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

进化生物学中一个长期存在且争论广泛的问题是，脊椎动物是如何从水过渡到陆地的。这种转变需要对骨骼的组成和结构进行戏剧性的改变，以使陆地上的摄食、呼吸和运动成为可能。尽管骨骼变化对脊椎动物进化至关重要，但稀少的化石记录使其进化轨迹和潜在机制在很大程度上不得而知。这个项目研究了肩带骨骼从鱼类到四足动物的骨骼转变的遗传机制。我们研究的综合性非常适合连接多个学科(比较解剖学、遗传学和基因组学)的培训，以创造一个学生通过动手研究活动和小组讨论学习的动态环境。为了将多学科科学纳入本科课程，PI于2018年与美国自然历史博物馆(AMNH)建立了一个新的教育项目，探索从鱼到四足动物的转变。学习这门课程的学生将参加罗格斯大学和AMNH的小组讨论，学习如何在不同的学科之间架起桥梁，并解决与鱼类到四足动物转变相关的问题。总体而言，这一综合研究和教育计划将相互促进对鱼类进化的深入了解，培养本科生和研究生，并向外行人提供引起社会广泛兴趣的“缺失环节”的谜团。该项目的主要目标是确定脊椎动物中发现的两种不同类型骨骼的规格机制，即膜内骨骼和软骨内骨骼，重点是胸(肩)带。对胸带的关注是基于一种既定的认识，即在鱼类进化为四足动物的过程中，两种骨骼类型的比例发生变化，使胸带与头骨断开，从而为陆地运动和生活方式铺平了道路。有趣的是，以前的研究表明，在胸带发育过程中，Gli3家族基因之一调节着膜内和软骨内成骨的平衡。先前的工作也确定了Gli3调节与成骨细胞和软骨细胞分化有关的基因。目的1鉴定Gli基因敲除鱼的膜内和软骨内成骨过程中Gli水平的作用，并对Gli家族基因敲除鱼类的胚胎和成体表型进行分析。目的2验证Gli含量调控成骨细胞和软骨细胞分化相关基因的假说。目的3利用转基因FISH验证Gli靶基因在骨化途径调控中的作用。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，并表明这一信号通路的变化可能是胸带从鱼类进化到四足动物的基础。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。

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.