Since the retina co-expresses both the Rpgr
ex1-19 and the Rpgr
ORF15 variants, we examined whether expression of Rpgr variants is altered in a temporal manner. We previously showed that Rpgr immunoreactivity is first detectable at the apices of the developing photoreceptor layer at postnatal day (P)3, which correlates with the timing and location of connecting cilia formation.
5 To further study the dynamics of Rpgr expression, we compared the expression of Rpgr
ex1-19 and Rpgr
ORF15 at specific phases of retinal development. In the mouse, much of retinal development takes place in the 3 weeks after birth in a process very similar to third-trimester human retinal development.
19 Proliferation, migration, and differentiation of neuronal precursor cells in the mouse retina is initiated at embryonic day (E)12 and continues through final neuronal differentiation and maturation at approximately P8.
20 The final stages of neuronal differentiation and retinal vascular development occur when mice open their eyes and vision is initiated at around P14. Since many factors critical to establishing this visual pathway are regulated during the first 2 postnatal weeks,
21 we analyzed Rpgr protein levels in retinal homogenates from P3, P7, P14, and adult (2-month) wild-type mice by using our anti-S1 antibody (
Fig. 2A), which recognizes both Rpgr variants. Rpgr
ex1-19 migrates as a 95- to 100-kDa band on Western blot analysis. Protein expression was detected at times of neuronal differentiation in the retina and decreased with age, with robust expression at P3 compared to adult expression levels (
Fig. 2B). In contrast, Rpgr
ORF15 migrated at approximately 200 kDa and the emergence of the Rpgr
ORF15 variants correlated with the maturation of photoreceptors (
Fig. 2B;
Fig. 3B). Relative intensities of the Rpgr
ex1-19 and Rpgr
OFR15 bands were quantified with the ImageJ software (
Fig. 2C). Thus, our data show a correlation between changes in the Rpgr isoform ratio and photoreceptor development and maturation.