Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.
PLoS Genet. 2011 May;7(5):e1002072. doi: 10.1371/journal.pgen.1002072. Epub 2011 May 26.
Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA-binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals.
脊椎动物中胚层的特化需要 Nodal 信号通路及其转录效应因子 FoxH1。然而，在几种物种中缺失 FoxH1 并不能可靠地导致完全丧失 Nodal 表型，这表明 Nodal 信号在早期发育过程中通过其他转录因子传递。我们使用一种称为 midway 的新型隐性斑马鱼 FoxH1 突变体研究了 Nodal 信号在中胚层模式形成过程中 FoxH1 依赖性和非依赖性作用，该突变体产生一种截短的 C 端 FoxH1 蛋白，缺乏 Smad 相互作用域，但保留 DNA 结合能力。通过凝胶迁移分析、Nodal 过表达实验和遗传上位性分析，我们证明 midway 比现有的斑马鱼 FoxH1 突变体 schmalspur 更准确地代表了 FoxH1 依赖性 Nodal 信号的完全缺失。母体合子 midway 突变体缺乏脊索，这与其他生物中 FoxH1 的缺失一致，但保留了内胚层和各种非轴中胚层命运的标记物的近野生型表达，包括轴旁中胚层和中间中胚层以及血液前体。我们发现 T 盒转录因子 Eomesodermin 的活性决定了 midway 胚胎中这些组织的特化。在 midway 突变体中抑制 Eomesodermin 严重减少了这些组织的特化，并有效地模拟了完全丧失 Nodal 信号时观察到的缺陷。我们的结果表明，在中胚层模式形成过程中，信号转导可用的转录因子的特定组合在确定 Nodal 途径输出方面发挥了关键和可分离的作用。我们的研究结果还为 Nodal 信号通路、脊索特化程序以及后生动物的后口动物家族的脊索动物分支的共同进化提供了新的见解。
