Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, 92093-0202, USA.
Evol Dev. 2012 Jul;14(4):338-50. doi: 10.1111/j.1525-142X.2012.00552.x.
To dissect the molecular mechanism of head specification in the basal chordate amphioxus, we investigated the function of Dkk3, a secreted protein in the Dickkopf family, which is expressed anteriorly in early embryos. Amphioxus Dkk3 has three domains characteristic of Dkk3 proteins-an N-terminal serine rich domain and two C-terminal cysteine-rich domains (CRDs). In addition, amphioxus Dkk3 has a TGFβ-receptor 2 domain, which is not present in Dkk3 proteins of other species. As vertebrate Dkk3 proteins have been reported to regulate either Nodal signaling or Wnt/β-catenin signaling but not both in the same species, we tested the effects of Dkk3 on signaling by these two pathways in amphioxus embryos. Loss of function experiments with an anti-sense morpholino oligonucleotide (MO) against amphioxus Dkk3 resulted in larvae with truncated heads and concomitant loss of expression of anterior gene markers. The resemblance of the headless phenotype to that from upregulation of Wnt/β-catenin signaling with BIO, a GSK3β inhibitor, suggested that Dkk3 might inhibit Wnt/β-catenin signaling. In addition, the Dkk3 MO rescued dorsal structures in amphioxus embryos treated with SB505124, an inhibitor of Nodal signaling, indicating that amphioxus Dkk3 can also inhibit Nodal signaling. In vitro assays in Xenopus animal caps showed that Nodal inhibition is largely due to domains other than the TGFβ domain. We conclude that amphioxus Dkk3 regulates head formation by modulating both Wnt/β-catenin and Nodal signaling, and that these functions may have been partitioned among various vertebrate lineages during evolution of Dkk3 proteins.
为了剖析头索动物文昌鱼头部特化的分子机制，我们研究了分泌型 Dickkopf 家族蛋白 Dkk3 的功能，该蛋白在文昌鱼早期胚胎中表达于前部。文昌鱼 Dkk3 具有 Dkk3 蛋白的三个特征结构域——富含丝氨酸的 N 端结构域和两个富含半胱氨酸的 C 端结构域（CRD）。此外，文昌鱼 Dkk3 具有一个 TGFβ 受体 2 结构域，而其他物种的 Dkk3 蛋白中没有这个结构域。由于脊椎动物 Dkk3 蛋白在同一物种中被报道可以调节 Nodal 信号或 Wnt/β-catenin 信号，但不能同时调节这两种信号，因此我们测试了 Dkk3 对文昌鱼胚胎中这两条信号通路的影响。针对文昌鱼 Dkk3 的反义 morpholino 寡核苷酸（MO）的功能丧失实验导致幼虫头部缩短，并且前部基因标记的表达丧失。无头表型与 Wnt/β-catenin 信号上调导致的表型相似，用 GSK3β 抑制剂 BIO 处理，表明 Dkk3 可能抑制 Wnt/β-catenin 信号。此外，Dkk3 MO 挽救了用 Nodal 信号抑制剂 SB505124 处理的文昌鱼胚胎中的背侧结构，表明文昌鱼 Dkk3 也可以抑制 Nodal 信号。在 Xenopus 动物帽的体外实验表明，Nodal 抑制主要归因于 TGFβ 结构域外的其他结构域。我们的结论是，文昌鱼 Dkk3 通过调节 Wnt/β-catenin 和 Nodal 信号来调节头部形成，并且这些功能可能在 Dkk3 蛋白的进化过程中在各种脊椎动物谱系中进行了分配。
