Expression patterns of ciliopathy genesARL3andCEP120reveal roles in multisystem development

Background

Joubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genesARL3andCEP120.

Methods

We sought to explore the developmental expression patterns ofARL3andCEP120in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characteriseARL3andCEP120expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource.

Results

BothARL3andCEP120are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes.

Conclusions

This study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development.

Joubert syndrome and related disorders (JSRD) are a group of autosomal inherited ciliopathies that are characterised as a cerebello-retinal-renal phenotype, and have an incidence rate of 1:80,000–100,000 live births [1,2,3]. The hallmark brain phenotype is a “molar tooth sign” shown on axial brain MRI, caused by cerebellar vermis hypoplasia and other mid and hindbrain malformations [4]. These defects often cause symptoms of hypotonia, ataxia and intellectual disability in patients [5]. The retinal and renal phenotypes associated with JSRD have a lower incidence rate and vary in severity. Renal disorders occur in ~ 25% of patients, often presenting as corticomedullary cysts, interstitial fibrosis, or tubulointerstitial kidney disease [5]. The renal component is progressive and can lead to end-stage renal disease [6]. Ocular phenotypes of retinitis dystrophy, retinitis pigmentosa, oculomotor apraxia, and ptosis are common in patients, and as with the renal aspects of JSRD are often progressive in nature [7].

Currently, there are more than 35 genes that are known to cause JSRD (https://www.omim.org/phenotypicSeries/PS213300). The syndrome is caused by defects of the primary cilia, which are found on most mammalian cells [8]. Primary cilia act as a cellular antenna to transduce extracellular signals such as mechanical flow, chemical stimulation, and key signalling pathways (including Hedgehog, Wnt, and PDGF) into the cell [9,10,11,12,13]. Due to the multi-organ involvement, varying phenotypes, and multitude of genes known to cause JSRD there is great heterogeneity within the syndrome and overlap with closely related ciliopathies including Bardet-Biedl syndrome (https://omim.org/phenotypicSeries/PS209900) and Jeune syndrome (https://omim.org/phenotypicSeries/PS208500) [14]. Recently discovered genetic causes of JSRD includeARL3[15] andCEP120[16,17]; the fact that their encoded proteins have such divergent roles within the primary cilium demonstrates the complexity underlying this group of related disorders.

ADP-ribosylation factor-like 3 (ARL3), a RAS superfamily member, is a low molecular weight GTP-binding protein [18] that cycles between inactive GDP-bound and active GTP-bound states to release cargo from their carriers in the cilium [19]. ARL3 interacts with its Guanine Exchange Factor ARL13B in the cilium [20] and GTPase Activating Protein RP2 at the basal body of the cell [21,22].Arl3knockout studies in mice demonstrate a multi-organ ciliopathy phenotype, including kidney cysts, liver fibrosis and retinal disease with photoreceptor cell degeneration [23,24,25,26]. Recently, two families affected by JSRD have been identified, presenting with ciliopathy phenotypes [15]. The underlying genetic cause was shown to be missense mutations inARL3, which affect an amino acid residue involved in the interaction between ARL3 and ARL13B [15].

Centrosomal protein of 120 kDa (CEP120) is a centrosomal protein involved in centriole biogenesis, including centriole duplication, assembly [27,28], elongation [29,30,31] and maturation [28]. CEP120 also interacts with other centrosomal proteins including CPAP [29,30], SPICE1 [29], Talpid3 [28,31] and C2CD3 [31]. CEP120 was found to be expressed ubiquitously in murine embryonic tissues such as the brain, kidney and lungs. Additionally, Cep120 was observed to be highly expressed in embryonic mouse brain compared to postnatal or adult mouse brain [32]. Inactivation ofCEP120in the mouse central nervous system results in hydrocephalous and cerebellar hypoplasia [28].CEP120突变已被证明导致JSRD和另一幅作品《年轻syndromes [16,17], and overlapping ciliopathy phenotypes such as tectocerebellar dysraphia with occipital encephalocele (TCDOE), Meckel syndrome (MKS) and oro-facial-digital (OFD) syndromes [17].

The developmental expression patterns ofARL3andCEP120in humans is not known. In order to explore this, we used an RNA in situ detection technique called RNAscope to compare and contrast the developmental spatial expression of these new and divergent causes of JSRD. We successfully characterisedARL3andCEP120expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR). This study provides insights into the potential pathogenesis of JSRD by uncovering the expression pattern of two JSRD-causative genes during normal human development.

RNAscope studies

Characterisation ofARL3andCEP120expression patterns was performed in human embryonic tissue using samples obtained from the MRC-Wellcome HDBR. Formalin fixed paraffin embedded sections of human embryonic and foetal tissue were prepared using 10% neutral buffered formalin and fixed for 32 h at room temperature. Samples were then prepared for the RNAscope assay, a RNA in situ detection platform for detection of target RNA within intact cells, as per manufacturers’ instructions [33,34]. An RNAscope 2.5 Assay RED was employed with 20 paired probes across nucleotides 169–1570 (NM_004311.3) and 115–1133 (NM_001166226.1) for detection ofARL3andCEP120, respectively and counterstained with Methyl Green.

Whole human embryo sections of 8 post-conception weeks (PCW), (equivalent to Carnegie Stage 23) were analysed, along with hindbrain (14PCW and 19PCW), eye (14PCW), kidney and adrenal gland (14PCW and 18PCW). A negative RNAscope 2.5 HD Assay Red control (dapB, a bacterial gene which is not expressed in human tissues) was performed (Supplementary Figure1). In addition, the RNAscope 2.5 HD Assay RED was performed with a positive control (KI67, a cell proliferation marker). KI67 is a nuclear protein commonly used as a proliferation marker, which is expressed in cycling cells and is associated with cellular proliferation. It is encoded by the gene marker of proliferation Ki-67,MKI67. ARL3andCEP120human expression patterns were analysed using the HDBR image server (Leica Biosystems).

Clinical phenotypes and sequence analysis

Reported clinical phenotypes associated withARL3andCEP120mutations were reviewed within OMIM (https://omim.org/). Putative Arl3 and Cep120 orthologues were identified using BLASTP, with human ARL3 (isoform a, NP_004302.1) and CEP120 (NP_694955.2) transcripts as the query sequences within NCBI (https://www.ncbi.nlm.nih.gov/). Additional databases including Flybasehttps://flybase.org/), Wormbase (https://www.wormbase.org/) and Phytozome (https://phytozome.jgi.doe.gov/) were also queried using BLAST.

Clinical phenotypes of ARL3 and CEP120 patients

Biallelic mutations in bothARL3andCEP120mutations are rare causes of ciliopathy syndromes. A comparison of the known phenotypes associated withARL3andCEP120mutations is shown in Table1. This overview reveals that mutations inCEP120are at present associated with severe phenotypes including MKS but also that single heterozygous changes inARL3are sufficient to cause retinal-limited phenotypes.ARL3is highly conserved, with homologs present inC. elegans,C. reinhardtiiandD. melanogasterwhereasCEP120appears not to have homologs within these lower organisms (Supplementary Table1). Known protein localisation within the cell of both ARL3 within the ciliary axoneme, and CEP120 in the centrosomes, are consistent with their role in ciliopathy syndromes (Supplementary Table2).

ARL3 and CEP120 are expressed in early human brain development

In 8PCW human brain tissue, the expression ofARL3andCEP120is remarkably similar. There is expression of both genes in the choroid plexus (Fig.1Ai and 1Bi), which appears to favour the luminal facing surface of the tissue, especially forARL3. The cell proliferation markerKI67does not share this same expression pattern in the choroid plexus (Fig.1 Ci). This specific expression pattern ofARL3andCEP120in luminal-facing cells is continued throughout the developing brain where both genes exhibit expression throughout the ventricular zone of the ganglionic eminences, cortical wall, and the hindbrain including the rhombic lip (Fig.1Aii-Aiv and 1Bii-Biv). There is specific expression ofARL3andCEP120in the layer of cells forming the apical surface in each tissue, facing into the ventricular space. Expression ofKI67is seen throughout these tissues (Fig.1Cii-Civ),与特定的表达式在顶端layer consistent with this being the major site of cell division (cells in G2/M1 phase of cell cycle) in the ventricular zone [37].

Expression of ARL3 and CEP120 is maintained in the developing cerebellum

In the human cerebellum at 14PCW there is expression ofARL3andCEP120. Both genes have strong expression in the external and internal granule cell layer (EGL and IGL) the developing cerebellum (Fig.2Ai and Ci). Expression in the EGL and IGL is seen at 19PCW forARL3(Fig2Bi) however,CEP120expression is predominantly localised in the EGL and the molecular layer (ML) of the cerebellum at 19PCW (Fig.2Di). Strong expression ofKI67is seen throughout the EGL in particular, but also the IGL at 19PCW, indicating the tissue is proliferative (Fig.2Ei).ARL3andCEP120are therefore widely expressed in the cerebellum during development, with specific expression ofCEP120in the ML which is predominantly occupied by the dendritic trees of Purkinje cells and the interacting parallel fibres of granule cells. As dendrites and axons contain low levels of mRNA, it is likely thatCEP120expression is predominantly located in the sparse population of interneurons found in the molecular layer [38] or in immature granule cells migrating from EGL to IGL [39] suggesting a role forCEP120in these cell types [38].

Expression of ARL3 and CEP120 in the developing eye

The human retina can be divided into nine layers based upon the cell types that occupy each layer (Fig.3a), with the retinal pigment epithelial (RPE) and photoreceptor layers at the outermost part of the eye [39]. At 8PCW, the retinal layers are not well defined with only a ganglion cell layer separated from a layer of mostly immature neuroblasts with a few photoreceptor cells by a thin inner plexiform layer [40]. At this stageARL3andCEP120show expression throughout the developing retina, with high expression within the retinal ganglion cells and the photoreceptor layer (Fig.3Bi and Di). At 14PCW, the retinal layers are maturing [39] which is reflected in the expression pattern of bothARL3andCEP120.Clear expression of both genes is still seen in all layers of the retina, although to a lesser extent in the plexiform and nerve fibre layers due to reduced cell density in these areas (Fig.3 Ci and Ei).

Expression of ARL3 and CEP120 in the developing dorsal root ganglia

The dorsal root ganglia are formed by migrating neural crest cells and contain most of the body’s sensory neurones [42,43]. BothARL3andCEP120show expression in cells of the dorsal root ganglia, which are post-mitotic primary sensory neurons (Fig.4Ai-Aii and Bi-Bii). There is strong expression ofKI67within limited number of cells in the dorsal root ganglia, presumably non-neuronal (Fig.4 Ci-Cii).

Expression of ARL3 and CEP120 in the developing kidney

In the developing human kidney at 8PCW, where there is strong renal cortical staining ofKI67indicating cell proliferation (Supplementary Figure2), there is expression ofARL3细胞在发展中皮质肾单位;this expression appears to be specifically oriented to the lumen of the structures (Fig.5Ai). This expression pattern is maintained at 14PCW (Fig.5Bi) and 18PCW (Fig.5 Ci). Expression ofCEP120is also seen in developing nephrons at 8PCW, however there is also expression in the renal cortex (Fig.5Di). This expression pattern ofCEP120is maintained at 14PCW (Fig.5Ei) and 18PCW, although overall expression appears to have decreased at this time point (Fig.5Fi).

Expression of ARL3 and CEP120 in other major organs

In the developing human heart, lung and gut at 8PCW, there is very low levels of expression ofARL3andCEP120(Supplementary Figure3). Expression ofARL3andCEP120is seen around the developing alveoli and at low levels in the developing bowel epithelia. The remaining organs of the developing embryo did not reveal prominent expression patterns.

Mutations inARL3andCEP120are 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 ofARL3andCEP120during 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. We usedKI67as a positive control although we recognise that expression ofKI67is not homogeneous throughout each tissue. Our data provide an insight into the developmental expression ofARL3andCEP120. 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 withARL3andCEP120mutations (Table1). A similar approach, using the valuable HDBR tissue bank has been performed, using in situ hybridisation for studying the expression ofARL13B[44],another cause of Joubert syndrome. HereARL13B发现在腋下和基底阶段CS16 plate of the myelencephalon, the mesencephalon and the metencephalon. At CS19ARL13Bwas 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 theCEP120andARL3data described here.

Expression of bothARL3andCEP120was minimal in developing cardiac, lung and gut tissues, consistent with lack of known phenotypes affecting these organ systems (Supplementary Figure3).ARL3andCEP120encode proteins that are expressed in the primary cilia and basal body respectively (Supplementary Table2) and pathogenic variants result in similar and overlapping phenotypes, including the cerebello-retinal-renal syndrome JSRD (Table1). The number of patients with pathogenic variants in eitherARL3orCEP120remains small, allowing a limited comparison of phenotypes, although skeletal manifestations (in particular short ribs/asphyxiating thoracic dystrophy phenotypes) seen in patients withCEP120mutations have not been documented in patients withARL3mutations.

There were notable differences in evolutionary conservation betweenARL3andCEP120(Supplementary Table1). The ARL3 human protein shares greater than 90% identity with its two orthologous sequences (there is genomic duplication ofarl3) inDanio 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 inDrosophila melanogaster,Caenorhabditis elegansandChlamydomonas reinhardtii. CEP120 is conserved in some vertebrate organisms but orthologues were not readily identified in invertebrates. There is a putativeCEP120orthologue, UNI2, found inChlamydomonas reinhardtii, but this has not as yet been confirmed as a functional ortholog [27,44]. ARL3 is described in diverse eukaryotic organisms such asLeishmania donovani[45] andCaenorhabditis elegans[46] [47] where it has a functional role in the cilium/flagella. Despite these differences in evolutionary conservation, our results show thatARL3andCEP120have 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 inARL3[15,16,17]. Similarly, the specific expression ofARL3andCEP120in the dorsal root ganglia hints at a role for both genes in primary sensory neurone differentiation. A recurring pattern was the expression of both mRNAs on the luminal facing surface of the cerebral tissue (seen in the choroid plexus and ventricular zones of the cerebral cortex ganglionic eminences, and hindbrain) which could suggest a sensory role for the gene products of both the genes within the cilium of the ventricular lining of the brain.

Expression ofARL3andCEP120changes during development notably in the cerebellum and kidney.ARL3andCEP120are expressed throughout the cerebellum at 14PCW however, at 19PCW, ARL3 expression was predominantly in the IGL whereasCEP120was expressed in the EGL and ML of the cerebellum. This could imply thatARL3andCEP120are expressed in different cell populations of the cerebellum,ARL3in both immature and mature granule cells, andCEP120in immature, migratory granule cells and ML interneurones [48]. It has been previously reported in mouse studies thatCep120is required for proliferation of cerebellar neural progenitor cells [28] and is required for correct development of the embryo. Taken with these results, it suggests thatCEP120expression is required for correct development of the cerebellum in humans.

Expression ofARL3andCEP120also differed in the developing kidney. The results showed thatARL3was specifically expressed in cells of the nephrons whereasCEP120was expressed in the nephrons as well as within cells in the developing renal cortex. This difference in expression could imply thatARL3has a more sensory/signalling function in luminal structures of the kidney, whereasCEP120has 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 Table2). As ARL3 is a trafficking protein involved in ciliary signalling [19,49], 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 [50,51].

In conclusion, we establish in human embryonic tissue expression patterns ofARL3andCEP120during development and provide insights into the wide phenotypic spectrum of mutations affectingARL3andCEP120in humans. These studies will allow further investigations into tissue-specific mechanistic roles ofARL3andCEP120in human health and disease.

All data generated or analysed during this study are included in this published article and its supplementary information files. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

EGL:

External granule cell layer

GCL:

Ganglion cell layer

HDBR:

Human Developmental Biology Resource

IGL:

Internal granule cell layer

INL:

Inner nuclear layer

IPL:

Inner plexiform layer

JATD:

Jeune asphyxiating thoracic dystrophy

JSRD:

Joubert syndrome and related disorders

MKS:

Meckel syndrome

ML:

Molecular layer

NFL:

Nerve fibre layer

OFD:

Oro-facial-digital

ONbL:

Outer neuroblastic layer

ONL:

Outer nuclear layer

OPL:

Outer plexiform layer

PCW:

Post-conception weeks

RPE:

Retinal pigment epithelium

TCDOE:

Tectocerebellar dysraphia with occipital encephalocele

Schematic diagrams were created usingBiorender.com

LP is funded by the Medical Research Council Discovery Medicine North Training Partnership. MB-G is funded by Kidney Research UK (ST_001_20171120) and Northern Counties Kidney Research Fund. EM is funded by Kidney Research UK (Paed_RP_20180925). SAR is Kidney Research UK post-doctoral fellow (PDF_003_20151124). CGM and JAS are funded by Kidney Research UK and Northern Counties Kidney Research Fund. The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data nor in the writing of the manuscript.

Author notes

1. L. Powell and M. Barroso-Gil contributed equally to this work.

Authors and Affiliations

1. Translational and Clinical Research Institute, Newcastle University, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK

   L. Powell, M. Barroso-Gil, L. A. Devlin, E. Molinari, S. A. Ramsbottom, C. G. Miles & J. A. Sayer

2. Biosciences Institute, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

   G. J. Clowry

3. The Newcastle Hospitals NHS Foundation Trust, Freeman Road, Newcastle upon Tyne, NE7 7DN, UK

   J. A. Sayer

4. National Institute for Health Research Newcastle Biomedical Research Centre, Newcastle upon Tyne, NE4 5PL, UK

   J. A. Sayer

Contributions

LP and MB-G analysed and interpreted the data regarding the RNA expression studies. GJC, LAD, SAR and CGM performed data analysis and contributed in writing the manuscript. JAS conceived the project. All authors read and approved the final manuscript.

Corresponding author

Correspondence toJ. A. Sayer.

Ethics approval and consent to participate

This study was conducted with full ethical approval. For human embryonic and foetal tissue samples, the samples were collected with appropriate written maternal consents and ethical approval by the Newcastle and North Tyneside 1 Research Ethics Committee, UK.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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鲍威尔L和Barroso-Gil M是共同第一作者

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