Downregulation of splicing regulator RBFOX1 compromises visual depth perception

<div><p>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 <i>Rbfox1</i> in adult <i>Rbfox1</i>^loxP/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 <i>Rbfox1</i> 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.</p></div

Differentially regulated genes in <i>Rbfox1</i> null mouse retinas that have been reported to be have neuronal function.

<p>Differentially regulated genes in <i>Rbfox1</i> null mouse retinas that have been reported to be have neuronal function.</p

Primers used for quantitative real-time PCR.

<p>Primers used for quantitative real-time PCR.</p

<i>Rbfox1</i> KO animals have normal retinal morphology.

<p>A and B. The Tg(UBC-cre/ERT2)1Ejb transgenic mouse was used to generate Rbfox1 KO animals. The images (modified from <a target="_blank">www.informatics.jax.org/recombinase/specificity?id=MGI:3707333&system=sensory+organs</a>) show beta-galactosidase expression in ocular tissues of an adult Tg(UBC-cre/ERT2)1Ejb transgenic mouse. The rationale for using this transgene is based on a strong Cre expression in RGCs, optic nerve, in the majority of dACs, and certain types of ACs adjacent to the IPL, as well as in the cornea. These locations are most relevant to study the effect of Rbfox1 downregulation on visual function. C-E. Colocalization of Rbfox1 with Rbpms (C), calbindin (D) and Rbfox2 (E) in retinas of wild-type and Rbfox1 KO animals. Very few Rbfox1-positive cells in the GCL were detected. Less dramatic (compared to the GCL), but evident downregulation of Rbfox1 is also observed among ACs in the INL. D. Calbindin immunoreactivity is decreased in Rbfox1 KO retinas; almost no calbindin staining is observed in the INL and very few calbindin-positive cells are present in the GCL. E. Rbfox2 expression in the GCL cells appears to be increased. F. Light microscopic examination of epoxy resin-embedded specimens also failed to identify any change in retinal morphology two months after Rbfox1 downregulation. G. Expression of glial fibrillary acidic protein (GFAP) in <i>Rbfox1</i> KO retinas. GFAP staining was used to evaluate potential “stress” in the retina associated with Rbfox1 downregulation. However, no significant difference in the levels of GFAP immunoreactivity between control and <i>Rbfox1</i> KO retinas was observed. OS, photoreceptor outer segments; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.</p

Rbfox1 expression pattern at embryonic day 12 (E12) of mouse ocular development.

<p>A. A distinctive Rbfox1 staining at E12 is clearly present in the surface ectoderm, lens and retina. B. Rbfox1 expression in retinal cells appears to be cytoplasmic, whereas, Rbfox2 expression is predominantly nuclear. The boxed area in is enlarged below. These cells are most likely RGCs, since at that developmental stage RGCs are the main type of differentiated retinal cells. L, lens; LC, lens capsule; R, retina; SE, surface ectoderm.</p

Visual cliff test reveals depth perception impairment in Rbfox1 KO mice.

<p>Rbfox1 KO and control mice were subjected to two modifications of the test: A. the first test determines the time the animal spends in deep versus shallow side of the chamber; B. in the second test animals were placed on a pedestal between deep and shallow sides and animal’s preference to step down on the perceived deep or shallow side was recorded. The test is designed to identify visual dysfunction that can alter animal’s avoidance of the deep side of the chamber. C. <i>Rbfox1</i> KO mice spent more time on the deep side than on the shallow side. The overall mean (+/-SD) time spent on the deep side was 179.7 +/- 58.8 seconds for all animals in the <i>Rbfox1</i> KO group and 42.3 +/- 28.8 seconds for all animals in the control group, respectively. There was a statistically significant mean difference in time spent on the deep side between two groups: 137.4 seconds (95% CI = 82.8–192.0 seconds; p = 0.002). D. <i>Rbfox1</i> KO mice were also less selective when choosing shallow vs deep side to step down. Control mice have clear preference for the shallow side of the box and avoid the deep side (C and D).</p

Rbfox1 distribution in the lens and cornea.

<p>A. Rbfox1 staining in the lens observed at E12 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200417#pone.0200417.g003" target="_blank">Fig 3A</a>) remained unchanged at P10, P12, P14, P15 and in adult animals. It was predominantly localized in the lens capsule (non-specific), although relatively faint expression can be clearly seen in the cytoplasm of lens epithelial cells from P10 throughout adulthood. Rbfox2 expression is localized to lens epithelial cells (appears to be nuclear) and fiber cells. B. In the cornea, strong Rbfox1 staining is observed in the stroma (non-specific) and endothelial cells. Less pronounced expression is present in epithelial cells. The pattern of this staining in the cornea did not undergo detectable change from P10 into adult stage. Rbfox2 expression was observed in corneal epithelial and endothelial cells, as well as keratocytes in the stroma. Strong Rbfox1 staining in lens capsule and corneal stroma is non-specific since it is present in negative control immunohistochemistry without Rbfox1 primary antibodies.</p

Identification of Rbfox1-regulated genes in the retina.

<p>A. Heatmap representing the top differentially expressed genes in <i>Rbfox1</i> KO vs control retinas after RNA sequencing. Rows are transcripts and each column is an experimental sample compared to three control samples. Red and green in the heat map indicate up- and down-regulation, respectively. B. Real-time PCR quantification of several differentially regulated genes identified by RNA-seq. Downregulation of prostaglandin D2 synthase (<i>Ptgds</i>), vesicle-associated membrane protein 1 (<i>Vamp1</i>), RPE-retinal G protein receptor (<i>Rgr</i>), calbindin 1 (<i>Calb1</i>) and upregulation of transient receptor potential cation channel, subfamily V, member 1 (<i>Trpv1</i>), estrogen receptor 1 (<i>Esr</i>), O-acyltransferase like protein (<i>Oacyl</i>) and synaptonemal complex central element protein 1 (<i>Syce1</i>) in the retinas of <i>Rbfox1</i> KO animals observed by quantitative real-time PCR are in agreement with the results of RNA-seq. The data are presented as the mean ± SEM. C. Immunohistochemical analysis of Vamp1 and Vamp2 in mouse retinal sections. Vamp1 is localized to a very small population of RGCs, whereas Vamp2 is widely expressed in the inner and outer plexiform layers. Vamp1 and Vamp2 were noticeably downregulated in retinas of <i>Rbfox1</i> KO animals, which is consistent with RNA-seq and real-time PCR data. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.</p

Dynamics of subcellular localization of Rbfox1 during early postnatal development of the retina.

<p>A. At P0, along with cells with cytoplasmic Rbfox1 expression, there are cells in the GCL with pronounced nuclear staining (pointed by arrows). Most of Rbfox1 and Rbpms expression is colocalized indicating that these cells are RGCs. B and C. At P5, Rbfox1 expression switches to be predominantly nuclear especially in RGCs and dACs. More cells with nuclear expression of Rbfox1 also appear in the INL. Arrows point at several cells in the GCL and INL with Rbfox1 nuclear staining. D. A significant overlap exists between Rbfox1 and Rbfox2 expression at P5, though in both, the GCL and INL, there are cells with specific expression of one or the other paralog. Rbfox1 staining in the IPL (B, C and D) is non-specific since it was also observed in a control experiment with only secondary antibodies. Blue and white arrows point at several Rbfox1-positive/Rbfox2-negative and Rbfox2-positive/Rbfox1-negative cells, respectively.</p

Association of differentially regulated genes in retinas of Rbfox KO animals with neurological diseases.

<p>Association of differentially regulated genes in retinas of Rbfox KO animals with neurological diseases.</p