The long term goal of this research on the basal chordate amphioxus, which diverged from the vertebrate lineage over 600 million years ago, is to elucidate the evolutionary origins of the patterning mechanisms that shape the vertebrate embryo. Amphioxus is vertebrate-like, but, unlike vertebrates, has neither duplicated nor lost many genes. Even so, many likely gene-regulatory sequences (enhancers) are conserved between the amphioxus and human genomes. Evolutionary changes in gene regulation are thought to be one way of effecting large-scale changes in animal body plans. The present research focuses on the 'engrailed' gene. In amphioxus, engrailed expression suggests functions in both specification of neuronal identity and in segmentation of the muscular somites. Engrailed expression in the amphioxus somites, lamprey velar muscles and some vertebrate jaw muscles led to the hypothesis that vertebrate jaw muscles evolved from the anterior muscular somites of an amphioxus-like jawless ancestor. Previous NSF-supported research revealed a 1.2 kb muscle enhancer from amphioxus engrailed that is highly conserved with the mouse engrailed-2 (en-2) jaw muscle enhancer. The present research has three specific aims: (1) to fully characterize the regulatory elements responsible for directing expression of amphioxus engrailed to muscle in amphioxus; (2) to determine the extent of evolutionary conservation of engrailed regulation in chordate muscle; and (3) to investigate evolutionary conservation of the gene network involving engrailed in segmentation of the muscles in amphioxus. The results should permit insights not only into the evolution of the engrailed gene but also into the evolution of gene regulation in general. In addition, it addresses the question of how gene function changes as new anatomical structures evolve. In terms of Broader Impacts the work is important in the general concept of how genome evolution leads to evolution of developmental mechanisms at the invertebrate to vertebrate transition. The results will be published in peer-reviewed journals and presented at scientific symposia. This research will provide training for one postdoctoral fellow, and will train undergraduates in hands-on research, who will be co-authors on research papers. Outreach to students, particularly those from Latin America, will be continued.
