BACKGROUND
skeletal tissues provide invaluable traits to document vertebrate evolution and to reveal the mechanistic basis for evolutionary change.
two main processes underlie skeletal development: histogenesis--histological differentiation of skeletal tissues--and morphogenesis--acquisition of skeletal element location, shape, and size.
skeletal histogenesis involves overt differentiation of cells that secrete the extracellular matrix of cartilage and bone , and follows the mesenchymal condensation of skeletogenic cells.
skeletal morphogenesis directs such differentiation events in space and time.
while many studies propose a molecular genetic basis for evolutionary changes to skeletal morphogenesis  <cit> , the evolution of skeletal histogenesis among vertebrates is fertile ground for additional molecular analyses  <cit> .
each cell type achieves and performs its function by employing a specific set of genes, recently termed its molecular fingerprint  <cit> .
are molecular fingerprints free to evolve in each vertebrate clade in response to selective pressures?
or were molecular fingerprints fixed shortly after tissues first evolved and since then have remained rather constant?
for example, skeletons of land animals evolved under greater gravitational stress than skeletons of aquatic animals  <cit> .
as a result, one might hypothesize that the molecular fingerprints of skeletal cells in tetrapods, for instance, would differ from those of teleosts.
alternatively, molecular fingerprints of skeletal cells may have been fixed at the evolutionary origin of the skeleton, and other mechanisms may adapt animals to lineage-specific selective pressures.
comparative studies can reveal the extent to which molecular fingerprints of skeletal cells evolved among vertebrate clades, although the few clades examined thus far are biased towards crown-groups of both ray-finned  and lobe-finned  fish .
therefore, analyses of more basally-diverging lineages, such as gar or lungfish, will provide evolutionary perspective to these studies.
comparisons among human, mouse, and chick skeletal tissues suggest that the molecular fingerprints of chondrocytes or osteoblasts do not vary greatly among tetrapods .
tetrapods exhibit one type of bone tissue, but they have three types of cartilage: elastic cartilage, hyaline cartilage, and fibrocartilage  <cit> .
here, we focus on the predominant type of cartilage in vertebrates, hyaline cartilage, which serves as the template for bone during endochondral ossification.
collagens are the most abundant proteinaceous skeletal matrix components, and tetrapod chondrocytes and osteoblasts typically express different fibrillar collagens.
in tetrapods, collagen type  <dig> alpha  <dig>  is expressed abundantly in bone and is absent from cartilage, while collagen type  <dig> alpha  <dig>  typifies cartilage and is not expressed in tetrapod bone  <cit> .
hyaline cartilage chondrocytes undergo a maturation process during development, when they express collagen type  <dig> alpha  <dig> , but tetrapod bone does not express col10a <dig>  <cit> .
despite these fundamental differences in collagen expression of tetrapod osteoblasts and chondrocytes, their molecular fingerprints also overlap; for example, both cell types express collagen type  <dig> alpha  <dig> .
skeletogenic transcription factors control molecular fingerprints of chondrocytes and osteoblasts; sox <dig> is required for chondrocyte differentiation, while runx <dig> is necessary for osteoblast differentiation  <cit> .
sox <dig> and runx <dig> dictate skeletal cell differentiation by binding to and promoting the transcription of genes that impart identity to skeletal tissues.
for instance, sox <dig> directly regulates col2a <dig> expression, while runx <dig> activates col1a <dig> transcription  <cit> .
much of the overlap in molecular fingerprints of tetrapod chondrocytes and osteoblasts can be attributed to runx <dig> activity, which is required for chondrocyte maturation in addition to its role in osteoblast differentiation  <cit> .
perhaps such overlap is not surprising, considering that tetrapod chondrocytes and osteoblasts differentiate from a bipotential progenitor cell, the osteochondroprogenitor, during both embryonic and adult stem cell development  <cit> .
indicative of the delicate balance required for these transcription factors to direct discrete cell lineages, sox <dig> can repress runx <dig> activity  <cit> .
although hyaline cartilage chondrocytes show conserved molecular fingerprints among vertebrate clades, a few studies in fish suggest that the molecular fingerprint of osteoblasts varies among vertebrates .
in contrast to tetrapod osteoblasts, zebrafish osteoblasts express col10a <dig>  and various teleosts show evidence of col <dig> in their bone matrix  <cit> .
a lineage-specific genome duplication event, the teleost genome duplication , occurred at the base of the teleost radiation, and genome duplications have been thought to facilitate the origin of new gene capabilities  <cit> .
for example, sox <dig> does not have a direct effect on osteoblast differentiation in tetrapods, but a sox <dig> duplicate in teleosts  has been reported to affect bone development  <cit> .
therefore, one of at least three evolutionary scenarios might explain differences in the molecular fingerprint of tetrapod and teleost osteoblasts .
hypothesis  <dig> proposes that a new function for these collagens in osteoblasts evolved in the teleost lineage, perhaps facilitated by the tgd .
hypothesis  <dig>  like hypothesis  <dig>  proposes that collagen gene neofunctionalization occurred, but that this event happened in the ancestral actinopterygian osteoblast, and hence ruling out the hypothesis that the tgd facilitated this novel gene function.
hypothesis  <dig> proposes that the last common ancestor to both tetrapods and teleosts had osteoblast expression patterns found in today's teleosts, but that these patterns were lost secondarily in the tetrapod lineage .
to distinguish among these possibilities, we compared molecular fingerprints of skeletogenic cells in the teleost danio rerio with the spotted gar lepisosteus oculatus, a member of a teleost sister group that diverged before the tgd.
currently, the molecular fingerprints of chondrocytes and osteoblasts are completely unknown for any non-teleost actinopterygian.
results demonstrated that gar and zebrafish share molecular fingerprints of both chondrocytes and osteoblasts.
as an indication of skeletal cell molecular fingerprints, we used in situ hybridization on developing gar and zebrafish embryos.
specifically, we analyzed expression of the structural collagen genes col1a <dig>  col2a <dig>  col10a <dig>  and col11a <dig> in well-developed cartilage and bone, and also revealed expression of the transcription factor genes sox <dig> and runx <dig> during mesenchymal condensation.
we found that, like osteoblasts in the teleost danio rerio, gar osteoblasts expressed col2a <dig> and col10a <dig>  therefore, these data refute by parsimony the role of the tgd in the origin of lineage-specific skeletal molecular fingerprints  and furthermore argue that the expression of "chondrocyte" genes in osteoblasts is a shared feature of actinopterygians.
more experiments will be required to distinguish between hypotheses  <dig> and  <dig>  in efforts to explain the actinopterygian expression patterns reported here, we found, surprisingly, that the "chondrogenic" transcription factor sox <dig> was expressed in developing gar and zebrafish osteoblasts.
we discuss these findings in a phylogenetic context and suggest that the molecular fingerprint of the primitive vertebrate osteoblast was less fixed than previously expected from studies of tetrapods.
methods
fish
all fish and embryos were maintained with iacuc approval, according to established protocols  <cit> .
wild-type zebrafish were of the ab strain; gar originated from animals collected in lafourche parish, louisiana .
histology and confocal microscopy
embryos and larvae were processed for alcian blue/alizarin red staining and sectioned for histology as described previously  <cit> .
for zebrafish, we used transgenic lines to help visualize the location and organization of specific populations of cells.
tgzc <dig> fish produce chondrocyte fluorescence  <cit> , tgb <dig> fish make fluorescent osteoblasts  <cit> , and tgy <dig> fish have fluorescence broadly among neural crest cells of the head  <cit> .
animals were imaged live under a confocal microscope while stained with the vital dye alizarin red, as described previously  <cit> .
molecular cloning and section in situ hybridization
whole-mount and section rna in situ hybridization were carried out as described  <cit> .
zebrafish probes used were runx2a, runx2b, sox9a, sox9b, col1a <dig>  col2a1a, col10a <dig>  and col11a <dig>  <cit> .
RESULTS
early and late stages of ceratohyal and dentary development
to elucidate molecular fingerprints of chondrocytes and osteoblasts in gar and zebrafish, we focused on two cranial skeletal elements: the ceratohyal, which in mammals forms the anterior horn of the hyoid bone and whose chondrocytes form directly from mesenchyme during endochondral ossification; and the dentary, which in mammals forms the mandible bone and whose osteoblasts differentiate directly from mesenchyme during intramembranous ossification.
to define equivalent developmental stages of cranial skeletogenesis between gar and zebrafish larvae, we performed whole-mount staining with alcian blue and alizarin red on a variety of stages for both species .
our histological analyses of ceratohyal development demonstrated a well-defined, alcian blue-stained cartilage rod in gar larvae by  <dig> days post-fertilization  and in zebrafish larvae by  <dig> dpf .
in the same region earlier in development, a faint alcian blue-stained condensation of mesenchyme was apparent in gar  and zebrafish  .
dentary bone formation was evidenced by substantial alizarin red staining adjacent to the anterolateral aspects of meckel's cartilage at  <dig> dpf for gar and at  <dig> dpf for zebrafish .
no alizarin red staining was found in the region of the dentary in gar at  <dig> dpf or in zebrafish at  <dig> dpf .
these results established the location and timing of equivalent stages of cartilage and bone development in gar and zebrafish.
chondrocyte molecular fingerprint
to analyze cellular and molecular features of developing chondrocytes in gar and zebrafish, we examined histologically stained sections, imaged transgenic zebrafish by confocal microscopy, and studied gene expression patterns in tissue sections.
at  <dig> dpf, the gar ceratohyal contained hundreds of chondrocytes, none of which showed evidence of hypertrophy .
by  <dig> dpf, however, the gar ceratohyal showed clearly hypertrophic chondrocytes in the mid-diaphyseal region .
at both  <dig> dpf and  <dig> dpf, aniline blue-stained bone matrix was apparent in an extremely thin layer of the perichondrium.
the  <dig> dpf zebrafish ceratohyal had dozens of chondrocytes, most of which already displayed evidence of hypertrophy .
alizarin red staining of bone matrix in the perichondrium was also evident.
therefore, well-developed ceratohyals of both the gar and zebrafish displayed equivalent cellular and histological features of chondrocyte  development.
we next sought to explore the molecular fingerprints of well-developed chondrocytes in gar and zebrafish.
in both gar and zebrafish, col1a <dig> expression was absent or very low in chondrocytes of the well-formed ceratohyal, although it was expressed clearly in cells of the perichondrium of both species .
ceratohyals in both gar and zebrafish had high levels of col2a <dig> transcripts , although more mature, mid-diaphyseal chondrocytes appeared to have down-regulated transcript levels , which is consistent with similar findings in tetrapods  <cit> .
in addition, col2a <dig> expression was detected in the perichondrium of the ceratohyal in both gar and zebrafish.
expression of col10a <dig> was high in mature chondrocytes and in perichondral cells of both gar and zebrafish ceratohyals, whereas surrounding, less mature chondrocytes did not express col10a <dig> .
transcripts for col11a <dig> were evident in both chondrocytes and perichondral cells of gar and zebrafish ceratohyals, although the levels of expression in mature chondrocytes appeared to be reduced relative to adjacent chondrocytes , which again is consistent with published reports in tetrapods  <cit> .
to help explain the collagen gene expression patterns observed in cells of the well-developed ceratohyal cartilage, we analyzed expression of genes encoding sox <dig> and runx <dig>  two transcription factors known to regulate these collagen genes in tetrapods  <cit> .
because specification of cell types occurs prior to their overt differentiation and transcription factor expression at this timepoint predicts skeletal cell fates  <cit> , we focused on the mesenchymal condensation phase of cartilage development.
progenitor cells of the gar ceratohyal had undergone mesenchymal condensation by  <dig> dpf, while chondrogenic condensation of the zebrafish ceratohyal had occurred by  <dig> hpf .
in gar, sox <dig> transcripts were abundant in chondrogenic cells, whereas levels of runx <dig> expression were only slightly above background .
due to the tgd, zebrafish has two co-orthologs of both sox <dig> and runx <dig>  <cit> .
expression patterns of sox <dig> and runx <dig> genes in cells of the zebrafish ceratohyal condensation at  <dig> hpf were similar to those seen in gar, although levels of runx <dig> gene expression were much higher .
in total, these data demonstrate that gar and zebrafish share molecular fingerprints of developing chondrocytes .
the detection of particular gene expression by in situ hybridization is indicated as follows: '+' = high detectable expression; '-' = undetectable expression; '+/-' = low detectable expression.
gar and zebrafish data are presented here, and chick and mouse data come from references cited in figure 1
osteoblast molecular fingerprint
to analyze the cellular and molecular features of developing osteoblasts in gar and zebrafish, we performed histological stains, confocal imaging of transgenic zebrafish, and in situ hybridization on tissue sections.
the dentaries of  <dig> dpf gar and  <dig> dpf zebrafish showed abundant bone matrix adjacent to meckel's cartilage .
in both gar and zebrafish, col1a <dig> expression was high in osteoblasts of the well-developed dentary .
transcripts for col2a <dig> were apparent in both gar and zebrafish dentaries, although levels detected in the zebrafish were relatively lower .
osteoblasts of both gar and zebrafish dentaries demonstrated abundant col10a <dig> expression, as well as high levels of col11a <dig> transcripts .
to help understand the expression patterns of collagen genes observed in osteoblasts of the well-developed dentary bone, we again analyzed sox <dig> and runx <dig> expression during mesenchymal condensation.
at  <dig> dpf, osteogenic cells of the gar dentary had undergone mesenchymal condensation, and were beginning to secrete aniline blue-stained bone matrix .
osteogenic cells of the zebrafish dentary condensation were visible at  <dig> hpf .
both sox <dig> and runx <dig> transcripts were apparent in osteogenic cells of the gar dentary .
the expression of sox <dig> in osteogenic cells of the gar dentary was dynamic during development.
while runx <dig> transcript levels were high in presumptive pre-osteoblasts lateral to meckel's cartilage at  <dig> dpf, sox <dig> expression was not detected in these cells .
these data argue that sox <dig> expression increased in osteoblasts as they began to differentiate.
similar to the single sox <dig> gene in gar, both sox9a and sox9b were expressed in osteogenic cells of the developing zebrafish dentary at  <dig> hpf .
also overlapping with the single runx <dig> expression in the gar dentary, runx2a and runx2b transcripts were abundant in the zebrafish dentary at  <dig> hpf .
in summary, these data demonstrate that gar and zebrafish share molecular fingerprints of developing osteoblasts .
the detection of particular gene expression by in situ hybridization is indicated as follows: '+' = high detectable expression; '-' = undetectable expression; '+/-' = low detectable expression.
gar and zebrafish data are presented here, and chick and mouse data come from references cited in figure 1
discussion
due to their preservation in the fossil record, cartilage and bone serve as invaluable traits in understanding vertebrate evolution.
evolutionary inferences, however, often assume that the histogenesis of skeletal tissues themselves remains constant among vertebrate lineages.
to be fair, cells that produce cartilage and bone  may have been free to evolve since their appearance roughly  <dig> million years ago  <cit> .
here, we ask explicitly: to what extent do vertebrate clades share expression of the sets of genes that characterize skeletogenic cell types ?
a molecular fingerprint that is shared among vertebrate clades would suggest evolutionary constraints on that skeletal cell type .
for instance, cells in cranial and appendicular skeletal tissues have different embryologic origins, and so developmental constraints may limit the molecular fingerprint of a skeletal cell that appears in both regions.
while future experiments can test this hypothesis more extensively, skeletogenic cells in different embryonic regions  of a given individual have been shown to exhibit a conserved molecular fingerprint  <cit> .
another interesting potential embryonic constraint is the fact that osteoblasts have two evolutionary and developmental origins within vertebrates.
during vertebrate phylogeny, bone originated in the dermis , and then later appeared in the perichondrium surrounding cartilage templates   <cit> .
while not a focus of this study, we did not find differences between molecular fingerprints of osteoblasts from the exoskeleton  and endoskeleton .
therefore, our results do not support the notion that the exoskeleton and endoskeleton have separate embryonic constraints on the molecular fingerprints of osteoblasts, but testing this hypothesis could be a fruitful avenue of future research.
a molecular fingerprint that varies among clades suggests relaxed constraints on the evolution of that cell type.
one might expect variation in molecular fingerprints of skeletogenic cells among various vertebrate lineages, especially given the different selective pressures to which each vertebrate clade has been exposed.
for example, the skeletons of land animals withstand a stronger effective gravitational force than do the skeletons of water-borne animals  <cit> .
some aquatic lineages, including sharks and other "cartilaginous" fish, and some antarctic fish, have even lost the majority of their bony skeleton at some point during phylogeny  <cit> .
are signatures of the embryonic response to these varied selective pressures seen in the molecular fingerprints of skeletogenic cells across vertebrates?
spurred by the reported and unexpected expression of col10a <dig> and col <dig>  two markers of tetrapod chondrocytes, in osteoblasts of teleosts , we pursued the hypothesis that molecular fingerprints of skeletogenic cells vary among vertebrate clades.
experiments revealed collagen and transcription factor gene expression in skeletal cells of hyaline cartilage and bone in the zebrafish--a teleost--and gar, which diverged in the actinopterygian lineage prior to the teleost-specific genome duplication .
specifically, our data distinguish among competing hypotheses to explain why osteoblasts of teleosts express col10a <dig> and col <dig>  which are not expressed in osteoblasts of tetrapods.
osteoblast expression of these collagens either represents a neofunctionalization event that was specific either to the teleost lineage subsequent to the tgd  or to the actinopterygian lineage , or they were expressed in osteoblasts of the common ancestor of tetrapods and teleosts and subsequently lost in tetrapods .
admittedly, evaluation of molecular fingerprints based upon expression of a few genes is a limited approach, but our findings on gene expression in chondrocytes and osteoblasts of the gar and zebrafish suggest evolutionary trends that could be embellished by massively parallel transcriptomics .
we demonstrate that gar and zebrafish share molecular fingerprints of both chondrocytes and osteoblasts .
despite evidence that genome duplication can facilitate the origin of new gene functions  <cit> , our data reject the proposed teleost neofunctionalization hypothesis for osteoblast evolution .
because both gar and zebrafish express col2a <dig> and col10a <dig> in their osteoblasts, the most parsimonious explanation is that these markers were present in the molecular fingerprint of the ancestral actinopterygian osteoblast.
therefore, parsimony favors hypothesis  <dig>  although our results do not reject hypothesis  <dig>  and more experiments are required to distinguish between these two possibilities.
the notion that osteoblasts achieved collagen neofunctionalization somewhere in the actinopterygian lineage  could be tested further by revealing the molecular fingerprint of osteoblasts in bichir, an actinopterygian diverging more basally than the gar lineage  <cit> .
similar studies of the lungfish or coelocanth, basally-diverging sarcopterygians, would test the possibility that col2a <dig> and col10a <dig> expression was present in osteoblasts of the ancestral bony fish and subsequently was lost somewhere in the sarcopterygian lineage leading to tetrapods .
our studies of skeletogenic transcription factors suggest a functional framework to explain why col2a <dig> and col10a <dig> are expressed in osteoblasts of actinopterygians, but not in osteoblasts of sarcopterygians .
in addition to runx <dig> expression, gar and zebrafish osteoblasts express sox <dig> during mesenchymal condensation of dermal bones.
developing osteoblasts of tetrapods typically express runx <dig> but not sox <dig> during mesenchymal condensation of dermal bones  <cit> .
we propose that the expression of sox <dig> in gar and zebrafish osteoblasts may explain the presence of col2a <dig> and col10a <dig> transcripts, given two assumptions.
first, actinopterygian osteoblasts would have to translate the sox <dig> transcript we observed into sox <dig> protein.
second, sox9-responsive cis-acting regulatory elements that drive col2a <dig> expression in tetrapods  <cit>  would have to operate similarly in actinopterygian lineages.
in support of this latter notion, col2a <dig> gene expression is extinguished in sox <dig> mutant zebrafish  <cit> .
currently, sox <dig> has not been shown to bind to the col10a <dig> promoter, but mis-expression of sox <dig> in developing avian osteoblasts causes ectopic col10a <dig> expression, and loss of sox <dig> can abrogate col10a <dig> expression in mouse  <cit> , showing that col10a <dig> is downstream of sox <dig> control.
deciphering the molecular mechanism by which sox <dig> expression in developing osteoblasts can vary among vertebrate clades will shed light on the evolution of cell type-specific molecular fingerprints.
if the primitive condition for osteoblasts in the common ancestor of actinopterygians and sarcopterygians included expression of sox <dig>  col2a <dig>  and col10a <dig>  then tetrapod osteoblasts would have lost expression of these genes secondarily, as outlined in hypothesis  <dig>  this possibility would give an entirely fresh phylogenetic context to reports of a transient chondrogenic phase during tetrapod dermal bone development  <cit> .
interestingly, sub-populations of chondrocytes in the zebrafish may lose sox <dig> and col2a <dig> expression as they transition to osteocytes in response to hh signaling  <cit> , so a developmental precedence may exist for the transitions in molecular fingerprints that hypothesis  <dig> proposes during evolution.
more broadly, we reveal fundamental differences between the molecular fingerprints of osteoblasts in actinopterygian and sarcopterygian clades, a finding consistent with the hypothesis that the primitive osteoblast-like cell was under reduced constraint  during early vertebrate phylogeny.
comparison of our data with published data for tetrapods further argues that, while the osteoblast has evolved differently between actinopterygian and sarcopterygian lineages, the molecular fingerprint of the chondrocyte appears to be conserved among vertebrates .
although sampling of vertebrate lineages in this manner is as yet too restricted to be confident of making generalizations, limited studies on chondrocytes of hyaline cartilage in amphibians and reptiles do support this conclusion  <cit> .
what mechanisms allow the osteoblast to vary among extant vertebrates, then, but constrain the chondrocyte?
we argue that cell types that appear earlier in phylogeny and ontogeny are less free to vary during subsequent evolution.
cartilage appeared in the fossil record in the primitive chordate haikouella  <dig> million years ago, and hyaline cartilage is a shared trait among chordates, hemichordates, and even some disparate protostome taxa  <cit> .
apart from two clades of diverged agnathans , all vertebrate lineages develop bone, which appeared in fossilized conodonts from  <dig> million years ago  <cit> .
therefore, cartilage appeared before bone during phylogeny.
in addition, cartilage appears before bone during ontogeny.
taken together, we suggest that because the chondrocyte appears before the osteoblast during both phylogeny and ontogeny, the molecular fingerprint of the chondrocyte is more constrained than that of the osteoblast.
as such, our interpretation is consistent with the notion of phyletic constraint  <cit>  and may provide a novel system by which to analyze molecular details of a developmental constraint.
CONCLUSIONS
while the molecular genetic basis for evolutionary changes to skeletal morphology has received much attention, similar studies on the evolution of skeletal cell types is limited.
the set of genes, or molecular fingerprint, expressed by a cartilage- or bone-forming cell  has been determined largely from human, mouse, and chick, thus providing an extremely limited sampling among vertebrate clades.
a couple of studies demonstrated that teleost osteoblasts express collagens that normally are expressed only in chondrocytes of tetrapods, allowing us to generate specific hypotheses on the evolution of the osteoblast among vertebrates .
here, we test the hypothesis that the molecular fingerprint of the osteoblast underwent neofunctionalization in the teleost lineage specifically, perhaps as a result of the teleost-specific genome duplication .
we compare expression of collagen and transcription factor genes during embryonic development of cartilage and bone in the teleost zebrafish danio rerio and the spotted gar lepisosteus oculatus, which diverged in the actinopterygian lineage prior to tgd.
we find equivalent expression patterns of these genes in chondrocytes and osteoblasts of zebrafish and gar, thus refuting by parsimony the hypothesis.
in addition, we show expression of the "chondrocyte" transcription factor sox <dig> in developing osteoblasts of zebrafish and gar, providing a molecular explanation for the expression of "chondrocyte" genes in fish osteoblasts.
finally, we argue from comparing our results to those of tetrapods that the molecular fingerprint of the osteoblast was not fixed during early vertebrate evolution, which supports previous work on bone and dentine tissues in the fossil record  <cit> , whereas the molecular fingerprint of the hyaline chondrocyte is constrained among vertebrate clades.
authors' contributions
bfe, aa, and jhp contributed to the conception of this study.
bfe, aa, yly, and jhp designed the experiments.
bfe stained bone and cartilage of staged specimens.
aa cloned gar genes.
yly performed in situ hybridizations.
bfe generated all images and wrote the manuscript.
all authors read, revised, and approved the final manuscript.
supplementary material
additional file 1
figure s <dig>  transcription factor expression in pre-osteoblasts of gar.
a, trichrome-stained coronal section.
b, c, in situ hybridization on coronal sections.
trichrome staining of  <dig> dpf gar  shows mesenchymal cells lateral to the condensation of meckel's cartilage.
these pre-osteoblasts do not express sox <dig> , but express high levels of runx <dig> .
scale bars: a-c =  <dig> μm.
abbreviations: dpf = days post-fertilization; mk = meckel's; po = pre-osteoblasts; tri = trichrome.
click here for file
acknowledgements
we wish to thank allyse ferrara and quentin fontenot of nicholls state university for gar embryos, poh kheng loi, adam burch, charles kimmel, and ruth bremiller for help with frozen sectioning, in situ hybridization, and section histology.
we also recognize john dowd, amanda rapp, and the fish facility at the university of oregon for tremendous fish housing and husbandry.
this work was supported in part by grant numbers f <dig> de016778- <dig>  and p <dig> hd <dig> and r <dig> rr <dig>  from the national institutes of health.

 skeletal tissues provide invaluable traits to document vertebrate evolution and to reveal the mechanistic basis for evolutionary change. two main processes underlie skeletal development: histogenesis--histological differentiation of skeletal tissues--and morphogenesis--acquisition of skeletal element location, shape, and size. skeletal histogenesis involves overt differentiation of cells that secrete the extracellular matrix of cartilage and bone, and follows the mesenchymal condensation of skeletogenic cells. skeletal morphogenesis directs such differentiation events in space and time. while many studies propose a molecular genetic basis for evolutionary changes to skeletal morphogenesis, the evolution of skeletal histogenesis among vertebrates is fertile ground for additional molecular analyses. each cell type achieves and performs its function by employing a specific set of genes, recently termed its molecular fingerprint. are molecular fingerprints free to evolve in each vertebrate clade in response to selective pressures? or were molecular fingerprints fixed shortly after tissues first evolved and since then have remained rather constant? for example, skeletons of land animals evolved under greater gravitational stress than skeletons of aquatic animals. as a result, one might hypothesize that the molecular fingerprints of skeletal cells in tetrapods, for instance, would differ from those of teleosts. alternatively, molecular fingerprints of skeletal cells may have been fixed at the evolutionary origin of the skeleton, and other mechanisms may adapt animals to lineage-specific selective pressures. comparative studies can reveal the extent to which molecular fingerprints of skeletal cells evolved among vertebrate clades, although the few clades examined thus far are biased towards crown-groups of both ray-finned and lobe-finned fish. therefore, analyses of more basally-diverging lineages, such as gar or lungfish, will provide evolutionary perspective to these studies. comparisons among human, mouse, and chick skeletal tissues suggest that the molecular fingerprints of chondrocytes or osteoblasts do not vary greatly among tetrapods. tetrapods exhibit one type of bone tissue, but they have three types of cartilage: elastic cartilage, hyaline cartilage, and fibrocartilage. here, we focus on the predominant type of cartilage in vertebrates, hyaline cartilage, which serves as the template for bone during endochondral ossification. collagens are the most abundant proteinaceous skeletal matrix components, and tetrapod chondrocytes and osteoblasts typically express different fibrillar collagens. in tetrapods, collagen type alpha is expressed abundantly in bone and is absent from cartilage, while collagen type alpha typifies cartilage and is not expressed in tetrapod bone. hyaline cartilage chondrocytes undergo a maturation process during development, when they express collagen type alpha, but tetrapod bone does not express col10a. despite these fundamental differences in collagen expression of tetrapod osteoblasts and chondrocytes, their molecular fingerprints also overlap; for example, both cell types express collagen type alpha. skeletogenic transcription factors control molecular fingerprints of chondrocytes and osteoblasts; sox is required for chondrocyte differentiation, while runx is necessary for osteoblast differentiation. sox and runx dictate skeletal cell differentiation by binding to and promoting the transcription of genes that impart identity to skeletal tissues. for instance, sox directly regulates col2a expression, while runx activates col1a transcription. much of the overlap in molecular fingerprints of tetrapod chondrocytes and osteoblasts can be attributed to runx activity, which is required for chondrocyte maturation in addition to its role in osteoblast differentiation. perhaps such overlap is not surprising, considering that tetrapod chondrocytes and osteoblasts differentiate from a bipotential progenitor cell, the osteochondroprogenitor, during both embryonic and adult stem cell development. indicative of the delicate balance required for these transcription factors to direct discrete cell lineages, sox can repress runx activity. although hyaline cartilage chondrocytes show conserved molecular fingerprints among vertebrate clades, a few studies in fish suggest that the molecular fingerprint of osteoblasts varies among vertebrates. in contrast to tetrapod osteoblasts, zebrafish osteoblasts express col10a and various teleosts show evidence of col in their bone matrix. a lineage-specific genome duplication event, the teleost genome duplication, occurred at the base of the teleost radiation, and genome duplications have been thought to facilitate the origin of new gene capabilities. for example, sox does not have a direct effect on osteoblast differentiation in tetrapods, but a sox duplicate in teleosts has been reported to affect bone development. therefore, one of at least three evolutionary scenarios might explain differences in the molecular fingerprint of tetrapod and teleost osteoblasts. hypothesis proposes that a new function for these collagens in osteoblasts evolved in the teleost lineage, perhaps facilitated by the tgd. hypothesis like hypothesis proposes that collagen gene neofunctionalization occurred, but that this event happened in the ancestral actinopterygian osteoblast, and hence ruling out the hypothesis that the tgd facilitated this novel gene function. hypothesis proposes that the last common ancestor to both tetrapods and teleosts had osteoblast expression patterns found in today's teleosts, but that these patterns were lost secondarily in the tetrapod lineage. to distinguish among these possibilities, we compared molecular fingerprints of skeletogenic cells in the teleost danio rerio with the spotted gar lepisosteus oculatus, a member of a teleost sister group that diverged before the tgd. currently, the molecular fingerprints of chondrocytes and osteoblasts are completely unknown for any non-teleost actinopterygian. results demonstrated that gar and zebrafish share molecular fingerprints of both chondrocytes and osteoblasts. as an indication of skeletal cell molecular fingerprints, we used in situ hybridization on developing gar and zebrafish embryos. specifically, we analyzed expression of the structural collagen genes col1a col2a col10a and col11a in well-developed cartilage and bone, and also revealed expression of the transcription factor genes sox and runx during mesenchymal condensation. we found that, like osteoblasts in the teleost danio rerio, gar osteoblasts expressed col2a and col10a therefore, these data refute by parsimony the role of the tgd in the origin of lineage-specific skeletal molecular fingerprints and furthermore argue that the expression of "chondrocyte" genes in osteoblasts is a shared feature of actinopterygians. more experiments will be required to distinguish between hypotheses and in efforts to explain the actinopterygian expression patterns reported here, we found, surprisingly, that the "chondrogenic" transcription factor sox was expressed in developing gar and zebrafish osteoblasts. we discuss these findings in a phylogenetic context and suggest that the molecular fingerprint of the primitive vertebrate osteoblast was less fixed than previously expected from studies of tetrapods. methods fish all fish and embryos were maintained with iacuc approval, according to established protocols. wild-type zebrafish were of the ab strain; gar originated from animals collected in lafourche parish, louisiana. histology and confocal microscopy embryos and larvae were processed for alcian blue/alizarin red staining and sectioned for histology as described previously. for zebrafish, we used transgenic lines to help visualize the location and organization of specific populations of cells. tgzc fish produce chondrocyte fluorescence, tgb fish make fluorescent osteoblasts, and tgy fish have fluorescence broadly among neural crest cells of the head. animals were imaged live under a confocal microscope while stained with the vital dye alizarin red, as described previously. molecular cloning and section in situ hybridization whole-mount and section rna in situ hybridization were carried out as described. zebrafish probes used were runx2a, runx2b, sox9a, sox9b, col1a col2a1a, col10a and col11a.  early and late stages of ceratohyal and dentary development to elucidate molecular fingerprints of chondrocytes and osteoblasts in gar and zebrafish, we focused on two cranial skeletal elements: the ceratohyal, which in mammals forms the anterior horn of the hyoid bone and whose chondrocytes form directly from mesenchyme during endochondral ossification; and the dentary, which in mammals forms the mandible bone and whose osteoblasts differentiate directly from mesenchyme during intramembranous ossification. to define equivalent developmental stages of cranial skeletogenesis between gar and zebrafish larvae, we performed whole-mount staining with alcian blue and alizarin red on a variety of stages for both species. our histological analyses of ceratohyal development demonstrated a well-defined, alcian blue-stained cartilage rod in gar larvae by days post-fertilization and in zebrafish larvae by dpf. in the same region earlier in development, a faint alcian blue-stained condensation of mesenchyme was apparent in gar and zebrafish. dentary bone formation was evidenced by substantial alizarin red staining adjacent to the anterolateral aspects of meckel's cartilage at dpf for gar and at dpf for zebrafish. no alizarin red staining was found in the region of the dentary in gar at dpf or in zebrafish at dpf. these results established the location and timing of equivalent stages of cartilage and bone development in gar and zebrafish. chondrocyte molecular fingerprint to analyze cellular and molecular features of developing chondrocytes in gar and zebrafish, we examined histologically stained sections, imaged transgenic zebrafish by confocal microscopy, and studied gene expression patterns in tissue sections. at dpf, the gar ceratohyal contained hundreds of chondrocytes, none of which showed evidence of hypertrophy. by dpf, however, the gar ceratohyal showed clearly hypertrophic chondrocytes in the mid-diaphyseal region. at both dpf and dpf, aniline blue-stained bone matrix was apparent in an extremely thin layer of the perichondrium. the dpf zebrafish ceratohyal had dozens of chondrocytes, most of which already displayed evidence of hypertrophy. alizarin red staining of bone matrix in the perichondrium was also evident. therefore, well-developed ceratohyals of both the gar and zebrafish displayed equivalent cellular and histological features of chondrocyte development. we next sought to explore the molecular fingerprints of well-developed chondrocytes in gar and zebrafish. in both gar and zebrafish, col1a expression was absent or very low in chondrocytes of the well-formed ceratohyal, although it was expressed clearly in cells of the perichondrium of both species. ceratohyals in both gar and zebrafish had high levels of col2a transcripts, although more mature, mid-diaphyseal chondrocytes appeared to have down-regulated transcript levels, which is consistent with similar findings in tetrapods. in addition, col2a expression was detected in the perichondrium of the ceratohyal in both gar and zebrafish. expression of col10a was high in mature chondrocytes and in perichondral cells of both gar and zebrafish ceratohyals, whereas surrounding, less mature chondrocytes did not express col10a. transcripts for col11a were evident in both chondrocytes and perichondral cells of gar and zebrafish ceratohyals, although the levels of expression in mature chondrocytes appeared to be reduced relative to adjacent chondrocytes, which again is consistent with published reports in tetrapods. to help explain the collagen gene expression patterns observed in cells of the well-developed ceratohyal cartilage, we analyzed expression of genes encoding sox and runx two transcription factors known to regulate these collagen genes in tetrapods. because specification of cell types occurs prior to their overt differentiation and transcription factor expression at this timepoint predicts skeletal cell fates, we focused on the mesenchymal condensation phase of cartilage development. progenitor cells of the gar ceratohyal had undergone mesenchymal condensation by dpf, while chondrogenic condensation of the zebrafish ceratohyal had occurred by hpf. in gar, sox transcripts were abundant in chondrogenic cells, whereas levels of runx expression were only slightly above background. due to the tgd, zebrafish has two co-orthologs of both sox and runx. expression patterns of sox and runx genes in cells of the zebrafish ceratohyal condensation at hpf were similar to those seen in gar, although levels of runx gene expression were much higher. in total, these data demonstrate that gar and zebrafish share molecular fingerprints of developing chondrocytes. the detection of particular gene expression by in situ hybridization is indicated as follows:'+' = high detectable expression;'-' = undetectable expression;'+/-' = low detectable expression. gar and zebrafish data are presented here, and chick and mouse data come from references cited in figure 1 osteoblast molecular fingerprint to analyze the cellular and molecular features of developing osteoblasts in gar and zebrafish, we performed histological stains, confocal imaging of transgenic zebrafish, and in situ hybridization on tissue sections. the dentaries of dpf gar and dpf zebrafish showed abundant bone matrix adjacent to meckel's cartilage. in both gar and zebrafish, col1a expression was high in osteoblasts of the well-developed dentary. transcripts for col2a were apparent in both gar and zebrafish dentaries, although levels detected in the zebrafish were relatively lower. osteoblasts of both gar and zebrafish dentaries demonstrated abundant col10a expression, as well as high levels of col11a transcripts. to help understand the expression patterns of collagen genes observed in osteoblasts of the well-developed dentary bone, we again analyzed sox and runx expression during mesenchymal condensation. at dpf, osteogenic cells of the gar dentary had undergone mesenchymal condensation, and were beginning to secrete aniline blue-stained bone matrix. osteogenic cells of the zebrafish dentary condensation were visible at hpf. both sox and runx transcripts were apparent in osteogenic cells of the gar dentary. the expression of sox in osteogenic cells of the gar dentary was dynamic during development. while runx transcript levels were high in presumptive pre-osteoblasts lateral to meckel's cartilage at dpf, sox expression was not detected in these cells. these data argue that sox expression increased in osteoblasts as they began to differentiate. similar to the single sox gene in gar, both sox9a and sox9b were expressed in osteogenic cells of the developing zebrafish dentary at hpf. also overlapping with the single runx expression in the gar dentary, runx2a and runx2b transcripts were abundant in the zebrafish dentary at hpf. in summary, these data demonstrate that gar and zebrafish share molecular fingerprints of developing osteoblasts. the detection of particular gene expression by in situ hybridization is indicated as follows:'+' = high detectable expression;'-' = undetectable expression;'+/-' = low detectable expression. gar and zebrafish data are presented here, and chick and mouse data come from references cited in figure 1 discussion due to their preservation in the fossil record, cartilage and bone serve as invaluable traits in understanding vertebrate evolution. evolutionary inferences, however, often assume that the histogenesis of skeletal tissues themselves remains constant among vertebrate lineages. to be fair, cells that produce cartilage and bone may have been free to evolve since their appearance roughly million years ago. here, we ask explicitly: to what extent do vertebrate clades share expression of the sets of genes that characterize skeletogenic cell types? a molecular fingerprint that is shared among vertebrate clades would suggest evolutionary constraints on that skeletal cell type. for instance, cells in cranial and appendicular skeletal tissues have different embryologic origins, and so developmental constraints may limit the molecular fingerprint of a skeletal cell that appears in both regions. while future experiments can test this hypothesis more extensively, skeletogenic cells in different embryonic regions of a given individual have been shown to exhibit a conserved molecular fingerprint. another interesting potential embryonic constraint is the fact that osteoblasts have two evolutionary and developmental origins within vertebrates. during vertebrate phylogeny, bone originated in the dermis, and then later appeared in the perichondrium surrounding cartilage templates. while not a focus of this study, we did not find differences between molecular fingerprints of osteoblasts from the exoskeleton and endoskeleton. therefore, our results do not support the notion that the exoskeleton and endoskeleton have separate embryonic constraints on the molecular fingerprints of osteoblasts, but testing this hypothesis could be a fruitful avenue of future research. a molecular fingerprint that varies among clades suggests relaxed constraints on the evolution of that cell type. one might expect variation in molecular fingerprints of skeletogenic cells among various vertebrate lineages, especially given the different selective pressures to which each vertebrate clade has been exposed. for example, the skeletons of land animals withstand a stronger effective gravitational force than do the skeletons of water-borne animals. some aquatic lineages, including sharks and other "cartilaginous" fish, and some antarctic fish, have even lost the majority of their bony skeleton at some point during phylogeny. are signatures of the embryonic response to these varied selective pressures seen in the molecular fingerprints of skeletogenic cells across vertebrates? spurred by the reported and unexpected expression of col10a and col two markers of tetrapod chondrocytes, in osteoblasts of teleosts, we pursued the hypothesis that molecular fingerprints of skeletogenic cells vary among vertebrate clades. experiments revealed collagen and transcription factor gene expression in skeletal cells of hyaline cartilage and bone in the zebrafish--a teleost--and gar, which diverged in the actinopterygian lineage prior to the teleost-specific genome duplication. specifically, our data distinguish among competing hypotheses to explain why osteoblasts of teleosts express col10a and col which are not expressed in osteoblasts of tetrapods. osteoblast expression of these collagens either represents a neofunctionalization event that was specific either to the teleost lineage subsequent to the tgd or to the actinopterygian lineage, or they were expressed in osteoblasts of the common ancestor of tetrapods and teleosts and subsequently lost in tetrapods. admittedly, evaluation of molecular fingerprints based upon expression of a few genes is a limited approach, but our findings on gene expression in chondrocytes and osteoblasts of the gar and zebrafish suggest evolutionary trends that could be embellished by massively parallel transcriptomics. we demonstrate that gar and zebrafish share molecular fingerprints of both chondrocytes and osteoblasts. despite evidence that genome duplication can facilitate the origin of new gene functions, our data reject the proposed teleost neofunctionalization hypothesis for osteoblast evolution. because both gar and zebrafish express col2a and col10a in their osteoblasts, the most parsimonious explanation is that these markers were present in the molecular fingerprint of the ancestral actinopterygian osteoblast. therefore, parsimony favors hypothesis although our results do not reject hypothesis and more experiments are required to distinguish between these two possibilities. the notion that osteoblasts achieved collagen neofunctionalization somewhere in the actinopterygian lineage could be tested further by revealing the molecular fingerprint of osteoblasts in bichir, an actinopterygian diverging more basally than the gar lineage. similar studies of the lungfish or coelocanth, basally-diverging sarcopterygians, would test the possibility that col2a and col10a expression was present in osteoblasts of the ancestral bony fish and subsequently was lost somewhere in the sarcopterygian lineage leading to tetrapods. our studies of skeletogenic transcription factors suggest a functional framework to explain why col2a and col10a are expressed in osteoblasts of actinopterygians, but not in osteoblasts of sarcopterygians. in addition to runx expression, gar and zebrafish osteoblasts express sox during mesenchymal condensation of dermal bones. developing osteoblasts of tetrapods typically express runx but not sox during mesenchymal condensation of dermal bones. we propose that the expression of sox in gar and zebrafish osteoblasts may explain the presence of col2a and col10a transcripts, given two assumptions. first, actinopterygian osteoblasts would have to translate the sox transcript we observed into sox protein. second, sox9-responsive cis-acting regulatory elements that drive col2a expression in tetrapods would have to operate similarly in actinopterygian lineages. in support of this latter notion, col2a gene expression is extinguished in sox mutant zebrafish. currently, sox has not been shown to bind to the col10a promoter, but mis-expression of sox in developing avian osteoblasts causes ectopic col10a expression, and loss of sox can abrogate col10a expression in mouse, showing that col10a is downstream of sox control. deciphering the molecular mechanism by which sox expression in developing osteoblasts can vary among vertebrate clades will shed light on the evolution of cell type-specific molecular fingerprints. if the primitive condition for osteoblasts in the common ancestor of actinopterygians and sarcopterygians included expression of sox col2a and col10a then tetrapod osteoblasts would have lost expression of these genes secondarily, as outlined in hypothesis this possibility would give an entirely fresh phylogenetic context to reports of a transient chondrogenic phase during tetrapod dermal bone development. interestingly, sub-populations of chondrocytes in the zebrafish may lose sox and col2a expression as they transition to osteocytes in response to hh signaling, so a developmental precedence may exist for the transitions in molecular fingerprints that hypothesis proposes during evolution. more broadly, we reveal fundamental differences between the molecular fingerprints of osteoblasts in actinopterygian and sarcopterygian clades, a finding consistent with the hypothesis that the primitive osteoblast-like cell was under reduced constraint during early vertebrate phylogeny. comparison of our data with published data for tetrapods further argues that, while the osteoblast has evolved differently between actinopterygian and sarcopterygian lineages, the molecular fingerprint of the chondrocyte appears to be conserved among vertebrates. although sampling of vertebrate lineages in this manner is as yet too restricted to be confident of making generalizations, limited studies on chondrocytes of hyaline cartilage in amphibians and reptiles do support this conclusion. what mechanisms allow the osteoblast to vary among extant vertebrates, then, but constrain the chondrocyte? we argue that cell types that appear earlier in phylogeny and ontogeny are less free to vary during subsequent evolution. cartilage appeared in the fossil record in the primitive chordate haikouella million years ago, and hyaline cartilage is a shared trait among chordates, hemichordates, and even some disparate protostome taxa. apart from two clades of diverged agnathans, all vertebrate lineages develop bone, which appeared in fossilized conodonts from million years ago. therefore, cartilage appeared before bone during phylogeny. in addition, cartilage appears before bone during ontogeny. taken together, we suggest that because the chondrocyte appears before the osteoblast during both phylogeny and ontogeny, the molecular fingerprint of the chondrocyte is more constrained than that of the osteoblast. as such, our interpretation is consistent with the notion of phyletic constraint and may provide a novel system by which to analyze molecular details of a developmental constraint.  while the molecular genetic basis for evolutionary changes to skeletal morphology has received much attention, similar studies on the evolution of skeletal cell types is limited. the set of genes, or molecular fingerprint, expressed by a cartilage- or bone-forming cell has been determined largely from human, mouse, and chick, thus providing an extremely limited sampling among vertebrate clades. a couple of studies demonstrated that teleost osteoblasts express collagens that normally are expressed only in chondrocytes of tetrapods, allowing us to generate specific hypotheses on the evolution of the osteoblast among vertebrates. here, we test the hypothesis that the molecular fingerprint of the osteoblast underwent neofunctionalization in the teleost lineage specifically, perhaps as a result of the teleost-specific genome duplication. we compare expression of collagen and transcription factor genes during embryonic development of cartilage and bone in the teleost zebrafish danio rerio and the spotted gar lepisosteus oculatus, which diverged in the actinopterygian lineage prior to tgd. we find equivalent expression patterns of these genes in chondrocytes and osteoblasts of zebrafish and gar, thus refuting by parsimony the hypothesis. in addition, we show expression of the "chondrocyte" transcription factor sox in developing osteoblasts of zebrafish and gar, providing a molecular explanation for the expression of "chondrocyte" genes in fish osteoblasts. finally, we argue from comparing our results to those of tetrapods that the molecular fingerprint of the osteoblast was not fixed during early vertebrate evolution, which supports previous work on bone and dentine tissues in the fossil record, whereas the molecular fingerprint of the hyaline chondrocyte is constrained among vertebrate clades. authors' contributions bfe, aa, and jhp contributed to the conception of this study. bfe, aa, yly, and jhp designed the experiments. bfe stained bone and cartilage of staged specimens. aa cloned gar genes. yly performed in situ hybridizations. bfe generated all images and wrote the manuscript. all authors read, revised, and approved the final manuscript. supplementary material additional file 1 figure s transcription factor expression in pre-osteoblasts of gar. a, trichrome-stained coronal section. b, c, in situ hybridization on coronal sections. trichrome staining of dpf gar shows mesenchymal cells lateral to the condensation of meckel's cartilage. these pre-osteoblasts do not express sox, but express high levels of runx. scale bars: a-c = μm. abbreviations: dpf = days post-fertilization; mk = meckel's; po = pre-osteoblasts; tri = trichrome. click here for file acknowledgements we wish to thank allyse ferrara and quentin fontenot of nicholls state university for gar embryos, poh kheng loi, adam burch, charles kimmel, and ruth bremiller for help with frozen sectioning, in situ hybridization, and section histology. we also recognize john dowd, amanda rapp, and the fish facility at the university of oregon for tremendous fish housing and husbandry. this work was supported in part by grant numbers f de016778- and p hd and r rr from the national institutes of health.
they either arose as neofunctionalization events after the teleost genome duplication, or they represent preserved ancestral functions that pre-date the tgd. here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. zebrafish and gar chondrocytes both expressed col10a as they matured, but both species' osteoblasts also expressed col10a which tetrapod osteoblasts do not express. results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. more broadly, our data suggest that the molecular fingerprint of the osteoblast, which largely is constrained among land animals, was not fixed during early vertebrate evolution. although the vertebrate skeleton arose in the sea million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. we compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish --a teleost--and the spotted gar --a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the tgd. this novel finding, along with sox and col2a expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. we analyzed developing embryos for expression of the structural collagen genes col1a col2a col10a and col11a in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox and runx during mesenchymal condensation. in fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions.
 although the vertebrate skeleton arose in the sea million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. in fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. they either arose as neofunctionalization events after the teleost genome duplication, or they represent preserved ancestral functions that pre-date the tgd. here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. we compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish --a teleost--and the spotted gar --a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the tgd. we analyzed developing embryos for expression of the structural collagen genes col1a col2a col10a and col11a in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox and runx during mesenchymal condensation.  results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. zebrafish and gar chondrocytes both expressed col10a as they matured, but both species' osteoblasts also expressed col10a which tetrapod osteoblasts do not express. this novel finding, along with sox and col2a expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint.  our surprising finding that the "chondrogenic" transcription factor sox is expressed in developing osteoblasts of both zebrafish and gar can help explain the expression of chondrocyte genes in osteoblasts of ray-finned fish. more broadly, our data suggest that the molecular fingerprint of the osteoblast, which largely is constrained among land animals, was not fixed during early vertebrate evolution. 