Microphthalmia gene expression in the vitiligo mutant mouse and its effect on neural retinal adhesion and photoreceptor cell deat

Abstract

The murine microphthalmia ·gene (Miff) encodes a basic-:-helix~loop:..helix transcription factor known to regulate transcription of genes encoding proteins· of the pigmentation pathway. It is also thoughtto promote pigment cell survival and development. One of the least severe Miff mutations occurs in the vitiligo mouse (Mitf vit) which has a G to A point mutation in the region of _Mitf encoding the first a-helix. This mutant has a normal eye size unlike many of the other mutant alleles and exhibits uneven pigmentation of the· retinal pigment epithelium (RPE) and slow, progressive photoreceptor cell (PC-) loss. Events leading to the retinal degeneration have not been elucidated. The purpose of the present study was three-fold. First, to determine whether apoptosis was the underlying mechanism of photoreceptor cell death in Mitfit. Second, to assess the adhesion bet\A/een neural retina and RPE of the Mitrit allele since retinal detachment is known to · induce PC cell apoptosis. Third, to establish the normal spatia-temporal expression of the Mitf. gene in . wild-type mic~ and to determine whether this pattern was disrupted in vitiligo mutant mice. To quantitate the level of PC apoptosis, the TUNEL assay was used to label cleaved chromatin, a hallmark of apoptosis. Results indicated that the number of apoptotic PCs was significantly higher in Mitfit during the initial" stages of .the d~g~t:l~.ration and remained higher than wild-type ·mice. These findings were confirmed by -transmission electron microscopy. To address RPE/neural reti"na adhesiveness, a peeling experiment was performed in which the RPE was examined for the amount of pigm~nt adherent to it as a direct ·assessment- of strength of adhesion. Scan.ning electron . microscopy was used to examine the level of damage sustained during the separation process.· Results indicated that adhesion of the neural retina in Mitf;t was diminished greatly in vivo compared to wild-type. In. addition, using an in vitro RPE/neural retina recombination assay, it was shown that, surprisingly, mutant RPE could attach similarly to control and mutant retinas at an early age, but not at later ages. Finally, to quantify Mitf expression, RT-PCR was performed and demonstrated a transient elevation of Mitf between E1 0.5-E13.5. in the Mitf;t mutant. 'In situ hybridization analysis of wild-type mice localized Mitf to· the neuroepithelium during ·onset of optic vesicle formation (E9.0-9.5) . ·and s~bsequently to the RPE during optic cup formation (E 1 0.0-E 11.5). The ·findings of this third study provided evidence that the Mitf gene is expressed early during . ocular development underscoring its importance in specifying the RPE. Its. specific expression in the RPE also provides molecular evidence that the primary site of the Mitf;t defect. is the RPE, a notion that had been hypothesized by several groups but not demonstrated. These studies suggest that the abnormal RPE defects are due to developmental changes as reflected by the transient elevation of Mitf expression in the RPE early in its formation. These defects render the RP~ unable to form a normal interaction with the neural retina which eventually detaches from the RPE leading to loss of photoreceptor cells by apoptosis.