Examination of the number of spinal motor neurons by using selective elimination of motor neurons

为了明确在发育过程中脊髓运动神经元的数量是如何控制的(神经发生和细胞死亡消除)，本研究进行了运动神经元选择性消除的实验研究。Bax是Bcl2家族中的促凋亡成员，被检测为运动神经元的刽子手。为了在早期PCD期间将Bax特异性地引入运动神经元，我们将其与TET调控系统相结合用于卵子电穿孔。在运动神经元特异性HB9启动子的控制下表达反向四环素调控的反式激活子，以靶向脊髓运动神经元的基因表达。用这种方法将获得功能的Bax突变体导入鸡颈运动神经元，与对照鸡胚相比，死亡运动神经元的数量显著增加。通过这种方法，朴素的Bax不影响运动神经元的存活，但使用构成启动子如CMV启动子来表达它们在脊髓中表达后立即显着地诱导大量细胞死亡。综上所述，本研究中研究的这些策略将允许在空间和时间上受控地消除脊髓运动神经元，并将特定地洞察脊髓运动神经元的数量在发育过程中是如何调节的。

To clear how the number of spinal motor neurons is controlled during development (neurogenesis and elimination by cell death), selective elimination of motor neurons has been experimentally developed in this study. Bax, a pro-apoptotic member of the Bcl-2 family, was examined as executioner for motor neurons. To introduce Bax specifically into motor neurons during the period of early PCD, we have used in ovo electroporation coupled with the tet regulatory system. The reverse tetracycline-controlled transactivator was expressed under the control of the motor neuron specific HB9 promoter to target gene expression to spinal motor neurons. When gain-of-function Bax mutants were introduced into chick cervical motor neurons by this method, the number of dying motor neurons was significantly increased compared with those of control chick embryos. Naive Bax did not affect motor neuron survival by this method but significantly induced a large amount of cell death as soon as expressed in the spinal cord using the constitutive promoter such as the CMV promoter for their expression. Taken together, these strategies examined in this study will allow spatially and temporally controlled elimination of spinal motor neurons and will give specific insight into how the number of spinal motor neurons is regulated during development.