Tunicates: not just little squirts? (2024)

Euan Brown
Heriot-Watt University, Edinburgh, UK

https://doi.org/10.36866/pn.99.31

Over the last 20 years whole-animal physiology has focused on an increasingly limited number of vertebrate models. However pressure to apply the 3R principles, a wish to uncover universal mechanisms, and a curiosity to understand where we come from (evolution), should mean that an occasional glance over the shoulder to what is available as alternatives is justified. Where better to look then than in the marine environment?

As animal life evolved first in the sea, it still contains a far greater diversity of key phyla than the land, and many of these are exclusively marine. Furthermore, the recent ‘genome revolution’ means that there are many more experimental resources available for what until recently, had been considered phylogenetic oddities. The good news then is that the occasional trip to the seaside to explore the use of alternatives is not an unreasonable activity for the serious physiologist! It is also worth pointing out that access to European Marine Laboratories for short visits to test out ideas has been greatly facilitated by the appearance of Research Infrastructure Initiatives funded by the European Union framework programmes. In the case of marine models, EMBRC (http://www.embrc.eu/) funds short term, timely projects. As support is available on site there is no need to become an expert on a particular organism to access it. Here, to encourage this process, I discuss some of the recent advances and opportunities in tunicates.

Tunicate relations

Tunicates occupy a key phylogenetic position, they are invertebrates, but unlike other invertebrates they belong to the phylum Chordata (Figure 1). Despite some fundamental differences, tunicates share several important common features with vertebrates; such as a basic chordate bauplan, including a dorsal nervous system with neural canal, notochord, hypophysis-pituitary complex, pineal eye and simple single chambered heart. The privileged phylogenetic position of ascidians is supported by most modern molecular phylogenies, establishing them as the sister group of vertebrates (implying that we shared a common ancestor around 500-600 million years ago). This makes tunicates particularly interesting for the study of the basic mechanisms that underpin the physiology of both groups.

What are tunicates?

Tunicates are a group of exclusively marine animals that include over 2,000 species divided into Ascidiacea (Aplousobranchia, Phlebobranchia, and Stolidobranchia) Thaliacea (Pyrosomida, Doliolida, and Salpida) and Appendicularia (Larvacea). Probably the most popular for experimental studies is the ascidian Ciona intestinalis, commonly known as the ‘sea squirt’. It is a solitary sessile tunicate that can be found growing in great clumps under piers around the world (Figure 2a). It can be collected with ease and maintained in simple seawater aquaria. Ciona is hermaphrodite (each individual contains eggs and sperm) and has a two stage life cycle. 18 hrs of development produce a free swimming vertebrate-like tadpole (Figure 2b) that transforms through metamorphosis within 6-12 hours after hatching into a miniature version of the tube-like adult (Figure 2c). Since the genome was sequenced in 2002, Ciona has become increasingly popular for evo/devo research. The genome is very compact (180 Mbp/haploid size), with around half of the number of genes estimated for vertebrates (~15,000). While vertebrate genomes have undergone at least three complete rounds of duplication (resulting in multiple copies of genes), invertebrate chordate genomes did not, and as a result are useful in single gene knock -down studies using morpholino, and siRNA techniques.

The larval nervous system

The nervous system, of ascidians and vertebrates seem to have followed two separate evolutionary trajectories. While the vertebrate CNS is has many cells and is anatomically complex, the CNS of the ascidian tadpole shows extreme parsimony, consisting of fewer than 100 identified neurones and no glia, perhaps holding the current ‘model animal record’ for fewest neurons in a CNS (Imai & Meinertzhagen 2007). Despite this, the larval nervous system conforms to the basic chordate CNS arrangement. A ‘forebrain’ or sensory vesicle integrates sensory information and contains a rudimentary ‘pineal-eye’ (~ 16 hyperpolarizing vertebrate-like photoreceptors and a tripartite lens) and a single otolith or gravity sensing organ. These organs can be clearly seen in the transparent larva as two black spots as they are heavily pigmented (Figure 2 b). The sensory vesicle is connected via a ‘midbrain’ neck region to a motor ganglion of ~10 cholinergic motoneurones that innervate the muscle of the tail. Rhythmic alternating activation of the tail drives a limited number of high frequency (30-40 Hz) swimming ‘gaits’. A number of neurotransmitter systems have been identified in the CNS (Figure 3). Hyperpolarising photoreceptors interact with dopaminergic interneurons that may prefigure retinal ganglion cells. Glutamatergic drive by interneurons impinges on cholinergic motoneurons in the motor ganglion which in turn activates muscle fibres by cholinergic synaptic transmission. GABAergic interneurons modulate the system at different levels. Probable glycinergic interneurons in the tail interact to control left-right alternation of swimming by acting on glycine receptors (Nishino et al. 2010). While the swimming output is experimentally tractable, unfortunately the cells of the nervous system have not proved to be amenable to microelectrode or patch clamp recording techniques (although cells can be dissociated and successfully cultured and patch clamped). A likely way forward for network analysis is the use of fluoro-genetic encodable voltage sensors targeted to specific neuronal classes.

Adult nervous system

It was thought until recently that the adult ‘brain’ ganglion was a primitive cell cluster unrelated to the larval chordate body plan, however recent work has established that it partly arises from stem cells that reside in neck region of the larva. Interestingly, these cells have some similarities to vertebrate cranial motoneurons as they express Ci-Phox2 and Ci-Tbx20, whose vertebrate orthologues define vertebrate cranial motoneurons (Dufour et al. 2006). Thus, the adult nervous system which has long been known to possess remarkable powers of regeneration also makes an attractive model for physiological studies.

Muscular system

The larval tail and adult body muscles are controlled by cholinergic synaptic transmission as are most chordates including vertebrates. In this respect they differ from the majority of invertebrate neuromuscular junctions which utilise glutamate as the excitatory transmitter. The adult muscles are involved in a strictly limited series of coordinated actions relating to filter-feeding and expulsion of eggs and sperm via the siphons. The larval neuromuscular system is organised into two ‘myotomes’ consisting of three rows of muscle on each side of the midline. Alternate activation of these muscle fibre groups drives rapid alternating swimming (Nishino et al. 2010).

Cardio–vascular system

The adult ascidian contains a vasculature and a simple single chambered heart that, curiously, pumps haemolymph in one direction then reverses periodically. It is much simpler than the single atrium and ventricle of fish heart and the four chambered mammalian system. It is absent in the tadpole larvae as there is no circulation. However in larval development, two cells that give rise to the heart can be clearly identified and followed during metamorphosis and into adult development (Davidson et al. 2006).

Because of rapid development, and destruction and remodelling of the neuromuscular system during and after metamorphosis and the rapid development of the single chambered heart, the muscular system is a potential model to study the physiology of heart and NMJ development.

Biophysics/biotechnology

The study of ion channels in ascidians has resulted in the recent striking discovery of two entirely new membrane protein classes –the voltage-sensing phosphatases (termed Ci-VSP) and the voltage sensitive proton channels (Ci-VSOP Ciona voltage sensing only protein) (Okamura 2007). These proteins were detected in silico in the Ciona genome sequence and subsequently found in mammals and most other organisms. In recent work, the voltage-sensing domain of Ci-VSP has been fused to a pair of fluorescent reporter proteins to generate genetically encodable florescent voltage reporting proteins.

In a recent extensive survey of the genome for evidence of theoretical proteins, it was found that the genome of Ciona contains genes coding for a minimal ‘chordate set ‘of ion channels and receptors. There was some evidence for gene loss and the appearance in tunicates of potentially chordate specific genes. There has been also apparent expansion of certain receptor families (e.g. GABA and ACh receptors).The main inhibitory neurotransmitter systems in vertebrates are GABAergic and Glycinergic. In invertebrates a more diverse system also includes inhibitory glutamate receptors which are so far unknown in chordates. All other receptors form part of the cys –loop superfamily class of ion channels which have been shown to have an ancient pre-bilateran origin. GABA and GABA receptors and their transporters have been identified in Ciona and the expression pattern of the GABA transporters indicates a presence in the motor ganglion.

Conclusions

Tunicates provide a simple chordate system with limited cell numbers and a single copy genome. This makes them ideal for the investigation of single gene issues. Their rapid development and small size render them ideal for imaging and for some whole animal techniques. Their close phylogenetic relationship to vertebrates makes them particularly useful in understanding the first steps in vertebrate evolution. Finally they have provided some biotechnologically useful proteins for cell physiology.

References

Davidson B, Shi W, Beh J, Christiaen L, Levine M. (2006). FGF signaling delineates the cardiac progenitor field in the simple chordate, Ciona intestinalis. Genes Dev 20(19), 2728-38

Dufour HD, Chettouh Z, Deyts C, de Rosa R, Goridis C, Joly JS, Brunet JF (2006). Precraniate origin of cranial motoneurons. Proc Natl Acad Sci USA 103(23), 8727-32

Imai JH, Meinertzhagen IA (2007). Neurons of the ascidian larval nervous system in Ciona intestinalis: I. Central nervous system. J Comp Neurol 501(3), 316-34

Nishino A, Okamura Y, Piscopo S, Brown ER (2010). A glycine receptor is involved in the organization of swimming movements in an invertebrate chordate. BMC Neurosci 11, 6

Okamura Y (2007) Biodiversity of voltage sensor domain proteins. Pflugers Arch 454(3), 361-71

Razy-Krajka F, Brown ER, Horie T, Callebert J, Sasakura Y, Joly JS, Kusakabe TG, Vernier P (2012). Monoaminergic modulation of photoreception in ascidian: evidence for a proto-hypothalamo-retinal territory. BMC Biol 10, 45