Department of Medicine, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Stem Cells. 2013 Jan;31(1):59-70. doi: 10.1002/stem.1254.
The paired box transcription factor Pax3 is well-known as a major regulator of embryonic myogenesis. Before Pax3 expression becomes restricted to the dermomyotome, this transcription factor is also expressed in the developing somites. The role of Pax3 at this early stage is unclear, in particular because of the scarce frequency of Pax3-positive cells in the early mouse embryo. Inducible gene expression in embryonic stem cells (ESCs) represents an excellent tool to overcome this limitation, since it can provide large quantities of otherwise rare embryonic populations expressing a factor of interest. Here we used engineered mouse ESCs to perform a functional analysis of Pax3 with the aim to identify the molecular determinants involved in the early functions of this transcription factor. We find that Pax3 induction during embryoid body differentiation results in the upregulation of genes expressed in the presomitic and somitic mesoderm. Moreover, we show that paraxial mesoderm induced by transient expression of Pax3 is not irreversibly committed to myogenesis rather requires sustained Pax3 expression. Using a series of deletion mutants of Pax3, which differentially affect its transcriptional activity, we map protein domains necessary for induction of paraxial mesoderm and induction of the myogenic program. The paired, homeo-, and transcriptional activation domains were each required for both processes, however, the paired-c-terminal RED domain showed a paraxial mesoderm-specific activity that was dispensable for myogenesis. These findings demonstrate and provide mechanistic insight into an early role for Pax3 in the generation of paraxial mesoderm.
配对盒转录因子 Pax3 是胚胎肌发生的主要调节因子,这是众所周知的。在 Pax3 表达被限制在真皮肌节之前,这种转录因子也在发育中的体节中表达。在这个早期阶段,Pax3 的作用尚不清楚,特别是由于早期小鼠胚胎中 Pax3 阳性细胞的频率很低。诱导胚胎干细胞(ESCs)中的基因表达是克服这一限制的极好工具,因为它可以提供大量否则罕见的表达感兴趣因子的胚胎群体。在这里,我们使用工程化的小鼠 ESCs 进行了 Pax3 的功能分析,目的是鉴定参与该转录因子早期功能的分子决定因素。我们发现,在胚状体分化过程中诱导 Pax3 会导致前体节和体节中胚层表达的基因上调。此外,我们表明,瞬时表达 Pax3 诱导的轴旁中胚层并非不可逆地向肌生成方向分化,而是需要持续的 Pax3 表达。我们使用一系列 Pax3 的缺失突变体,这些突变体差异影响其转录活性,从而绘制出诱导轴旁中胚层和诱导肌生成程序所需的蛋白结构域图谱。配对、同源和转录激活结构域对于这两个过程都是必需的,但是配对-C 端 RED 结构域表现出轴旁中胚层特异性活性,对于肌生成是可有可无的。这些发现证明并提供了 Pax3 在产生轴旁中胚层中的早期作用的机制见解。