Evolution of the thyroid: Anterior–posterior regionalization of the Oikopleura endostyle revealed by Otx, Pax2/5/8, and Hox1 expression

Authors


Abstract

The thyroid in vertebrates and its homolog, the endostyle in nonvertebrate chordates, share a molecular code for dorsoventral patterning. Little is yet known, however, about mechanisms that pattern the endostyle's anterior–posterior (AP) axis. To extend our understanding of thyroid development and evolution, we studied Oikopleura dioica, a larvacean urochordate that retains a chordate body plan as adults. Transcription factor expression domains revealed AP regionalization of the endostyle, with expression of Otx rostrally, Hox1 caudally, and two Pax2/5/8 paralogs centrally. Comparative analysis suggested that the endostyle of stem chordates expressed orthologs of these genes and that ancestral subfunctions partitioned differentially among lineages. Because the ordered expression of Otx, Pax2/5/8, and Hox1 displays patterning in both the endodermally derived endostyle and the ectodermally derived central nervous system, we propose that this gene set belonged to the developmental genetic toolkit of stem bilaterians and repeatedly provided AP positional information in various developmental situations. Developmental Dynamics 237:1490-1499, 2008. © 2008 Wiley-Liss, Inc.

INTRODUCTION

The thyroid, located in the neck ventral to the pharynx, is one of the largest endocrine glands in humans and other vertebrates. The thyroid regulates energy production and growth by synthesizing tyrosine-based, iodine-containing hormones (e.g., thryroxine [T4] and triiodothyronine [T3]) and the peptide hormone calcitonin, which regulates levels of calcium and phosphate in the blood. Thyroid dysfunction leads to diseases such as hypothyroidism and hyperthyroidism, thereby deregulating metabolism, causing birth defects, reproductive problems, and behavioral disorders. The thyroid is composed of three main cell types: follicular cells, which absorb iodine and synthesize thyroid hormone; thyroid epithelial cells, which surround and support the follicular cells; and parafollicular cells, which secrete calcitonin. In embryos of humans and other vertebrates, the thyroid primordium develops from the ventral aspect of the second pharyngeal pouch as an out-pocketing of the floor of the pharynx; this primordium ultimately detaches from the pharyngeal epithelium, migrates ventrally, and differentiates into hormone-synthesizing follicular cells in the mature thyroid (Norris,1918; Macchia,2000; De Felice and Di Lauro,2004; Graham et al.,2005). Analysis of mouse knockout mutations and human patients with hereditary thyroid disorders showed that the TTF, PAX2/5/8, TSHR, HOX, TPO, TG, HYT, and FOXE gene families are key players in the genetic regulatory network that controls thyroid development (see Macchia,2000; De Felice and Di Lauro,2004; Parlato et al.,2004; Trueba et al.,2005 and references therein).

The presumed homolog of the thyroid in filter feeding, nonvertebrate chordates (i.e., cephalochordates and urochordates [tunicates]) is the endostyle, an organ located ventral to the pharyngeal floor that secretes food-trapping mucus. This homology, originally based on the reorganization of the larval endostyle into the adult thyroid in lampreys (Muller,1873), is supported by iodine binding, peroxidase activity, thyroglobulin, and calcitonin-like cells both in endostyles and in thyroids (Gorbman and Creaser,1943; Barrington,1957; Thorpe et al.,1972; Thorndyke,1978; Thorndyke and Probert,1979; Kobayashi et al.,1983; Fredriksson et al.,1985,1988).

As in larval lampreys, the endostyles of nonvertebrate chordates are organized into functional zones distributed in the dorsoventral (or mediolateral) axis (Barrington,1958). In a cross-section of the endostyle, these zones are numbered bilaterally from midventral to dorsolateral, and although the exact number and configuration of the zones differs among species, the endostyles in various chordates generally share an overall organization that includes midventral glandular zones, dorsolateral iodine-binding zones, and intervening zones of support cells (Hiruta et al.,2005).

Comparative analyses of the expression patterns of endostyle genes (e.g., Tpo, Ttf-1, Pax2/5/8, FoxE4, FoxQ1, FoxA, Ci-Vwfl, and Ci-Ends) in amphioxus and ascidians help us to understand the evolutionary origin of the vertebrate thyroid (Kozmik et al.,1999; Ogasawara et al.,1999; Ristoratore et al.,1999; Venkatesh et al.,1999; Ogasawara,2000; Murakami et al.,2001; Ogasawara and Satou,2003; Sasaki et al.,2003; Hiruta et al.,2005; Mazet et al.,2005). Results show that a combinatorial code of molecular markers defines each zone of the endostyle across its dorsoventral axis (Hiruta et al.,2005). The conservation of this code of molecular markers between cephalochordates and urochordates reveals structural homologies that support a common evolutionary origin (Hiruta et al.,2005).

Most studies on the evolution of the endostyle and thyroid have focused on the dorsoventral axis, but little attention has been paid to the mechanisms that pattern the anterior–posterior (AP) axis of the endostyle. In this work, we investigated the molecular regionalization of the endostyle in the AP axis during the development of the larvacean urochordate Oikopleura dioica. Larvaceans (or appendicularians) diverged basally in the Urochordate subphylum (Christen and Braconnot,1998; Wada,1998; Swalla et al.,2000; but see a different evolutionary interpretation in Stach and Turbeville,2002). Because the ontogeny of the larvacean endostyle from embryo to adult is not interrupted by metamorphosis as it is in ascidians, the development of the endostyle can be followed directly in Oikopleura from its origin in the pharyngeal epithelium during early embryonic stages until the fully functional endostyle in adult stages less than 24 hr after fertilization; this trajectory can serve as a marker for developmental progression (Troedsson et al.,2007). Our analysis of the developmental expression patterns of the O. dioica orthologs of Otx, Pax2/5/8, and Hox1 revealed an AP axial regionalization of the endostyle in which the most anterior region expresses Otx, the most posterior region expresses Hox1, and the central region expresses Pax2/5/8. Of interest, the central nervous system (CNS) of chordates and some lophotrochozoans and even the diffuse ectodermal nerve net of hemichordates use the same order of expression of the Otx-Pax2/5/8-Hox1 gene set to pattern the AP axis in what has been called a “tripartite” regionalization (Wada et al.,1998; Holland and Holland,1999; Wada and Satoh,2001; Hirth et al.,2003; Lowe et al.,2003). We suggest the hypothesis that the Otx-Pax2/5/8-Hox1 molecular set has been used repeatedly in evolution to provide positional information in the AP axis in the development of a variety of structures, including the endostyle and central nervous system.

RESULTS AND DISCUSSION

Endostyle Morphology in Oikopleura dioica

In 1872, Fol described how the pharynx of larvaceans traps food particles in a mucus secreted by the endostyle (Fol,1872,1876). During the first half of the twentieth century, several authors described the histology and morphology of the larvacean endostyle (Salensky,1904; Ihle,1907; Martini,1909; Lohmann,1933; Berrill,1950). Decades later, high-resolution light and electron micrographs and cytochemistry revealed the cytological structure and distribution of histochemical activities in the larvacean endostyle (Olsson,1963,1965; Fredriksson et al.,1985). To complement past observations on fixed specimens, we investigated live animals by differential interference contrast (DIC) microscopy. Our studies confirmed the presence of typical glandular cells, supporting cells, and iodine-binding cells previously described (Fig. 1; Olsson,1965). The endostyle resides ventral to the pharynx (ph) in the anterior part of the trunk, a position comparable to that of the thyroid in vertebrates (Fig. 1A). The most prominent feature of the Oikopleura endostyle is a single row of large glandular cells (GLC, in gray in Fig. 1B) on each side of the lumen (L), with obvious, large, basally located cell nuclei (N in Fig. 1B, and inset in Fig. 1D) and apically positioned secretory vesicles (S in Fig. 1B, and inset in Fig. 1D) that secrete into the lumen food-trapping mucus rich in mucins and sulfate-containing proteins (Olsson,1965; Fig. 1B–D). A thin epithelial floor made of ciliated cells (CIC) and nonciliated cells (VMC) forms a ventral support for glandular cells (Fig. 1B,C; Olsson,1965). Dorsal to the glandular cells, a single paired row of connecting cells (CNC) links with four rows of corridor cells (COC) that narrow the lumen of the endostyle into a passageway and form a slit that connects the endostyle lumen to the pharynx lumen (PFC; Fig. 1B,C). The two central rows of the corridor cells correspond to the iodine-binding zones (Fredriksson et al.,1985). The corridor contains numerous giant cilia (GC) that help direct and wind the food-trapping secretions of the endostyle into a strand that enters the pharynx lumen (Fig. 1B–D). Previous cytochemical studies suggest that the iodination of proteins secreted by the glandular cells is probably catalyzed by extracellular peroxidases when the secreted substance passes the corridor cells toward the pharynx (Fredriksson et al.,1985). Overall, our observations in live animals complement past comparative studies between larvaceans and other chordates, and are consistent with the idea that the endostyle of Oikopleura shares a ground plan with all other known endostyles in chordates with respect to the organization of various types of cells involved in food-trapping and iodinating functions, and the fact that urochordates, cephalochordates, and vertebrates share the genetic machinery that patterns different functional zones in the dorsoventral axis (Hiruta et al.,2005).

Figure 1.

Endostyle morphology and cell-type distribution in Oikopleura dioica. A: Lateral left view of a juvenile of O. dioica showing the location of the endostyle (dashed-rectangle) ventral to the pharynx. B: Schematic representation of a cross-section of the Oikopleura endostyle of an adult animal showing its different cellular components across the dorsoventral axis (modified after Olsson,1965; see Fredikson,1985, for image details). C,D: Ventrolateral (C) and ventral (D) views of the endostyle taken by differential interference contrast (DIC) microscopy in live Oikopleura juvenile, showing cellular characteristics of the endostyle. Inset in D shows higher magnification of a glandular cell with large nucleus, secretion vesicles, giant cilia, and the lumen of the organ. See main text for details. b, brain; bg, bucal glands; CIC, ciliated cells; CNC, connecting cells; COC, corridor cells; cr, ciliary ring; GC, giant cilia; GLC, glandular cells; es, esophagus; g, gonad; L, lumen; m, mouth; N, cellular nuclei; ph, pharynx; PC, pharynx cells; PFC, pharynx floor cells; r, rectum; st, stomach; t, tail; VMC, ventromedian cells; vo, ventral organ. Scale bar = 100 μm.

A ventral view of the Oikopleura endostyle shows that topology is not uniform along its AP axis: the lumen is enlarged rostrally; the frontal wall of the endostyle contains specialized cells that project giant cilia caudally; and the most posterior part of the endostyle narrows substantially (Fig. 1D). To determine whether this morphological regionalization follows from an earlier molecular genetic AP regionalization, we examined the expression patterns of transcription factors differentially expressed along the AP axis in the endostyle.

Otx, Pax2/5/8a, Pax2/5/8b, and Hox1 Expression During Endostyle Development in Oikopleura dioica

In a previous study, we found that genes involved in AP patterning of the central nervous system in O. dioica are also expressed in non-neural tissues (Cañestro et al.,2005). In the current work, we investigated the expression of Otx, Pax2/5/8, and Hox genes in the endodermal precursor of the endostyle. Oikopleura has three Otx paralogs (Otxa, Otxb, and Otxc) that arose from gene duplication events in the larvacean lineage after it diverged from the lineage of ascidians, which, like amphioxus, have a single Otx gene (Wada et al.,1996; Williams and Holland,1996; Hinman and Degnan,2000; Cañestro et al.,2005; Edvardsen et al.,2005). Vertebrates also have three Otx family genes (OTX1, OTX2, and CRX), which arose in the vertebrate lineage after it diverged from the urochordate lineage (Germot et al.,2001). In the case of the Pax2/5/8 gene family, Oikopleura and ascidians both have two Pax2/5/8 paralogs (Pax2/5/8a and Pax2/5/8b), which arose from the duplication of an ancestral urochordate gene before the divergence of the larvacean and ascidian lineages (Cañestro et al.,2005). Amphioxus has just one Pax2/5/8 gene, and most vertebrates have three paralogs (Pax2, Pax5, and Pax8), which likely arose during two rounds of whole genome duplication after the divergence of urochordate and vertebrate lineages (Kozmik et al.,1999; Dehal and Boore,2005). Oikopleura, like ascidians and amphioxus, has a single Hox1 gene, in contrast to three found in vertebrates (Hoxa1, Hoxb1, Hoxd1), again arising in the vertebrate genome duplication events (Seo et al.,2004; Cañestro et al.,2005; Dehal and Boore,2005).

Oikopleura develops rapidly, and in less than 24 hr after fertilization, juveniles inflate their first house and initiate their free-swimming, filter-feeding adult lifestyle. By the early hatchling stage at approximately 6 hours postfertilization (hpf), cavities first begin to become delineated in the trunk of the Oikopleura embryo, and the presumptive primordium of the floor of the pharynx and endostyle first become recognizable. At this stage, Otxc expression appeared as a medial domain, probably in one single cell, in the most anterior part of the presumptive endostyle (Fig. 2A). Otxa and Otxb are expressed in the CNS and epidermis, but not in the endostyle (Cañestro et al.,2005; Cañestro and Postlethwait,2007). Posterior to the Otxc expression domain, a bilateral dorsal domain expressed Pax2/5/8a (Fig. 2B) and a bilateral ventral domain expressed Pax2/5/8b (Fig. 2C). Posterior to the Pax2/5/8b expression domain, Hox1 expression was detected in a bilateral domain in the most posterior part of the presumptive endostyle primordium (Fig. 2D).

Figure 2.

A–D: Expression analysis by whole-mount in situ hybridization of Otxc (A), Pax2/5/8a (B), Pax2/5/8b (C), and Hox1 (D) in presumptive endostyle cell precursors (pink arrowheads) of Oikopleura dioica at the early hatchling developmental stage (approximately 6 hr postfertilization). Top row: lateral left view. Bottom row: ventral view at the level of the dashed-line in the lateral view (anterior toward the left). Scale bar = 100 μm.

By late hatchling stages (20 hpf), the endostyle begins to attain its definitive shape, and endostyle cilia have begun to flutter. At this stage, the expression patterns of Otxc, Pax2/5/8a, Pax2/5/8b, and Hox1 exhibited the definitive AP molecular regionalization of the endostyle: (1) the anterior portion of the endostyle expressed Otxc (Fig. 3A); (2) the central portion expressed the two Pax2/5/8 paralogs, with Pax2/5/8a expressed dorsally in cells lining the endostyle corridor, which connects the endostyle lumen with the pharyngeal cavity (Fig. 3B), and Pax2/5/8b expressed ventrally in a patch of medial, anterior cells in the floor of the endostyle (Fig. 3C); and (3) the posterior part of the endostyle expressed Hox1 (Fig. 3D). These expression domains demarcate functional domains, as interpreted below.

Figure 3.

A–D: Whole-mount in situ hybridization for the expression of Otxc(A), Pax2/5/8a (B), Pax2/5/8b (C), and Hox1 (D) in the presumptive endostyle primordium (pink arrowheads) of Oikopleura dioica at the late hatchling stage (approximtely 20 hr postfertilization). The top row shows lateral left (A–D) and frontal (A′–D′) perspectives of the trunk at the level of the vertical dashed-line in the lateral view. These views orient higher magnifications shown in the rows below: second row, lateral left view magnified from the region labeled with a dashed-rectangle in A–D; third row, frontal view magnified from the region labeled with a dashed-square in A′–D′; and bottom row, ventral view at the level of the horizontal dashed-line in the lateral view A–D (anterior is toward the left). Black arrowheads in C indicate Pax2/5/8b expression in the pharyngeal epithelium. Some cells types have been labeled in the magnified frontal view in A to facilitate orientation and comparison with Figure 1B. The differences in the morphology between these frontal views and the schematic representation shown in Figure 1B are due mainly to the relative enlargement of the gland cells and thinning of the ventral floor cells that occur as the animals age from juvenile to adult. bg, bucal glands; GC, giant cilia; GLC, glandular cells; L, endostyle lumen; PFC, pharynx floor cells. Scale bar = 100 μm.

Three-Dimensional Reconstruction of AP Axial Patterning in the Oikopleura Endostyle

Analysis of the expression patterns of transcription factor genes allows us to reconstruct the AP patterning of the Oikopleura endostyle in three dimensions over developmental time (Fig. 4), and to relate each expression domain to functional domains. (1) The most anterior part of the endostyle that expresses Otxc (green in Fig. 4B) coincides with the region that, according to Olsson (1965), includes specialized cells that form the giant cilia (Fig. 1C,D). (2) In the central portion of the endostyle, the dorsal, Pax2/5/8a-expressing region (red in Fig. 4B) includes the rows of corridor cells that show peroxidase activity and bind iodine in Oikopleura (Fredriksson et al.,1985). Ventrally, the central portion expresses Pax2/5/8b (purple in Fig. 4B) in a region that may include ciliated support cells and medial cells that form the rostral part of the epithelial floor of the endostyle, and (3) the most posterior part of the endostyle that expresses Hox1 (yellow in Fig. 4B) may include supporting and medial cells that form the posterior half of the epithelial floor of the endostyle and a proliferative zone identified that supplies new glandular cells as the animal and endostyle grow (Troedsson et al.,2007). The early, organized expression of the Otx-Pax2/5/8-Hox1 gene set in presumptive endostyle precursor cells (Fig. 2), well before these cells differentiate their final function (Fig. 3), would be predicted by the hypothesis that these transcription factors help pattern the endostyle primordium into specific regions along the AP axis.

Figure 4.

Ordered expression of Otx, Pax2/5/8, and Hox1 in the anterior–posterior (AP) regionalization of the endostyle of Oikopleura dioica. A: Differential interference contrast microscopy photograph of a live larva of Oikopleura dioica at late hatchling stage, showing a ventral perspective of the endostyle (dashed-rectangle) in relation to other organs. bg, bucal glands; cr, ciliary ring; lr, langerhand receptor; m, mouth; s, stomach. B: Schematic three-dimensional reconstruction of the endostyle summarizing the AP regionalization revealed by the expression domains of Otxc (green), Pax2/5/8a (red), Pax2/5/8b (blue), and Hox1 (yellow), and showing virtual representations of cross-sections at different levels of the anterior–posterior axis. Scale bar = 10 μm.

Subfunctionalization of Pax2/5/8 and Evolution of Endostyle and Thyroid

The last common ancestor of extant chordates probably had a single Pax2/5/8 gene that possessed a variety of functions. Because this ancestral gene apparently did not duplicate in the cephalochordate lineage (Kozmik et al.,1999; Kreslova et al.,2002), the single amphioxus Pax2/5/8 gene may reflect the ancestral chordate condition. According to recent investigations of chordate phylogeny, urochordates are the living sister group of vertebrates, with which they form a clade called Olfactores, while cephalochordates diverge basally among chordates (Blair and Hedges,2005; Bourlat et al.,2006; Delsuc et al.,2006; Vienne and Pontarotti,2006; reviewed in Cañestro et al.,2007); thus, amphioxus serves as an outgroup to infer the evolution of the Pax2/5/8 gene family in urochordates and vertebrates. Urochordates have two Pax2/5/8 paralogs that originated in an ancient gene duplication event that occurred in a stem urochordate before the divergence of the larvacean and the ascidian lineages (Wada et al.,2003; Cañestro et al.,2005). The nonoverlapping and sometimes complementary, coordinated expression patterns of the two Pax2/5/8 paralogs in urochordates observed in this and other work (Mazet et al.,2003; Cañestro et al.,2005) is expected from the process of subfunctionalization, an evolutionary mechanism that initially preserves duplicated genes, followed by a process of independent subfunction partitioning, an evolutionary mechanism that can occur at any time after a gene duplication event independent of the forces that initially preserved the gene duplicates (Force et al.,1999; Postlethwait et al.,2004). Comparative analysis of Pax2/5/8a and Pax2/5/8b expression domains in the endostyle of Oikopleura and other chordates helps to establish cell type homologies and to discover Pax2/5/8 subfunctions related to the development of the endostyle and thyroid.

The Pax2/5/8a expression domain in the iodine-binding corridor cells in the dorsal endostyle in Oikopleura is homologous to the expression of Pax2 or Pax8 in the follicular cells in the vertebrate thyroid (Plachov et al.,1990; Zannini et al.,1992; Fabbro et al.,1994; van der Kallen et al.,1996; Macchia et al.,1998; Mansouri et al.,1998; Heller and Brandli,1999; Wendl et al.,2002; Trueba et al.,2005). Therefore, our results support the previous hypothesis that corridor cells in the endostyle in Oikopleura are homologous to follicular cells in the vertebrate thyroid (Rall et al.,1964; Fredriksson et al.,1985).

In contrast to Pax2/5/8a, which is expressed in the central, dorsal part of the Oikopleura endostyle, Pax2/5/8b is expressed ventrally. Of interest, summing the Pax2/5/8a expression domain in the iodine-binding dorsal zone and the Pax2/5/8b expression domain in the supportive zone in the ventral endostyle recapitulates the expression of the single amphioxus Pax2/5/8 gene in the iodine-binding dorsal zones 5a, 5b, and 6, and the ventral supportive zone 3 (Hiruta et al.,2005). This fact suggests that ancestral chordate subfunctions of Pax2/5/8, as judged by the outgroup amphioxus gene, have partitioned between the two OikopleuraPax2/5/8 genes. This conclusion predicts the existence of independent ancestral regulatory elements driving Pax2/5/8 in each of these cell types in the endostyle. Interestingly, although the duplication of Pax2/5/8 in the urochordate lineage occurred before the divergence of ascidian and larvacean lineages (Cañestro et al.,2005), the ascidian endostyle expresses Pax2/5/8a, but not Pax2/5/8b, in both the dorsal iodine-binding zone 7 and the ventral supportive zone 5 (for details on zone numbering, see Hiruta et al.,2005). Differences in expression patterns of orthologous Pax2/5/8 duplicates in larvaceans and ascidians suggest that the two Pax2/5/8 paralogs experienced subfunction partitioning independently in the two urochordate lineages after they diverged from one another.

In vertebrates, expression analysis of Pax2/5/8 family genes in the thyroid reveals multiple subfunctions and suggests that multiple independent subfunction partitioning events probably occurred after the Pax2/5/8 family expanded early in the vertebrate radiation. In mouse, for instance, Pax8 is expressed during thyroid development but Pax2 is not (Plachov et al.,1990; Wendl et al.,2002); reciprocally, in frogs the roles of these genes are reversed, as Pax2 but not Pax8 is expressed in the thyroid (Heller and Brandli,1999). These results suggest that the ancestral thyroid regulatory subfunctions of Pax2/5/8 were preserved by both Pax2 and Pax8 in stem amniotes, and resolved independently in the amphibian and mammalian lineages. In zebrafish, pax8 and pax2a (previously called pax2.1) are both expressed in the thyroid, although at different times in development (Wendl et al.,2002). Analysis of mutant zebrafish embryos revealed that pax2a function is required for the development of thyroid follicular cells, as is Pax8 in mouse (Wendl et al.,2002). In conclusion, gene expression data suggest that the Pax2/5/8 gene in stem chordates had separate regulatory elements for the dorsal and ventral expression domains in the endostyle, and that, after gene duplication in urochordates, these elements partitioned to Pax2/5/8a or Pax2/5/8b and, just as in vertebrates, they partitioned to different duplicates (Pax2 or Pax8) in different lineages.

Functional analysis of Pax2/5/8 genes in the development of the endostyle of nonvertebrate chordates will provide new insights into our understanding of the evolution and development of the vertebrate thyroid. Analysis of regulatory elements and identification of transcription factors that bind them to control the expression of Pax2/5/8 paralogs in various parts of the endostyle in urochordates will further our understanding of divergent subfunctionalization events between Oikopleura and ascidians, and it may help untangle unknown Pax2/5/8 subfunctions present in the last common chordate ancestor that might also be preserved in vertebrates. Comparative analysis of the specification and early differentiation of the endostyle primordium from the floor of the pharynx, which occurs during embryonic development in Oikopleura as in vertebrates and cephalochordates, and endostyle differentiation in ascidians, which is delayed until after metamorphosis, might help to distinguish between the roles of Pax2/5/8 in early molecular patterning and later roles related to the regulation of different cell types in the functional adult endostyle.

Is the Ordered Expression of Otx-Pax2/5/8-Hox in the AP Axis of Oikopleura Endostyle a Shared Chordate Feature?

Although patterning along the AP axis of the endostyle in other chordates has not yet been systematically investigated, we wondered if the ordered Otx-Pax2/5/8-Hox expression in the endostyle is unique to Oikopleura, or if available evidence suggests that it may be shared with other chordates. In addition to the well-studied role of Pax2/5/8 orthologs during follicular cell development, evidence suggests that Otx and Hox genes are also expressed during development of the thyroid and endostyle in other chordates.

In ascidians, Otx is expressed in the pharynx primordium in premetamorphic larvae, and in the most rostral third of the endostyle of Herdmania curvata, anterior to the Pax2/5/8a expression domain in postmetamorphic 5-day-old juveniles (Hinman and Degnan,2000). The expression of Otx in the ascidian endostyle, at the time and location predicted from our observations in Oikopleura, suggests a conserved role for Otx in the endostyle among urochordates, and supports structural homology between the most rostral region of the endostyle in both classes of urochordates. In ascidians, Hox1 expression has been reported in endodermal cells at larval stages (Ikuta et al.,2004), but the fate of these Hox1-expressing endodermal cells relative to endostyle development in ascidians has not yet been determined; thus, whether the most posterior part of the Oikopleura and ascidian endostyles are homologous and share Hox1 expression remains to be investigated.

In vertebrates, the thyroid develops from the endoderm of the second pouch (Norris,1918; Graham et al.,2005). In mouse, Otx1 and Otx2 are expressed in the thyroid duct and thyroid rudiment during development (Simeone et al.,1993; Acampora et al.,1995). The early embryonic lethality of homozygous mutant Otx2−/− mice, and redundant functions shared by Otx1 and Otx2 genes make it difficult to study the role of Otx paralogs in the development of the murine thyroid (Ang et al.,1996; Acampora et al.,1999).

In mouse embryos, the Hox1 co-orthologs Hoxa1 and Hoxb1 are both expressed in the pharyngeal endoderm, and Hoxb1 at least is expressed in the second pharyngeal pouch (Frohman et al.,1990; Murphy and Hill,1991). Furthermore, the double disruption of Hoxb1 and Hoxa1 leads to selective loss of the second pharyngeal pouch, and the variable deletion of part or all of the thyroid (Rossel and Capecchi,1999). This result would be expected if Hox1 genes are important for development of the posterior portion of the thyroid in mouse. These expression patterns and mutant analyses in mouse, taken with the expression pattern of Hox1 in Oikopleura, are as would be expected if Hox1 played a role in patterning the posterior of the endostyle of the last common ancestor of vertebrates and urochordates.

In cephalochordates at approximately 30 hpf, Pax2/5/8 expression appears in the ventral part and right side of the pharynx in the region that eventually differentiates into endostyle (Kozmik et al.,1999). Of interest, in later developmental stages, Pax2/5/8 expression diminishes in the most anterior part of the amphioxus endostyle at the time and location predicted from our observations in Oikopleura, corresponding to the stage at which Pax2/5/8 expression becomes restricted to the central part of the endostyle (Kozmik et al.,1999). Although Otx and Hox1 expression have not yet been investigated in the differentiated endostyle of amphioxus, Otx and Hox1 are expressed early in the pharyngeal endoderm from which the endostyle differentiates (Williams and Holland,1996; Schubert et al.,2005). In the pharynx of 30-hpf amphioxus embryos, Otx is expressed anteriorly, and Hox1 is expressed posteriorly (Schubert et al.,2005), following the same order that we observed in the expression of the orthologs in Oikopleura when endostyle precursors differentiate from the pharyngeal primordium. It is thus possible that the genetic mechanisms that pattern the AP axis of the pharyngeal endoderm from which the endostyle derives also pattern the endostyle. The mechanism of AP patterning of the pharyngeal endoderm in amphioxus is shared with urochordates and vertebrates, which also express Otx in stomodeal and anterior pharyngeal endoderm (Simeone et al.,1993; Williams and Holland,1996; Hinman and Degnan,2000; Cañestro and Postlethwait,2007), and express a combinatorial code of Hox genes to define AP identity in posterior pharyngeal endoderm (Hunt et al.,1991; Holland and Holland,1996; Couly et al.,2002; Miller et al.,2004; Schubert et al.,2005; Crump et al.,2006). Thus, expression of Otx and Hox genes in anterior and posterior endostyle regions in Oikopleura suggests the hypothesis that the endostyle uses genetic mechanisms that regionalize the anterior endoderm in the pharynx (Grapin-Botton and Melton,2000; Shivdasani,2002).

Recurrent Utilization of the Ordered Expression of the Otx-Pax2/5/8-Hox Gene Set to Provide AP Positional Information

It is striking that the order of Otx-Pax2/5/8-Hox gene expression that we discovered here for the endostyle of Oikopleura also patterns the AP axis of the CNS in various organisms, including chordates, hemichordates, and flies (Wada et al.,1998; Holland and Holland,1999; Wada and Satoh,2001; Hirth et al.,2003; Lowe et al.,2003). Originally, Wada et al. (1998) used the expression pattern of this Otx-Pax2/5/8-Hox gene set to recognize the “tripartite” AP organization of the CNS, consisting of an Otx-positive anterior domain, a Hox1-positive posterior domain, and a Pax2/5/8-positive central domain, as an ancestral feature of the chordate CNS. Further studies in other organisms have extended the origin of this tripartite organization of the CNS to stem bilaterians (Hirth et al.,2003). Our finding of the same order of expression of the Otx-Pax2/5/8-Hox gene set during development of the endostyle in Oikopleura suggests that it may be part of a previously unrecognized patterning cassette present in the developmental genetic toolkit of stem bilaterians, and suggests that it has been deployed multiple times during animal evolution to provide AP positional information during the development of multiple structures. One could speculate that an ancestral overarching mechanism could simultaneously establish the coordinated AP patterning of Otx-Pax2/5/8-Hox1 in both the endoderm and CNS, or alternatively, that vertical communication might coordinate expression of this gene set in both tissue layers. These ideas agree with the analysis of the expression of Cdx genes in both the posterior part of the neural tube and the posterior part of the digestive tube in ascidians and other chordates (Hinman et al.,2000; and references therein). Future investigations may well uncover additional organs that use the Otx-Pax2/5/8-Hox gene set to pattern their AP axis.

EXPERIMENTAL PROCEDURES

Biological Materials

O. dioica were collected at the Oregon Institute of Marine Biology (Charleston, OR). Animals were cultured in the laboratory at the University of Oregon (Eugene, OR) at 13°C in 10-μm filtered sea-water for several generations. The transparency of Oikopleura embryos and adults allows noninvasive study of internal morphology at the level of individual cells. For some images, we merged DIC optical sections using Adobe Photoshop software to achieve integrated visualization of structures in different focal planes (Fig. 1C,D).

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization was performed as described previously (Bassham and Postlethwait,2000; Cañestro and Postlethwait,2007) with minor modifications: fixed, dehydrated embryos were dechorionated manually with glass needles before re-hydration; Tween-20 concentration was increased from 0.1% to 0.15% in the hybridization buffer, in the PBT solution and in the posthybridization washing buffers; and embryos were mounted in 80% glycerol for microscopy. Riboprobes for detecting the expression of Otxc (GenBank accession no. AY897557), Hox1 (GenBank accession no. AY871214), Pax2/5/8a (GenBank accession no. AY870648), and Pax2/5/8b (GenBank accession no. AY870649) genes are described in (Cañestro et al.,2005).

Acknowledgements

We thank skipper B. Young of the “Charming Polly” for help in collecting larvaceans. We thank E. Sanders and M. Fajer for help experiments, and three anonymous reviewers for thoughtful comments.

Ancillary