Penetrance of eye defects in mice heterozygous for mutation of Gli3 is enhanced by heterozygous mutation of Pax6

Abstract

Background: Knowledge of the consequences of heterozygous mutations of developmentally important genes is important for understanding human genetic disorders. The Gli3 gene encodes a zinc finger transcription factor and homozygous loss-of-function mutations of Gli3 are lethal. Humans heterozygous for mutations in this gene suffer Greig cephalopolysyndactyly or Pallister-Hall syndromes, in which limb defects are prominent, and mice heterozygous for similar mutations have extra digits. Here we examined whether eye development, which is abnormal in mice lacking functional Gli3, is defective in Gli3+/- mice.

Results: We showed that Gli3 is expressed in the developing eye but that Gli3+/- mice have only very subtle eye defects. We then generated mice compound heterozygous for mutations in both Gli3 and Pax6, which encodes another developmentally important transcription factor known to be crucial for eye development. Pax6+/-; Gli3+/- eyes were compared to the eyes of wild-type, Pax6+/- or Gli3+/- siblings. They exhibited a range of abnormalities of the retina, iris, lens and cornea that was more extensive than in single Gli3+/- or Pax6+/- mutants or than would be predicted by addition of their phenotypes.

Conclusion: These findings indicate that heterozygous mutations of Gli3 can impact on eye development. The importance of a normal Gli3 gene dosage becomes greater in the absence of a normal Pax6 gene dosage, suggesting that the two genes co-operate during eye morphogenesis.

Figure 1

(A) Gli3 mRNA expression in the developing eye at E14.5. mRNA expression is observed in the neural retinal, retinal pigment epithelium, lens and surface ectoderm. (B-E) Pax6 protein expression in (B) wild type, (C)Gli3^+/-, (D) Pax6^+/- and (E) Pax6^+/-; Gli3^+/- eyes. In wild type embyos (B), Pax6 is expressed in cells of the neural retina, lens epithelium and surface ectoderm. Gli3^+/-embryos (C) show identical Pax6 expression to wild type with no overt retinal dysmorphology. Eyes of the developing Pax6^+/- embryo (D) show a similar Pax6 expression to wild type but cells forming the persistent lens stalk (arrow) can be clearly seen to be Pax6 expressing. Pax6^+/-;Gli3^+/- eyes (E) exhibit Pax6 expression localised to the neural retina, retinal pigment epithelium and lens epithelium but show overt distal retinal dysplasia. Rpe, retinal pigment epithelium; nr, neural retina; L, lens; se, surface ectoderm. Scale bar = 200 μm.

Figure 2

Masses of wild-type (n = 18), Gli3^+/- (n = 30), Pax6^+/- (n = 24) and Pax6^+/-; Gli3^+/- (n = 25) adult eyes.

Figure 3

Retinal morphology of wild-type (A), Gli3^+/- (B, E), Pax6^+/- (C) and Pax6^+/-;Gli3^+/- (D, F-H) animals. Although a majority of Gli3^+/- retinae exhibit normal lamination (B), mild focal dysplasia is seen (E). Pax6^+/-; Gli3^+/- retinal phenotypes include thinning of the retina (D) and dysplasia (F-H). gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; ipl, inner plexiform layer; opl, outer plexiform layer; r, rosette. Scale bars = 100 μm.

Figure 4

Retinal layer thickness in wild-type, Gli3^+/-, Pax6^+/- and Pax6^+/-; Gli3^+/- eyes. (A) Schematic representation of areas of retinal layer measurements in wild-type and mutant eyes. Measurement of retinal layers for (B) whole retinal thickness, (C) outer nuclear layer (ONL), (D) inner nuclear layer (INL), (E) outer plexiform layer (OPL), (F) inner plexiform layer (IPL) showing average thickness (μm) (+/- s.e.m.) for each genotype group. Significant differences between genotypes are shown with brackets. l, lens; r, retina; on, optic nerve. * P = < 0.05; ** P = 0.001.

Figure 5

Anterior segment abnormalities in Pax6^+/-; Gli3^+/- eyes. Abnormal contacts between cornea and iris (A-C), lens and cornea (E) and iris and lens (D) were observed in Pax6^+/-; Gli3^+/- eyes. (A-C) Iris hyperplasia and formation of cyst-like structures involving iris, retina and cornea were also observed (C). (D, E) Some lenses of Pax6^+/-; Gli3^+/- animals were highly dysgenic. Some Gli3^+/- animals had iris tissue attached to the lens (arrow in F). i, iris; cs, corneal stroma; L, lens; c, cyst. Scale bars = 100 μm.

Figure 6

Corneal abnormalities in Pax6^+/- and Pax6^+/-; Gli3^+/- eyes. Corneal morphology of (A) wild-type, (B) Gli3^+/-, (C) Pax6^+/- and (D) Pax6^+/-; Gli3^+/- animals. (C) Pax6^+/- and (D) Pax6^+/-; Gli3^+/- eyes have a thinner corneal epithelium and more hypercellular stroma (arrows in C) than wild-type (A) and Gli3^+/- (B) eyes. Graphs show relative thicknesses of (E) corneal epithelium and (F) corneal stroma of the various genotype groups. The corneal epithelium of Pax6^+/- and Pax6^+/-; Gli3^+/- eyes was significantly thinner than that of wild-type and Gli3^+/-eyes, both centrally and peripherally. The peripheral stroma of Pax6^+/-; Gli3^+/- eyes was thinner than that of wild-type and Gli3^+/- eyes, thus making the mean stromal thickness (average of peripheral and central) of Pax6^+/-; Gli3^+/- eyes significantly less than that of wild-type, Pax6^+/- and Gli3^+/- eyes. * = significantly different to wild-type; † = significantly different to Gli3^+/-; ‡ = significantly different to Pax6^+/-. ce, corneal epithelium; cs, corneal stroma. Scale bar = 50 μm.

Figure 7

Summary of possible interactions between Gli3 and Pax6 in Shh signalling. The processing of Gli3 to its cleaved form (Gli3CL) from its full-length form (Gli3FL) is inhibited by Shh. Levels of Pax6 are regulated by levels of Gli3CL by a process of double repression, perhaps involving Pax2 and Vax1, such that lowering levels of Gli3CL lowers Pax6 levels. Pax6 negatively regulates Shh expression: thus, lowered Pax6 levels increase Shh's repression of Gli3CL production.