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 kidney failure 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, this data supports conserved roles for CEP164 throughout the development of numerous organs, which we suggest accounts for the multi-system disease phenotype of CEP164 mediated NPHP-RC.


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PCR products were run on a 1.5% agarose gel, with GelRed Nucleic Acid GelStain

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Sections of tissues were cut (10-12μm) using a cryostat and mounted on charged glass slides.

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These were incubated in 0.2% glutaraldehyde fix (0.2% glutaraldehyde, 2mM MgCl 2 , 5mM 192 EGTA in PBS) for 5 min, washed in PBS for 10 min, and then washed in X-Gal wash ( with incision in the skull down the sagittal plane and a slit in the abdomen at the umbilicus.

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Tissues were transferred to methacarn until further processed. This same protocol was used 221 for processing of foetal eye, brain and kidney; the embryonic and foetal kidney were cut 222 sagittally, to allow the fix to penetrate. Human embryonic and foetal tissues were washed in 223 increasing concentrations of ethanol, and then incubated in 3 changes of xylene before 224 embedding in paraffin wax. Sections, on positively charged glassed slides, were de-waxed in 225 two washes of xylene, and then rinsed in two changes of absolute ethanol. Slides were 226 incubated in methanol peroxide solution (0.5% H 2 O 2 ) for 10 min to block endogenous 227 peroxidase. Slides were rinsed in tap water and then antigen retrieval was completed using 228 citrate buffer. After rinsing in Tris-buffered saline (TBS), slides were incubated with 10% 229 goat serum (Vector) for 10 min at room temperature and then incubated with the primary 230 antibody in goat serum with TBS overnight at 4 °C (S1 Table). After washing with TBS, 231 slides were incubated with the goat anti rabbit BA-1000 biotinylated secondary antibody 232 (Vector Laboratories) diluted in goat serum with TBS (1/500), for 30 min at room 233 temperature. The secondary was washed with TBS and then slides were incubated with 234 VECTASTAIN Elite ABC kit PK6100 tertiary complex (Vector Laboratories), for 30 min at 235 room temperature. After TBS washes, the stain was developed for 10 min at room 236 temperature using ImmPACT DAB peroxidase substrate (SK-6100) solution (Vector 237 Laboratories). The slides were washed thoroughly in water, and counterstained with 238 haematoxylin, dehydrated, cleared and mounted.

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An indirect, two-step, method was utilised for the foetal kidney sections. Samples were 241 incubated with a HRP goat anti-rabbit IgG peroxidase secondary (Vector) (1/500). After TBS 242 washes, the stain was developed using DAB peroxidase substrate solution, as described 243 previously. No primary controls sections were utilised as negative controls, anti-PAX6 was 244 utilised as a positive control for the brain and cerebellum (S1 Table).        Table, S3 Table, S4 Table).

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Our results demonstrate that during human and murine development CEP164 is expressed 543 widely, in multiple organs. Notably, CEP164 expression is clearly defined within tissues. In 544 the developing human kidney, CEP164 is expressed in the apical epithelium membrane of 545 metanephric-derived renal vesicles and subsequent nephron tubules (8PCW-18PCW), 546 correlating with the presence of primary cilia [37]. Primary cilia are vital in the kidney for 547 mechanosensation and chemical signal transduction, which is required for orientated cellular 548 divisions during development and kidney maintenance. As CEP164 expression seems to be 549 low/not present in the cap mesenchyme, it could be speculated that CEP164 expression is 550 switched on during mesenchymal-epithelial transition, which coincides with the formation of 551 the primary cilium during establishment of apical-basal polarity, cell junctions and lumen 552 formation [37]. CEP164 is expressed within the glomerulus of the renal corpuscle, with a reduction in expression with maturity. Interestingly, this correlates with the loss of primary 554 cilium in podocytes seen with glomeruli maturity in rats [38]. It could be postulated that in 555 the developing human kidney, primary cilium, and thus CEP164 expression, is lost in 556 maturing glomeruli [38]. This could protect against overstimulation of calcium-mediated 557 signalling due to an increase in glomerular filtration rate [38]. CEP164 is also expressed in 558 the uteric bud, which develops into the primary ciliated collecting duct network (8PCW-559 18PCW), consistent with numerous IMCD3 expression studies [1,19,20,25,32]. The human 560 CEP164 expression pattern is conserved in the murine kidney (P0.5-P29.5); there may be 561 cell-specific Cep164 expression within nephron tubular segments, however this would need 562 to be further studied.  It is also well established that CEP164 is present at the basal body of retinal pigment 599 epithelial cells, clarifying the results from our study [1,2,15,17,23]. It can be hypothesised 600 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 602 trigger cell loss, leading to a retinal degeneration phenotype, as seen in some CEP164 603 NPHP-RC patients [1,9,44]. Primary cilia are vital in the developing brain for Shh signalling, which is needed for 618 proliferation of neuronal granular cell precursors in vertebrates, being a major driver of 619 cerebellar precursors [45,46]. Shh is also required for the cell-specific expansion of postnatal 620 progenitors [40]. Wnt signalling, transduced by the primary cilia, is also required for neuronal 621 patterning, cell proliferation and neuronal migration [46][47][48]. In the adult nervous system, 622 primary cilia are thought to be required in stem cell regulation and tissue regeneration, 623 however the full extent of primary cilia function in the adult central nervous system is still to 624 be determined [40,45]. It could be suggested that CEP164 has a functional role in human brain development. Potentially, aberrant function of CEP164, may lead to brain dysplasia via 626 abnormal primary cilia functioning in neuronal precursors. Some CEP164 NPHP-RC 627 patients show neurological phenotypes, including abnormal developmental delay, intellectual 628 disability and in one patient, cerebellar vermis aplasia, an archetypal feature of Joubert 629 syndrome. Another patient also experiences seizures [1,9].

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In the murine cerebellum, Cep164 expression is widespread including the migrating 632 molecular, ganglion and Purkinje cell layers, as well as the white matter (P0.5-P29.5).

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Wholemount expression studies from the international phenotyping consortium have shown 634 that Cep164 is expressed strongly throughout the adult murine brain [36]. It may be that 635 Cep164 RNA expression does not directly correlate with CEP164 protein expression.

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Although there is believed to be high conservation between the human and murine 637 transcriptomic networks, CEP164 may be part of the divergent pathway in the human-murine 638 brain transcriptomics [46].    CEP164 is also expressed in other developing tissues that contain a primary cilium, (human 8 675 PCW, mouse P15.5-30.5); including cardiomyocytes of the heart, cholangiocytes of the liver 676 bile ducts, smooth muscle cells, cartilage, spermatogonia and spermatocytes, bone primordia 677 and the epithelium lining and smooth muscles of the GI tract [50][51][52][53][54][55][56][57]. Accordingly, CEP164 678 is not present in the hepatocytes, which do not contain primary cilium [58]. In summary, CEP164 demonstrates widespread yet defined expression throughout human and 689 murine development, which is predominantly maintained into adult life. Human and murine 690 data largely correlate and CEP164 function is likely to be conserved between the two species.

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CEP164 is expressed in tissues affected in CEP164-NPHP-RC patients, however clinical 692 heterogeneity, commonly seen in ciliopathies, needs to be further investigated.