The long term goal of this research is to elucidate the evolutionary origins of the patterning mechanisms that shape the vertebrate body plan by using amphioxus (Branchiostoma floridae) as a model for the ancestral vertebrate. Amphioxus, the closest living invertebrate relative of the vertebrates, has both a genome and a body plan that are vertebrate-like, but less complex. This relative simplicity uniquely positions amphioxus for comparisons, on the one hand, with animals more basal in the tree of life and, on the other hand, with the vertebrates. The present proposal focuses on the initial mechanisms establishing anterior/posterior (A/P) patterning and regional identities within the amphioxus central nervous system (CNS). These mechanisms include both an evolutionarily ancient mechanism and an evolutionarily more recent one. The first, which was evidently present in the bilaterian ancestor, is specification of posterior identity by Wnt/bets catenin signaling. The second, limited to chordates, is retinoic acid (RA)-signaling that establishes regional identities along the A/P axis. In vertebrates, these two pathways interact. This raises the question of when interactions between the two pathways evolved. The proposed study will test the hypothesis, based on preliminary data, that substantial interaction between these pathways evolved only after the split between amphioxus and the vertebrates. The specific aims are (1) to determine if Wnt/beta-catenin signals from the amphioxus blastopore mediate anterior/posterior patterning of the neuroectoderm (2) to test whether anterior identity of the CNS is maintained by signals mediated, in part, by Wnt-suppressing signals from Hex-expressing anterior endoderm and (3) to identify interactions between Wnt/beta-catenin and RA-signaling pathways. Methods to address the first aim include manipulating Wnt-signaling in amphioxus with lithium, which blocks the downstream component GSK3beta, with the cell-permeable GSK3beta inhibitor Myr-N-GKEAPPAPPQSP-NH2 and with antisense morpholino-oligonucleotides against specific Wnts. For the second aim, Hex will be upregulated by over-expression of Hex mRNA and function blocked with morpholino-oligonucleotides. Amphioxus homologs of Wnt-antagonists will be cloned and, depending on their expression, their function will be tested. For the third specific aim, expression of genes implicated as mediating Wnt-signaling will be determined in amphioxus embryos treated with either RA or an RA-antagonist. Regulatory regions of putative targets of both Wnt- and RA-signaling will be examined for retinoic acid receptor response elements (RAREs) and binding sites for Tcf, which mediates Wnt/beta-catenin signaling. This will be facilitated by the draft sequence of the amphioxus genome, which will be completed by the Joint Genome Institute early in 2005. Selected sites will be mutated and expression of reporter constructs containing them will show if they are essential for normal gene expression. Intellectual merit: Understanding how mechanisms for anterior suppression of Wnt/beta-catenin signals evolved and crosstalk between the Wnt/ beta catenin and RA-signaling pathways evolved will help reconstruct the evolution of developmental mechanisms in early vertebrates. In addition, this research will give insight into the molecular mechanisms for patterning the chordate, including vertebrate embryos. Broader impact: The work is also important in the broader concept of questions of the evolution of structures, evolution of developmental mechanisms and gene duplication. This research will provide training for two graduate students and undergraduates.
