Abstract
Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.
Highlights
Glycine is a major inhibitory neurotransmitter in the vertebrate spinal cord, brainstem, and retina, where it activates ionotropic glycine receptors (GlyR) [1,2,3,4]
Differences in glycine transporter subtype expression are observed at the cell type level: GlyT1 is found on astroglial cells and glutamatergic neurons, whereas GlyT2 localizes to glycinergic presynaptic terminals [5]
At least in B. floridae and B. lanceolatum, the GlyT2.1 and GlyT2.2 genes were located on the same scaffold in the genome and only about 100 kb apart (Table 1), suggesting that the amphioxus GlyT2.1 and GlyT2.2 genes originated from a lineage-specific duplication event
Summary
Glycine is a major inhibitory neurotransmitter in the vertebrate spinal cord, brainstem, and retina, where it activates ionotropic glycine receptors (GlyR) [1,2,3,4]. Shpak and coworkers analyzed genomic databases of selected invertebrate deuterostomes (a cephalochordate, an ascidian, and a sea urchin) and retrieved, for each species, two glycine transporter paralogs, which they called GlyT1-like and GlyT2-like. The paucity of data on glycinergic neurotransmission among invertebrates makes the evolutionary history of this neurotransmitter difficult to trace To address this issue, we analyzed the glycine transporters complement of amphioxus, an invertebrate with a key phylogenetic position within the chordates, to define the evolutionary events that took place during the early diversification of vertebrates [32]. Partial sequences of B. lanceolatum glycine transporter genes were amplified by RT-PCR with specific primers (Supplementary Table S1), cloned into the pCRII-TOPO Vector (Life Technologies, Carlsbad, CA, USA), and used as templates to synthesize sense and antisense riboprobes with the DIG RNA Labeling Kit (Roche, Penzberg, Germany) following the manufacturer’s instructions. The hu-elav and glutamic acid decarboxylase (GAD) clones have previously been obtained [54,55]
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