Activation of Wnt signaling reduces ipsilaterally projecting retinal ganglion cells in pigmented retina

Abstract

In mammalian albinism, disrupted melanogenesis in the retinal pigment epithelium (RPE) is associated with fewer retinal ganglion cells (RGCs) projecting ipsilaterally to the brain, resulting in numerous abnormalities in the retina and visual pathway, especially binocular vision. To further understand the molecular link between disrupted RPE and a reduced ipsilateral RGC projection in albinism, we compared gene expression in the embryonic albino and pigmented mouse RPE. We found that the Wnt pathway, which directs peripheral retinal differentiation and, generally, cell proliferation, is dysregulated in the albino RPE. Wnt2b expression is expanded in the albino RPE compared with the pigmented RPE, and the expanded region adjoins the site of ipsilateral RGC neurogenesis and settling. Pharmacological activation of Wnt signaling in pigmented mice by lithium (Li⁺) treatment in vivo reduces the number of Zic2-positive RGCs, which are normally fated to project ipsilaterally, to numbers observed in the albino retina. These results implicate Wnt signaling from the RPE to neural retina as a potential factor in the regulation of ipsilateral RGC production, and thus the albino phenotype.

Keywords: Ciliary margin zone; Retinal pigment epithelium; Wnt2b; Zic2.

Fig. 1.

Embryonic pigmented and albino RPE have differential gene expression. (A) GSEA for pigmented versus albino RPE. Sets have a false discovery rate (FDR; q value)<0.05 and were hand curated into thematic categories. (B) GSEA for pigmented versus albino RPE focused on signaling pathways. Bars indicate the number of gene sets with FDR<0.25. Parentheses indicate the number of gene sets with FDR<0.05. In pigmented RPE, no signaling gene set was detected with FDR<0.05 cut-off. (C) Wnt2b expression in peripheral RPE is expanded centrally in albino retina compared with pigmented RPE at E15.5 (arrowhead). (C1,C2) Ventrotemporal (VT) retina sections from a different embryo at E15.5 at higher magnification. d, dorsal; v, ventral. n=8 (E13.5), n=5 embryos (E15.5). (D) Quantification of line tracing of the Wnt2b-positive area in the total perimeter of RPE. Four retinal sections (two rostral, one caudal and one where the optic nerve exits the eye) were analyzed. ON, optic nerve. Unpaired t-test (n=5 embryos). Bars represent mean. Error bars: ±s.e.m. *P<0.05; **P<0.01. (E) Wif1 expression in peripheral neural retina (arrowheads) at E13.5 (n=4 embryos) and E15.5 (n=3 embryos). (F) Fzd7 is enriched in the ventral peripheral retina compared with dorsal peripheral retina (arrowheads) at E13.5 and E15.5 in both genotypes (n=3 embryos). (F1,F2) Higher magnification of E15.5 retina. Scale bars: 100 µm.

Fig. 2.

Expression of Cx43, but not of other genes in the CMZ, is enriched in embryonic albino retina. (A) Otx1 and Bmp4 are expressed similarly in the CMZ albino and pigmented retina (n=3 embryos for Otx1, n=5 embryos for Bmp4). In situ hybridization following melanin bleaching. (B) Otx1, Bmp4 and Msx1 are expressed similarly in the CMZ at E15.5 in both genotypes (n=3 embryos for Otx1 and Msx1, n=4 embryos for Bmp4). (C) Cx43 expression in the CMZ is more intense in an expanded area in albino compared with pigmented retina (arrowhead) at E13.5 but this difference is less evident after E14.5 (n=5 embryos for E13.5 and E15.5, n=3 embryos for E12.5 and E14.5). (D) The neural precursor cell markers Sox2 and Fzd5 are both expressed similarly in pigmented and albino retina (n=3 embryos). Scale bars: 100 µm.

Fig. 3.

Lithium treatment of pigmented mice leads to a reduction in the number of Zic2-positive RGCs. (A) Timeline of LiCl or control (NaCl) injection into pregnant dams (arrowheads) and analysis. (B-E) There were fewer Zic2-positive cells (arrowheads) in lithium-treated pigmented retina, similar to numbers in control (NaCl-treated) albino retina. (F) The number of Zic2 and Isl1/2 double-positive cells decreased in lithium-treated pigmented VT retina, but not in lithium-treated albino retina or control (NaCl-treated) pigmented and albino retina at E15.5. (G) The number of Isl1/2-positive cells (all RGCs) in lithium-treated and control (NaCl-injected) pigmented and albino VT retina is unchanged at E15.5. (F,G) One-way ANOVA (n=7 embryos from five or six litters, two retinal sections/embryo). Bars represent mean. Error bars: ±s.e.m. *P<0.05; **P<0.01; ***P<0.001. ns, not significantly different. (H) Ectopic expression of Otx1 was not observed after lithium treatment at E15.5; n=7 embryos. (I,J) Wnt target genes, Axin2 (I, in situ hybridization) and Lef1 (J, immunohistochemistry) were upregulated after lithium treatment in the RPE (arrowheads in albino retina) and CMZ (arrows), but not in control (NaCl-treated) retina at E15.5. Melanin masks immunohistochemistry and in situ hybridization signal in pigmented RPE (melanin bleaching was not compatible with Axin2 and Lef1 staining). n=3 embryos. Scale bars: 100 µm in B-E,I,J; 200 µm in H.

Fig. 4.

Lithium treatment does not affect retinal proliferation, as measured by PH3 expression. (A) VT retinal sections at E15.5 labeled with PH3 (green), Isl1/2 (magenta) and Hoechst (blue). (B) For cell quantification, the domain of the ciliary marginal zone (CMZ) was delineated by the border of Isl1/2 expression (magenta). Two sectors in the CMZ domain and four sectors in the neural retina (NR) were quantified. (C) The numbers of PH3-positive cells in the ventrotemporal and dorsotemporal CMZ and neural retina. One-way ANOVA (n=8 embryos from six litters). Bars represent mean. Error bars: ±s.e.m. ns, not significantly different. Scale bar: 100 µm.