Article Figures & Data
Figures
- Figure 1.
Tbr2 is expressed in a distinct subset of RGCs. Sections through P8 eyes were immunostained with the antibodies indicated; the color of the text matches the color of the label. Blue is DAPI, showing the location of retinal nuclei, and a GFP antibody was used to increase signal. Few Tbr2+ RGCs express Brn3a (2.4%, n = 3235 cells; A, B), GABA (3%, n = 96 cells; C), DRD4-GFP (12%; n = 75 cells; E), CB2-GFP (3%; n = 30 cells; F), or CART (9%; n = 80 cells; I). However, most Cdh3-GFP (98%; n = 44 cells; D), Melanopsin+ (91%; n = 32 cells; G), and Unc5d+ (68%; n = 107 cells; H) RGCs also express Tbr2. B shows a flat-mounted retina, RGC side up. Yellow arrowheads indicate examples of Tbr2+ cells that are colabeled with the indicated marker. White arrowheads indicate examples of Tbr2+ cells that are not colabeled. ONL, Outer nuclear layer; IPL, inner plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: A, 50 μm; B–I, 25 μm.
- Figure 2.
Tbr2 is expressed in at least three largely nonoverlapping sets of RGCs. Panels are labeled as in Figure 1. Tbr2 does not colocalize with Isl2-GFP, which projects to image-forming areas (5%; n = 136 cells; A), and few cells show coincident expression of Melanopsin and Unc5d (1.5%; n = 67 cells; B) or Cdh3-GFP and Unc5d (0%; n = 44 cells; C). Tbr2-GFP lamination patterns show that many labeled cells project to the inner ON sublamina (D) with a few projecting to the outer OFF lamina (D, E). A majority of Tbr2+ cells were labeled by CB2-Cre (56%; n = 116 cells; F) and, although all Tbr2-GFP+ cells express Tbr2 (100%; n = 50 cells; G), and a majority express melanopsin (76%; n = 62 cells; H), only 6% (n = 765) of Tbr2+ RGCs are labeled by Tbr2-GFP. I is a diagram showing the degree of coexpression of Tbr2+ RGC subclasses. GCL, Ganglion cell layer; IPL, inner plexiform layer. Scale bar, 25 μm.
- Figure 3.
Tbr2-GFP labels axons that project to a subset of the non-image-forming retinorecipient areas of the brain. Sections through an adult (P20) mouse brain show Tbr2-GFP labeled (A, D, G, J, L, N) and all contralateral eye inputs (B, E, H) in different retinorecipient areas. Merged images are shown in C, F, I, K, M, and O. The retinorecipent areas shown are as follows: OPN (A–C); vLGN, IGL, and dLGN (D–F); NOT and PPN (G–I), SCN (J, K), MTN (L, M), and SC (N, O). Arrowheads in N show sparse, GFP-positive axons in the deep SC. Each of five mice examined shared the same projection patterns. Scale bars, 100 μm for all panels.
- Figure 4.
Conditional deletion of Tbr2 using CB2-Cre leads to the loss of Tbr2+ RGCs. Sections through P8 littermate control (A–C) and Tbr2fl/fl;CB2Cre/+ (D–F) retinas. Tbr2+ cells are reduced by 68% in the Tbr2 mutant (WT: 194.4/section, n = 8 sections; KO: 61.4/section, n = 12 sections) and Unc5d+ (Control: 40/section, n = 5 sections; KO:3.9/section, n = 10 sections) and melanopsin+ RGCs (Control: 33/section, n = 3 sections; KO: 6.8/section, n = 6 sections) are also reduced and staining in the inner ON sublaminae is missing (white arrowheads; B, E). The numbers of Brn3a+ (Control: 423/section, n = 3 sections; KO: 467/section, n = 3 sections) and CART+ (Control: 94/section, n = 3 sections; KO: 88/section, n = 3 sections;) cells are not significantly different between controls and Tbr2 mutants (C, F). Arrowheads in B and E show the location of melanopsin+- and Unc5d+ dendrites in ON sublimae in controls and conditional mutants. Retinas from 3 mice of each genotype were used. Scale bar, 25 μm.
- Figure 5.
Tbr2 mutants show decreased axonal projections to non-image-forming areas. Axon tracing of the contralateral eye with CtB-555 in control (A, B, E, F, G) and Tbr2fl/fl;CB2Cre/+ mice (C, D, H, I, J) was used to visualize projections to the retinorecipient areas labeled. Tbr2 mutant mice have decreased total retinal projections to the vLGN, IGL, OPN, and PPN (yellow arrowheads) but not to the dLGN, NOT, SC MTN or SCN. Scale bar, 100 μm. Fluorescence intensity was calculated for each target region (n = 3–5 mice; K) and significant was calculated using t tests. **p < 0.05; ***p < 0.01. p-values are as follows: vLGN = 0.043; IGL = 0.049; dLGN = 0.3162; OPN = 0.0006; NOT = 0.636; PPN = 0.008; SC = 0.254, MTN = 0.585, and SCN = 0.386.
- Figure 6.
Pupillary light reflex in Tbr2 mutants is attenuated. A–F, Adult control or Tbr2fl/fl;CB2Cre/+ mice were dark adapted for 1 min and then either low (172 mW/cm2) or high (2.9 mW/cm2) light intensity was presented for 30 s to the contralateral eye and monitored with an infrared camera. G, Graph showing the percent pupil constriction in control and Tbr2 mutant mice (n = 6 of each) and a significant decrease in response of the mutant at both light intensities. Using one-way ANOVA, p = 0.00014 for high light and p = 2.2 × 10−8 for low light. ***p < 0.001. Scale bar, 500 μm.
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