Kwiatkowski Adam V, Garner Craig C, Nelson W James, Gertler Frank B
Department of Biology, Stanford University, Stanford, CA 94305, USA.
Mol Cell Neurosci. 2009 May;41(1):44-50. doi: 10.1016/j.mcn.2009.01.008. Epub 2009 Feb 5.
A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based method to analyze cell autonomy by generating two-color chimeric mouse embryos. Ena/VASP-deficient mutant and control embryonic stem (ES) cells were labeled with different fluorescent chimeric proteins (EGFP and mCherry) that were modified to bind to the plasma membrane. These labeled ES cells were used to generate two-color chimeric embryos possessing two genetically distinct populations of cortical cells, permitting multiple aspects of neuronal morphogenesis to be analyzed and compared between the two cell populations. We observed little difference between the ability of control and Ena/VASP-deficient cells to contribute to cortical organization during development. In contrast, we observed axon fiber tracts originating from control neurons but not Ena/VASP-deficient neurons, indicating that loss of Ena/VASP causes a cell autonomous defect in cortical axon formation. This technique could be applied to determine other cell autonomous functions in different stages of cortical development.
一个由细胞内在和外在信号组成的复杂程序引导着皮质发育。尽管对小鼠的遗传学研究已经揭示了众多基因和基因家族在皮质发生的多个方面所起的作用，但确定它们的细胞自主功能往往因多效性缺陷而变得复杂。在这里，我们描述了一种基于慢病毒的新方法，通过生成双色嵌合小鼠胚胎来分析细胞自主性。用修饰后可结合质膜的不同荧光嵌合蛋白（EGFP和mCherry）标记Ena/VASP缺陷型突变体和对照胚胎干细胞（ES细胞）。这些标记的ES细胞被用于生成具有两种基因不同的皮质细胞群体的双色嵌合胚胎，从而能够分析和比较这两个细胞群体之间神经元形态发生的多个方面。我们观察到，在发育过程中，对照细胞和Ena/VASP缺陷型细胞对皮质组织的贡献能力几乎没有差异。相比之下，我们观察到轴突纤维束起源于对照神经元，而不是Ena/VASP缺陷型神经元，这表明Ena/VASP的缺失导致皮质轴突形成出现细胞自主缺陷。该技术可用于确定皮质发育不同阶段的其他细胞自主功能。
