In embryos of indirectly developing echinoids, the secondary (oral-aboral) larval axis is established after fertilization by an as yet undiscovered process. One of the earliest manifestations of this axis is an asymmetry in mitochondrial respiration, with the prospective oral side of the embryo exhibiting a higher rate of respiration than the prospective aboral side. We show here that respiratory asymmetry can be experimentally induced within embryos by immobilizing them in tight clusters of four ("rosettes"). Within such clusters a redox gradient is established from the inside to the outside of the rosette. Vital staining of clustered embryos demonstrates that the side of the embryo facing the outside of the rosette (i.e., the most oxidizing) tends to become the oral side, while the side facing the inside tends to become the aboral side. Effective entrainment of the oral-aboral axis requires that the embryos remain immobilized in rosettes until the hatching blastula stage. To begin to investigate the molecular mechanisms underlying this effect we made use of P3A2, a transcriptional regulatory protein whose activity is spatially modulated along the oral-aboral axis. When synthetic mRNA encoding P3A2 fused to the VP16 activation domain is injected into eggs, it activates embryonic expression of a green fluorescent protein reporter gene containing a basal promoter and a single strong P3A2 target site. In embryo rosettes, such activation occurs predominantly on the outside of the rosette, suggesting that the activity of the P3A2 protein is spatially regulated by the respiratory asymmetry established by clustering the embryos. These findings are discussed with reference to earlier work on both oral-aboral axis specification and P3A2 and used to develop a testable model of the mechanism of oral-aboral axis specification in the sea urchin embryo.
Copyright 2001 Academic Press.