It has become clear that modifications in the interactions among developmental regulatory genes are an important source of phenotypic change during the course of evolution. Recruitment is an evolutionary process where an existing regulatory gene acquires a new developmental role. One of the first cases of recruitment to be recognized was the acquisition of a pair-rule patterning role for even-skipped during insect evolution. Many other probable cases have since been identified from various phyla, suggesting that recruitment is a relatively common and important evolutionary process -- yet almost nothing is known about the mechanistic basis for recruitment. This proposal focuses on three putative cases of recruitment in echinoderms. One involves the expression of distal-less in the imaginal rudiment of echinoids, before and during ectodermal invagination. The other two are apparently independent cases, in holothuroids and echinoids, where orthodenticle expression has become tightly correlated with shifts in the cilated bands of larvae. The proposed research examines the genetic, developmental, and evolutionary bases for these three cases of recruitment. The first goal is to define the phylogenetic context. This involves analyzing gene expression in several species, including ones with morphologies that differ in ways that are likely to be informative. Comparative expression data are used to identify when during the diversification of echinoderms the new role appeared. These data are also used to identify species that closely bracket the transition for experimental characterizations. The second goal is to examine the altered developmental role experimentally. This involves analyzing the phenotypic consequences of various manipulations of gene expression: misexpression, overexpression, and blocked expression. These experiments are done with species that have the new developmental role as well as those that lack it. Phenotypes resulting from these experiments will provide functional information about the new role. The third goal is to seek the genetic basis for each new role. This involves characterizing the genetic basis for the expression domain associated with the new developmental role. Transient expression assays are used to investigate this, involving promoter/reporter constructs that are swapped between species. These experiments will reveal whether the new expression domain is due to changes in the sequence of the 5' flanking region (cis) or in the array of transcription factors present in the nucleus (trans). These studies are expected to provide important insights into the evolution of gene regulatory systems.
