Developmental expression of three small GTPases in the mouse eye
Developmental expression of three small GTPases in the mouse eye
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发表时间：
2007-07
期刊：
影响因子：
2.2
通讯作者：
D. Mitchell;B. Bryan;Jinping Liu;Wen-bin Liu;Lan Zhang;J. Qu;Xiangtian Zhou;Mingyao Liu;D. Li
中科院分区：
文献类型：
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作者：
D. Mitchell;B. Bryan;Jinping Liu;Wen-bin Liu;Lan Zhang;J. Qu;Xiangtian Zhou;Mingyao Liu;D. Li
目的小GTP酶作为“分子开关”通过结合和释放GTP介导下游信号传导。Rho家族的GTP酶在调节细胞分化和细胞骨架变化中起着重要作用。由于眼睛的发育需要全面的形态发生运动和广泛的细胞分化，我们推测，不同的小GTP酶可能在眼睛发育的形态发生过程中发挥重要作用。为了探索这种可能性，我们研究了三个主要的Rho-GTPases的表达模式：RhoA，Rac 1和Cdc 42在胚胎，出生后（出生后一天），和成年（两个月大）小鼠的眼睛。方法收集胚胎、出生后和成年C57 BL/6小鼠的各种眼组织。使用从成年小鼠的角膜、视网膜、透镜上皮细胞和透镜纤维细胞或大鼠晶状体的不同部分提取的总蛋白进行蛋白质印迹。通过11.5 pc、14.5 pc、17.5 pc、出生后和成年小鼠的眼球区域切割6 μm厚切片进行免疫组织化学（IHC）。使用兔免疫前抗体和用饱和水平的相应肽抗原封闭的GTP酶特异性抗体进行平行对照。结果RhoA和Cdc 42在胚胎小鼠眼的三个隔室中的表达在11.5pc时开始检测。然而，Rac 1在这些隔室中在14.5 pc时变得容易检测。RhoA，Rac 1和Cdc 42的水平增加，在17.5 pc的三个隔室中检测到，RhoA，Rac 1和Cdc 42的最强信号在17.5 pc的初级透镜纤维细胞中观察到。在出生后的小鼠眼中，这三种小GTP酶在小鼠角膜的内皮细胞和上皮细胞、眼透镜的上皮细胞、光感受器、水平/无长突/Muller细胞和视网膜的一些神经节细胞中显著表达。在角膜基质成纤维细胞、透镜纤维细胞以及小鼠视网膜的内丛状层和外丛状层中观察到低得多的表达水平。在成年小鼠眼中，所有三种Rho-GTP酶均在角膜上皮细胞和视网膜中表达。然而，在角膜内皮细胞中仅检测到RhoA蛋白，在眼透镜中检测到Rac 1蛋白。结论3种小GTP酶在小鼠胚胎17.5 pc和出生后的角膜、透镜和视网膜中均有较强的表达，提示它们在小鼠眼各部分的形态发生中起重要作用。特别地，RhoA、Rac 1和Cdc 42在胚胎透镜纤维细胞中的高水平表达表明它们参与初级透镜纤维细胞的分化。在成年小鼠眼中，所有三种Rho-GTP酶似乎都参与角膜上皮细胞和视网膜的分化，然而，RhoA单独可能是内皮细胞分化所需的，Rac 1可能在支持连续透镜生长和维持透镜透明度中起重要作用。
Purpose The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye. Methods Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen. Results In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens. Conclusions The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.