Acid-sensing ion channels (ASICs) are proton-gated ion channels broadly expressed in the vertebrate nervous system, converting decreased extracellular pH into excitatory sodium current. ASICs were previously thought to be a vertebrate-specific branch of the DEG/ENaC family, a broadly conserved but functionally diverse family of channels. Here, we provide phylogenetic and experimental evidence that ASICs are conserved throughout deuterostome animals, showing that ASICs evolved over 600 million years ago. We also provide evidence of ASIC expression in the central nervous system of the tunicate, Furthermore, by comparing broadly related ASICs, we identify key molecular determinants of proton sensitivity and establish that proton sensitivity of the ASIC4 isoform was lost in the mammalian lineage. Taken together, these results suggest that contributions of ASICs to neuronal function may also be conserved broadly in numerous animal phyla.

Using RNA interference, we have selectively perturbed neurotransmitter-related features of the larval swimming behavior of Oikopleura dioica, a tunicate with a central nervous system comprising about 130 neurons. We injected dsRNA into fertilized eggs to knockdown the expression of the genes, respectively, encoding ChAT (choline acetyltransferase) and GAD (glutamic acid decarboxylase), enzymes critical for the biosynthesis of acetylcholine and GABA. These two neurotransmitters have conserved roles during evolution, particularly within chordate motor systems, where they mediate respectively neuromuscular and central inhibitory signals. In Oikopleura, interference with ChAT expression prevented the normal bidirectional, propagating tail movement characteristic of swimming, permitting only repeated unilateral tail bends. Proper swimming was never observed, and the resting period between episodes of activity was lengthened. This phenotype is most likely caused by the reduction of transcription observed for both the targeted ChAT gene and the VAChT gene (Vesicular Acetylcholine Transporter), both genes being transcribed from the same operon. Interference with GAD expression led to an uncoordinated version of swimming with a spiral movement trajectory, but with episodes similar in duration and cycle frequency to those of normal swimming. Our results suggest locomotor functions for ChAT and GABA that are more subtle than previously proposed for tunicates and opens the way for a genetic dissection of Oikopleura neuronal circuits, which are likely to be among the most simplified in the chordate phylum.

The mechanisms driving innovations that distinguish large taxons are poorly known and essentially accessible via a candidate gene approach. A spectacular acquisition by tunicate larvaceans is the house, a complex extracellular filtration device. Its components are secreted by the oikoplastic epithelium which covers the animal trunk. Here we describe the development of this epithelium in larvae through the formation of specific cellular territories known to produce distinct sets of house proteins (Oikosins). It involves cell divisions and morphological differentiation but very limited cell migration. A diverse set of homeobox genes, most often duplicated in the genome, are transiently and site-specifically expressed in the trunk epithelium at early larval stages. Using RNA interference, we show that two prop duplicates are involved in the differentiation of a region on and around the dorsal midline, regulating morphology and the production of a specific oikosin. Our observations favor a scenario in which multiple homeobox genes and most likely other developmental transcription factors were recruited for this innovation. Their frequent duplications probably predated, but were not required for the emergence of the house.

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