PDF(Pubmed)
Abstract：
Photoreceptors are sensory neurons that capture light within their outer segment, a narrow cylindrical organelle stacked with disc-shaped membranes housing the visual pigment. Photoreceptors are the most abundant neurons in the retina and are tightly packed to maximize the capture of incoming light. As a result, it is challenging to visualize an individual cell within a crowded photoreceptor population. To address this limitation, we developed a rod-specific mouse model that expresses tamoxifen-inducible cre recombinase under the control of the Nrl promoter. We characterized this mouse using a farnyslated GFP (GFPf) reporter mouse and found mosaic rod expression throughout the retina. The number of GFPf-expressing rods stabilized within 3 days post tamoxifen injection. At that time, the GFPf reporter began to accumulate in basal disc membranes. Using this new reporter mouse, we attempted to quantify the time course of photoreceptor disc renewal in WT and Rd9 mice, a model of X-linked retinitis pigmentosa previously proposed to have a reduced disc renewal rate. We measured GFPf accumulation in individual outer segments at 3 and 6 days post-induction and found that basal accumulation of the GFPf reporter was unchanged between WT and Rd9 mice. However, rates of renewal based on the GFPf measurements were inconsistent with historical calculations from radiolabeled pulse-chase experiments. By extending GFPf reporter accumulation to 10 and 13 days we found that this reporter had an unexpected distribution pattern that preferentially labeled the basal region of the outer segment. For these reasons the GFPf reporter cannot be used for measuring rates of disc renewal. Therefore, we used an alternative method that labels newly forming discs with a fluorescent dye to measure disc renewal rates directly in the Rd9 model and found it was not significantly different from WT. Our study finds that the Rd9 mouse has normal rates of disc renewal and introduces a novel Nrl:CreERT2 mouse for gene manipulation of individual rods.
摘要：
光感受器是感觉神经元，在它们的外段捕获光，一种狭窄的圆柱形细胞器，上面有圆盘状的膜来容纳视觉色素。光感受器是视网膜中最丰富的神经元，并且紧密堆积以最大程度地捕获入射光。因此,在拥挤的光感受器群体中可视化单个细胞是具有挑战性的。为了解决这个限制，我们开发了一种杆特异性小鼠模型，该模型在Nrl启动子的控制下表达他莫昔芬诱导型cre重组酶。我们使用farnyslatedGFP（GFPf）报告小鼠对该小鼠进行了表征，并在整个视网膜中发现了镶嵌杆表达。在他莫昔芬注射后3天内稳定表达GFPf的棒的数量。当时,GFPf报告分子开始在基盘膜中积累。用这个新的记者鼠标，我们试图量化WT和Rd9小鼠的光感受器盘更新的时间过程，以前提出的X连锁色素性视网膜炎模型具有降低的椎间盘更新率。我们在诱导后3天和6天测量了个体外节段中的GFPf积累,并且发现GFPf报道分子的基础积累在WT和Rd9小鼠之间没有变化。然而,基于GFPf测量的更新率与放射性标记脉冲追踪实验的历史计算不一致.通过将GFPf报道分子的积累延长至10天和13天,我们发现该报道分子具有意想不到的分布模式,其优先标记外节的基底区域。由于这些原因,GFPf报告器不能用于测量光盘更新的速率。因此,我们使用另一种方法,用荧光染料标记新形成的椎间盘,直接在Rd9模型中测量椎间盘更新率,发现它与WT没有显著差异.我们的研究发现Rd9小鼠具有正常的椎间盘更新率，并引入了一种新型的Nrl：CreERT2小鼠用于单个棒的基因操纵。
