Maternal Gdf3 is an obligatory cofactor in Nodal signaling for embryonic axis formation in zebrafish.
Maternal Gdf3 is an obligatory cofactor in Nodal signaling for embryonic axis formation in zebrafish.
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DOI：
10.7554/elife.28534
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发表时间：
2017-11-15
期刊：
影响因子：
7.7
通讯作者：
Yost HJ
中科院分区：
文献类型：
--
作者：
Bisgrove BW;Su YC;Yost HJ
斑马鱼Gdf 3（Dvr 1）是细胞信号配体TGFβ超家族的成员，包括非洲爪蟾Vg 1和哺乳动物Gdf 1/3。令人惊讶的是，斑马鱼中的工程纯合突变体没有明显的表型。消除Gdf 3的母合子突变体的卵母细胞中的胚胎致死率，可以完全挽救与gdf 3 RNA，证明Gdf 3是必需的，只有在早期的发展，超过此突变体是可行的和可育的。Gdf 3突变体对Nodal配体和Nodal阻遏物Lefty 1是难治的。由TGFβ配体激活素和组成型活性受体Alk 4和Alk 2驱动的信号传导在gdf 3突变体中保持完整，表明Gdf 3在与Nodal相同的途径步骤中起作用。将gdf 3和ndr 2 RNA靶向特定谱系表明，外源gdf 3仅在与内源Nodal共表达时才能够完全拯救突变体。总之，这些发现表明，Gdf 3是在胚胎轴的建立过程中Nodal信号传导的重要辅因子。所有的脊椎动物--像鱼和人类这样有脊椎的动物--都有三个清晰的轴：头到尾、后到前和左到右。当信号分子与细胞表面的受体结合时，动物在胚胎中就制定了这些计划。这些信号分子包括称为“Nodal”和“生长和分化因子”的相关蛋白质。然而，在发育生物学领域，关于这些蛋白质在脊椎动物的早期发育中是一起工作还是独立工作，一直存在很大争议。斑马鱼经常用于研究动物发育，Bisgrove等人决定通过使用基因组编辑删除它来测试这些鱼是否需要称为Gdf 3的生长和分化因子。事实证明，斑马鱼可以在没有Gdf 3基因的情况下正常生存和发育，只要它们的母亲仍然有该基因的工作拷贝。然而，当突变雌性的后代没有从母亲那里继承制造Gdf 3的指令时，它们在几天内死亡。即使后代从父亲那里继承了该基因的工作拷贝，情况也是如此。Bisgrove等人接着表明，来自突变母亲的胚胎可以通过注射短寿命RNA分子来挽救，这些RNA分子包括制造一些Gdf 3蛋白的指令。注射的突变胚胎可以活到成年。这表明Gdf 3仅在胚胎早期发育期间需要。进一步的实验表明，Gdf 3本身不能激活其受体。相反，Gdf 3很可能与Nodal相互作用，形成一种激活受体的双蛋白复合物。另外两组研究人员也独立地报告了类似的发现。在先天性心脏病患者中发现了影响与Gdf 3非常相似的蛋白质的突变。通过揭示Gdf 3和Nodal之间的相互作用，这些新发现可以帮助科学家更详细地了解这种疾病的遗传原因。使用突变斑马鱼的进一步研究也可用于探索其他发育疾病的原因。
Zebrafish Gdf3 (Dvr1) is a member of the TGFβ superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFβ ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis. All vertebrates – animals with backbones like fish and humans – have body plans with three clear axes: head-to-tail, back-to-front and left-to-right. Animals lay down these plans as embryos, when signaling molecules bind to receptors on the surface of their cells. These signaling molecules include related proteins called “Nodal” and “Growth and Differentiation Factors”. However, there has been much debate in the field of developmental biology about whether these proteins work together or independently during the early development of vertebrates. Zebrafish are often used to study animal development, and Bisgrove et al. decided to test whether these fish need a Growth and Differentiation Factor known as Gdf3 by deleting it using genome editing. It turns out that zebrafish can survive and develop as normal without the gene for Gdf3, just as long as their mothers still had a working copy of the gene. Yet, when the offspring of mutant females did not inherit the instructions to make Gdf3 from their mothers, they died within a couple of days. This was true even if the offspring inherited a working copy of the gene from their fathers. Bisgrove et al. then went on to show that embryos from a mutant mother could be saved with an injection of short-lived RNA molecules that include the instructions to make some Gdf3 proteins. The injected mutant embryos could live to adulthood. This shows that Gdf3 is only needed during the embryo’s early development. Further experiments suggested that Gdf3 does cannot activate its receptors on its own. Instead, it is likely that Gdf3 interacts with Nodal to form a two-protein complex that activates the receptors. Two other groups of researchers have independently reported similar findings. Mutations affecting proteins very similar to Gdf3 have been found in people with congenital heart defects. By revealing the interaction between Gdf3 and Nodal, these new findings could help scientists to understand the genetic causes of this condition in more detail. Further studies using the mutant zebrafish could also be used to explore the causes of other developmental diseases.