The endostyle is present in urochordates, cephalochordates, and larval lampreys (ammocoetes). This organ secretes mucoproteins for filter feeding and incorporates iodine into iodoproteins thyroglobulin in the lamprey and unidentified iodoproteins in amphioxus and tunicates (e.g., Fredriksson et al., 1985, 1988). Because of these thyroid-like property and because in lamprey it is transformed into the follicular thyroid during metamorphosis (Wright and Youson, 1976), the endostyle is thought to be the precursor of the vertebrate follicular thyroid (e.g., Salvatore, 1969).
In ascidians and amphioxus, the endostyle primordium originates from the anteroventral part of the pharyngeal endoderm and form a groove on the ventral surface of the pharyngeal epithelium. Differentiated endostyles in invertebrate chordates are histologically divided in numbers of functional units called “zones” that extend anteroposteriorly in the epithelium. As summarized in Figure 4, both ascidian and amphioxus endostyles are numbered bilaterally from midventral to dorsolateral (Thomas, 1956; Barrington, 1957, 1958; Thorpe et al., 1972; Dunn, 1974; Fujita and Sawano, 1979; Fredriksson et al., 1985). The ascidian endostyle consists of nine zones, including supporting zones (zones 1, 3, and 5), three-pairs of glandular zones (zones 2, 4, and 6), and iodine-binding zones (zones 7, 8, and 9). The amphioxus endostyle contains seven functional compartments such as supporting (zones 1 and 3), two pairs of glandular (zones 2 and 4), and iodine-binding zones (zones 5a, 5b, and 6). On the other hand, the endostyle of the larval lamprey has a complicated structure, which possesses two-types of glandular cells (type 1v and 1d) and thyroid-equivalent cells (type 2c and 3; e.g., Egeberg, 1965; Fujita and Honma, 1968). All the endostyles have a similar configuration of glandular components found midventrally and of iodine-binding components dorsolaterally, suggesting their common origin.
In previous studies, we characterized in the ascidian endostyle the expression patterns of several thyroid-related genes. For instance, in Ciona, the ortholog of TPO (thyroid peroxidase), a differentiation marker of the vertebrate thyroid was found to be expressed exclusively in the thyroid-like region of zone 7 (Ogasawara et al., 1999a), and the ortholog of TTF-1 (thyroid transcription factor-1) was expressed in the endostyle primordium and zones 1, 3, and 5 of the adult endostyle (Ogasawara et al., 1999b). Likewise, amphioxus TPO was expressed in zones 5 and 6, and TTF-1 expression was detected mainly in zones 1, 3, 5, and 6 of the endostyle (Ogasawara, 2000). In lamprey, TTF-1 was detected in the zone 2a, 2b, and 2c cells in the endostyle (Ogasawara et al., 2001b). These analyses further support the common origin of the invertebrate endostyle and glandular follicles of the vertebrate thyroid from a molecular point of view.
On the other hand, TTF-2 (thyroid transcription factor-2) regulates the expression of several thyroid differentiation marker genes (Zannini et al., 1997; Damante et al., 2001). TTF-2 belongs to the FoxE class Fox gene family, which is divided into three groups, E1 (TTF-2), E2, and E3 (Kaestner et al., 2000). Of interest, the other FoxE genes are not expressed in the thyroid but in tissues like eye lens, brain, and some ganglia of intestine (Blixt et al., 2000; Brownell et al., 2000). Yu and colleagues (2002) characterized an amphioxus TTF-2 ortholog that they called AmphiFoxE4. Of interest, expression of AmphiFoxE4 in larval Branchiostoma floridae was detected in the club-shaped gland, an amphioxus-specific organ located near the endostyle. However, Ci-FoxE, which is phylogenetically close to AmphiFoxE4, was expressed in zone 7, which is the iodine-binding region of the adult ascidian endostyle (Ogasawara and Satou, 2003). Therefore, we characterized the FoxE4 expression pattern in the adult amphioxus endostyle.
Pax2/5/8, a transcription factor gene with paired-box, in ascidians and amphioxus retains nucleotide sequence similar each to vertebrate Pax2, Pax5, and Pax8. Although Pax2 and Pax5 have no evidence of functional relationships with thyroid development, Pax8 is associated with development and function of the mammalian thyroid (Damante et al., 2001). Amphioxus AmphiPax2/5/8 (Kozmik et al., 1999) and lamprey LjPax2/5/8 (Murakami et al., 2001) are expressed in their larval endostylar primordiums, as well as at midbrain–hindbrain boundary of the neural tube. In ascidians, similar expression patterns of Hr-Pax258 and Ci-Pax2/5/8 in the neural tube were reported (Wada et al., 1998; Imai et al., 2002). However, little is known about expressions of Pax2/5/8 orthologs in their adult endostyles. Therefore, comparative expression analysis of Pax2/5/8 ortholog is also intriguing.
In the present study, we investigated the expression patterns of several thyroid-related transcription factor genes such as FoxE4 and Pax2/5/8 in amphioxus Branchiostoma belcheri. We also observed the expression patterns of Fox class genes such as FoxQ1 and FoxA, which are intensively expressed in the endostyle (or have intensive expression domains in the endostyle), to understand phylogenetic relationship of each zone in the ascidian and amphioxus endostyles.
RESULTS AND DISCUSSION
Identification of FoxE4 and FoxQ1 in Amphioxus Branchiostoma belcheri
An amphioxus ortholog of FoxE4 was obtain by reverse transcriptase polymerase chain reaction (RT-PCR) from a pooled mRNA expressed in B. belcheri endostyle using primers designed from the B. floridae FoxE4 sequence. Because the nucleotide sequence of this amplified cDNA was nearly identical to the sequence of AmphiFoxE4, we named it BbFoxE4 (DDBJ/EMBL/GenBank accession no. AB193514). Overall identity of the nucleotide and amino acid sequences of the forkhead domain between the two species were 97.0% and 100%, respectively. Molecular phylogenetic analysis based on the amino acid sequences of the forkhead domain confirmed that BbFoxE4 makes a cluster with AmphiFoxE4 and Ci-FoxE. These proteins form a clade at the base of the vertebrate FoxE proteins such as FoxE1, FoxE2, and FoxE3 (Fig. 1A).
FoxQ1 genes have been isolated from mouse (Hong et al., 2001) and an ascidian C. intestinalis (Ogasawara and Satou, 2003). Recently, amphioxus FoxQ1 was also reported in B. floridae (Mazet et al., 2005). By using a set of degenerated primers targeting the forkhead domain of these proteins, we amplified a fragment from endostyle mRNA of B. belcheri. We subsequently screened an amphioxus endostyle cDNA library and isolated a full-length cDNA clone, which encodes a protein with a forkhead domain similar to mouse FoxQ1 and Ciona Ci-FoxQ with 86.6% and 91.2% identities, respectively (boxed in Fig. 1B). A neighbor-joining phylogenetic analysis confirmed that this clone groups with the ascidian Ci-FoxQ protein and with the mouse FoxQ1 (Fig. 1A). These clades were supported by bootstrap values of 71% and 100%, respectively. We named the gene BbFoxQ1. The nucleotide sequences will appear under the DDBJ/EMBL/GenBank accession no. AB193512.
FoxE4 Is Expressed in the Adult Endostyle of Amphioxus
In situ hybridization of BbFoxE4 showed that expression of FoxE4 ortholog in amphioxus larvae was localized in the club-shaped gland (data not shown), as Yu and colleagues (2002) have reported. However, in adults, it was detected mainly in the endostyle and localized in zone 6 (Fig. 2A,B) without exceptions, with occasional expression in zone 5a (inset in Fig. 2B). In ascidian, expression of Ci-FoxE was not detected in larva but was observed in the developing endostyle of juveniles. In the adult endostyle, Ci-FoxE was expressed mainly in zone 7 that corresponds to the iodine-binding component (Ogasawara and Satou, 2003). These observations confirm that FoxE4 is coexpressed with TTF-1 and TPO in the iodine-binding region of the invertebrate chordate endostyles.
Yu and colleagues (2002) proposed an interesting scenario about the function of FoxE genes related to the thyroid evolution, in which the early morphogenetic role of FoxE class genes is the budding of organ rudiments off from the pharyngeal epithelium, because AmphiFoxE4 associates with the evagination of the club-shaped gland from the pharyngeal endoderm. They also suggested that the genetic pathway, including AmphiFoxE4 might have been recruited from a neighboring region to the endostyle during the rise of the vertebrates. Their alternative interpretation is the genetic transfer from the endostyle to the club-shaped gland in the cephalochordate lineage. A third possibility is that the thyroid results from the fusion of the endostyle and a club-shaped gland at the dawn of the vertebrate evolution (Mazet, 2002). Our observations of BbFoxE4 and Ci-FoxE expression in the iodine-binding regions of the adult endostyles strongly suggest that the role of FoxE genes in the thyroid arose before the vertebrate lineage and that their function in the pharyngeal tissue might not be associated to a budding process but rather to regulation of thyroid-specific genes to be expressed later in development. To specify the role of FoxE4 in genetic mechanisms underlying the evolution of the vertebrate thyroid, further study on developing and differentiated endostyles of chordates is required.
FoxQ1 Is a Marker Gene for Phylogenetic Estimation of Endostylar Components
Because the allocation of the functional components are divergent between the amphioxus and Ciona endostyles (Fig. 4), further comparative expression analyses using orthologous genes, in particular genes expressed in zone-specific manner in the endostyle, is indispensable to assess phylogenetic relationship of each zone between both lineages. A candidate gene is FoxQ1, which has such a zonal expression pattern in zones 3, 5, 7, and the lateral part of zone 8 in the ascidian endostyle (Ogasawara and Satou, 2003). In adult amphioxus, a similar expression pattern was observed in zones 3, 5a, and 6 of the endostyle (Fig. 2C,D). Furthermore, expression of BbFoxQ1 in the dorsal and ventral blood vessels of amphioxus larvae (data not shown) and that of Ci-FoxQ in the blood vessels of ascidian juveniles (Ogasawara and Satou, 2003) resemble each other. These similarities suggest that the regulatory mechanisms governing FoxQ1 expression may be conserved in both animals.
Comparative Analysis of Pax2/5/8 and FoxA Expressions Between Ascidian and Amphioxus Endostyles
Comparative expression studies of transcription factors in different taxa facilitates the understanding of development and function of the endostyle. In addition, from an evolutionary viewpoint, the expression pattern of thyroid markers is essential to understand the origin of this organ and possible phylogenetic relationships between thyroid and endostyle.
A paired-box gene, Pax2/5/8, is comparable to Pax8, which plays a role in development and function of the thyroid (Damante et al., 2001). In invertebrate chordates, although embryonic expression of Hr-Pax258 and Ci-Pax2/5/8 in ascidians and AmphiPax2/5/8 in amphioxus have been reported, expressions in their adult endostyle have not yet been observed. The sequencing of the C. intestinalis genome and gene analyses for other urochordates revealed that, in this animal group, Pax2/5/8 has been duplicated into two genes, in general (Wada et al., 2003). In C. intestinalis, expression of a paralog, Ci-Pax2/5/8-A, was detected in zones 3 and 5 of the differentiated endostyle of juveniles (Fig. 3A) and was found in zones 5 and 7 (Fig. 3B) but not in zone 3 in adults. These domains of expression, thus, seem to correspond to those of amphioxus BbPax2/5/8. Expression of the other paralog, Pax2/5/8-B, was not observed in juvenile and adult endostyle (data not shown). To examine the expression pattern of the B. belcheri counterpart both in larval and adult states, we obtained a PCR fragment of BbPax2/5/8 for B. belcheri (DDBJ/EMBL/GenBank accession no. AB193513). As in B. floridae, BbPax2/5/8 was found in the larval endostylar primordium (data not shown) and localized in zones 3, 5a, 5b, and 6 in adult state (Fig. 3C,D).
Multiple whole-mount in situ hybridization approaches using Ciona juveniles reported previously (Ogasawara et al., 2002) yielded several candidates of marker genes helpful to analyze the phylogenetic relationship between ascidian and amphioxus endostyles. One of them is the Ciona FoxA ortholog named Ci-fkh/FoxA-a (Corbo et al., 1997; Imai et al., 2004), which was expressed in the juvenile endostyle (Fig. 3E). In the adult endostyle, FoxA transcripts were detected mainly in zones 3 and the medial part of zone 8, as well as faintly in zone 1 (Fig. 3F). In amphioxus B. belcheri, FoxA ortholog was isolated and named BbHNF-3-1 (Terazawa and Satoh, 1997). BbHNF-3-1 expression was detected in the endostylar primordium of larvae (Fig. 3G). In adults, it was expressed mainly in zone 3 and the region lateral to zone 6, as well as faintly in zone 1 (Fig. 3H,I). Similarities in expression patterns of FoxA between the ascidian and amphioxus endostyles suggest that common mechanisms of FoxA expression might be retained in these animals.
Histological Similarity and Diversity Between Endostyles
Although the zonal pattern of endostyles differs between ascidian and amphioxus (Fig. 4A,B), both show a common symmetrical pattern, including supporting, glandular, and iodine-binding zones. The alignment of each zone in the half of the endostyle suggests some phylogenetic relations. Figure 4C,D summarizes the gene expression patterns examined in the present study, including TPO (Ogasawara et al., 1999a; Ogasawara, 2000). Histological characters and gene expression patterns are almost identical in zones 1 to 4 of both ascidian and amphioxus endostyles, suggesting some shared mechanisms of gene regulation. Expression analyses of amphioxus orthologs to Ciona CiEnds3, Ci-vWFL1, and Ci-vWFL2, which have specific expression patterns in each glandular zone (Ogasawara and Satoh, 1998; Sasaki et al., 2003), might give a phylogenetic story for these zones between the two animals, whereas the region lateral to zone 4 appears to be more complicated and difficult to compare. For instance, (1) the ascidian endostyle has a surplus pair of glandular zones, which have no counterparts in amphioxus; (2) the ascidian TTF-1 is not expressed in the iodine-binding region; and (3) expression patterns of Pax2/5/8 and TPO are different between ascidian and amphioxus. Despite these complexities, almost identical expressions of FoxE4, FoxQ1, and FoxA suggest a phylogenetic relationship at least between zones 5 to 8 of the ascidian endostyle and zones 5a to the region lateral to zone 6 of the amphioxus endostyle.
In the agnathan vertebrate lamprey, the structure of the endostyle seems to be more complicated with increasing number of zones; however, two types of glandular zones and expression of the lamprey TTF-1 ortholog suggest a phylogenetic connection with the amphioxus endostyle (Ogasawara et al., 2001b). In some thaliaceans such as doliolid species of Dolioletta gegenbauri and Doliolum 2nationalis, two pairs of glandular zones (zones 2 and 4) are located medial to the iodine-binding zone (zone 5b), being separated by zone 5a (Fredriksson et al., 1988), which corresponds to the amphioxus configuration. However, other thaliaceans such as salp species of Salpa fusiformis and Thalia democratica have three pairs of glandular zones as seen in Ciona. In the larvacean lineage, Oikopleura dioica possesses only a pair of glandular zones. Owing to these variations, the basic structure of the endostyle in the latest common ancestor of chordates is still unclear, and further comparative expression analyses in these species might provide more opportunities to figure out the phylogenetic relationship of the endostylar architecture. In particular, Ciona genes expressed in glandular zones such as CiEnds1, CiEnds2 (Ogasawara and Satoh, 1998), and genes mentioned above might elucidate the addition and/or elimination process of glandular component during the evolution of chordates.
Adult specimens of amphioxus B. belcheri were collected at Ariake-Kai near the Aizu Marine Biological Station of Kumamoto University, Kumamoto, Japan, and fixed for in situ hybridization as described in Ogasawara (2000). Collection and fixation of amphioxus larvae was carried out as described in Yasui et al. (2000). Juveniles and adults specimens of ascidian C. intestinalis were prepared as described in Ogasawara et al. (2002).
Isolation and Sequencing of cDNA Clones for B. belcheri FoxE4, Pax2/5/8 and FoxQ1 Genes
Extraction of endostyle poly(A)+ RNA and construction of a cDNA library of B. belcheri were carried out as described in Tanaka et al. (1996). Fragments of FoxE4 and Pax2/5/8 were amplified by RT-PCR from endostyle mRNA, and PCR products of 507-bp and 563-bp were obtained, respectively. Oligonucleotide primers for FoxE4 were designed of the basis of the AmphiFoxE4 sequence: FoxE4-F, 5′-TACAGCTACATCGCGCTGATCTCGATG-3′; and FoxE4-R, 5′-GATGATGTTGTCGATGCTGAACAT-3′. Oligonucleotide primers for Pax2/5/8 were designed based on the sequence of AmphiPax2/5/8: Pax2/5/8-F, 5′-TGTGACAACGACACAGTTCCCAG -3′; and Pax2/5/8-R, 5′-GGAACAGCAACTGGATAGTGGCC- 3′. The sense-strand oligonucleotide primer for FoxQ1 (FoxQ1-F, 5′-TA(CT)AT(ACT)GCI(CT)TIAT(ACT)GCIATGGC-3′, which corresponds to the amino acid sequence YIALIAMA), and the antisense oligonucleotide primer FoxQ1-R (5′-AANACICC(AG)TCNGC(AG)AAIGT(AG)TAITC-3′, which corresponds to the amino acid sequence EYTFADGVF), were synthesized based on the sequence of the conserved forkhead domains of Ci-FoxQ and mouse FoxQ1. A fragment of 257-bp was amplified by RT-PCR from the endostyle mRNA and subcloned into pGEM-T vector (Promega). Several cDNA clones for FoxQ1 were screened using 1.2 × 105 phages of the B. belcheri endostyle cDNA library as described by Ogasawara (2000). Amplified cDNA fragments and full-length cDNA clones were sequenced using an ABI PRISM 310 DNA Sequencer (Perkin Elmer). Digoxigenin (DIG) -labeled RNA probe were synthesized and purified using a centrifugal ultrafilter as described in Ogasawara et al. (2001a).
Sequence Comparison and Molecular Phylogenetic Analysis
Amino acid sequences of the Fox proteins were aligned using the SeqQpp 1.9 manual aligner for Macintosh (Gilbert, 1993). Phylogenetic analyses were performed using the deduced amino acid sequences of the forkhead domain of Fox family gene products using the neighbor-joining method (Saitou and Nei, 1987) included in the CLUSTAL V software (Higgins et al., 1992). Confidence in the phylogeny was assessed by bootstrap resampling of the data (×100; Felsenstein, 1985).
In Situ Hybridization
Whole-mount in situ hybridization was carried out as described by Ogasawara (2000) and Sasaki et al. (2003). Antisense DIG-RNA probes were synthesized by following the instructions from the supplier of the labeling kit (DIG RNA Labeling kit; Roche). After whole-mount in situ hybridization, some specimens were embedded in polyester wax (BDH Chemicals, Ltd.) and sectioned at 10-μm intervals for observation at high magnification.
We thank Dr. G. Satoh for providing the BbHNF-3-1 cDNA clone, and Dr. T. Kusakabe for providing Ciona Pax2/5/8-A and Pax2/5/8-B cDNA clones. We thank Prof. Y. Henmi, Mr. Shimazaki, and Mr. Kai of the Aizu Marine Biological Station of Kumamoto University for collecting specimens of adult amphioxus.