Jankowski Jakob, Holst Martin I, Liebig Christian, Oberdick John, Baader Stephan L
Institute of Anatomy, Anatomy and Cell Biology, University of Bonn, 53115 Bonn, Germany.
J Comp Neurol. 2004 Apr 19;472(1):87-99. doi: 10.1002/cne.20059.
The transcription factor Engrailed-2 is expressed in cerebellar Purkinje cells (PCs) throughout embryonic development but is downregulated in PCs after birth. Since the onset of PC differentiation coincides with this change of gene expression, we asked whether downregulation of Engrailed-2 is necessary for proper timing of PC differentiation. To investigate this, we used an L7En-2 transgenic mouse model in which Engrailed-2 expression in PCs is maintained beyond the day of birth. In these L7En-2 mice the onset of parvalbumin expression was delayed in all PCs by about 3 days; the spatial expression pattern, however, remained comparable to wildtype cerebella. Furthermore, parvalbumin expression resembled the known pattern of normal PC maturation, suggesting a direct link between parvalbumin expression and PC differentiation. Consistent with a delay of PC differentiation, we found that PCs of L7En-2 cerebella displayed a reduced tendency to align in the typical monolayer. The average size of L7En-2 PCs was reduced and the dendritic arbor developed more slowly than in wildtype PCs. In contrast, major morphological features of PCs were comparable in L7En-2 and wildtype cerebella after postnatal day 11. In addition, we observed a transient reduction of PC survival in organotypic slice cultures of L7En-2 cerebella in comparison with wildtype slice cultures. Since PC survival parallels PC differentiation in vitro, we propose that the observed delay in PC differentiation upon Engrailed-2 overexpression is an intrinsic property of Engrailed-2 activity, and that downregulation of Engrailed-2 in wildtype PCs around the day of birth is critical for the timing of distinct steps of PC differentiation.
转录因子En-2在整个胚胎发育过程中均在小脑浦肯野细胞（PCs）中表达，但在出生后PCs中表达下调。由于PC分化的开始与这种基因表达变化相吻合，我们探究了En-2的下调对于PC分化的正确时间是否必要。为了研究这一点，我们使用了一种L7En-2转基因小鼠模型，其中PCs中En-2的表达在出生后仍得以维持。在这些L7En-2小鼠中，所有PCs中小清蛋白表达的开始均延迟了约3天；然而，其空间表达模式与野生型小脑仍具有可比性。此外，小清蛋白的表达类似于正常PC成熟的已知模式，这表明小清蛋白表达与PC分化之间存在直接联系。与PC分化延迟一致，我们发现L7En-2小脑的PCs在典型单层中排列的趋势降低。L7En-2 PC的平均大小减小，并且树突状分支的发育比野生型PC更慢。相比之下，出生后第11天之后，L7En-2和野生型小脑中PC的主要形态特征具有可比性。此外，与野生型脑片培养相比，我们观察到L7En-2小脑的器官型脑片培养中PC存活出现短暂减少。由于体外PC存活与PC分化平行，我们提出，在En-2过表达时观察到的PC分化延迟是En-2活性的内在特性，并且出生当天左右野生型PCs中En-2的下调对于PC分化不同步骤的时间安排至关重要。
