BACKGROUND
antarctic notothenioids are endemic to the southern ocean and probably evolved in situ from a sluggish, benthic perciform species beginning 40- <dig> mya in the then temperate waters of the antarctic continental shelf  <cit> .
with the opening of the drake passage , the establishment of the antarctic circumpolar current, and a sharp drop in atmospheric carbon dioxide levels, the southern ocean became thermally isolated, began to cool, and attained its present frigid temperatures  by the mid-miocene   <cit> .
the rich, shallow-water, temperate fish fauna characteristic of the late eocene  became largely extinct due to habitat destruction and changes in trophic structure, thus freeing ecological niches into which the notothenioids radiated  <cit> .
the hallmark of the antarctic notothenioid radiation is the evolution of secondary pelagicism, which is prominent in the family nototheniidae  <cit>  and common in the family channichthyidae  <cit> .
about 50% of notothenioid species now either live in or actively exploit pelagic habitats  <cit> .
a significant obstacle facing notothenioids as they radiated into pelagic habitats was the ancestral absence of a swim bladder, the gas-filled chamber that most teleosts use to maintain buoyancy.
in pelagic notothenioids, natural selection favored compensatory changes in the musculoskeletal system to achieve neutral buoyancy, including the replacement of densely mineralized bone with cartilage and connective tissue, decreased bone mineralization, and the accumulation of lipid deposits in muscle and connective tissues.
changes in habitat were also accompanied by the evolution of distinct oral jaw morphologies and body shapes that accommodate shifts in diet and foraging behavior  <cit> .
trophic evolution among notothenioids resulted in two distinct pelagic ecotypes: true pelagics and benthopelagics  <cit> .
true pelagics spend most of their time in the water column where they feed on microinvertebrates.
they possess relatively short, protractile jaws for suction feeding and have few, large oral teeth arranged along a single tooth row .
in contrast, benthopelagics spend much of their time on or close to the ocean floor but venture into the pelagic zone to forage on schools of small fish and macroinvertebrates.
most benthopelagic notothenioids have non-protractile, elongate jaws, a wide gape, and many, small oral teeth .
this morphology enables benthopelagics to feed efficiently on krill and schools of small fishes by expanding their buccal cavity, and overtaking and sifting large mouthfuls of prey .
extant benthic notothenioids preserve the ancestral condition, exhibiting robust skeletons and heavily fortified jaws bearing several rows of large teeth .
notothenioids achieved secondary pelagicism by pedomorphism, the retention of ancestrally juvenile traits by adults in a descendant taxon  <cit> .
pedomorphism arises from heterochronic processes that alter the schedule of developmental events  <cit> .
striking examples of pedomorphic characters in pelagic and benthopelagic notothenioids include delayed and reduced skeletal ossification, retention of the notochord, reduced numbers of teeth and tooth rows, and reduction of the pterygoid process of the palatoquadrate  <cit> .
as a first step toward understanding the molecular mechanisms that underlie skeletal reduction and morphological change in this group, we characterized early craniofacial development in a true pelagic species, p. antarcticum , and a benthopelagic species, c. aceratus .
we describe shared derived aspects of craniofacial development in pelagic notothenioids that differ significantly from those observed in a benthic notothenioid species, n. coriiceps , another percomorph species, gasterosteus aculeatus , and a more distantly related outgroup, danio rerio .
we found that pelagic notothenioid larvae exhibit a delay in osteogenic development, and that this heterochronic shift is associated with altered gene expression, including delayed expression of the osteogenic markers, col1a <dig> and col10a <dig>  and prolonged expression of the cartilage differentiation gene, col2a <dig>  these data provide a discrete molecular inroad into the mechanisms that underlie the evolution of this pedomorphic character.
we also show that other changes in the patterning of the notothenioid trophic apparatus are due to apparent shifts in the relative timing of development of particular skeletal elements.
these data are consistent with the hypothesis that heterochronic shifts in early craniofacial skeletal development have played significant roles in the adaptive diversification of antarctic notothenioids.
RESULTS
development of skeletal mineralization is delayed in antarctic fish
skeletal preparations were made of pelagic notothenioid larvae beginning at developmental stages just before hatching and continuing until just before yolk absorption and the onset of exogenous feeding , and compared to those of the benthic notothenioid and outgroup species.
the results showed that pelagic notothenioid pharyngeal skeletons lack any mineralized tissues at developmental stages during which other species have begun to differentiate a well-formed bony skeleton .
formation of mineralized tissue was not detected using alizarin red staining, although developing cartilages had accumulated alcian-staining cartilaginous material.
we conclude that bone mineralization is delayed in developing pelagic, osteopenic antarctic notothenioid embryos relative to outgroup fishes.
to investigate the molecular genetic basis for this delayed ossification, we cloned and examined the developmental expression of genes involved in skeletal matrix formation.
nucleotide sequence similarity and phylogenetic reconstruction of collagen genes
to examine the expression of genes directing cartilage and bone formation, we cloned notothenioid derived collagen genes specific for the development of cartilage  and bone .
the predicted amino acid sequence similarities among fishes were similar for all three collagen genes, with highest identities exhibited between the two notothenioids, the osteopenic c. aceratus and the strongly mineralized n. coriiceps , whereas the lowest identities were generally found between fishes and tetrapod outgroups .
the exception to this pattern was col2a1b, in which the d. rerio ortholog was the most highly divergent sequence for all comparisons, including tetrapods.
the nucleotide sequences of col2a1b from chaenocephalus and notothenia differed at only two of  <dig> positions, a remarkably low rate of synonymous substitution in two lineages thought to have separated approximately  <dig> mya  <cit> .
phylogenetic trees reflected accepted relationships among fish species, with danio the most distantly related lineage to the takifugu +  clade .
d. rerio col2a1b exhibited an exceptionally long branch relative to other orthologs in this tree, reflecting accelerated amino acid sequence divergence in this lineage.
altered patterns of gene expression underlie reduced mineralization in pelagic notothenioid larvae
we observed prolonged developmental expression of the early cartilage marker col2a <dig> and reduced expression of the later developmental markers col10a <dig> and col1a <dig> in pelagic notothenioid larvae as compared to similarly staged stickleback and zebrafish.
by  <dig> and  <dig> days post-fertilization , respectively, stickleback and zebrafish larvae exhibited very little col2a <dig> expression in the pharyngeal skeleton .
in contrast, both pelagic notothenioid species exhibited strong expression of this cartilage marker through extended periods of larval development .
the hypertrophic cartilage marker, col10a <dig>  is normally expressed in terminally differentiated chondrocytes just before apoptosis and the onset of endochondral ossification  <cit> .
this marker was discretely expressed throughout the pharyngeal skeleton in stickleback and zebrafish  but was limited to the cleithrum  of the pectoral girdle, and to the upper and lower jaws  in pelagic notothenioids .
the osteoblast differentiation marker col1a <dig> was likewise ubiquitously expressed throughout the pharyngeal skeleton in both stickleback and zebrafish larvae .
the same marker was only weakly expressed in the pharyngeal skeleton of the benthopelagic notothenioid, c. aceratus, and was virtually absent from the pelagic species, p. antarcticum .
this pattern of delayed osteogenic gene expression was also evident in sectioned specimens .
pelagic notothenioids exhibited strong col2a <dig> gene expression throughout the ceratohyal cartilage , whereas in similarly staged stickleback and zebrafish col2a <dig> expression was restricted to the proximal and distal ends of this skeletal element .
expression of col10a <dig> was absent from the ceratohyal cartilage in notothenioid larvae , but was strongly expressed in the putative hypertrophic domain of the ceratohyal in stickleback and zebrafish .
likewise, strong expression of col1a <dig> was observed in the perichondrium surrounding the ceratohyal cartilage in both stickleback and zebrafish , while only weak perichondrial col1a <dig> expression was observed in notothenioid larvae .
although only single representative staged specimens are presented here, the observed trends were consistent over extended periods of larval development.
in toto, these data are consistent with a delay in the osteogenic developmental program relative to the chondrogenic program in the two pelagic notothenioid species, relative to the other fishes examined.
in contrast to the pharyngeal skeleton, the development of the appendicular skeleton was relatively conserved among fish species .
in all species examined, col2a <dig> expression was observed in the scapulocoracoid , col10a <dig> was expressed in the cleithrum , and col1a <dig> expression was observed in the fin fold .
the only notable difference among species was strong expression of col10a <dig> in the endochondral disk  of c. aceratus .
we did not observe expression of col10a <dig> in this tissue in the other species at any stage, and expression of this marker appeared in the fins of c. aceratus only at later stages of larval development.
the implications of potential novel gene expression in the fins of certain notothenioid species are interesting given the importance of fins in the adaptive radiation of this group  <cit> , but the anatomical consequences of this expression pattern are not yet known.
these data suggest a tradeoff between the chondrogenic and osteogenic programs during craniofacial  skeletal development.
skeletal development is highly conserved among vertebrates, and pathways leading to either cartilage or bone formation are tightly linked.
condensing cartilage progenitor cells express sox <dig>  which regulates col2a <dig> expression  <cit>  and is necessary and sufficient for cartilage differentiation  <cit> .
osteoprogenitor cells express runx <dig>  which modulates the expression of bone matrix genes including col1a <dig> and is necessary and sufficient for bone formation  <cit> .
both sox <dig> and runx <dig> can be expressed in the same cells, and it is the relative amount of each that specifies cell fate  <cit> .
runx <dig> binds to col10a <dig> and ihh promoters  <cit>  to activate their expression in terminally differentiated hypertrophic chondrocytes  <cit> .
secreted ihh stimulates surrounding perichondrial osteoblasts to secrete pthrp  <cit> , which diffuses back to prehypertrophic chondrocytes, binds to its receptor pth1r, and blocks further chondrocyte maturation.
thus, pedomorphic bone development and changes in collagen gene expression patterns in pelagic notothenioid species could be due to one or more mutations affecting these regulatory genes.
in addition to early pattering mechanisms, evolved bone loss in notothenioids might involve later developmental events.
bone development continues throughout the life of an individual, through balanced processes of bone growth and bone remodeling.
fish regulate skeletal growth and remodeling in much the same way as other vertebrates, through coordinated activities of bone forming cells  and bone resorbing cells   <cit> .
it is possible that decreased bone density in notothenioids, which begins early in life, may be maintained later in development by modulating osteoblast and osteoclast activities.
because osteoporosis in humans results from an unfavorable balance between bone deposition by osteoblasts and bone resorption by osteoclasts, these considerations provide fertile ground for future investigations.
pedomorphosis in notothenioid skeletal patterning
morphological analyses revealed several pedomorphic traits in notothenioid species relative to stickleback and zebrafish.
the most conspicuous differences between notothenioid and outgroup pharyngeal skeletons were the absence of the pterygoid process of the palatoquadrate and the concomitant expansion of ventral cartilages.
this was most pronounced in the benthopelagic species c. aceratus, where the anterior and ventral pharyngeal cartilage elements  were well formed and elongated before any other element appeared .
this developmental sequence is distinct from that observed in both stickleback and zebrafish, where the palatoquadrate  forms almost simultaneously with meckel's cartilage , followed soon after by the ceratohyal  <cit> .
the pterygoid process of the palatoquadrate forms relatively late in zebrafish and stickleback, after most of the other pharyngeal cartilages have begun to develop, but is well formed at the beginning of larval development.
in notothenioids, the pterygoid process is conspicuously absent throughout extended periods of larval development, and in adults this structure  is greatly reduced in size  <cit> .
factors that pattern the pharyngeal skeleton along the d-v axis have been well studied in a variety of models, and include endothelin , an important signaling molecule for d-v patterning of the zebrafish pharyngeal skeleton  <cit> .
the edn <dig> gene is expressed in the ventral pharyngeal endoderm and is thought to signal through hand <dig> to promote the development of ventral arch structures , while inhibiting the expression of bapx <dig> and eng <dig>  which are expressed in the dorsal pharyngeal pouch  <cit> .
an enticing model of notothenioid jaw development would predict that over-expression of ventral factors  in the pharyngeal endoderm leads to the attenuation of dorsal signaling molecules, and the development of longer lower jaws and reduced upper jaw elements.
alternatively, the observed attenuation of the dorsal pterygoid process in notothenioids might be due to more localized signals, such as those regulating stomodeal development.
in zebrafish, the pterygoid process develops from a subset of cranial neural crest cells that condense along the dorsal ectoderm of the stomodeum, the presumptive mouth opening  <cit> .
abrogation of hedgehog signaling at the developing midline disrupts the specification of the stomodeum, and the condensation of cells fated to become the pterygoid, suggesting that the stomodeum may be a critical signaling center for pterygoid development.
notothenioid larvae were also distinguished from outgroup species by the conspicuous absence of the most posterior pharyngeal cartilage, the fifth ceratobranchial .
this bilaterally paired element forms the pharyngeal 'jaw' in both percomorph  and cypriniform  fishes, bears teeth, and is used to processes prey gathered by the oral jaws.
in most bony fishes, development of this cartilage is accelerated; cb <dig> forms at the same time as cb <dig> and before cb <dig>  and is one of the first elements to be mineralized in the head  <cit> .
in notothenioid adults, however, cb <dig> is significantly reduced  <cit> , which we show here to be associated with a developmental delay in the formation of cb <dig>  notothenioid larvae possess only a rudimentary cb <dig> , and it is the last pharyngeal cartilage to form, well after the development of more anterior cb cartilages.
at the latest developmental stages examined, only a few pharyngeal teeth had formed in c. aceratus and n. coriiceps larvae, and no teeth had developed on the oral or pharyngeal jaws of p. antarcticum larvae, whereas comparably staged stickleback and zebrafish possessed well-mineralized cb5s bearing several teeth .
a-p patterning of the pharyngeal skeleton is determined by nested, overlapping expression of various hox genes  <cit> , and it is possible that mis-expression of posterior hox genes might play a role in the delayed development of cb <dig> in notothenioid larvae.
alternatively, the primary mechanism leading to the reduction of cb <dig> could be due to signals emanating from tissues surrounding hox-positive cranial neural crest cells, including the pharyngeal endoderm or adjacent mesoderm  <cit> .
each of these hypotheses remains to be tested in a more thorough analysis of a-p patterning of the notothenioid pharyngeal skeleton.
each notothenioid ecotype examined here exhibited relatively elongated lower jaws, absent pterygoid processes, and reduced cb5s as larvae , suggesting that these traits were present in the common notothenioid ancestor and evolved independently from the bone mineralization phenotype.
the pterygoid process of the palatoquadrate and the pharyngeal jaw play significant roles in the collecting and processing of pelagic prey  <cit> .
the pterygoid process articulates with the maxilla and is predicted to play an important role during jaw protrusion  <cit> .
the observation that the ectopterygoid is one of the few bones in the head where an early osteogenic program is retained in pelagic notothenioids , likely reflects the importance for this bone during suction feeding.
the pharyngeal jaw is used for both the mastication and laceration of prey in fishes  <cit> .
while pharyngeal mastication is likely not important for suction feeding notothenioids  <cit> , a reduced fifth ceratobranchial may have affected the efficiency to which the pharyngeal jaws are able process prey via laceration .
whether a delay in the development of either of these structures represents a developmental constraint that has shaped patterns of diversification as nototheniods radiated to fill pelagic foraging niches remains an open question.
CONCLUSIONS
notothenioids dominate the near-shore habitat of antarctica and are an important example of an adaptive radiation in marine fishes  <cit> .
while the ecological and environmental shifts that have precipitated these events are well understood, virtually nothing is known about the molecular and developmental changes that underlie adaptive variation in notothenioid feeding architecture.
we propose that salient aspects of the evolution of the notothenioid craniofacial skeleton can be explained by discrete shifts in early developmental genetic scheduling .
a small but growing number of studies have described heterochrony at the molecular level.
the evolution and development of the vertebrate limb, for example, provides several examples of developmental genetic heterochrony.
shifts in the relative timing of expression of shh in the zone of polarizing activity are correlated with decreased digit number in lizards  <cit> , while prolonged fgf <dig> expression in the apical ectodermal ridge is correlated with the development of extra phalangeal elements in dolphins  <cit> .
excellent opportunities to study genetic heterochrony are also provided by naturally occurring wing color mutations in butterflies  <cit> , and by induced mutations in c. elegans that affect developmental timing  <cit> .
genetic heterochrony may also explain differential expression of cone opsin genes in cichlid fishes, providing a potentially potent mechanism through which visual sensitivities can evolve in this group  <cit> .
in spite of these putative exemplars the number of described molecular mechanisms that underlie evolutionary change by heterochrony are disproportionately small given the prominence of this process in discussions of morphological evolution  <cit> .
here we show that the evolution of bone loss in antarctic notothenioids can be explained in part by the prolongation of the early chondrogenic developmental pathway through extended periods of larval development.
these data offer a molecular inroad into the larger signaling cascades that modulate bone density, a trait that is linked to the adaptive radiation of antarctic notothenioids  <cit> .
in addition to the evolutionary implications, understanding these mechanisms could provide insights into the development of human osteopenia and suggest effective therapies to prevent and/or treat this disease  <cit> .

 antarctic notothenioids are endemic to the southern ocean and probably evolved in situ from a sluggish, benthic perciform species beginning 40- mya in the then temperate waters of the antarctic continental shelf. with the opening of the drake passage, the establishment of the antarctic circumpolar current, and a sharp drop in atmospheric carbon dioxide levels, the southern ocean became thermally isolated, began to cool, and attained its present frigid temperatures by the mid-miocene. the rich, shallow-water, temperate fish fauna characteristic of the late eocene became largely extinct due to habitat destruction and changes in trophic structure, thus freeing ecological niches into which the notothenioids radiated. the hallmark of the antarctic notothenioid radiation is the evolution of secondary pelagicism, which is prominent in the family nototheniidae and common in the family channichthyidae. about 50% of notothenioid species now either live in or actively exploit pelagic habitats. a significant obstacle facing notothenioids as they radiated into pelagic habitats was the ancestral absence of a swim bladder, the gas-filled chamber that most teleosts use to maintain buoyancy. in pelagic notothenioids, natural selection favored compensatory changes in the musculoskeletal system to achieve neutral buoyancy, including the replacement of densely mineralized bone with cartilage and connective tissue, decreased bone mineralization, and the accumulation of lipid deposits in muscle and connective tissues. changes in habitat were also accompanied by the evolution of distinct oral jaw morphologies and body shapes that accommodate shifts in diet and foraging behavior. trophic evolution among notothenioids resulted in two distinct pelagic ecotypes: true pelagics and benthopelagics. true pelagics spend most of their time in the water column where they feed on microinvertebrates. they possess relatively short, protractile jaws for suction feeding and have few, large oral teeth arranged along a single tooth row. in contrast, benthopelagics spend much of their time on or close to the ocean floor but venture into the pelagic zone to forage on schools of small fish and macroinvertebrates. most benthopelagic notothenioids have non-protractile, elongate jaws, a wide gape, and many, small oral teeth. this morphology enables benthopelagics to feed efficiently on krill and schools of small fishes by expanding their buccal cavity, and overtaking and sifting large mouthfuls of prey. extant benthic notothenioids preserve the ancestral condition, exhibiting robust skeletons and heavily fortified jaws bearing several rows of large teeth. notothenioids achieved secondary pelagicism by pedomorphism, the retention of ancestrally juvenile traits by adults in a descendant taxon. pedomorphism arises from heterochronic processes that alter the schedule of developmental events. striking examples of pedomorphic characters in pelagic and benthopelagic notothenioids include delayed and reduced skeletal ossification, retention of the notochord, reduced numbers of teeth and tooth rows, and reduction of the pterygoid process of the palatoquadrate. as a first step toward understanding the molecular mechanisms that underlie skeletal reduction and morphological change in this group, we characterized early craniofacial development in a true pelagic species, p. antarcticum, and a benthopelagic species, c. aceratus. we describe shared derived aspects of craniofacial development in pelagic notothenioids that differ significantly from those observed in a benthic notothenioid species, n. coriiceps, another percomorph species, gasterosteus aculeatus, and a more distantly related outgroup, danio rerio. we found that pelagic notothenioid larvae exhibit a delay in osteogenic development, and that this heterochronic shift is associated with altered gene expression, including delayed expression of the osteogenic markers, col1a and col10a and prolonged expression of the cartilage differentiation gene, col2a these data provide a discrete molecular inroad into the mechanisms that underlie the evolution of this pedomorphic character. we also show that other changes in the patterning of the notothenioid trophic apparatus are due to apparent shifts in the relative timing of development of particular skeletal elements. these data are consistent with the hypothesis that heterochronic shifts in early craniofacial skeletal development have played significant roles in the adaptive diversification of antarctic notothenioids.  development of skeletal mineralization is delayed in antarctic fish skeletal preparations were made of pelagic notothenioid larvae beginning at developmental stages just before hatching and continuing until just before yolk absorption and the onset of exogenous feeding, and compared to those of the benthic notothenioid and outgroup species. the results showed that pelagic notothenioid pharyngeal skeletons lack any mineralized tissues at developmental stages during which other species have begun to differentiate a well-formed bony skeleton. formation of mineralized tissue was not detected using alizarin red staining, although developing cartilages had accumulated alcian-staining cartilaginous material. we conclude that bone mineralization is delayed in developing pelagic, osteopenic antarctic notothenioid embryos relative to outgroup fishes. to investigate the molecular genetic basis for this delayed ossification, we cloned and examined the developmental expression of genes involved in skeletal matrix formation. nucleotide sequence similarity and phylogenetic reconstruction of collagen genes to examine the expression of genes directing cartilage and bone formation, we cloned notothenioid derived collagen genes specific for the development of cartilage and bone. the predicted amino acid sequence similarities among fishes were similar for all three collagen genes, with highest identities exhibited between the two notothenioids, the osteopenic c. aceratus and the strongly mineralized n. coriiceps, whereas the lowest identities were generally found between fishes and tetrapod outgroups. the exception to this pattern was col2a1b, in which the d. rerio ortholog was the most highly divergent sequence for all comparisons, including tetrapods. the nucleotide sequences of col2a1b from chaenocephalus and notothenia differed at only two of positions, a remarkably low rate of synonymous substitution in two lineages thought to have separated approximately mya. phylogenetic trees reflected accepted relationships among fish species, with danio the most distantly related lineage to the takifugu + clade. d. rerio col2a1b exhibited an exceptionally long branch relative to other orthologs in this tree, reflecting accelerated amino acid sequence divergence in this lineage. altered patterns of gene expression underlie reduced mineralization in pelagic notothenioid larvae we observed prolonged developmental expression of the early cartilage marker col2a and reduced expression of the later developmental markers col10a and col1a in pelagic notothenioid larvae as compared to similarly staged stickleback and zebrafish. by and days post-fertilization, respectively, stickleback and zebrafish larvae exhibited very little col2a expression in the pharyngeal skeleton. in contrast, both pelagic notothenioid species exhibited strong expression of this cartilage marker through extended periods of larval development. the hypertrophic cartilage marker, col10a is normally expressed in terminally differentiated chondrocytes just before apoptosis and the onset of endochondral ossification. this marker was discretely expressed throughout the pharyngeal skeleton in stickleback and zebrafish but was limited to the cleithrum of the pectoral girdle, and to the upper and lower jaws in pelagic notothenioids. the osteoblast differentiation marker col1a was likewise ubiquitously expressed throughout the pharyngeal skeleton in both stickleback and zebrafish larvae. the same marker was only weakly expressed in the pharyngeal skeleton of the benthopelagic notothenioid, c. aceratus, and was virtually absent from the pelagic species, p. antarcticum. this pattern of delayed osteogenic gene expression was also evident in sectioned specimens. pelagic notothenioids exhibited strong col2a gene expression throughout the ceratohyal cartilage, whereas in similarly staged stickleback and zebrafish col2a expression was restricted to the proximal and distal ends of this skeletal element. expression of col10a was absent from the ceratohyal cartilage in notothenioid larvae, but was strongly expressed in the putative hypertrophic domain of the ceratohyal in stickleback and zebrafish. likewise, strong expression of col1a was observed in the perichondrium surrounding the ceratohyal cartilage in both stickleback and zebrafish, while only weak perichondrial col1a expression was observed in notothenioid larvae. although only single representative staged specimens are presented here, the observed trends were consistent over extended periods of larval development. in toto, these data are consistent with a delay in the osteogenic developmental program relative to the chondrogenic program in the two pelagic notothenioid species, relative to the other fishes examined. in contrast to the pharyngeal skeleton, the development of the appendicular skeleton was relatively conserved among fish species. in all species examined, col2a expression was observed in the scapulocoracoid, col10a was expressed in the cleithrum, and col1a expression was observed in the fin fold. the only notable difference among species was strong expression of col10a in the endochondral disk of c. aceratus. we did not observe expression of col10a in this tissue in the other species at any stage, and expression of this marker appeared in the fins of c. aceratus only at later stages of larval development. the implications of potential novel gene expression in the fins of certain notothenioid species are interesting given the importance of fins in the adaptive radiation of this group, but the anatomical consequences of this expression pattern are not yet known. these data suggest a tradeoff between the chondrogenic and osteogenic programs during craniofacial skeletal development. skeletal development is highly conserved among vertebrates, and pathways leading to either cartilage or bone formation are tightly linked. condensing cartilage progenitor cells express sox which regulates col2a expression and is necessary and sufficient for cartilage differentiation. osteoprogenitor cells express runx which modulates the expression of bone matrix genes including col1a and is necessary and sufficient for bone formation. both sox and runx can be expressed in the same cells, and it is the relative amount of each that specifies cell fate. runx binds to col10a and ihh promoters to activate their expression in terminally differentiated hypertrophic chondrocytes. secreted ihh stimulates surrounding perichondrial osteoblasts to secrete pthrp, which diffuses back to prehypertrophic chondrocytes, binds to its receptor pth1r, and blocks further chondrocyte maturation. thus, pedomorphic bone development and changes in collagen gene expression patterns in pelagic notothenioid species could be due to one or more mutations affecting these regulatory genes. in addition to early pattering mechanisms, evolved bone loss in notothenioids might involve later developmental events. bone development continues throughout the life of an individual, through balanced processes of bone growth and bone remodeling. fish regulate skeletal growth and remodeling in much the same way as other vertebrates, through coordinated activities of bone forming cells and bone resorbing cells. it is possible that decreased bone density in notothenioids, which begins early in life, may be maintained later in development by modulating osteoblast and osteoclast activities. because osteoporosis in humans results from an unfavorable balance between bone deposition by osteoblasts and bone resorption by osteoclasts, these considerations provide fertile ground for future investigations. pedomorphosis in notothenioid skeletal patterning morphological analyses revealed several pedomorphic traits in notothenioid species relative to stickleback and zebrafish. the most conspicuous differences between notothenioid and outgroup pharyngeal skeletons were the absence of the pterygoid process of the palatoquadrate and the concomitant expansion of ventral cartilages. this was most pronounced in the benthopelagic species c. aceratus, where the anterior and ventral pharyngeal cartilage elements were well formed and elongated before any other element appeared. this developmental sequence is distinct from that observed in both stickleback and zebrafish, where the palatoquadrate forms almost simultaneously with meckel's cartilage, followed soon after by the ceratohyal. the pterygoid process of the palatoquadrate forms relatively late in zebrafish and stickleback, after most of the other pharyngeal cartilages have begun to develop, but is well formed at the beginning of larval development. in notothenioids, the pterygoid process is conspicuously absent throughout extended periods of larval development, and in adults this structure is greatly reduced in size. factors that pattern the pharyngeal skeleton along the d-v axis have been well studied in a variety of models, and include endothelin, an important signaling molecule for d-v patterning of the zebrafish pharyngeal skeleton. the edn gene is expressed in the ventral pharyngeal endoderm and is thought to signal through hand to promote the development of ventral arch structures, while inhibiting the expression of bapx and eng which are expressed in the dorsal pharyngeal pouch. an enticing model of notothenioid jaw development would predict that over-expression of ventral factors in the pharyngeal endoderm leads to the attenuation of dorsal signaling molecules, and the development of longer lower jaws and reduced upper jaw elements. alternatively, the observed attenuation of the dorsal pterygoid process in notothenioids might be due to more localized signals, such as those regulating stomodeal development. in zebrafish, the pterygoid process develops from a subset of cranial neural crest cells that condense along the dorsal ectoderm of the stomodeum, the presumptive mouth opening. abrogation of hedgehog signaling at the developing midline disrupts the specification of the stomodeum, and the condensation of cells fated to become the pterygoid, suggesting that the stomodeum may be a critical signaling center for pterygoid development. notothenioid larvae were also distinguished from outgroup species by the conspicuous absence of the most posterior pharyngeal cartilage, the fifth ceratobranchial. this bilaterally paired element forms the pharyngeal'jaw' in both percomorph and cypriniform fishes, bears teeth, and is used to processes prey gathered by the oral jaws. in most bony fishes, development of this cartilage is accelerated; cb forms at the same time as cb and before cb and is one of the first elements to be mineralized in the head. in notothenioid adults, however, cb is significantly reduced, which we show here to be associated with a developmental delay in the formation of cb notothenioid larvae possess only a rudimentary cb, and it is the last pharyngeal cartilage to form, well after the development of more anterior cb cartilages. at the latest developmental stages examined, only a few pharyngeal teeth had formed in c. aceratus and n. coriiceps larvae, and no teeth had developed on the oral or pharyngeal jaws of p. antarcticum larvae, whereas comparably staged stickleback and zebrafish possessed well-mineralized cb5s bearing several teeth. a-p patterning of the pharyngeal skeleton is determined by nested, overlapping expression of various hox genes, and it is possible that mis-expression of posterior hox genes might play a role in the delayed development of cb in notothenioid larvae. alternatively, the primary mechanism leading to the reduction of cb could be due to signals emanating from tissues surrounding hox-positive cranial neural crest cells, including the pharyngeal endoderm or adjacent mesoderm. each of these hypotheses remains to be tested in a more thorough analysis of a-p patterning of the notothenioid pharyngeal skeleton. each notothenioid ecotype examined here exhibited relatively elongated lower jaws, absent pterygoid processes, and reduced cb5s as larvae, suggesting that these traits were present in the common notothenioid ancestor and evolved independently from the bone mineralization phenotype. the pterygoid process of the palatoquadrate and the pharyngeal jaw play significant roles in the collecting and processing of pelagic prey. the pterygoid process articulates with the maxilla and is predicted to play an important role during jaw protrusion. the observation that the ectopterygoid is one of the few bones in the head where an early osteogenic program is retained in pelagic notothenioids, likely reflects the importance for this bone during suction feeding. the pharyngeal jaw is used for both the mastication and laceration of prey in fishes. while pharyngeal mastication is likely not important for suction feeding notothenioids, a reduced fifth ceratobranchial may have affected the efficiency to which the pharyngeal jaws are able process prey via laceration. whether a delay in the development of either of these structures represents a developmental constraint that has shaped patterns of diversification as nototheniods radiated to fill pelagic foraging niches remains an open question.  notothenioids dominate the near-shore habitat of antarctica and are an important example of an adaptive radiation in marine fishes. while the ecological and environmental shifts that have precipitated these events are well understood, virtually nothing is known about the molecular and developmental changes that underlie adaptive variation in notothenioid feeding architecture. we propose that salient aspects of the evolution of the notothenioid craniofacial skeleton can be explained by discrete shifts in early developmental genetic scheduling. a small but growing number of studies have described heterochrony at the molecular level. the evolution and development of the vertebrate limb, for example, provides several examples of developmental genetic heterochrony. shifts in the relative timing of expression of shh in the zone of polarizing activity are correlated with decreased digit number in lizards, while prolonged fgf expression in the apical ectodermal ridge is correlated with the development of extra phalangeal elements in dolphins. excellent opportunities to study genetic heterochrony are also provided by naturally occurring wing color mutations in butterflies, and by induced mutations in c. elegans that affect developmental timing. genetic heterochrony may also explain differential expression of cone opsin genes in cichlid fishes, providing a potentially potent mechanism through which visual sensitivities can evolve in this group. in spite of these putative exemplars the number of described molecular mechanisms that underlie evolutionary change by heterochrony are disproportionately small given the prominence of this process in discussions of morphological evolution. here we show that the evolution of bone loss in antarctic notothenioids can be explained in part by the prolongation of the early chondrogenic developmental pathway through extended periods of larval development. these data offer a molecular inroad into the larger signaling cascades that modulate bone density, a trait that is linked to the adaptive radiation of antarctic notothenioids. in addition to the evolutionary implications, understanding these mechanisms could provide insights into the development of human osteopenia and suggest effective therapies to prevent and/or treat this disease. 
our data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of antarctic notothenioids into pelagic habitats. notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy. relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. we compared craniofacial skeletal development in two pelagic notothenioids, chaenocephalus aceratus and pleuragramma antarcticum, to that in a benthic species, notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements. pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon.
 pedomorphism is the retention of ancestrally juvenile traits by adults in a descendant taxon. despite its importance for evolutionary change, there are few examples of a molecular basis for this phenomenon. notothenioids represent one of the best described species flocks among marine fishes, but their diversity is currently threatened by the rapidly changing antarctic climate. notothenioid evolutionary history is characterized by parallel radiations from a benthic ancestor to pelagic predators, which was accompanied by the appearance of several pedomorphic traits, including the reduction of skeletal mineralization that resulted in increased buoyancy.  we compared craniofacial skeletal development in two pelagic notothenioids, chaenocephalus aceratus and pleuragramma antarcticum, to that in a benthic species, notothenia coriiceps, and two outgroups, the threespine stickleback and the zebrafish. relative to these other species, pelagic notothenioids exhibited a delay in pharyngeal bone development, which was associated with discrete heterochronic shifts in skeletal gene expression that were consistent with persistence of the chondrogenic program and a delay in the osteogenic program during larval development. morphological analysis also revealed a bias toward the development of anterior and ventral elements of the notothenioid pharyngeal skeleton relative to dorsal and posterior elements.  our data support the hypothesis that early shifts in the relative timing of craniofacial skeletal gene expression may have had a significant impact on the adaptive radiation of antarctic notothenioids into pelagic habitats. 