Introduction

Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter that regulates many basal brain functions including sleep, feeding and mood. A serotonergic nervous system is found in the embryos and larvae of almost all animal groups suggesting an early origin of a nervous system using this neurotransmitter (Hay-Schmidt 2000). Ascidians are marine sessile animals, which develop through a swimming larva. Together with pelagic larvaceans, salps and doliolids they form the Tunicata sub-phylum, a group at the base of the chordate lineage. In the last decade, the ascidian Ciona intestinalis has become a model organism for developmental and genetic studies. Its tadpole larva shows the basic chordate features, comprising a notochord, which runs the length of the tail, and a dorsal tubular central nervous system (CNS). The tail musculature of the larva of this species comprises two sets of striated muscle cells, each set of muscle cells is aligned in three longitudinal bands flanking the notochord (Bone 1992). The CNS is formed by about 330 cells most of which are within the sensory vesicle, and because of its relative simplicity it was considered a miniaturised model of vertebrate nervous system. In fact, ascidian CNS can be divided into three anatomical regions, which are characterised by the expression of specific genes and which are considered to be a homolog to the forebrain, the mid-hindbrain boundary (MHB) and the hindbrain of vertebrates. Respectively, these regions are: the sensory vesicle, expressing Ci-Otx, an otx homolog; the neck, expressing Ci-Pax 2/5/8, ortholog of Pax2, Pax5 and Pax8 and the visceral ganglion, expressing Ci-Hox genes (see review Meinertzhagen et al. 2004). Recently, Dufour et al. (2006) revised the tripartite model of ascidian CNS on the base of the expression of the paired-like homeobox gene Ci-Phox2. These authors confirmed the status of the forebrain to the rostral sensory vesicle and equated the hindbrain with the neck, rather than to the visceral ganglion, which, in turn, was homologised to the spinal cord of vertebrates. Despite increasing data regarding the molecular characterisation of C. intestinalis larva nervous system, there is little evidence about neurotransmitter presence and function in this model organism (Meinertzhagen et al. 2004). Serotonin-like immunoreactivity was described in the larval nervous system of some different ascidian species (Pennati et al. 2001; Stach 2005).

TPH encodes tryptophan hydroxylase, the rate limiting enzyme in the biosynthesis of serotonin and is considered the most reliable marker of serotonin-producing neurons (Goridis and Rohrer 2002). Together with tyrosine hydroxylase (TH) and phenylalanine hydroxylase (PAH), TPH makes a superfamily of aromatic amino acid hydroxylases, catalysing key steps in important metabolic pathways (Kaufman 1995; Kappock and Caradonna 1996). These enzymes are characterised by a regulatory N-terminal domain, a catalytic domain and a C-terminal oligomerisation domain; they exhibit extensive sequence similarity at the catalytic domains. While PAH is mainly a liver and kidney enzyme, TPH and TH are involved in neurotransmitter synthesis and are expressed in the nervous system (Kappock and Caradonna 1996). In vertebrates, at least 2 isoforms of TPH are present, called TPH1 and TPH2. In mammals, TPH1 is found in both serotonin-producing cells in peripheral organs and neurons in the brain, while distribution of TPH2 is limited to the brain (Patel et al. 2004).

In the present work, we cloned C. intestinalis TPH-encoding gene and analysed its spatial and temporal expression pattern by in situ hybridisation to describe the development of the serotonergic system in this species.

Materials and methods

Animals and embryos

Adults of C. intestinalis were collected in the Gulf of Naples, Italy and reared in aquaria at 15°C. Gametes were obtained dissecting the gonoducts of at least three adults and were used for in vitro fertilisation. Developing embryos were maintained at 18°C in a thermostatic chamber.

Cloning of Ci-TPH cDNAs

C. intestinalis embryos and larvae were collected, immediately submerged in RNA later (Ambion Europe, UK) and stored at −20°C. For RNA preparation, samples were homogenised and then RNA was extracted using the Trizol Ls reagent (Invitrogen, San Diego, CA). Synthesis of first-strand cDNA was performed with 5 μg RNA using the SuperScript first-strand synthesis (Invitrogen) and oligo(dT) primers. Then, to isolate the tryptophan hydroxylase fragment sequence, we first designed two specific primers (F1: 5′-ATGTATGGAGCTGAACTTGATGC-3′; R1: 5′-TCAGCAAACTTCAATCTTTCG-3′) using the information available on JGI C. intestinalis genomic database (JGI model ci0100149739). PCR was carried out using 1 μl Ciona cDNA in a 50 μl reaction mixture using Hot Master mix according to the instructions (Eppendorf, Italy). To obtain the 5′ end of Ci-TPH, RNA ligase-mediated rapid amplification of 5′ ends (RLM-RACE) (Ambion Europe, UK) strategy was conducted with the following primers: 5′ RACE outer specific primer, 5′-AGTATGGATCGCTGTGGTGTCTGA-3′ and 5′ RACE inner specific primer, 5′-AGAACTCCGATAGTAATGGTAGA-3′. Amplified PCR products were cloned using the TA Cloning Kit (Invitrogen) and sequenced using an Applied Biosystems Big Dye Terminator Cycle Sequencing Kit. The complete sequence of Ci-TPH was submitted to the GenBank under accession number: DQ856593.

Sequence and phylogenetic analysis

For sequence analysis, the Vector NTI Suite, version 9.0 (Informax, North Bethesda, MD) software package was used. Amino acid alignments were performed using ClustalX software and optimised manually. Distances were computed by the neighbour-joining method implemented in ClustalX and trees constructed with the NJplot program. Bootstrap analysis was made on 100 sampling steps.

The phylogenetic tree was visualised with Tree View. Accession numbers of the hydroxylase sequences used in the alignment and in the phylogenetic tree are: Homo sapiens HsTPH1, HsTPH2, HsTH and HsPAH (NP_004170, AAI14500, P07101, NP_000268); Mus musculus MmTPH1, MmTPH2, MmTH and MmPAH (NP_033440, NP_775567, NP_033403, P16331); Gallus gallus GgTPH1, GgTpH2, GgTH (NP_990287, NP_001001301, NP_990136); Danio rerio DrTpH1, DrTPH2 (AAH59550, NP_999960); Anguilla anguilla AaTH (O42091); Branchiostoma floridae BfPAH and Branchiostoma lanceolatum BlTH (CAA04917 and CAE12259); C. intestinalis CiTH and CiPAH (NP_001027967 and ci0100145352 JGI databank); Drosophila melanogaster DmTH (CAA53802); Caenorhabditis elegans CeTPH, CeTH and CePAH (AAD30115,P90986 and AAD31643); Carassius auratus CaGAD67 (AAG33932).

Whole mount in situ hybridisation

RNA antisense and sense probes were synthesised following the instructions supplied with the DIG RNA labeling kit (Roche Diagnostics, Italy). Templates for the probes corresponded to the deduced open reading frame of Ci-TPH cDNA sequence. Gastrulae, neurulae, early and late tailbud embryos and swimming larvae were used for whole mount in situ hybridisation, following the protocol described by Gionti et al. (1998). Labeled samples were counter-stained with 1% of Ponceau S in 1% acetic acid, embedded in resin and sectioned at 3 μm.

Results and discussion

Sequence analysis and phylogenetic analysis of Ci-TPH

The Ci-TPH cDNA sequence from C. intestinalis is 1,397-bp long, including a coding region of 1,347-bp and a 5′ untranslated region 50-bp long with an in-frame stop codon upstream from the putative start codon. The longest open reading frame codes for 448 amino acids (Fig. 1a). The molecular weight predicted for Ci-TPH by sequence analysis is 51 kDa.

Fig. 1
figure 1

Sequence and phylogenetic analysis of Ci-TPH. a Aligned protein sequences (ClustalX program) of Ci-TPH and THP enzymes from human, mouse, chicken, zebrafish and C. elegans. Shared colors indicate conserved (identical or similar) residues. Core catalytic domain is underlined in red. b Phylogenetic analysis of Ci-TPH with aromatic amino acid hydroxylases proteins from several organisms. The tree was constructed using sequences from the core conserved domains and a part of the N terminus, excluding positions with gaps. Numbers at the nodes are bootstrap values based on 100 replicates. Scale bar of 0.1 at the bottom left corner means 0.1 nucleotide substitutions for the site. Caurassius auratus GAD67 was used as outgroup. PAH phenylalanine hydroxylase, TPH tryptophan hydroxylase, TH tyrosine hydroxylase

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The analysis on JGI genomic databases performed using Ci-TPH cDNA sequence reveals 12 introns that interrupt the coding sequence http://genome.jgi-psf.org/Cioin2/Cioin2.home.html).

Blast analysis of Ci-TPH predicted protein sequence reveals similarity to tryptophan hydroxylase (TPH), tyrosine hydroxylase (TH) and phenylalanine hydroxylase (PAH) enzymes. In particular, the carboxyl-terminal two thirds of the Ci-TPH sequence shares a high similarity with vertebrate TPH sequences (61–63%), and low similarity with TH (48–55%) and PAH (54–59%). In contrast, the Ci-TPH N-terminal region and the last 65 aa at the C terminus region are less homologous and also vary in length. ScanProsite analysis http://www.expasy.org/prosite/) reveals that Ci-TPH presents the conserved core region where the catalytic domain is located (Fig. 1a). Furthermore, several structural motifs of the TPH/TH/PAH sub-family are also present: a potential iron-binding site (His275, His280 and Glu295) (Goodwill et al. 1998) and the biopterin-dependent aromatic amino acid hydroxylases signature peptide PDicHEILGHVP (located at 270–281 aa).

Finally, phylogenetic analysis demonstrates that Ci-TPH is more related to TPH sequences than to those of the other two aromatic amino acid hydroxylase, even if it does not cluster clearly with vertebrates TPH (Fig. 1b), whereas PAH and TH Ciona orthologs showed higher affinity to vertebrate orthologs. Ci-TPH sequence seems quite divergent from other TPH proteins and this could be related to the accelerated sequence divergence in the Ciona lineage (Holland and Gibson-Brown 2003). However, Ci-TPH was more closely related to the CeTPH sequence, which is a well-known marker of serotonin producing-cells in Caenorhabditis elegans (Sze et al. 2000).

Developmental expression of Ci-TPH

In C. intestinalis, Ci-TPH expression was not detectable until the neurula stage (Fig. 2a). At the early tailbud stage, the expression of this gene was first detected with a punctuated distribution along the tail (Fig. 2b). The number of positive spots in the tail increased at the middle tailbud stage (Fig. 2c) and, in dorsal view, they were clearly visible aligned in two rows flanking the notochord (Fig. 2d). At this stage, Ci-TPH expression first appeared in the central nervous system (CNS) in a few cells at the level of the differentiating visceral ganglion (Fig. 2e–f). Also in larval CNS, Ci-TPH expression was restricted to a few cells (Fig. 2h) grouped into two distinct clusters (Fig. 2i,j) in the anterior visceral ganglion. Histological sections of hybridised samples revealed that Ci-TPH-expressing cells were always situated in the floor of the neural tube, just above the underlying notochord (Fig. 2k,l). The visceral ganglion has long been considered homologous to the hindbrain of vertebrates (Imai et al. 2002). Considering this comparison, the serotonin containing neurons can be equated to those of vertebrate raphe nuclei, localised in the ventral hindbrain and containing most of CNS serotonergic neurons (Bellipanni et al. 2002). If the visceral ganglion is to be considered homologous to the spinal cord, according to Dufour et al. (2006), then Ci-TPH-expressing neurons in C. intestinalis CNS could be compared to TPHD1-positive neurons in the zebrafish spinal cord (Bellipanni et al. 2002). In any case, serotonergic neurons of the ascidian larva are located in a division of the CNS ventral and posterior to the MHB (mid-hindbrain boundary), as in vertebrates.

Fig. 2
figure 2

Expression of Ci-TPH during C. intestinalis development. a Dorsal view of neurula embryo. No positive signal is present. b Lateral view of early tailbud embryo. Some positive cells are scattered in the tail. c–e Middle tailbud embryo. c Lateral view showing intensely stained cells in the tail. d Dorsal view with stained cells aligned in two rows flanking the notochord. e Dorsal view at deeper focal depth than c, showing labeled cells in the visceral ganglion. f Antero-dorsal view with a Ci-TPH expression in cells of the CNS. g–q Newly hatched larva. g Whole mount larva showing intense hybridisation signal in the visceral ganglion and in numerous, regularly spaced spots in the tail. h Lateral view of the trunk region. i Dorsal view of the posterior trunk region. j Magnification of the sensory vesicle region showing positive cells in the visceral ganglion arranged into two clusters. k Transverse section of a larva at the level of the visceral ganglion where a positive cell lying just above the notochord is visible. l Schematic drawing of k. m Magnification of a portion of the tail, showing labeled spots at the level of all three bands of muscle cell on the left side of the larva. n Longitudinal section of the tail showing the signal at the level of two adjacent muscle cells. o–p Transverse sections of the tail with Ci-TPH transcripts at the level of a dorsal and a ventral (o) and two dorsal (p) muscle cells. q Schematic drawing of p. Muscle cells are stained in orange. Arrows indicate the intensely stained cells in the ganglion. en endoderm, ep epidermis, mc muscle cell, nc notochord, nt neural tube, oc ocellus, ot otolith, sv sensory vesicle. Scale bars: a, b, c, d, e, f, i, j, k 25 μm; g, h, m 50 μm; n, o, p 10 μm

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TPH is generally considered the most reliable marker for serotonergic neurons (Goridis and Rohrer 2002). However, we were not able to label serotonin in C. intestinalis larvae using a specific antibody. By this technique we localised serotonin in the CNS of metamorphosing larvae and in some cells of the endostyle and in the chromaffin-like cells of juveniles (unpublished data), as previously observed in Phallusia mammillata (Pennati et al. 2001). We suppose that serotonin content in Ciona swimming larvae was too low to be detected by this technique. Recently, Stach (2005) reported the presence of 5-HT-like immunoreactivity in the nervous structure of C. intestinalis larvae, but the morphology of labeled structures was unclear. Moreover, he reported the presence of serotonin in the posterior sensory vesicle of Herdmania momus and of Ascidia interrupta. In P. mammillata, larvae serotonin was localised by immunohistochemistry in the sensory vesicle, strictly associated with the ocellus, in palps sensory neurons and in the caudal epidermal neurons (Pennati et al. 2001). From these results, it emerged that serotonin positive cells are present in the anterior region of the CNS of ascidian larvae in a position that can vary from species to species and that the presence of 5-HT in the peripheral nervous system seem to be specific of P. mammillata.

In C. intestinalis larva, Ci-TPH transcripts were also present at the level of the muscle cells of the tail. In whole mount specimens, the hybridisation signal appeared as single spots, smaller than the muscle cells and regularly spaced along the tail. By comparing several hybridised larvae, it was found that TPH-positive spots in the tail were 22 ± 2, over 36 muscle cells in the larva tail. In transverse sections, it was clear that muscle cells of ventral (Fig. 2o,q), dorsal (Fig. 2p,q) and median bands (data not shown) could contain Ci-TPH transcripts. Histological sections revealed also that hybridisation signal was not spread in the entire cytoplasm but it seemed to be confined to one end of the cell, usually near the area of contact between two positive cells (Fig. 2n). This peculiar pattern suggested that Ci-TPH expression at the level of the muscle cells might be indeed present at the level of the neuro-muscular junctions. Indeed, motor neurons projections, so far described, reach all muscle cells of the dorsal bands and the first rows of ventral muscle cells (Bone 1992). Moreover, only cholinergic neuro-muscular junctions were described in Ciona larvae (Meedel and Whittaker 1979), but we cannot rule out the possibility that additional neuron projections from the visceral ganglion may contact muscle cells in the tail.

In vertebrates, serotonin is able to modulate rhythmic locomotor activity via the central pattern generating (CPG) networks, responsible for coordinating motor activity in the absence of sensory input from peripheral receptors (Branchereau et al. 2000). We propose that in C. intestinalis larvae, serotonin is also produced close to the muscle cells to modulate the continuous left–right alternate tail contractions during swimming. It was demonstrated that a high level of 5-HT can shorten the swimming phase of ascidian larvae, promoting metamorphosis (Zega et al. 2005). It could be that in Ciona larvae, serotonin signaling might also modulate this important phase of the life cycle.

The anatomy and the function of the Ci-TPH expressing system in C. intestinalis larvae is to be further investigated to shed light into the serotonergic signaling pathway linking the visceral ganglion to the tail muscles.