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

在鸟类胚胎脊髓中，运动神经元经历了两个不同的时期的程序性细胞死亡(PCD)。一种是在相对较晚的阶段(雏鸡的E6-E10)观察到的，涉及脊髓的所有节段。相反，另一种则发生在相对较早的阶段(E4-E5)，并且仅发生在支配颈髓的非肢体部位。为了确定垂死MN在早期细胞死亡类型中的同一性，我们检测了鸡胚胎垂死MN中亚群特异性MN标志物(Is1、Is12、Lim3和MNR2)的表达。定量分析表明，PCD仅发生在表达Isl1、Isl2和MNR2但不表达Lim3的MN的亚群中。这个亚群的MN在E3(-)和E3.5之间成为有丝分裂后，在失去Lim3表达后的E4出现在腹角背外侧区的一个独特的亚群。然而，它们在E5之前就消失了。逆转录病毒载体导入Bcl2基因后，宫颈MN免于细胞死亡。在细胞死亡后(E5)，我们观察到，在感染的胚胎中，有一个额外的MN亚群表达Isl1和Isl2，但不表达Lim3。类似的MN也出现在胸区，并发育成支配肋间肌肉的MN。这些结果表明，这种类型的颈髓MN的PCD只发生在LIM3阴性的MN亚群中，对应于支配肋间肌肉的MN。为了进一步研究Lim3表达与细胞死亡的关系，我们用携带鸡Lim3基因的逆转录病毒载体通过电穿孔技术感染了一侧脊髓。在感染侧，死亡的MN数量在E4.5时显著减少，而健康MN的数量在细胞死亡后增加(E5.5)。这些结果表明，在颈髓早期类型的细胞死亡中，Lim3的下调参与了决定细胞死亡的机制。

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.