Integration of FGF and TWIST in calvarial bone and suture development

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.     

Abnormalities in cartilage and bone development in the Apert syndrome FGFR2(+/S252W) mouse

Apert syndrome is an autosomal dominant disorder characterized by malformations of the skull, limbs and viscera. Two-thirds of affected individuals have a S252W mutation in fibroblast growth factor receptor 2 (FGFR2). To study the pathogenesis of this condition, we generated a knock-in mouse model with this mutation. The Fgfr2(+/S252W) mutant mice have abnormalities of the skeleton, as well as of other organs including the brain, thymus, lungs, heart and intestines. In the mutant neurocranium, we found a midline sutural defect and craniosynostosis with abnormal osteoblastic proliferation and differentiation. We noted ectopic cartilage at the midline sagittal suture, and cartilage abnormalities in the basicranium, nasal turbinates and trachea. In addition, from the mutant long bones, in vitro cell cultures grown in osteogenic medium revealed chondrocytes, which were absent in the controls. Our results suggest that altered cartilage and bone development play a significant role in the pathogenesis of the Apert syndrome phenotype.     

Compound deletion of Fgfr3 and Fgfr4 partially rescues the Hyp mouse phenotype

Uncertainty exists regarding the physiologically relevant fibroblast growth factor (FGF) receptor (FGFR) for FGF23 in the kidney and the precise tubular segments that are targeted by FGF23. Current data suggest that FGF23 targets the FGFR1c-Klotho complex to coordinately regulate phosphate transport and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production in the proximal tubule. In studies using the Hyp mouse model, which displays FGF23-mediated hypophosphatemia and aberrant vitamin D, deletion of Fgfr3 or Fgfr4 alone failed to correct the Hyp phenotype. To determine whether FGFR1 is sufficient to mediate the renal effects of FGF23, we deleted Fgfr3 and Fgfr4 in Hyp mice, leaving intact the FGFR1 pathway by transferring compound Fgfr3/Fgfr4-null mice on the Hyp background to create wild-type (WT), Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. We found that deletion of Fgfr3 and Fgfr4 in Fgfr3(-/-)/Fgfr4(-/-) and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice induced an increase in 1,25(OH)(2)D. In Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, it partially corrected the hypophosphatemia (P(i) = 9.4 ± 0.9, 6.1 ± 0.2, 9.1 ± 0.4, and 8.0 ± 0.5 mg/dl in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), increased Na-phosphate cotransporter Napi2a and Napi2c and Klotho mRNA expression in the kidney, and markedly increased serum FGF23 levels (107 ± 20, 3,680 ± 284, 167 ± 22, and 18,492 ± 1,547 pg/ml in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), consistent with a compensatory response to the induction of end-organ resistance. Fgfr1 expression was unchanged in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice and was not sufficient to transduce the full effects of FGF23 in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. These studies suggest that FGFR1, FGFR3, and FGFR4 act in concert to mediate FGF23 effects on the kidney and that loss of FGFR function leads to feedback stimulation of Fgf23 expression in bone.     

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2(S252W) mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2(S252W) and β-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.     

The mouse seminal vesicle shape mutation is allelic with Fgfr2

The mouse seminal vesicle shape (svs) mutation is a spontaneous recessive mutation that causes branching morphogenesis defects in the prostate gland and seminal vesicles. Unlike many other mutations that reduce prostatic and/or seminal vesicle branching, the svs mutation dramatically reduces branching without reducing organ growth. Using a positional cloning approach, we identified the svs mutant lesion as a 491 bp insertion in the tenth intron of Fgfr2 that results in changes in the pattern of Fgfr2 alternative splicing. An engineered null allele of Fgfr2 failed to complement the svs mutation proving that a partial loss of FGFR2(IIIb) isoforms causes svs phenotypes. Thus, the svs mutation represents a new type of adult viable Fgfr2 allele that can be used to elucidate receptor function during normal development and in the adult. In the developing seminal vesicles, sustained activation of ERK1/2 was associated with branching morphogenesis and this was absent in svs mutant seminal vesicles. This defect appears to be the immediate downstream effect of partial loss of FGFR2(IIIb) because activation of FGFR2(IIIb) by FGF10 rapidly induced ERK1/2 activation, and inhibition of ERK1/2 activation blocked seminal vesicle branching morphogenesis. Partial loss of FGFR2(IIIb) was also associated with down-regulation of several branching morphogenesis regulators including Shh, Ptch1, Gli1, Gli2, Bmp4, and Bmp7. Together with previous studies, these data suggest that peak levels of FGFR2(IIIb) signaling are required to induce branching and sustain ERK1/2 activation, whereas reduced levels support ductal outgrowth in the prostate gland and seminal vesicles.     

Generation of Fgfr3 Conditional Knockout Mice

Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.     

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology

Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form.     

Loss of Fgfr2 leads to partial XY sex reversal

In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.     

Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells

Fibroblast growth factor-2 (FGF2) is a powerful promoter of bone growth. We demonstrate here that brief exposure to FGF2 enhances mineralized nodule formation in cultured rat osteoprogenitor cells due to an expansion of cells that subsequently mineralize. This mitogenic effect is mediated via sulfated glycosaminoglycans (GAGs), FGFR1, and the extracellular signal-regulated kinase (ERK) pathway. The GAGs involved in this stimulation are chondroitin sulfates (CS) rather than heparan sulfates (HS). However, continuous FGF2 treatment reduces alkaline phosphatase (ALP) activity, downregulates collagen Ialpha1 (ColIalpha1) and FGFR3 expression, upregulates the expression and secretion of osteopontin (OPN) and inhibits mineralization. The inhibitory effects of FGF2 on FGFR3 expression and ALP activity are also mediated by the ERK pathway, although the effects of FGF2 on ColIalpha1 and OPN expression are mediated by GAGs and PKC activity. Thus short-term activation of FGF2/FGFR1 promotes osteoprogenitor proliferation and subsequent differentiation, while long-term activation of FGF2 signaling disrupts mineralization by modulating osteogenic marker expression. This study thus establishes the central role of sulfated GAGs in the osteogenic progression of osteoprogenitors.     

Roles of FGFR3 during morphogenesis of Meckel's cartilage and mandibular bones

To address the functions of FGFR2 and FGFR3 signaling during mandibular skeletogenesis, we over-expressed in the developing chick mandible, replication-competent retroviruses carrying truncated FGFR2c or FGFR3c that function as dominant negative receptors (RCAS-dnFGFR2 and RCAS-dnFGFR3). Injection of RCAS-dnFGFR3 between HH15 and 20 led to reduced proliferation, increased apoptosis, and decreased differentiation of chondroblasts in Meckel's cartilage. These changes resulted in the formation of a hypoplastic mandibular process and truncated Meckel's cartilage. This treatment also affected the proliferation and survival of osteoprogenitor cells in osteogenic condensations, leading to the absence of five mandibular bones on the injected side. Injection of RCAS-dnFGFR2 between HH15 and 20 or RCAS-dnFGFR3 at HH26 did not affect the morphogenesis of Meckel's cartilage but resulted in truncations of the mandibular bones. RCAS-dnFGFR3 affected the proliferation and survival of the cells within the periosteum and osteoblasts. Together these results demonstrate that FGFR3 signaling is required for the elongation of Meckel's cartilage and FGFR2 and FGFR3 have roles during intramembranous ossification of mandibular bones.     

Fibroblast growth factor receptor 1 signaling in the osteo-chondrogenic cell lineage regulates sequential steps of osteoblast maturation

Mutations in fibroblast growth factor receptors (Fgfrs) 1-3 cause skeletal disease syndromes in humans. Although these Fgfrs are expressed at various stages of chondrocyte and osteoblast development, their function in specific skeletal cell types is poorly understood. Using conditional inactivation of Fgfr1 in osteo-chondrocyte progenitor cells and in differentiated osteoblasts, we provide evidence that FGFR1 signaling is important for different stages of osteoblast maturation. Examination of osteogenic markers showed that inactivation of FGFR1 in osteo-chondro-progenitor cells delayed osteoblast differentiation, but that inactivation of FGFR1 in differentiated osteoblasts accelerated differentiation. In vitro osteoblast cultures recapitulated the in vivo effect of FGFR1 on stage-specific osteoblast maturation. In immature osteoblasts, FGFR1 deficiency increased proliferation and delayed differentiation and matrix mineralization, whereas in differentiated osteoblasts, FGFR1 deficiency enhanced mineralization. Furthermore, FGFR1 deficiency in differentiated osteoblasts resulted in increased expression of Fgfr3, a molecule that regulates the activity of differentiated osteoblasts. Mice lacking Fgfr1, either in progenitor cells or in differentiated osteoblasts, showed increased bone mass as adults. These data demonstrate that signaling through FGFR1 in osteoblasts is necessary to maintain the balance between bone formation and remodeling through a direct effect on osteoblast maturation.     

Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2 +P253R mice

Background

Apert syndrome is characterized by craniosynostosis and limb abnormalities and is primarily caused by FGFR2 +/P253R and +/S252W mutations. The former mutation is present in approximately one third whereas the latter mutation is present in two-thirds of the patients with this condition. We previously reported an inbred transgenic mouse model with the Fgfr2 +/S252W mutation on the C57BL/6J background for Apert syndrome. Here we present a mouse model for the Fgfr2+/P253R mutation.

Results

We generated inbred Fgfr2 ^+/P253R mice on the same C56BL/6J genetic background and analyzed their skeletal abnormalities. 3D micro-CT scans of the skulls of the Fgfr2 ^+/P253R mice revealed that the skull length was shortened with the length of the anterior cranial base significantly shorter than that of the Fgfr2 ^+/S252W mice at P0. The Fgfr2 ^+/P253R mice presented with synostosis of the coronal suture and proximate fronts with disorganized cellularity in sagittal and lambdoid sutures. Abnormal osteogenesis and proliferation were observed at the developing coronal suture and long bones of the Fgfr2 ^+/P253R mice as in the Fgfr2 ^+/S252W mice. Activation of mitogen-activated protein kinases (MAPK) was observed in the Fgfr2 ^+/P253R neurocranium with an increase in phosphorylated p38 as well as ERK1/2, whereas phosphorylated AKT and PKCα were not obviously changed as compared to those of wild-type controls. There were localized phenotypic and molecular variations among individual embryos with different mutations and among those with the same mutation.

Conclusions

Our in vivo studies demonstrated that the Fgfr2 +/P253R mutation resulted in mice with cranial features that resemble those of the Fgfr2 ^+/S252W mice and human Apert syndrome. Activated p38 in addition to the ERK1/2 signaling pathways may mediate the mutant neurocranial phenotype. Though Apert syndrome is traditionally thought to be a consistent phenotype, our results suggest localized and regional variations in the phenotypes that characterize Apert syndrome.     

Regulation of cranial morphogenesis and cell fate at the neural crest-mesoderm boundary by engrailed 1

The characterization of mesenchymal progenitors is central to understanding development, postnatal pathology and evolutionary adaptability. The precise identity of the mesenchymal precursors that generate the coronal suture, an important structural boundary in mammalian skull development, remains unclear. We show in mouse that coronal suture progenitors originate from hedgehog-responsive cephalic paraxial mesoderm (Mes) cells, which migrate rapidly to a supraorbital domain and establish a unidirectional lineage boundary with neural crest (NeuC) mesenchyme. Lineage tracing reveals clonal and stereotypical expansion of supraorbital mesenchymal cells to form the coronal suture between E11.0 and E13.5. We identify engrailed 1 (En1) as a necessary regulator of cell movement and NeuC/Mes lineage boundary positioning during coronal suture formation. In addition, we provide genetic evidence that En1 functions upstream of fibroblast growth factor receptor 2 (Fgfr2) in regulating early calvarial osteogenic differentiation, and postulate that it plays an additional role in precluding premature osteogenic conversion of the sutural mesenchyme.     

FGF/FGFR signaling coordinates skull development by modulating magnitude of morphological integration: evidence from Apert syndrome mouse models

The fibroblast growth factor and receptor system (FGF/FGFR) mediates cell communication and pattern formation in many tissue types (e.g., osseous, nervous, vascular). In those craniosynostosis syndromes caused by FGFR1-3 mutations, alteration of signaling in the FGF/FGFR system leads to dysmorphology of the skull, brain and limbs, among other organs. Since this molecular pathway is widely expressed throughout head development, we explore whether and how two specific mutations on Fgfr2 causing Apert syndrome in humans affect the pattern and level of integration between the facial skeleton and the neurocranium using inbred Apert syndrome mouse models Fgfr2(+/S252W) and Fgfr2(+/P253R) and their non-mutant littermates at P0. Skull morphological integration (MI), which can reflect developmental interactions among traits by measuring the intensity of statistical associations among them, was assessed using data from microCT images of the skull of Apert syndrome mouse models and 3D geometric morphometric methods. Our results show that mutant Apert syndrome mice share the general pattern of MI with their non-mutant littermates, but the magnitude of integration between and within the facial skeleton and the neurocranium is increased, especially in Fgfr2(+/S252W) mice. This indicates that although Fgfr2 mutations do not disrupt skull MI, FGF/FGFR signaling is a covariance-generating process in skull development that acts as a global factor modulating the intensity of MI. As this pathway evolved early in vertebrate evolution, it may have played a significant role in establishing the patterns of skull MI and coordinating proper skull development.     

Differential Fibroblast Growth Factor 8 (FGF8)-Mediated Autoregulation of Its Cognate Receptors,Fgfr1 and Fgfr3, in Neuronal Cell Lines

Fibroblast growth factors (FGFs) mediate a vast range of CNS developmental processes including neural induction, proliferation, migration, and cell survival. Despite the critical role of FGF signaling for normal CNS development, few reports describe the mechanisms that regulate FGF receptor gene expression in the brain. We tested whether FGF8 could autoregulate two of its cognate receptors, Fgfr1 and Fgfr3, in three murine cell lines with different lineages: fibroblast-derived cells (3T3 cells), neuronal cells derived from hippocampus (HT-22 cells), and neuroendocrine cells derived from hypothalamic gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). GnRH is produced by neurons in the hypothalamus and is absolutely required for reproductive competence in vertebrate animals. Several lines of evidence strongly suggest that Fgf8 is critical for normal development of the GnRH system, therefore, the GT1-7 cells provided us with an additional endpoint, Gnrh gene expression and promoter activity, to assess potential downstream consequences of FGF8-induced modulation of FGF receptor levels. Results from this study suggest that the autoregulation of its cognate receptor represents a common downstream effect of FGF8. Further, we show that Fgfr1 and Fgfr3 are differentially regulated within the same cell type, implicating these two receptors in different biological roles. Moreover, Fgfr1 and Fgfr3 are differentially regulated among different cell types, suggesting such autoregulation occurs in a cell type-specific fashion. Lastly, we demonstrate that FGF8b decreases Gnrh promoter activity and gene expression, possibly reflecting a downstream consequence of altered FGF receptor populations. Together, our data bring forth the possibility that, in addition to the FGF synexpression group, autoregulation of FGFR expression by FGF8 represents a mechanism by which FGF8 could fine-tune its regulatory actions.     

Nedd4‐1 binds and ubiquitylates activated FGFR1 to control its endocytosis and function

Fibroblast growth factor receptor 1 (FGFR1) has critical roles in cellular proliferation and differentiation during animal development and adult homeostasis. Here, we show that human Nedd4 (Nedd4‐1), an E3 ubiquitin ligase comprised of a C2 domain, 4 WW domains, and a Hect domain, regulates endocytosis and signalling of FGFR1. Nedd4‐1 binds directly to and ubiquitylates activated FGFR1, by interacting primarily via its WW3 domain with a novel non‐canonical sequence (non‐PY motif) on FGFR1. Deletion of this recognition motif (FGFR1‐Δ6) abolishes Nedd4‐1 binding and receptor ubiquitylation, and impairs endocytosis of activated receptor, as also observed upon Nedd4‐1 knockdown. Accordingly, FGFR1‐Δ6, or Nedd4‐1 knockdown, exhibits sustained FGF‐dependent receptor Tyr phosphorylation and downstream signalling (activation of FRS2α, Akt, Erk1/2, and PLCγ). Expression of FGFR1‐Δ6 in human embryonic neural stem cells strongly promotes FGF2‐dependent neuronal differentiation. Furthermore, expression of this FGFR1‐Δ6 mutant in zebrafish embryos disrupts anterior neuronal patterning (head development), consistent with excessive FGFR1 signalling. These results identify Nedd4‐1 as a key regulator of FGFR1 endocytosis and signalling during neuronal differentiation and embryonic development.