Evolution of the thyroid: Anterior–posterior regionalization of the

DEVELOPMENTAL DYNAMICS 237:1490 –1499, 2008
PATTERNS & PHENOTYPES
Evolution of the Thyroid: Anterior–Posterior
Regionalization of the Oikopleura Endostyle
Revealed by Otx, Pax2/5/8, and Hox1
Expression
Cristian Cañestro,1† Susan Bassham,2† and John H. Postlethwait1*
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.
Key words: thyroid; endostyle; subfunctionalization; evolution of chordate development; tunicate; appendicularian; Otx;
Pax; Hox; developmental genetic toolkit
Accepted 20 February 2008
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, iodinecontaining hormones (e.g., thryroxine
[T4] and triiodothyronine [T3]) and
the peptide hormone calcitonin, which
regulates levels of calcium and phosphate in the blood. Thyroid dysfunc-
tion 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
1
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
Institute of Neuroscience, University of Oregon, Eugene, Oregon
Center of Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon
Grant sponsor: National Science Foundation; Grant number: IOB-0719577.
†
Drs. Cañestro and Bassham contributed equally to this study.
*Correspondence to: John H. Postlethwait, Institute of Neuroscience, University of Oregon, Eugene, OR, 97403.
E-mail: [email protected]
2
DOI 10.1002/dvdy.21525
Published online 3 April 2008 in Wiley InterScience (www.interscience.wiley.com).
© 2008 Wiley-Liss, Inc.
REGIONALIZATION OF OIKOPLEURA ENDOSTYLE IN THE AP AXIS 1491
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 iodinebinding 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 iodinebinding 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
1492 CAÑESTRO ET AL.
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).
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, filterfeeding 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).
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.
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/
REGIONALIZATION OF OIKOPLEURA ENDOSTYLE IN THE AP AXIS 1493
Fig. 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 ␮m.
Fig. 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 ␮m.
1494 CAÑESTRO ET AL.
Fig. 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 ␮m.
Fig. 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 ␮m.
REGIONALIZATION OF OIKOPLEURA ENDOSTYLE IN THE AP AXIS 1495
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.
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 Oikopleura Pax2/5/8
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
1496 CAÑESTRO ET AL.
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 (GrapinBotton 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 OtxPax2/5/8-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 or-
REGIONALIZATION OF OIKOPLEURA ENDOSTYLE IN THE AP AXIS 1497
ganization 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 OtxPax2/5/8-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-␮m
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).
ACKNOWLEDGMENTS
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.
REFERENCES
Acampora D, Mazan S, Lallemand Y,
Avantaggiato V, Maury M, Simeone A,
Brulet P. 1995. Forebrain and midbrain
regions are deleted in Otx2-/- mutants
due to a defective anterior neuroectoderm specification during gastrulation.
Development 121:3279 –3290.
Acampora D, Avantaggiato V, Tuorto F,
Barone P, Perera M, Choo D, Wu D,
Corte G, Simeone A. 1999. Differential
transcriptional control as the major molecular event in generating Otx1-/- and
Otx2-/- divergent phenotypes. Development 126:1417–1426.
Ang SL, Jin O, Rhinn M, Daigle N, Stevenson L, Rossant J. 1996. A targeted mouse
Otx2 mutation leads to severe defects in
gastrulation and formation of axial mesoderm and to deletion of rostral brain.
Development 122:243–252.
Barrington EJW. 1957. The distribution
and significance of organically bound iodine in the ascidian Ciona intestinalis
Linnaeus. J Mar Biol Assoc UK 36:1–16.
Barrington EJW. 1958. The localization of
organically bound iodine in the endostyle
of amphioxus. J Mar Biol Assoc UK 37:
117–126.
Bassham S, Postlethwait J. 2000.
Brachyury (T) expression in embryos of a
larvacean urochordate, Oikopleura dioica, and the ancestral role of brachyury.
Dev Biol 220:322–333.
Berrill N. 1950. The tunicata. London: Ray
Society Monograph.
Blair JE, Hedges SB. 2005. Molecular phylogeny and divergence times of deuterostome animals. Mol Biol Evol 22:2275–
2284.
Bourlat SJ, Juliusdottir T, Lowe CJ, Freeman R, Aronowicz J, Kirschner M,
Lander ES, Thorndyke M, Nakano H,
Kohn AB, Heyland A, Moroz LL, Copley
RR, Telford MJ. 2006. Deuterostome
phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida. Nature 444:85–88.
Cañestro C, Postlethwait JH. 2007. Development of a chordate anterior-posterior
axis without classical retinoic acid signaling. Dev Biol 305:522–538.
Cañestro C, Bassham S, Postlethwait JH.
2005. Development of the central nervous system in the larvacean Oikopleura
dioica and the evolution of the chordate
brain. Dev Biol 285:298 –315.
Cañestro C, Yokoi H, Postlethwait JH.
2007. Evolutionary developmental biology and genomics. Nat Rev Genet 8:932–
942.
Christen R, Braconnot J-C. 1998. Molecular phylogeny of tunicates. A preliminary
study using 28S ribosomal RNA partial
sequences: important implications in
terms of evolution and ecology. In: Bone
Q, editor. The biology of pelagic tunicates. New York: Oxford University
Press. p 265–271.
Couly G, Creuzet S, Bennaceur S, Vincent
C, Le Douarin NM. 2002. Interactions
between Hox-negative cephalic neural
crest cells and the foregut endoderm in
patterning the facial skeleton in the vertebrate head. Development 129:1061–
1073.
Crump JG, Swartz ME, Eberhart JK, Kimmel CB. 2006. Moz-dependent Hox expression controls segment-specific fate
maps of skeletal precursors in the face.
Development 133:2661–2669.
De Felice M, Di Lauro R. 2004. Thyroid
development and its disorders: genetics
and molecular mechanisms. Endocr Rev
25:722–746.
Dehal P, Boore JL. 2005. Two rounds of
whole genome duplication in the ancestral vertebrate. PLoS Biol 3:e314.
Delsuc F, Brinkmann H, Chourrout D,
Philippe H. 2006. Tunicates and not
cephalochordates are the closest living
relatives of vertebrates. Nature 439:965–
968.
Edvardsen RB, Seo HC, Jensen MF, Mialon A, Mikhaleva J, Bjordal M, Cartry J,
Reinhardt R, Weissenbach J, Wincker P,
Chourrout D. 2005. Remodelling of the
homeobox gene complement in the tunicate Oikopleura dioica. Curr Biol 15:R12–
R13.
Fabbro D, Di Loreto C, Beltrami CA, Belfiore A, Di Lauro R, Damante G. 1994.
Expression of thyroid-specific transcription factors TTF-1 and PAX-8 in human
thyroid neoplasms. Cancer Res 54:4744 –
4749.
Fol H. 1872. Etudes sur les Appendiculaires du De troit de MEssine. Mem Soc
Physique Hist Nat Geneve 21:445.
Fol H. 1876. Uber die Schleimdruse oder
den Endostyl der Tunicatem. Morph
Jahrb 1:222.
Force A, Lynch M, Pickett FB, Amores A,
Yan Y-L, Postlethwait J. 1999. Preservation of duplicate genes by complemen-
1498 CAÑESTRO ET AL.
tary, degenerative mutations. Genetics
151:1531–1545.
Fredriksson G, Ofverholm T, Ericson LE.
1985. Ultrastructural demonstration of
iodine binding and peroxidase activity in
the endostyle of Oikopleura dioica (Appendicularia). Gen Comp Endocrinol 58:
319 –327.
Fredriksson G, Ofverholm T, Ericson LE.
1988. Iodine binding and peroxidase
activity in the endostyle of Salpa fusiformis, Thalia democratica, Dolioletta
gegenbauri and Doliolum nationalis (Tunicata, Thaliacea). Cell Tissue Res 253:
403–411.
Frohman MA, Boyle M, Martin GR. 1990.
Isolation of the mouse Hox-2.9 gene;
analysis of embryonic expression suggests that positional information along
the anterior-posterior axis is specified by
mesoderm. Development 110:589 –607.
Germot A, Lecointre G, Plouhinec JL, Le
Mentec C, Girardot F, Mazan S. 2001.
Structural evolution of Otx genes in craniates. Mol Biol Evol 18:1668 –1678.
Gorbman A, Creaser CW. 1943. Accumulation of radioactive iodine by the endostyle of larval lampreys and the problem of homology of the thyroid. J Exp
Zool 89:391–405.
Graham A, Okabe M, Quinlan R. 2005. The
role of the endoderm in the development
and evolution of the pharyngeal arches.
J Anat 207:479 –487.
Grapin-Botton A, Melton DA. 2000.
Endoderm development: from patterning
to organogenesis. Trends Genet 16:124 –
130.
Heller N, Brandli AW. 1999. Xenopus
Pax2/5/8 orthologues: novel insights into
Pax gene evolution and identification of
Pax-8 as the earliest marker for otic and
pronephric cell lineages. Dev Genet 24:
208 –219.
Hinman VF, Degnan BM. 2000. Retinoic
acid perturbs Otx gene expression in the
ascidian pharynx. Dev Genes Evol 210:
129 –139.
Hinman VF, Becker E, Degnan BM. 2000.
Neuroectodermal and endodermal expression of the ascidian Cdx gene is separated by metamorphosis. Dev Genes
Evol 210:212–216.
Hirth F, Kammermeier L, Frei E, Walldorf
U, Noll M, Reichert H. 2003. An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila. Development 130:2365–2373.
Hiruta J, Mazet F, Yasui K, Zhang P, Ogasawara M. 2005. Comparative expression analysis of transcription factor
genes in the endostyle of invertebrate
chordates. Dev Dyn 233:1031–1037.
Holland LZ, Holland ND. 1996. Expression
of AmphiHox-1 and AmphiPax-1 in amphioxus embryos treated with retinoic
acid: insights into evolution and patterning of the chordate nerve cord and pharynx. Development 122:1829 –1838.
Holland LZ, Holland ND. 1999. Chordate
origins of the vertebrate central nervous
system. Curr Opin Neurobiol 9:596 –602.
Hunt P, Gulisano M, Cook M, Sham MH,
Faiella A, Wilkinson D, Boncinelli E,
Krumlauf R. 1991. A distinct Hox code
for the branchial region of the vertebrate
head. Nature 353:861–864.
Ihle JEW. 1907. Uber den endostyl und die
systematische Stellung der Appendicularien. Zool Anz 31:770.
Ikuta T, Yoshida N, Satoh N, Saiga H.
2004. Ciona intestinalis Hox gene cluster: its dispersed structure and residual
colinear expression in development. Proc
Natl Acad Sci U S A 101:15118 –15123.
Kobayashi H, Tsuneki K, Akiyoshi H,
Kobayashi Y, Nozaki M, Ouji M. 1983.
Histochemical distribution of peroxidase
in ascidians with special reference to the
endostyle and the branchial sac. Gen
Comp Endocrinol 50:172–187.
Kozmik Z, Holland ND, Kalousova A,
Paces J, Schubert N, Holland L. 1999.
Characterization of an amphioxus paried
box gene, amphiPax2/5/8: developmental expression patterns in optic support
cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in
the midbrain boundary region. Development 126:1295–1304.
Kreslova J, Holland LZ, Schubert M, Burgtorf C, Benes V, Kozmik Z. 2002. Functional equivalency of amphioxus and vertebrate Pax258 transcription factors
suggests that the activation of mid-hindbrain specific genes in vertebrates occurs
via the recruitment of Pax regulatory elements. Gene 282:143–150.
Lohmann H. 1933. Erste Klasse der Tunicaten: Appendiculariae. In: Kukenthal
W, Krumbach T, editors. Handbuch der
Zoologie. Berlin and Leipzig: Walter De
Gruyter and Co.
Lowe CJ, Wu M, Salic A, Evans L, Lander
E, Stange-Thomann N, Gruber CE, Gerhart J, Kirschner M. 2003. Anteroposterior patterning in hemichordates and the
origins of the chordate nervous system.
Cell 113:853–865.
Macchia PE. 2000. Recent advances in understanding the molecular basis of primary congenital hypothyroidism. Mol
Med Today 6:36 –42.
Macchia PE, Lapi P, Krude H, Pirro MT,
Missero C, Chiovato L, Souabni A,
Baserga M, Tassi V, Pinchera A, Fenzi
G, Gruters A, Busslinger M, Di Lauro R.
1998. PAX8 mutations associated with
congenital hypothyroidism caused by
thyroid dysgenesis. Nat Genet 19:83–86.
Mansouri A, Chowdhury K, Gruss P. 1998.
Follicular cells of the thyroid gland require Pax8 gene function. Nat Genet 19:
87–90.
Martini E. 1909. Studien uber die Konstanz histologischer Elemente, I. Oikopleura longicauda. Z Wiss Zool 92:563–
626.
Mazet F, Hutt JA, Millard J, Shimeld SM.
2003. Pax gene expression in the developing central nervous system of Ciona
intestinalis. Gene Expr Patterns 3:743–
745.
Mazet F, Hutt JA, Milloz J, Millard J, Graham A, Shimeld SM. 2005. Molecular evidence from Ciona intestinalis for the
evolutionary origin of vertebrate sensory
placodes. Dev Biol 282:494 –508.
Miller CT, Maves L, Kimmel CB. 2004.
moz regulates Hox expression and pharyngeal segmental identity in zebrafish.
Development 131:2443–2461.
Muller W. 1873. Uber die hypobranchialrinne der tunicaten und deren vorhandensein bei amphioxus und den cyklostomen. Jena Z Med Naturw 7:327–332.
Murakami Y, Ogasawara M, Sugahara F,
Hirano S, Satoh N, Kuratani S. 2001.
Identification and expression of the lamprey Pax6 gene: evolutionary origin of
the segmented brain of vertebrates. Development 128:3521–3531.
Murphy P, Hill RE. 1991. Expression of the
mouse labial-like homeobox-containing
genes, Hox 2.9 and Hox 1.6, during segmentation of the hindbrain. Development 111:61–74.
Norris EH. 1918. The early morphogenesis
of the human thyroid gland. Am J Anat
24:443–465.
Ogasawara M. 2000. Overlapping expression of amphioxus homologs of the thyroid transcription factor-1 gene and thyroid peroxidase gene in the endostyle:
insight into evolution of the thyroid
gland. Dev Genes Evol 210:231–242.
Ogasawara M, Satou Y. 2003. Expression
of FoxE and FoxQ genes in the endostyle
of Ciona intestinalis. Dev Genes Evol 213:
416 –419.
Ogasawara M, Di Lauro R, Satoh N. 1999.
Ascidian homologs of mammalian thyroid peroxidase genes are expressed in
the thyroid-equivalent region of the endostyle. J Exp Zool 285:158 –169.
Olsson R. 1963. Endostyles and endostylar
secretions: a comparative histochemical
study. Acta Zool 44:299 –329.
Olsson R. 1965. The cytology of the endostyle of Oikopleura dioica. Ann N Y
Acad Sci 118:1038 –1051.
Parlato R, Rosica A, Rodriguez-Mallon A,
Affuso A, Postiglione MP, Arra C, Mansouri A, Kimura S, Di Lauro R, De Felice
M. 2004. An integrated regulatory network controlling survival and migration
in thyroid organogenesis. Dev Biol 276:
464 –475.
Plachov D, Chowdhury K, Walther C, Simon D, Guenet JL, Gruss P. 1990. Pax8,
a murine paired box gene expressed in
the developing excretory system and thyroid gland. Development 110:643–651.
Postlethwait J, Amores A, Cresko W,
Singer A, Yan YL. 2004. Subfunction
partitioning, the teleost radiation and
the annotation of the human genome.
Trends Genet 20:481–490.
Rall JE, Robbins J, Lewallen CG. 1964.
The thyroid. New York/London: Academic Press.
Ristoratore F, Spagnuolo A, Aniello F, Branno M, Fabbrini F, Di Lauro R. 1999.
Expression and functional analysis of
Cititf1, an ascidian NK-2 class gene, suggest its role in endoderm development.
Development 126:5149 –5159.
Rossel M, Capecchi MR. 1999. Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and
defects in craniofacial development. Development 126:5027–5040.
REGIONALIZATION OF OIKOPLEURA ENDOSTYLE IN THE AP AXIS 1499
Salensky W. 1904. Etudes anatomiques
sur les Appendiculaires. Mem Acad Sci
St Petesbourg Ser 8:13–14.
Sasaki A, Miyamoto Y, Satou Y, Satoh N,
Ogasawara M. 2003. Novel endostylespecific genes in the ascidian Ciona intestinalis. Zoolog Sci 20:1025–1030.
Schubert M, Yu JK, Holland ND, Escriva
H, Laudet V, Holland LZ. 2005. Retinoic
acid signaling acts via Hox1 to establish
the posterior limit of the pharynx in the
chordate amphioxus. Development 132:
61–73.
Seo HC, Edvardsen RB, Maeland AD,
Bjordal M, Jensen MF, Hansen A, Flaat
M, Weissenbach J, Lehrach H, Wincker
P, Reinhardt R, Chourrout D. 2004. Hox
cluster disintegration with persistent
anteroposterior order of expression in
Oikopleura dioica. Nature 431:67–71.
Shivdasani R. 2002. Molecular regulation
of vertebrate early endoderm development. Dev Biol 249:191–203.
Simeone A, Acampora D, Mallamaci A,
Stornaiuolo A, D’Apice MR, Nigro V,
Boncinelli E. 1993. A vertebrate gene related to orothodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the
gastrulating mouse embryo. EMBO J 11:
2735–2747.
Stach T, Turbeville JM. 2002. Phylogeny of
Tunicata inferred from molecular and
morphological characters. Mol Phylogenet Evol 25:408 –428.
Swalla B, Cameron C, Corley L, Garey J.
2000. Urochordates are monophyletic
within the deuterostomes. Syst Biol 49:
52–64.
Thorndyke MC. 1978. Evidence for a ’mammalian’ thyroglobulin in endostyle of the
ascidian Styela clava. Nature 271:61–62.
Thorndyke MC, Probert L. 1979. Calcitonin-like cells in the pharynx of the ascidian Styela clava. Cell Tissue Res 203:301–
309.
Thorpe A, Thorndyke MC, Barrington EJ.
1972. Ultrastructural and histochemical
features of the endostyle of the ascidian
Ciona intestinalis with special reference
to the distribution of bound iodine. Gen
Comp Endocrinol 19:559 –571.
Troedsson C, Ganot P, Bouquet JM, Aksnes DL, Thompson EM. 2007. Endostyle
Cell Recruitment as a Frame of Reference for Development and Growth in the
Urochordate Oikopleura dioica. Biol Bull
213:325–334.
Trueba SS, Auge J, Mattei G, Etchevers H,
Martinovic J, Czernichow P, Vekemans
M, Polak M, Attie-Bitach T. 2005. PAX8,
TITF1, and FOXE1 gene expression patterns during human development: new
insights into human thyroid development and thyroid dysgenesis-associated
malformations. J Clin Endocrinol Metab
90:455–462.
Van der Kallen CJ, Spierings DC, Thijssen
JH, Blankenstein MA, de Bruin TW.
1996. Disrupted co-ordination of Pax-8
and thyroid transcription factor-1 gene
expression in a dedifferentiated rat thyroid tumour cell line derived from
FRTL-5. J Endocrinol 150:377–382.
Venkatesh TV, Holland ND, Holland LZ,
Su MT, Bodmer R. 1999. Sequence and
developmental expression of amphioxus
AmphiNk2–1: insights into the evolutionary origin of the vertebrate thyroid
gland and forebrain. Dev Genes Evol 209:
254 –259.
Vienne A, Pontarotti P. 2006. Metaphylogeny of 82 gene families sheds a new light
on chordate evolution. Int J Biol Sci 2:
32–37.
Wada H. 1998. Evolutionary history of
free-swimming and sessile lifestyles in
urochordates as deduced from 18S rDNA
molecular phylogeny. Mol Biol Evol 15:
1189 –1194.
Wada H, Satoh N. 2001. Patterning the
protochordate neural tube. Curr Opin
Neurobiol 11:16 –21.
Wada S, Katsuyama Y, Sato Y, Itoh CS,
Aiga H. 1996. Hroth, an orthodenticlerelated homeobox gene of the ascidian,
Halocynthia roretzi: its expression and
putative roles in the axis formation during embryogenesis. Mech Dev 60:59 –71.
Wada H, Saiga H, Satoh N, Holland PWH.
1998. Tripartite organization of the ancestral chordate brain and the antiquity
of placodes: insights from ascidian Pax2/
5/8, Hox and Otx genes. Development 125:
1113–1122.
Wada S, Tokuoka M, Shoguchi E, Kobayashi K, Di Gregorio A, Spagnuolo A,
Branno M, Kohara Y, Rokhsar D, Levine
M, Saiga H, Satoh N, Satou Y. 2003. A
genomewide survey of developmentally
relevant genes in Ciona intestinalis. II.
Genes for homeobox transcription factors. Dev Genes Evol 213:222–234.
Wendl T, Lun K, Mione M, Favor J, Brand
M, Wilson SW, Rohr KB. 2002. Pax2.1 is
required for the development of thyroid
follicles in zebrafish. Development 129:
3751–3760.
Williams NA, Holland PWH. 1996. Old
head on young shoulders. Nature 383:
490.
Zannini M, Francis-Lang H, Plachov D, Di
Lauro R. 1992. Pax-8, a paired domaincontaining protein, binds to a sequence
overlapping the recognition site of a homeodomain and activates transcription
from two thyroid-specific promoters. Mol
Cell Biol 12:4230 –4241.