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Published on: April 29, 2011
Expression and localization of bestrophin during normal mouse development
Benjamin Bakall1, Lihua Y Marmorstein, George Hoppe
1Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Purpose:
Best macular dystrophy is caused by mutations in the VMD2 gene, which encodes the protein bestrophin. The purpose of this study was to determine the postnatal onset of expression of bestrophin mRNA and protein in the mouse retinal pigment epithelium (RPE).
Methods:
Rabbit anti-mouse bestrophin polyclonal antisera designated Pab-003 was generated against a peptide derived from the C terminus of mouse bestrophin and characterized by Western blot and immunofluorescence staining of transfected cells. Expression of bestrophin mRNA during ocular development was studied with quantitative PCR. Bestrophin protein expression in the developing eye was observed by using immunohistochemistry. The onset of mouse phototransduction was determined by conventional electroretinography (ERG).
Results:
Bestrophin mRNA was detected at embryonic day 15 in whole mouse eyes by RT-PCR. Real-time quantification of mouse bestrophin mRNA levels indicated that the highest levels of mRNA were present in the early postnatal period. In contrast, bestrophin in the RPE was first detected at postnatal day (P)10 by immunohistochemistry. Phototransduction, as determined by the presence of an ERG a-wave, was first observed at P10.
Conclusions:
The results of this study show that mouse bestrophin mRNA is present in the eye during embryogenesis and significantly precedes the onset of bestrophin protein expression at P10. The appearance of bestrophin in the basolateral plasma membrane of the RPE is coincident with the first detectable ERG a-wave. Because bestrophin is thought to play a role in generating the light peak, a late response of the ERG, these data support a temporal role for bestrophin in RPE responses to light. Furthermore, bestrophin protein appears to be a very late marker of RPE differentiation and to be subject to strong translational control.
Insights
Bestrophin mRNA appears early in mouse eye development, but bestrophin protein expression in the retinal pigment epithelium (RPE) begins at postnatal day 10, coinciding with the onset of phototransduction.
Area of Science:
- Ophthalmology
- Molecular Biology
- Developmental Biology
Background:
- Best macular dystrophy is linked to mutations in the VMD2 gene, encoding the protein bestrophin.
- Understanding bestrophin expression is crucial for elucidating its role in retinal function and disease.
Purpose of the Study:
- To determine the postnatal onset of bestrophin mRNA and protein expression in the mouse retinal pigment epithelium (RPE).
Main Methods:
- Generated rabbit anti-mouse bestrophin polyclonal antisera.
- Quantified bestrophin mRNA using quantitative PCR during ocular development.
- Assessed bestrophin protein expression via immunohistochemistry.
- Determined phototransduction onset using electroretinography (ERG).
Main Results:
- Bestrophin mRNA detected by embryonic day 15; highest levels in early postnatal period.
- Bestrophin protein first detected in RPE at postnatal day 10.
- Phototransduction (ERG a-wave) also first observed at postnatal day 10.
Conclusions:
- Mouse bestrophin mRNA expression precedes protein appearance by several days.
- Bestrophin protein onset in RPE coincides with the first detectable ERG a-wave.
- Suggests a temporal role for bestrophin in RPE light responses and indicates late RPE differentiation and translational control.
