Fox (forkhead/winged helix) family genes in the cephalochordate amphioxus

The cephalochordate amphioxus is the closest living invertebrate relative of the vertebrates, and is the key to understanding the evolution of developmental mechanisms during the invertebrate-to-vertebrate transition. My research focused on the amphioxus Fox genes. The Fox genes encode a family of evolutionary conserved transcription factors which are characterized by an approximately 100 amino acid DNA binding domain. I analyzed the molecular evolution and developmental expression of three novel amphioxus Fox genes, namely the AmphiFoxD, AmphiFoxE4 and AmphiFoxQ2. Phylogenetic analysis indicates that both FoxD and FoxE subfamilies have undergone multiple rounds of gene duplication in the vertebrate lineage. The analysis of AmphiFoxD shows that its major expression domains are in somites, notochord, hindgut, and the cerebral vesicle. Each of the five vertebrate FoxD paralogous genes is expressed in one or more of these domains. However, one of the vertebrate duplicates, FoxD3, has also evolved a new expression domain in neural crest. This suggests that after gene duplication, the evolution of new regulatory elements could have facilitated the co-option of this gene for neural crest development during early vertebrate evolution. To test this hypothesis, I began identifying the tissue specific enhancers of AmphiFoxD. Using microinjection and examining reporter construct expression patterns, I have identified the cis-regulatory DNA regions that are essential for tissue specific expression of AmphiFoxD. The comparison of expression patterns of AmphiFoxE4 and vertebrate FoxE genes suggests that a FoxE4-like ancestral gene might have functioned in pharyngeal development. Early in vertebrate evolution, this gene was co-opted for an additional function of lens formation. Later in vertebrate evolution, FoxE4 duplicated into FoxE1 and FoxE3, and its two functions were subdivided between paralogous genes: pharyngeal differentiation to FoxE1 and lens formation for FoxE3. Phylogenetic analysis of AmphiFoxQ2 and its related sequences indicates that they form a separate group from the known FoxQ1 genes. During development, AmphiFoxQ2 is exclusively expressed in the anterior end of the embryo, suggesting that it might play an important role in establishing and/or maintaining the anterior-posterior axis in amphioxus embryos.