Expression and localization of bestrophin during normal mouse development
Expression and localization of bestrophin during normal mouse development
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DOI：
10.1167/iovs.03-0030
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发表时间：
2003-08-01
影响因子：
4.4
通讯作者：
Marmorstein, AD
中科院分区：
文献类型：
--
作者：
Bakall, B;Marmorstein, LY;Marmorstein, AD
目的。最佳黄斑营养不良是由 VMD2 基因突变引起的，该基因编码黄斑蛋白。本研究的目的是确定小鼠视网膜色素上皮 (RPE) 中黄斑黄蛋白 mRNA 和蛋白的出生后表达起始情况。兔抗小鼠斑黄蛋白多克隆抗血清（命名为 Pab-003）是针对源自小鼠斑黄蛋白 C 末端的肽生成的，并通过蛋白质印迹和转染细胞的免疫荧光染色进行表征。通过定量 PCR 研究了眼部发育过程中 bestropin mRNA 的表达。通过免疫组织化学观察发育中眼睛中的卵黄蛋白表达。通过传统的视网膜电图（ERG）确定小鼠光转导的开始。 结果。在胚胎第 15 天，通过 RT-PCR 在小鼠全眼中检测到 Bestropin mRNA。小鼠斑黄蛋白 mRNA 水平的实时定量表明，mRNA 水平最高出现在出生后早期。相比之下，RPE 中的黄斑黄蛋白首次在出生后第 10 天通过免疫组织化学检测到。根据 ERG a 波的存在确定的光转导首次在 P10 时观察到。结论。这项研究的结果表明，在胚胎发生过程中，小鼠的眼内出现了卵黄蛋白 mRNA，并且在 P10 时明显先于卵黄蛋白蛋白表达的开始。 R-PE 基底外侧质膜中黄斑黄蛋白的出现与第一个可检测到的 ERG a 波一致。由于黄斑病蛋白被认为在产生光峰（ERG 的晚期反应）中发挥作用，因此这些数据支持黄斑病蛋白在 RPE 对光的反应中的时间作用。此外，黄斑黄蛋白似乎是 RPE 分化的晚期标志物，并且受到强有力的翻译控制。
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 R-PE 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.