Lang Susanne, von Philipsborn Anne C, Bernard André, Bonhoeffer Friedrich, Bastmeyer Martin
Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076, Tübingen, Germany.
Anal Bioanal Chem. 2008 Feb;390(3):809-16. doi: 10.1007/s00216-007-1363-3. Epub 2007 Jun 8.
A microfluidic network (microFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (microCP) step, the protein was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5 were fabricated spanning a total distance of 320 microm. We tested the response of chick retinal ganglion cell (RGC) axons, which are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population, all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5 concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during the detection of a concentration gradient of ephrinA5.
蚀刻在硅片上的微流体网络（microFN）用于将含有不同浓度轴突导向分子ephrinA5的蛋白质溶液输送到硅胶印章上。在随后的微接触印刷（microCP）步骤中，蛋白质被转移到聚苯乙烯培养皿上。通过这种方式，制备了总距离为320微米的ephrinA5的逐步底物结合浓度梯度。我们测试了在体内由ephrin梯度引导的鸡视网膜神经节细胞（RGC）轴突对这些体外梯度的反应。颞侧而非鼻侧轴突在梯度中的一个特定区域停止，该区域覆盖有一定表面密度的底物结合ephrinA5。在颞侧RGC群体中，所有轴突对测试的梯度反应一致。停止区的位置取决于梯度的斜率，浅梯度中轴突比陡梯度中向梯度内生长得更远。然而，浅梯度中轴突在比陡梯度更低的ephrinA5浓度下停止，这表明生长锥在检测ephrinA5浓度梯度期间可以调节其敏感性。
