Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, Poland.
PLoS One. 2013 Sep 3;8(9):e74346. doi: 10.1371/journal.pone.0074346. eCollection 2013.
In this study, we investigated the distribution, localization and several various functions of TrkC receptors during development of the Monodelphisopossum brain. Western blotting analysis showed that two different forms of the TrkC receptor, the full-length receptor and one of its truncated forms, are abundantly expressed in the opossum brain. The expression of TrkC receptors was barely detected in the brain of newborn opossums. At postnatal day (P) 3, the expression of full-length TrkC remained at low levels, while moderate expression of the TrkC truncated form was detected. The expression levels of both forms of this protein gradually increased throughout development, peaking at P35. We found that in different neocortical areas located both at the rostral and caudal regions of the cortex, up to 98% of BrdU-labeled cells forming cortical layers (II-VI) had prominently expressed TrkC. To assess which developmental processes of cortical cells are regulated by TrkC receptors, three different shRNAs were constructed. The shRNAs were individually tested in transfected cortical progenitor cells grown on culture plates for 2 days. The effects of the shRNA-TrkC constructs were similar: blockade of TrkC receptors decreased the number of Ki67-positive and apoptotic cells, and it did not change the number of TUJ-positive neurons in vitro. Thus, the lack of TrkC receptors in cultured progenitor cells provided insight on the potential role of these receptors in the regulation of proliferation and cell survival but not in the differentiation of cortical cells.
在这项研究中,我们调查了 Monodelphisopossum 大脑发育过程中 TrkC 受体的分布、定位和多种功能。Western blot 分析表明,两种不同形式的 TrkC 受体,全长受体和其截断形式之一,在负鼠脑中大量表达。TrkC 受体在新生负鼠脑中几乎检测不到。在出生后第 3 天(P3),全长 TrkC 的表达水平保持在低水平,而 TrkC 截断形式的表达水平适中。这种蛋白质的两种形式的表达水平在整个发育过程中逐渐增加,在 P35 时达到峰值。我们发现,在位于大脑皮质前、后区域的不同新皮质区域,高达 98%的 BrdU 标记细胞形成皮质层(II-VI)显著表达 TrkC。为了评估 TrkC 受体调控皮质细胞的哪些发育过程,构建了三种不同的 shRNA。这些 shRNA 分别在培养板上培养的转染皮质祖细胞中进行了 2 天的测试。shRNA-TrkC 构建体的作用相似:阻断 TrkC 受体减少了 Ki67 阳性和凋亡细胞的数量,但没有改变体外 TUJ 阳性神经元的数量。因此,培养的祖细胞中缺乏 TrkC 受体为这些受体在调节增殖和细胞存活中的潜在作用提供了线索,但对皮质细胞的分化没有影响。