Clinical phenotypes of ARL3 and CEP120 patients
Biallelic mutations in both ARL3 and CEP120 mutations are rare causes of ciliopathy syndromes. A comparison of the known phenotypes associated with ARL3 and CEP120 mutations is shown in Table 1. This overview reveals that mutations in CEP120 are at present associated with severe phenotypes including MKS but also that single heterozygous changes in ARL3 are sufficient to cause retinal-limited phenotypes. ARL3 is highly conserved, with homologs present in C. elegans, C. reinhardtii and D. melanogaster whereas CEP120 appears not to have homologs within these lower organisms (Supplementary Table 1). 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 Table 2).
ARL3 and CEP120 are expressed in early human brain development
In 8PCW human brain tissue, the expression of ARL3 and CEP120 is remarkably similar. There is expression of both genes in the choroid plexus (Figure 1Ai and 1Bi), which appears to favour the luminal facing surface of the tissue, especially for ARL3. The cell proliferation marker KI67 does not share this same expression pattern in the choroid plexus (Figure 1Ci). This specific expression pattern of ARL3 and CEP120 in 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 (Figure 1Aii-Aiv and 1Bii-Biv). There is specific expression of ARL3 and CEP120 in the layer of cells forming the apical surface in each tissue, facing into the ventricular space. Expression of KI67 is seen throughout these tissues (Figure 1Cii-Civ), with specific expression in the apical layer consistent with this being the major site of cell division (cells in G2/M1 phase of cell cycle) in the ventricular zone (35).
Expression of ARL3 and CEP120 is maintained in the developing cerebellum
In the human cerebellum at 14PCW there is expression of ARL3 and CEP120. Both genes have strong expression in the external and internal granule cell layer (EGL and IGL) the developing cerebellum (Figure 2Ai and Ci). Expression in the EGL and IGL is seen at 19PCW for ARL3 (Figure 2Bi) however, CEP120 expression is predominantly localised in the EGL and the molecular layer (ML) of the cerebellum at 19PCW (Figure 2Di). Strong expression of KI67 is seen throughout the EGL in particular, but also the IGL at 19PCW, indicating the tissue is proliferative (Figure 2Ei). ARL3 and CEP120 are therefore widely expressed in the cerebellum during development, with specific expression of CEP120 in 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 that CEP120 expression is predominantly located in the sparse population of interneurons found in the molecular layer (36) or in immature granule cells migrating from EGL to IGL (37) suggesting a role for CEP120 in these cell types (36).
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 (Figure 3A), with the retinal pigment epithelial (RPE) and photoreceptor layers at the outermost part of the eye (37). 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 (38). At this stage ARL3 and CEP120 show expression throughout the developing retina, with high expression within the retinal ganglion cells and the photoreceptor layer (Figure 3Bi and Di). At 14PCW, the retinal layers are maturing (37) which is reflected in the expression pattern of both ARL3 and CEP120. 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 (Figure 3Ci 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 (39, 40). Both ARL3 and CEP120 show expression in cells of the dorsal root ganglia, which are post-mitotic primary sensory neurons (Figure 4Ai-Aii and Bi-Bii). There is strong expression of KI67 within limited number of cells in the dorsal root ganglia, presumably non-neuronal (Figure 4Ci-Cii).
Expression of ARL3 and CEP120 in the developing kidney
In the developing human kidney at 8PCW, where there is strong renal cortical staining of KI67 indicating cell proliferation (Supplementary Figure 2), there is expression of ARL3 in cells within the developing cortical nephrons; this expression appears to be specifically oriented to the lumen of the structures (Figure 5Ai). This expression pattern is maintained at 14PCW (Figure 5Bi) and 18PCW (Figure 5Ci). Expression of CEP120 is also seen in developing nephrons at 8PCW, however there is also expression in the renal cortex (Figure 5Di). This expression pattern of CEP120 is maintained at 14PCW (Figure 5Ei) and 18PCW, although overall expression appears to have decreased at this time point (Figure 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 of ARL3 and CEP120 (Supplementary Figure 3). Expression of ARL3 and CEP120 is 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.