Collaborative Research: Enhancers and the Convergent Evolution of Limb Deduction in Squamates

虽然手臂和腿的发展是一项关键的进化创新，使动物能够迁徙到陆地上，但许多蛇和蜥蜴物种随后失去了这些肢体。事实上，肢体丧失在蜥蜴中至少进化了24倍。如何在蜥蜴谱系中反复发生肢体丧失是一个问题，可以通过重复的自然实验来解决，这将是本研究的重点。这个项目将揭示在这些不同的蜥蜴群体中，是否相同或不同的基因变化与肢体丧失有关。这项研究将以新颖的方式结合不同生物学领域的技术，探索这一重复进化的不同寻常的例子，并对基因组功能获得新的见解。该项目将极大地提高人们对参与肢体发育的基因和遗传控制元素的了解。此外，在这个项目的过程中，将对人类和其他动物共享的数千个基因区域进行表征。因此，项目数据将提供可用于更好地理解人类和不同动物基因组中的其他功能元件的见解。开发的方法将为其他希望利用自然进化实验的力量来了解基因组功能的研究人员铺平道路。最后，将为教育工作者开发材料，用于教授学生如何将信息编码在基因组中，以及如何使用自然进化实验来揭示遗传序列如何产生独特的生物多样性。该项目将使用染色质免疫沉淀和高通量DNA测序(CHIP-SEQ)来识别鳞类基因组中控制肢体胚胎发育的调节元件(增强子)。这些信息将被用来设计针对这些调控区域的特定鳞状DNA序列捕获诱饵。将设计更多的捕获诱饵，以瞄准正在发育的肢体中活跃的基因的编码区。总而言之，这些诱饵将被用来调查每一种截肢鳞类的关键发育基因的非编码调控元件和编码外显子。肢体增强剂和肢体发育基因的DNA序列变化将在肢体减少的物种及其全肢近亲中进行比较，调节活性的功能变化将通过转基因小鼠报告试验在体内进行评估。这将深入了解与导致肢体丧失进化的基因编码序列变化相比，调控变化的相对频率。最后，Tbx4基因的古代羊膜增强子的功能进化将通过利用减少肢体的物种的多个谱系的序列变异来从分子上进行剖析。这将使人们深入了解失去监管角色的增强者的命运，并确定这些元素是否被招募到新的职能中。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Although the development of arms and legs is a key evolutionary innovation that allowed animals to move onto land, many species of snakes and lizards subsequently lost these limbs. Indeed, limb loss has evolved at least 24 times among lizards. How limb loss has occurred repeatedly in lizard lineages is a question that can be addressed with the replicated natural experiments that will be the focus of this study. This project will reveal whether the same or different genetic changes are associated with limb loss among these different lizard groups. This research will combine technologies from different fields of biology in novel ways to explore this extraordinary example of repeated evolution and gain new insight into genome function. This project will greatly improve knowledge of the genes and genetic control elements involved in limb development. Additionally, in the course of this project, thousands of genetic regions that are shared by humans and other animals will be characterized. Therefore, project data will deliver insights that can be applied to better understand other functional elements within the genomes of humans and different animals. The methods developed will pave the way for other researchers who wish to leverage the power of natural evolutionary experiments to understand genome function. Finally, materials for educators will be developed for use in teaching students how information is encoded in genomes and how natural evolutionary experiments can be used to reveal how genetic sequences yield unique biological diversity.This project will use chromatin-immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify regulatory elements (enhancers) in squamate genomes that control the embryonic development of the limbs. This information will be used to design squamate-specific DNA sequence capture baits targeting these regulatory regions. Additional capture baits will be designed to target the coding regions of genes active in the developing limbs. Together, these baits will be used to survey both noncoding regulatory elements and coding exons of key developmental genes from every lineage of limb-reduced squamates. DNA sequence changes in limb enhancers and limb development genes will be compared in limb-reduced species and their fully limbed relatives, and functional changes in regulatory activity will be assessed in vivo with transgenic mouse reporter assays. This will provide insight into the relative frequency of regulatory changes compared to gene coding sequence changes contributing to the evolution of limb loss. Finally, the functional evolution of an ancient amniote limb-genital enhancer for the gene Tbx4 will be dissected molecularly by taking advantage of sequence variation across the multiple lineages of limb-reduced species. This will grant insight into the fate of enhancers that have lost their regulatory roles and determine whether these elements are recruited to new functions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.