Zolessi F R, Arruti C
Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay.
Brain Res Dev Brain Res. 2001 Oct 24;130(2):257-67. doi: 10.1016/s0165-3806(01)00251-6.
MARCKS, a substrate for several kinases, has critical functions in morphogenetic processes involved in the development of the nervous system. We previously described the purification of MARCKS from chick embryo brain, using a monoclonal antibody (mAb 3C3), raised against embryonic neural retina. Here we show that mAb 3C3 is an antibody sensitive to phosphorylation state. We used it to explore the appearance and developmental progression of phospho-MARCKS (ph-MARCKS) during initial stages of neurogenesis in retina and spinal cord, and compared its distribution with total MARCKS. Before the onset of neural differentiation, MARCKS protein was already accumulated in neural and non-neural embryonic tissues, while ph-MARCKS immunoreactivity was weak, although ubiquitous too. A sudden increase of ph-MARCKS, paralleling a total MARCKS augmentation, was particularly noticeable in the earliest differentiating neurons in the neural retina. Ganglion cells displayed a high ph-MARCKS signal in the soma, as well as in the growing axon. A short time thereafter, a similar increase of ph-MARCKS was present across the entire width of the neural retina, where the differentiation of other neurons and photoreceptors occurs. The increase of ph-MARCKS in cells took place before the detection of the transcription factor Islet-1/2, an early neuronal differentiation molecular marker, in cells of the same region. Analogous phenomena were observed in cervical regions of the spinal cord, where motor neurons were differentiating. Neurogenic regions in the spinal cord contained higher amounts of ph-MARCKS than the floor plate. Taken together, these results strongly suggest that the appearance and relatively long-lasting presence of ph-MARCKS polypeptides are related to specific signaling pathways active during neurogenesis.
MARCKS是几种激酶的底物，在神经系统发育所涉及的形态发生过程中具有关键作用。我们之前描述了从鸡胚脑中纯化MARCKS的方法，使用的是一种针对胚胎神经视网膜产生的单克隆抗体（mAb 3C3）。在此我们表明mAb 3C3是一种对磷酸化状态敏感的抗体。我们用它来探究视网膜和脊髓神经发生初始阶段磷酸化MARCKS（ph-MARCKS）的出现和发育进程，并将其分布与总MARCKS进行比较。在神经分化开始之前，MARCKS蛋白已在神经和非神经胚胎组织中积累，而ph-MARCKS免疫反应性较弱，尽管也是普遍存在的。ph-MARCKS的突然增加与总MARCKS的增加同时出现，在神经视网膜最早分化的神经元中尤为明显。神经节细胞在胞体以及生长中的轴突中显示出高ph-MARCKS信号。此后不久，在神经视网膜的整个宽度上都出现了类似的ph-MARCKS增加，其他神经元和光感受器在此处发生分化。细胞中ph-MARCKS的增加发生在同一区域细胞中检测到转录因子Islet-1/2（一种早期神经元分化分子标记）之前。在脊髓颈段也观察到了类似现象，运动神经元在此处分化。脊髓中的神经发生区域比底板含有更多的ph-MARCKS。综上所述，这些结果强烈表明ph-MARCKS多肽的出现和相对持久的存在与神经发生过程中活跃的特定信号通路有关。
