Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673.
Development. 2011 Feb;138(4):755-65. doi: 10.1242/dev.062521.
Different levels and timing of Hedgehog (Hh) signalling activity have been proposed to specify three distinct cell types in the zebrafish myotome. Two of these, the medial fast-twitch fibres (MFFs) and the slow-twitch muscle pioneers (MPs) are characterised by expression of eng1a, -1b and -2a and require the highest levels of Hh for their specification. We have defined a minimal eng2a element sufficient to drive reporter expression specifically in MPs and MFFs. This element binds both Gli2a, a mediator of Hh signalling, and activated Smads (pSmads), mediators of bone morphogenic protein (BMP) signalling, in vivo. We found a strict negative correlation between nuclear accumulation of pSmad, and eng2a expression in myotomal cells and show that abrogation of pSmad accumulation results in activation of eng2a, even when Hh signalling is attenuated. Conversely, driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. Nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. We show that a synthetic form of the Gli2 repressor interacts with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signalling activity. Our results demonstrate that the eng2a promoter integrates repressive and activating signals from the BMP and Hh pathways, respectively, to limit its expression to MPs and MFFs. We suggest a novel basis for crosstalk between the Hh and BMP pathways, whereby BMP-mediated repression of Hh target genes is promoted by a direct interaction between Smads and truncated Glis, an interaction that is abrogated by Hh induced depletion of the latter.
不同水平和时间的 Hedgehog (Hh) 信号活动被提出用于在斑马鱼肌节中指定三种不同的细胞类型。其中两种,即内侧快肌纤维(MFFs)和慢肌先驱物(MPs),其特征在于表达 eng1a、-1b 和 -2a,并需要最高水平的 Hh 来指定。我们已经定义了一个最小的 eng2a 元件,足以驱动报告基因在 MPs 和 MFFs 中特异性表达。该元件在体内与 Hedgehog 信号的介质 Gli2a 和骨形态发生蛋白 (BMP) 信号的介质激活的 Smads (pSmads) 结合。我们发现核内 pSmad 积累与肌节细胞中 eng2a 表达之间存在严格的负相关,并表明 pSmad 积累的消除导致 eng2a 的激活,即使 Hh 信号减弱。相反,即使 Hh 途径活性最大,驱动 pSmad 的核内积累也会抑制 eng 表达的诱导。pSmads 的核内积累通过最大的 Hh 途径激活而耗尽。我们表明,Gli2 抑制剂的合成形式与 Smad1 特异性相互作用在发育中的胚胎肌节细胞的核中,并且这种相互作用取决于 BMP 信号活性。我们的结果表明,eng2a 启动子整合了来自 BMP 和 Hh 途径的抑制和激活信号,分别将其表达限制在 MPs 和 MFFs 中。我们提出了 Hh 和 BMP 途径之间相互作用的新基础,其中 BMP 介导的 Hedgehog 靶基因的抑制通过 Smads 和截短的 Glis 之间的直接相互作用来促进,这种相互作用被 Hh 诱导的后者耗尽所消除。