developing telencephalon (Tel) (Fig 3A and 3B) (Fig 3A and 3B). At 8 PCW, CEP164 is also present in the developing neuroepithelium of the diencephalon (Di) (Fig 3A and 3B.), mesencephalon (Mes) (Fig 3A.I), metencephalon (Met) (Fig 3A) and myencephalon (Mye) (Fig 3A, B.I.IV.V), with expression strongest in the apical epithelium lining the brain ventricles as highlighted by a black arrow (Fig 3A and 3B). At 8PCW there is strong, well-defined CEP164 expression in the midline of the myencephalon (Fig 3B), as well as cells of the nasal epithelium (Fig 3B). 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 (Fig 3A, 3B,  3D and 3E). The developing human cerebellar folds show defined CEP164 expression in the migrating molecular cell layer at 16 PCW (Fig 3D), but this seems to be reduced by 18PCW (Fig 3E). CEP164 is expressed at the apical membrane of the ventricular surface of the pons and cerebellum (Fig 3A, 3D and 3E) as well as the myencephalon, specifically in the axon tracts of the medulla oblongata (Fig 3D and 3E).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 (Fig 3A), but this appears to be lost with maturation of the cerebellum (Fig 3D and 3E). Notably the human foetal brain negative controls demonstrate very low background staining, specifically in the choroid plexus region (Fig 3C,  3F and S4D-S4F Fig). A PAX6 antibody was used as a positive control (Fig 3F and S4 Fig).
At P0.5 to P15.5 Cep164 is expressed in the migrating molecular cell layer and Purkinje layer of the cerebellum (Fig 3H and 3I). Later in development, Cep164 is also expressed in the ganglion cell layer (P30.5) (Fig 3J). Cep164 is also expressed at the apical membrane of the ventricular surface of the cerebellum at P0.5 and P15.5 (Fig 3H). 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 (Fig 3G, 3H, 3I and 3J). Cep164 is also expressed in the 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 Expression patterns of the ciliopathy gene CEP164 ependymal cells of the choroid plexus, which is demonstrated in P0.5 sections (Fig 3G and  3H). 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 (Fig 3K  and S5 Fig).

CEP164 expression during human and murine development in other tissues
CEP164 is expressed widely throughout the developing human embryo at 8PCW (Fig 4A.). In the 8PCW lung, CEP164 is expressed most strongly in the epithelial cells lining the lumen of the bronchi and bronchioles (Fig 4A). Weaker CEP164 expression is also present in the bronchiole smooth muscle, and the alveoli primordia (Fig 4A). In the 8PCW developing heart, CEP164 is expressed in cardiomyocytes (Fig 4A). 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 (Fig 4A). At 8PCW in the developing gonads, CEP164 is expressed weakly in the germline epithelium, but strongly in the seminiferous cord tubules (Fig  4A). Additionally, CEP164 is expressed strongly in the dorsal root ganglia of the spinal cord, with weak expression in the developing vertebrae (Fig 4A). CEP164 is also expressed in developing bone primordia of the limbs at 8 PCW (Fig 4A). Due to endogenous beta galactosidase activity the liver (S4 Fig) it cannot be determined whether CEP164 is expressed in human embryonic hepatocytes. The other tissues show no endogenous beta galactosidase staining (S4 Fig).
Murine postnatal tissues also demonstrate widespread Cep164 expression. At P0.5 Cep164 is expressed in the developing costal cartilage primordia and intercostal muscles (Fig 4B). 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 (Fig 4C and 4D), as well as the smooth muscle and cartilage of the trachea and bronchi (Fig  4C and 4D) Weak Cep164 expression is seen in the alveoli at P15.5 (Fig 4C), which seems to be reduced by P29.5 (Fig 4D). Like human embryonic tissues, Cep164 is present in cardiomyocytes at P15.5 and P30.5 (Fig 4C and 4D). In postnatal P15.5 murine testes, Cep164 is expressed in the connecting tubules and Leydig cells (Fig 4C and S7 Fig). In the seminiferous tubules, Cep164 is expressed in the smooth muscle cells and weakly in developing spermatogonia and spermatocytes (Fig 4C and S7 Fig). Strong Cep164 expression is present in the spermatid tails (Fig 4C and S7 Fig). By P30.5 Cep164 expression is defined to developing spermatogonia, spermatocytes, and spermatids (Fig 4D and S5 Fig). 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 (Fig 4C). 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 (S5 Fig). There is low endogenous beta galactosidase activity in the bronchioles at P15.5, but not at P30.5 (S5 Fig).
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 (S6 Fig). 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.
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][47][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 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. 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