Oxygen, pH, and oral-aboral axis specification in the sea urchin embryo

Abstract

The oral-aboral axis of the sea urchin embryo is specified via the redox-regulated activation of nodal (Coffman et al., 2009, and references therein). Embryos cultured at high density under cover glass develop a radialized phenotype owing to inhibition of nodal expression (Coffman et al. 2004), an effect that we attributed to hypoxia. Recently Agca et al. (2009) reported that controlled hypoxia does inhibit nodal expression as well as ectoderm differentiation, but only when it is maintained until pluteus stage. However, we found that embryos developed under cover glass become radialized even when the cover glass is removed at early blastula stage (Supplemental Figure 1). We therefore asked whether other factors might contribute to this teratogenic effect. Since embryos cultured under otherwise identical conditions develop normally, it is clear that the relevant effect of cover glass is to limit gas exchange, which in addition to hypoxia will promote acidification by preventing escape of CO2. To determine which of these factors causes radialized development, embryos were developed to hatching blastula stage under normoxic (∼10 ppm O2) or hypoxic (≤1 ppm O2) conditions in culture medium buffered to pH 8, 7.5, 7, or 6.5. While acidic pH was mildly radializing, hypoxia alone produced significant radialization, which was enhanced by acidification (Fig. 1 A, Supplemental Table 1 and Supplemental Fig. 2). Since the embryos were released from hypoxia at blastula stage, these results differ from those of Agca et al. (2009), for reasons that remain unclear. One difference that may be relevant is that we determined the level of hypoxia by measuring dissolved oxygen, whereas Agca et al. (2009) monitored atmospheric oxygen. In addition, we scored morphology at prism stage, whereas Agca et al. did so at pluteus stage, which may have allowed for recovery by regulative development. Finally, Agca et al. did not quantify the phenotypes they reported. Figure 1 Hypoxia induces radialized development and reduces mitochondrial H2O2 levels in sea urchin embryos. (A) Differential effects of controlled hypoxia and pH on development of oral-aboral polarity. Embryos were developed under the indicated conditions (see ... Ratiometric imaging was used to measure the effect of limiting gas exchange on intracellular H2O2 and pH (Fig. 1 B, Supplemental Fig. 3). Both mitochondrial and cytoplasmic H2O2 were reduced by this treatment, with a decrease in mitochondrial H2O2 comparable in magnitude to that produced by a mitochondrially-targeted catalase (Mt-Cat) shown previously to both inhibit nodal activation and entrain oral-aboral polarity (Coffman et al. 2009) (Fig. 1B). This decrease in H2O2, a major reactive oxygen species (ROS), contradicts the untested assumption of Agca et al. (2009) that hypoxia elevates ROS. As would be expected, limiting gas exchange also lowers intracellular pH, but less so than the decrease produced by pH buffering that did not induce radialization (Supplemental Fig. 3), suggesting that the relevant pH effects are extracellular. We conclude that hypoxia is the major cause of the radialization caused by development under cover glass. This is probably attributable at least in part to decreased H2O2 levels, as the window of vulnerability corresponds to the initial H2O2-dependent activation phase of nodal expression (Supplemental Figure 1; Coffman et al., 2009). Extracellular acidification appears to play an ancillary role, perhaps weakening ligand-receptor interactions and thus by dampening the feedback amplification of nodal expression.