Research Program for Experimental Ophthalmology and Glaucoma Research, Department of Ophthalmology and Optometry, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria.
Spinal Cord Injury and Tissue Regeneration Center, Salzburg, Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Salzburg, Austria.
Curr Eye Res. 2022 Apr;47(4):590-596. doi: 10.1080/02713683.2021.2002910. Epub 2022 Mar 21.
Pericytes (PCs), located abluminal of endothelial cells on capillaries, are essential for vascular development and stability. They display a heterogeneous morphology depending on organ localization, differentiation state, and function. Consequently, PCs show a diverse gene expression profile, impeding the usage of a unique PC marker and therefore the distinct identification of PCs. Inducible reporter mouse models represent an important tool for investigating the fate of PCs under physiological and pathophysiological conditions. PC-specific expression efficiency of the fluorescence reporter tdTomato following tamoxifen induction was analyzed and compared in two inducible Cre recombinase-expressing mouse models under control of the NG2 and PDGFRb promotor.
The NG2-CreER™-tdTomato and the PDGFRb-P2A-CreER-tdTomato mice were treated with tamoxifen at three defining time points of retinal vascular development: post-natal days (P)5, P10/11/12, and P48/49/50/51. TdTomato reporter induction efficiency was determined by analyzing retinal whole mounts utilizing confocal microscopy, using the antibodies Anti-neural/glial antigen 2 (PCs), Anti-Collagen IV (basement membrane), and Anti-Glutamine Synthetase (Müller glial cells).
Tamoxifen induction at the three different time points resulted in PC-specific expression of tdTomato in both reporter models. In the NG2-CreER™-tdTomato mouse, the induction efficiency ranged from 21.9 to 35.5%. In the PDGFRb-P2A-CreER-tdTomato mouse, an induction efficiency between 78.9 and 94.1% was achieved. TdTomato expression in the retina was restricted to PCs and vascular smooth muscle cells in the NG2-CreER™-tdTomato mouse, however, in the PDGFRb-P2A-CreER-tdTomato mouse, tdTomato was also expressed in Müller glial cells.
Both reporter mouse models represent promising tools for fate-mapping studies of PCs. While the NG2-CreER™-tdTomato mouse reveals very specific labeling of PCs in the retina, its induction efficiency is lower compared to the PDGFRb-P2A-CreER-tdTomato mouse. Although the latter revealed a high percentage of tdTomato-positive PCs in the retina, additional labeling of Müller cells potentially hampers analysis of reporter-positive PCs.
位于毛细血管内皮细胞管腔侧的周细胞对于血管发育和稳定性至关重要。它们的形态取决于器官定位、分化状态和功能而表现出异质性。因此，周细胞表现出不同的基因表达谱，这阻碍了使用独特的周细胞标记物，从而无法明确识别周细胞。诱导型报告小鼠模型是研究生理和病理条件下周细胞命运的重要工具。本研究在两种由 NG2 和 PDGFRb 启动子驱动的诱导型 Cre 重组酶表达小鼠模型中，分析并比较了经他莫昔芬诱导后荧光报告基因 tdTomato 的周细胞特异性表达效率。
NG2-CreER™-tdTomato 和 PDGFRb-P2A-CreER-tdTomato 小鼠在视网膜血管发育的三个定义时间点（出生后第 5 天、第 10/11/12 天和第 48/49/50/51 天）接受他莫昔芬处理。通过分析利用抗神经/胶质抗原 2（周细胞）、抗胶原蛋白 IV（基底膜）和抗谷氨酰胺合成酶（Müller 胶质细胞）抗体进行共聚焦显微镜检查的视网膜全片，确定 tdTomato 报告基因的诱导效率。
在两种报告小鼠模型中，在三个不同时间点用他莫昔芬诱导均可导致 tdTomato 在周细胞中特异性表达。在 NG2-CreER™-tdTomato 小鼠中，诱导效率范围为 21.9%至 35.5%。在 PDGFRb-P2A-CreER-tdTomato 小鼠中，实现了 78.9%至 94.1%的诱导效率。在 NG2-CreER™-tdTomato 小鼠中，tdTomato 在视网膜中的表达仅限于周细胞和血管平滑肌细胞，但在 PDGFRb-P2A-CreER-tdTomato 小鼠中，tdTomato 也在 Müller 胶质细胞中表达。
两种报告小鼠模型均代表研究周细胞命运的有前途的工具。虽然 NG2-CreER™-tdTomato 小鼠在视网膜中显示出非常特异性的周细胞标记，但与 PDGFRb-P2A-CreER-tdTomato 小鼠相比，其诱导效率较低。虽然后者在视网膜中显示出较高比例的 tdTomato 阳性周细胞，但 Müller 细胞的额外标记可能会阻碍对报告阳性周细胞的分析。
