Post-gastrulation transition from whole-body to tissue-specific intercellular calcium signaling in the appendicularian tunicate Oikopleuradioica.

We recently described calcium signaling in the appendicularian tunicate Oikopleura dioica during pre-gastrulation stages, and showed that regularly occurring calcium waves progress throughout the embryo in a characteristic spatiotemporal pattern from an initiation site in muscle lineage blastomeres. Here, we have extended our observations to the period spanning from gastrulation to post-hatching stages. We find that repetitive Ca2+ waves persist throughout this developmental window, albeit with a gradual increase in frequency. The initiation site of the waves shifts from muscle cells at gastrulation and early tailbud stages, to the central nervous system at late tailbud and post-hatching stages, indicating a transition from muscle-driven to neurally driven events as tail movements emerge. At these later stages, both the voltage gated Na+ â€‹channel blocker tetrodotoxin (TTX) and the T-type Ca2+ channel blocker and nAChR antagonist mecamylamine eliminate tail movements. At late post-hatching stages, mecamylamine blocks Ca2+ signals in the muscles but not the central nervous system. Post-gastrulation Ca2+ signals also arise in epithelial cells, first in a haphazard pattern in scattered cells during tailbud stages, evolving after hatching into repetitive rostrocaudal waves with a different frequency than the nervous system-to-muscle waves, and insensitive to mecamylamine. The desynchronization of Ca2+ waves arising in different parts of the body indicates a shift from whole-body to tissue/organ-specific Ca2+ signaling dynamics as organogenesis occurs, with neurally driven Ca2+ signaling dominating at the later stages when behavior emerges.

Acid-sensing ion channels emerged over 600 Mya and are conserved throughout the deuterostomes.

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, Oikopleura dioica 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.

Gap junction-dependent coordination of intercellular calcium signalling in the developing appendicularian tunicate Oikopleura dioica.

We characterized spontaneous Ca2+ signals in Oikopleura dioica embryos from pre-fertilization to gastrula stages following injection of GCaMP6 mRNA into unfertilized eggs. The unfertilized egg exhibited regular, transient elevations in intracellular Ca2+ concentration with an average duration of 4-6 s and an average frequency of about 1 every 2.5 min. Fertilization was accompanied by a longer Ca2+ transient that lasted several minutes. Thereafter, regular Ca2+ transients were reinstated that spread within seconds among blastomeres and gradually increased in duration (by about 50%) and decreased in frequency (by about 20%) by gastrulation. Peak amplitudes also exhibited a dynamic, with a transitory drop occurring at about the 4-cell stage and a subsequent rise. Each peak was preceded by about 15 s by a smaller and shorter Ca2+ increase (about 5% of the main peak amplitude, average duration 3 s), which we term the "minipeak". By gastrulation, Ca2+ transients exhibited a stereotyped initiation site on either side of the 32-64-cell embryo, likely in the nascent muscle precursor cells, and spread thereafter symmetrically in a stereotyped spatial pattern that engaged blastomeres giving rise to all the major tissue lineages. The rapid spread of the transients relative to the intertransient interval created a coordinated wave that, on a coarse time scale, could be considered an approximate synchronization. Treatment with the divalent cations Ni2+ or Cd2+ gradually diminished peak amplitudes, had only moderate effects on wave frequency, but markedly disrupted wave synchronization and normal development. The T-type Ca2+ channel blocker mibefradil similarly disrupted normal development, and eliminated the minipeaks, but did not affect wave synchronization. To assess the role of gap junctions in calcium wave spread and coordination, we first characterized the expression of two Oikopleura connexins, Od-CxA and Od-CxB, both of which are expressed during pre-gastrulation and gastrula stages, and then co-injected double-stranded inhibitory RNAs together with CGaMP6 to suppress connexin expression. Connexin mRNA knockdown led to a gradual increase in Ca2+ transient peak width, a decrease of interpeak interval and a marked disruption of wave synchronization. As seen with divalent cations and mibefradil, this desynchronization was accompanied by a disruption of normal development.

Development of the house secreting epithelium, a major innovation of tunicate larvaceans, involves multiple homeodomain transcription factors.

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.

Modification of the larval swimming behavior in Oikopleura dioica, a chordate with a miniaturized central nervous system by dsRNA injection into fertilized eggs.

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.

A miniaturized nigrostriatal-like circuit regulating locomotor performance in a protochordate.

To gain insight into the evolution of motor control systems at the origin of vertebrates, we have investigated higher-order motor circuitry in the protochordate Oikopleura dioica. We have identified a highly miniaturized circuit in Oikopleura with a projection from a single pair of dopaminergic neurons to a small set of synaptically coupled GABAergic neurons, which in turn exert a disinhibitory descending projection onto the locomotor central pattern generator. The circuit is reminiscent of the nigrostriatopallidal system in the vertebrate basal ganglia, in which disinhibitory circuits release specific movements under the modulatory control of dopamine. We demonstrate further that dopamine is required to optimize locomotor performance in Oikopleura, mirroring its role in vertebrates. A dopamine-regulated disinhibitory locomotor control circuit reminiscent of the vertebrate nigrostriatopallidal system was thus already present at the origin of ancestral chordates and has been maintained in the face of extreme nervous system miniaturization in the urochordate lineage.