Efflux Transporters in Sea Urchin Development

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Project Summary for IBN-0446384Efflux Transporters in Sea Urchin DevelopmentEfflux transporters in embryos can have both housekeeping and developmental roles. The housekeeping role can protect the embryo from external toxicants during embryogenesis. The developmental role can pump endogenous signaling molecules out of cells to influence adjacent cells or to feedback on the cell itself in an autocrine fashion. Preliminary work reveals members of the mrp family of ABC efflux transporters are dormant in the unfertilized sea urchin egg and become active as part of the response to fertilization. The increased activity appears to result from movement of vesicles containing these transporters to the egg surface after fertilization and insertion of the proteins into the membrane. Proposed experiments will prove or disprove the vesicle movement hypothesis using antibodies to follow any changes in the location of the transport proteins.Preliminary results suggest several developmental roles. One is a role in forwarding cell division during the cleavage period. The proposed research will examine specificity of the effect by injection of antibodies to the active site of the transporter. If indicated as a specific effect a search will be made for the active substance effluxed by the transporter. A plausible endogenous substrate are leukotrienes and levels of these compounds will be measured during cleavage and later development. The other transporter function is a role in signaling in primary mesenchyme cell differentiation, perhaps in the cross-talk between ectoderm and underlying mesenchyme cells. The proposed studies will describe the expression of new transporters during development, localize them by in situ hybridization and assess their role using morpholinos to specifically prevent new transport expression. This work also broadens the view of efflux transporters, which has in the past been primarily centered on their medical relevance in terms of evading drug therapy during treatment against cancer. The proposed research looks at these ubiquitous molecules in new ways; in terms of a fertilization response leading to re-organization of the plasma membrane, in terms of a role in normal cell functions such as mitosis and in terms of a signaling role for integrating morphogenetic responses. The proposed research also has broad impact on education, as many of these experimental results can be integrated into laboratory exercises used in introductory biology labs at Stanford and in high school and college exercises across the country. To facilitate this, exercises suggested by this research will be disseminated on the widely used (last year received 4 million hits) Sea Urchin Embryology web site (http://www.stanford.edu/group/Urchin/ ).This combination of environmental work with cell and developmental biology also attracts numerous undergraduate students to the P.I's lab (often from under-represented groups); these students often receive University recognition for outstanding undergraduate research and many of these students later go on to graduate study in developmental biology and environmental sciences