Distribution of prototypical primary cilia markers in subtypes of retinal ganglion cells

Abstract

Loss of retinal ganglion cells (RGCs) underlies several forms of retinal disease including glaucomatous optic neuropathy, a leading cause of irreversible blindness. Several rare genetic disorders associated with cilia dysfunction have retinal degeneration as a clinical hallmark. Much of the focus of ciliopathy associated blindness is on the connecting cilium of photoreceptors; however, RGCs also possess primary cilia. It is unclear what roles RGC cilia play, what proteins and signaling machinery localize to RGC cilia, or how RGC cilia are differentiated across the subtypes of RGCs. To better understand these questions, we assessed the presence or absence of a prototypical cilia marker Arl13b and a widely distributed neuronal cilia marker AC3 in different subtypes of mouse RGCs. Interestingly, not all RGC subtype cilia are the same and there are significant differences even among these standard cilia markers. Alpha-RGCs positive for osteopontin, calretinin, and SMI32 primarily possess AC3-positive cilia. Directionally selective RGCs that are CART positive or Trhr positive localize either Arl13b or AC3, respectively, in cilia. Intrinsically photosensitive RGCs differentially localize Arl13b and AC3based on melanopsin expression. Taken together, we characterized the localization of gold standard cilia markers in different subtypes of RGCs and conclude that cilia within RGC subtypes may be differentially organized. Future studies aimed at understanding RGC cilia function will require a fundamental ability to observe the cilia across subtypes as their signaling protein composition is elucidated. A comprehensive understanding of RGC cilia may reveal opportunities to understanding how their dysfunction leads to retinal degeneration.

Characterization of Primary Cilia Formation in Human ESC-Derived Retinal Organoids

“…Since little is known regarding cilia in nonphotoreceptor cells in retinal organoids [46,52], we focused our analysis on the inner retina during organoid development. Consistent with our previous reports in mouse retinas [31,45], many ciliated cells (excluding photoreceptors) appeared in the inner parts of retinal organoids at all developmental time points (Figure 1(a)). Next, we assessed the number of cells (excluding photoreceptors) with cilia (ciliation) at different retinal organoid developmental stages.…”

“…AP2α-positive amacrine cells in retinal organoids demonstrated primary cilia at all developmental stages, from day 44 to day 176 (Figure 4(a)). Quantitative analysis showed that the frequency (ciliation) and length of primary cilia displayed no We and others have previously reported the presence of primary cilia within RGCs and their subtypes in mice and zebrafish [25,45]. In this study, our efforts focused on characterizing the frequency and length of primary cilia during the development and differentiation of Brn3b-positive RGCs in retinal organoids.…”

“…After successful culture and organoid formation, immunostaining was performed on cryopreserved sections of the retinal organoids collected from day 44 to day 266. We employed antibodies that specifically detect two ciliary protein markers, Arl13b and Centrin3: Arl13b identifies ciliary axonemes, Centrin3 identifies basal bodies [45,55]. An Arl13b fluorescence signal that aligned with a Centrin3 signal in confocal Zstack images was considered a primary cilium (Supplementary Figure 1.…”

“…Interestingly, we found that majority of apoptotic RGCs (caspase3-positive and Brn3b-positive) were nonciliated, with only 12:04 ± 12:53% of cells were ciliated (Supplementary Figure 3). Many different subtypes of RGCs have been identified in the mammalian retina, a third of which express the calciumbinding protein, calretinin [45,64]. We further assessed primary cilia in calretinin-positive; Brn3b-positive RGCs during retinal organoid development.…”

“…The loss of AC3 in olfactory neurons disrupts glomeruli formation and proper olfactory axon projection in mice [44]. Recently, we have also demonstrated that most retinal ganglion cells (RGC) have AC3-positive primary cilia in adult mice, suggesting that AC3 may play a key role in the postnatal development and maintenance of RGC homeostasis [45].…”