Characterization of primary cilia during the differentiation of retinal ganglion cells in the zebrafish.

Paola Lepanto,Jose L Badano,Camila Davison,Gabriela Casanova,Flavio R Zolessi

doi:10.1186/s13064-016-0064-z

Abstract

BackgroundRetinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. The primary cilium, as a signaling hub in the cell, may have a role during this process but its presence and localization during RGC generation, and its contribution to the process of cell differentiation, have not been previously assessed in vivo.MethodsIn this work we analyzed the distribution of primary cilia in vivo using laser scanning confocal microscopy, as well as their main ultrastructural features by transmission electron microscopy, in the early stages of retinal histogenesis in the zebrafish, around the time of RGC generation and initial differentiation. In addition, we knocked-down ift88 and elipsa, two genes with an essential role in cilia generation and maintenance, a treatment that caused a general reduction in organelle size. The effect on retinal development and RGC differentiation was assessed by confocal microscopy of transgenic or immunolabeled embryos.ResultsOur results show that retinal neuroepithelial cells have an apically-localized primary cilium usually protruding from the apical membrane. We also found a small proportion of sub-apical cilia, before and during the neurogenic period. This organelle was also present in an apical position in neuroblasts during apical process retraction and dendritogenesis, although between these stages cilia appeared highly dynamic regarding both presence and position. Disruption of cilia caused a decrease in the proliferation of retinal progenitors and a reduction of neural retina volume. In addition, retinal histogenesis was globally delayed albeit RGC layer formation was preferentially reduced with respect to the amacrine and photoreceptor cell layers.ConclusionsThese results indicate that primary cilia exhibit a highly dynamic behavior during early retinal differentiation, and that they are required for the proliferation and survival of retinal progenitors, as well as for neuronal generation, particularly of RGCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0064-z) contains supplementary material, which is available to authorized users.

Highlights

Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors
We found evident axonemes in only 19.5 % of the cases (18/92; Fig. 6d and f ), and invariably, these cilia were positioned at the base of the RGC dendritic tree, where most were pointing basally
By using transmission electron microscopy (TEM), we evaluated the effect of the morpholino oligomer (MO) in the differentiating neural retina, where we observed a significant reduction in apical primary cilia length (Fig. 8d)

Summary

Introduction

Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. To achieve this unique organization, dividing neuroepithelial cells must give rise to Both cell type-specific expression of transcription factors and tissue-derived positional and trophic factors are likely to interact to achieve a mature and fully functional retina. The basal lamina of the neuroepithelium (the “inner limiting membrane”) has been shown to play a critical role as RGC axon extension and orientation depends on the presence of Laminin [2] Another contributing signaling molecule is Sonic Hedgehog (Shh), which is needed for spreading the wave of RGC and amacrine cell differentiation across the retina, as well as later on for photoreceptor cell differentiation [3,4,5,6,7]. The tissue impinges constraints on the inherited differentiation program, guiding it to achieve the mature functional structure

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