Loss of Ptdsr activity is associated with defects in ocular development and can lead to formation of ectopic eye structures
By gross morphology we could differentiate two classes of Ptdsr mutants: those that appeared normal with both eyes present (Figure 4) and those that were severely affected and displayed uni- or bilateral anophthalmia (Figure 5).
Analysis of normal or mildly affected embryos revealed no differences between mutant and wild-type embryos in the differentiation of the developing eye until E16.5. In both genotypes, inner and outer layers of the retina displayed a comparable differentiation status, as shown, for example, at E12.5 (Figure 4a,e). At day E16.5, however, retinal layers in Ptdsr -/- embryos were much thinner than in wild-type embryos, contained fewer cells and were greatly reduced in size (Figure 4b,f). Comparison of the retinal structures of Ptdsr +/+ and Ptdsr -/- embryos revealed that all four retinal layers were present in Ptdsr-knockout mice at E16.5 (Figure 4b,f). At E18.5 (Figure 4c,g) and in neonatal animals (postnatal day P0; Figure 4d,h), the differences in retinal differentiation between Ptdsr+/+ and Ptdsr -/- mice were still evident, but the size reduction of the retinal layers was less pronounced in the knockout mice. Ptdsr-deficient animals seem to have compensated for the marked delay in cellular differentiation and expansion of retinal layers. Close examination of retinal structures revealed that the inner granular layer was still less expanded in Ptdsr-deficient animals, however, and that it contained fewer cells and was still severely underdeveloped in comparison with the corresponding retinal layer in control animals (Figure 4c,4g and 4d,4h). Thus, even mildly affected Ptdsr -/- mutants had ocular malformations with defects in differentiation of retinal structures.
We next examined Ptdsr -/- embryos that displayed unilateral or bilateral absence of eyes (Figure 5a) by serial sectioning of whole embryos. These embryos showed complex malformations of the optical cup, including absence of the lens (Figure 5b). Most surprisingly, we found pigmented epithelial cells in the nasal cavity of all Ptdsr-knockout mice with anophthalmia that were analyzed histopathologically. We could identify black-colored pigmented cells embedded in the epithelium of the maxillary sinus that resembled presumptive retinal-pigmented epithelium (Figure 5b,c). Examination of consecutive serial sections revealed the formation of a primitive eye structure, with induction and subsequent proliferation of ectopic mesenchymal tissue immediately adjacent to the displaced pigmented epithelium (Figure 5d). This structure was clearly induced ectopically, and we failed to identify similar changes in any of the wild-type embryos. In summary, we observed a wide range of ocular malformations in Ptdsr-deficient mice that ranged from differentiation defects in retinal cell layers (for example, the inner granular layer) in mildly affected homozygotes to anophthalmia in severely affected Ptdsr -/- mice that was associated with induction of ectopic eye structures in nasal cavities.