BACKGROUND
ohno's classical model to explain the fate of genes after gene duplication  <cit>  predicts that one gene duplicate preserves the original gene function while its paralog either disappears by accumulation of detrimental mutations  or occasionally acquires beneficial mutations that confer novel, positively selected functions .
the duplication-degeneration-complementation  model predicts a third alternative, in which the two duplicate genes become permanently preserved as a consequence of complementary, degenerative mutations that result in partitioning of ancestral subfunctions, so that the sum of the functions of two paralogs equals the functions of the original gene prior to the duplication  <cit> .
the ddc model also predicts that after the initial preservation of the two duplicates, whether by subfunctionalization or from neofunctionalization, further partitioning of redundant subfunctions may occur.
studies show that functional constraints on genes duplicated in whole-genome duplications are relaxed, compared with singletons, for tens of millions of years  <cit> .
in addition, novel functions may originate over time, their evolution facilitated by the relaxation of pleiotropy occasioned by fewer tasks in each descendent gene duplicate compared with its single copy gene ancestor  <cit> .
understanding the evolution of duplicated genes is important because of the hypothesis that gene duplicates provide opportunities for the evolution of reproductive barriers that lead to lineage divergence  <cit> , and for the origin of evolutionary novelties  <cit> .
in vertebrates, many sets of paralogous genes arose in two rounds of genome duplication  that took place in early vertebrate evolution  <cit> .
an additional round of genome duplication  occurred in the teleost lineage after ray-fin fish diverged from lobe-fin fish, and provided additional gene family members observed in many fish models  <cit> .
it has been suggested that the r <dig> and r <dig> genome amplifications facilitated the origin of vertebrate innovations  <cit> , and the r <dig> event may have facilitated the teleost species radiation  <cit> .
non-vertebrate chordates often have single copies of vertebrate gene families because their lineages diverged from the vertebrate lineage before the r <dig> and r <dig> genome duplication events.
recent phylogenomic analyses converge on the conclusion that the chordate subphylum urochordata, which includes the classes larvacea and ascidiacea, are the closest living relatives of the vertebrates, constituting the group olfactores , while the subphylum cephalochordata, including the amphioxus, diverged basally among chordates .
as gene duplication is pervasive  <cit> , non-vertebrate chordates sometimes have genes that duplicated independently in the cephalochordate  <cit>  or urochordate lineages  <cit> .
we propose that the comparative analysis of gene expression patterns in a gene family that experienced lineage-specific, independent duplication events, interpreted in a phylogenetic context and with respect to subfunction partitioning, can help in the inference of ancient gene functions and in the identification of gene functions that arose by neofunctionalization, and thus may be important for lineage divergence and the origin of developmental novelties.
to test this proposition, we examined the pax2/5/ <dig> gene family.
pax2/5/ <dig> genes encode transcription factors with conserved motifs, including a paired domain, homeodomain, and octapeptide, and are associated with mechanosensory development in mammals  <cit> , frogs  <cit> , fish  <cit> , flies  <cit> , ascidians  <cit>  and in mollusks  <cit> , while in a cnidarians, the apparent pax2/5/ <dig> homolog is associated with nerve and sensory cell differentiation  <cit> .
pax2/5/ <dig> genes duplicated independently in different chordate lineages.
within the vertebrates, tetrapods have three members of the pax2/5/ <dig> gene family , and teleosts additionally have duplicate pax <dig> genes .
in contrast to vertebrates, the basally diverging cephalochordate amphioxus possesses a single pax2/5/ <dig> gene that is equally related to pax <dig>  pax <dig> and pax <dig>  <cit> , and urochordates have two pax2/5/ <dig> genes   <cit>  that originated in a duplication event prior to the divergence of larvacean and ascidian lineages  <cit> .
the chordate pax2/5/ <dig> family embodies a full spectrum of gene evolutionary events: non-duplication ; independent duplications ; gene loss ; neofunctionalization ; and ancestral subfunctions appear to have been partitioned between paralogs within a lineage and, further, differently partitioned between lineages .
the independent duplication of the stem olfactores' pax2/5/ <dig> gene in vertebrate and urochordate lineages provides replicate evolutionary experiments to explore the principles of subfunction partitioning and the origin of novel functions.
to exploit this opportunity, we provide here a detailed description of expression patterns for pax2/5/ <dig> paralogs during development of the larvacean urochordate oikopleura dioica.
we then compare our results with expression patterns of pax2/5/ <dig> orthologs in ascidians, with independently duplicated pax2/5/ <dig> paralogs in vertebrates, and with the non-duplicated pax2/5/ <dig> gene in amphioxus as an outgroup.
we discuss the expression of the pax2/5/ <dig> gene family during development of the heart, endostyle, pharynx, and sensory organs, and provide new insights that reconcile previous conflicting hypotheses about the homology of the ascidian atrial primordia and the vertebrate otic placode.
this work thus illustrates the power of comparative analyses of independently duplicated genes to infer ancestral gene subfunctions, modules that can segregate independently from each other to evolving gene duplicates.
RESULTS
functional motif variation and gene structure of chordate pax2/5/ <dig> paralogs
pax2/5/ <dig> proteins are transcription factors, whose sequence is poorly conserved across chordata outside of two functional motifs: the paired domain, which interacts with the dna of target genes , and an octapeptide motif , which is conserved with other pax proteins  <cit> , functions in repression of pax transactivation  <cit> , and interacts with other transcriptional cofactors  <cit> .
sequence alignment of various chordate pax2/5/ <dig> proteins  reveals that while the sequence of the dna-interacting paired domain is highly conserved across all chordate pax2/5/ <dig> proteins analyzed, the octapeptide motif is less conserved.
in urochordates, for instance, the octapeptide is present in the ascidian halocynthia roretzi  <cit> , and, contrary to a previous report  <cit> , our alignment reveals it is also present in ciona intestinalis pax2/5/8a .
the octapeptide sequence of the oikopleura pax2/5/8a gene is poorly conserved, and the octapeptide motif is absent from the expected position in all urochordate pax2/5/8b proteins .
interestingly, we have identified a new motif  that is conserved among all ascidian pax2/5/8b paralogs, located further toward the carboxyl end.
as this newly recognized sequence shows some similarities with the octapeptide motif, we have called it the 'octapeptide-like' motif .
we could not identify an octapeptide-like motif in the oikopleura pax2/5/8b.
our alignment also reveals a lysine-arginine-rich domain  that is conserved in vertebrate pax <dig> and amphioxus pax2/5/ <dig>  is variable among vertebrate pax <dig> and pax <dig> paralogs, and is present in urochordate pax2/5/8b but not pax2/5/8a.
this protein motif marks the beginning of an amino acid range identified as important for greatly increasing vertebrate pax <dig> protein's transactivation activity  <cit> .
comparison of gene structures revealed that, while most chordate pax2/5/ <dig> genes have  <dig> to  <dig> exons, ascidian pax2/5/8b paralogs have  <dig> exons, which code for a protein of about  <dig> amino acids, three times longer than the approximately  <dig> amino acid-long pax2/5/ <dig> proteins from other chordates.
this difference in size is due mainly to an exon more than  <dig> nucleotides long located upstream of the paired domain.
our analysis of publicly available est sequences confirmed the presence of this large exon in gene models predicted for pax2/5/8b in ciona intestinalis  and in ciona savignyi  .
this large exon is absent from o. dioica pax2/5/ <dig> paralogs and may have evolved in the ascidian lineage after the divergence of urochordates.
in conclusion, various pax2/5/ <dig> paralogs appear to have lost ancestral features and evolved new structural motifs or new exons, as would be predicted by the ddc model applied to pax2/5/ <dig> paralogs evolving after independent gene duplication events.
the identification of these structural features focuses attention on regions to test for function to learn the roles each motif may have played in the origin of lineage-specific morphologies.
developmental expression of the oikopleura paralogs pax2/5/8a and pax2/5/8b
tailbud stages
at the end of gastrulation, pax2/5/8a and pax2/5/8b were both broadly expressed in the oikopleura embryo.
pax2/5/8a was expressed mainly in the ectoderm of the trunk , while pax2/5/8b was expressed primarily in the interior of the trunk .
in early tailbud stages, pax2/5/8b expression continued in its broad internal expression domain , but pax2/5/8a expression became restricted to two domains: a few medial cells at the boundary of the anterior brain and the pharynx , and a bilateral pair of ectodermal cells in the posterior trunk .
by mid- and late-tailbud stage, the signal of the pax2/5/8a expression domains strengthened , and a new domain appeared in the rostral-most ectoderm of the trunk , probably labeling the first two cells fated to invaginate into the stomodeum and form the oral epithelium .
early hatchling and mid-hatchling stages
at the early hatchling stage, pax2/5/8a expression maintained its late-tailbud stage pattern.
the rostral-most expression domain in the stomodeal region expanded internally from the surface to form a group of contiguous internal cells , which were separated from the internal expression domain in presumptive pharynx/endostyle precursor cells .
the bilateral pax2/5/8a expression domain in the posterior part of the trunk included at least four ectodermal cells in the area where the langerhans mechanoreceptors eventually develop , at a level slightly anterior to the tip of the notochord and adjacent to the homolog of the vertebrate hindbrain  <cit> .
during early hatchling stages, the internal broad expression domain of pax2/5/8b that was present at tailbud stages began to fade at the same time that two separate expression domains became distinct, one in the ectoderm at about the same bilateral position as the pax2/5/8a ectodermal domain  and the other within the pharynx .
although pax2/5/8a and pax2/5/8b were both expressed in the ectoderm at the eventual position of the langerhans receptors, close inspection revealed differences in the number and morphology of the pax2/5/8a and pax2/5/8b positive cells .
by mid-hatchling stages, the bilateral pax2/5/8a expression domain expanded to at least six ectodermal cells , while the brief flash of pax2/5/8b expression in the ectoderm became undetectable .
simultaneously, the distal portion of each gill pouch began to express pax2/5/8b.
it is not known if ectodermal cells contribute to the formation of the gill pouches at these early stages when the ectoderm is still forming its epithelial character.
expression of the two oikopleura pax2/5/ <dig> paralogs during mid-hatchling stages appeared to be complementary in the pharynx in time and space: while pax2/5/8a expression was diminishing in the stomodeum and the rostral pharynx , new pax2/5/8b expression domains appeared .
the proximal endoderm of the gill pouches began to express pax2/5/8a while the distal portion of the gill pouches expressed pax2/5/8b .
presumptive dorsal precursor cells of the endostyle first began to express pax2/5/8a, while presumptive ventral endostyle cells expressed pax2/5/8b .
late hatchling stages
by the late hatchling stage, organs are distinct and internal cavities have started to expand.
at this stage, the gills and mouth have opened to the outside, and the heart and the ciliary rings of the gills have both begun to beat.
the direct development of larvacean urochordates permits the tracing of gene expression domains in organ rudiments from hatchlings until fully mature adults.
in contrast, in ascidian urochordates, many organs begin to develop only at metamorphosis, just as many chordate-specific characters disappear.
pax2/5/8a expression in late hatchling stages overlaps pax2/5/8b in some domains: in the distal part of the gills from the ciliary rings  to the external gill opening , and, transiently, in the posterior trunk epidermis .
other domains are unique to each gene, and in several regions expression of the paralogs appears complementary.
for example, while the lips of the mouth express pax2/5/8a , the pharynx just internal to the lips strongly expresses pax2/5/8b ; while dorsal cells of the endostyle express pax2/5/8a , ventral cells of the endostyle express pax2/5/8b ; and while the most posterior section of the rectum expresses pax2/5/8a , the anus expresses pax2/5/8b .
except for the distal part of the gills, the posterior pharynx exclusively expresses pax2/5/8b, including the gill endoderm where the two gill pouches meet medially .
and, strikingly, only one layer of the two-ply heart, the pericardium, expresses pax2/5/8b ; the muscular myocardium does not.
a subtle metamorphic event called 'tailshift', the rapid reorientation of the tail from a posterior to a ventral position with respect to the trunk, signals the beginning of the juvenile stage in oikopleura.
just prior to tailshift, the two pax2/5/ <dig> paralogs continue to show mostly non-overlapping expression patterns, now in clearly differentiated organs.
pax2/5/8a  is still expressed around the mouth , in the dorsal, iodine-binding corridor cells of the endostyle, in the gill endoderm, in the rectum, and in a row of cells at the posterior margin of the oikoplastic epithelium .
expression of pax2/5/8b  is also a continuation of domains identified earlier, including the pharynx and gills, the ventral endostyle, the anus, and the pericardium.
new domains, however, now appear in dorsal, ventral and lateral fields of the oikoplastic epithelium, the tissue that will soon secrete the first filter-feeding 'house' the animal inhabits  <cit> .
figure 5c is a schematic diagram summarizing the expression domains of both oikopleura pax2/5/ <dig> paralogs in late hatchlings.
table  <dig> summarizes by tissue and developmental stage all pax2/5/ <dig> expression domains.
a, pax2/5/8a; b, pax2/5/8b; a/b, expression of paralogs does not spatially overlap or only partly overlaps; a+b, both paralogs spatially overlap.
discussion
these investigations of the structure and expression of pax2/5/ <dig> gene duplicates in oikopleura, when analyzed in a comparative context with shared and independent pax2/5/ <dig> gene duplications in other chordate lineages, illuminate a variety of problems in the evolution of chordate developmental mechanisms and the principles that govern change in duplicated gene function over time.
the work resolves alternative hypotheses for the homologies of the ascidian atrial siphon, illuminates evolution of the thyroid, identifies candidates for ancestral gene subfunctions, and defines the origin of functional innovations in this gene family.
pax2/5/ <dig> and the evolution and development of the chordate heart
the shared expression of cardiac developmental genes  supports the homology of bilaterian hearts from chordates to flies  <cit> .
the evolutionary relationship of the single-chambered heart of non-vertebrate chordates to the individual chambers of the innovative multi-chamber vertebrate heart, however, is still unclear .
our work reveals unprecedented pax2/5/ <dig> expression in the developing heart of a chordate, and may provide a late ontogenetic marker for assessing homology of tissue layers among simple, urochordate hearts.
in contrast to the single-layered heart of cephalochordates, urochordates have a two-layered heart.
the simple heart of oikopleura dioica consists of two epithelial layers lying just medial to the left stomach lobe.
in oikopleura species, the lateral wall of the heart contains the muscle fibers while the medial wall is a thin pericardial membrane .
peristaltic heart contractions, which periodically reverse, cause the hemolymph to course between the heart and the stomach wall  <cit> .
in comparison, the ascidian heart rolls up to become tubular rather than planar, but also has a contractile layer and a non-muscular, pericardial layer .
the larvacean pericardial membrane, therefore, is the likely homolog of the ascidian pericardium.
in ascidians, heart development can be broadly divided in two phases .
in the first phase, heart cell precursors become fated during early cleavage and embryonic stages, and after a complex process of muscle cell migration from the tail to the trunk, heart development temporarily arrests.
in the second phase, after metamorphosis, heart development re-initiates and after final differentiation, the heart starts to beat and becomes functional.
in larvaceans, the transparency and absence of a drastic metamorphosis provides a complementary model system for the study of heart development.
in o. dioica, the heart starts beating less than  <dig> hours post fertilization, before tailshift.
in late larvacean hatchlings, when heart differentiation is at or nearing completion, the pericardium expresses pax2/5/8b.
in ascidians, however, neither pax2/5/ <dig> paralog has been shown to be expressed in the heart, perhaps because most published analyses of pax2/5/ <dig> expression in ciona, halocynthia and herdmania  <cit>  have not included late developmental stages after metamorphosis .
therefore, whether pax2/5/ <dig> expression in the heart – and more specifically in the pericardium – is a shared feature among urochordates remains to be investigated.
cephalochordates have a single-layered, muscularized blood vessel ventral to the gut that propels the hemolymph by peristalsis  <cit> .
during amphioxus development, the primordium of this muscularized vessel expresses a homolog of vertebrate nk <dig> class genes – a group that includes several genes necessary for vertebrate cardiac development – suggesting that the amphioxus vessel is homologous to the vertebrate heart  <cit> .
pax2/5/ <dig> is not reported to be expressed in the circulatory vessels of amphioxus  <cit> , and likewise, amphioxus has no structure that appears to be morphologically comparable to the pax2/5/8-expressing larvacean pericardium or to the pericardia of ascidians or vertebrates.
in vertebrates, mouse pax <dig> and pax <dig> are not expressed in the heart  <cit> , and although pax <dig> expression has been detected in the heart of newborn mice, its level was equal to or less than all other tissues tested except kidney  <cit> , suggesting that it may represent background levels.
no role for pax <dig>  pax <dig>  or pax <dig> has been demonstrated in the heart in knockout mutations in mouse  <cit> .
consistent with these results, our survey of est databases in ncbi  reveals the absence of pax2/5/ <dig> genes in the heart transcriptomes of human, mouse, frogs and zebrafish.
thus, we can infer that pax2/5/ <dig> expression in the heart was either already present in the last common ancestor of olfactores  and lost in the vertebrate lineage, or was a neofunctionalization event within the urochordates, and perhaps within larvaceans.
evidence of pax2/5/ <dig> subfunction partitioning in thyroid homologs
the thyroid is an endocrine gland located in the neck ventral to the pharynx in humans and other vertebrates, and it regulates energy production and growth by synthesizing tyrosine-based, iodine-containing hormones .
the presumed homolog of the thyroid in filter-feeding, non-vertebrate chordates is the endostyle, an organ located ventral to the pharyngeal floor that functions in iodine binding and secretion of food-trapping mucus  <cit> .
the homology between the thyroid and the endostyle is further supported by the expression of similar molecular markers, including members of the pax2/5/ <dig> family .
our results show that the larvacean endostyle expresses pax2/5/ <dig> genes in a pattern similar to that described for amphioxus  <cit> , ascidians  <cit> , and vertebrates  <cit> .
the last common ancestor of all three chordate subphyla, therefore, probably employed a pax2/5/ <dig> gene in the development of an endostyle-like organ that evolved into the endostyle of cephalochordates and urochordates and the thyroid of vertebrates .
pax2/5/ <dig> subfunctions related to vertebrate thyroid development have likely suffered independent processes of subfunction partitioning among paralogs .
for instance, in mouse, pax <dig> is expressed during thyroid development but pax <dig> is not  <cit> ; in frogs, however, subfunctions of these genes are reversed, as pax <dig> but not pax <dig> is involved in the development of the thyroid  <cit> .
these results suggest that ancestral vertebrate thyroid regulatory subfunctions of pax2/5/ <dig> were preserved by both pax <dig> and pax <dig> in stem amniotes, and resolved independently in the amphibian and mammalian lineages.
in zebrafish, both pax <dig> and pax2a  retain thyroid expression, although they are activated at different times in development  <cit> .
although the duplication of pax2/5/ <dig> paralogs in urochordates occurred before the divergence of the larvacean and ascidian lineages  <cit>  , the endostyle of oikopleura expresses both pax2/5/ <dig> paralogs , while the endostyle of ascidians does not express pax2/5/8b, but expresses pax2/5/8a in both the dorsal and ventral domains, the sum of the pattern for the two oikopleura duplicates  <cit> .
these lineage-specific differences in expression patterns of orthologous pax2/5/ <dig> duplicates suggest that in the last common ancestor of larvaceans and ascidians, both pax2/5/8a and pax2/5/8b were expressed both dorsally and ventrally, and that after lineages diverged, in larvaceans the dorsal and ventral endostyle subfunctions partitioned to different pax2/5/ <dig> genes, but in ascidians both subfunctions remained associated with the pax2/5/8a paralog and both were lost by the pax2/5/8b paralog.
as vertebrate pax <dig> can directly bind the promoters of thyroid follicle cell-specific genes such as thyroperoxidase  <cit> , we raise the hypothesis that urochordate pax2/5/8a genes, which are expressed in peroxidase-producing, dorsal cells of the endostyle  <cit> , play a homologous role while urochordate pax2/5/8b genes have lost this function.
evidence of pax2/5/ <dig> subfunction partitioning in the anterior pharynx and stomodeum
comparison between ascidians, larvaceans and cephalochordates suggests that stem chordates expressed pax2/5/ <dig> in the stomodeum and anterior pharynx, but that this expression domain was lost in the vertebrate lineage.
larvaceans and ascidians both have ciliated mechanosensory organs that ring the mouth and are involved in a filter-feeding, particle-rejection response  <cit> .
although the oral sensory cell types themselves are morphologically different in the two urochordate classes, these organs may share a common origin in a stomodeal sensory placode that has been lost in the vertebrate lineage  <cit> .
although both larvacean pax2/5/ <dig> paralogs are expressed in this stomodeal domain, their patterns are distinct: pax2/5/8a expression in the rostral pharynx of early hatchlings is replaced with pax2/5/8b by mid-hatchling stages.
in mid-hatchlings, pax2/5/8a expression is in the external ectoderm surrounding the mouth opening, including the two bristle-bearing cells of the upper lip, while pax2/5/8b is expressed just inside the mouth, including the ciliated sensory cells of the circumoral organ.
although the two mechanosensory cell types fall into expression domains of different pax2/5/ <dig> paralogs, they are innervated by the same branched sensory axons emanating from a pair of rostral brain cells  <cit> .
the single amphioxus pax2/5/ <dig> gene is expressed both externally around the developing mouth and internally in the pharynx  <cit> ; this expression domain corresponds to the sum of expression domains occupied by different larvacean paralogs, as expected by subfunction partitioning  <cit>  if the last stem chordate had separate regulatory elements governing the contiguous external and internal pharyngeal domains that partitioned to different larvacean paralogs after the pax2/5/ <dig> duplication event.
ascidian pax2/5/8a is expressed in the invaginating stomodeum but pax2/5/8b is expressed in the buccal cavity, a pattern comparable with the late hatchling expression domains of the oikopleura orthologs.
at least some subfunctions, therefore, appear to have partitioned between pax2/5/ <dig> paralogs before the larvacean and ascidian lineages diverged.
the ascidian pax2/5/8a gene, however, appears to lack expression comparable with the early, internal expression of oikopleura pax2/5/8a in the rostral pharynx, a difference that may derive from the developmental delay and incomplete development of endodermal organs experienced by ascidian larvae compared with the uninterrupted and complete endodermal ontogeny in larvaceans.
pax2/5/ <dig> expression and the langerhans receptor, ascidian atrial siphon, and vertebrate otic placode
the expression of pax2/5/ <dig> genes in the langerhans receptors of oikopleura helps us understand apparently conflicting interpretations of the ancestral role of pax2/5/ <dig> in the origin of the vertebrate otic placode.
previous evidence supports the notion that larvacean langerhans receptors are homologous to hair cell-like sensory organs in the ascidian atrium, the chamber surrounding the branchial basket  <cit> .
expression of pax2/5/8a in the ascidian atrium and expression of pax <dig> and pax <dig> in the vertebrate hair cell-producing otic placode suggested the hypothesis that the atrium of ascidians is homologous to the otic vesicles of vertebrates  <cit> .
an alternative to the 'placode hypothesis', however, arose from the finding that in amphioxus, the developing gill slits and mouth express pax2/5/ <dig>  this alternative suggests that pax2/5/ <dig> expression in the atrial primordium of ascidians reflects an ancient gene function in the perforation, adhesion and fusion of epithelial layers, such as in the epithelia of gill openings, rather than the homology of atrial primordia and vertebrate placodes  <cit> .
the 'epithelial fusion hypothesis' is consistent with the finding that among vertebrates, pax <dig> apparently plays a role in gill slit perforation in xenopus  <cit>  and pax <dig> is necessary for vaginal opening in mouse  <cit> .
furthermore, pax <dig> and pax <dig> regulate genes that control the composition of the extracellular matrix in epithelial fusions  <cit> .
evidence to resolve this dilemma comes from examination of gene expression and organ structure in a phylogenetic context.
our analysis of larvacean pax2/5/ <dig> expression teases apart epithelial fusion from placode formation and suggests that pax2/5/ <dig> likely functioned in both processes in ancient chordates.
in oikopleura, several pax2/5/ <dig> expression domains can be grouped into two overlapping categories: sites of epithelial fusion and sites of sensory cell development.
at the sites of epithelial fusion, larvacean pax2/5/ <dig> paralogs are expressed at the junction of the gill pouch with the epidermis, the joining of the rectum to the epidermis at the anus, and the fusion of the stomodeum and pharynx at the mouth.
at sites of sensory cell development, oikopleura's pax2/5/ <dig> paralogs are expressed at the stomodeal placode and the putative acousticolateralis placode homolog .
therefore, in contrast to ascidians, in which hair cell-like organs and sites of perforation are conflated in the atrium  <cit> , oikopleura's paired mechanosensory organs are topographically separate from the gill openings.
in agreement with the perforation argument  <cit> , expression of oikopleura pax2/5/ <dig> as in amphioxus, in the gill openings and anus, which lack sensory cell types, supports the hypothesis of an ancient function for pax2/5/ <dig> in the adhesion and fusion of epithelial layers and in the promotion of epithelial perforations.
on the other hand, expression of oikopleura pax2/5/8a at early tailbud stage in the primordia of the langerhans organs occurs long before the fusion of the gill endoderm with the ectoderm, which happens at the late hatchling stage.
this timing gap argues against the interpretation that early expression of pax2/5/ <dig> in the langerhans domain is associated only with remodeling the extracellular matrix for gill perforation.
instead, early pax2/5/ <dig> expression defines the location of paired, thickened sensory organ primordia, in agreement with the hypothesis of wada et al.
<cit>  that pax2/5/ <dig> marks a urochordate placode.
larvacean pax2/5/ <dig> expression adds to a growing body of morphological and gene expression data that supports the origin of cranial placodes in early chordates, rather than in vertebrates, as had been long assumed  <cit> ,  <cit> , and strengthens the case for an early origin specifically of the otic or acousticolateralis placode.
therefore, analysis of pax2/5/ <dig> paralogs in oikopleura has allowed us to reconcile the 'placode hypothesis' and 'epithelial fusion hypothesis', supporting an evolutionary scenario in which the role of pax2/5/ <dig> in epithelial fusions and perforations was already present in stem chordates, and the role of pax2/5/ <dig> related to placode development is likely also ancient, though perhaps restricted to olfactores .
evolution of pax2/5/ <dig> genetic pathways
pax, eya, six and dach genes form a genetic network in several biological processes, including in sensory placode development .
pax <dig>  pax <dig>  pax <dig>  eya <dig>  six <dig>  and dach <dig> are co-expressed during fish otic vesicle development , where they interact to specify otic tissue and maintain its continued ontogenesis.
larvacean pax2/5/ <dig> genes are expressed in presumptive sensory tissues previously reported also to express eya and six orthologs  <cit> , namely around the mouth and in the mechanosensory langerhans receptors.
the two pax2/5/ <dig> paralogs may interact with different subsets of eya-six-dach genes in different tissues.
for instance, in developing sensory tissues, pax2/5/8a expression in tailbud and early hatchling stages is similar to that of eya and six3/6a in the developing langerhans receptor primordia, and to six1/ <dig> and six3/6a expression around the mouth, including sensory cilia-bearing cells of the upper lip.
endodermal pax2/5/8b expression, on the other hand, most strongly overlaps that of six3/6a in the rostral pharynx and eya in the gill endoderm in mid-hatchling stages.
similarly, ascidian pax2/5/8a expression overlaps eya and six1/ <dig> in the atrial primordia and eya, six1/ <dig>  and six3/ <dig> in the stomodeal domain, but pax2/5/8b exhibits broad expression in the ectoderm and may also overlap eya, six1/ <dig>  and six3/ <dig> in a way that larvacean pax2/5/8b does not  <cit> .
such differences between presumed pax2/5/ <dig> paralogs and orthologs is further evidence that, though the pax-six-eya-dach 'module' is conserved at the level of gene families, paralogs within each module may differ both within a developmental program and between lineages  <cit> .
not surprisingly, then, sequence analysis shows that several known protein interaction domains differ between the larvacean pax2/5/ <dig> paralogs and between the larvacean and ascidian orthologs , consistent with divergence of the molecular pathways in which these pax proteins participate.
pax2/5/ <dig> gene duplications and the evolution of gene functions
a requirement for partitioning of ancestral gene functions is that the ancestral gene must have multiple independently mutable functions either in protein-coding domains or in regulatory elements that drive restricted expression: in other words, units that are by definition 'subfunctions'.
direct evidence for independently mutable pax2/5/ <dig> subfunctions in an extant gene comes from the drosophila ortholog, called d-pax <dig>  in which mutations in separate regulatory elements affect development of either ommatidia or sensory bristles  <cit> .
the known functions of vertebrate pax2/5/ <dig> genes are diverse and include the establishment of the midbrain-hindbrain border  <cit> , specification and mature function of thyroid follicular cells  <cit> , specification and morphogenesis in the pronephros  <cit> , differentiation of interneuron subtypes in the central nervous system , promotion of correct axon guidance in the optic nerves  <cit> , and morphogenesis and sensory cell specification in the epibranchial, otic and lateral line sensory placodes .
it is not known, however, how many of these functions, segregated among extant genes, resulted from independently mutable ancestral subfunctions.
from the partially overlapping expression patterns of vertebrate pax2/5/ <dig> genes, we can infer that subfunction partitioning has occurred in this gene family.
next, comparative analysis of expression domains in the chordate phylogenetic context helps us infer when subfunctions arose.
parallel, independent partitioning of pax2/5/ <dig> endostyle/thyroid functions in both vertebrate and urochordate lineages suggests that the single pax2/5/ <dig> gene of stem olfactores had already acquired independently mutable regulation for endostyle/thyroid expression.
likewise, paralleling segregation of mammalian otic placode subfunctions to pax <dig> and pax <dig>  <cit> , larvacean otic placode-like tissues express only one pax2/5/ <dig> paralog early and in a pattern overlapping orthologs of other vertebrate placode markers of the six and eya gene families  <cit>  .
it remains possible, though, that despite the similarity between vertebrate and urochordate pax2/5/ <dig> gene expression patterns, each lineage separately evolved independently mutable functions in the endostyle/thyroid and otic placode development and that the ancestral gene did not already bear these subfunctions.
within the urochordate lineage, further parsing of gene functions may represent subfunctions that were present in the ancestral urochordate pax2/5/ <dig> gene.
for example, at some sites of epithelial fusion, larvacean pax2/5/ <dig> paralogs exhibit complementary expression, for example just outside  or just inside  the mouth and just outside  or just inside  the anus.
therefore, though a role in epithelial fusions is probably ancient in chordata, urochordate paralogs may have taken on separate refinements of the same function.
in addition to the partitioning of ancestral functions, we might also expect to detect apparent neofunctionalizations of gene duplicates, particularly when we compare deeply diverging lineages such as vertebrates and urochordates.
the role of vertebrate pax <dig> in the b-lymphoid lineage of the immune system might be one example of a vertebrate neofunctionalization  <cit> .
similarly, because no comparable expression can be found outside of urochordata, a role for pax2/5/8b in the pericardium of the heart could be a novel deployment of pax2/5/ <dig> exclusive to the urochordates or even to the larvacean lineage.
though observed pax2/5/ <dig> expression patterns are consistent with a hypothesis of ancestral subfunction partitioning, this analysis detects only transcriptional regulatory differences and might underestimate the extent of actual subfunction partitioning.
for example, mrna expression of the paralogs may overlap in a given tissue, but the structurally different proteins the genes encode may not have redundant functions in those tissues.
in addition, post-transcriptional regulation would also be missed in our analysis.
partitioned genes likely continue to evolve after their initial partitioning, and such divergence could complicate distinguishing subfunctionalization from neofunctionalization when comparing gene functions in deeply diverging lineages.
nonetheless, functional analysis of urochordate pax2/5/ <dig> genes could help distinguish between ancestral chordate subfunction partitioning and convergent patterns of neofunctionalization.
for example, if pax2/5/ <dig> proteins carry out a similar function in the vertebrate thyroid as in the urochordate endostyle, this would be more concrete evidence that an ancient function was partitioned to paralogs in both chordate subphyla.
CONCLUSIONS
the present work shows how analyzing the evolution of gene families that have experienced multiple independent gene duplication events in related lineages can improve understanding of the evolution of the genetic mechanisms underlying the development of homologous structures of anatomically divergent organisms , and can help to identify gene subfunctions that otherwise may be difficult to recognize because of extensive pleiotropy .
analysis of the evolution of subfunctions in a phylogenetic context identifies lineage specific innovations .
the modular fashion in which gene subfunctions partition after independent gene duplication events in various lineages implies the existence of independent regulatory elements that control temporal and spatial expression for each subfunction.
for instance, comparison of pax2/5/ <dig> expression patterns in the endostyles of amphioxus, oikopleura, and ascidians predicts the presence of separable regulation for expression in the iodine-binding dorsal component and the supporting ventral component of the endostyle/thyroid, a hypothesis that remains to be tested.
the identification of the complete set of gene orthologs and paralogs in gene families in an increasing number of completely sequenced genomes will likely reveal additional cases of independent gene duplications and subfunctionalization events, the analysis of which will improve our understanding of the evolution of gene functions and the implications in the evolution and diversity of living forms.

 ohno's classical model to explain the fate of genes after gene duplication predicts that one gene duplicate preserves the original gene function while its paralog either disappears by accumulation of detrimental mutations or occasionally acquires beneficial mutations that confer novel, positively selected functions. the duplication-degeneration-complementation model predicts a third alternative, in which the two duplicate genes become permanently preserved as a consequence of complementary, degenerative mutations that result in partitioning of ancestral subfunctions, so that the sum of the functions of two paralogs equals the functions of the original gene prior to the duplication. the ddc model also predicts that after the initial preservation of the two duplicates, whether by subfunctionalization or from neofunctionalization, further partitioning of redundant subfunctions may occur. studies show that functional constraints on genes duplicated in whole-genome duplications are relaxed, compared with singletons, for tens of millions of years. in addition, novel functions may originate over time, their evolution facilitated by the relaxation of pleiotropy occasioned by fewer tasks in each descendent gene duplicate compared with its single copy gene ancestor. understanding the evolution of duplicated genes is important because of the hypothesis that gene duplicates provide opportunities for the evolution of reproductive barriers that lead to lineage divergence, and for the origin of evolutionary novelties. in vertebrates, many sets of paralogous genes arose in two rounds of genome duplication that took place in early vertebrate evolution. an additional round of genome duplication occurred in the teleost lineage after ray-fin fish diverged from lobe-fin fish, and provided additional gene family members observed in many fish models. it has been suggested that the r and r genome amplifications facilitated the origin of vertebrate innovations, and the r event may have facilitated the teleost species radiation. non-vertebrate chordates often have single copies of vertebrate gene families because their lineages diverged from the vertebrate lineage before the r and r genome duplication events. recent phylogenomic analyses converge on the conclusion that the chordate subphylum urochordata, which includes the classes larvacea and ascidiacea, are the closest living relatives of the vertebrates, constituting the group olfactores, while the subphylum cephalochordata, including the amphioxus, diverged basally among chordates. as gene duplication is pervasive, non-vertebrate chordates sometimes have genes that duplicated independently in the cephalochordate or urochordate lineages. we propose that the comparative analysis of gene expression patterns in a gene family that experienced lineage-specific, independent duplication events, interpreted in a phylogenetic context and with respect to subfunction partitioning, can help in the inference of ancient gene functions and in the identification of gene functions that arose by neofunctionalization, and thus may be important for lineage divergence and the origin of developmental novelties. to test this proposition, we examined the pax2/5/ gene family. pax2/5/ genes encode transcription factors with conserved motifs, including a paired domain, homeodomain, and octapeptide, and are associated with mechanosensory development in mammals, frogs, fish, flies, ascidians and in mollusks, while in a cnidarians, the apparent pax2/5/ homolog is associated with nerve and sensory cell differentiation. pax2/5/ genes duplicated independently in different chordate lineages. within the vertebrates, tetrapods have three members of the pax2/5/ gene family, and teleosts additionally have duplicate pax genes. in contrast to vertebrates, the basally diverging cephalochordate amphioxus possesses a single pax2/5/ gene that is equally related to pax pax and pax, and urochordates have two pax2/5/ genes that originated in a duplication event prior to the divergence of larvacean and ascidian lineages. the chordate pax2/5/ family embodies a full spectrum of gene evolutionary events: non-duplication; independent duplications; gene loss; neofunctionalization; and ancestral subfunctions appear to have been partitioned between paralogs within a lineage and, further, differently partitioned between lineages. the independent duplication of the stem olfactores' pax2/5/ gene in vertebrate and urochordate lineages provides replicate evolutionary experiments to explore the principles of subfunction partitioning and the origin of novel functions. to exploit this opportunity, we provide here a detailed description of expression patterns for pax2/5/ paralogs during development of the larvacean urochordate oikopleura dioica. we then compare our results with expression patterns of pax2/5/ orthologs in ascidians, with independently duplicated pax2/5/ paralogs in vertebrates, and with the non-duplicated pax2/5/ gene in amphioxus as an outgroup. we discuss the expression of the pax2/5/ gene family during development of the heart, endostyle, pharynx, and sensory organs, and provide new insights that reconcile previous conflicting hypotheses about the homology of the ascidian atrial primordia and the vertebrate otic placode. this work thus illustrates the power of comparative analyses of independently duplicated genes to infer ancestral gene subfunctions, modules that can segregate independently from each other to evolving gene duplicates.  functional motif variation and gene structure of chordate pax2/5/ paralogs pax2/5/ proteins are transcription factors, whose sequence is poorly conserved across chordata outside of two functional motifs: the paired domain, which interacts with the dna of target genes, and an octapeptide motif, which is conserved with other pax proteins, functions in repression of pax transactivation, and interacts with other transcriptional cofactors. sequence alignment of various chordate pax2/5/ proteins reveals that while the sequence of the dna-interacting paired domain is highly conserved across all chordate pax2/5/ proteins analyzed, the octapeptide motif is less conserved. in urochordates, for instance, the octapeptide is present in the ascidian halocynthia roretzi, and, contrary to a previous report, our alignment reveals it is also present in ciona intestinalis pax2/5/8a. the octapeptide sequence of the oikopleura pax2/5/8a gene is poorly conserved, and the octapeptide motif is absent from the expected position in all urochordate pax2/5/8b proteins. interestingly, we have identified a new motif that is conserved among all ascidian pax2/5/8b paralogs, located further toward the carboxyl end. as this newly recognized sequence shows some similarities with the octapeptide motif, we have called it the'octapeptide-like' motif. we could not identify an octapeptide-like motif in the oikopleura pax2/5/8b. our alignment also reveals a lysine-arginine-rich domain that is conserved in vertebrate pax and amphioxus pax2/5/ is variable among vertebrate pax and pax paralogs, and is present in urochordate pax2/5/8b but not pax2/5/8a. this protein motif marks the beginning of an amino acid range identified as important for greatly increasing vertebrate pax protein's transactivation activity. comparison of gene structures revealed that, while most chordate pax2/5/ genes have to exons, ascidian pax2/5/8b paralogs have exons, which code for a protein of about amino acids, three times longer than the approximately amino acid-long pax2/5/ proteins from other chordates. this difference in size is due mainly to an exon more than nucleotides long located upstream of the paired domain. our analysis of publicly available est sequences confirmed the presence of this large exon in gene models predicted for pax2/5/8b in ciona intestinalis and in ciona savignyi. this large exon is absent from o. dioica pax2/5/ paralogs and may have evolved in the ascidian lineage after the divergence of urochordates. in conclusion, various pax2/5/ paralogs appear to have lost ancestral features and evolved new structural motifs or new exons, as would be predicted by the ddc model applied to pax2/5/ paralogs evolving after independent gene duplication events. the identification of these structural features focuses attention on regions to test for function to learn the roles each motif may have played in the origin of lineage-specific morphologies. developmental expression of the oikopleura paralogs pax2/5/8a and pax2/5/8b tailbud stages at the end of gastrulation, pax2/5/8a and pax2/5/8b were both broadly expressed in the oikopleura embryo. pax2/5/8a was expressed mainly in the ectoderm of the trunk, while pax2/5/8b was expressed primarily in the interior of the trunk. in early tailbud stages, pax2/5/8b expression continued in its broad internal expression domain, but pax2/5/8a expression became restricted to two domains: a few medial cells at the boundary of the anterior brain and the pharynx, and a bilateral pair of ectodermal cells in the posterior trunk. by mid- and late-tailbud stage, the signal of the pax2/5/8a expression domains strengthened, and a new domain appeared in the rostral-most ectoderm of the trunk, probably labeling the first two cells fated to invaginate into the stomodeum and form the oral epithelium. early hatchling and mid-hatchling stages at the early hatchling stage, pax2/5/8a expression maintained its late-tailbud stage pattern. the rostral-most expression domain in the stomodeal region expanded internally from the surface to form a group of contiguous internal cells, which were separated from the internal expression domain in presumptive pharynx/endostyle precursor cells. the bilateral pax2/5/8a expression domain in the posterior part of the trunk included at least four ectodermal cells in the area where the langerhans mechanoreceptors eventually develop, at a level slightly anterior to the tip of the notochord and adjacent to the homolog of the vertebrate hindbrain. during early hatchling stages, the internal broad expression domain of pax2/5/8b that was present at tailbud stages began to fade at the same time that two separate expression domains became distinct, one in the ectoderm at about the same bilateral position as the pax2/5/8a ectodermal domain and the other within the pharynx. although pax2/5/8a and pax2/5/8b were both expressed in the ectoderm at the eventual position of the langerhans receptors, close inspection revealed differences in the number and morphology of the pax2/5/8a and pax2/5/8b positive cells. by mid-hatchling stages, the bilateral pax2/5/8a expression domain expanded to at least six ectodermal cells, while the brief flash of pax2/5/8b expression in the ectoderm became undetectable. simultaneously, the distal portion of each gill pouch began to express pax2/5/8b. it is not known if ectodermal cells contribute to the formation of the gill pouches at these early stages when the ectoderm is still forming its epithelial character. expression of the two oikopleura pax2/5/ paralogs during mid-hatchling stages appeared to be complementary in the pharynx in time and space: while pax2/5/8a expression was diminishing in the stomodeum and the rostral pharynx, new pax2/5/8b expression domains appeared. the proximal endoderm of the gill pouches began to express pax2/5/8a while the distal portion of the gill pouches expressed pax2/5/8b. presumptive dorsal precursor cells of the endostyle first began to express pax2/5/8a, while presumptive ventral endostyle cells expressed pax2/5/8b. late hatchling stages by the late hatchling stage, organs are distinct and internal cavities have started to expand. at this stage, the gills and mouth have opened to the outside, and the heart and the ciliary rings of the gills have both begun to beat. the direct development of larvacean urochordates permits the tracing of gene expression domains in organ rudiments from hatchlings until fully mature adults. in contrast, in ascidian urochordates, many organs begin to develop only at metamorphosis, just as many chordate-specific characters disappear. pax2/5/8a expression in late hatchling stages overlaps pax2/5/8b in some domains: in the distal part of the gills from the ciliary rings to the external gill opening, and, transiently, in the posterior trunk epidermis. other domains are unique to each gene, and in several regions expression of the paralogs appears complementary. for example, while the lips of the mouth express pax2/5/8a, the pharynx just internal to the lips strongly expresses pax2/5/8b; while dorsal cells of the endostyle express pax2/5/8a, ventral cells of the endostyle express pax2/5/8b; and while the most posterior section of the rectum expresses pax2/5/8a, the anus expresses pax2/5/8b. except for the distal part of the gills, the posterior pharynx exclusively expresses pax2/5/8b, including the gill endoderm where the two gill pouches meet medially. and, strikingly, only one layer of the two-ply heart, the pericardium, expresses pax2/5/8b; the muscular myocardium does not. a subtle metamorphic event called'tailshift', the rapid reorientation of the tail from a posterior to a ventral position with respect to the trunk, signals the beginning of the juvenile stage in oikopleura. just prior to tailshift, the two pax2/5/ paralogs continue to show mostly non-overlapping expression patterns, now in clearly differentiated organs. pax2/5/8a is still expressed around the mouth, in the dorsal, iodine-binding corridor cells of the endostyle, in the gill endoderm, in the rectum, and in a row of cells at the posterior margin of the oikoplastic epithelium. expression of pax2/5/8b is also a continuation of domains identified earlier, including the pharynx and gills, the ventral endostyle, the anus, and the pericardium. new domains, however, now appear in dorsal, ventral and lateral fields of the oikoplastic epithelium, the tissue that will soon secrete the first filter-feeding'house' the animal inhabits. figure 5c is a schematic diagram summarizing the expression domains of both oikopleura pax2/5/ paralogs in late hatchlings. table summarizes by tissue and developmental stage all pax2/5/ expression domains. a, pax2/5/8a; b, pax2/5/8b; a/b, expression of paralogs does not spatially overlap or only partly overlaps; a+b, both paralogs spatially overlap. discussion these investigations of the structure and expression of pax2/5/ gene duplicates in oikopleura, when analyzed in a comparative context with shared and independent pax2/5/ gene duplications in other chordate lineages, illuminate a variety of problems in the evolution of chordate developmental mechanisms and the principles that govern change in duplicated gene function over time. the work resolves alternative hypotheses for the homologies of the ascidian atrial siphon, illuminates evolution of the thyroid, identifies candidates for ancestral gene subfunctions, and defines the origin of functional innovations in this gene family. pax2/5/ and the evolution and development of the chordate heart the shared expression of cardiac developmental genes supports the homology of bilaterian hearts from chordates to flies. the evolutionary relationship of the single-chambered heart of non-vertebrate chordates to the individual chambers of the innovative multi-chamber vertebrate heart, however, is still unclear. our work reveals unprecedented pax2/5/ expression in the developing heart of a chordate, and may provide a late ontogenetic marker for assessing homology of tissue layers among simple, urochordate hearts. in contrast to the single-layered heart of cephalochordates, urochordates have a two-layered heart. the simple heart of oikopleura dioica consists of two epithelial layers lying just medial to the left stomach lobe. in oikopleura species, the lateral wall of the heart contains the muscle fibers while the medial wall is a thin pericardial membrane. peristaltic heart contractions, which periodically reverse, cause the hemolymph to course between the heart and the stomach wall. in comparison, the ascidian heart rolls up to become tubular rather than planar, but also has a contractile layer and a non-muscular, pericardial layer. the larvacean pericardial membrane, therefore, is the likely homolog of the ascidian pericardium. in ascidians, heart development can be broadly divided in two phases. in the first phase, heart cell precursors become fated during early cleavage and embryonic stages, and after a complex process of muscle cell migration from the tail to the trunk, heart development temporarily arrests. in the second phase, after metamorphosis, heart development re-initiates and after final differentiation, the heart starts to beat and becomes functional. in larvaceans, the transparency and absence of a drastic metamorphosis provides a complementary model system for the study of heart development. in o. dioica, the heart starts beating less than hours post fertilization, before tailshift. in late larvacean hatchlings, when heart differentiation is at or nearing completion, the pericardium expresses pax2/5/8b. in ascidians, however, neither pax2/5/ paralog has been shown to be expressed in the heart, perhaps because most published analyses of pax2/5/ expression in ciona, halocynthia and herdmania have not included late developmental stages after metamorphosis. therefore, whether pax2/5/ expression in the heart – and more specifically in the pericardium – is a shared feature among urochordates remains to be investigated. cephalochordates have a single-layered, muscularized blood vessel ventral to the gut that propels the hemolymph by peristalsis. during amphioxus development, the primordium of this muscularized vessel expresses a homolog of vertebrate nk class genes – a group that includes several genes necessary for vertebrate cardiac development – suggesting that the amphioxus vessel is homologous to the vertebrate heart. pax2/5/ is not reported to be expressed in the circulatory vessels of amphioxus, and likewise, amphioxus has no structure that appears to be morphologically comparable to the pax2/5/8-expressing larvacean pericardium or to the pericardia of ascidians or vertebrates. in vertebrates, mouse pax and pax are not expressed in the heart, and although pax expression has been detected in the heart of newborn mice, its level was equal to or less than all other tissues tested except kidney, suggesting that it may represent background levels. no role for pax pax or pax has been demonstrated in the heart in knockout mutations in mouse. consistent with these results, our survey of est databases in ncbi reveals the absence of pax2/5/ genes in the heart transcriptomes of human, mouse, frogs and zebrafish. thus, we can infer that pax2/5/ expression in the heart was either already present in the last common ancestor of olfactores and lost in the vertebrate lineage, or was a neofunctionalization event within the urochordates, and perhaps within larvaceans. evidence of pax2/5/ subfunction partitioning in thyroid homologs the thyroid is an endocrine gland located in the neck ventral to the pharynx in humans and other vertebrates, and it regulates energy production and growth by synthesizing tyrosine-based, iodine-containing hormones. the presumed homolog of the thyroid in filter-feeding, non-vertebrate chordates is the endostyle, an organ located ventral to the pharyngeal floor that functions in iodine binding and secretion of food-trapping mucus. the homology between the thyroid and the endostyle is further supported by the expression of similar molecular markers, including members of the pax2/5/ family. our results show that the larvacean endostyle expresses pax2/5/ genes in a pattern similar to that described for amphioxus, ascidians, and vertebrates. the last common ancestor of all three chordate subphyla, therefore, probably employed a pax2/5/ gene in the development of an endostyle-like organ that evolved into the endostyle of cephalochordates and urochordates and the thyroid of vertebrates. pax2/5/ subfunctions related to vertebrate thyroid development have likely suffered independent processes of subfunction partitioning among paralogs. for instance, in mouse, pax is expressed during thyroid development but pax is not; in frogs, however, subfunctions of these genes are reversed, as pax but not pax is involved in the development of the thyroid. these results suggest that ancestral vertebrate thyroid regulatory subfunctions of pax2/5/ were preserved by both pax and pax in stem amniotes, and resolved independently in the amphibian and mammalian lineages. in zebrafish, both pax and pax2a retain thyroid expression, although they are activated at different times in development. although the duplication of pax2/5/ paralogs in urochordates occurred before the divergence of the larvacean and ascidian lineages, the endostyle of oikopleura expresses both pax2/5/ paralogs, while the endostyle of ascidians does not express pax2/5/8b, but expresses pax2/5/8a in both the dorsal and ventral domains, the sum of the pattern for the two oikopleura duplicates. these lineage-specific differences in expression patterns of orthologous pax2/5/ duplicates suggest that in the last common ancestor of larvaceans and ascidians, both pax2/5/8a and pax2/5/8b were expressed both dorsally and ventrally, and that after lineages diverged, in larvaceans the dorsal and ventral endostyle subfunctions partitioned to different pax2/5/ genes, but in ascidians both subfunctions remained associated with the pax2/5/8a paralog and both were lost by the pax2/5/8b paralog. as vertebrate pax can directly bind the promoters of thyroid follicle cell-specific genes such as thyroperoxidase, we raise the hypothesis that urochordate pax2/5/8a genes, which are expressed in peroxidase-producing, dorsal cells of the endostyle, play a homologous role while urochordate pax2/5/8b genes have lost this function. evidence of pax2/5/ subfunction partitioning in the anterior pharynx and stomodeum comparison between ascidians, larvaceans and cephalochordates suggests that stem chordates expressed pax2/5/ in the stomodeum and anterior pharynx, but that this expression domain was lost in the vertebrate lineage. larvaceans and ascidians both have ciliated mechanosensory organs that ring the mouth and are involved in a filter-feeding, particle-rejection response. although the oral sensory cell types themselves are morphologically different in the two urochordate classes, these organs may share a common origin in a stomodeal sensory placode that has been lost in the vertebrate lineage. although both larvacean pax2/5/ paralogs are expressed in this stomodeal domain, their patterns are distinct: pax2/5/8a expression in the rostral pharynx of early hatchlings is replaced with pax2/5/8b by mid-hatchling stages. in mid-hatchlings, pax2/5/8a expression is in the external ectoderm surrounding the mouth opening, including the two bristle-bearing cells of the upper lip, while pax2/5/8b is expressed just inside the mouth, including the ciliated sensory cells of the circumoral organ. although the two mechanosensory cell types fall into expression domains of different pax2/5/ paralogs, they are innervated by the same branched sensory axons emanating from a pair of rostral brain cells. the single amphioxus pax2/5/ gene is expressed both externally around the developing mouth and internally in the pharynx; this expression domain corresponds to the sum of expression domains occupied by different larvacean paralogs, as expected by subfunction partitioning if the last stem chordate had separate regulatory elements governing the contiguous external and internal pharyngeal domains that partitioned to different larvacean paralogs after the pax2/5/ duplication event. ascidian pax2/5/8a is expressed in the invaginating stomodeum but pax2/5/8b is expressed in the buccal cavity, a pattern comparable with the late hatchling expression domains of the oikopleura orthologs. at least some subfunctions, therefore, appear to have partitioned between pax2/5/ paralogs before the larvacean and ascidian lineages diverged. the ascidian pax2/5/8a gene, however, appears to lack expression comparable with the early, internal expression of oikopleura pax2/5/8a in the rostral pharynx, a difference that may derive from the developmental delay and incomplete development of endodermal organs experienced by ascidian larvae compared with the uninterrupted and complete endodermal ontogeny in larvaceans. pax2/5/ expression and the langerhans receptor, ascidian atrial siphon, and vertebrate otic placode the expression of pax2/5/ genes in the langerhans receptors of oikopleura helps us understand apparently conflicting interpretations of the ancestral role of pax2/5/ in the origin of the vertebrate otic placode. previous evidence supports the notion that larvacean langerhans receptors are homologous to hair cell-like sensory organs in the ascidian atrium, the chamber surrounding the branchial basket. expression of pax2/5/8a in the ascidian atrium and expression of pax and pax in the vertebrate hair cell-producing otic placode suggested the hypothesis that the atrium of ascidians is homologous to the otic vesicles of vertebrates. an alternative to the'placode hypothesis', however, arose from the finding that in amphioxus, the developing gill slits and mouth express pax2/5/ this alternative suggests that pax2/5/ expression in the atrial primordium of ascidians reflects an ancient gene function in the perforation, adhesion and fusion of epithelial layers, such as in the epithelia of gill openings, rather than the homology of atrial primordia and vertebrate placodes. the'epithelial fusion hypothesis' is consistent with the finding that among vertebrates, pax apparently plays a role in gill slit perforation in xenopus and pax is necessary for vaginal opening in mouse. furthermore, pax and pax regulate genes that control the composition of the extracellular matrix in epithelial fusions. evidence to resolve this dilemma comes from examination of gene expression and organ structure in a phylogenetic context. our analysis of larvacean pax2/5/ expression teases apart epithelial fusion from placode formation and suggests that pax2/5/ likely functioned in both processes in ancient chordates. in oikopleura, several pax2/5/ expression domains can be grouped into two overlapping categories: sites of epithelial fusion and sites of sensory cell development. at the sites of epithelial fusion, larvacean pax2/5/ paralogs are expressed at the junction of the gill pouch with the epidermis, the joining of the rectum to the epidermis at the anus, and the fusion of the stomodeum and pharynx at the mouth. at sites of sensory cell development, oikopleura's pax2/5/ paralogs are expressed at the stomodeal placode and the putative acousticolateralis placode homolog. therefore, in contrast to ascidians, in which hair cell-like organs and sites of perforation are conflated in the atrium, oikopleura's paired mechanosensory organs are topographically separate from the gill openings. in agreement with the perforation argument, expression of oikopleura pax2/5/ as in amphioxus, in the gill openings and anus, which lack sensory cell types, supports the hypothesis of an ancient function for pax2/5/ in the adhesion and fusion of epithelial layers and in the promotion of epithelial perforations. on the other hand, expression of oikopleura pax2/5/8a at early tailbud stage in the primordia of the langerhans organs occurs long before the fusion of the gill endoderm with the ectoderm, which happens at the late hatchling stage. this timing gap argues against the interpretation that early expression of pax2/5/ in the langerhans domain is associated only with remodeling the extracellular matrix for gill perforation. instead, early pax2/5/ expression defines the location of paired, thickened sensory organ primordia, in agreement with the hypothesis of wada et al. that pax2/5/ marks a urochordate placode. larvacean pax2/5/ expression adds to a growing body of morphological and gene expression data that supports the origin of cranial placodes in early chordates, rather than in vertebrates, as had been long assumed,, and strengthens the case for an early origin specifically of the otic or acousticolateralis placode. therefore, analysis of pax2/5/ paralogs in oikopleura has allowed us to reconcile the'placode hypothesis' and'epithelial fusion hypothesis', supporting an evolutionary scenario in which the role of pax2/5/ in epithelial fusions and perforations was already present in stem chordates, and the role of pax2/5/ related to placode development is likely also ancient, though perhaps restricted to olfactores. evolution of pax2/5/ genetic pathways pax, eya, six and dach genes form a genetic network in several biological processes, including in sensory placode development. pax pax pax eya six and dach are co-expressed during fish otic vesicle development, where they interact to specify otic tissue and maintain its continued ontogenesis. larvacean pax2/5/ genes are expressed in presumptive sensory tissues previously reported also to express eya and six orthologs, namely around the mouth and in the mechanosensory langerhans receptors. the two pax2/5/ paralogs may interact with different subsets of eya-six-dach genes in different tissues. for instance, in developing sensory tissues, pax2/5/8a expression in tailbud and early hatchling stages is similar to that of eya and six3/6a in the developing langerhans receptor primordia, and to six1/ and six3/6a expression around the mouth, including sensory cilia-bearing cells of the upper lip. endodermal pax2/5/8b expression, on the other hand, most strongly overlaps that of six3/6a in the rostral pharynx and eya in the gill endoderm in mid-hatchling stages. similarly, ascidian pax2/5/8a expression overlaps eya and six1/ in the atrial primordia and eya, six1/ and six3/ in the stomodeal domain, but pax2/5/8b exhibits broad expression in the ectoderm and may also overlap eya, six1/ and six3/ in a way that larvacean pax2/5/8b does not. such differences between presumed pax2/5/ paralogs and orthologs is further evidence that, though the pax-six-eya-dach'module' is conserved at the level of gene families, paralogs within each module may differ both within a developmental program and between lineages. not surprisingly, then, sequence analysis shows that several known protein interaction domains differ between the larvacean pax2/5/ paralogs and between the larvacean and ascidian orthologs, consistent with divergence of the molecular pathways in which these pax proteins participate. pax2/5/ gene duplications and the evolution of gene functions a requirement for partitioning of ancestral gene functions is that the ancestral gene must have multiple independently mutable functions either in protein-coding domains or in regulatory elements that drive restricted expression: in other words, units that are by definition'subfunctions'. direct evidence for independently mutable pax2/5/ subfunctions in an extant gene comes from the drosophila ortholog, called d-pax in which mutations in separate regulatory elements affect development of either ommatidia or sensory bristles. the known functions of vertebrate pax2/5/ genes are diverse and include the establishment of the midbrain-hindbrain border, specification and mature function of thyroid follicular cells, specification and morphogenesis in the pronephros, differentiation of interneuron subtypes in the central nervous system, promotion of correct axon guidance in the optic nerves, and morphogenesis and sensory cell specification in the epibranchial, otic and lateral line sensory placodes. it is not known, however, how many of these functions, segregated among extant genes, resulted from independently mutable ancestral subfunctions. from the partially overlapping expression patterns of vertebrate pax2/5/ genes, we can infer that subfunction partitioning has occurred in this gene family. next, comparative analysis of expression domains in the chordate phylogenetic context helps us infer when subfunctions arose. parallel, independent partitioning of pax2/5/ endostyle/thyroid functions in both vertebrate and urochordate lineages suggests that the single pax2/5/ gene of stem olfactores had already acquired independently mutable regulation for endostyle/thyroid expression. likewise, paralleling segregation of mammalian otic placode subfunctions to pax and pax, larvacean otic placode-like tissues express only one pax2/5/ paralog early and in a pattern overlapping orthologs of other vertebrate placode markers of the six and eya gene families. it remains possible, though, that despite the similarity between vertebrate and urochordate pax2/5/ gene expression patterns, each lineage separately evolved independently mutable functions in the endostyle/thyroid and otic placode development and that the ancestral gene did not already bear these subfunctions. within the urochordate lineage, further parsing of gene functions may represent subfunctions that were present in the ancestral urochordate pax2/5/ gene. for example, at some sites of epithelial fusion, larvacean pax2/5/ paralogs exhibit complementary expression, for example just outside or just inside the mouth and just outside or just inside the anus. therefore, though a role in epithelial fusions is probably ancient in chordata, urochordate paralogs may have taken on separate refinements of the same function. in addition to the partitioning of ancestral functions, we might also expect to detect apparent neofunctionalizations of gene duplicates, particularly when we compare deeply diverging lineages such as vertebrates and urochordates. the role of vertebrate pax in the b-lymphoid lineage of the immune system might be one example of a vertebrate neofunctionalization. similarly, because no comparable expression can be found outside of urochordata, a role for pax2/5/8b in the pericardium of the heart could be a novel deployment of pax2/5/ exclusive to the urochordates or even to the larvacean lineage. though observed pax2/5/ expression patterns are consistent with a hypothesis of ancestral subfunction partitioning, this analysis detects only transcriptional regulatory differences and might underestimate the extent of actual subfunction partitioning. for example, mrna expression of the paralogs may overlap in a given tissue, but the structurally different proteins the genes encode may not have redundant functions in those tissues. in addition, post-transcriptional regulation would also be missed in our analysis. partitioned genes likely continue to evolve after their initial partitioning, and such divergence could complicate distinguishing subfunctionalization from neofunctionalization when comparing gene functions in deeply diverging lineages. nonetheless, functional analysis of urochordate pax2/5/ genes could help distinguish between ancestral chordate subfunction partitioning and convergent patterns of neofunctionalization. for example, if pax2/5/ proteins carry out a similar function in the vertebrate thyroid as in the urochordate endostyle, this would be more concrete evidence that an ancient function was partitioned to paralogs in both chordate subphyla.  the present work shows how analyzing the evolution of gene families that have experienced multiple independent gene duplication events in related lineages can improve understanding of the evolution of the genetic mechanisms underlying the development of homologous structures of anatomically divergent organisms, and can help to identify gene subfunctions that otherwise may be difficult to recognize because of extensive pleiotropy. analysis of the evolution of subfunctions in a phylogenetic context identifies lineage specific innovations. the modular fashion in which gene subfunctions partition after independent gene duplication events in various lineages implies the existence of independent regulatory elements that control temporal and spatial expression for each subfunction. for instance, comparison of pax2/5/ expression patterns in the endostyles of amphioxus, oikopleura, and ascidians predicts the presence of separable regulation for expression in the iodine-binding dorsal component and the supporting ventral component of the endostyle/thyroid, a hypothesis that remains to be tested. the identification of the complete set of gene orthologs and paralogs in gene families in an increasing number of completely sequenced genomes will likely reveal additional cases of independent gene duplications and subfunctionalization events, the analysis of which will improve our understanding of the evolution of gene functions and the implications in the evolution and diversity of living forms. 
separate from the vertebrate genome expansions, a duplication also created two pax2/5/ genes in the common ancestor of ascidian and larvacean urochordates. to better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate oikopleura dioica, the embryonic gene expression patterns of pax2/5/ paralogs. we compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication pax2/5/ gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of pax2/5/ gene subfunctions in the common ancestor of olfactores. comparative analysis of expression patterns of chordate pax2/5/ duplicates, rooted on the single-copy pax2/5/ gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that pax2/5/ functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings. after the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the pax pax and pax genes of most vertebrates, and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. expression pattern differences of larvacean and ascidian pax2/5/ orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. expression of larvacean pax2/5/ in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of pax pax and pax in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor. gene duplication provides opportunities for lineage diversification and evolution of developmental novelties.
 gene duplication provides opportunities for lineage diversification and evolution of developmental novelties. duplicated genes generally either disappear by accumulation of mutations, or are preserved either by the origin of positively selected functions in one or both duplicates, or by the partitioning of original gene subfunctions between the duplicates. the pax2/5/ family of important developmental regulators has undergone parallel expansion among chordate groups. after the divergence of urochordate and vertebrate lineages, two rounds of independent gene duplications resulted in the pax pax and pax genes of most vertebrates, and an additional duplication provided the pax2a and pax2b duplicates in teleost fish. separate from the vertebrate genome expansions, a duplication also created two pax2/5/ genes in the common ancestor of ascidian and larvacean urochordates.  to better understand mechanisms underlying the evolution of duplicated genes, we investigated, in the larvacean urochordate oikopleura dioica, the embryonic gene expression patterns of pax2/5/ paralogs. we compared the larvacean and ascidian expression patterns to infer modular subfunctions present in the single pre-duplication pax2/5/ gene of stem urochordates, and we compared vertebrate and urochordate expression to infer the suite of pax2/5/ gene subfunctions in the common ancestor of olfactores. expression pattern differences of larvacean and ascidian pax2/5/ orthologs in the endostyle, pharynx and hindgut suggest that some ancestral gene functions have been partitioned differently to the duplicates in the two urochordate lineages. novel expression in the larvacean heart may have resulted from the neofunctionalization of a pax2/5/ gene in the urochordates. expression of larvacean pax2/5/ in the endostyle, in sites of epithelial remodeling, and in sensory tissues evokes like functions of pax pax and pax in vertebrate embryos, and may indicate ancient origins for these functions in the chordate common ancestor.  comparative analysis of expression patterns of chordate pax2/5/ duplicates, rooted on the single-copy pax2/5/ gene of amphioxus, whose lineage diverged basally among chordates, provides new insights into the evolution and development of the heart, thyroid, pharynx, stomodeum and placodes in chordates; supports the controversial conclusion that the atrial siphon of ascidians and the otic placode in vertebrates are homologous; and backs the notion that pax2/5/ functioned in ancestral chordates to engineer epithelial fusions and perforations, including gill slit openings. 