Adult Expression of Tbr2 Is Required for the Maintenance but Not Survival of Intrinsically Photosensitive Retinal Ganglion Cells

Abstract

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). ipRGCs regulate subconscious non-image-forming behaviors such as circadian rhythms, pupil dilation, and light-mediated mood. Previously, we and others showed that the transcription factor Tbr2 (EOMES) is required during retinal development for the formation of ipRGCs. Tbr2 is also expressed in the adult retina leading to the hypothesis that it plays a role in adult ipRGC function. To test this, we removed Tbr2 in adult mice. We found that this results in the loss of melanopsin expression in ipRGCs but does not lead to cell death or morphological changes to their dendritic or axonal termination patterns. Additionally, we found ectopic expression of Tbr2 in conventional RGCs does not induce melanopsin expression but can increase melanopsin expression in existing ipRGCs. An interesting feature of ipRGCs is their superior survival relative to conventional RGCs after an optic nerve injury. We find that loss of Tbr2 decreases the survival rate of ipRGCs after optic nerve damage suggesting that Tbr2 plays a role in ipRGC survival after injury. Lastly, we show that the GABAergic amacrine cell marker Meis2, is expressed in the majority of Tbr2-expressing displaced amacrine cells as well as in a subset of Tbr2-expressing RGCs. These findings demonstrate that Tbr2 is necessary but not sufficient for melanopsin expression, that Tbr2 is involved in ipRGC survival after optic nerve injury, and identify a marker for Tbr2-expressing displaced amacrine cells.

Keywords: EOMES; cell maintenance; melanopsin; optic nerve crush; transcription factor.

FIGURE 1

Tbr2^CreER recapitulates endogenous Tbr2 expression and loss of Tbr2 does not alter dendritic stratification or axon projections of Tbr2 expressing neurons. (A) Schematic of genetic strategy used to remove Tbr2 from Tbr2⁺ cells in adulthood while simultaneously fluorescently labeling them. Black boxes are exons, thin lines are non-coding regions. (B,C) Flatmount view, GCL side up, of a retina derived from a Tbr2^CreER/+;tdT (control, B) or Tbr2^CreER/flox;tdT (Tbr2^KO, C) P60 mouse immunostained to reveal expression of Tomato (red), Tbr2 (green), RBPMS (blue) with the first image being a merge of all markers; solid circles represent Tbr2⁺ Tomato-labeled RGCs, solid arrows point to Tbr2⁺ Tomato-labeled amacrine cells (lack RBPMS expression), dashed circles indicate Tbr2⁺ RGCs that do not express Tomato, dotted circles indicate Tomato-labeled RGCs that do not express Tbr2, and dashed arrows point to Tomato-labeled amacrine cells that do not express Tbr2; scale bar = 25 μm. While 98% of wildtype Tomato⁺ RGCs express Tbr2 (B), only 15% express Tbr2 in the mutant (C). (D,E) Section of a control (D) and Tbr2^KO (E) mouse retina immunostained to reveal expression of Tomato (magenta), RBPMS (green), DAPI (cyan); scale bar = 25 μm. There is no difference in the localization of Tomato⁺dendrites between these mice (arrows indicate dendrites of Tomato⁺ RGCs (asterisks) in the innermost and outermost sublaminae of the IPL). (F,G) Comparison of the axonal trajectories of Tomato-labeled RGCs in control (F) and Tbr2^KO (G) mice. Coronal sections reveal that Tomato⁺ RGCs innervate the SCN, dLGN, IGL, vLGN, OPN, PPN, and deep SC in controls (F) and maintain this innervation in Tbr2^KO mice (G). Scale bars = 250 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; SCN, suprachiasmatic nucleus; dLGN, dorsal lateral geniculate nucleus; IGL, intergeniculate leaflet; vLGN, ventral lateral geniculate nucleus; OPN, olivary pretectal nucleus; PPN, posterior pretectal nucleus; SC, superior colliculus.

FIGURE 2

Loss of Tbr2 in adulthood results in loss of melanopsin expression but not cell death of Tomato⁺ RGCs. (A) Flatmounted control (Tbr2^CreER/+;tdT) retina (GCL side up) immunostained to reveal expression of Tomato (red), melanopsin (green), and RBPMS (blue); circled cells are Tomato⁺ RGCs that do not express melanopsin, arrows point to melanopsin⁺Tomato⁺ RGCs, scale bar = 25 μm. (B) Flatmounted Tbr2^KO (Tbr2^CreER/flox;tdT) retina immunostained to reveal expression of Tomato (red), melanopsin (green), and RBPMS (blue); circled cells are Tomato⁺ RGCs that do not express melanopsin, arrows point to melanopsin⁺Tomato⁺ RGCs. Tomato⁺ cells lose melanopsin expression. (C) Quantification of cell densities (cells/mm²) of different cell populations in control and Tbr2^KO mice (n = 3 mice in each group; each dot represents the sum of four fields of view (FOVs) for 1 retina; mean ± SEM displayed; two-way ANOVA with Sidak’s multiple comparisons test; no significant differences). (D) Quantification of the percentages of Tomato⁺ cells that belong to cell populations of interest in control and Tbr2^KO mice (n = 3 mice in each group; each dot represents the percentage for 4 summed FOVs of 1 retina; mean ± SEM displayed; two-way ANOVA with Sidak’s multiple comparisons test; *** P < 0.001).

FIGURE 3

Ectopic expression of Tbr2 does not induce melanopsin expression in non-Tbr2 RGCs. (A) Experimental overview of intravitreal AAV2 injection. One eye of an adult mouse (∼P40) was injected with a control virus, GFP-AAV2, while the contralateral eye was injected with a Tbr2-expressing virus, Tbr2-GFP-AAV2. (B) Flatmounted retinas of eyes injected with GFP-AAV2 (left) or Tbr2-GFP-AAV2 (right) are immunostained with the antibody indicated: Tbr2 (magenta), GFP (green), and RBPMS (magenta); the left images for each virus condition are showing overlap of GFP and Tbr2 expression while the right images are showing overlap of GFP and RBPMS expression; white arrowheads indicate example virus-infected RGCs that do not express Tbr2 and yellow arrowheads indicate example virus-infected RGCs that express Tbr2; scale bar = 25 μm. In the Tbr2-GFP-AAV2-infected retina (right), all virus-infected RGCs express Tbr2. (C) Flatmounted retinas of a wildtype mouse infected with GFP-AAV2 (left) and Tbr2-GFP-AAV2 (middle) and immunostained to reveal expression of melanopsin (grayscale); magenta arrowheads point to melanopsin⁺ cells, scale bar = 25 μm. Right panel is showing the quantification of melanopsin⁺ cells in these retinas (n = 3 mice for each group; each dot represents the sum of 4 FOVs per retina; mean ± SEM displayed; Student’s t-test; **P < 0.01). There is a significant increase in melanopsin⁺ cells in the Tbr2-GFP-AAV2-infected retina. (D) Flatmounted retinas of an Isl1^Cre;Tbr2^flox/flox mouse infected with GFP-AAV2 (left) and Tbr2-GFP-AAV2 (middle) immunostained to reveal expression of melanopsin (magenta), GFP (green), RBPMS (blue); white arrowheads point to example melanopsin-negative RGCs infected with virus, yellow arrowheads point to rare melanopsin⁺ cells, scale bar = 25 μm. Right panel is showing the quantification of melanopsin⁺ cells (n = 4 mice in each group; each dot represents the total number of melanopsin⁺ cells in 1 retina; mean ± SEM displayed; Student’s t-test; ns = P > 0.05). There is no change in melanopsin expression in Tbr2-GFP-AAV2-infected retinas in Isl1^Cre;Tbr2^flox/flox mice. (E) Flatmounted retinas of control (Tbr2^CreER/+;tdT, top) and Tbr2^KO (Tbr2^CreER/flox;tdT, bottom) mice immunostained to reveal tomato (red), GFP (green), RBPMS (blue), and melanopsin (gray) expression in GFP-AAV2-infected (left) and Tbr2-GFP-AAV2-infected (middle) retinas. The first image of each is a merge of all color channels, others are showing melanopsin in combination with the other markers indicated. Magenta arrowheads point to virus-infected Tomato⁺ RGCs that express melanopsin and white arrowheads point to virus-infected Tomato⁺ RGCs that do not express melanopsin. Scale bar = 25 μm. Right, quantification of the percentage of virus-infected Tomato⁺ RGCs that express melanopsin in control and Tbr2^KO mice infected with GFP-AAV2 and Tbr2-GFP-AAV2 (n = 4 mice for each group, each dot represents the percentage for the sum of 7 or more FOVs in one retina; mean ± SEM displayed; two-way ANOVA with Sidak’s multiple comparisons, *P < 0.05, ns = P > 0.05). There is increased melanopsin expression in Tbr2-GFP-AAV2-infected Tomato⁺ RGCs.

FIGURE 4

Tbr2-expressing RGCs preferentially survive optic nerve injury and Tbr2 influences their survival. (A) Flatmounted retinas of a wildtype mouse 2 weeks after optic nerve crush in one eye (right, “optic nerve crushed”). The optic nerve of the contralateral control eye was not crushed (left, “uncrushed”). Both retinas were stained to reveal expression of Tbr2 (magenta), melanopsin (cyan), and RBPMS (green); solid white circles represent Tbr2⁺ RGCs that do not express melanopsin, yellow arrows point to melanopsin⁺Tbr2⁺ RGCs, dashed white circles indicate Tbr2⁺ amacrine cells, scale bar = 25 μm. (B) Quantification of percent survival of cell populations of interest in ONC eyes normalized to uncrushed control eyes (n = 3 mice, each dot represents the percentage for one mouse-4 FOVs/retina; mean ± SEM displayed; one-way ANOVA with Dunnett’s multiple comparisons, comparing each group to RGCs; ***P < 0.001, ns = P > 0.05). A significantly greater percentage of melanopsin⁺Tbr2⁺ RGCs survive ONC compared to other RGCs. (C) Left, flatmounted retinas of uncrushed control (Tbr2^CreER/+;tdT, top) and Tbr2^KO (Tbr2^CreER/flox, bottom) eyes. Right, flatmounted retinas of optic nerve crushed control (top) and Tbr2^KO (bottom) eyes immunostained to reveal expression of Tomato (magenta), melanopsin (cyan), and RBPMS (green). Scale bar = 25 μm. (D) Quantification of percent survival of Tomato⁺ RGCs (purple, pattern) and all other RGCs (black, solid) in ONC eyes normalized to uncrushed control eyes in control and Tbr2^KO mice (n = 3 mice for each group, each dot represents the percentage for one mouse-4 FOVs/retina; mean ± SEM displayed; two-way ANOVA with Sidak’s multiple comparisons, ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05). Tomato⁺ RGCs have increased survival relative to other RGCs in both control and Tbr2^KO mice but show decreased survival in Tbr2^KO mice.

FIGURE 5

Meis2 is expressed in the majority of Tbr2-expressing displaced amacrine cells and in a subset of Tbr2-expressing RGCs. (A) Flatmounted retina of a control (Tbr2^CreER/+;tdT) mouse immunostained to reveal expression of tomato (red), Meis2 (green), and RBPMS (blue). First image is a merge of all color channels, others are showing each channel independently; arrows point to Tomato⁺ ACs expressing Meis2; closed white circles represent Tomato⁺ RGCs expressing Meis2; dashed white circles indicate Tomato⁺ RGCs that do not express Meis2. All Tomato⁺ ACs in this image express Meis2. (B) Quantification of the density (cells/mm²) of cell populations of interest in control and Tbr2^KO mice (n = 3 mice in each group, each dot represents the sum of 4 FOVs per retina, mean ± SEM displayed; two-way ANOVA with Sidak’s multiple comparisons test; no significant differences). There is a trend toward a decrease in the density of labeled amacrine cells in Tbr2^KO mice. (C) Quantification of the percentage of Tomato⁺ cells that belong to cell populations of interest in control and Tbr2^KO mice (n = 3 mice in each group; each dot represents the percentage for 4 summed FOVs of 1 retina; mean ± SEM displayed; two-way ANOVA with Sidak’s multiple comparisons test; no significant differences).