Two ribeye genes in teleosts: the role of Ribeye in ribbon formation and bipolar cell development

Abstract

Ribeye is the only known protein specific to synaptic ribbon, but its function is unclear. We show that the teleost fish, Fugu and zebrafish, have two ribeye genes, ribeye a and ribeye b. Whole-mount in situ hybridization revealed that ribeye a is expressed in tissues containing synaptic ribbons, including the pineal gland, inner ear, and retina. Ribeye b is absent in the pineal gland. In the retina, ribeye a is expressed in both photoreceptors and bipolar cells, whereas ribeye b is detected only in photoreceptors. To study the function of Ribeye a in retina, we depleted it by morpholino antisense oligos. Fish deficient in Ribeye a lack an optokinetic response and have shorter synaptic ribbons in photoreceptors and fewer synaptic ribbons in bipolar cells. Their bipolar cells still target Syntaxin-3 proteins to the inner plexiform layer and have abundant vsx1 mRNA. However, they lack large synaptic terminals and show increased apoptosis. Rod bipolar cells are fewer in number and/or deficient in PKCalpha. Recovery of Ribeye a levels rescues the optokinetic response, increases the number of PKCalpha-positive bipolar cells, and stops apoptosis. We conclude that Ribeye a is important for late steps in bipolar cell development.

Figure 1.

Expression of the two zebrafish ribeye genes during early development. A, RT-PCR analysis. The expression of ribeye a and ribeye b was detected at 24 hpf, whereas ribeye a2 expression was not detected until 72 hpf. β-Actin was used as a control. B, D, Whole-mount in situ hybridization analysis of ribeye a at 48 hpf (B) and 80 hpf (D). The transcripts of ribeye a were found in the inner ears (arrow), pineal gland (arrowhead and inset), and retina. C, E, Whole-mount in situ hybridization analysis of ribeye b at 48 hpf (C) and 80 hpf (E). ribeye b mRNA was identified in the inner ears (arrow) and the retina but not the pineal gland. F, G, Ribeye mRNA distribution by in situ hybridization in transverse eye sections (80 hpf). Ribeye a mRNA was found in the outer half of the inner nuclear layer, presumably bipolar cells, and in the outer nuclear layer in photoreceptor cells (F). Ribeye b was found in photoreceptor cells only (G). H, I, Immunofluorescence labeling of CtBP and Ribeye in transverse eye sections. Ribeye proteins were preferentially localized in the outer and inner plexiform layers, likely in the presynaptic terminals of photoreceptors and bipolar cells. Retinal layers are indicated as follows: IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Figure 2.

Western blot analysis confirms the predicted teleost Ribeye a and a2. The antibody detected CtBP1/2 (50 kDa) in all samples and also detected Ribeye (120 kDa) in retinal extract from adult rat (Rat). In addition, a 144 kDa protein similar to the size of predicted Ribeye a was revealed in retinal extract of adult goldfish (GF), adult zebrafish (ZF), and in extract from zebrafish whole embryos at 48 hpf (ZF48 h). Furthermore, the antibody also detected a 86 kDa protein similar to the size of predicted Ribeye a2 in adult fish retina but not in 48 hpf embryos.

Figure 3.

Ribeye a is essential for OKR. Embryos injected with water (H₂O), mismatch-MO (Mis4), MO1, or MO2 were tested for OKR at 84 hpf. The bars represent the percentage of embryos that were tested positive; the number of tested embryos is given in parentheses.

Figure 4.

MO suppresses Ribeye a expression. Western blot analysis of Ribeye a levels. A, MO-injected embryos contained markedly decreased Ribeye a compared with those injected with H₂O or Mis4. However, in MO-injected embryos that were OKR positive, Ribeye a levels were not significantly decreased. B, Bar graph representing quantitative analysis of Western data. Ribeye a was reduced by >60% in MO-injected, OKR-negative embryos. In contrast, it was decreased only ∼30% in MO-injected, OKR-positive embryos. ^**p < 0.01.

Figure 5.

Ribeye a deficiency alters retinal ribbon structures. A, Synaptic ribbon in the OPL (photoreceptor layer) of a control (Mis4-injected) embryo, which associates with the presynaptic membrane via an arciform density. Nearby are the densely stained membranes of postsynaptic cells, probably two horizontal cells and one bipolarcell. B, As in A but from an MO1-injected embryo; the ribbon is shorter. C, Ribbon in the IPL (bipolar cell layer) from a Mis4-injected embryo, which has a pentalaminar appearance and is associated with densely staining synaptic membranes. D, A sin C but from an MO1-injected embryo; pentalaminar structure is absent. The arrows indicate synaptic membranes. R, Ribbons; D, dense bodies. Scale bar, 100 nm.

Figure 6.

Development of bipolar cells in Ribeye a-deficient larvae. A, B, Expression of vsx-1 in retinal bipolar cells at 84 hpf embryos by in situ hybridization in Mis4-injected (A) and MO1-injected (B) embryos. C, D, Immunofluorescence of Syntaxin-3 (green) and synaptophysin (red) in OPL (top panel) and IPL (bottom panel) in Mis4-injected (C) and MO1-injected (D) embryos. Throughout, MO1-injected larvae tested negative for OKR. The arrowheads indicate probable presynaptic terminals. Scale bar, 10 μm.

Figure 7.

PKCα expression in rod bipolar cells requires Ribeye a. A-D, Cell-specific immunostaining of retinal ganglion cells (Zn-5; navy blue next to the lens), bipolar cells (PKCα; brown), and photoreceptor cells (Zpr-1; navy blue next to pigment epithelium) in embryos injected with water (A), Mis4 (B), MO1 and OKR positive (C), or MO1 and OKR negative (D). Note the diminished PKCα staining in D. Larvae are at 84 hpf throughout.

Figure 8.

Ribeye a deficiency induces cell death. A, B, Cell death in the retina at 84 hpf was limited in embryos injected with Mis4 (A) and MO1 but OKR positive (B). C, In contrast, significant more cell death, particularly in the inner nuclear layer (INL), was seen in the retina of MO1-injected OKR-negative embryos. GCL, Ganglion cell layer; ONL, outer nuclear layer.

Figure 9.

Recovery of Ribeye a expression restores retinal defects. A, Western blot analysis of Ribeye a levels in recovered embryos and in OKR-negative embryos. B, Bargraph indicating that significantly more Ribeye a is present in the recovered animals (^**p < 0.01; ttest) (B). C,D, Immunohistological graphs indicating that recovered embryos also have more PKCα-positive bipolar cells (C) than OKR-negative embryos (D). E, F, Fluorescence micrographs showing that apoptosis is limited in retinas of recovered embryos (E) but extensive in retinas of nonrecovered animals (F).