Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: Figure1. Expressionofthetwozebrafishribeyegenesduringearlydevelopment.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. -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 mRNAwasfoundintheouterhalfoftheinnernuclearlayer,presumablybipolarcells,andinthe 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 pre- synapticterminalsofphotoreceptorsandbipolarcells.Retinallayersareindicatedasfollows:IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, In Situ Hybridization, Immunofluorescence, Labeling

Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: Figure2. WesternblotanalysisconfirmsthepredictedteleostRibeyeaanda2.Theantibody detected CtBP1/2 (50 kDa) in all samples and also detected Ribeye (120 kDa) in retinal extract fromadultrat(Rat).Inaddition,a144kDaproteinsimilartothesizeofpredictedRibeyeawas revealed in retinal extract of adult goldfish (GF), adult zebrafish (ZF), and in extract from ze- brafish 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.

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Injection

Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: Figure 5. Ribeye a deficiency alters retinal ribbon structures. A, Synaptic ribbon in the OPL (photoreceptorlayer)ofacontrol(Mis4-injected)embryo,whichassociateswiththepresynap- tic membrane via an arciform density. Nearby are the densely stained membranes of postsyn- apticcells,probablytwohorizontalcellsandonebipolarcell.B,AsinAbutfromanMO1-injected embryo; the ribbon is shorter. C, Ribbon in the IPL (bipolar cell layer) from a Mis4-injected embryo,whichhasapentalaminarappearanceandisassociatedwithdenselystainingsynaptic membranes.D,AsinCbutfromanMO1-injectedembryo;pentalaminarstructureisabsent.The arrows indicate synaptic membranes. R, Ribbons; D, dense bodies. Scale bar, 100 nm.

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Injection, Membrane, Staining

Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: 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-injectedlarvaetestednegativeforOKR.Thearrowheadsindicateprobablepresynapticterminals.Scalebar,10m.

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Expressing, In Situ Hybridization, Injection, Immunofluorescence

Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: Figure 7. PKC expression in rod bipolar cells requires Ribeye a. A–D, Cell-specific immu- nostaining of retinal ganglion cells (Zn-5; navy blue next to the lens), bipolar cells (PKC; brown), and photoreceptor cells (Zpr-1; navy blue next to pigment epithelium) in embryos injectedwithwater(A),Mis4(B),MO1andOKRpositive(C),orMO1andOKRnegative(D).Note the diminished PKC staining in D. Larvae are at 84 hpf throughout.

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Expressing, Staining

Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: 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)andMO1butOKRpositive(B).C,Incontrast,significantmorecelldeath,particularlyintheinnernuclearlayer(INL),was seen in the retina of MO1-injected OKR-negative embryos. GCL, Ganglion cell layer; ONL, outer nuclear layer.

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Injection

Journal: Journal of Neuroscience

Article Title: Two ribeye Genes in Teleosts: The Role of Ribeye in Ribbon Formation and Bipolar Cell Development

doi: 10.1523/jneurosci.4657-04.2005

Figure Lengend Snippet: Figure 9. Recovery of Ribeye a expression restores retinal defects. A, Western blot analysis of Ribeye a levels in recovered embryosandinOKR-negativeembryos.B,BargraphindicatingthatsignificantlymoreRibeyeaispresentintherecoveredanimals (**p0.01;ttest)(B).C,D,ImmunohistologicalgraphsindicatingthatrecoveredembryosalsohavemorePKC-positivebipolar cells(C)thanOKR-negativeembryos(D).E,F,Fluorescencemicrographsshowingthatapoptosisislimitedinretinasofrecovered embryos (E) but extensive in retinas of nonrecovered animals (F).

Article Snippet: Antibodies and their dilutions used in this study are as follows: rabbit anti-Xenopus CtBP antibody for Ribeye (1:200; kindly provided by Dr. Arie P. Otte, University of Amsterdam, Amsterdam, The Netherlands); monoclonal antibody Zpr1 for red/blue cones (1:100; University of Oregon, Eugene, OR); rabbit anti-PKC antibody for rod bipolar cells (1:100; s.c.-208 rabbit; Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal antibody Zn5 for retinal ganglion cells (RGCs) (1:200; University of Oregon); rabbit anti-Syntaxin3 antibody for presynaptic plasma membrane of ribbon synapse (1:100; 110-033; Synaptic Systems, Gottingen, Germany); mouse anti-synaptophysin 1 monoclonal antibody for synaptic vesicle (1:200; 101-011; Synaptic Systems).

Techniques: Expressing, Western Blot