Nitric oxide (NO) signaling inTrichoplaxand related species: Microchemical characterization and the lineage-specific diversification

AbstractNitric oxide (NO) is a free radical gaseous messenger with a broad distribution across the animal kingdom. However, the early evolution of nitric oxide-mediated signaling in animals is unclear due to limited information about prebilaterian metazoans such as placozoans. Here, we analyzed NO synthases (NOS) in four different species of placozoans (haplotypes H1, H2, H4, H13). In contrast to all other invertebrates studied,HoilungiaandTrichoplaxhave three distinct NOS genes, including PDZ domain-containing NOS. To characterize NOS activity inTrichoplax adhaerens,we used capillary electrophoresis for microchemical assays of NO-related metabolites. Specifically, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional NOS. Next, using fluorescent single-moleculein situhybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions ofTrichoplax.These data suggest the compartmentalized release of this messenger and a greater diversity of cell types in placozoans than anticipated. We also revealed a dramatic diversification of NO receptor machinery, including identification of both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, althoughTrichoplaxis considered to be one of the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa.Short AbstractNitric oxide (NO) is a ubiquitous gaseous messenger, but we know little about its early evolution. Here, we analyzed NO synthases (NOS) in four different species of placozoans – one of the early-branching animal lineages. In contrast to other invertebrates studied,TrichoplaxandHoilungiahave three distinct NOS genes, including PDZ domain-containing NOS. Using ultra-sensitive capillary electrophoresis assays, we quantified nitrites (products of NO oxidation) and L-citrulline (co-product of NO synthesis from L-arginine), which were affected by NOS inhibitors confirming the presence of functional enzymes inTrichoplax. Using fluorescent single-moleculein situhybridization, we showed that distinct NOSs are expressed in different subpopulations of cells, with a noticeable distribution close to the edge regions ofTrichoplax.These data suggest both the compartmentalized release of NO and a greater diversity of cell types in placozoans than anticipated. NO receptor machinery includes both canonical and novel NIT-domain containing soluble guanylate cyclases as putative NO/nitrite/nitrate sensors. Thus, althoughTrichoplaxandHoilungiaexemplify the morphologically simplest free-living animals, the complexity of NO-cGMP-mediated signaling in Placozoa is greater to those in vertebrates. This situation illuminates multiple lineage-specific diversifications of NOSs and NO/nitrite/nitrate sensors from the common ancestor of Metazoa.