Mutations in ARL3 and CEP120 are rare and relatively new causes of JSRD and other related ciliopathies. Human protein atlas data suggests that tissue expression of ARL3 protein is widely expressed, with highest expression scores seen in cerebellum and lowest in heart and skeletal muscle (https://www.proteinatlas.org/ENSG00000138175-ARL3/tissue). RNA expression is high in cerebral cortex, cerebellum, retina and kidney consistent with its known phenotypes. CEP120 protein expression is not annotated within the human protein atlas, whereas RNA is strongly expressed in the cerebellum (https://www.proteinatlas.org/ENSG00000168944-CEP120/tissue). We aimed to define expression of ARL3 and CEP120 during human development using the HDBR tissue bank employing a relatively new in situ hybridisation assay called RNAscope for the detection of target RNA within intact cells. Our data provide an insight into the developmental expression of ARL3 and CEP120. We show that both of these genes are expressed in key tissues (including retina, cerebellum and kidney) during development. This expression pattern fits with the multisystem disease phenotypes seen in patients with ARL3 and CEP120 mutations (Table 1). A similar approach, using the valuable HDBR tissue bank has been performed, using in situ hybridisation for studying the expression of ARL13B (41), another cause of Joubert syndrome. Here ARL13B was detected at stage CS16 in the alar and basal plate of the myelencephalon, the mesencephalon and the metencephalon. At CS19 ARL13B was seen in the ventricular layer of the diencephalon and myelencephalon, the tegmentum of the pons and the cerebellar rhombic lips as well as the dorsal root ganglia. This pattern of expression is remarkably similar to the CEP120 and ARL3 data described here.
Expression of both ARL3 and CEP120 was minimal in developing cardiac, lung and gut tissues, consistent with lack of known phenotypes affecting these organ systems (Supplementary Fig. 3). ARL3 and CEP120 encode proteins that are expressed in the primary cilia and basal body respectively (Supplementary Table 2) and pathogenic variants result in similar and overlapping phenotypes, including the cerebello-retinal-renal syndrome JSRD (Table 1). The number of patients with pathogenic variants in either ARL3 or CEP120 remains small, allowing a limited comparison of phenotypes, although skeletal manifestations (in particular short ribs/asphyxiating thoracic dystrophy phenotypes) seen in patients with CEP120 mutations have not been documented in patients with ARL3 mutations.
There were notable differences in evolutionary conservation between ARL3 and CEP120 (Supplementary Table 1). The ARL3 human protein shares greater than 90% identity with its two orthologous sequences (there is genomic duplication of arl3) in Danio renio (zebrafish), a well-studied model species in vertebrates. In contrast, CEP120 human protein only shares 57% identity with its single orthologous sequence found in zebrafish. Moreover, human ARL3 protein shares > 60% identity with its orthologues found in Drosophila melanogaster, Caenorhabditis elegans and Chlamydomonas reinhardtii. CEP120 is conserved in some vertebrate organisms but orthologues were not readily identified in invertebrates. There is a putative CEP120 orthologue, UNI2, found in Chlamydomonas reinhardtii, but this has not as yet been confirmed as a functional ortholog (27, 42). ARL3 is described in diverse eukaryotic organisms such as Leishmania donovani (43) and Caenorhabditis elegans (44, 45) where it has a functional role in the cilium/flagella. Despite these differences in evolutionary conservation, our results show that ARL3 and CEP120 have similar expression patterns during human development, specifically in the eye and dorsal root ganglia as well as during early brain development. Both genes are expressed throughout the retina during development, with expression in the RPE and photoreceptor layers, suggesting a role for both genes during retinal development. This is further supported by the numerous retinal phenotypes associated with mutations in ARL3 (15–17). Similarly, the specific expression of ARL3 and CEP120 in the dorsal root ganglia hints at a role for both genes in primary sensory neurone migration and differentiation. A recurring pattern was the expression of both mRNAs on the luminal facing surface of the cerebral tissue (seen in the choroid plexus, cortical plate, ganglionic eminence, and hindbrain) which could suggest a sensory role for both the genes and within the cilium of the ventricular lining of the brain.
Expression of ARL3 and CEP120 changes during development notably in the cerebellum and kidney. ARL3 and CEP120 are expressed throughout the cerebellum at 14PCW however, at 19PCW CEP120 was also expressed in the ML of the cerebellum. This could imply that ARL3 and CEP120 are expressed in different cell populations of the cerebellum as the ML contains parallel fibres and the dendrites of Purkinje cells, whereas the rest of the cerebellum is largely made up of granule cells (36). It has been previously reported in mouse studies that Cep120 is required for proliferation of cerebellar neural progenitor cells (28) and is required for correct development of the embryo. Taken with these results, it suggests that CEP120 is required for correct development of the cerebellum in humans.
Expression of ARL3 and CEP120 also differed in the developing kidney. The results showed that ARL3 was specifically expressed in cells of the nephrons whereas CEP120 was expressed in the nephrons as well as within cells in the developing renal cortex. This difference in expression could imply that ARL3 has a more sensory/signalling function in luminal structures of the kidney, whereas CEP120 has a more universal role in all cells as it is expressed more ubiquitously throughout the tissue.
The differences in gene expression may reflect the divergent functions of ARL3 and CEP120 proteins (Supplementary Table 2). As ARL3 is a trafficking protein involved in ciliary signalling (19, 46), it may only be expressed in actively signalling cells during certain points in development such as nephron progenitors and cells in the IGCL. In contrast, CEP120 is involved in building the centriole, and therefore cilium, (27, 29) and so will be expressed more widely within tissues, especially those with ciliated epithelia (47, 48).
In conclusion, we establish in human embryonic tissue expression patterns of ARL3 and CEP120 during development and provide insights into the wide phenotypic spectrum of mutations affecting ARL3 and CEP120 in humans. These studies will allow further investigations into tissue-specific mechanistic roles of ARL3 and CEP120 in human health and disease.