Development of neuronal type specific cDNA libraries

由于神经细胞从分化开始就有其自身的特点，因此正确的寻径到正确的靶细胞是建立神经网络的关键。选择性轴突生长被认为是通过沿通路提供的信号和生长锥上的识别分子之间的积极相互作用来实现的。为了识别这些线索和识别分子，我们将重点放在鸡胚胎腰椎运动神经元上，因为我们可以非常详细地分析它们的轴突生长，并且我们可以从胚胎中获得足够的材料。我们试图利用针对运动神经元细胞表面唯一表达的SC1抗体，从纯化的胚胎第5天（E5）鸡运动神经元cDNA文库中鉴定运动神经元或其亚型上唯一表达的分子。然后，我们对受体酪氨酸激酶（rtk）和mRNA差异显示进行了RT-PCR。迄今为止，根据其结构基序，rtk至少包括16个亚科。从富集的运动神经元中，我们鉴定出12个RTK亚家族，并通过原位杂交发现Eph亚家族成员Cek8在支配肢体肌肉的脊髓臂段和腰椎段的运动神经元上特异性表达。此外，同一家族的Cek4在支配轴肌的运动神经元上表达，而Ret RTK在所有运动神经元中表达。从mRNA差异显示中，我们克隆了近40个cDNA克隆，发现至少有3个新分子在运动神经元的所有或亚群中唯一表达。我们目前正在分析Cek4， Cek8， Ret和运动神经元上表达的新分子的生物学功能。

Since neuronal cells have own characteristics from the beginning of their differentiation, the correct path finding to their correct target cells is essential for establishing neural network. Selective axonal growth is considered to be achieved by active interactions between the cues provided along the pathway and the recognition molecules localized on the growth cones. To identify such cues and recognition molecules, we focused on the chick embryonic lumbar motoneurons, because their axonal growth is analyzed in great detail and we can obtain enough materials from the embryos. We tried to identify molecules that are uniquely expressed on motoneurons or their subtypes from the cDNA libraries made from the purified embryonic day 5 (E5) chick motoneurons by panning, using antibody against SC1 that is uniquely expressed on motoneuron cell surface. We then performed RT-PCR for receptor tyrosine kinases (RTKs) and mRNA differential display. To date, RTKs comprise at least 16 subfamilies based upon their structural motifs. From enriched motoneurons, we identified 12 RTK subfamilies and found by in situ hybridization that Cek8, a member of Eph subfamily, is specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles. Furthermore, Cek4 of the same family is expressed on motoneurons innervating axial muscles, whereas Ret RTK is expressed in all motoneurons. From the mRNA differential display we cloned nearly 40 cDNA clones and found at least 3 novel molecules are uniquely expressed on all or subpopulation of motoneurons. We are now analyzing the biological functions of Cek4, Cek8, Ret and the novel molecules expressed on the motoneurons.