The function of Gli proteins in the specification of ossification pathways in pectoral girdle development and evolution

在进化生物学中，脊椎动物是如何从水中过渡到陆地上的，这是一个长期存在且备受争议的问题。这种转变需要骨骼的组成和结构发生巨大的变化，以便能够在陆地上进食、呼吸和运动。尽管骨骼变化对脊椎动物的进化至关重要，但化石记录的稀疏使得其进化轨迹和潜在机制在很大程度上未知。本项目研究鱼类肩带骨向四足动物骨骼转变的遗传机制。我们研究的综合性质非常适合跨学科（比较解剖学、遗传学和基因组学）的培训，以创造一个动态的环境，让学生通过实践研究活动和小组讨论学习。为了将多学科科学融入本科课程，PI于2018年与美国自然历史博物馆（AMNH）建立了一个新的教育计划，探索鱼类向四足动物的转变。参加本课程的学生将参加罗格斯大学和AMNH的小组讨论，学习如何连接不同的学科，并解决与鱼类到四足动物过渡相关的问题。总的来说，这一综合研究和教育计划将相互促进对鱼类进化的深刻理解，培养本科生和研究生，并向外行人提供“缺失环节”之谜的答案，这一谜团吸引了我们社会的大量兴趣。该项目的主要目标是确定脊椎动物中膜内和软骨内两种不同类型的骨骼的具体机制，重点是胸（肩）带。对胸带的关注是基于一种公认的认识，即在向四足动物进化的过程中，鱼类两种骨骼比例的变化使胸带与头骨分离，从而为陆地运动和生活方式铺平了道路。有趣的是，先前的研究表明，glii家族基因之一gli3调节胸带发育过程中膜内和软骨内成骨的平衡。先前的工作还确定Gli3调节参与成骨细胞和软骨细胞分化的基因。目的1将确定Gli水平在膜内和软骨内成骨中的作用，并表征Gli家族基因敲除鱼的胚胎和成体表型。目的2将验证Gli的量调节参与成骨细胞和软骨细胞分化的一系列基因的假设。目的3将利用转基因鱼测试Gli靶基因在骨化途径规范中的功能。这些目标将揭示脊椎动物胸带中两种不同类型的骨骼是如何被指定的，并表明这种信号通路的变化可能是鱼到四足动物胸带进化的基础。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。

One of the long-standing, and extensively debated, questions in evolutionary biology is how vertebrates transitioned from water to land. The transition required dramatic changes to the composition and architecture of the skeleton to enable terrestrial feeding, respiration, and locomotion. Despite the critical importance of the skeletal changes for vertebrate evolution, the sparse fossil record has rendered its evolutionary trajectory and the underlying mechanisms largely unknown. This project investigates the genetic mechanisms of the skeletal transition of shoulder girdle bones from fish to tetrapods. The integrative nature of our research is well-suited for a training of bridging multiple disciplines (comparative anatomy, genetics, and genomics) to create a dynamic environment where students learn through hands-on research activities and group discussion. Towards the goal of embedding multidisciplinary science into undergraduate curriculum, the PI established a new educational program that explores fish-to-tetrapod transition with the American Museum of Natural History (AMNH) in 2018. Students that take this course will participate in group discussions at Rutgers U and AMNH, learn how to bridge different disciplines, and address questions related to the fish-to-tetrapod transition. Collectively, this integrated research and educational program will reciprocally facilitate the deep understanding of fish evolution, foster undergraduates and graduate students, and inform lay people with the answers to the mysteries of the “missing link” which attracts large amount of interest in our society. The main goal of this project is to identify the specification mechanisms of the two distinct types of bones found in vertebrates, intramembranous and endochondral, focusing on the pectoral (shoulder) girdle. Focus on the pectoral girdle is based on the established appreciation that during the evolution into tetrapods changes in the ratios of two bone types in fish disconnected the pectoral girdle from the skull, thus paving the way to the terrestrial locomotion and way of life. Intriguingly, previous studies showed that one of the gli family gene gli3 regulates a balance of intramembranous and endochondral ossification in pectoral girdle development. Prior work also determined that Gli3 regulates genes involved in osteoblast and chondrocyte differentiation. Objective 1 will identify the roles of the Gli level in the specification of intramembranous and endochondral ossification, and characterize the embryonic and adult phenotypes of gli family gene knockout fish. Objective 2 will test the hypothesis that the amount of Gli regulates the battery of genes involved in osteoblast and chondrocyte differentiation. Objective 3 will test the function of Gli target genes in the specification of ossification pathways using transgenic fish. These objectives will reveal how two distinct types of bones are specified in the vertebrate pectoral girdle and suggest that changes in this signaling pathway could underly the pectoral girdle evolution from fish to tetrapods.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.