Functional roles of reelin signaling cascades In formation of cerebral neocortex

摇动大鼠(SRK)是一种常染色体隐性遗传突变，表现出类似绞股式的异常运动行为。小鼠Reeler突变是由名为reelin的特定基因的几个突变引起的，这些突变导致大脑皮层和中枢神经系统其他区域的reelin表达或分泌缺陷。为了解决SRK突变是否也是由reelin突变引起的问题，我们分析了SRK中的reelin基因。Northern分析表明，正常大鼠大脑和小脑均表达一条12kb的转录本，而SRK大鼠脑组织中的表达显著降低。原位杂交结果显示，SRK大鼠大脑皮质边缘区Cajal-Retzius细胞和小脑皮质外颗粒细胞表达Reelin mRNA，表达方式与正常对照组相似，但表达水平明显降低。用CR-50单抗进行免疫组织化学分析，大脑皮层和小脑皮质均未发现免疫反应产物。从cDNA序列中，我们发现SRK reelin有一个61个碱基的插入，在阅读框中包含一个终止密码子。基因组DNA分析表明，SRK基因组reelin基因发生了10个碱基的缺失，其中包含一个预测的外显子-内含子边界，导致SRK reelin基因的插入序列从下一个内含子开始整合到SRK reelin基因中。因此，SRK突变是另一种类型的突变，它缺乏功能性Reelin蛋白的表达，因此导致卷轴表型。

The shaking rat Kawasaki (SRK) is an autosomal recessive mutant which exhibits reeler-like abnormal locomotor behaviors. The murine reeler mutants arise from several mutations in the specific gene called reelin, which result in defects of Reelin expression or secretion in the cerebral cortex and other regions of CNS. To address the issue whether the SRK mutation also arises from a mutation in reelin, we analyzed the reelin gene in SRK. Northern analysis of reelin mRNA from normal rats showed that rat reelin was expressed as a 〜12-kb transcript in both the cerebrum and cerebellum, while reelin expression was markedly reduced in SRK brains. In situ hybridization analysis showed that reelin mRNA in SRK brains was expressed in Cajal-Retzius cells in the marginal zone of the cerebral cortex and outer granular cells in the cerebellar cortex in similar manners to normal controls, but its expression was considerably reduced. The immunohistochemical analysis using the CR-50 monoclonal antibody, no immunoproduct was recognized in the cerebral and cerebellar cortices. From the cDNA sequences, we found a 61-base insertion in SRK reelin, which contains a termination codon in the reading frame. Furthermore, genomic DNA analysis revealed that a 10-base deletion, which contains a predicted exon-intron boundary, occurred in the SRK genomic reelin gene, resulting in incorporation of the insertion sequence originated from the following intron into SRK reelin mRNA. Thus, the SRK mutation is another type of mutations which lacks expression of the functional Reelin protein and, therefore, causes the reeler phenotype.