Intellectual merit To explore the degree to which the developmental architecture of an organism constrains its evolution, the proposed work exploits the multiple "evolutionary experiments" provided by homoplasy, i.e. the evolution of similar features in independent species lineages. In the absence of strong developmental constraints, chances are that homoplastic changes should not occur in the same gene or even the same genetic pathway. On the other hand, if the same mechanism is involved in each independent appearance of a feature, developmental constraint is indicated. Experiments are designed to test whether or not homoplasy exists at a particular level in the developmental hierarchy: i.e., does morphological homoplasy involve the same developmental process, the same genetic pathway, and/or the same part of the pathway? From the phylogenetic studies of rhabditid nematodes related to Caenorhabditis elegans, homoplasy is found in two kinds of structures in the male tail, a well-studied, genetically accessible model for elucidating developmental pathways and genes involved in patterning and morphogenesis. Specifically, during rhabditid evolution, multiple independent changes have occurred (1) between anterior and posterior positions of the chemoreceptive sensilla called phasmids, relative to 3 mechanosensory sensilla called rays, and (2) between the pointed leptoderan and rounded peloderan forms of male tail tips. These characters serve as models for general classes of morphological evolution, such as heterotopy and heterochrony. To assess the degree of homoplasy, the developmental processes involved will first be examined at the level of individual cells (cell lineages, fusions, morphogenesis) to see if development changes the same way during each homoplastic change in morphology. Secondly, at least one candidate genetic pathway will be tested to see if it is conserved in controlling the homoplastic morphological feature. If it is, then expression patterns of indicator genes will be analyzed to determine if the same part of the pathway (i.e., upstream or downstream of the tested gene) changed each time. (Determining the exact causal molecular or genetic change is not required simply to address at what level homoplasy occurred; only a "read-out" of a couple points in the pathway is needed to test where expression is conserved or where it differs between species.) Preliminary results suggest that the evolution of phasmid position involves switches in the polarity of the asymmetric division of a single blast cell (T), providing a novel mechanism for heterotopy (evolutionary change in relative positioning). The Wnt signaling pathway is a good candidate for being involved, as it controls this polarity in C. elegans. Preliminary data also suggest a potential role for the micro-RNA-controlled heterochronic pathway in the evolution of tail tip morphology. Although many evo-devo studies focus on transcriptional regulation, male tail tip evolution may provide a novel model for posttranscriptional regulation in developmental evolution. This model is also likely to provide a deeper mechanistic understanding into how heterochrony (evolutionary change in relative timing) can result in morphological variation. Regardless of whether or not homoplasy exists at any particular mechanistic level in the evolution of these two features, it will be important eventually (beyond the current proposal) to determine what kinds of genes and molecular changes underlie the evolutionary developmental changes. Thus, whatever is discovered, the proposed studies will provide a foundation for future work to understand many aspects of evolutionary developmental mechanisms. Broader impact A major resource used by other scientists and educators for comparative biology, the NYU Rhabditid Collection, will be maintained in the course of this research without additional cost. More efficient methods for gene knockdown will be developed to empower nematode systems for other studies by other investigators in comparative functional genomics and evolutionary development. New reagents and antibodies will be developed. Results of the work will be published in well-regarded journals. Diverse undergraduates will be recruited and individually mentored in new research. A PhD student and a postdoctoral associate will receive cross-disciplinary training in evolution, development, and molecular genetics.
