Wang Y, Jones F S, Krushel L A, Edelman G M
Department of Neurobiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):1892-6. doi: 10.1073/pnas.93.5.1892.
During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.
在脊椎动物神经系统发育过程中，神经细胞黏附分子（N-CAM）以特定的时空模式表达。我们提出，N-CAM的表达部分受同源框基因编码的蛋白质控制。这一假说得到了先前体外实验的支持，这些实验表明同源框基因的产物能够通过两个同源结构域结合位点HBS-I和HBS-II与N-CAM启动子结合并使其反式激活。我们现在通过使用含有与β-半乳糖苷酶报告基因相连的天然和突变N-CAM启动子构建体的转基因小鼠，来检验N-CAM基因在体内是同源结构域蛋白的靶标的假说。小鼠N-CAM基因5'侧翼区域的片段足以指导报告基因在中枢神经系统中的表达，其模式与内源性N-CAM基因的表达模式一致。例如，在胚胎第11天（E11），在脊髓背外侧和腹外侧区域的有丝分裂后神经元中发现了β-半乳糖苷酶染色；在E14.5时，整个脊髓的这些神经元中都可见染色。相比之下，携带在两个同源结构域结合位点（HBS-I和HBS-II）都有突变的N-CAM启动子-报告基因构建体的小鼠，其脊髓中的表达模式发生了改变。在E11时，在脊髓腹外侧可见β-半乳糖苷酶表达，但在背外侧区域则没有，而在E14.5时，在脊髓的任何细胞中都不再检测到β-半乳糖苷酶表达。因此，N-CAM启动子中的同源结构域结合位点在确定发育中的脊髓背腹轴上N-CAM的特定表达模式中似乎很重要。这些实验表明，N-CAM基因是脊椎动物体内同源框基因产物的靶标。
