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Isolation and characterization of endostyle-specific genes in the ascidian Ciona intestinalis.

Introduction

The endostyle is a specialized organ in the pharynx of tunicates, cephalochordates, cyclostomates, and certain prosobranchiates (Orton, 1912). The ascidian endostyle forms a trough-shaped structure in the ventral wall of the pharynx which extends from the fore-part of the pharynx to the esophagus [ILLUSTRATION FOR FIGUREs 1A AND 2D OMITTED]. In 1834, Lister first investigated how food particles in the feeding current are trapped in the pharynx of appendicularians. Fol (1876) found that the food is trapped by a mucous substance produced from the endostyle of tunicates. Thereafter, the ascidian endostyle has been intensively investigated by histological and ultrastructural observations (Olsson, 1963, 1965; Aros and Viragh, 1969; Fujita and Nanba, 1971); by the examination of 125I incorporation (Thorpe et al., 1972; Dunn, 1974); by histochemical detection of thyroperoxidase (Fujita and Sawano, 1979); and by partial purification of thyroperoxidase and its enzyme activity (Dunn, 1980).

It is commonly considered that the endostyle of lower chordates may be a homolog and primitive antecedent of the vertebrate thyroid gland, mainly because the organ incorporates iodine (Barrington, 1957, 1958; Salvatore, 1969). However, the endostyle is a mucus-secreting and food-collecting organ, and the ability to concentrate iodine is restricted to a small region of the organ (Olsson, 1963). The general organization of the ascidian endostyle is depicted in Fig. 1B. The cells of this organ are differentiated into eight or nine strips, or zones, that run parallel to one another in longitudinal orientation. The cells of each zone are highly specialized in morphology and function. The cells of zones 7, 8, and 9, like the thyroid cells of higher vertebrates, have an iodine-concentrating activity. The cells of zones 2, 4, and 6 have numerous secretory granules. These cells are believed to secrete the proteins or mucoprotein related to the digestion of food. The cells of zones 1, 3, and 5 are considered supporting elements and also as elements that might play a role in catching and transporting food.

We are interested in molecular developmental mechanisms that permitted or accelerated the advent of chordates. The phylum Chordata consists of the subphyla Urochordata (tunicates), Cephalochordata (amphioxus), and Vertebrata. Chordates are categorized as deuterostomes, along with two other invertebrate groups, echinoderms and hemichordates, as was supported by molecular phylogenic studies (Wada and Satoh, 1994; Turbeville et al., 1994). Chordates share several characteristic features including a notochord, a dorsal hollow nerve cord, and pharyngeal gill slits. In addition, lower chordates (including tunicates, amphioxus, and lampreys) share an endostyle. We have emphasized that these hallmarks of the chordate body plan apparently evolved with the emergence of creatures resembling tadpole larvae (Satoh, 1995; Satoh and Jeffery, 1995). Therefore, investigations of the organization of these structures are of salient importance in attempts to understand the origin of chordates.

Coincidently with this change in the mode of larval locomotion, most of the primitive chordates or chordate ancestors may have shifted their feeding system to the use of pharyngeal gill slits for extracting suspended food from the water and of an endostyle for secreting mucus to catch the food particles (e.g., Brusca and Brusca, 1990). This possibility suggests that, in addition to the notochord and nerve cord, the pharyngeal gill and endostyle are key organs that can be used to explore molecular mechanisms involved in the emergence of chordates. In other words, we believe that the origin and evolution of chordates may be approached by isolating genes specific to the pharyngeal gill or endostyle and by analyzing how these genes are organized during evolution in various deuterostomes. In previous studies, we isolated cDNA clones for pharyngeal gill-specific genes (HrPhG1 and HrPhG2; Tanaka et al., 1996). and for endostyle-specific genes (HrEnds1 and HrEnds2; Ogasawara et al., 1996) from the ascidian Halocynthia roretzi, which belongs to the order Pleurogona. Both endostyle-specific genes are expressed in zone 6 and encode secreted proteins. In the present study, we attempted the isolation of cDNA clones for endostyle-specific genes from Ciona intestinalis, which belongs to the order Enterogona. If both ascidian species conserve endostyle-specific structural genes, they may serve as models for the investigation of the molecular mechanisms involved in the organization of other chordate groups.

Materials and Methods

Biological materials

Adults of Ciona intestinalis, C. savignyi, and Styela clava were collected near the Marine BioSource Education Center of Tohoku University, Onagawa, Miyagi and Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo, Iwate, Japan. After the dissection of adult specimens, tissues and organs were quickly frozen in liquid nitrogen, and kept at -80 [degrees] C until use.

Isolation of RNAs and construction of cDNA libraries

Total RNA was extracted from the endostyle and pharyngeal gill of C. intestinalis by the AGPC method (Chomczynski and Sacchi, 1987). [Poly(A).sup.+] RNA was purified with oligotex dT30 beads (Roche Japan, Tokyo). Complementary DNA was synthesized and cDNA libraries were constructed as described in a previous report (Ogasawara et al., 1996). An endostyle cDNA library was constructed using a uni-ZAP-II vector (Stratagene, La Jolla, CA).

Isolation and sequencing of cDNA clones for endostyle-specific genes

The endostyle cDNA libraries were screened differentially. Duplicate filters of the library were made; one was hybridized with a [32P]-labeled total cDNA probe prepared from 5 [[micro]gram] of [poly(A).sup.+] RNA of endostyle under high-stringency conditions, and the other was hybridized with a [32P]-labeled total cDNA probe of pharyngeal gill under the same conditions. Plaques that showed positive hybridization with the endostyle probe but were negative for the pharyngeal-gill probe were selected and isolated by two rounds of screening. The specificity of the clones positive for the endostyle was confirmed by a Northern blot analysis. The clones were prepared for sequencing by controlled nested deletion from either the T3 or T7 side and sequenced using the ABI PRISM 377 DNA Sequencer (Perkin Elmer, Norwalk, CT).

Northern blot analysis

The Northern blot hybridization was carried out by the standard procedure (Sambrook et al., 1989), and the filters were washed under high-stringency conditions. DNA probes for blot hybridizations were labeled with [32P]-dCTP using a random primed labeling kit (Boehringer Mannheim, Heidelberg, Germany).

In situ hybridization

Juveniles of C. intestinalis, C. savignyi, and Styela clava were fixed in 4% paraformaldehyde in 0.5 M NaCl, 0.1 M MOPS buffer at 4 [degrees] C for 12 h. Probes were synthesized by following the instructions from the supplier of the kit (DIG RNA Labeling kit; Boehringer Mannheim). The in situ hybridization of whole-mount specimens was carried out basically as described previously (Ogasawara et al., 1996). For the in situ hybridization of sectioned specimens, samples were dehydrated with a graded series of alcohol, embedded in polyester wax (BDH), and sectioned at 6 [[micro]meter].

Results

As in the case of the isolation of cDNA clones for the endostyle-specific genes of H. roretzi (Ogasawara et al., 1996), differential screenings of a C. intestinalis endostyle cDNA library with total cDNA probes for the endostyle and pharyngeal gill yielded several cDNA clones specific to or enriched in the endostyle library. The preliminary in situ hybridization analysis of sectioned specimens demonstrated that transcripts of four cDNA clones were specific to the endostyle. We named the corresponding genes CiEnds1 (Ciona intestinalis endostyle gene 1), CiEnds2, CiEnds3, and CiEnds4. During the screening procedures, we noticed that the CiEnds1 transcript was abundant in the library, representing nearly 10% of the library clones. When 1 [[micro]meter] of [poly(A).sup.+] RNA of the endostyle was electrophoresed, we detected the transcript as a band stained with 0.5 [[micro]gram]/ml ethidium bromide (data not shown).

Characterization of cDNA clone for CiEnds 1

As shown in Figure 2C, the Northern blot analysis of the CiEnds1 transcript in various tissues and organs of a C. intestinalis adult detected the transcript of about 2.3 kb only in the endostyle. Hybridization signals were not detected in the pharyngeal gill, body-wall muscle, intestine, or gonad.

The nucleotide sequence of the cDNA clone for CiEnds1 will appear under the accession number of AB010895 in the DDBJ, EMBL, and GenBank nucleotide sequence databases. The insert of the cDNA clone consisted of 2265 nucleotides, including 17 adenyl residues at the 3[prime] end. The clone contained a single open reading frame (ORF) of 1950 nucleotides, which predicted a polypeptide of 650 amino acids [ILLUSTRATION FOR FIGURE 2A OMITTED]. The calculated molecular mass (Mr) of the CiEnds1-encoded protein (CiENDS1) was 75.5 k.

CiENDS1 did not show any sequence motifs shared by transcriptional factors, a transmembrane domain, nuclear localization signals, or motifs found in growth factor proteins. However, as shown in Figure 2B, the mean hydropathy profiles of CiENDS1 showed that the N-terminus was highly hydrophobic. This region had a typical signal peptide sequence that consisted of a positively charged residue (amino acid position 2; K, Lys), a hydrophobic (3-13) region of 10-15 residues, a charged residue (position 15; S, Ser), and a residue containing the short side chain (position 16; A, Ala). A predicted cleavage site of the signal peptide was evident behind the Ala (position 16). This sequence motif strongly suggests that CiENDS1 is a secretory protein, with a probability of 82% determined by using the PSORT Program (Online. PSORT World Wide Web Server: Available: http://psort.nibb.ac.jp). In addition, CiENDS1 contained four putative N-linked glycosylation sites [ILLUSTRATION FOR FIGURE 2A OMITTED].

The in situ hybridization of whole-mount specimens demonstrated that the signals were restricted to the endostyle [ILLUSTRATION FOR FIGURE 2D OMITTED]. In addition, the in situ hybridization of sectioned specimens demonstrated that the signals were not distributed over the entire regions of the endostyle but rather were restricted to zone 6 [ILLUSTRATION FOR FIGURE 2E OMITTED]. No signals above background level were found in the control specimen hybridized with the sense probe (data not shown).

Characterization of cDNA clone for CiEnds2

The nucleotide sequence of cDNA clone for CiEnds2 will appear under the accession number of AB010896 in the DDBJ, EMBL, and GenBank nucleotide sequence databases. The insert of the cDNA clone consisted of 2107 nucleotides, including 25 adenyl residues at the 3[prime] end. The occurrence of a 2.3-kb-long CiEnds2 transcript only in the endostyle [ILLUSTRATION FOR FIGURE 3C OMITTED] suggested that the cDNA was close to full-length. The clone contained a single ORF of 1950 nucleotides, which also predicted a polypeptide of 650 amino acids [ILLUSTRATION FOR FIGURE 3A OMITTED]. The calculated Mr of the CiEnds2-encoded protein (CiENDS2) was 77.3 k.

CiENDS2 may also be a secretory protein. As shown in Figure 3B, the mean hydropathy profiles of CiENDS2 showed that the N-terminus was highly hydrophobic. This region had a typical signal peptide sequence that consisted of a positively charged residue (amino acid position 2; K, Lys), a hydrophobic (3-13) region of 10-15 residues, a charged residue (position 15; N, Asp), and a residue containing the short side chain (position 16; A, Ala). A predicted cleavage site of the signal peptide was evident behind the Ala (position 16). However, the results of the PSORT Program calculation showed that CiENDS2 seems to have an uncleaved N-terminal signal sequence. In addition, CiENDS2 contained a putative N-linked glycosylation site [ILLUSTRATION FOR FIGURE 3A OMITTED]. CiENDS2 showed 48.3% identity at the amino acid level with CiENDS1, a finding that will be discussed later.

The in situ hybridization of whole-mount specimens demonstrated that the signals were restricted to the endo-style [ILLUSTRATION FOR FIGURE 3D OMITTED], whereas that for sectioned specimens demonstrated that the signal was restricted to zone 6 [ILLUSTRATION FOR FIGURE 3E OMITTED]. No signals above the background level were found in the other zones.

Characterization of cDNA clone for CiEnds3

The nucleotide sequence of cDNA clone for CiEnds3 will appear under the accession number of AB010897 in the DDBJ, EMBL, and GenBank nucleotide sequence databases. The insert of the CiEnds3 cDNA was 1319 nucleotides, including 17 adenyl residues at the 3[prime] end. The Northern blot analysis demonstrated the occurrence of a 1.4-kb long CiEnds3 transcript, which was detected only in the endostyle [ILLUSTRATION FOR FIGURE 4C OMITTED]. This suggested that the cDNA was close to full-length. The clone contained a single ORF of 945 nucleotides, which predicted a polypeptide of 315 amino acids [ILLUSTRATION FOR FIGURE 4A OMITTED]. The calculated Mr of the CiEnds3-encoded protein (CiENDS3) was 35.3 k.

CiENDS3 may also be a secretory protein. As shown in Figure 4B, the mean hydropathy profiles of CiENDS3 showed that the N-terminus was highly hydrophobic. This region had a typical signal peptide sequence that consisted of a positively charged residue (amino acid position 2; R), a hydrophobic (3-13) region of 10-15 residues, a charged residue (position 15; T), and a residue containing the short side chain (position 16; C). A predicted cleavage site of the signal peptide was evident behind the Cys (position 16). This sequence motif strongly suggests that CiENDS3 is a secretory protein, with a probability of 58% determined by the PSORT Program.

In addition, CiENDS3 contained a unique repeat of 10 amino acids [ILLUSTRATION FOR FIGURE 4A OMITTED]. The repeat consisted of R(QPCI)(RRPC)I. This type of repeat has not been reported to date in the PDB, SWISSPROT, and PIR databases surveyed.

The in situ hybridization of whole-mount specimens demonstrated that the signals were restricted to the endostyle [ILLUSTRATION FOR FIGURE 4D OMITTED]. In addition, the in situ hybridization of sectioned specimens demonstrated that the signal was restricted to zone 2 [ILLUSTRATION FOR FIGURE 4E OMITTED]. No signals above the background level were found in the other zones.

Characterization of cDNA clone for CiEnds4

The in situ hybridization to isolate endostyle-specific cDNA clones demonstrated that the transcript of another clone (CiEnds4) was restricted to the zones 3, 5, and 7 [ILLUSTRATION FOR FIGURE 5A OMITTED]. Strong signals were evident in zones 3 and 5, supporting elements of the endostyle; weak signals were also evident in zone 7, a putative iodine-concentrating element [ILLUSTRATION FOR FIGURE 5A OMITTED].

The insert of the CiEnds4 cDNA was 1541 nucleotides, including 19 adenyl residues in the 3[prime] end. The clone contained a single ORF of 1125 nucleotides, which predicted a polypeptide of 375 amino acids (data not shown). A cDNA clone for C. intestinalis cytoplasmic actin has been isolated and characterized in the laboratory of Dr. Takahito Nishikata of Konan University, Kobe, Japan (pers. comm.). The determination of the partial nucleotide sequence of CiEnds4 cDNA clone revealed that CiEnds4 encodes a cytoplasmic actin.

The above-mentioned results suggested that the gene or genes for one or more cytoplasmic actin are actively expressed in the supporting elements of the endostyle. We therefore examined, using the CiEnds4 probe, whether the cytoplasmic actin gene is expressed in the supporting elements of the endostyle of two other ascidian species, C. savignyi and Styela clava. As shown in Figure 5C and D, the CiEnds4 probe detected cytoplasmic actin transcripts in zone 3 of both species.

In addition, the Northern blot analysis demonstrated the occurrence of a 1.6-kb-long CiEnds4 transcript not only in the endostyle but also in other organs including the pharyngeal gill, body-wall muscle, intestine, and gonad [ILLUSTRATION FOR FIGURE 5B OMITTED].

Discussion

In the present study, we isolated cDNA clones for four genes (CiEnds1, CiEnds2, CiEnds3, and CiEnds4), which are expressed in different zones of the ascidian endostyle. CiEnds1 and CiEnds2 are expressed in zone 6 and encode peptides with similar amino acid sequences. CiEnds3 is expressed in zone 2 and encodes a polypeptide with a novel repeat of 10 amino acids, tentatively called "ends-repeat." CiEnds4 encodes a cytoplasmic actin that is expressed mainly in zones 3 and 5.

In a previous study, we characterized cDNA clones for two endostyle-specific genes, HrEnds1 and HrEnds2, from the ascidian H. roretzi (Ogasawara et al., 1996). Both genes are expressed in zone 6 and encode secreted proteins. Transcripts of both genes are abundant in the endostyle library; each represents about 10% of the cDNA clones of the library. As revealed by the present study, CiEnds1 is also expressed in zone 6 and encodes a secreted protein. This transcript also represents the most abundant species in the library. These results strongly suggest that zone 6 plays a major role in the secretion of mucus by the endostyle and has functions different from those of zones 2 and 4. This notion is consistent with a previous ultrastructural observation that the size and structure of the secretory granules in zone 6 differ from those of the other glandular zones 2 and 4 (Aros and Viragh, 1969). Zone 6 occupies the largest area in the endostyle and is characterized as the most developed glandular zone, containing abundant endoplasmic reticulum (Fujita and Nanba, 1971). Aros and Viragh (1969) and Fujita and Nanba (1971) reported that zone 6 contains at least two types of secretory granule - a large electrondense granule and a smaller granule. Directly facing the pharynx in zone 6 is a wide exit for secretion; in contrast, zones 2 and 4 have only a very limited exit for secretion (Thorpe et al., 1972).

When the amino acid sequences are compared for CiENDS1 and CiENDS2 [ILLUSTRATION FOR FIGURE 6 OMITTED] and for CiENDS1, HrENDS2, and CiENDS2 [ILLUSTRATION FOR FIGURE 7 OMITTED], these three polypeptides closely resemble each other: the sequence identity was 48.3% between CiEndsl and CiEnds2, 22.2% between CiENDS1 and HrENDS2 (similarity 43.7%), and 22.8% between CiENDS2 and HrENDS2 (similarity 42.6%). Our previous genomic Southern blotting analysis of HrEnds2 suggested that there are some other genes in the H. roretzi genome that contain a sequence similar to that of HrEnds2. In the present study, we isolated two cDNA clones from C. intestinalis that contained a sequence similar to that of HrEnds2. It is therefore likely that CiENDS1, CiENDS2, and HrENDS2 are members of the same protein family, and it is possible that these genes were derived from a common ancestral gene...

The first aim of our studies is to isolate genes that are expressed in certain zones of secretory function, these genes being common in different species that belong to different orders of ascidians. In the present study, we first attempted the screening of C. intestinalis homologs of HrEnds1 and HrEnds2 with low-stringency hybridization conditions, using these genes as probes. Unfortunately, we could not isolate any homologs with the conditions we adopted. Therefore, we next tried the differential screening we used in a previous study (Ogasawara et al., 1996), because we thought that if the nature of the endostyle is the same between C. intestinalis and H. roretzi, we might isolate homologous genes easily using this method. In the present screening, we were able to isolate an endostyle-specific cDNA clone for CiEnds1; this clone was highly expressed in zone 6 and contained a sequence similar to that of HrEnds2. In the further screening, we isolated CiEnds2, which was also expressed in zone 6 and contained sequences similar to that of HrEnds2. Ciends1, CiEnds2, and HrEnds2 may be related to each other and play an important role in the ascidian endostyle, and therefore they are good candidates for future studies.

Interestingly, the CiEnds3 encodes a polypeptide with a novel repeat of 10 amino acids [the core repeat is 8 amino acids (QPCI)(RRPC)]. We tentatively call this the "ends-repeat." Because of its uniqueness and conservation among the repeats, this gene is also a good candidate as a probe to explore common mechanisms involved in development and evolution of the endostyle.

There are some differences in the functions of endostyles among ascidian species. The zones that have iodine-concentrating activities (Kobayashi et al., 1983) and thyroperoxidase activities (Dunn, 1974) are different between H. roretzi, C. intestinalis, and some other ascidians. However, we think that the functions and gene expressions of the endostyle are basically the same. Indeed, the present study isolated related genes, the nature of which (spatiotemporal expression and amount of the transcript) is the same in C. intestinalis and H. roretzi. Furthermore, CiEnds4, which encoded a cytoplasmic actin, was strongly expressed in zone 3 of C. intestinalis, C. savignyi, and S. clava [ILLUSTRATION FOR FIGURE 5A, C, AND D OMITTED]. In future studies, we should use these probes to isolate endostyle-specific genes from cephalochordates, cyclostomes, and hemichordates.

In addition to zones 2, 4, and 6 that secrete mucus, zones 8 and 9 are of special interest because other research has indicated that the vertebrate thyroid gland may be homologous with the endostyle of tunicates (Dunn, 1974; Fujita and Sawano, 1979; Kobayashi et al., 1983), cephalochordates (Tsuneki et al., 1983; Fredriksson et al., 1985; Ericson et al., 1985), and larval lampreys (Egeberg, 1965; Fujita and Honma, 1969). In this and previous studies, we were not able to isolate cDNA clones for genes expressed in zones 8 and 9. The isolation of cDNA clones for genes specific to the thyroid-equivalent elements is very important for elucidating the evolutionary relationship of these elements among ascidians. In addition, the isolation and characterization of genes that are involved in the structure and function of vertebrate thyroid glands are of particular interest for future studies.

Acknowledgments

We thank all of the staff members of the Marine BioSource Education Center of Tohoku University, Onagawa, Miyagi and the Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo for their hospitality. We also thank Kazuko Hirayama for her technical assistance. M.O. is a predoctoral fellow of JSPS with a Monbusho research grant (No. 3535). This research was also supported by a Grant-in-Aid for Specially Promoted Research (No. 07102012) from the Monbusho, Japan, to N.S.

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Author:Ogasawara, Michio; Satoh, Noriyuki
Publication:The Biological Bulletin
Date:Aug 1, 1998
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