Article Figures & Data
Figures
- Figure 1.
LRFN2 is located at the DBC signalplex in mouse retina. A, Mass spectrometry analysis of proteins copurified with antibodies to TRPM1. B, Schematic diagram of the domain structure of LRFN2 predicted by the SMART® program. LRFN2 contains a signal sequence (purple box), extracellular LRR, Ig and FN3 domains, a transmembrane segment (TM, blue box), and intracellular C terminus containing a PDZ binding domain (green ball). C, Mass spectrometry analysis of proteins copurified with antibodies to LRFN2. For full list of proteins see Mendeley Data, V1, doi: 10.17632/wphby2csr5.1.
- Figure 2.
LRFN2 is localized to cone synapses in the OPL of mouse retina. A, Representative confocal images after immunohistochemical staining for LRFN2 (green) in retina slices from control (left panels) and Lrfn2−/− retinas (right panels). Scale bar A = 5 µm; B, Representative confocal images of the OPL after immunohistochemical staining for LRFN2 (green) and Ribeye (magenta) in retina slices from control (left panels) and Lrfn2−/− retinas (right panels). Scale bar = 20 µm. C, Western blot showing presence of LRFN2 in control retinal lysates, and its absence in Lrfn2−/− retinal lysates. β-actin was used as a loading control. 100 µg protein from retinal lysates was loaded/lane. D, Immunohistochemical localization of LRFN2 (green) with the cone terminal maker PNA (red) in retinal sections from control and Lrfn2−/− retinas. Scale bar = 5 µm.
- Figure 3.
Lrfn2 mRNA is expressed in the ONL and INL. In situ hybridization of mRNAs from Polr2 (RNA polymerase II as a positive control), Grm6 as a representative DBC signalplex member, and Lrfn2. The dashed lines shows the location of the OPL (outer plexiform layer), ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Dot plots from scRNAseq are shown in Extended Data Figure 3-1.
- Figure 4.
Synaptic proteins are localized normally in Lrfn2−/− mouse retina. Representative confocal images showing immunohistochemical localization of synaptic proteins TRPM1, LRIT3, mGluR6 and GPR179 in the OPL of transverse retinal slices from control and Lrfn2−/− retinas. Scale bar = 5 µm. Circles indicate cone synapses. OPL, outer plexiform layer. Validation of the mGluR6 antibody is shown in Extended Data Figure 4-1.
- Figure 5.
Synaptic proteins pikachurin, ribeye and ELFN2 are localized normally in absence of LRFN2. A, Representative confocal images after immunohistochemistry for Pikachurin (top row), Ribeye (middle row), and ELFN2 (bottom row) in transverse retinal sections from control and Lrfn2−/− mouse retina. Scale bar = 5 µm. OPL, outer plexiform layer. Bi, Representative western blots of retinal lysates for DBC signalplex proteins, Bii, Quantification of signalplex protein levels from western blots of retinal lysates from 3 mice of each genotype (mean ± 95% CI). Data were normalized to the control sample. β-actin was used as a loading control.
- Figure 6.
LRFN2 is expressed normally in control, Lrit3−/−, Trpm1−/−, Grm6−/−, Gpr179−/−, Nyxnob and Elfn2−/− retinas. Images are from representative of the OPL from retinal sections (n = 3) stained for LRFN2 (green) and PNA (magenta). Scale bar = 5 µm. OPL, outer plexiform layer.
- Figure 7.
Photopic ERG b-waves of Lrfn2−/− mice have a reduced amplitude. Electroretinogram waveforms for a single animal under A, scotopic and B, photopic conditions at different intensity flashes for a control (black) and a Lrfn2−/− (red) eye. Average stimulus-response plots for the ERG a-wave and b-wave amplitudes under C, scotopic and D, photopic conditions for control (n = 7, black) and Lrfn2−/− (n = 8, red). Statistics: comparison of control vs Lrfn2−/− groups, *padj < 0.05 for stimuli, 2-way repeated measures ANOVA. Between the groups, there are no differences in b-wave amplitude under scotopic conditions, and in a-wave amplitude under either scotopic or photopic conditions (2-way repeated measures ANOVA, padj > 0.05). 95% confidence intervals of the differences between genotypes are shown in Extended Data Figure 7-1.
Tables
Antigen Dilution Source Catalogue # LRFN2 1:1,000 Sigma SAB3500015 LRIT3 1:1,000 Hasan et al. (2019) N/A GPR179 1:2,000 Peachey et al. (2012) N/A TRPM1 1:1,000 Hasan et al. (2019) N/A mGluR6 1:1,000 Current paper N/A Pikachurin 1:2,000 Wako Chemicals 011-22631; PNA 1:1,000 Molecular probes L-32460; ELFN2 1:2,000 Invitrogen PA5-43521,
Extended Data Figure 3-1
Single cell RNAseq data shows a relatively low level expression of Lrfn2 in retina cells. A. Dot plot for select genes in P14 mouse retina. Lrfn2 expression is highest in cones. Data set: C57B6 wild-type P14 retina by drop-seq (Macosko EZ, et al. (2015) Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell 161:1202-1214.) B, Dotplot of Lrfn2 and select gene mRNA expression in retinal bipolar cells. Data set: Retinal Bipolar Neuron Drop-seq Shekhar K et al. (2016) Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics. Cell 166:1308-1323 e1330. C, Dotplot of Lrfn2 and select gene mRNA expression in retinal amacrine cells. All data are from the Single Cell Portal (https://singlecell.broadinstitute.org/single_cell). Data set: Mouse Retinal Cell Atlas: Molecular Identification of over Sixty Amacrine Cell Types (Yan W et al. (2020) Mouse Retinal Cell Atlas: Molecular Identification of over Sixty Amacrine Cell Types. J Neurosci 40:5177-5195.). Download Extended Data Figure 3-1, TIF file.
Extended Data Figure 4-1
mGluR6 antibody is specific. We developed an antibody to mGluR6 as described in the methods section. Serum was collected and mGluR6 antibody affinity purified. A, The purified mGluR6 antibody (green) was used to stain Control and Grm6-/- retina sections. The only staining was as puncta at the OPL. B, High power images of staining with mGluR6 antibody (green) and the cone terminal marker PNA (magenta). The data demonstrate punctate staining in the outer plexiform layer (OPL). The merged image shows green puncta from rod to rod BC synapses, and the large magenta/white puncta staining is from cone terminals. Scale bar in A = 10µm and in B = 5µm. Download Extended Data Figure 4-1, TIF file.
Extended Data Figure 7-1
Confidence intervals of differences between Control and Lrfn2-/-. Data were analyzed using 2-way ANOVA, with post hoc comparisons adjusted using Šídák's multiple comparisons test for multiple testing adjustment. The only values showing a significant difference at Padj ≤ 0.05 are shown in red. Download Extended Data Figure 7-1, TIF file.
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