Animals and embryos

Mature adults of C. intestinalis were collected from harbors in Murotsu and Aioi, Hyogo, Japan. The adults were maintained in indoor tanks of artificial seawater (Marine Art BR, Senju Seiyaku, Osaka, Japan) at 18°C. The embryos were prepared using gametes obtained from the gonoducts, as described previously (Nakagawa et al., 1999).

Isolation and sequencing of cDNA clones encoding G protein α subunits

The total RNA was prepared from the C. intestinalis larvae, and a cDNA library was constructed with a λZAP vector (Stratagene, La Jolla, USA), as described previously (Iwasa et al., 2000). The cDNA library was directly used as a template to amplify the cDNA fragments of about 500 bp encoding the G protein α subunits by polymerase chain reaction (PCR), using a pair of degenerate oligo-nucleotide primers corresponding to two conserved amino acid sequences, KQM(K/R)IIH and KWI(H/Q)CF, respectively. The 5′-and 3′- portions of cDNAs were amplified from the cDNA library by PCR using a gene-specific primer and a vector primer, as described previously (Iwasa et al., 2000). Full-length cDNA clones were amplified by PCR using a thermostable DNA polymerase bearing proofreading activity (Takara LA Taq; Takara Shuzo, Japan), with primers corresponding to the 5′- and 3′-untranslated region (UTR) sequences of cDNAs (5′-ATACGAGCAAGCACAGCGGGAA-3′ for 5′-UTR, and 5′-TATGCATGCGATGACGTCAC-3′ for 3′-UTR). The PCR products were subcloned into plasmid vectors and sequenced on both strands with an automatic DNA sequencer (Shinadzu DSQ 1000L, Shimadzu, Kyoto, Japan).

Molecular phylogenetic analysis

The deduced amino acid sequences of Gα encoded by the 500-bp cDNA fragments amplified by PCR from C. intestinalis larvae were aligned with the amino acid sequences of Gα from other animals. A neighbor-joining tree was constructed with the alignment using the Clustal W program (Thompson et al., 1994). The evolutionary distances were estimated using Kimura's empirical method. The sequences used were: eleven Homo sapiens Gα isoforms (Gα_i XM_011603, Gα_q NM_002072, Gα_s X04408, Gα_olf L10665, Gα_z XM_009867, Gα₁₁ AF011497, Gα₁₂ L01694, Gα₁₃ L22075, Gα₁₅ XM_009220, cone transducin D10377, rod transducin X63749), two Rattus norvegicus isoforms (Gα_o M17526, gustducin X65741), four Octopus vulgaris Gα isoforms (Gα_i AB025780, Gα_q AB025782, Gα_o AB025781, Gα_s AB025783), and Drosophila melanogaster Gα_f L09700.

In situ hybridization

Digoxigenin-labeled RNA probes were synthesized using a DIG RNA labeling kit (Roche, Japan), according to the manufacturer's protocol. For CiGα_i2, CiGα_q, and CiGα_x, the 500-bp cDNA fragments obtained by PCR were used as templates to synthesize the probes. For CiGα_i1a and CiGα_i1b, the RNA probe was synthesized from the 136-bp coding region (nt 607–742 for CiGα_i1a and nt 497–632 for CiGα_i1b) corresponding to the alternatively-spliced exon of each transcript. To detect all transcripts from the CiGα_i1 gene, an RNA probe was also synthesized from a cDNA fragment containing 3′-coding and untranslated regions (spanning from the nt 603 to the 1925 of CiGα_i1a).

Whole-mount in situ hybridization was carried out basically according to the protocol by Wada et al. (1995). After the coloring reaction, the embryos and larvae were dehydrated in an ethanol series and incubated in ethanol for 5–10 min. Following rehydration with PBST, the embryos were incubated in 25% glycerol in PBST for 5 min, and then transferred to 50% glycerol in PBST. The embryos were photographed and stored in 50% glycerol/PBST.

Analysis of partial genomic structure of the CiGα _i1 gene

The genomic DNA of C. intestinalis was extracted from the sperm of one individual, according to a standard method (Sambrook et al., 1989). A genomic DNA fragment containing exons for both CiGα_i1a and CiGα_i1b was amplified by PCR using LA Taq with a pair of gene-specific primers 5′-TGGGAGACTGCATGAAACGAAT-3′ (corresponding to nt 520–541 of the CiGα_i1a cDNA), and 5′-GCTACACAGAAGATGATAGCAG-3′ (corresponding to nt 808–829 of the CiGα_i1a cDNA). The PCR products were cloned into pBluescript II SK (+) (Stratagene). The nucleotide sequence was determined on both strands with an automatic DNA sequencer (Shimadzu DSQ 1000L, Shimadzu).

Isolation and characterization of C. intestinalis cDNAs encoding Gα subunits

Five DNA fragments, each encoding a central part (164–166 aa) of Gα with a distinct amino acid sequence, were amplified from a C. intestinalis larval cDNA library by PCR with the degenerate primers. The Gα isoforms encoded by the cDNA fragments were designated as CiGα_x, CiGα_q, CiGα_i1a, CiGα_i1b, and CiGα_i2. The deduced amino acid sequences of the five isoforms were shown in Fig. 1.

Fig. 1

Comparison of amino acid sequences among C. intestinalis Gα isoforms. Amino acid sequences encoded by the 500-bp cDNA fragments of five C. intestinalis Gα are aligned. Dashes indicate gaps introduced in the sequence to optimize the alignment. Asterisks indicate the positions where all isoforms exhibit the same amino acid. Dots indicate the positions where all of the residues are similar to each other. Arrows above the CiGα_x sequence indicate the positions of the two primers used to amplify the cDNA fragments by PCR.

To investigate the structural and evolutionary relationships among CiGα_x, CiGα_q, CiGα_i1a, CiGα_i1b, CiGα_i2, and known metazoan Gα isoforms, phylogenetic analysis was performed by the neighbor-joining method (Saitou and Nei, 1987; Fig. 2). CiGα_q is most closely related to vertebrate Gα_q/Gα₁₁ isoforms. CiGα_x is also a member of the G_q class. Within the G_q class, however, CiGα_x is fairly diverged from both the Gα_q/Gα₁₁ subfamily and the Gα₁₅/Gα₁₆ subfamily. Therefore, CiGα_x may represent a novel subfamily, the members of which have not been identified in other animals. CiGα_i1a, CiGα_i1b, and CiGα_i2 were closely related to vertebrate and invertebrate Gα_i isoforms. Among these, the C. intestinalis Gα_i isoforms CiGα_i1a and CiGα_i1b are most closely related to each other.

Fig. 2

Molecular phylogenetic tree of G protein α subunits. A phylogenetic tree was inferred from the amino acid sequences by the neighbor-joining method. The scale bar indicates 0.1 amino acid replacements per site. The numbers at the nodes are bootstrap values based on 1,000 replicates. The five C. intestinalis Gα isoforms are boxed. The four major classes (G_i, G_q, G_s, and G₁₂) of Gα (Simon et al., 1991) are indicated at the right of the corresponding branches.

Messenger RNA encoding CiGα_i1a and CiGα_i1b are produced by alternative splicing from the CiGα_i1 gene

Between CiGα_i1a and CiGα_i1b, only 12 residues were different out of 165 amino acids encoded by the cDNA fragment described above. The diverged positions were limited within a 33-amino acid portion of the polypeptides, and outside this variable region there were few synonymous nucleotide substitutions between their cDNA sequences. These features of CiGα_i1a and CiGα_i1b indicate the possibility that transcripts for these isoforms are splicing variants of a single gene. To assess this possibility, we first determined the entire cDNA sequences of CiGα_i1a and CiGα_i1b. The full-length cDNAs encode a 354-amino acid polypeptide, and the predicted amino acid sequences are identical between CiGα_i1a and CiGα_i1b, except for the 12 positions found by the analysis of the PCR fragments (Fig. 3A). The 5′ and 3′UTR sequences are almost identical between the CiGα_i1a and CiGα_i1b transcripts (identity >98%, data not shown).

Fig. 3

Two splicing variants of the CiGα_i1 gene: CiGα_i1a and CiGα_i1b. (A) The deduced amino acid sequences of CiGα_i1a and CiGα_i1b. The entire sequence of CiGα_i1a is shown. Dots represent the amino acid residues of CiGα_i1b identical to those of CiGα_i1b, and the letters represent the variable positions in CiGα_i1b. The variable region encoded by the alternatively spliced exons is boxed. (B) Genomic structure of a part of the CiGα_i1 gene containing alternatively spliced exons for CiGα_i1a and CiGα_i1b. The exons are indicated by uppercase letters, whereas the introns are indicated by lowercase letters. The predicted amino acid sequences are indicated beneath the corresponding codons by the single-letter codes.

To further confirm that the CiGα_i1a and CiGα_i1b tran-scripts originate from a single gene, we then examined the structure of the gene. Genomic DNA containing the coding regions that had different sequences between CiGα_i1a and CiGα_i1b were amplified from the C. intestinalis sperm DNA by PCR. A 2.2-kb genomic DNA fragment was amplified and cloned. The entire nucleotide sequence of the genomic DNA fragment revealed that two homologous exons were tandemly aligned on the same DNA strand (Fig. 3B). One of the exons that locates upstream to the other encodes the variable region of CiGα_i1a, and the downstream exon encodes that of CiGα_i1b. The positions of the introns adjacent to the isoform-specific exons of CiGαi1a/b are conserved with respect to the Gα sequences in mammals and insects (Fig. 3A,B). The genomic organization of the CiGα_i1 gene strongly suggests that the CiGα_i1a and CiGα_i1b mRNAs are produced by alternative splicing.

Expression patterns of Gα mRNAs in C. intestinalis embryos

The expression patterns of mRNAs for C. intestinalis Gα isoforms were examined in tailbud embryos by whole-mount in situ hybridization (Fig. 4). The CiGα_x expression was widely observed throughout the trunk and tail of the embryos, and the signals were stronger in the epidermis, mesenchyme, and tail muscle cells (Fig. 4A,B). The CiGα_q transcripts were expressed in the anterior and dorsal trunk epidermis as well as the dorsal side of the tip of the tail (Fig. 4C). The anterior CiGα_q-expressing regions seem to contain the developing adhesive organ. On the dorsal trunk epidermis, the CiGα_q-expressing regions were bilaterally located as two pairs of patches (Fig. 4D). Both of the two splicing variants of CiGα_i1, CiGα_i1a, and CiGα_i1b, were specifically expressed in the brain and adhesive organ (Fig. 4E, F). A difference in expression patterns was not clear between CiGα_i1a and CiGα_i1b, although the hybridization signals were much weaker for CiGα_i1b. We failed to detect clear hybridization signals for CiGα_i2, even after observing the staining reaction for three days; only faint signals were observed in the brain (Fig. 4G).

Fig. 4

Spatial expression patterns of mRNAs for five Gα isoforms in C. intestinalis tailbud embryos. Transcripts of each Gα isoform were detected at the mid tailbud stage by whole-mount in situ hybridization. (A, B) Lateral (A) or dorsal view (B) of an tailbud embryo hybridized with a CiGα_x probe. (C, D) Lateral (C) or dorsal (D) view of a tailbud embryo hybridized with a CiGα_q probe. (E–G) Lateral view of tailbud embryos hybridized with a CiGα_i1a (E), CiGα_i1b (F), or CiGα_i2 (G) probe. See text for details of the expression patterns. Scale bar, 100 μm.

The distinct expression of the CiGα_i1 gene in the nervous system of tailbud embryos prompted us to further examine the expression patterns of this gene throughout embryonic development. Probably due to the small size (136 bp) of the splice variant-specific probes, the hybridization signals were very weak, especially for CiGα_i1b (Fig. 4E,F). Therefore, we used a longer probe that can be hybridized with both CiGα_i1a and CiGα_i1b mRNAs (see Materials and Methods). The CiGα_i1 transcripts were present in the eggs and cleavage stage embryos as maternal messages (Fig. 5B–F). The transcripts were ubiquitously distributed in the eggs and early embryos (Fig. 5B–G). The ubiquitous distribution of the CiGα_i1 mRNA was observed until the gastrula stage (Fig. 5G). At the neurula stage, the CiGα_i1 expression was restricted to the anterior ectoderm, especially the presumptive brain vesicle, although the hybridization signal was ambiguous (Fig. 5H). The CiGα_i1 expression became restricted to the palps (adhesive organ), the central nervous system (CNS), including the brain vesicle and the visceral ganglion, and the dorso-distal part of the tail (Fig. 5I–K). At the mid tailbud stage, the hybridization signals were very strong and were restricted to the palps, the entire brain vesicle, and the visceral ganglion (Fig. 5L,M). The gene expression persisted to the CNS until the tadpole larva stage (Fig. 5N).

Fig. 5

Whole-mount in situ hybridization using a CiGα_i1 sense (A) or antisense (B–N) probe. (A, C) Four-cell embryo. (B) Fertilized egg. (D) Eight-cell embryo. (E) 16-cell embryo. (F) 64-cell embryo. (G) Gastrula. (H) Neurula. (I–K) Ventral (I), lateral (J), or dorsal (K) view of an early tailbud embryo. (L, M) Lateral (L) or dorsal (M) view of mid tailbud embryo. (N) Tadpole larva. Scale bar, 100 μm.

Diversity of Gα isoforms in ascidians

In the present study, we showed that mRNAs encoding at least five different Gα isoforms are present in ascidian embryos. Mammals have at least 17 functional Gα genes, several of which are spliced alternatively, that encode 23 distinct protein products (Nürnberg et al., 1995). The presence of multiple Gα isoforms has also been demonstrated in various invertebrates, including insects, nematodes, octopus, hydra, and sponges (Wilkie and Yokoyama, 1994; Suga et al., 1999; Iwasa et al., 2000). Metazoan Gα sub-units can be classified into G_s, G_i, G_q, and G₁₂ classes (Simon et al., 1991; Suga et al., 1999). The present analysis identified two members of the G_q class and three of the G_iclass in C. intestinalis embryos. Although we have not found Gα_s isoforms in Ciona, we recently identified a cDNA clone encoding Gα closely related to vertebrate Gα_s isoforms in another ascidian species, Halocynthia roretzi (Iwasa et al., 2001). To date, however, no Gα isoforms have been assigned to the G₁₂ class in ascidians.

Among the G_q class members, CiGα_q is closely related to the vertebrate Gα_q and Gα₁₁. However, the vertebrate Gα_q and Gα₁₁ are more closely related to each other than to CiGα_q, suggesting that the vertebrate Gα_q and Gα₁₁ iso-forms originated by gene duplication during vertebrate evolution after the divergence between the vertebrate and the urochordate. The metazoan G_q-class isoforms are further classified into Gα_q and Gα15/16 subfamilies. Another Ciona G_q-class isoform, CiGα_x, is fairly diverged both from the Gα_qand Gα15/16 subfamilies. Therefore, CiGα_x may be a member of a novel Gα subfamily. It will be interesting to see whether Gα closely related to CiGα_x, and is present in other animals, including vertebrates.

Based on their primary structure, G_i-class isoforms are further classified into four distinct subfamilies: Gα_i, Gα_o, Gα_t, and Gα_z (Suga et al., 1999). All three G_i class members of Ciona belong to the Gα_i subfamily, and so far no ascidian Gα isoforms have been assigned to the Gα_t, Gα_o, and Gα_z subfamilies. Since Gα_o isoforms have been reported in diverse invertebrate phyla, including sponges (Suga et al., 1999), arthropods (Thambi et al., 1989; Horgan et al., 1995), and molluscs (Kojima et al., 1997; Iwasa et al., 2000), it is likely that ascidians also have this isoform. G_o is abundant in the CNS both in vertebrates (Strathmann et al., 1990) and in insects (Thambi et al., 1989; Horgan et al., 1995), and play important roles in the function and development of the nervous systems (Offermanns 2001). Therefore, future studies are needed to clarify whether Gα_o isoforms are present in ascidians.

Alternative splicing of CiGα _i1 transcripts

A comparison of the CiGα_i1a and CiGα_i1b cDNA sequences and the genomic structure of the CiGα_i1 gene strongly suggest that CiGα_i1a and CiGα_i1b are products of alternative splicing. In vertebrate, splice variants of Gα_s, Gα_i, and Gα_o are known. Alternative splicing of the mammalian Gα_o gene generates two different isoforms of Gα (Tsukamoto et al., 1991). These isoforms, Gα_o1 and Gα_o2, are identical to each other in the N-terminal 248 amino acids; the sequences thereafter diverge. Gα_o1 and Gα_o2 result from alternative splicing of exons 7 and 8. The diverged carboxyl-terminus contains an effector-interacting domain. Therefore, Gα_o1 and Gα_o2 exhibit different properties in signal transduction (Kleuss et al., 1991). There are four splice variants of mammalian Gα_s (Kozasa et al., 1988). These Gα_s variants directly activate adenylate cyclases and calcium channels (Mattera et al., 1989). The relative proportion and tissue distribution of the two variants of Gα_s change during cellular differentiation, development, aging, and adaptive processes (Ihnatovych et al., 2001). Mammalian Gα_i proteins are encoded by three different genes, Gα_i1, Gα_i2, and Gα_i3, which are closely-related to each other (Itoh et al., 1988). Transcripts of the mammalian Gα_i2 gene undergo alternative splicing, which in turn give rise to two distinct proteins with different carboxyl-terminal amino acid sequences (Montmayeur and Borrelli 1994). The Gα_i2 vari-ants exhibit differential cellular localization and function. Multiple Gα isoforms are also produced by alternative splicing of a single Gα_o gene in Drosophila (de Sousa et al., 1989). Therefore, alternative splicing seems to be a common mechanism to produce diversity of Gα isoforms in a wide variety of animals. Interestingly, the way alternative splicing occurs differs between CiGα_i1 and vertebrate Gα genes, suggesting that these alternative splicing events evolved independently in ascidians and in vertebrates. In future studies, it will be very important to investigate and learn the functional differences between CiGα_i1a and CiGα_i1b in the regulation of signal transduction during ascidian development.

Spatially restricted expression of Ciona Gα genes and roles of G-protein signaling in ascidian embryos and larvae

The present study demonstrated that multiple Gα genes are expressed with distinct expression patterns in Ciona embryos. Although autonomous cell-fate specification is a dominant mechanism in ascidian embryos, the importance of cell-cell communications has increasingly become evident in the determination and differentiation of embryonic cells in ascidians, especially in their nervous systems (Meinertzhagen and Okamura 2001; Wada and Satoh 2001; Darras and Nishida 2001). It is quite probable that G proteins mediate signaling in these cell-cell interactions during ascidian development. It is also known that G proteins are located on intracellular membranes, and are involved in membrane trafficking and vesicular transport mechanisms of the cell (Nürnberg et al., 1995). Therefore, some of the Ciona Gα isoforms may participate in these cellular activities.

Among the four Gα genes identified in this study, CiGα_q and CiGα_i1 showed distinct and spatially restricted expression patterns. In the tailbud stage, CiGα_q is expressed in the anterior and dorsal trunk epidermis and the tail tip. Trunk regions expressing CiGα_q seem to include the future adhesive organ and siphon rudiments (Nakayama et al., 2001). The CiGα_q protein may be involved in cell signaling during development of these organs.

From fertilization to the gastrula stage, CiGα_i1 mRNA is present ubiquitously as maternal messages. Therefore, CiGα_i1 may mediate signaling between blastomeres during the cleavage stages. Later in embryogenesis, the CiGα_i1 expression is restricted to the CNS and the adhesive organ. This expression pattern suggests that CiGα_i1 isoforms play important roles in the development and function of the nervous systems which require intracellular signaling in various aspects.

Recently, we have reported that Ciona larvae express a vertebrate-type opsin gene Ciopsin1 in the photoreceptor cells of the ocellus (Kusakabe et al., 2001). In vertebrates, G_t (transducin) are responsible for signal transduction in the photoreceptor cells of the retina by coupling with rhodopsin, while invertebrate opsins activate G_q and G_o (Tsuda and Tsuda, 1990). However, our extensive search has failed to find Gα_t in the Ciona EST and genome sequence databases. Interestingly, both G_t and G_o belong to the G_i class and the vertebrate rhodopsin can activate G_i in vitro (Terakita et al., 2002). We have shown that CiGα_i1 is expressed in the brain vesicle including photoreceptor cells. Therefore, CiGα_i1 may interact with Ci-opsin1 in phototransduction of the ascidian larval ocellus. Since no Gα_t has been reported in invertebrates to date, Gα_t may have appeared during early vertebrate evolution after the separation between vertebrates and urochordates.