Expression of photoreceptor-associated molecules during human fetal eye development.

PURPOSE: A characteristic feature of the human retina is the early differentiation of foveal cells followed by a central to peripheral wave of maturation. This can obscure the true onset of differentiation when regions other that the fovea are sampled, or when methods based on whole retina or whole eye tissue are employed, such as reverse transcription-polymerase chain technique (RT-PCR). In order to assess the suitability of RT-PCR based approaches during human retinal development and to gain insight into the developmental progression of photoreceptor differentiation and maturation in the human, we analyzed the expression of several photoreceptor-associated genes by immunocytochemical labeling (ICC) of the foveal region as well as by RT-PCR of total RNA from whole fetal eyes from different developmental stages.
METHODS: Expression of phosphodiesterase beta (PDEB), interphotoreceptor binding protein (IRBP), tubby-like protein (TULP), short wavelength specific (S) opsin, long and medium wavelength specific (L/M) opsin, rod opsin and the transcription factors Crx and Nrl were assessed by RT-PCR from total RNA prepared from snap frozen intact human fetal eyes ranging from fetal week 9 (Fwk 9) to Fwk 18. ICC labeling was performed in a large number of eyes within an age group for IRBP, TULP, Nrl, S opsin, L/M opsin and rod opsin on frozen sections that included the fovea centralis.
RESULTS: All ICC markers appeared first in or around the fovea. We detected PDEB and Crx expression as early as Fwk 10, by RT-PCR. TULP and IRBP were first observed with ICC in a small number of foveal cones at Fwk 9, although the first transcripts were not detected until Fwk 12. Nrl-positive nuclei appeared around the fovea by Fwk 11 and S opsin-positive cones by Fwk 12. L/M opsin-positive cones and rod opsin-positive rods were first detected between Fwk 15-16. In general, ICC labeling in the fovea was present for most genes up to 2 weeks before the corresponding transcripts could be successfully amplified by RT-PCR from whole eye tissue.
CONCLUSIONS: Our results indicate that in order to pinpoint exactly when and where a molecule appears, ICC labeling of the fovea is a more reliable indicator. RT-PCR was prone to underestimate the exact onset of expression of the molecules tested, yet it faithfully recapitulated the sequence in which they appeared. In addition, our data show that in the human fetal retina, Crx and Nrl are both expressed when the first rod photoreceptors are being generated. This agrees well with previous in vitro results suggesting a synergistic action of both proteins during differentiation of human rod photoreceptors.