Oral-aboral axis specification in the sera urchin embryo I. Axis entrainment by respiratory asymmetry

In embryos of indirectly developing echinoids, the secondary (oral-aboral) larval axis is established after fertilization by an as yet undiscovered pr ocess. One of the earliest manifestations of this axis is an asymmetry in m itochondrial respiration, with the prospective oral side of the embryo exhi biting a higher rate of respiration than the prospective aboral side. We sh ow here that respiratory asymmetry can be experimentally induced within emb ryos by immobilizing them in tight clusters of four ("rosettes"). Within su ch clusters a redox gradient is established from the inside to the outside of the rosette. Vital staining of clustered embryos demonstrates that the s ide of the embryo facing the outside of the rosette (i.e., the most oxidizi ng) tends to become the oral side, while the side facing the inside tends t o become the aboral side. Effective entrainment of the oral-aboral axis req uires that the embryos remain immobilized in rosettes until the hatching bl astula stage. To begin to investigate the molecular mechanisms underlying t his 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 egg s, it activates embryonic expression of a green fluorescent protein reporte r gene containing a basal promoter and a single strong P3A2 target site. In embryo rosettes, such activation occurs predominantly on the outside of th e rosette, suggesting that the activity of the P3A2 protein is spatially re gulated by the respiratory asymmetry established by clustering the embryos. These findings are discussed with reference to earlier work on both oral-a boral axis specification and P3A2 and used to develop a testable model of t he mechanism of oral-aboral axis specification in the sea urchin embryo. (C ) 2001 Academic Press.