Testing Models of Jaw Joint Evolution Using the Sea Lamprey, Xenopus laevis, and zebrafish

有颌脊椎动物是5亿年前从无颌鱼类进化而来的，从那以后，它们成为了地球上最成功的动物群体之一。这个项目旨在了解有颌脊椎动物是如何从无颌鱼类进化而来的。这将通过比较现代无颌鱼类——七鳃鳗——与两种有颌脊椎动物——非洲爪蛙和斑马鱼的发展来完成。为了做到这一点，操纵基因功能的现代方法将被用来改变参与头部骨骼发育的基因的活性。这些操作的目的是部分概括进化过程，并确定哪些基因对颌骨进化最重要。这项工作将揭示一个重大的进化事件，它使现在生活在地球上的大多数脊椎动物（包括人类）的进化成为可能。该项目还将有助于阐明所有脊椎动物的头骨和下颚是如何发育的，并有助于了解影响面部、头骨和下颚的疾病的发育和遗传基础。此外，该项目将为一名博士后学者、两名研究生和几名本科生和高中生提供现代基因组工程方法的培训。它还将支持一个关于动物发育和进化的巡回博物馆展览，并由首席研究员进行公开讲座。早期脊椎动物化石和基部分化的现代脊椎动物的形态学表明，颌骨的进化涉及头部骨骼结构的两个主要变化；形态学上不同的背侧和腹侧骨骼元素的进化，以及这些元素之间的柔性关节组织的定位。这些变化发生的机制尚不清楚。在颌口动物中，头部骨骼前体在发育早期通过Jagged-Notch、BMP和内皮素（Edn）信号通路的相互作用分离为背侧、腹侧和中间结构域。这些信号调节Dlx、Msx和Hand转录因子的组合表达，进而激活形成骨骼元件和指定关节的形态发生和分化程序。为了更好地了解噬口鱼头部骨骼进化背后的发育变化，本研究将利用活的七鳃鳗作为研究对象。七鳃鳗是现存的唯一一种可以进行常规胚胎操作的无颌脊椎动物。初步的基因表达研究表明，七鳃鳗在头部骨骼前体中缺乏Dlx同源物的背-腹侧限制性表达。随后的研究意外地揭示了在七鳃鳗头部形成过程中Edn信号组分、Dlx、Msx和Hand的颚口样表达。此外，研究还发现关节组织指示物的七鳃鳗同源物Gdf5在粘液软骨中表达，粘液软骨是一种柔软的间充质骨骼组织，在组织学上类似于颌口关节组织。这些数据支持这样一个模型：1)一个复杂的颌口类头部骨骼模式系统早于颌骨的进化；2)关节的进化是由在这个保守的前模式中，用于软骨组织分化的古老基因程序的重新定位所驱动的。这项工作将利用最近优化的CRISPR/Cas9诱变七鳃鳗，在海洋七鳃鳗Petromyzon marinus和青蛙爪蟾（Xenopus laevis）中验证该模型的关键预测。

Jawed vertebrates evolved from jawless fish a half-billion years ago and have since become one of the most successful animal groups on the planet. This project aims to understand how jawed vertebrates evolved from jawless fish. This will be done by comparing the development of the modern jawless fish, lamprey, with that of two jawed vertebrates, the african clawed frog, and the zebrafish. To do this, modern methods for manipulating gene function will be used to alter the activity of genes involved in head skeleton development. These manipulations are aimed at partially recapitulating the evolutionary process and pinpointing which genes were most important for jaw evolution. The work will shed light on a major evolutionary event that made possible the evolution of the majority of vertebrates now living on the planet, including humans. The project will also help elucidate how the head skeleton and jaw develops in all vertebrates and contribute to efforts to understand the developmental and genetic basis of diseases effecting the face, skull, and jaw. Additionally, the project will provide training in modern genome engineering methods for a postdoctoral scholar, two graduate students, and several undergraduate and high school students. It will also support the maintenance of a traveling museum exhibit about animal development and evolution, and accompanying public lectures by the lead investigator.The morphology of early vertebrate fossils, and basally-diverging modern vertebrates, suggests that jaw evolution involved two major changes in head skeleton structure; the evolution of morphologically distinct dorsal and ventral skeletal elements, and the positioning of flexible joint tissue between these elements. How, mechanistically, these changes occurred, is unclear. In gnathostomes, head skeleton precursors are segregated early in development into dorsal, ventral, and intermediate domains by interactions between Jagged-Notch, BMP, and Endothelin (Edn) signaling pathways. These signals regulate the combinatorial expression of Dlx, Msx, and Hand transcription factors which, in turn, activate the morphogenetic and differentiation programs that shape skeletal elements and specify joints. To better understand the developmental changes underlying gnathostome head skeleton evolution, this research will utilize the living agnathan, lamprey. Lamprey is the only extant jawless vertebrate amenable to routine embryonic manipulations. Initial gene expression studies suggested that lamprey lacks dorso-ventral restricted expression of Dlx paralogs in head skeleton precursors. Subsequent work revealed unexpectedly gnathostome-like expression of Edn signaling components, Dlx, Msx, and Hand in the forming lamprey head. Furthermore, it was found that a lamprey homolog of the joint tissue specifier, Gdf5, is expressed in mucocartilage, a soft, mesenchymal skeletal tissue histologically similar to gnathostome joint tissue. These data supported a model in which; 1) a sophisticated gnathostome-type head skeleton patterning system predates the evolution of jaws and 2) the evolution of joints was driven by the repositioning of ancient gene programs for soft skeletal tissue differentiation within this conserved pre-pattern. The proposed work will take advantage of recent optimization of CRISPR/Cas9 mutagenesis in lamprey to test key predictions of this model in the sea lamprey Petromyzon marinus, and the frog Xenopus laevis.