Puech Pierre-Henri, Taubenberger Anna, Ulrich Florian, Krieg Michael, Muller Daniel J, Heisenberg Carl-Philipp
Center of Biotechnology, TU Dresden, Cellular Machines, Tatzberg 49, 01307 Dresden, Germany.
J Cell Sci. 2005 Sep 15;118(Pt 18):4199-206. doi: 10.1242/jcs.02547.
During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.
在脊椎动物原肠胚形成过程中，不同胚层的祖细胞获得特定的黏附特性，这有助于胚层的形成和分离。有研究表明，Wnt信号通过调节胚层间不同程度的黏附在这一过程中发挥作用，然而，这方面的直接证据仍然缺乏。在这里，我们表明，Wnt11是一种调节原肠胚运动的关键信号，对于斑马鱼中内胚层祖细胞与纤连蛋白（原肠胚形成过程中一种丰富的细胞外基质成分）的黏附是必需的。为了测量这种效应，我们开发了一种检测方法，使用原子力显微镜（AFM）来量化单个斑马鱼原代中内胚层祖细胞的黏附情况。当在纤连蛋白包被的底物上进行测试时，我们观察到来自野生型胚胎和slb/wnt11突变胚胎（wnt11基因存在功能缺失突变）的培养中内胚层祖细胞在脱离力和功方面存在显著差异。这些差异可能是由于对纤连蛋白底物的黏附减少，因为野生型和突变型细胞的整体细胞形态和细胞弹性并没有明显不同。此外，在存在纤连蛋白 - 整合素结合抑制剂（如RGD肽）的情况下，黏附力和功显著降低，表明整合素参与了我们检测中中内胚层祖细胞的结合。这些发现表明，AFM可用于定量测定培养的原代原肠胚细胞的底物黏附情况，并深入了解Wnt11信号在单细胞水平上调节细胞黏附中的作用。
