Expression of Wnt/β-catenin pathway genes in wild-type and apc mutant eyes. A,B: In 32 and 36 hours postfertilization (hpf) wild-type (wt) eyes, axin2 is expressed in the lens and in peripheral cells of the eye (arrows indicate central border of expression). C: By 48 hpf, only a few most peripherally located cells express axin2. D,E: In 32 and 36 hpf apc mutant eyes, axin2 is up-regulated in cells that express axin2 in wt eyes. F: In 48 hpf apc mutant eyes, axin2 expression is expanded, as it fails to become restricted to only a few peripheral cells. Even in apc mutants axin2 is never activated in the central retina. G-J: in 24-48 hpf wt eyes, only cells in the anterior lens epithelium express the Wnt inhibitor and Wnt target dkk1. K: In contrast, in 24 hpf apc mutant eyes dkk1 is strongly up-regulated in dorsal peripheral regions of the retina. L-N: Beginning at 32 hpf, expression is also observed in the ventral retina. N: Between 36 and 48 hpf dkk1 is down-regulated in the most peripheral cells (arrowheads). Throughout all stages observed dkk1 is also up-regulated in the lens. The dorsal retinal pigmented epithelium is expanded adjacent to the Wnt/β-catenin activation domain (arrow).

Peripheral fate markers are expanded in apc mutant eyes. A: otx1 is only expressed in the most peripheral cells in wild-type 48 hours postfertilization (hpf) eyes. B: in 48 hpf apc mutant eyes otx1 is centrally expanded. C: follistatin labels even fewer cells in the periphery of 48 hpf wild-type eyes than otx1. D: Like otx1, follistatin is expanded in 48 hpf apc mutant eyes.

Wnt/β-catenin signaling inhibits retinal identity and proneural gene expression. A: The neural competence factor sox2 is expressed adjacent to the domain of Wnt/β-pathway activation and is absent from the most peripheral cells (arrows). B: The sox2 domain is centrally shifted in 48 hours postfertilization (hpf) apc mutant eyes. The more peripheral, expanded zone of Wnt/β-pathway activation is devoid of sox2 expression. C: The proneural gene atoh7 is expressed in a wedge adjacent, and more central to the sox2 domain. D: In 48 hpf apc mutant eyes, the atoh7 domain is centrally displaced. E,G: Double fluorescent in situ analysis of sox2 in red and atoh7 in green in 38 and 48 hpf wild-type eyes reveals that sox2 and atoh7 are expressed in adjacent, mutually exclusive domains. F,H: In 38 hpf apc eyes, the sox2 and atoh7 expression domains are shifted toward the central retina. In 48 hpf apc eyes, sox2 and atoh7 expression becomes more disorganized. I,J: Immunofluorescence with Zn8 shows that retinal ganglion cells differentiate in 48 hpf mutant eyes, even though they are restricted to the central retina.

Wnt/β-catenin causes up-regulation of genes that inhibit neural differentiation. A: The transcriptional repressor her6 is expressed in the most peripheral cells in 48 hours postfertilization (hpf) wild-type eyes, as well as in cells surrounding the optic stalk. B: In apc mutant eyes, the her6 expression domain is expanded. C,D: her6 is likely a target of stat3 that is also up-regulated in apc mutant eyes.

Wnt/β-catenin signaling is not involved in the initiation of vsx2 and rx1 expression but affects their maintenance. A,B: At 32 hours postfertilization (hpf), rx1 is broadly expressed in wild-type and apc mutant eyes, even though Wnt/β-catenin target genes are already expanded at this stage. C,D: In 32 hpf wild-type eyes, vsx2 is more strongly expressed in all cells of the dorsal, whereas in apc mutant eyes, it is also present in the ventral domain (D). E: In 48 hpf wild-type eyes, rx1 is expressed in all peripheral cells, encompassing the otx1, sox2, and atoh7 domain, as well as differentiating photoreceptor cells. G: In 48 hpf wild-type eyes, vsx2 is expressed in an overlapping domain with the exception of photoreceptor cells. F,H: In 48 hpf apc mutant cells, rx1 and vsx2 are expressed in more central cells but are down-regulated in the expanded CMZ.

Proliferation is reduced in cells with active Wnt/β-catenin signaling. A: In 48 hours postfertilization (hpf) wild-type eyes, cyclinD1 is expressed in the sox2 and atoh7 domain but is excluded from the most peripheral cells. B: myca is expressed in the sox2 domain and is also excluded from the most peripheral cells. C,D: In 48 hpf apc eyes, cyclinD1 and myca are excluded from the expanded Wnt/β-catenin activation domain but are expressed in the central retina. E: In 50 hpf bromodeoxyuridine (BrdU) -treated wild-type embryos chased for 1 hr, BrdU is incorporated in cells of the sox2, atoh7, and cyclinD domain. Cells peripheral to these zones are mostly devoid of BrdU labeled cells (arrow heads). F: In 50 hpf apc mutant eyes, the expanded peripheral zone is BrdU-negative suggest that cells in the Wnt/β-catenin pathway domain are postmitotic or slowly cycling (outlined by arrowheads). G: In 50 hpf BrdU treated wild-type embryos chased for 2 hr, most cells in the periphery of the eye incorporated BrdU, suggesting that they are more slowly cycling than central cells. However, a few very peripheral cells are still BrdU negative. The very bright cells in the periphery are macrophages and not part of the developing retina. H: In 50 hpf apc mutant eyes that have been treated with BrdU and chased for 2 hr, BrdU is incorporated in single cells in 35% of all sections counted (n = 31; white arrowhead); demonstrating that not all cells are postmitotic.

A-D: meis1 and pax6a act upstream of Wnt/β-catenin pathway activation, as these genes are normally expressed in the periphery of 48 hours postfertilization (hpf) apc mutant eyes.

Cell death is not responsible for gene expression changes in apc mutant eyes. A,B: TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) staining of 48 hours postfertilization (hpf) wild-type (A) and apc mutant (B) eyes. In apc mutant eyes, more cells are undergoing cell death than in wild-type eyes. C,D: In situ hybridization of 48 hpf p53 morpholino injected wild-type and apc mutant embryos with the Wnt/β-catenin pathway target lef1. D: Even in the absence of cell death, apc mutant eyes show an expansion of Wnt/β-catenin target gene expression.