Conservation and Non-Conservation of Engrailed Gene Regulation in Metazoan Evolution: Amphioxus as a Model for the Ancestral Vertebrate

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0236171HollandThe long term goal of this research is to elucidate the evolutionary origins of the patterning mechanisms that led to the vertebrate body plan. This proposal focuses on the evolution of cis-regulatory elements of the engrailed gene in relation to the evolution of body plans using amphioxus (Branchiostoma) as a proxy for the ancestral vertebrate. Amphioxus is vertebrate-like but lacks the extensive gene duplications characteristic of vertebrates. Thus, developmental gene cascades in amphioxus are proving to be similar to those in vertebrates, but less complex and easier to study. Evolutionary changes in developmental gene regulation are one important way to effect large-scale changes in animal body plans. An understanding of the evolution of cis-regulation of the engrailed gene can give insights into the ancestral roles of engrailed in development, in particular, and the evolutionary conservation of the roles of developmental genes versus their co-option for new roles in newly evolved structures, in general. Theories regarding the ancestral role(s) of engrailed include functions in (1) boundary formation, (2) specification of particular nerve cells, (3) specification of subsets of muscle cells and (4) segmentation. The last is particularly controversial. To test these ypotheses, expression of lacZ reporter constructs of cis-regulatory DNA from amphioxus engrailed will be compared in amphioxus, the zebrafish, a tunicate (Ciona), Drosophila and the lamprey. In preliminary work, a construct including 8.5 kb of amphioxus engrailed regulatory DNA, which shares many transcription factor binding sites with the mouse engrailed-2 region that directs expression to jaw muscle precursors, was expressed in the first 4 of these organisms. Initial results suggest that (1) engrailed regulation may be conserved during evolution of the vertebrate jaw muscles from the anterior somites of an amphioxus-like ancestor and (2) a role for engrailed in specification of muscle cell identity and/or properties may be conserved in Drosophila, amphioxus and the vertebrates. In the present research, smaller constructs will be expressed in the 5 organisms to determine if the same constellation of transcription factor binding sites directs expression to muscle and the nerve cord in all organisms. Comparative experiments will be done with the mouse engrailed-2 regulatory regions known to direct expression to the vertebrate jaw muscles. Finally, by overexpressing and blocking expression of amphioxus engrailed in amphioxus embryos, it will be determined if engrailed really does function in segmentation of the amphioxus mesoderm as its expression suggests. The combined results from these experiments will give insights into the evolution of regulation of the engrailed gene in particular, and the evolution of cis-regulation of genes in general. An understanding of the evolution of gene regulation is central to an understanding of how life evolved on earth. This research will continue a history of training undergraduate students, including woman, under-represented minorities and those with physical disabilities, in basic techniques of molecular biology and embryology. It will also provide advanced training for a postdoctoral fellow in experimental embryology and molecular biology. The results will be published in peer-reviewed journals and presented at scientific meetings.