Primary Cilia in Amacrine Cells in Retinal Development

Abstract

Primary cilia are conserved organelles found in polarized cells within the eye that regulate cell growth, migration, and differentiation. Although the role of cilia in photoreceptors is well-studied, the formation of cilia in other retinal cell types has received little attention. In this study, we examined the ciliary profile focused on the inner nuclear layer of retinas in mice and rhesus macaque primates. METHODS.Retinal sections or flatmounts from Arl13b-Cetn2 tg transgenic mice were immunostained for cell markers (Pax6, Sox9, Chx10, Calbindin, Calretinin, ChaT, GAD67, Prox1, TH, and vGluT3) and analyzed by confocal microscopy. Primate retinal sections were immunostained for ciliary and cell markers (Pax6 and Arl13b). Optical coherence tomography (OCT) and ERGs were used to assess visual function of Vift88 mice. RESULTS.During different stages of mouse postnatal eye development, we found that cilia are present in Pax6-positive amacrine cells, which were also observed in primate retinas. The cilia of subtypes of amacrine cells in mice were shown by immunostaining and electron microscopy. We also removed primary cilia from vGluT3 amacrine cells in mouse and found no significant vision defects. In addition, cilia were present in the outer limiting membrane, suggesting that a population of Müller glial cells forms cilia. CONCLUSIONS.We report that several subpopulations of amacrine cells in inner nuclear layers of the retina form cilia during early retinal development in mice and primates.

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.…”

“…Interestingly, we did not detect ciliated cells that expressed Prox 1, a known marker for horizontal cells [47,54,58], in retinal organoids at day 176. This result suggests that Prox1-positive horizontal cells in retinal organoids do not harbor primary cilia at 3 Stem Cells International least at this time, which is consistent with our previous mouse data [31]. Lastly, using a GFAP antibody to visualize astrocytes [48], we observed expression of primary cilia in GFAP-positive astrocytes in day 266 retinal organoids.…”

“…Amacrine Cells and Their Subtypes in hESC-Derived Retinal Organoids. We previously found that amacrine cells in mouse and primate retinas contain primary cilia [31]. Amacrine cells are localized to the inner part of retina, where ciliated cells in retinal organoids were observed to be most abundant.…”

“…It is also well known that eye patterning and morphogenesis of mouse eyes are dependent upon proper ciliogenesis [26][27][28][29][30]. Most of our knowledge comes from investigations of cilia within photoreceptors, and much less is known about the role of cilia in nonphotoreceptors in the retina [6,28,31].…”

Light-induced asymmetries in embryonic retinal gene expression are mediated by the vascular system and extracellular matrix

“…The ECM is connected to ion activity and to the asymmetric movement of primary cilia, that drives asymmetries in mice and other vertebrate species 23 . Moreover, cilia are present in the retina at the level of photoreceptors and in other retinal cell types 28 , 29 , acting in the detection and transduction of visual signals, macromolecules and other signals 108 . At the molecular level, we found enrichment of ion activity, including Ca 2+ ion binding, that is connected to the activity of cilia.…”