Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein
Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein
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DOI：
10.1006/dbio.2001.0390
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发表时间：
2001-10-15
影响因子：
2.7
通讯作者：
Zohar, Y
中科院分区：
文献类型：
--
作者：
Du, SJ;Frenkel, V;Zohar, Y
斑马鱼最近已经成为发育生物学家的首选模型。这一独特的模型使现代分子和遗传学研究得以进行，以确定参与各种发育过程的基因。遗传方法的成功在很大程度上取决于一种简单而有效的筛选方法的应用，以确定有趣的突变。为了开发一种适合于筛选骨骼突变体的斑马鱼胚胎中骨骼结构的可视化方法，我们研究了荧光生色团Calcein的使用，它专门结合到钙化的骨骼结构上。利用这种方法，我们跟踪了斑马鱼胚胎从受精后第1天到第21天的骨骼结构的发育，并分析了骨形态发生蛋白-2(BMP2)对中轴骨骼发育的影响。我们发现钙化骨骼结构的发育是从头到尾的渐进方式。头部(即颌骨)的钙化结构首先发育，然后是躯干的中轴骨。值得注意的是，轴骨内的椎骨钙化似乎有两个区域。前三个椎骨在第一个区域，其余的在第二个区域。与阿尔辛蓝染色相比，钙黄绿素染色确实标记了钙化的骨骼结构，而且，它是一种更灵敏和包容的骨骼结构可视化方法。为了确定钙调素染色是否也可以用来检测骨骼发育异常，我们在斑马鱼脊索细胞中异位表达了BMP2。我们证明BMP2在脊索细胞中的异位表达抑制了中轴骨的发育。综上所述，这些结果清楚地证明了钙黄素染色在斑马鱼胚胎发育中显示骨结构的敏感性，以及它在筛选具有骨结构缺陷的突变体方面的有效性。(C)2001年学术出版社。
Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects. (C) 2001 Academic Press.