Conservation and Diversification of an Ancestral Chordate Gene Regulatory Network for Dorsoventral Patterning

Formation of a dorsoventral axis is a key event in the early development of most animal embryos. It is well established that bone morphogenetic proteins (Bmps) and Wnts are key mediators of dorsoventral patterning in vertebrates. In the cephalochordate amphioxus, genes encoding Bmps and transcription factors downstream of Bmp signaling such as Vent are expressed in patterns reminiscent of those of their vertebrate orthologues. However, the key question is whether the conservation of expression patterns of network constituents implies conservation of functional network interactions, and if so, how an increased functional complexity can evolve. Using heterologous systems, namely by reporter gene assays in mammalian cell lines and by transgenesis in medaka fish, we have compared the gene regulatory network implicated in dorsoventral patterning of the basal chordate amphioxus and vertebrates. We found that Bmp but not canonical Wnt signaling regulates promoters of genes encoding homeodomain proteins AmphiVent1 and AmphiVent2. Furthermore, AmphiVent1 and AmphiVent2 promoters appear to be correctly regulated in the context of a vertebrate embryo. Finally, we show that AmphiVent1 is able to directly repress promoters of AmphiGoosecoid and AmphiChordin genes. Repression of genes encoding dorsal-specific signaling molecule Chordin and transcription factor Goosecoid by Xenopus and zebrafish Vent genes represents a key regulatory interaction during vertebrate axis formation. Our data indicate high evolutionary conservation of a core Bmp-triggered gene regulatory network for dorsoventral patterning in chordates and suggest that co-option of the canonical Wnt signaling pathway for dorsoventral patterning in vertebrates represents one of the innovations through which an increased morphological complexity of vertebrate embryo is achieved.背腹轴的形成是多数动物胚胎早期发育过程中的关键事件。已有充分研究证实，骨形态发生蛋白（Bone Morphogenetic Proteins，BMPs）与Wnt蛋白（Wnts）是脊椎动物背腹模式建成的核心调控介质。在头索动物文昌鱼（cephalochordate amphioxus）中，编码BMPs以及BMP信号下游转录因子如Vent（Vent）的基因，其表达模式与脊椎动物中的直向同源基因（orthologues）高度相似。然而，核心科学问题在于：调控网络组分的表达模式保守是否意味着其功能网络互作也保守？若是如此，功能复杂性的提升又是如何通过演化实现的？本研究采用异源系统研究策略，即通过哺乳动物细胞系中的报告基因实验（reporter gene assays）以及青鳉鱼（medaka fish）的转基因技术（transgenesis），比较了基础脊索动物文昌鱼与脊椎动物背腹模式建成相关的基因调控网络。研究发现，BMP信号而非经典Wnt信号，可调控同源框蛋白（homeodomain proteins）编码基因AmphiVent1与AmphiVent2的启动子。进一步实验显示，AmphiVent1与AmphiVent2的启动子在脊椎动物胚胎的内源环境中能够被正确调控。最后，本研究证实AmphiVent1可直接抑制AmphiGoosecoid与AmphiChordin基因的启动子活性。爪蟾（Xenopus）与斑马鱼（zebrafish）的Vent基因通过抑制背侧特异性信号分子Chordin（Chordin）的编码基因以及转录因子Goosecoid（Goosecoid），是脊椎动物轴形成过程中的关键调控互作环节。我们的数据表明，脊索动物背腹模式建成的核心BMP触发型基因调控网络具有高度的进化保守性，并提示脊椎动物中通过招募经典Wnt信号通路参与背腹模式建成，是实现脊椎动物胚胎形态复杂性提升的创新演化机制之一。