The Evolution of Developmental Mechanisms for Anteroposterior Pattening: Amphioxus as a Model for the Ancestral Vertebrate

本研究的长期目标是通过使用文昌鱼（佛罗里达Branchiostoma floridae）作为脊椎动物祖先的模型来阐明塑造脊椎动物身体计划的模式机制的进化起源。文文鱼是与脊椎动物最接近的无脊椎动物，它的基因组和身体结构都与脊椎动物相似，但没有脊椎动物那么复杂。这种相对简单的特点使得文昌鱼一方面可以与生命树中更基础的动物进行比较，另一方面也可以与脊椎动物进行比较。本研究的重点是文昌鱼中枢神经系统（CNS）中建立前/后（A/P）模式和区域特征的初始机制。这些机制既包括一个进化上古老的机制，也包括一个进化上较近的机制。第一个特征明显存在于双侧祖先中，即通过Wnt/ bettcatenin信号来确定后验身份。第二种，仅限于脊索动物，是维甲酸（RA）——沿着A/P轴建立区域特征的信号。在脊椎动物中，这两种途径相互作用。这就提出了这两种途径之间的相互作用是何时进化的问题。根据初步数据，这项拟议的研究将验证这样一个假设，即这些途径之间的实质性相互作用是在文文鱼和脊椎动物分裂之后才进化出来的。具体目的是：(1)确定文文鱼胚孔的Wnt/ β -catenin信号是否介导神经外胚层的前后模式；(2)测试CNS的前身份是否部分由表达hexx的前内胚层的Wnt抑制信号介导；(3)确定Wnt/ β -catenin和ra信号通路之间的相互作用。实现第一个目标的方法包括用锂来控制文文鱼中的wnt信号，锂可以阻断下游组分gsk3 β，使用细胞渗透性gsk3 β抑制剂Myr-N-GKEAPPAPPQSP-NH2，以及使用针对特定wnt的反义氨基寡核苷酸。对于第二个目标，Hex将通过hexmrna的过表达而上调，并被morpholinoolignucleotides阻断功能。文昌鱼wnt拮抗剂的同源物将被克隆，并根据其表达进行功能测试。对于第三个特定目的，将确定在类风湿关节炎或类风湿关节炎拮抗剂处理的文昌鱼胚胎中涉及介导wnt信号的基因的表达。Wnt-和ra -信号的预期靶点的调控区域将检查维甲酸受体反应元件（RAREs）和Tcf的结合位点，Tcf介导Wnt/ β -连环蛋白信号传导。联合基因组研究所将于2005年初完成文昌鱼基因组序列草图，这将为这一工作提供便利。选定的位点将发生突变，含有这些位点的报告基因结构的表达将显示它们是否对正常基因表达至关重要。智力价值：了解Wnt/ β -catenin信号前抑制机制的进化以及Wnt/ β -catenin和ra信号通路之间的串音的进化将有助于重建早期脊椎动物发育机制的进化。此外，这项研究将深入了解脊索动物，包括脊椎动物胚胎的分子机制。更广泛的影响：这项工作对结构进化、发育机制进化和基因复制等问题的更广泛概念也很重要。本研究将为两名研究生和本科生提供培训。

Project Summary The long term goal of this research is to elucidate the evolutionary origins of the patterning mechanisms that shape the vertebrate body plan by using amphioxus (Branchiostoma floridae) as a model for the ancestral vertebrate. Amphioxus, the closest living invertebrate relative of the vertebrates, has both a genome and a body plan that are vertebrate-like, but less complex. This relative simplicity uniquely positions amphioxus for comparisons, on the one hand, with animals more basal in the tree of life and, on the other hand, with the vertebrates. The present proposal focuses on the initial mechanisms establishing anterior/posterior (A/P) patterning and regional identities within the amphioxus central nervous system (CNS). These mechanisms include both an evolutionarily ancient mechanism and an evolutionarily more recent one. The first, which was evidently present in the bilaterian ancestor, is specification of posterior identity by Wnt/bets catenin signaling. The second, limited to chordates, is retinoic acid (RA)-signaling that establishes regional identities along the A/P axis. In vertebrates, these two pathways interact. This raises the question of when interactions between the two pathways evolved. The proposed study will test the hypothesis, based on preliminary data, that substantial interaction between these pathways evolved only after the split between amphioxus and the vertebrates. The specific aims are (1) to determine if Wnt/beta-catenin signals from the amphioxus blastopore mediate anterior/posterior patterning of the neuroectoderm (2) to test whether anterior identity of the CNS is maintained by signals mediated, in part, by Wnt-suppressing signals from Hex-expressing anterior endoderm and (3) to identify interactions between Wnt/beta-catenin and RA-signaling pathways. Methods to address the first aim include manipulating Wnt-signaling in amphioxus with lithium, which blocks the downstream component GSK3beta, with the cell-permeable GSK3beta inhibitor Myr-N-GKEAPPAPPQSP-NH2 and with antisense morpholino-oligonucleotides against specific Wnts. For the second aim, Hex will be upregulated by over-expression of Hex mRNA and function blocked with morpholino-oligonucleotides. Amphioxus homologs of Wnt-antagonists will be cloned and, depending on their expression, their function will be tested. For the third specific aim, expression of genes implicated as mediating Wnt-signaling will be determined in amphioxus embryos treated with either RA or an RA-antagonist. Regulatory regions of putative targets of both Wnt- and RA-signaling will be examined for retinoic acid receptor response elements (RAREs) and binding sites for Tcf, which mediates Wnt/beta-catenin signaling. This will be facilitated by the draft sequence of the amphioxus genome, which will be completed by the Joint Genome Institute early in 2005. Selected sites will be mutated and expression of reporter constructs containing them will show if they are essential for normal gene expression. Intellectual merit: Understanding how mechanisms for anterior suppression of Wnt/beta-catenin signals evolved and crosstalk between the Wnt/ beta catenin and RA-signaling pathways evolved will help reconstruct the evolution of developmental mechanisms in early vertebrates. In addition, this research will give insight into the molecular mechanisms for patterning the chordate, including vertebrate embryos. Broader impact: The work is also important in the broader concept of questions of the evolution of structures, evolution of developmental mechanisms and gene duplication. This research will provide training for two graduate students and undergraduates.