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

尽管手臂和腿的发育是使动物能够在陆地上移动的关键进化创新，但许多种类的蛇和蜥蜴后来失去了这些四肢。事实上，在蜥蜴中，肢体丧失至少进化了24次。肢体丧失是如何在蜥蜴谱系中反复发生的，这是一个可以通过重复的自然实验来解决的问题，这将是本研究的重点。这个项目将揭示相同或不同的基因变化是否与这些不同蜥蜴群体的肢体丧失有关。这项研究将以新颖的方式结合来自不同生物学领域的技术，探索这个重复进化的非凡例子，并获得对基因组功能的新见解。该项目将极大地提高对参与肢体发育的基因和遗传控制因素的认识。此外，在这个项目的过程中，人类和其他动物共有的数千个遗传区域将被表征。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其他功能元素的见解。所开发的方法将为其他希望利用自然进化实验的力量来理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，用于教授学生如何在基因组中编码信息，以及如何使用自然进化实验来揭示基因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）来鉴定控制四肢胚胎发育的鳞状动物基因组中的调控元件（增强子）。这些信息将用于设计针对这些调控区域的鳞片特异性DNA序列捕获诱饵。额外的捕获诱饵将被设计为针对在发育肢体中活跃的基因编码区域。总之，这些诱饵将用于调查非编码调控元件和关键发育基因的编码外显子来自四肢减少鳞片的每一个谱系。将比较肢体增强基因和肢体发育基因在肢体减少物种及其完全肢体亲缘物种中的DNA序列变化，并通过转基因小鼠报告基因试验在体内评估调节活性的功能变化。这将有助于深入了解与基因编码序列变化相比，调控变化的相对频率有助于肢体丧失的进化。最后，通过利用肢体减少物种的多个谱系中的序列变异，将对古代羊膜动物肢体生殖增强子Tbx4基因的功能进化进行分子解剖。这将有助于深入了解失去调控作用的增强子的命运，并确定这些元素是否被招募到新的功能中。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.