Functional roles of reelin signaling cascades In formation of cerebral neocortex

摇鼠川崎（SRK）是一种常染色体隐性突变体，表现出摇鼠样异常运动行为。小鼠reeler突变是由特定基因reelin的几个突变引起的，这些突变导致reelin在大脑皮层和中枢神经系统其他区域的表达或分泌缺陷。为了解决SRK突变是否也由reelin突变引起的问题，我们分析了SRK中的reelin基因。对正常大鼠的reelin mRNA的Northern分析显示，大鼠的reelin在大脑和小脑中都以约12 kb的转录本表达，而在SRK脑中，reelin的表达明显减少。原位杂交分析显示，SRK脑reelin mRNA在大脑皮层边缘区Cajal-Retzius细胞和小脑皮层外颗粒细胞中的表达与正常对照组相似，但表达量明显降低。使用CR-50单克隆抗体进行免疫组化分析，在大脑和小脑皮层未识别出免疫产物。从cDNA序列中，我们在SRK reelin中发现了一个61碱基的插入，该插入在阅读框中包含一个终止密码子。此外，基因组DNA分析显示，SRK基因组reelin基因中存在10个碱基的缺失，其中包含预测的外显子-内含子边界，导致来自以下内含子的插入序列合并到SRK reelin mRNA中。因此，SRK突变是另一种缺乏功能Reelin蛋白表达的突变，因此导致了reeler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.