BACKGROUND
bone development can occur in two distinct ways: 1) through endochondral ossification where the mesenchymal cells differentiate into chondrocytes and lay down a cartilaginous template that is later replaced by bone; or 2) through intramembranous ossification where mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate.
during endochondral ossification, the transcription factors sox <dig>  sox <dig> and/or sox <dig> are expressed and are involved in the induction of chondrocytes  <cit> .
chondrocytes in the growth plate are subsequently induced to exit the cell cycle and commit to terminal differentiation.
the prehypertrophic chondrocytes mature into hypertrophic chondrocytes, which lay down a matrix rich in collagen x, and secrete vegf  <cit> .
vegf promotes the invasion of blood vessels from the perichondrium, bringing in both the bone forming osteoblasts and the bone resorbing osteoclasts.
the hypertrophic chondrocytes then undergo apoptosis, and are replaced by trabecular bone and bone marrow.
in contrast, the flat bones of the skull, the frontal and parietal bones, form by intramembranous ossification.
cranial mesenchymal cells directly differentiate into osteoblasts that initiate mineralization and secrete an extracellular matrix rich in collagen i  <cit> .
growth of these calvarial bones occurs through proliferation and differentiation of osteoblasts at the margins or sutures.
the molecular pathways that dictate these alternative ossification programs are not yet well defined.
in particular, it is not known whether intramembranous ossification is prespecified by the ontogeny of the cranial mesenchyme or is a response to local environmental signals.
fibroblast growth factors  appear to be important for both types of ossification  <cit> .
fgfs comprise a large family of proteins that includes at least  <dig> known members  <cit> .
fgfs bind and signal through low and high affinity fgf receptors  <cit> .
the four known high affinity receptors  are structurally similar transmembrane receptor tyrosine kinases.
during intramembranous ossification of the flat bones, fgfr1– <dig> are expressed by the differentiating osteoblasts at the osteogenic fronts and also by the adjacent cartilage  <cit> .
fgfr <dig> is expressed in cells close to and within the osteoid; fgfr <dig> is expressed in the proliferating osteogenic stem cells; fgfr <dig> expression is seen in the thin layer of cartilage underlying the lower part of the coronal suture  <cit> .
as fgfr <dig> null mice do not show defects in calvarial development, it has been hypothesized that intramembranous bone formation is controlled primarily by fgfr <dig> and fgfr <dig>  <cit> .
mutations in fgfr <dig>  fgfr <dig> and fgfr <dig> in humans that affect skeletal growth are consistent with this hypothesis  <cit> .
craniosynostosis is mainly associated with mutations in fgfr <dig> and fgfr <dig>  <cit> .
mutations that affect the growth of long bones resulting in syndromes such as achondroplasia  and thanatophoric dysplasia  are mainly localized to fgfr <dig>  these autosomal dominant disorders are believed to reflect either an enhancement of receptor activity or a neomorphic gain-of-function effect  <cit> .
during long bone development, fgf receptors are expressed in the epiphyseal growth plates: fgfr <dig> is expressed in the proliferating chondrocytes; fgfr <dig> is expressed in the hypertrophic chondrocytes; fgfr <dig> and fgfr <dig> are expressed in the perichondrium  <cit> .
fgfr <dig> is expressed in early mesenchymal condensates and in the periosteal collar around the cartilage models  <cit> .
targeted deletion of fgfr2iiic suggests that fgfr2iiic is a positive regulator of ossification in both the osteoblasts and chondrocytic lineages  <cit> .
targeted deletion of fgfr <dig> results in mice that show overgrowth of the long bones and abnormal proliferation of chondrocytes suggesting that fgfr <dig> stimulation inhibits chondrocyte proliferation  <cit> .
these studies describe roles for fgf receptor-mediated signaling during differentiation/maturation of chondrocytes.
the roles of fgf ligands in skeletal development are unclear.
during calvarial development, fgf <dig> is expressed in the osteoblasts; fgf <dig> and fgf <dig> are expressed in the sutural mesenchyme; fgf <dig> is initially expressed in the cranial mesenchymal cells and later, in the differentiating osteoblasts  <cit> .
fgf <dig> is expressed in the sutural mesenchyme and is upregulated in the endocranial portions of the mesenchyme and is downregulated during postnatal development  <cit>  <cit> .
the specific in vivo functions of these different fgfs remain undefined.
in vitro, cephalic neural crest cells from quail embryos have been shown to respond to exogenous fgf- <dig> in a dose dependant manner; lower doses induce proliferation and higher doses induce cartilage differentiation  <cit> .
during long bone development, at the time of initiation of endochondral differentiation, fgf <dig> is expressed in the condensing mesenchyme  <cit> .
fgf <dig>  fgf <dig>  fgf <dig> and fgf <dig> are expressed in loose mesenchyme outside the condensation  <cit> .
however, mice lacking these fgfs show no apparent defects in skeletal development  <cit> .
functional redundancy between these fgfs may, in part, account for the lack of phenotype.
therefore, roles for these fgfs in the early stages of chondrogenesis have not yet been defined.
in this study, we have analyzed transgenic mice that express fgf <dig> in their cranial mesenchymal cells.
these mice show abnormal head development and are born with a pronounced bulge in their skulls.
skeletal preparations of these mice revealed dramatic changes in parietal bone formation.
in the region where the parietal bones normally form, cranial mesenchymal cells are induced to differentiate into chondrocytes that proliferate, hypertrophy and subsequently differentiate as bone.
correlative changes in expression of marker genes sox <dig>  col2a <dig>  col10a <dig>  ihh, cbfai and osteocalcin occur in conjunction with the altered differentiation program.
thus the parietal bones in these mice form by endochondral ossification rather than by the usual intramembranous ossification route.
fate mapping studies indicate that the ectopic cartilagenous precursors in these mice are derived from the mesoderm.
based on these results we suggest that cranial mesenchymal cells are competent to initiate endochondral ossification, and can be switched to this alternative developmental program by ectopic expression, or overexpression, of fgf <dig>
RESULTS
changes in cranial morphology in the fgf <dig> transgenic mice
transgenic mice were generated by microinjection of a construct with the αa-crystallin promoter linked to a mouse fgf <dig> cdna   <cit> .
eleven founders carrying the transgene were identified.
five of them had cataracts .
in the transgenic line ove <dig>  in addition to lens defects, there were defects in the development of the skull .
mice in this family that are heterozygous for the transgene are born with 'dome-shaped' heads .
this phenotype is even more pronounced in homozygous transgenic mice .
calvarial development in fgf <dig> transgenic mice
the skeletal defects were examined by alcian blue and alizarin red staining of e <dig>  p <dig>  p <dig> and p <dig> mice .
the skeletal preparations show a dramatic expansion of cartilage in the head region by e <dig> .
parietal bone development was particularly affected .
at p <dig>  the phenotypic differences between the nontransgenic and fgf <dig> transgenic mice were still pronounced.
at this age, the calvarial skull in nontransgenic mice is mostly ossified except at the sutures .
in contrast, there is an expanded cartilage territory  in the fgf <dig> transgenic mice .
the transgenic mice also show a central hole in the skull where there is neither cartilage nor bone .
development of the occipital bones appeared normal .
at p <dig>  the alcian blue stained tissues in the transgenic cranium were interspersed with alizarin red stained regions suggesting that the cartilage territory was being replaced by bony structures .
by  <dig> month of age, the cartilage territory was completely replaced by bone .
the alterations in skeletal development in the fgf <dig> transgenic mice were examined at the histological level by analyses of hematoxylin and eosin stained sections of the cranium.
no distinctive differences in morphology were seen at e <dig> .
by e <dig>  more cranial mesenchymal cells were seen in the skull region of the fgf <dig> transgenic mice  and these cells were organized differently from nontransgenic mice .
by e <dig>  the cranial mesenchymal cells in the nontransgenic mice had initiated intramembranous ossification .
in contrast, the mesenchymal cells in the fgf <dig> transgenic skulls differentiated into cells resembling chondrocytes .
within the next few days these cells increased in size and became hypertrophic .
by p <dig>  perichondrial cells  had begun to invade the cartilage territory .
by p <dig>  the cartilage territory was transformed into trabecular bone and bone marrow .
these results considered together suggest that the cranial mesenchymal cells in the parietal region in these mice recapitulate the sequence of events that occurs during endochondral ossification.
ectopic expression of the fgf <dig> transgene
to test if the alterations seen in the skulls of the ove <dig> mice were due to ectopic expression of the transgene, in situ hybridizations were performed using an s35-labelled riboprobe that recognizes the sv <dig> portion of the transgene .
transgene expression was seen in the lens  <cit> , the dorsal portion of the retinal pigmented epithelium  <cit>  and also in the cranial mesenchymal cells beginning at e <dig> .
expression levels peak by e <dig> and start to decrease by e <dig> .
transgene expression is extinguished by e <dig> .
section and whole mount in situ hybridizations show that the extraocular transgene expression is restricted to the mesenchymal cells overlying the future mid and hind brain regions .
these results argue that the defects in skeletal development in the ove <dig> line are attributable to ectopic and transient expression of the fgf <dig> transgene.
expression of chondrocyte specific markers
the different stages of endochondral ossification are characterized by expression of different marker genes.
expression of sox <dig>  a member of the sox family of transcription factors, has been shown to be essential for chondrocyte condensation  <cit> .
in particular, sox <dig> is required for the expression of cartilage-specific extracellular matrix components such as collagen ii   <cit> .
in the long bone growth plates, col2a <dig> is expressed in the resting and proliferating chondrocytes, ihh in prehypertropic chondrocytes and col10a <dig> in hypertropic chondrocytes  <cit> .
expression of the runt domain transcription factor cbfai has been shown to be essential for differentiation of the cells in the osteoblastic lineage  <cit> .
osteocalcin, one of the downstream targets of cbfai, is expressed exclusively in the osteoblasts  <cit> .
expression of these six marker genes was examined by in situ hybridization using s35-labelled riboprobes .
no appreciable differences in expression of sox <dig> between nontransgenic and fgf <dig> transgenic mice were seen at e <dig> .
however, significant induction of sox <dig> expression was seen in the cranial mesenchymal cells of the transgenic mice by e <dig> .
sox <dig> expression was seen to persist in the developing chondrocytes .
cbfai and osteocalcin are expressed during the normal program of intramembranous ossification in nontransgenic mice, but their expression was not seen in the corresponding regions of the embryonic transgenic heads .
col2a <dig> expression was seen in the developing chondrocytes in the transgenic cranium but not the wildtype, at e <dig>  e <dig> and p <dig> .
by p <dig> expression levels were found to be reduced in transgenic hypertropic chondrocytes .
in situ hybridizations at p <dig> showed that col10a <dig> and ihh are expressed in the hypertropic chondrocytes .
expression of col10a <dig> was not seen in the chondrocytes at p <dig> or before .
these results demonstrate that the transgenic cranial mesenchymal cells undergo endochondral ossification instead of intramembranous ossification.
expression of fgf <dig>  fgfr <dig> and fgfr3
fgf <dig> has been reported to be expressed in cranial mesenchymal cells as well as in the dural mesoderm during embryonic development  <cit> .
expression levels of the fgf <dig> transgene and endogenous fgf <dig> were compared by in situ hybridizations using an -labelled fgf <dig> riboprobe .
at e <dig> and e <dig>  fgf <dig> was not expressed at detectable levels in the cranial mesenchymal cells in nontransgenic mice.
in contrast, fgf <dig> expression was clearly present in the transgenic mice .
as fgf <dig> binds to and signals through fgfr <dig> and fgfr <dig>  in situ hybridizations were performed to examine their expression levels .
low levels of fgfr <dig> expression can be seen in cranial mesenchymal cells at e <dig> in both the nontransgenic and transgenic mice .
fgfr <dig> expression is maintained in the induced chondrocytes in the transgenic mice .
in contrast, fgfr <dig> expression was not detected at e <dig> either in transgenic or in nontransgenic cranial mesenchyme .
fgfr <dig> was expressed at low levels in the differentiating chondrocytes of the fgf <dig> transgenic mice .
in addition, crossing the ove <dig> mice with fgfr <dig> null mice  <cit>  does not alter the "dome-head" phenotype .
these results taken together suggest that the developmental switch in the transgenic mice may initially be mediated by ectopic activation of fgfr <dig>
fgf <dig> induces proliferation of the cranial mesenchymal cells
to test if the cranial mesenchymal cells proliferate in response to fgf <dig> expression, sections of e <dig> and e <dig> skulls were assayed for brdu incorporation .
at e <dig> and at  <dig>  the cranial mesenchymal cells in the parietal region of fgf <dig> transgenic mice show a significant increase in brdu incorporation .
these results suggest that fgf <dig> expression in the cranial mesenchymal cells results in increased proliferation.
fate mapping of cells in the parietal region in the fgf <dig> transgenic mice
cell lineage tracing studies performed in mice and in chicks show that the cranial skeletogenic mesenchyme is derived from two sources, neural crest and mesoderm  <cit> .
to determine if the cells in the parietal region that form ectopic cartilage in the ove <dig> mice are neural crest or mesodermal derivatives, we performed fate mapping experiments using wnt1-cre and r26r mice  <cit> .
as has been described previously, wnt1-cre/r26r compound heterozygotes stably express β-galactosidase in neural crest cells and their descendants but not in the mesodermal derivatives  <cit> .
ove <dig> mice were crossed with wnt1-cre/r26r mice and heads of e <dig> embryos were stained with x-gal .
in the absence of wnt1-cre, lacz positive cells were not detected in the r26r embryo  or in the fgf9/r26r embryo .
in the wnt1-cre/r26r embryo , the parietal region  of the skull showed no evidence for lacz activity, implying that neural crest derivatives do not contribute to this region of the skull.
in the fgf9/wnt1-cre/r26r embryo , there is also no blue staining in the  parietal region.
these results establish that the cells in the parietal region that form ectopic cartilage in the ove <dig> mice are not neural crest derived but are for the most part, derived from mesoderm.
in summary, in family ove <dig>  ectopic fgf <dig> expression switches the differentiation and proliferation properties of cranial mesodermal cells, resulting in inhibition of intramembranous ossification and conversion to a program of endochondral ossification.
discussion
in this study, we have analyzed a line of transgenic mice where the differentiation program of a portion of the cranial mesenchyme is converted from intramembranous to endochondral ossification.
the transgenic mice ectopically express fgf <dig> in their cranial mesenchymal cells.
expression of fgf <dig> correlates with localized proliferation of the mesenchymal cells and with upregulation of sox <dig> expression and downregulation of cbfai and osteocalcin.
the mesenchymal cells consequently differentiate into chondrocytes that express col2a <dig>  these chondrocytes proliferate, then hypertrophy, express col10a1and are later replaced by trabecular bone and bone marrow.
therefore, parietal bone formation in these mice recapitulates the sequence of events that occurs during endochondral ossification.
these results demonstrate that fgf <dig> expression is sufficient to convert the differentiation program of  the cranial mesenchymal cells from intramembranous to endochondral ossification.
ectopic expression of the transgene
the skeletal defects in this transgenic line  occur in mice that are either heterozygous or homozygous for the fgf <dig> transgene.
it is, therefore, unlikely that the skull defects in this line of mice are due to disruption of a gene essential for membranous ossification of the parietal bones.
the transgene expression pattern provides compelling evidence that the skull defects in these mice are due to ectopic expression of the transgene.
in situ hybridization analyses show that the transgene is expressed in an appropriate spatial and temporal pattern to induce the developmental changes.
in addition, the transgene is not expressed in regions where membranous ossification still occurs .
furthermore, transgene expression precedes sox <dig> expression and condensation of the mesenchymal cells.
also, our results are consistent with the results of other studies.
for example, recent studies of a mouse model for apert syndrome, with a single amino acid change in fgfr <dig>  support the notion that enhanced activation of fgfr <dig> can cause some cranial mesenchyme to convert to a chondrocyte differentiation program  <cit> .
in addition, fgf <dig> expression in a chondrocytic cell line is sufficient to induce expression of sox <dig>  <cit> .
addition of fgf- <dig> in vitro to cranial mesenchymal cells from quail embryos can induce cartilage differentiation at high doses  <cit> .
taken together, these results suggest that initiation of chondrogenesis in the cranial mesenchymal cells in this line of mice is due to ectopic expression of the fgf <dig> transgene.
in the ove <dig> family, expression of the fgf <dig> transgene is not only ectopic but also transient.
expression in the cranial mesenchyme was not detectable after e <dig>  this implies that stimulation by fgf <dig> is sufficient to induce  cranial mesoderm to switch to a chondrocytic differentiation program, but sustained expression of fgf <dig> is not required for the later stages of differentiation.
therefore, the endochondral ossification program, once initiated by fgf <dig>  appears to become autonomous.
the reason for the ectopic fgf <dig> expression is not clear at present.
it is possible the transgene array has integrated in the neighborhood of an endogenous enhancer that directs expression to the cranial mesenchymal cells.
some of the integrated copies of the transgene still retain the αa-crystallin promoter since the transgene is still expressed in the lens  <cit> .
the transgene is also expressed in the dorsal margins of the retinal pigmented epithelium  and this expression transforms the rpe into neuroretina  <cit> .
nature of fgf <dig> induction
expression of fgf <dig> in growth plate chondrocytes using the col2a <dig> promoter results in reduced proliferation and terminal differentiation of chondrocytes  <cit> .
in addition, targeted deletion of fgfr <dig>  one of the receptors through which fgf <dig> signals, results in mice with overgrowth of the long bones.
these results suggest that a primary role of fgf signaling in the long bones is to act as a negative regulator of chondrocyte proliferation.
these results are in contrast to our results that show that ectopic fgf <dig> expression in embryonic cranial mesenchymal cells induces overproliferation followed by endochondral ossification.
how can these disparate observations be reconciled?
one possible explanation is the differential timing of fgf <dig> expression.
the col2a <dig> promoter is active in the differentiated chondrocytes, while our transgenic fgf <dig> is expressed in undifferentiated cranial mesenchymal cells.
second, there are differences in the responding tissues.
chondrocytes in the long bones originate from the lateral plate mesoderm while the parietal mesenchyme is derived from cranial mesoderm.
since the stimulated cells have different developmental origins, there is no reason to expect that the responses will be identical.
for example, although lens and corneal epithelial cells are related to each other, both morphologically and developmentally, they respond differently to fgf stimulation  <cit> .
the reduction in proliferation in the long bones in response to fgf stimulation may be a unique property of the growth plate chondrocytes  <cit> .
in contrast, the fgf9-induced proliferation of cranial mesenchymal cells is consistent with the notion that fgfs, in general, act as mitogens during normal development.
endogenous role for fgf9?
though fgf <dig> appears to be sufficient to induce chondrogenesis in the skull, the role of fgf <dig> during normal development of cartilage in the cranium is unclear.
our in situ hybridizations did not show any detectable expression of fgf <dig> in the calvarial mesenchyme at e <dig> or e <dig>  expression is seen later during embryonic development in the endocranial portions of the mesenchyme and is downregulated during postnatal development  <cit> .
this expression pattern suggests that fgf <dig> is not important for initial specification of the cells in the chondrocytic lineage in the cranium.
consistent with this model, targeted deletion of fgf <dig> does not result in any visible skeletal abnormalities in the skull  <cit> .
in contrast, in the transgenic mice, the ectopic fgf <dig> plays an instructive role and initiates chondrogenesis.
fgf <dig> is known to signal through fgfr <dig> and fgfr <dig>  <cit> .
signaling by fgf <dig> in the transgenic heads is likely to be mediated initially through fgfr <dig> rather than fgfr <dig> as the parietal fate switch in the fgf <dig> transgenic mice is not rescued by the loss of fgfr <dig>  after sox <dig> expression has been induced and the chondrocytic program has been initiated , expression of the fgf <dig> transgene is no longer required.
in contrast to the normal chondrocytic differentiation program in long bones, ectopic chondrogenesis in the cranium proceeds at a much slower rate.
though the reasons for this are presently unclear, we speculate that this may be due to the multi-step nature of the endochondral differentiation program.
respecification and reprogramming of the parietal environment is required, and this is accomplished in a less timely fashion than at the normal sites of differentiation.
fgfrs and intramembranous ossification
our findings are in apparent conflict with some of the current models for the roles of fgf receptors in intramembranous ossification.
autosomal dominant mutations in fgfr <dig> or fgfr <dig> lead to premature differentiation and fusion of the skull sutures in humans and these effects are thought to be the consequence of enhanced receptor activity  <cit> .
based upon such a model, our transgenic mice would be predicted to exhibit an analogous phenotype, i.e.
craniosynostosis.
inappropriate activation of the receptors in the cranial mesenchyme would be predicted to lead to premature differentiation.
however, our studies indicate that ligand-mediated activation of fgfr <dig> induces proliferation of embryonic cranial mesenchymal cells and subsequent differentiation into cartilage.
by extrapolation then, our results imply that mutations in the fgfr <dig> gene in human patients do not result in ligand-independent constitutive activation of the receptor.
this prediction is supported by the finding that targeted inactivation of fgfr <dig> in mice causes developmental alterations in cell types that are not affected by the putative gain-of-function mutations.
an alternative explanation is that transient stimulation of fgfr <dig>  may lead to a qualitatively different cellular response compared to sustained ligand-independent stimulation of fgfr <dig> .
in either case, our results show that a single fgf can function as an instructive signal, inducing a specific cell type to switch from one differentiation program to an autonomous, alternative differentiation pathway.
CONCLUSIONS
although the mechanistic details of fgf <dig> stimulation of the cranial mesenchymal cells remain to be elucidated, the ove <dig> transgenic mice provide the first demonstration in vivo that an fgf can switch the differentiation program of immature cranial mesoderm.
these results also demonstrate that mesoderm-derived cranial mesenchymal cells are developmentally flexible and can undergo either intramembranous or endochondral ossification in response to extracellular signals.
these transgenic mice provide a model system in which to elucidate the molecular connection between stimulation of an fgfr  and induction of expression of a cell-fate-determining transcription factor .

 bone development can occur in two distinct ways: 1) through endochondral ossification where the mesenchymal cells differentiate into chondrocytes and lay down a cartilaginous template that is later replaced by bone; or 2) through intramembranous ossification where mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. during endochondral ossification, the transcription factors sox sox and/or sox are expressed and are involved in the induction of chondrocytes. chondrocytes in the growth plate are subsequently induced to exit the cell cycle and commit to terminal differentiation. the prehypertrophic chondrocytes mature into hypertrophic chondrocytes, which lay down a matrix rich in collagen x, and secrete vegf. vegf promotes the invasion of blood vessels from the perichondrium, bringing in both the bone forming osteoblasts and the bone resorbing osteoclasts. the hypertrophic chondrocytes then undergo apoptosis, and are replaced by trabecular bone and bone marrow. in contrast, the flat bones of the skull, the frontal and parietal bones, form by intramembranous ossification. cranial mesenchymal cells directly differentiate into osteoblasts that initiate mineralization and secrete an extracellular matrix rich in collagen i. growth of these calvarial bones occurs through proliferation and differentiation of osteoblasts at the margins or sutures. the molecular pathways that dictate these alternative ossification programs are not yet well defined. in particular, it is not known whether intramembranous ossification is prespecified by the ontogeny of the cranial mesenchyme or is a response to local environmental signals. fibroblast growth factors appear to be important for both types of ossification. fgfs comprise a large family of proteins that includes at least known members. fgfs bind and signal through low and high affinity fgf receptors. the four known high affinity receptors are structurally similar transmembrane receptor tyrosine kinases. during intramembranous ossification of the flat bones, fgfr1– are expressed by the differentiating osteoblasts at the osteogenic fronts and also by the adjacent cartilage. fgfr is expressed in cells close to and within the osteoid; fgfr is expressed in the proliferating osteogenic stem cells; fgfr expression is seen in the thin layer of cartilage underlying the lower part of the coronal suture. as fgfr null mice do not show defects in calvarial development, it has been hypothesized that intramembranous bone formation is controlled primarily by fgfr and fgfr. mutations in fgfr fgfr and fgfr in humans that affect skeletal growth are consistent with this hypothesis. craniosynostosis is mainly associated with mutations in fgfr and fgfr. mutations that affect the growth of long bones resulting in syndromes such as achondroplasia and thanatophoric dysplasia are mainly localized to fgfr these autosomal dominant disorders are believed to reflect either an enhancement of receptor activity or a neomorphic gain-of-function effect. during long bone development, fgf receptors are expressed in the epiphyseal growth plates: fgfr is expressed in the proliferating chondrocytes; fgfr is expressed in the hypertrophic chondrocytes; fgfr and fgfr are expressed in the perichondrium. fgfr is expressed in early mesenchymal condensates and in the periosteal collar around the cartilage models. targeted deletion of fgfr2iiic suggests that fgfr2iiic is a positive regulator of ossification in both the osteoblasts and chondrocytic lineages. targeted deletion of fgfr results in mice that show overgrowth of the long bones and abnormal proliferation of chondrocytes suggesting that fgfr stimulation inhibits chondrocyte proliferation. these studies describe roles for fgf receptor-mediated signaling during differentiation/maturation of chondrocytes. the roles of fgf ligands in skeletal development are unclear. during calvarial development, fgf is expressed in the osteoblasts; fgf and fgf are expressed in the sutural mesenchyme; fgf is initially expressed in the cranial mesenchymal cells and later, in the differentiating osteoblasts. fgf is expressed in the sutural mesenchyme and is upregulated in the endocranial portions of the mesenchyme and is downregulated during postnatal development. the specific in vivo functions of these different fgfs remain undefined. in vitro, cephalic neural crest cells from quail embryos have been shown to respond to exogenous fgf- in a dose dependant manner; lower doses induce proliferation and higher doses induce cartilage differentiation. during long bone development, at the time of initiation of endochondral differentiation, fgf is expressed in the condensing mesenchyme. fgf fgf fgf and fgf are expressed in loose mesenchyme outside the condensation. however, mice lacking these fgfs show no apparent defects in skeletal development. functional redundancy between these fgfs may, in part, account for the lack of phenotype. therefore, roles for these fgfs in the early stages of chondrogenesis have not yet been defined. in this study, we have analyzed transgenic mice that express fgf in their cranial mesenchymal cells. these mice show abnormal head development and are born with a pronounced bulge in their skulls. skeletal preparations of these mice revealed dramatic changes in parietal bone formation. in the region where the parietal bones normally form, cranial mesenchymal cells are induced to differentiate into chondrocytes that proliferate, hypertrophy and subsequently differentiate as bone. correlative changes in expression of marker genes sox col2a col10a ihh, cbfai and osteocalcin occur in conjunction with the altered differentiation program. thus the parietal bones in these mice form by endochondral ossification rather than by the usual intramembranous ossification route. fate mapping studies indicate that the ectopic cartilagenous precursors in these mice are derived from the mesoderm. based on these results we suggest that cranial mesenchymal cells are competent to initiate endochondral ossification, and can be switched to this alternative developmental program by ectopic expression, or overexpression, of fgf  changes in cranial morphology in the fgf transgenic mice transgenic mice were generated by microinjection of a construct with the αa-crystallin promoter linked to a mouse fgf cdna. eleven founders carrying the transgene were identified. five of them had cataracts. in the transgenic line ove in addition to lens defects, there were defects in the development of the skull. mice in this family that are heterozygous for the transgene are born with'dome-shaped' heads. this phenotype is even more pronounced in homozygous transgenic mice. calvarial development in fgf transgenic mice the skeletal defects were examined by alcian blue and alizarin red staining of e p p and p mice. the skeletal preparations show a dramatic expansion of cartilage in the head region by e. parietal bone development was particularly affected. at p the phenotypic differences between the nontransgenic and fgf transgenic mice were still pronounced. at this age, the calvarial skull in nontransgenic mice is mostly ossified except at the sutures. in contrast, there is an expanded cartilage territory in the fgf transgenic mice. the transgenic mice also show a central hole in the skull where there is neither cartilage nor bone. development of the occipital bones appeared normal. at p the alcian blue stained tissues in the transgenic cranium were interspersed with alizarin red stained regions suggesting that the cartilage territory was being replaced by bony structures. by month of age, the cartilage territory was completely replaced by bone. the alterations in skeletal development in the fgf transgenic mice were examined at the histological level by analyses of hematoxylin and eosin stained sections of the cranium. no distinctive differences in morphology were seen at e. by e more cranial mesenchymal cells were seen in the skull region of the fgf transgenic mice and these cells were organized differently from nontransgenic mice. by e the cranial mesenchymal cells in the nontransgenic mice had initiated intramembranous ossification. in contrast, the mesenchymal cells in the fgf transgenic skulls differentiated into cells resembling chondrocytes. within the next few days these cells increased in size and became hypertrophic. by p perichondrial cells had begun to invade the cartilage territory. by p the cartilage territory was transformed into trabecular bone and bone marrow. these results considered together suggest that the cranial mesenchymal cells in the parietal region in these mice recapitulate the sequence of events that occurs during endochondral ossification. ectopic expression of the fgf transgene to test if the alterations seen in the skulls of the ove mice were due to ectopic expression of the transgene, in situ hybridizations were performed using an s35-labelled riboprobe that recognizes the sv portion of the transgene. transgene expression was seen in the lens, the dorsal portion of the retinal pigmented epithelium and also in the cranial mesenchymal cells beginning at e. expression levels peak by e and start to decrease by e. transgene expression is extinguished by e. section and whole mount in situ hybridizations show that the extraocular transgene expression is restricted to the mesenchymal cells overlying the future mid and hind brain regions. these results argue that the defects in skeletal development in the ove line are attributable to ectopic and transient expression of the fgf transgene. expression of chondrocyte specific markers the different stages of endochondral ossification are characterized by expression of different marker genes. expression of sox a member of the sox family of transcription factors, has been shown to be essential for chondrocyte condensation. in particular, sox is required for the expression of cartilage-specific extracellular matrix components such as collagen ii. in the long bone growth plates, col2a is expressed in the resting and proliferating chondrocytes, ihh in prehypertropic chondrocytes and col10a in hypertropic chondrocytes. expression of the runt domain transcription factor cbfai has been shown to be essential for differentiation of the cells in the osteoblastic lineage. osteocalcin, one of the downstream targets of cbfai, is expressed exclusively in the osteoblasts. expression of these six marker genes was examined by in situ hybridization using s35-labelled riboprobes. no appreciable differences in expression of sox between nontransgenic and fgf transgenic mice were seen at e. however, significant induction of sox expression was seen in the cranial mesenchymal cells of the transgenic mice by e. sox expression was seen to persist in the developing chondrocytes. cbfai and osteocalcin are expressed during the normal program of intramembranous ossification in nontransgenic mice, but their expression was not seen in the corresponding regions of the embryonic transgenic heads. col2a expression was seen in the developing chondrocytes in the transgenic cranium but not the wildtype, at e e and p. by p expression levels were found to be reduced in transgenic hypertropic chondrocytes. in situ hybridizations at p showed that col10a and ihh are expressed in the hypertropic chondrocytes. expression of col10a was not seen in the chondrocytes at p or before. these results demonstrate that the transgenic cranial mesenchymal cells undergo endochondral ossification instead of intramembranous ossification. expression of fgf fgfr and fgfr3 fgf has been reported to be expressed in cranial mesenchymal cells as well as in the dural mesoderm during embryonic development. expression levels of the fgf transgene and endogenous fgf were compared by in situ hybridizations using an -labelled fgf riboprobe. at e and e fgf was not expressed at detectable levels in the cranial mesenchymal cells in nontransgenic mice. in contrast, fgf expression was clearly present in the transgenic mice. as fgf binds to and signals through fgfr and fgfr in situ hybridizations were performed to examine their expression levels. low levels of fgfr expression can be seen in cranial mesenchymal cells at e in both the nontransgenic and transgenic mice. fgfr expression is maintained in the induced chondrocytes in the transgenic mice. in contrast, fgfr expression was not detected at e either in transgenic or in nontransgenic cranial mesenchyme. fgfr was expressed at low levels in the differentiating chondrocytes of the fgf transgenic mice. in addition, crossing the ove mice with fgfr null mice does not alter the "dome-head" phenotype. these results taken together suggest that the developmental switch in the transgenic mice may initially be mediated by ectopic activation of fgfr fgf induces proliferation of the cranial mesenchymal cells to test if the cranial mesenchymal cells proliferate in response to fgf expression, sections of e and e skulls were assayed for brdu incorporation. at e and at the cranial mesenchymal cells in the parietal region of fgf transgenic mice show a significant increase in brdu incorporation. these results suggest that fgf expression in the cranial mesenchymal cells results in increased proliferation. fate mapping of cells in the parietal region in the fgf transgenic mice cell lineage tracing studies performed in mice and in chicks show that the cranial skeletogenic mesenchyme is derived from two sources, neural crest and mesoderm. to determine if the cells in the parietal region that form ectopic cartilage in the ove mice are neural crest or mesodermal derivatives, we performed fate mapping experiments using wnt1-cre and r26r mice. as has been described previously, wnt1-cre/r26r compound heterozygotes stably express β-galactosidase in neural crest cells and their descendants but not in the mesodermal derivatives. ove mice were crossed with wnt1-cre/r26r mice and heads of e embryos were stained with x-gal. in the absence of wnt1-cre, lacz positive cells were not detected in the r26r embryo or in the fgf9/r26r embryo. in the wnt1-cre/r26r embryo, the parietal region of the skull showed no evidence for lacz activity, implying that neural crest derivatives do not contribute to this region of the skull. in the fgf9/wnt1-cre/r26r embryo, there is also no blue staining in the parietal region. these results establish that the cells in the parietal region that form ectopic cartilage in the ove mice are not neural crest derived but are for the most part, derived from mesoderm. in summary, in family ove ectopic fgf expression switches the differentiation and proliferation properties of cranial mesodermal cells, resulting in inhibition of intramembranous ossification and conversion to a program of endochondral ossification. discussion in this study, we have analyzed a line of transgenic mice where the differentiation program of a portion of the cranial mesenchyme is converted from intramembranous to endochondral ossification. the transgenic mice ectopically express fgf in their cranial mesenchymal cells. expression of fgf correlates with localized proliferation of the mesenchymal cells and with upregulation of sox expression and downregulation of cbfai and osteocalcin. the mesenchymal cells consequently differentiate into chondrocytes that express col2a these chondrocytes proliferate, then hypertrophy, express col10a1and are later replaced by trabecular bone and bone marrow. therefore, parietal bone formation in these mice recapitulates the sequence of events that occurs during endochondral ossification. these results demonstrate that fgf expression is sufficient to convert the differentiation program of the cranial mesenchymal cells from intramembranous to endochondral ossification. ectopic expression of the transgene the skeletal defects in this transgenic line occur in mice that are either heterozygous or homozygous for the fgf transgene. it is, therefore, unlikely that the skull defects in this line of mice are due to disruption of a gene essential for membranous ossification of the parietal bones. the transgene expression pattern provides compelling evidence that the skull defects in these mice are due to ectopic expression of the transgene. in situ hybridization analyses show that the transgene is expressed in an appropriate spatial and temporal pattern to induce the developmental changes. in addition, the transgene is not expressed in regions where membranous ossification still occurs. furthermore, transgene expression precedes sox expression and condensation of the mesenchymal cells. also, our results are consistent with the results of other studies. for example, recent studies of a mouse model for apert syndrome, with a single amino acid change in fgfr support the notion that enhanced activation of fgfr can cause some cranial mesenchyme to convert to a chondrocyte differentiation program. in addition, fgf expression in a chondrocytic cell line is sufficient to induce expression of sox. addition of fgf- in vitro to cranial mesenchymal cells from quail embryos can induce cartilage differentiation at high doses. taken together, these results suggest that initiation of chondrogenesis in the cranial mesenchymal cells in this line of mice is due to ectopic expression of the fgf transgene. in the ove family, expression of the fgf transgene is not only ectopic but also transient. expression in the cranial mesenchyme was not detectable after e this implies that stimulation by fgf is sufficient to induce cranial mesoderm to switch to a chondrocytic differentiation program, but sustained expression of fgf is not required for the later stages of differentiation. therefore, the endochondral ossification program, once initiated by fgf appears to become autonomous. the reason for the ectopic fgf expression is not clear at present. it is possible the transgene array has integrated in the neighborhood of an endogenous enhancer that directs expression to the cranial mesenchymal cells. some of the integrated copies of the transgene still retain the αa-crystallin promoter since the transgene is still expressed in the lens. the transgene is also expressed in the dorsal margins of the retinal pigmented epithelium and this expression transforms the rpe into neuroretina. nature of fgf induction expression of fgf in growth plate chondrocytes using the col2a promoter results in reduced proliferation and terminal differentiation of chondrocytes. in addition, targeted deletion of fgfr one of the receptors through which fgf signals, results in mice with overgrowth of the long bones. these results suggest that a primary role of fgf signaling in the long bones is to act as a negative regulator of chondrocyte proliferation. these results are in contrast to our results that show that ectopic fgf expression in embryonic cranial mesenchymal cells induces overproliferation followed by endochondral ossification. how can these disparate observations be reconciled? one possible explanation is the differential timing of fgf expression. the col2a promoter is active in the differentiated chondrocytes, while our transgenic fgf is expressed in undifferentiated cranial mesenchymal cells. second, there are differences in the responding tissues. chondrocytes in the long bones originate from the lateral plate mesoderm while the parietal mesenchyme is derived from cranial mesoderm. since the stimulated cells have different developmental origins, there is no reason to expect that the responses will be identical. for example, although lens and corneal epithelial cells are related to each other, both morphologically and developmentally, they respond differently to fgf stimulation. the reduction in proliferation in the long bones in response to fgf stimulation may be a unique property of the growth plate chondrocytes. in contrast, the fgf9-induced proliferation of cranial mesenchymal cells is consistent with the notion that fgfs, in general, act as mitogens during normal development. endogenous role for fgf9? though fgf appears to be sufficient to induce chondrogenesis in the skull, the role of fgf during normal development of cartilage in the cranium is unclear. our in situ hybridizations did not show any detectable expression of fgf in the calvarial mesenchyme at e or e expression is seen later during embryonic development in the endocranial portions of the mesenchyme and is downregulated during postnatal development. this expression pattern suggests that fgf is not important for initial specification of the cells in the chondrocytic lineage in the cranium. consistent with this model, targeted deletion of fgf does not result in any visible skeletal abnormalities in the skull. in contrast, in the transgenic mice, the ectopic fgf plays an instructive role and initiates chondrogenesis. fgf is known to signal through fgfr and fgfr. signaling by fgf in the transgenic heads is likely to be mediated initially through fgfr rather than fgfr as the parietal fate switch in the fgf transgenic mice is not rescued by the loss of fgfr after sox expression has been induced and the chondrocytic program has been initiated, expression of the fgf transgene is no longer required. in contrast to the normal chondrocytic differentiation program in long bones, ectopic chondrogenesis in the cranium proceeds at a much slower rate. though the reasons for this are presently unclear, we speculate that this may be due to the multi-step nature of the endochondral differentiation program. respecification and reprogramming of the parietal environment is required, and this is accomplished in a less timely fashion than at the normal sites of differentiation. fgfrs and intramembranous ossification our findings are in apparent conflict with some of the current models for the roles of fgf receptors in intramembranous ossification. autosomal dominant mutations in fgfr or fgfr lead to premature differentiation and fusion of the skull sutures in humans and these effects are thought to be the consequence of enhanced receptor activity. based upon such a model, our transgenic mice would be predicted to exhibit an analogous phenotype, i.e. craniosynostosis. inappropriate activation of the receptors in the cranial mesenchyme would be predicted to lead to premature differentiation. however, our studies indicate that ligand-mediated activation of fgfr induces proliferation of embryonic cranial mesenchymal cells and subsequent differentiation into cartilage. by extrapolation then, our results imply that mutations in the fgfr gene in human patients do not result in ligand-independent constitutive activation of the receptor. this prediction is supported by the finding that targeted inactivation of fgfr in mice causes developmental alterations in cell types that are not affected by the putative gain-of-function mutations. an alternative explanation is that transient stimulation of fgfr may lead to a qualitatively different cellular response compared to sustained ligand-independent stimulation of fgfr. in either case, our results show that a single fgf can function as an instructive signal, inducing a specific cell type to switch from one differentiation program to an autonomous, alternative differentiation pathway.  although the mechanistic details of fgf stimulation of the cranial mesenchymal cells remain to be elucidated, the ove transgenic mice provide the first demonstration in vivo that an fgf can switch the differentiation program of immature cranial mesoderm. these results also demonstrate that mesoderm-derived cranial mesenchymal cells are developmentally flexible and can undergo either intramembranous or endochondral ossification in response to extracellular signals. these transgenic mice provide a model system in which to elucidate the molecular connection between stimulation of an fgfr and induction of expression of a cell-fate-determining transcription factor. 
at these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. fate mapping studies show that the cranial mesenchymal cells in the parietal region that show a switch in cell fate are likely to be derived from the mesoderm. these results demonstrate that fgf expression is sufficient to convert the differentiation program of mesoderm-derived cranial mesenchyme cells from intramembranous to endochondral ossification. this type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone. the parietal bones in these mice show a switch from intramembranous to endochondral ossification. the flat bones of the skull normally form through intramembranous ossification. we have analyzed a line of transgenic mice that expresses fgf a member of the fibroblast growth factor family, in cranial mesenchymal cells.
 the flat bones of the skull normally form through intramembranous ossification. at these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. this type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone.  we have analyzed a line of transgenic mice that expresses fgf a member of the fibroblast growth factor family, in cranial mesenchymal cells. the parietal bones in these mice show a switch from intramembranous to endochondral ossification. cranial cartilage precursors are induced to proliferate, then hypertrophy and are later replaced by bone. these changes are accompanied by upregulation of sox ihh, col2a col10a and downregulation of cbfai and osteocalcin. fate mapping studies show that the cranial mesenchymal cells in the parietal region that show a switch in cell fate are likely to be derived from the mesoderm.  these results demonstrate that fgf expression is sufficient to convert the differentiation program of mesoderm-derived cranial mesenchyme cells from intramembranous to endochondral ossification. 