Expression and localization of bestrophin during normal mouse development.
OBJECTIVE
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.
Bakall
Benjamin
B
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
Marmorstein
Lihua Y
LY
Hoppe
George
G
Peachey
Neal S
NS
Wadelius
Claes
C
Marmorstein
Alan D
AD
D000818:Animals; D000075002:Bestrophins; D015153:Blotting, Western; D004596:Electroretinography; D005123:Eye / Q000196:embryology* / Q000254:growth & development; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D007124:Immunoenzyme Techniques; D007473:Ion Channels; D008027:Light; D051379:Mice; D008807:Mice, Inbred BALB C; D010786:Photoreceptor Cells / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000196:embryology* / Q000254:growth & development; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014785:Vision, Ocular
C491856:Best1 protein, mouse; D000075002:Bestrophins; D005136:Eye Proteins; D007473:Ion Channels; D012333:RNA, Messenger