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

RUI：脊椎动物配对附肢基因调控网络的进化起源

• 批准号: 1853949
• 负责人: Marcus Davis
• 金额: $ 31.64万
• 依托单位: James Madison University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1853949&HistoricalAwards=false
• 关键词: RUI; Evolutionary; origins; vertebrate; paired

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.

鱼类的鳍和陆生脊椎动物的四肢在解剖学上是不同的：鳍在一系列射线中“封顶”，而四肢在自足（手/脚）中终止。这些差异支持了鳍鱼和自足动物是由截然不同的基因和基因“回路”形成的观点。如果这是真的，对鳍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中，包括对人类的研究。然而，该项目的初步数据表明，相同的基因回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/肢体发育程序，首先表征两种鱼类发育鳍中关键基因的活性，这两种鱼类非常适合解决这些问题，它们是美洲白鲟和小斑点猫鲨。这些结果将指导测试基因功能的实验，并将其与发育肢体的已知数据进行比较。结果将表明，一个共同的遗传程序调节鳍和肢体的形成，并证明鱼鳍是研究肢体的有力研究工具。该项目成功的部分原因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。在进化生物学中，鳍向肢过渡的发育基础仍然是一个长期存在的问题。在目前的模型中，鳍由不同的近端和远端发育模块构成，分别产生含有软骨内或真皮成分的成人骨骼隔室。强调骨骼类型导致假设鳍折叠和自足不是同源的，由不同的模块图案，尽管相似的远端位置。然而，新的发现提出了一个有趣的替代假设：自足类动物与皮肤鳍具有深层的调节同源性。该项目将建立远端配对附属物基因调控网络（GRN）的进化起源模型，扩展在两种系统发育良好的脊椎动物，美洲白鲟（Polyodon spathula）和小斑点猫鲨（Scyliorhinus canicula）中的发现。为了测试这一模型，RNA-seq将用于在白鲟的鳍室中建立一个全面的转录图谱。这些数据集将与现有的转录组资源进行比较，以确定候选的远端附属物GRN基因，这些基因将通过白鲟的药理学扰动试验和猫鲨的互补基因表达试验进行测试。CRISPR-Cas9介导的基因敲低将测试白鲟鳍中HoxA基因的功能。总之，本项目的结果将阐明远端附属物GRN的关键成分，催化调节早期骨骼发生的新研究方向，并重新激发对使用鳍研究肢体出生缺陷和再生研究的兴趣。拟议的工作将培养多样化的研究生和本科生群体，包括通过少数民族参与项目招收的学生。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项目，在亚特兰大地区的公立高中开展推广项目。

项目成果

Deep evolutionary origin of limb and fin regeneration

• DOI: 10.1073/pnas.1900475116
• 发表时间: 2019-07-23
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Darnet, Sylvain;Dragalzew, Aline C.;Schneider, Igor
• 通讯作者: Schneider, Igor