Hayashi M, Ninomiya Y, Parsons J, Hayashi K, Olsen B R, Trelstad R L
J Cell Biol. 1986 Jun;102(6):2302-9. doi: 10.1083/jcb.102.6.2302.
We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.
我们采用了一种高度特异的原位杂交方法,可对鸡胚组织石蜡切片中I型和II型胶原的mRNA进行差异检测。所有探针均为编码前α链C-前肽区部分的cDNA限制性片段,其中一些片段还编码I型或II型胶原mRNA的3'-非翻译区。用3H标记的I型或II型胶原mRNA探针,对17日龄鸡胚的肌腱成纤维细胞涂片、胸骨软骨细胞涂片以及10日龄全胚石蜡切片和6.5日龄胚角膜进行杂交。放射自显影片显示,I型胶原探针标记的肌腱成纤维细胞以及II型胶原探针标记的胸骨软骨细胞中有明显的标记;在10日龄胚胎巩膜和四肢软骨中,I型探针显示围绕软骨的成纤维细胞层以及软骨中的少数软骨细胞有强烈标记,而II型探针则强烈标记软骨细胞,仅少量标记成纤维细胞;在6.5日龄胚胎角膜中,I型探针大量标记基质中的上皮细胞和成纤维细胞,少量标记内皮细胞,而II型探针几乎只标记上皮细胞,内皮细胞仅有轻微标记。这些数据表明胚胎组织分别和/或同时表达这两种胶原基因,并为细胞外基质成分的细胞调控研究提供了新方法。