Thompson J R, Chen S W, Ho L, Langston A W, Gudas L J
Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.
Dev Dyn. 1998 Jan;211(1):97-108. doi: 10.1002/(SICI)1097-0177(199801)211:1<97::AID-AJA9>3.0.CO;2-2.
The murine Hoxa1 gene is a member of the vertebrate Hox complex and plays a role in defining the body plan during development. At day 8.0-9.0 post coitus, Hoxa1 transcripts are detected extensively throughout the embryo in the neural tube, adjacent mesenchyme, paraxial mesoderm, somites and gut epithelium; expression extends from the most caudal region of the embryo to the rhombomere 3/4 border. This spatiotemporal expression of Hoxa1 mRNA is critical for normal embryonic development. We have previously identified a 10 bp element, called CE2, which is located approximately 3 kilobases 3' of the Hoxa1 coding region in the RAIDR5 enhancer, and which binds to an approximately 170 kd protein in retinoic acid treated P19 embryonal carcinoma cells. CE2 elements were also identified 3' of the murine Hoxb1 gene, the chicken Hoxb1 gene and the human Hoxa1 gene. To examine the role of this CE2 element in regulating Hoxa1 expression in vivo, transgenic mice were generated which express a Hoxa1 beta-galactosidase reporter gene that contains a mutation in the CE2 element. Relative to transgenic mice bearing a wild type CE2 element, the mutant CE2 construct recapitulated rhombomeric, neural, and gut epithelium expression but failed to show beta-galactosidase expression in somites and adjacent mesenchymal tissue. Gel shift analysis showed that binding activity similar to that detected in extracts prepared from retinoic acid treated P19 cells was present in nuclear extracts prepared from day 9.0 embryos. However, an additional binding complex not detected in P19 cells was also observed. These results indicate that in transgenic animals, the evolutionary conserved CE2 element is a somite and adjacent mesenchymal enhancer of Hoxa1 expression.
小鼠Hoxa1基因是脊椎动物Hox复合体的成员，在发育过程中对确定身体结构起着作用。在交配后第8.0 - 9.0天，Hoxa1转录本在整个胚胎的神经管、相邻间充质、轴旁中胚层、体节和肠上皮中广泛检测到；表达从胚胎最尾端区域延伸至菱脑节3/4边界。Hoxa1 mRNA的这种时空表达对正常胚胎发育至关重要。我们之前鉴定出一个10 bp的元件，称为CE2，它位于RAIDR5增强子中Hoxa1编码区下游约3千碱基处，并且在视黄酸处理的P19胚胎癌细胞中与一种约170 kd的蛋白质结合。在小鼠Hoxb1基因、鸡Hoxb1基因和人类Hoxa1基因的下游也鉴定出了CE2元件。为了研究这个CE2元件在体内调节Hoxa1表达中的作用，构建了转基因小鼠来表达一个Hoxa1β - 半乳糖苷酶报告基因，该基因在CE2元件处有一个突变。相对于携带野生型CE2元件的转基因小鼠，突变的CE2构建体重现了菱脑节、神经和肠上皮的表达，但在体节和相邻间充质组织中未显示β - 半乳糖苷酶表达。凝胶迁移分析表明，从第9.0天胚胎制备的核提取物中存在与从视黄酸处理的P19细胞制备的提取物中检测到的类似结合活性。然而，还观察到了一种在P19细胞中未检测到的额外结合复合物。这些结果表明，在转基因动物中，进化保守的CE2元件是Hoxa1表达的体节和相邻间充质增强子。
