Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by Müller Cells in Adult Mammalian Retina

BackgroundEvidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells.
Methodology/Principal FindingsWe demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception.
Conclusion/SignificanceIn summary, our results provide a proof of principle for non-neurotoxin-mediated activation of Müller cells through Notch and Wnt signaling toward the regeneration of rod photoreceptors.

背景：来自不同物种的多种研究方法所获得的证据表明，米勒胶质细胞（Müller cell）的功能可能不止于维持视网膜稳态（retinal homeostasis），在成体视网膜中还可充当祖细胞。体外富集培养的米勒胶质细胞，或是在体内因神经毒素造成视网膜损伤后重新进入细胞周期的米勒胶质细胞，均展现出多能性与自我更新能力——这是干细胞（stem cell）的核心特征。
方法/主要发现：本研究证实，Notch与Wnt信号通路可通过其经典信号通路激活米勒胶质细胞，且少量被激活的米勒胶质细胞可在外核层（outer nuclear layer）沿视杆感光细胞谱系（rod photoreceptor lineage）分化。我们通过多种实验手段验证了激活的米勒胶质细胞向感光细胞谱系的分化过程，包括Hoechst染料外排分析、利用Nrl-GFP小鼠视网膜开展的遗传分析，以及在野生型小鼠和rd小鼠的体外实验中使用GS-GFP慢病毒（lentivirus）进行的谱系示踪，还在S334ter大鼠体内开展了相关验证。通过对S334ter大鼠进行视觉刺激下的头颈部追踪实验——这是检测光感知能力的行为学方法——结果显示，接受米勒胶质细胞激活处理的动物，其光感知能力得到了显著提升。处理组中具有视杆感光细胞表型的激活米勒胶质细胞数量，与其光感知能力的改善程度呈显著正相关。
结论与意义：综上，本研究结果为通过Notch与Wnt经典信号通路激活米勒胶质细胞、进而实现视杆感光细胞再生的非神经毒素介导策略提供了原理验证。