Kil S H, Lallier T, Bronner-Fraser M
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
Dev Biol. 1996 Oct 10;179(1):91-101. doi: 10.1006/dbio.1996.0243.
Although it is well-established that beta1 integrins play a functional role in the migration of cranial neural crest cells, little is known about the number or importance of their associated alpha subunits. Here, we have utilized antisense oligonucleotides (aONs) against various mammalian integrin alpha subunits to functionally "knock out" integrins in vitro and in vivo. First, we examined the attachment in vitro of cranial neural crest cells to fibronectin and laminin in the presence of antisense or reversed-sense oligonucleotides using a quantitative adhesion assay. We found three alpha integrin aONs that blocked attachment to fibronectin substrates only, one that blocked attachment to laminin substrates only, and one that blocked attachment to both fibronectin and laminin. As expected, an aON to chick beta1 integrin reduced attachment to both fibronectin and laminin substrates. These results suggest that there are three or more functionally distinct integrin heterodimers on avian cranial neural crest cells. Second, we examined the ability of aONs against various alpha integrin subunits to perturb cranial neural crest migration in vivo by injecting the oligonucleotides into the cranial mesenchyme through which neural crest cells migrate. Those alpha aONs that inhibited cell attachment in vitro also caused neural crest and/or neural tube abnormalities after injection in vivo. In addition, two aONs that had no effect in vitro did affect emigration of neural crest cells in vivo. Immunoprecipitations revealed that some integrin subunits were depleted after treatment with antisense but not reversed-sense oligonucleotides both in vivo and in vitro. The results suggest that integrin alpha subunits are required for cranial neural crest cell attachment and emigration.
尽管β1整合素在颅神经嵴细胞迁移中发挥功能作用已得到充分证实，但关于其相关α亚基的数量或重要性却知之甚少。在此，我们利用针对各种哺乳动物整合素α亚基的反义寡核苷酸（aONs）在体外和体内对整合素进行功能性“敲除”。首先，我们使用定量黏附试验，在存在反义或正义寡核苷酸的情况下，检测颅神经嵴细胞在体外与纤连蛋白和层粘连蛋白的黏附情况。我们发现三种α整合素aONs仅阻断与纤连蛋白底物的黏附，一种仅阻断与层粘连蛋白底物的黏附，还有一种阻断与纤连蛋白和层粘连蛋白两者的黏附。正如预期的那样，针对鸡β1整合素的aON降低了与纤连蛋白和层粘连蛋白底物的黏附。这些结果表明，禽类颅神经嵴细胞上存在三种或更多功能不同的整合素异二聚体。其次，我们通过将寡核苷酸注射到神经嵴细胞迁移通过的颅间充质中，检测针对各种α整合素亚基的aONs在体内干扰颅神经嵴迁移的能力。那些在体外抑制细胞黏附的α aONs在体内注射后也会导致神经嵴和/或神经管异常。此外，两种在体外无作用的aONs在体内确实影响神经嵴细胞的迁出。免疫沉淀显示，在体内和体外，用反义而非正义寡核苷酸处理后，一些整合素亚基会减少。结果表明，整合素α亚基是颅神经嵴细胞黏附和迁出所必需的。
