Rezaie Tayebeh, Stoilov Ivaylo, Sarfarazi Mansoor
Molecular Ophthalmic Genetics Laboratory, Department of Surgery, University of Connecticut Health Center, Farmington, CT 06030-1110, USA.
Mol Vis. 2007 Aug 27;13:1446-50.
To analyze optineurin (Optn) gene expression in various embryonic stages of mouse development by whole mount in situ hybridization.
FVB/NcrlBR mouse embryos (10.5 and 13.5 dpc) were collected by timed breeding experiments. A 712 bp Optn cDNA fragment was amplified by PCR and cloned into a transcription vector pCRII-TOPO. Digoxigenin labeled sense and antisense RNA probes were generated by in vitro transcription. The labeled RNA probe was localized using an anti-digoxigenin antibody conjugated with alkaline phosphatase. Colorimetric detection was performed with substrate solution containing, 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
This study revealed that the developing eye represents a major expression site for Optn. At both 10.5 and 13.5 dpc a strong specific expression was detected in the outer layer of the optic cup (future pigment layer of the retina). This is in contrast to the expression of another glaucoma gene, Cyp1b1, the expression of which at this state is only limited to the inner (neural) layer of the optic cup (future nervous layer of the retina). Inspection of sections from the cephalic region of whole mounts also revealed limited Optn staining in the lens as well as in the optic nerve. A second Optn expression domain was detected at the base of the developing forelimb. The biological significance of this observation is not clear and remains to be determined.
Eye and forelimb were identified as two major sites for expression of the Optn gene. These findings suggest that Optn expression is triggered during early stages of eye development. Expression of the Optn gene in ocular tissues during mouse embryogenesis correlates with the presence and distribution of the optineurin protein, as previously reported in adult ocular tissues. These findings are also in agreement with the predicted function of Optn protein in the eye and the role of its ortholog in human glaucoma. Further investigations are required to determine the molecular mechanisms of Optn in the developing murine forelimb.
通过全胚胎原位杂交分析小鼠发育各胚胎阶段视神经病相关蛋白（Optn）基因的表达情况。
通过定时配种实验收集FVB/NcrlBR小鼠胚胎（胚胎龄10.5天和13.5天）。通过聚合酶链反应（PCR）扩增出712bp的Optn cDNA片段，并克隆到转录载体pCRII-TOPO中。通过体外转录生成地高辛标记的正义和反义RNA探针。使用与碱性磷酸酶偶联的抗地高辛抗体对标记的RNA探针进行定位。用含有4-硝基蓝四唑氯化物（NBT）和5-溴-4-氯-3-吲哚磷酸（BCIP）的底物溶液进行比色检测。
本研究表明，发育中的眼睛是Optn的主要表达部位。在胚胎龄10.5天和13.5天时，在视杯外层（视网膜未来色素层）均检测到强烈的特异性表达。这与另一个青光眼相关基因Cyp1b1的表达情况相反，Cyp1b1在此阶段的表达仅局限于视杯内层（视网膜未来神经层）。对全胚胎头部区域切片的检查还显示，晶状体和视神经中也有有限的Optn染色。在发育中的前肢基部检测到第二个Optn表达域。这一观察结果的生物学意义尚不清楚，有待确定。
眼睛和前肢被确定为Optn基因的两个主要表达部位。这些发现表明，Optn基因的表达在眼睛发育早期就被触发。小鼠胚胎发育过程中Optn基因在眼组织中的表达与视神经病相关蛋白的存在和分布相关，正如先前在成年眼组织中所报道的那样。这些发现也与Optn蛋白在眼睛中的预测功能及其直系同源物在人类青光眼中的作用相一致。需要进一步研究以确定Optn在发育中小鼠前肢中的分子机制。
