RUI: Evolutionary origins of the vertebrate paired appendage gene regulatory network

鱼类的鳍和陆生脊椎动物的四肢在解剖学上是不同的：鳍被一系列的射线“覆盖”，而四肢则终止于一只独足类动物（手/脚）。这些差异支持了鳍条和直足类是由明显独立的基因和遗传“回路”形成的观点。 如果这是真的，鳍条生长和再生的研究可能不能直接转移到肢体出生缺陷和再生研究，包括人类。 然而，该项目的初步数据显示，相同的遗传电路确实形成了鳍和四肢的骨骼。 该项目将确认这一共同的鳍/肢建设计划，首先表征两种鱼的鳍发育中的关键基因的活动，非常适合解决这些问题，美国白鲟和小斑点猫鲨。 这些结果将指导实验来测试基因功能，并与已知的肢体发育数据进行比较。 结果将表明，一个共同的遗传程序调节鳍和四肢的形成，并证明鱼鳍是研究四肢的有力工具。 这个项目的成功部分将是对新研究人员的培训-一个多样化的研究生和本科生群体将把他们的才能带到这个项目中，并为亚特兰大地区公立学校系统的公共推广经验做出贡献。鳍到肢体过渡的发展基础仍然是进化生物学中一个长期存在的问题。 在目前的模型中，鳍的图案由不同的近端和远端的发展模块，生成成人骨骼隔间分别含有endochondrium或真皮元素。 强调骨骼类型导致的假设，鳍折和autopods是不同源的，模式由不同的模块，尽管类似的远端位置。 然而，新的发现提出了一个有趣的替代假设：即autopods共享一个深的监管同源性与真皮鳍射线。 该项目将建立一个远端成对附属物基因调控网络（GRN）的进化起源模型，扩大在两种遗传定位良好的脊椎动物，美国白鲟（Polyodon spathula）和小斑点猫鲨（Scyliorhinus canicula）中的发现。为了测试这个模型，RNA-seq将用于在白鲟的鳍区室中构建一个全面的转录图谱。将这些数据集与现有的转录组学资源进行比较，以确定候选的远端附肢GRN基因，这些基因将通过白鲟中的药理学扰动测定和猫鲨中的互补基因表达测定进行测试。 CRISPR-Cas9介导的基因敲除将测试白鲟鳍中的HoxA基因功能。 总之，该项目的成果将阐明远端附肢GRN的关键组成部分，催化调节早期骨骼发生的新研究方向，并重新激发人们对使用鳍研究肢体出生缺陷和再生研究的兴趣。拟议的工作将培训一批多样化的研究生和本科生，包括通过少数民族参与计划招募的学生。PI和学生将通过虚拟访问PI的实验室，课堂参观和暑期实习计划，在亚特兰大地区的公立高中开展外展计划。

The fins of fishes and the limbs of terrestrial (land living) vertebrates are anatomically distinct: Fins are 'capped' in a series of rays, while limbs terminate in an autopod (hands/feet). These differences support the view that fin rays and autopods are patterned by distinctly separate genes and genetic 'circuits'. If this were true, studies of fin ray growth and regeneration may not be directly transferable to studies of limb birth defects and regeneration research, including in humans. However, preliminary data from this project shows that the same genetic circuits do form the skeletons of both fins and limbs. This project will confirm this common fin/limb building program, first characterizing the activity of key genes in the developing fins of two fish ideally suited to address these questions, the American paddlefish and the small spotted catshark. These results will then guide experiments to test gene function, which will be compared to known data for developing limbs. Outcomes will show that a shared genetic program regulates formation of fins and limbs and demonstrate that fish fins are a powerful research tool for the study of limbs. Part of the success of this program will be the training of new researchers - a diverse group of graduate and undergraduates will bring their talents to this project and contribute to public outreach experiences in the Atlanta area public school system.The developmental basis of the fin-to-limb transition remains a longstanding question in evolutionary biology. In current models, fins are patterned by distinct proximal and distal developmental modules, generating adult skeletal compartments containing endochondral or dermal elements respectively. Emphasis on skeletal type led to the hypothesis that fin-folds and autopods are not homologous, patterned by different modules, despite similar distal positions. However, new findings raise an intriguing alternative hypothesis: that autopods share a deep regulatory homology with dermal fin rays. This project will build a model for the evolutionary origin of the distal paired appendage gene regulatory network (GRN), expanding on discoveries in two phylogenetically well-positioned vertebrates, the American paddlefish (Polyodon spathula) and the small spotted catshark (Scyliorhinus canicula). To test this model, RNA-seq will be used to build a comprehensive transcriptional map in the fin compartments of paddlefish. These datasets will be compared to existing transcriptomic resources to identify candidate distal appendage GRN genes, which will be tested via pharmacological perturbation assays in paddlefish and complementary gene expression assays in catshark. CRISPR-Cas9 mediated gene knockdowns will test HoxA gene function in paddlefish fins. Together, the outcomes of this project will elucidate key components of the distal appendage GRN, catalyze new research directions that regulate early skeletogenesis, and reinvigorate interest in the use of fins for the study of limb birth defects and regeneration studies. The proposed work will train a diverse group of graduate and undergraduate students, including those recruited through minority participation programs. The PI and students will collaborate on outreach programs to Atlanta area public high schools through virtual access to the PI's lab, classroom visits, and summer intern programs.