This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Coffman Lab has used its INBRE support (startup funds awarded to James Coffman, PI) to carry out research on the mechanism underlying developmental specification of the oral- aboral (OA) axis of the sea urchin larva. Axis specification is the initial symmetry breaking event in development that sets up the spatial coordinates for the body plan of an organism. Based on previous work, we have hypothesized that the OA axis of the sea urchin larva is initially specified by a redox gradient established by asymmetric distribution of mitochondria in the egg and early embryo, with the side of the embryo inheriting the highest density of mitochondria being pre-disposed to activate expression of the TGF-beta signaling molecule Nodal and thence develop as oral ectoderm. We further hypothesize that the specification of OA polarity involves mitochondrial H2O2-mediated inactivation of a phosphatase that otherwise inactivates p38-MAPK, activity of which is required for Nodal expression. During this funding period we have developed tools to test these hypotheses, and have used them to generate preliminary data for a pending NIH/NIEHS grant application. These tools include morpholino antisense oligonucleotides targeted to a redox-regulated protein phosphatase, PP2A, which our preliminary data suggest lies upstream of p38-MAPK and Nodal expression; a mitochondrially targeted GFP, which we are using to track the distribution of mitochondria in living embryos; and a mitochondrially targeted catalase, an enzyme that depletes H2O2 which we are using to test the hypothesis that mitochondrial H2O2 is a signaling intermediate required for p38 activation and consequent specification of oral ectoderm. These reagents will also be used to test the hypothesis that the well known sensitivity of OA axis specification to environmental toxicants such as heavy metals is attributable to alterations in redox state and mitochondrial oxidant production, which is the focus of our pending NIEHS grant application.