The Orphan Gene dauerless Regulates Dauer Development and Intraspecific Competition in Nematodes by Copy Number Variation

Many nematodes form dauer larvae when exposed to unfavorable conditions, representing an example of phenotypic plasticity and a major survival and dispersal strategy. In Caenorhabditis elegans, the regulation of dauer induction is a model for pheromone, insulin, and steroid-hormone signaling. Recent studies in Pristionchus pacificus revealed substantial natural variation in various aspects of dauer development, i.e. pheromone production and sensing and dauer longevity and fitness. One intriguing example is a strain from Ohio, having extremely long-lived dauers associated with very high fitness and often forming the most dauers in response to other strains´ pheromones, including the reference strain from California. While such examples have been suggested to represent intraspecific competition among strains, the molecular mechanisms underlying these dauer-associated patterns are currently unknown. We generated recombinant-inbred-lines between the Californian and Ohioan strains and used quantitative-trait-loci analysis to investigate the molecular mechanism determining natural variation in dauer development. Surprisingly, we discovered that the orphan gene dauerless controls dauer formation by copy number variation. The Ohioan strain has one dauerless copy causing high dauer formation, whereas the Californian strain has two copies, resulting in strongly reduced dauer formation. Transgenic animals expressing multiple copies do not form dauers. dauerless is exclusively expressed in CAN neurons, and both CAN ablation and dauerless mutations increase dauer formation. Strikingly, dauerless underwent several duplications and acts in parallel or downstream of steroid-hormone signaling but upstream of the nuclear-hormone-receptor daf-12. We identified the novel or fast-evolving gene dauerless as inhibitor of dauer development. Our findings reveal the importance of gene duplications and copy number variations for orphan gene function and suggest daf-12 as major target for dauer regulation. We discuss the consequences of the novel vs. fast-evolving nature of orphans for the evolution of developmental networks and their role in natural variation and intraspecific competition.许多线虫在遭遇不良环境条件时会形成dauer幼虫（dauer larvae），这是表型可塑性的典型范例，同时也是其核心的生存与扩散策略。在秀丽隐杆线虫（Caenorhabditis elegans）中，dauer幼虫诱导的调控通路是信息素、胰岛素以及类固醇激素信号转导研究的经典模型。近期对太平洋普氏线虫（Pristionchus pacificus）的研究显示，其dauer发育的多个维度存在显著的自然变异，涵盖信息素的产生与感知、dauer幼虫的寿命与适合度。其中一个颇具研究价值的案例来自俄亥俄州的一个品系：该品系的dauer幼虫寿命极长且适合度极高，并且在响应其他品系（包括加州参考品系）的信息素时，往往能产生最多的dauer幼虫。尽管这类案例被认为反映了不同品系间的种内竞争，但目前学界尚未阐明这些与dauer发育相关表型背后的分子机制。我们通过加州与俄亥俄州品系杂交构建了重组自交系（recombinant inbred lines, RIL），并采用数量性状位点（quantitative trait loci, QTL）分析方法，探究决定dauer发育自然变异的分子机制。令人意外的是，我们发现孤儿基因（orphan gene）dauerless通过拷贝数变异调控dauer幼虫的形成：俄亥俄州品系仅携带1个dauerless拷贝，因此dauer幼虫形成率极高；而加州品系携带2个拷贝，导致dauer幼虫形成率显著降低。携带多个dauerless拷贝的转基因线虫则无法形成dauer幼虫。dauerless仅在CAN神经元（CAN neurons）中特异性表达，对CAN神经元进行消融或引入dauerless突变均会提升dauer幼虫的形成率。值得注意的是，dauerless经历了多次复制事件，其调控通路位于类固醇激素信号转导的下游或与之平行，并作用于核激素受体daf-12的上游。我们将这个属于新基因或快速进化基因的dauerless鉴定为dauer发育的抑制因子。本研究结果阐明了基因复制与拷贝数变异对于孤儿基因功能的重要性，并指出daf-12是dauer发育调控的核心靶点。我们还讨论了孤儿基因的新基因/快速进化属性对发育网络演化的影响，以及其在自然变异和种内竞争中发挥的作用。