Downregulation of splicing regulator RBFOX1 compromises visual depth perception.

Lei Gu,Fei Yu,Joseph Caprioli,Dean Bok,Natik Piri

doi:10.1371/journal.pone.0200417

Abstract

Rbfox1 is a splicing regulator that has been associated with various neurological conditions such as autism spectrum disorder, mental retardation, epilepsy, attention-deficit/hyperactivity disorder and schizophrenia. We show that in adult rodent retinas, Rbfox1 is expressed in all types of retinal ganglion cells (RGCs) and in certain subsets of amacrine cells (ACs), within the inner nuclear (INL) and ganglion cell (GCL) layers. In the INL, all Rbfox1-positive cells were colocalized with GABAergic ACs, however not all GABAergic ACs were immunostained for Rbfox1. In the GCL, a vast majority of GABAergic dACs were Rbfox1-immunopositive. Furthermore, all cholinergic starburst ACs (SACs) in the INL (type a) and in the GCL (type b) were Rbfox1 positive. The expression of Rbfox1 in the retina significantly overlapped with expression of Rbfox2, another member of Rbfox family of proteins. Rbfox2, in addition to RGCs and ACs, was also expressed in horizontal cells. In developing retinas at E12 and E15, Rbfox1 is localized to the cytoplasm of differentiating RGCs and ACs. Between P0 and P5, Rbfox1 subcellular localization switched from cytoplasmic to predominantly nuclear. Downregulation of Rbfox1 in adult Rbfox1loxP/loxP mice had no detectable effect on retinal gross morphology. However, the visual cliff test revealed marked abnormalities of depth perception of these animals. RNA sequencing of retinal transcriptomes of control and Rbfox1 knockout animals identified a number of Rbfox1-regulated genes that are involved in establishing neuronal circuits and synaptic transmission, including Vamp1, Vamp2, Snap25, Trak2, and Slc1A7, suggesting the role of Rbfox1 in facilitating synaptic communications between ACs and RGCs.

Highlights

Rbfox1 (RNA binding protein, fox-1 homolog 1) and two other members of the Rbfox family, Rbfox2 and Rbfox3, are evolutionarily conserved RNA-binding proteins that regulate tissuespecific alternative splicing
In the inner nuclear (INL), as stated above, Rbfox1 was mostly expressed in amacrine cells (ACs) that were adjacent to the inner plexiform layer (IPL), whereas Rbfox2 expression was more broadly distributed among ACs
If not all, Rbfox1-positive cells in the INL were colocalized with GABAergic ACs, whereas not all GABAergic ACs were immunostained with Rbfox1 (Fig 2A)

Summary

Introduction

Rbfox (RNA binding protein, fox-1 homolog 1) and two other members of the Rbfox family, Rbfox and Rbfox, are evolutionarily conserved RNA-binding proteins that regulate tissuespecific alternative splicing. Genes that were underrepresented (downregulated) in RGC-deficient retinas compared to the controls, including Rbfox and Rbfox, were subjected to further analysis as RGC-expressed genes. From this pool of RGC-expressed genes, we had earlier identified and characterized a new RGC marker —Rbpms (RNA binding protein with multiple splicing) and Nell (neural epidermal growth factor-like 2) [11, 12]. We analyze the expression pattern of Rbfox 1 in adult and differentiating retinal neurons, evaluate its role in visual function with Rbfox KO animals, and identify potential targets of Rbfox in RGCs and ACs by comparing retinal transcriptomes of Rbfox KO and control animals

Results

Discussion

Experimental procedures Animals