Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA
* Author for correspondence (email: jpostle{at}uoneuro.uoregon.edu)
Accepted 7 July 2005
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SUMMARY |
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Key words: Appendicularia, Larvacean, Tunicate, Chordate evolution
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Introduction |
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Because no placode-like structures or their derivatives have been
recognized without controversy in non-vertebrate chordates, biologists
generally assume that various placodes rapidly arose together within the early
vertebrates. Consistent with this hypothesis, placodes broadly share several
characteristics: they originate from an arc of ectoderm around the anterior
neural plate, form from thickened epithelia, generate some overlapping cell
types (e.g. sensory neurons) and most produce migratory cells by delamination
(Baker and Bronner-Fraser,
1997). In addition, placodes express paralogs of a number of gene
families (Streit, 2004
).
Paralogs of eyes absent (eya) are expressed in the lens and
adenohypophyseal placodes and all of the neurogenic placodes
(David et al., 2001
;
Xu et al., 1997
). Pitx genes
mark the most anterior placodes, the olfactory and adenohypophyseal, as well
as the stomodeum (Gage and Camper,
1997
; Lanctot et al.,
1997
). Like Eya genes, Six family paralogs are expressed in all
placodes (Baker and Bronner-Fraser,
2001
; Kawakami et al.,
2000
; Oliver et al.,
1995
; Schlosser and Ahrens,
2004
). Significantly, Six genes are also important in the
developing forebrain, including: the olfactory bulbs and neurohypophysis
(posterior pituitary), which become physically and functionally connected to
the olfactory and adenohypophyseal organs (e.g.
Ghanbari et al., 2001
;
Jean et al., 1999
). Despite
examples of shared gene expression and the prevailing opinion that placodes
develop from a common primordium (Streit,
2004
), Begbie and Graham
(Begbie and Graham, 2001
) have
reasoned that the diversity of placode-derived organs, and the diversity of
ways in which they are induced to form indicate that placodes should not be
considered closely related embryonic structures and might not all share a
common evolutionary origin limited to Vertebrata.
Gans and Northcutt (Gans and Northcutt,
1983) argued that, while the chordate common ancestor probably had
a neural crest/placode precursor involved in sensory tissue development, this
precursor was part of a diffuse ectodermal nerve plexus like that seen in some
modern deuterostomes, including echinoderms and hemichordates, and did not
form focal condensations characteristic of vertebrate placodes. Evidence in
favor of an ancient, eochordate origin for placodes, however, has come from
analysis of ascidian and cephalochordate molecular embryology
(Baker and Bronner-Fraser,
1997
; Holland and Holland,
2001
; Manni et al.,
2004b
; Mazet et al.,
2005
; Shimeld and Holland,
2000
). For example, ascidians have been proposed to possess an
otic placode homolog: the paired, thickened, ectodermal primordia of the
exhalent siphon (atrium) in Ciona embryos express Pax2/5/8,
an ascidian ortholog of the vertebrate otic placode-marking genes
Pax2 and Pax8 (Wada et
al., 1998
), and the adult atrium wall contains `cupular' cells
that are morphologically similar to hair cells of the vertebrate inner ear and
lateral line (Bone and Ryan,
1978
). Cupular cells, unlike vertebrate hair cells, however, are
primary sensory cells [sending axons to the central nervous system (CNS)
rather than being separately innervated], and the role of ascidian
Pax2/5/8 in the atrium might instead be interpreted as a non-placodal
requirement in outer gill embryogenesis as has been proposed for amphioxus
Pax2/5/8 (Kozmik et al.,
1999
). Many ascidians are found to have rows of `coronal' organs
in the incurrent, oral siphon; Burighel et al. and Manni et al.
(Burighel et al., 2003
;
Manni et al., 2004a
) propose
that these are a lateral line homolog, but they are located inside the
equivalent of the stomodeum, which does not contain lateral line receptors in
vertebrates.
Cephalochordates and ascidians may have a homolog of a non-neurogenic
placode, the pituitary. In amphioxus embryos, the primordium of Hatschek's pit
expresses the adenohypophyseal marker pitx
(Boorman and Shimeld, 2002;
Yasui et al., 2000
), and adult
pit cells are immunoreactive for several vertebrate pituitary hormones
(Nozaki and Gorbman, 1992
).
Because the ascidian neurohypophyseal duct, which connects the cerebral
ganglion to the oral cavity, is neurogenic and produces migratory cells, it is
a candidate homolog for both neural crest and for the combined olfactory and
adenohypophyseal placodes (Burighel et
al., 2003
; Manni et al.,
2004a
). Although the neurohypophyseal duct resembles Rathke's
pouch in topography and in the expression of pitx in the associated
ciliated funnel (Boorman and Shimeld,
2002
; Christiaen et al.,
2002
), the structure is unpaired, unlike the vertebrate olfactory
placodes. Ruppert (Ruppert,
1990
), who observed phagocytotic rather than secretory cells in
the ascidian duct walls, proposed that the duct regulates blood volume, and is
unrelated to the adenohypophysis.
Developmental morphologists proposed that the `ventral organ' and the
`ciliary funnel' in the larvacean urochordate Oikopleura dioica, are
homologs of the olfactory organ and the pituitary
(Bollner et al., 1986;
Holmberg, 1982
;
Olsson, 1969
). The dilated
cilia of the sensory cells of the ventral organ protrude into the environment
from a slit-like pocket of ectoderm
(Bollner et al., 1986
). These
cells are primary sensory cells like vertebrate olfactory receptor cells, and
their axons integrate into the rostral-most CNS
(Bollner et al., 1986
). To
reflect these structural similarities to the vertebrate olfactory epithelium,
they were called olfactory or chemosensory cells by various authors, although
their function is unknown (Bollner et al.,
1986
; Lohmann,
1933
).
The connection of the larvacean ciliary funnel between the brain and the
roof of the anterior pharynx topographically resembles the developing
vertebrate pituitary when Rathke's pouch invaginates in the roof of the
stomodeum and contacts the primordial hypothalamus. In addition, dorsal funnel
cells appear to secrete a substance periodically into the trunk haemocoel
(Holmberg, 1982). Because of
these characteristics, Olsson (Olsson,
1969
) and Holmberg (Holmberg,
1982
) argued that the larvacean ciliary funnel is homologous to
both the adeno- and neurohypophyses. This hypothesis mirrors the proposed
pituitary homology of Hatschek's pit in cephalochordates and the
neurohypophyseal duct or neural gland complex in ascidians
(Gorbman, 1995
;
Olsson, 1990
).
At least three other ciliated sensory cell types exist around the mouth and
in the lateral trunk in Oikopleura
(Olsson et al., 1990).
Although they have not been suggested to be homologous to vertebrate placode
derivatives, these mechanoreceptors are organized into paired sensory organs,
and like the ventral organ, they form thickened areas in otherwise monolayer
epithelia.
Several questions remain unanswered regarding the origin of placodes. Do non-vertebrate chordates have placodes? If so, are they orthologous to specific vertebrate placodes? Are they composites of vertebrate placodes? Or did vertebrate and non-vertebrate placodes independently evolve from a common ancestral placode precursor? Given the diversity of morphologies among chordate taxa - which challenges the clear assignment of placode homologies - do different lineages of non-vertebrate chordates (i.e. cephalochordates, ascidians, thaliaceans, and larvaceans) have different or overlapping sets of placode homologs? To infer the nature of developmental mechanisms in the last common ancestor of extant chordates and to reassess the role the origin of placodes might have played in the vertebrate transition, we must try to answer these questions.
We present the isolation and developmental expression patterns of eya, pitx and three Six genes from Oikopleura dioica in the context of the origin of placodes. In this study, we show that homologs of genes important for vertebrate olfactory and pituitary placodes are expressed in the primordia of the larvacean ventral organ and ciliary funnel. We show that these larvacean organs conform to morphological criteria that characterize vertebrate placodes, and report that the primordia of paired larvacean peripheral mechanoreceptor organs express placode-marking genes. Finally, we discuss the relationship of these organs to ascidian mechanoreceptors that have been proposed to be homologs of otic or lateral line hair cells.
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Materials and methods |
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Cloning and sequence analysis
Gene fragments were obtained by degenerate PCR amplification from genomic
or cDNA templates using primers based on alignment of homologous genes. Primer
sequences can be provided on request.
cDNA sequences were obtained by RACE PCR (SMART RACE cDNA Amplification
Kit, Clontech), and by amplification from first-strand synthesis cDNA pools.
Poly-A mRNA was purified (Micro-FastTrack 2.0 mRNA isolation kit, Invitrogen)
from 1600 hatchlings ranging from just hatching to tailshift stage, just
before house building.
A genomic fosmid library was constructed using the CopyControl Fosmid Library Production Kit (EpiCentre) and DNA from about 50 ripe O. dioica males. Fosmids containing eya, pitx, six1/2, six3/6a and six3/6b were isolated by screening the arrayed library with gene-specific PCR primers. Fosmids were fully sequenced by the Joint Genome Institute, Walnut Creek, California. GenBank accession numbers for Oikopleura genes are: eya (DQ011272, fosmid), (DQ011273, cDNA); pitx (DQ011274, fosmid), (DQ011275, DQ011276, DQ011277, DQ011278, cDNAs); six1/2 (DQ011279, fosmid), (DQ011280, cDNA); six3/6a (DQ011281, fosmid), (DQ011282, cDNA); six3/6b (DQ011283, fosmid), (DQ011284, cDNA).
Sequences were aligned using ClustalW software (http://sf01.bic.nus.edu.sg/clustalw/). Unrooted neighbor-joining phylogenies were calculated using PAUP 4.0b software. GenBank Accession Numbers for protein sequences used in Fig. 3B can be provided on request.
In situ hybridization
Embryos for in situ analysis were treated as in Bassham and Postlethwait
(Bassham and Postlethwait,
2000), or sometimes the probe hybridization time was shortened
from overnight to 2.5 to 4 hours.
Scanning electron microscopy
Animals were fixed 2 hours at 4°C in 2% glutaraldehyde, 0.1 M
cacodylate and 0.27 M NaCl (pH 7.3), rinsed in 0.1 M cacodylate buffer,
postfixed for 1 hour at 4°C in 2% osmium tetroxide, 0.1 M cacodylate
buffer, rinsed in distilled water, dehydrated through an ethanol series, and
dried in a Quorum Technologies E3100 Critical Point Drying Apparatus.
Sputter-coated samples were imaged on a JEOL JSM-6400FV scanning electron
microscope.
Light microscopy
Animals were anaesthetized with 0.02% MESAB and photographed on a Leica
DMLB compound microscope with a SPOT RT Color digital camera (Diagnostic
Instruments). Because high magnification and DIC microscopy greatly limit
depth of field, two or more focal planes of some images have been digitally
merged using Adobe Photoshop software to clearly illustrate three-dimensional
relationships of organs and gene expression domains:
Fig. 1B-D;
Fig. 5C,D inset;
Fig. 6A.
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Results |
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Four cells line and ensheath each of the paired rostral nerves
(Fig. 1C,F) (Bollner et al., 1986); these
cells form synapses with processes from the brain and ventral organ
(Bollner et al., 1986
).
Thinking the paired cell clusters were outgrowths of the brain, Bollner et al.
(Bollner et al., 1986
) called
them the `rostral brain bulbs' and tentatively suggested their homology to the
vertebrate olfactory bulbs. Delsman
(Delsman, 1912
), by contrast,
described the development of these bulbs from the rostral ectoderm, not from
the brain: he observed that a transverse band of epithelial ectoderm overlying
the mouth thickens and protrudes inward and ultimately separates from the
epidermis. Delsman's almost century-old descriptive diagrams are here
confirmed and recreated with DIC microscopy
(Fig. 2). This cell behavior is
similar to the delamination of placode cells in vertebrates
(Baker and Bronner-Fraser,
1997
).
The ciliary funnel is a cone that slopes rostrally and ventrally from the
right side of the brain (Fig.
1B), similar to the right-handed infundibular extension and
Hatschek's pit in amphioxus (Gorbman et
al., 1999). The base of the funnel forms an opening into the
right, dorsal wall of the pharynx (Fig.
1A-C,E,F). Holmberg (Holmberg,
1982
) described three distinct funnel segments. In the pharyngeal
wall, a ring of cells guards the funnel opening against particles with a
screen of static cilia. In the ventral part of the funnel, the walls are
one-cell thick and bear long cilia that constantly beat towards the dorsal end
of the funnel. The dorsal funnel is unciliated, and several brain cells extend
processes that contribute to the dorsal funnel wall. At this blind, dorsal
end, the `tip' cells appear to secrete a substance into the haemocoel
(Holmberg, 1982
).
Larvaceans have at least three kinds of ciliated mechanosensory organs: (1)
the Langerhans receptors, which are bilaterally paired in the posterior trunk;
(2) two upper lip cells which we here report to bear a stiff, bristle-like
cilium previously unrecognized because the cilia are absent in adults
(Fig. 1E); and (3) lying around
the circumference of the mouth, a band of ciliated mechanosensory cells which
each protrude clusters of cilia from a groove formed by overlapping epithelial
cells (Olsson et al.,
1990).
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Eya transcription factors contain a unique `Eya-domain' that interacts with
other proteins, including Six gene products
(Hanson, 2001;
Li et al., 2003
;
Ohto et al., 1999
;
Pignoni et al., 1997
). The
Oikopleura Eya-domain is 65.7 to 71.2% identical to the four human
EYA proteins and 71.7% identical to Ciona Eya. A catalytic motif
critical for Eya phosphatase activity that releases vertebrate Six1 from
inhibition (Li et al., 2003
)
is identical in larvacean, vertebrate and protostome Eyas. A gene phylogeny of
Eya amino acid sequences is consistent with the presence of a single
Eya gene in the last common ancestor of urochordates and vertebrates,
followed by duplications in the vertebrate lineage
(Fig. 3B).
Vertebrate Pitx genes display several isoforms resulting from alternative
splicing and alternative promoters, and these isoforms differ in tissue and
time of expression, and in function (Cox
et al., 2002; Essner et al.,
2000
; Gage and Camper,
1997
; Schweickert et al.,
2000
; Tremblay et al.,
2000
). Oikopleura pitx RACE products also displayed two
distinct 5' UTRs and translation start sites; if these distinct 5'
ends are transcribed from separate promoters, as in Ciona pitx
(Christiaen et al., 2005
),
then Oikopleura pitx promoter 1 is approximately 3.4 kb upstream of
promoter 2. To assess transcript diversity, PCR primers in each 5' UTR
were paired with a primer in the common 3' UTR and products were
amplified from embryonic cDNA pools. We observed at least six splice forms,
four of which are schematized in Fig.
3A. Long transcripts from promoter 1 are similar to vertebrate
Pitx2 isoforms a and b and to Ciona
pitxa/b (Christiaen et al.,
2002
; Cox et al.,
2002
; Essner et al.,
2000
). Vertebrate Pitx2 isoforms differentially affect the
development of left-right asymmetry (Liu
et al., 2001
; Schweickert et
al., 2000
); a thorough analysis of Oikopleura pitx
isoforms in the context of larvacean anatomical asymmetries will be published
separately. The Oikopleura transcript from Promoter 2 is similar to
vertebrate and Ciona isoform c, except that the C terminus
is truncated by alternative splicing. This is the first report of naturally
occurring Pitx isoforms with truncated C-termini.
In transcripts from promoter 1, the short 5'UTR was preceded by a
sequence that did not match genomic flanking sequence but that is identical to
a trans-spliced leader present in many Oikopleura transcripts
(Ganot et al., 2004).
Trans-splicing of pitx transcripts might be associated with
enhancement of translation, as in other organisms
(Maroney et al., 1995
;
Zeiner et al., 2003
).
Ciona pitx transcripts also have a trans-spliced leader
(Christiaen et al., 2002
).
Pitx genes belong to the Aristaless-related subfamily
within the Paired-class homeobox gene superfamily. In addition to the
DNA-binding homeodomain, Aristaless-related proteins share a protein
interaction domain called the OAR domain
(Furukawa et al., 1997;
Meijlink et al., 1999
). We
detected no OAR domain in Oikopleura Pitx by alignment with other
proteins. Despite this difference from even the reported ascidian
pitx cDNA sequences (Boorman and
Shimeld, 2002
; Christiaen et
al., 2002
), gene phylogeny
(Fig. 3B), GenBank BLAST
results and protein alignment indicate this Oikopleura gene is
otherwise an indisputable Pitx ortholog.
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Six genes are clustered in both fly and vertebrate genomes, and the common
ancestor of these lineages may have had a single cluster containing one of
each of the three classes of Six genes
(Gallardo et al., 1999;
Kawakami et al., 2000
).
Although Six1/2 and Six3/6 were probably adjacent genes in
the ancestral state, the genomic regions flanking each Oikopleura Six
gene on our completely sequenced fosmids contain segments with strong BLAST
similarities only to non-Six-related genes (not shown), implying that
the larvacean Sixes are not tightly clustered. If this larvacean gene
arrangement represents the breaking of an ancestral Six gene cluster in the
larvacean lineage, it would parallel the fragmentation of the
Oikopleura Hox cluster (Seo et
al., 2004
).
pitx and six3/6a expression and the ANR
Having isolated Oikopleura homologs of vertebrate `placode genes',
we tested the hypothesis that Oikopleura possesses homologs of the
most anterior vertebrate placodes by investigating whether the expression of
Eya, Pitx and Six genes may mark a pre-placodal domain at the anterior margin
of the embryonic CNS.
In vertebrate embryos, the primordia of the stomodeum and of the olfactory
and adenohypophyseal placodes lie in adjacent parts of the anterior neural
ridge (ANR), an arc of ectoderm surrounding the rostral margin of the neural
plate (Couly and Le Douarin,
1985; Kawamura and Kikuyama,
1992
). Genes important for stomodeal and placode development,
including Pitx, Eya and Six family genes, are expressed in the ANR (e.g.
Baker and Bronner-Fraser, 2001
;
Schlosser and Ahrens, 2004
;
Streit, 2004
). Similar to
their vertebrate counterparts, larvacean Eya, Six and Pitx genes are expressed
early in overlapping patterns at the anterior margin of the developing CNS: in
rostral ectoderm, in the presumptive mouth and in the rostral-most pharynx
(Fig. 4).
Because Oikopleura embryos undergo neurulation when they have few
cells and limited anatomical landmarks, we began our analysis when the
presumptive CNS has just completed internalization
(Cañestro et al.,
2005). At incipient-tailbud stage, the morphological division
between the trunk and tail first becomes apparent, and ectoderm cells begin to
transition from a rounded, cleavage stage morphology to an epithelial
morphology. The anterior-most CNS is marked by homologs of pax6 and
otx at this and later stages (S. Bassham, PhD Thesis, University of
Oregon, 2002) (Cañestro et al.,
2005
). A rectangular field of ectodermal cells just rostral to the
CNS strongly expresses six3/6a
(Fig. 4A,C). Two cells probably
in the presumptive rostral pharynx, adjacent to the anterior CNS, express
pitx (Fig. 4B,C). We
did not detect eya expression above background in incipient-tailbuds,
and six1/2 expression was broad in the ectoderm (not shown).
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As with the expression of vertebrate Pitx1, Pitx2 and
Six3 genes (Oliver et al.,
1995; Schweickert et al.,
2000
), Oikopleura pitx and six3/6a expression
begins early in overlapping domains at the rostral border of the CNS, far
preceding the terminal differentiation of cell types such as sensory neurons.
The region marked by these larvacean genes is probably homologous to the
vertebrate ANR and rostral pre-placodal region, although, unlike the
vertebrate ANR, these Oikopleura eya, pitx and six genes do
not all overlap in a `pan-placodal' domain (see Discussion).
Eya, Pitx and Six gene expression in the ventral organ
The hypothesis that the larvacean ventral organ is homologous to the
vertebrate olfactory epithelium (Bollner et
al., 1986) predicts that Oikopleura orthologs of
vertebrate genes important for olfactory placode development should also be
expressed in the developing larvacean ventral organ. To test this prediction,
we analyzed the expression of larvacean eya, pitx and six
genes in Oikopleura embryos and hatchlings.
In early larvacean hatchlings, when organogenesis is still ongoing in the
trunk, eya, pitx and six1/2 genes appear to be co-expressed
in ectodermal cells concentric with cells immediately surrounding the
presumptive mouth (Fig. 5A-D).
For eya and pitx, this expression is a continuation of
rostral ectodermal expression from mid-tailbud stages (discussed above).
Ventrally, paired patches of six1/2 expression meet at the midline
(Fig. 5B). Similar patterns are
observed for both eya and pitx (not shown and
Fig. 5C). The ventral organ,
which forms in this region, develops from ectodermal cells that sink into a
groove perpendicular to the body axis
(Bollner et al., 1986), and
although the ventral organ appears unpaired in adults, this early gene
expression pattern together with bilateral innervation reveals that the organ
originates as a paired structure. In lateral view, six1/2 expression
is just rostral to the thickened ectodermal epithelium at the anterior border
of the underlying endostyle; if these six1/2-expressing cells are
presumptive ciliated receptor cells, then this represents a stage before their
involution (Fig. 5B).
Pitx expression around the presumptive mouth in hatchlings includes cells in the ring domain shared with six1/2 and eya, and also cells immediately adjacent to the mouth (Fig. 5C,D), but excludes at least the upper lip cells, which in late hatchling stages carry sensory bristles. six1/2 is also expressed in cells immediately adjacent to the mouth (Fig. 5B), in the region where the bristle-bearing cells and other ciliated mechanoreceptors will form (discussed below).
In late hatchling stages, the cells lining the rostral nerves express both pitx (Fig. 5E,F) and six3/6a (Fig. 5G).
Oikopleura eya, pitx and six gene expression in the
developing ventral organ is consistent with the homology of this organ to the
vertebrate olfactory placodes, which express orthologs of these genes.
Although vertebrate Eya and Six genes are found in all other placode types,
only the olfactory, adenohypophyseal and lens placodes express Pitx genes
(Baker and Bronner-Fraser,
2001; Gage et al.,
1999
). Because in larvacean embryos the dorsal and lateral arc of
expression around the mouth is contiguous with ventral expression, we propose
that bilateral halves of this ring of expression mark ventral organ placodes
that include not only the ventral ciliated receptors but also the delaminating
cells that will ensheath the paired rostral nerves. eya, pitx and
six1/2 expression appears to include more than just the delaminating
and receptor cells, however, because expression persists in the ectoderm
overlying these structures until mid- or late hatchling stages (e.g.
Fig. 5E,F); this expression may
be important for development of epidermal cells that remain in close physical
association with derivatives of the ventral organ placodes.
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Duplicate larvacean homologs of six3/6 are expressed in the
rostral pharynx. In young hatchlings, the expression domain of
six3/6b is nested within the broader six3/6a expression
domain (Fig. 6A-D). While
six3/6a is expressed in the entire roof of the anterior pharynx, in
the rostral brain, in the esophagus and in posterior ectodermal domains,
six3/6b appears only in a trio of cells on the right-hand side
(Fig. 6B,D); these cells may
give rise to the three or four cells that make up the most ventral part of the
adult ciliary funnel (Holmberg,
1982) and, later, the ventral part of the morphologically distinct
funnel expresses six3/6b (Fig.
6F). Six3/6a expression in later stages marks the entire
ciliary funnel from its base to the brain (not shown) and is therefore
presumably in both pharynx-derived and CNS-derived parts of the funnel.
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The expression patterns of larvacean pitx and six3/6
genes are consistent with the orthology of the ciliary funnel and the
vertebrate pituitary rather than other placode-derived organs, particularly
because vertebrate Pitx genes are expressed in only the most anterior placodes
(pituitary, olfactory and lens) (Baker and
Bronner-Fraser, 2001). Pitx is expressed in the
ventral-most part of the funnel, while six3/6 paralogs are expressed
in both the ventral and more dorsal parts of the funnel. As vertebrate Pitx
genes are expressed in the adenohypophysis while Six3 and
Six6 are expressed in both the adenohypophyseal placode and the
hypothalamic primordium (e.g. Ghanbari et
al., 2001
), the Pitx-expressing and non-Pitx-expressing domains of
the larvacean funnel might correspond to adeno- and neurohypophyseal
components, respectively.
eya and six3/6a in the Langerhans and circumoral ciliated mechanoreceptors
Oikopleura has other paired peripheral mechanoreceptor organs, the
Langerhans and oral sensory organs. The two Langerhans organs include a
receptor cell bearing a long rigid cilium, supporting cells, and a neuron in
the caudal ganglion that sends processes to each of the Langerhans organs and
towards the anterior brain (Holmberg,
1986). In the rostral part of the anterior brain, two neurons
innervate respectively right and left sides of the oral sensory complex; e.g.
with a single branching axon, the left neuron innervates both the left upper
lip cell and the left side of the circumoral ring of ciliated cells
(Olsson et al., 1990
).
Are the larvacean sensory cells homologous to either ascidian cupular or coronal receptor cell types? Are they homologous to vertebrate hair cells? Hypotheses that the Langerhans organ is homologous to the inner ear neuroepithelium and that the larvacean oral sensory system is homologous to the lateral line neuromasts predict that larvacean homologs of Eya and Six genes important for otic and lateral line placode development should be expressed in the primordia of these larvacean peripheral mechanosensory organs. We found larvacean eya and six3/6a expression in lateral paired patches in the trunk of tailbud embryos and early hatchlings where the Langerhans organs develop (Fig. 7A-F), and eya and six1/2 expression in the oral region in late hatchlings where the sensory cells of the lips develop (Fig. 7H,I).
In tailbud stages, paired lateral ectodermal patches at the junction of the
trunk and tail express eya in the region where the Langerhans
mechanoreceptors will develop (Fig.
7A,B). Expression narrows to a pair of cells at the posterior,
ventral trunk in early hatchlings (Fig.
7D). The pharyngeal endoderm of the proximal gill pouches also
expresses eya (Fig.
4A); similarly, murine Eya1 is expressed in the
pharyngeal pouch endoderm (Xu et al.,
1997).
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Cells immediately surrounding the presumptive mouth express
six3/6a and pitx in tailbud stages
(Fig. 4A-G); they express
six1/2 in early hatchlings (Fig.
4B), and they express both six1/2 and eya in
late hatchling stages (Fig.
7H). In addition, the lower lip and the stomodeal roof, presumably
including the precursors of the ciliated cells, express pitx in
hatchling stages (Fig. 4B,E; Fig. 5C,D,F;
Fig. 6H). Interestingly, the
two bristle-bearing cells of the upper lip
(Fig. 6H) in these stages
appear not to express pitx, but pitx is expressed in a
prominent cell bulging from the left rostral brain
(Fig. 5E); this brain
expression is almost certainly one of the paired cells described by Olsson et
al. (Olsson et al., 1990) that
conspicuously bulge from the left rostral brain and originate nerve n2 to both
the upper lip and ciliated cells of the mouth.
Although neither the larvacean Langerhans nor the circumoral mechanoreceptor organs have been proposed on morphological grounds to be homologous to vertebrate placode-derived sensory organs, the embryonic primordia of these tissues appear to express orthologs of vertebrate placode-marking genes. These larvacean sensory systems could have independently evolved from a placode precursor in the common ancestor of chordates and may have no counterpart in vertebrates. Alternatively, the Langerhans organ primordia may be orthologous to both the ascidian atrial primordia and the vertebrate otic placodes.
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Discussion |
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In vertebrates, evidence suggests that specific placodes individualize from
a pan-placodal field by a series of inductive fate restrictions
(Streit, 2004), and, at least
in zebrafish, placode precursor cells move to condense from an initially broad
field (Whitlock and Westerfield,
2000
). By contrast, larvacean placode specification probably
occurs when the embryo consists of fewer than 200 cells (before incipient
tailbud stage). In concert with reduced cell number and mosaic development,
the gradual cascade of placode specification could have become secondarily
simplified in the urochordate lineage, leaving no broad pan-placodal field
marked by simultaneous expression of Eya, Pitx and Six genes, but rather the
expression of at least some of these markers first appears when placode
subtypes are already spatially separated and distinguished by unique, although
overlapping, sets of markers.
The ventral organ and the vertebrate olfactory organ
Eya and Six proteins (excluding Six3 and Six6) interact synergistically,
both in vertebrates and in flies, to activate transcription of downstream
genes (Ohto et al., 1999).
Oikopleura six1/2 and eya expression domains overlap in a
ring of ectodermal cells centered on the presumptive mouth
(Fig. 4). The overlap of
six1/2 and eya would be predicted if their protein products
interact in the same way as their homologs do in vertebrates and flies.
Larvacean pitx expression also overlaps eya and
six1/2 in a ring of ectodermal cells around the mouth
(Fig. 4E,I;
Fig. 5C,D). Vertebrate
Pitx1 and Pitx2 genes are expressed early in the stomodeum,
this gene expression persists in the olfactory and adenohypophyseal placodes
as these invaginate, and Pitx1 may directly regulate vertebrate olfactory
receptor genes (Hoppe et al.,
2003). The location and timing of larvacean six1/2, eya
and pitx expression in the rostral ectoderm are appropriate to mark
two cell groups (the ciliary cells of the ventral organ and the ensheathing
`rostral brain bulb' cells) that form by a placode-like process. Consistent
with Delsman's (Delsman, 1912
)
and our observations that the ensheathing cells along the rostral nerves
delaminate from the ectoderm, initially ectodermal epithelial cells express
pitx and then the rostral brain bulb cells express the gene
(Fig. 6C-F). The rostral nerves
also express six3/6a at the stage when the separation of the bulb
cells from the rostral epidermis is visible
(Fig. 6G), consistent with the
expression of vertebrate Six3 genes in the olfactory placode (e.g.
Ghanbari et al., 2001
).
We propose, based on the expression of eya, six1/2, six3/6a and
pitx, that the rostral bulbs represent part of the ventral organ
placode, and that this placode is the ortholog of the vertebrate olfactory
placode. In vertebrates, the olfactory placode produces not only ciliary
sensory cells, but also several other cell types, some of which are ultimately
located at a distance from the olfactory epithelium; these types include
supporting, basal and ensheathing cells
(Farbman, 1992).
Bollner et al. (Bollner et al.,
1986) sorted the four ensheathing cells of the larvacean rostral
bulbs into three classes, one of which had mesaxon-like structures similar to
those typical of vertebrate olfactory ensheathing cells (OECs), which bundle
the unmyelinated axons of the receptor cells. Unlike the Schwann cells of
other sensory nerves, OECs originate in the placode itself rather than from
neural crest, and, like the larvacean rostral bulb cells, they embrace bundles
of axons, rather than enveloping axons one at a time
(Farbman, 2000
). However, the
bulb cells are also like vertebrate terminal nerve cells, some of which appear
to originate in the olfactory placode
(Wirsig-Wiechmann, 2001
).
Terminal nerve cells exert a neuromodulatory influence on the olfactory
neurons; similarly, the larvacean bulb cells exhibit immunoreactivity for
GABA, an inhibitory neurotransmitter
(Bollner et al., 1991
).
There is no structure morphologically comparable with the ventral organ
described in cephalochordates or ascidians. In cephalochordates, although the
rostral epidermis that has been suggested to be olfactory is marked by
AmphiPax-6 in post-gastrula Branchiostoma floridae
(Glardon et al., 1997), the
corresponding tissue does not appear also to express pitx
(Boorman and Shimeld, 2002
).
Furthermore, individual ciliated sensory cells identical to those that form in
the Pax6 domain are also scattered throughout the trunk epidermis and
are not discretely clustered (Holland and
Yu, 2002
; Northcutt,
1996
), making them unlike placode-derived olfactory organs.
Burighel et al. (Burighel et al.,
1998
) have proposed that the ascidian neural gland complex is both
an olfactory and pituitary homolog
(Burighel et al., 1998
). The
idea that a single organ served for both olfaction and hormone regulation in a
chordate ancestor and that this organ split into separate olfactory and
pituitary organs in vertebrates (Gorbman,
1995
) is weakened by the existence of separate probable olfactory
and adenohypophyseal organs in Oikopleura, and by the finding of
Whitlock et al. (Whitlock et al.,
2003
) that cells of the terminal nerve that produce GnRH
(otherwise a hormone of the pituitary) are products of neural crest rather
than the olfactory placode, as was originally thought. Mazet et al.
(Mazet et al., 2005
) recently
proposed that the ascidian palps might be an olfactory organ homolog, based on
expression of eya and neuronal marker COE; it seems likely,
on the basis of gene expression and topography, that the Oikopleura
ventral organ placode is homologous to the ectoderm of the ascidian palps,
although, unlike the larvacean ventral organ, no ciliated sensory cells have
been identified on the palps. Other olfactory markers in non-vertebrate
chordates should be analyzed to resolve these apparent differences among
chordates.
The ciliary funnel and the vertebrate pituitary
Like early developmental stages of the vertebrate adenohypophysis, the
larvacean ciliary funnel forms a pouch that connects the pharyngeal roof with
the brain. Much of the larvacean funnel, however, is an outgrowth of the brain
(Delsman, 1912). The neural
origin and secretory behavior of the ciliary funnel prompted Holmberg
(Holmberg, 1982
) to propose
that this larvacean organ represents the combined homolog of the
adenohypophysis and neurohypophysis. Fate-mapping in chick
(Takor and Pearse, 1975
) and
amphibians (Kawamura and Kikuyama,
1992
) shows that immediately adjacent regions give rise to the
hypothalamus and the adenohypophysis, though they separate after neural tube
closure, and the authors argue that the pituitary should be regarded as a
single developmental entity rather than as a composite organ. Expression of
vertebrate Six3 in both the hypothalamus and Rathke's pouch is a
continuation from the earlier expression of a gene in the anterior neural
plate (Oliver et al., 1995
;
Ghanbari et al., 2001
;
Kobayashi et al., 1998
).
Expression of larvacean Six3/6 homologs in the ciliary funnel,
therefore, supports this organ's homology with the vertebrate adeno- and also
neurohypophysis. Larvaceans may embody either an ancestral or a derived state
in which the adenohypophyseal placode maintains a physical connection to the
brain from the neural plate stage when they are contiguous.
Although Rathke's pouch falls within the ectoderm of the stomodeum in most
vertebrates, the ciliary funnel in Oikopleura connects to the roof of
the pharynx; in addition, larvacean six3/6 genes are expressed in the
pharyngeal roof before the funnel is morphologically distinguishable. Although
there are vertebrates in which the adenohypophyseal placode contacts the
foregut endoderm before it contacts the stomodeum [e.g. in hagfish
(Gorbman, 1983)], it is also
possible that the roof of the larvacean rostral pharynx is of ectodermal
origin. Larvacean pax2/5/8 is expressed in the rostral pharynx (S.
Bassham, PhD Thesis, University of Oregon, 2002), while Ciona
Pax2/5/8 is expressed in the ectoderm of the stomodeum
(Wada et al., 1998
).
Similarly, in the ascidian Botryllus, the dorsal pharynx wall that
will fuse to the stomodeum and form the ciliated duct secretes tunic material,
an ectodermal product (Manni et al.,
1999
). A fate map of early larvacean development is needed to
assess whether the rostral pharynx and the ventral part of the ciliary funnel
develop from an endodermal or ectodermal lineage.
In addition to the Six genes, the developing vertebrate pituitary also
expresses Pitx genes. Vertebrate Pitx1 is an early marker of the
stomodeum and the foregut endoderm, and its expression persists in the
olfactory and adenohypophyseal epithelia into late development
(Lanctot et al., 1997). In the
pituitary, Pitx1 ultimately acts to regulate transcription of hormone
genes, and Pitx2 is important for proper morphogenesis of Rathke's pouch
(Drouin et al., 1998
;
Suh et al., 2002
). Larvacean
pitx is also expressed in the mouth and rostral foregut, where it
overlaps with the expression of six3/6a and six3/6b. pitx,
however, does not appear to be expressed in dorsal parts of the funnel. This
is similar to ascidian development, where only the ciliated funnel in the
pharyngeal wall expresses pitx but the ciliated duct that connects to
the brain does not (Boorman and Shimeld,
2002
).
Arguments that the origin of the adenohypophysis predated the origin of the
vertebrates and was originally chemosensory
(Gorbman, 1995;
Manni et al., 1999
;
Nozaki and Gorbman, 1992
;
Olsson, 1990
) are based in
part on the location of Rathke's pouch, Hatschek's pit and the ciliated funnel
in the buccal cavity, where, in an aquatic organism, these organs would be in
constant communication with waterborne chemicals from the environment. Because
of the importance of synchronizing sexual maturation and reproductive
behavior, the systems for collecting olfactory information, including sex
pheromones, and for eliciting maturational and behavioral changes could have
been anciently linked (Muske,
1993
). Because ascidian pitx is not expressed in the part
of the duct proximal to the brain and because vertebrate Pitx is
eventually downregulated in the olfactory organ, Boorman and Shimeld
(Boorman and Shimeld, 2002
)
tentatively proposed that the ascidian duct could be homologous to the
olfactory component of a combined adenohypophyseal/olfactory organ; this
hypothesis, similar to that of Burighel et al.
(Burighel et al., 1998
),
implies a conflation of organs in ascidians that is at odds with the separate
olfactory and adenophypophyseal homologies that we propose in Oikopleura
dioica and Mazet et al. (Mazet et
al., 2005
) propose for ascidians.
In any case, both ascidian and larvacean urochordates apparently differ from vertebrates, which continually use Pitx in adenohypophyseal development through adult pituitary function. Functional tests are needed to determine if the ability of Pitx to regulate transcription of hormone genes is conserved in urochordates.
Larvacean mechanosensory organs and placode-marking genes
In the ascidian Ciona, the walls of the exhalent atrial siphon
contain hair cell-like cupular organs that Bone and Ryan
(Bone and Ryan, 1978) have
proposed are homologous to the acoustico-lateralis (otic + lateral line)
system. Wada et al. (Wada et al.,
1998
) proposed that the atrial primordia are, more specifically,
orthologs of vertebrate otic placodes. Larvaceans also have peripheral
ciliated mechanoreceptors. In the posterior larvacean trunk, the paired
ciliated Langerhans receptors elicit an escape response when stimulated
(Bone and Ryan, 1979
). Several
characteristics make these organs plausible homologs of vertebrate otic
placode derivatives. The larvacean Langerhans cells are unusual for
non-vertebrate sensory cells in that they are secondary receptors, meaning
they do not themselves send an axon to the CNS but instead are separately
innervated (Bone and Ryan,
1979
). In this respect, they are more like hair cells than are
cupular cells, which are primary receptors
(Bone and Ryan, 1978
).
Vertebrate Eya1 and Six1 are expressed in the otic placode
where they are crucial for the development of the sensory epithelium
(Zheng et al., 2003
). Paired
patches of larvacean eya expression appear in tailbud stage at the
junction between the trunk and the tail
(Fig. 7A,B) where the
Langerhans receptors will develop. The paired larvacean eya
expression pattern is adjacent to the caudal ganglion, the larvacean homolog
of the vertebrate hindbrain
(Cañestro et al.,
2005
), where the axons that innervate the receptors originate
(Holmberg, 1986
). This
topographic relationship is similar to vertebrate otic development in which a
combination of signals from the nearby hindbrain and mesendoderm may induce
otic placode formation (e.g. Liu et al.,
2003
) and the otic neuron axons project to the hindbrain.
Oikopleura has linearly arranged ciliated mechanoreceptors that
ring the mouth (Fig. 7I);
similar to the vertebrate lateral line organs, they are secondary receptors
and lie in a groove formed by overlying epithelial cells
(Olsson et al., 1990).
Touching these cilia causes a reversal of the feeding current driven by the
spiracle (gill) cilia ring, a response predicted to reject particles too big
for the fixed-gape mouth (Galt and Mackie,
1971
; Lohmann,
1933
). Eya1 is important for survival of hair cells in
the developing ear and lateral line placodes of zebrafish
(Kozlowski et al., 2005
), and
Six1 is expressed in lateral line hair cells in amphibians and fish
(Bessarab et al., 2004
;
Pandur and Moody, 2000
). The
developing larvacean stomodeum expresses both eya and
six1/2, suggesting that, as in vertebrates, these genes could play a
role in the development of ciliated mechanosensory cells.
Superficial structural parallels between urochordate and vertebrate
ciliated mechanosensory systems suggest that the common chordate ancestor
already had lateral line and otic placodes. The variety of morphologies among
mechanoreceptor cells and organs in larvaceans, ascidians and vertebrates,
however, might indicate that, rather than necessarily being specific otic and
lateral line orthologs, these organs could have evolved by the independent
proliferation in chordate lineages of a mechanosensory placode precursor.
Although Eya1 is expressed in vertebrate otic placodes, Six3
and Six6 are not. The unpredicted expression of a larvacean
six3/6a in the developing Langerhans receptor could represent a
larvacean co-option of this homeobox gene into otic development, or it could
be evidence that the Langerhans organs are paralogs, rather than orthologs of
the otic placodes. The fact that Ciona six1/2 is expressed in the
atrial primordia (Mazet et al.,
2005) is evidence in favor of the first of these hypotheses.
The projection of axons from the larvacean circumoral sensory cells into
the rostral brain and the projection of axons from the ascidian coronal organ
into the cerebral ganglion (Burighel et
al., 2003) are not predicted for a lateral line ortholog, whose
nerves should enter the hindbrain as in vertebrates
(Gompel et al., 2001
). The
presence of ciliated, secondary mechanoreceptors in the oral ectoderm is
common to larvaceans, ascidians and cephalochordates
(Lacalli and Hou, 1999
),
however, and a common ancestral organ cannot be excluded, despite differing
cell morphologies. Vertebrate Eya1 and Six1 expression is
persistent in the stomodeum (Schlosser and
Ahrens, 2004
), suggesting that the stomodeum of the
vertebrate/urochordate common ancestor expressed Eya and
Six1/2 and a role for these genes in oral mechanosensory organ
development may have been retained in filter-feeding non-vertebrate chordates.
Expression analysis of Eya and Six1/2 in coronal
organ-bearing ascidians and in amphioxus, and functional analysis of the genes
are needed to test this hypothesis.
Conclusions
In summary, we found that developmental expression of larvacean eya,
pitx and six genes corresponds with predictions stemming from
morphology-based homology assignments between larvacean and vertebrate
placode-derived organs, making a strong case not only for placodes, but for
specific placodes as plesiomorphies, or primitive character states, of the
phylum Chordata. We propose that separate olfactory and pituitary organs were
already present in the common ancestor of modern chordates
(Fig. 8).
In addition, mechanosensory organs orthologous to the otic placode, represented in urochordates by the larvacean Langerhans and ascidian atrial placodes, might also be ancestral to all modern chordates. Finally, a fourth, oral mechanosensory placode may be ancestral in the chordates, but lost or modified in the vertebrates. We caution, however, that there could have been an independent proliferation and parallel evolution of mechanosensory placodes in the different chordate lineages, and that, although these placodes retain ancestral gene expression of placode markers, they might be paralogous organs. Likewise, the presence of both orthologous and paralogous placodes in vertebrates with respect to urochordates implies independent proliferation and diversification of some placodes in the vertebrates, but this is not incompatible with a common placodal primordium in early vertebrate development. Differences in morphology and complement of sensory organs among larvaceans and ascidians highlight the need for broad sampling among chordate clades before drawing generalizations about the inferred chordate ancestor.
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ACKNOWLEDGMENTS |
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