mechanisms of early motor neuron cell death in cervical segments of avian embryos

在鸟类胚胎脊髓中，运动神经元（MN）在两个不同的时期经历程序性细胞死亡（PCD）。一种是在相对较晚的阶段（E6-E10，在小鸡中）观察到的，涉及脊髓的所有节段。相反，另一个发生在相对较早的阶段（E4-E5），仅在非肢体支配颈脊髓。为了确定在细胞死亡的早期类型的垂死的MN的身份，我们检查表达的亚组特异性MN标记（Isl 1，Isl 2，Lim 3和MNR 2）在垂死的MN的鸡胚。定量分析显示，PCD仅发生在表达Isl 1、Isl 2和MNR 2但不表达Lim 3的MN亚组中。这个亚组的MN在E3（-）和E3.5之间成为有丝分裂后，在失去Lim 3表达后，在E4前角的背外侧区域出现一个独特的亚组。但是，它们在E5时消失。用逆转录病毒载体导入Bcl-2基因后，宫颈MN免于细胞死亡。在细胞死亡期（E5）后，我们观察到，在感染的胚胎中，有一个额外的MN亚组表达Isl 1和Isl 2，但不表达Lim 3。类似的MN也出现在胸部区域，并发展成神经支配肋间肌的MN。这些结果表明，这种类型的PCD的MN在颈脊髓中只发生在Lim 3阴性亚组的MN，对应于MN支配肋间肌。为了进一步研究Lim 3表达与细胞死亡之间的关系，我们利用电穿孔技术将携带小鸡Lim 3 cDNA的逆转录病毒载体感染一侧脊髓。在感染侧，死亡MN的数量在E4.5时显著减少，而健康MN的数量在细胞死亡期（E5.5）后增加。这些结果表明，Lim 3的下调参与了决定颈髓早期细胞死亡类型中细胞死亡的机制。

In the avian embryo spinal cord, motoneurons (MNs) undergo programmed cell death (PCD) at two distinct periods. One is observed at relatively later stages (E6-E10, in the chick) and involves all segments of the spinal cord. By contrast, the other occurs at relatively earlier stages (E4-E5) and only in the non-limb innervating cervical spinal cord. To determine the identity of the dying MNs in the early type of cell death, we examined expression of subgroup-specific MN markers (Isl1, Isl2, Lim3 and MNR2) in the dying MNs of the chick embryo. Quantitative analysis revealed that PCD occurs only in a subgroup of MNs that express Isl1, Isl2 and MNR2 but do not express Lim3. MNs of this subgroup become postmitotic between E3 (-) and E3.5 and appear as a distinct subgroup in the dorsolateral region of the ventral horn by E4 after losing Lim3 expression. However, they disappear by E5. Following introducing Bcl-2 gene with retroviral vector, cervical MNs were rescued from cell death. After the period of cell death (E5), we observed that, in the infected embryos, there was an additional subgroup of MNs that expressed Isl1 and Isl2 but did not express Lim3. Similar MNs appear also in the thoracic region and develop into MNs that innervate intercostal muscles. These results suggest that this type of PCD of MN in the cervical spinal cord occurs only in the Lim3-negative subgroup of MNs that correspond to MNs innervating intercostal muscles. To further examine the relations between Lim3 expression and cell death, we infected one side of the spinal cord with the retrovirus vector carrying chick Lim3 cDNA using electroporation technique. On the infected side, the number of dying MNs markedly decreased at E4.5 and the number of healthy MNs increased after the period of cell death (E5.5). These results suggest that the downregulation of Lim3 is involved in mechanisms that determine cells to die in the early type of cell death in the cervical spinal cord.