Multiphoton imaging of chick retinal development in relation to gap junctional communication.
Multiphoton imaging of chick retinal development in relation to gap junctional communication.
复制标题
与间隙连接通讯相关的小鸡视网膜发育的多光子成像。
DOI：
10.1113/jphysiol.2007.138776
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Becker DL
中科院分区：
文献类型：
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作者：
Becker DL
发育中的视网膜中的神经祖细胞延伸从基底玻璃体表面延伸至顶脑室表面的突起。在细胞周期中，细胞核经历运动间核迁移（INM），在G1期间沿玻璃体方向移动，在其高峰期穿过S期，然后在进入G2时返回心室表面，在那里它进入M期并分裂。我们以前已经表明，个别跳跃运动的细胞核与这些祖细胞内的胞浆钙浓度的瞬时变化，这些事件传播到邻近的祖细胞通过连接蛋白43（Cx43）间隙连接通道，从而协调迁移的耦合集群的细胞。阻断与药物、Cx43特异性反义寡脱氧核苷酸（asODNs）或显性负性Cx43（dnCx43）的偶联可抑制钙事件的共享，减少每个细胞经历的数量并显著减缓INM。我们已经开发出的协议，成像迁移祖细胞的共聚焦显微镜在相对较短的时间内，并通过多光子显微镜在更长的时间内，包括完整的细胞周期。我们发现，干扰缝隙连接通讯不仅减缓了祖细胞的INM，而且明显地阻止了它们在细胞周期的关键阶段改变方向。它还破坏了幼神经元在终末分裂后向适当层的迁移，并导致其异位分化。使用多光子显微镜在活体视网膜中的3D体积上进行延长的延时成像的能力现在应该允许详细探索控制视网膜神经上皮发育的基本机制。
Neural progenitor cells in the developing retina extend processes that stretch from the basal vitread surface to the apical ventricular surface. During the cell cycle, the nucleus undergoes interkinetic nuclear migration (INM), moving in a vitread direction during G1, passing through S‐phase at its peak and then, on entering G2, returning towards the ventricular surface where it enters M‐phase and divides. We have previously shown that individual saltatory movements of the nucleus correlate with transient changes in cytosolic calcium concentration within these progenitor cells and that these events spread to neighbouring progenitors through connexin43 (Cx43) gap junction channels, thereby coordinating the migration of coupled clusters of cells. Disrupting coupling with pharmacological agents, Cx43‐specific antisense oligodeoxynucleotides (asODNs) or dominant negative Cx43 (dnCx43) inhibits the sharing of calcium events, reducing the number that each cell experiences and significantly slowing INM. We have developed protocols for imaging migrating progenitor cells by confocal microscopy over relatively short periods, and by multiphoton microscopy over more extended periods that include complete cell cycles. We find that perturbing gap junctional communication not only slows the INM of progenitor cells but also apparently prevents them from changing direction at critical phases of the cell cycle. It also disrupts the migration of young neurons to their appropriate layers after terminal division and leads to their ectopic differentiation. The ability to perform extended time‐lapse imaging over 3D volumes in living retina using multiphoton microscopy should now allow fundamental mechanisms governing development of the retinal neuroepithelium to be probed in detail.