FGFs,their receptors,and human limb malformations: Clinical and molecular correlations

Fibroblast growth factors (FGFs) comprise a family of 22 distinct proteins with pleiotropic signaling functions in development and homeostasis. These functions are mediated principally by four fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase family, with heparin glycosaminoglycan as an important cofactor. Developmental studies in chick and mouse highlight the critical role of FGF‐receptor signaling in multiple phases of limb development, including the positioning of the limb buds, the maintenance of limb bud outgrowth, the detailed patterning of the limb elements, and the growth of the long bones. Corroborating these important roles, mutations of two members of the FGFR family (FGFR1 and FGFR2) are associated with human disorders of limb patterning; in addition, mutations of FGFR3 and FGF23 affect growth of the limb bones. Analysis of FGFR2 mutations in particular reveals a complex pattern of genotype/phenotype correlation, which will be reviewed in detail. Circumstantial evidence suggests that the more severe patterning abnormalities are mediated by illegitimate paracrine signaling in the mesoderm, mediated by FGF10 or by a related FGF, and this is beginning to gain some experimental support. A further test of this hypothesis is provided by a unique family segregating two FGFR2 mutations in cis (S252L; A315S), in which severe syndactyly occurs in the absence of the craniosynostosis that typically accompanies FGFR2 mutations. © 2002 Wiley‐Liss, Inc.     

FGF9 mutation causes craniosynostosis along with multiple synostoses

Craniosynostosis is commonly caused by mutations in fibroblast growth factor receptors (FGFRs), highlighting the essential role of FGF‐mediated signaling in skeletal development. We set out to identify the molecular defect in a family referred for craniosynostosis and in whom no mutation was previously detected. Using next‐generation sequencing, we identified a novel missense mutation in FGF9. Modeling based upon the crystal structure and functional studies confirmed its pathogenicity showing that it impaired homodimerization and FGFR3 binding. Only one FGF9 mutation has been previously reported in a multigeneration family with multiple synostoses (SYNS3) but no signs of craniosynostosis. In contrast, our family has a greater phenotypic resemblance to that observed in the Fgf9 spontaneous mouse mutant, elbow‐knee‐synostosis, Eks, with both multiple synostoses and craniosynostosis. We have demonstrated for the first time that mutations in FGF9 cause craniosynostosis in humans and confirm that FGF9 mutations cause multiple synostoses.     

FGFR2, FGF8, FGF10 and BMP7 as candidate genes for hypospadias

Hypospadias is a common malformation, which results from failure of urethral tube closure, and whose molecular mechanisms are still largely unknown. The normal genital development is orchestrated by the urethral plate epithelium (UPE), at the genital tubercle (GT), which has polarizing activity, controlling a network of epithelial-mesenchymal interactions, which, when disturbed, may lead to hypospadias. Homeobox proteins (HOXs), fibroblast growth factors (FGFs) and bone morphogenic proteins (BMPs) are essential in this process. Hypospadias in the Hoxa13 -/- mice occurs as a result of the combined loss of Fgf8 and Bmp7 expression in the UPE. In both Fgf10 and Fgfr2 deficient mutant hypospadic male mice, cell proliferation is arrested prematurely and the maturation of the urethral epithelium is disrupted. Fgf8, Fgf10, and their receptor Fgfr2 are downstream targets of androgens (AR) during external genital development, an important fact given the pivotal role of AR in male sex differentiation. Therefore, we examined FGFR2, FGF10, FGF8, and BMP7 as candidate genes for hypospadias. DNA from 60 boys with familial, isolated, hypospadias was screened for mutations in FGFR2, FGF10, FGF8, and BMP7 genes, using DHPLC and DNA sequence analysis. The sequence variations c.590C>G and c.582-62G>A in FGF8, and, c.550+27C>T, c.727+180T>G, c.830T>C (p.Me186Thr), and c.2454C>T in FGFR2 were found uniquely in patients with hypospadias, as compared with 96 controls. No genetic variant in the other genes was detected. These results indicate that mutations are rare in FGF8 and FGFR2 in hypospadias, but gene variants may influence the risk.     

FGFs,their receptors,and human limb malformations: clinical and molecular correlations

Fibroblast growth factors (FGFs) comprise a family of 22 distinct proteins with pleiotropic signaling functions in development and homeostasis. These functions are mediated principally by four fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase family, with heparin glycosaminoglycan as an important cofactor. Developmental studies in chick and mouse highlight the critical role of FGF-receptor signaling in multiple phases of limb development, including the positioning of the limb buds, the maintenance of limb bud outgrowth, the detailed patterning of the limb elements, and the growth of the long bones. Corroborating these important roles, mutations of two members of the FGFR family (FGFR1 and FGFR2) are associated with human disorders of limb patterning; in addition, mutations of FGFR3 and FGF23 affect growth of the limb bones. Analysis of FGFR2 mutations in particular reveals a complex pattern of genotype/phenotype correlation, which will be reviewed in detail. Circumstantial evidence suggests that the more severe patterning abnormalities are mediated by illegitimate paracrine signaling in the mesoderm, mediated by FGF10 or by a related FGF, and this is beginning to gain some experimental support. A further test of this hypothesis is provided by a unique family segregating two FGFR2 mutations in cis (S252L; A315S), in which severe syndactyly occurs in the absence of the craniosynostosis that typically accompanies FGFR2 mutations.     

Gain of FGF4 is a frequent event in KIT/PDGFRA/SDH/RAS‐P WT GIST

Gastrointestinal stromal tumors (GIST) lacking mutations in KIT/PDGFRA or RAS pathways and retaining an intact SDH complex are usually referred to as KIT/PDGFRA/SDH/RAS‐P WT GIST or more simply quadruple WT GIST (~5% of all GIST). Despite efforts made, no recurrent genetic event in quadruple WT GIST has been identified so far. To further investigate this disease, we performed high throughput copy number analysis on quadruple WT GIST specimens identifying a recurrent focal gain in band 11q13.3 (involving FGF3/FGF4) in 6/8 cases. This event was not found in the other molecular GIST subgroups. FGF3/FGF4 duplication was associated with high expression of FGF4, both at mRNA and protein level, a growth factor normally not expressed in adult tissues or in KIT/PDGFRA‐mutated GIST. FGFR1 was found to be the predominant FGF receptor expressed and phosphorylation of AKT was detected, suggesting that a FGF4‐FGFR1 autocrine loop could stimulate downstream signaling in quadruple WT GIST. Together with the recent reports of quadruple WT cases carrying FGFR1 activating alterations, these findings strengthen the hypothesis of a potential involvement of FGFR pathway deregulation in quadruple WT GIST, which may represent a rationale for novel therapeutic approaches.     

Conditional gene inactivation reveals roles for Fgf10 and Fgfr2 in establishing a normal pattern of epithelial branching in the mouse lung

Fibroblast growth factor 10 (FGF10) signaling through FGF receptor 2 (FGFR2) is required for lung initiation. While studies indicate that Fgf10 and Fgfr2 are also important at later stages of lung development, their roles in early branching events remain unclear. We addressed this question through conditional inactivation of both genes in mouse subsequent to lung initiation. Inactivation of Fgf10 in lung mesenchyme resulted in smaller lobes with a reduced number of branches. Inactivation of Fgfr2 in lung epithelium resulted in disruption of lobes and small epithelial outgrowths that arose arbitrarily along the main bronchi. In both mutants, there was an increase in cell death. Also, the expression patterns of key signaling molecules implicated in branching morphogenesis were altered and a proximal lung marker was expanded distally. Our results indicate that both Fgf10 and Fgfr2 are required for a normal branching program and for proper proximal–distal patterning of the lung.Developmental Dynamics 238:1999–2013, 2009. © 2009 Wiley‐Liss, Inc.     

Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18

Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. The ligands interacting with FGF receptors (FGFRs) in developing bone have remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are not known. Here we show that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibited expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone.     

Expression of Fgf receptors 1, 2, and 3 in the developing mid- and hindbrain of the mouse.

Fibroblast growth factor 8 (FGF8) mediates the function of the midbrain-hindbrain organizer (MHO). FGF signals are transmitted by means of four known FGF receptors (FGFRs). Studies of Fgfr expression in early vertebrate development have shown that Fgfr1 is expressed along the entire neural tube, whereas Fgfr2 and Fgfr3 expression has been shown to spare the tissue adjacent to the MHO. The FGF8 signal from the MHO, therefore, was believed to be transmitted by FGFR1 exclusively. However, incongruent results from conditional mutants of Fgf8 and Fgfr1 in the midbrain-hindbrain (MHB) region contradict this hypothesis. Therefore, we reexamined the expression of the Fgfrs in this region. Fgfr1 is expressed all over the neural tube. Strikingly, Fgfr2 is expressed throughout the floor plate of the MHB region. In the basal plate, Fgfr2 directly abuts the Fgf8 expression domain at the MHO, anteriorly and posteriorly. Fgfr3 expression is in contact with the Fgf8 expression domain only in the rostroventral hindbrain. Based on these findings, we postulate a role for FGFR2 and FGFR3 in FGF signaling in the ventral midbrain and hindbrain.     

Genetic regulatory pathways of split-hand/foot malformation

Split-hand/foot malformation (SHFM) is caused by mutations in TP63, DLX5, DLX6, FGF8, FGFR1, WNT10B, and BHLHA9. The clinical features of SHFM caused by mutations of these genes are not distinguishable. This implies that in normal situations these SHFM-associated genes share an underlying regulatory pathway that is involved in the development of the central parts of the hands and feet. The mutations in SHFM-related genes lead to dysregulation of Fgf8 in the central portion of the apical ectodermal ridge (AER) and subsequently lead to misexpression of a number of downstream target genes, failure of stratification of the AER, and thus SHFM. Syndactyly of the remaining digits is most likely the effects of dysregulation of Fgf-Bmp-Msx signaling on apoptotic cell death. Loss of digit identity in SHFM is hypothesized to be the effects of misexpression of HOX genes, abnormal SHH gradient, or the loss of balance between GLI3A and GLI3R. Disruption of canonical and non-canonical Wnt signaling is involved in the pathogenesis of SHFM. Whatever the causative genes of SHFM are, the mutations seem to lead to dysregulation of Fgf8 in AER cells of the central parts of the hands and feet and disruption of Wnt-Bmp-Fgf signaling pathways in AER.     

Homozygous FIBP nonsense variant responsible of syndromic overgrowth,with overgrowth,macrocephaly, retinal coloboma and learning disabilities

The acidic fibroblast growth factor (FGF) intracellular binding protein (FIBP) interacts directly with the fibroblast growth factor FGF1. Although FIBP is known to be implicated in the FGF signaling pathway, its precise function remains unclear. Gain‐of‐function variants in several FGF receptors (FGFRs) are implicated in a wide spectrum of growth disorders from achondroplasia to overgrowth syndromes. In a unique case from a consanguineous union presenting with overgrowth, macrocephaly, retinal coloboma, large thumbs, severe varicose veins and learning disabilities, exome sequencing identified a homozygous nonsense FIBP variant. The patient's fibroblasts exhibit FIBP cDNA degradation and an increased proliferation capacity compared with controls. The phenotype defines a new multiple congenital abnormalities (MCA) syndrome, overlapping with the heterogeneous group of overgrowth syndromes with macrocephaly. The different clinical features can be explained by the alteration of the FGFR pathway. Taken together, these results suggest the implication of FIBP in a new autosomal recessive MCA.     

Regulation of external genitalia development by concerted actions of FGF ligands and FGF receptors

Members of the fibroblast growth factor (FGF) family play diverse roles during the development and patterning of various organs. In human and mice, 22 FGFs and four receptors derived from several splice variants are present. Redundant expression and function of FGF genes in organogenesis have been reported, but their roles in embryonic external genitalia, genital tubercle (GT), development have not been studied in detail. To address the role of FGF during external genitalia development, we have analyzed the expression of FGF genes (Fgf8, 9, 10) and receptor genes (Fgfr1, r2IIIb, r2IIIc) in GT of mice. Furthermore, Fgf10 and Fgfr2IIIb mutant mice were analyzed to elucidate their roles in embryonic external genitalia development. Fgfr2IIIb was expressed in urethral plate epithelium during GT development. Fgfr2IIIb mutant mice display urethral dysmorphogenesis. Marker gene analysis for urethral plate and bilateral mesenchymal formation suggests the existence of epithelial-mesenchymal interaction during urethral morphogenesis. Therefore, FGF10/FGFR2IIIb signals seem to constitute a developmental cascade for such morphogenesis.Y. Satoh and R. Haraguchi contributed equally to this work     

Effect of FGF/FGFR pathway blocking on lung adenocarcinoma and its cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are known to promote tumourigenesis through various mechanisms. Fibroblast growth factor (FGF)/FGF receptor (FGFR)-dependent lung cancers have been described. We have developed a mouse model of lung adenocarcinoma that was constructed through the induction of Fgf9 overexpression in type 2 alveolar cells. The expression of Fgf9 in adult lungs resulted in the rapid development of multiple adenocarcinoma-like tumour nodules. Here, we have characterised the contribution of CAFs and the Fgf/Fgfr signalling pathway in maintaining the lung tumours initiated by Fgf9 overexpression. We found that CAF-secreted Fgf2 contributes to tumour cell growth. CAFs overexpressed Tgfb, Mmp7, Fgf9, and Fgf2; synthesised more collagen, and secreted inflammatory cell-recruiting cytokines. CAFs also enhanced the conversion of tumour-associated macrophages (TAMs) to the tumour-supportive M2 phenotype but did not influence angiogenesis. In vivo inhibition of Fgfrs during early lung tumour development resulted in significantly smaller and fewer tumour nodules, whereas inhibition in established lung tumours caused a significant reduction in tumour size and number. Fgfr inhibition also influenced tumour stromal cells, as it significantly abolished TAM recruitment and reduced tumour vascularity. However, the withdrawal of the inhibitor caused a significant recurrence/regrowth of Fgf/Fgfr-independent lung tumours. These recurrent tumours did not possess a higher proliferative or propagative potential. Our results provide evidence that fibroblasts associated with the Fgf9-induced lung adenocarcinoma provide multiple means of support to the tumour. Although the Fgfr blocker significantly suppressed the tumour and its stromal cells, it was not sufficient to completely eliminate the tumour, probably due to the emergence of alternative (resistance/maintenance) mechanism(s). This model represents an excellent tool to further study the complex interactions between CAFs, their related chemokines, and the progression of lung adenocarcinoma; it also provides further evidence to support the need for a combinatorial strategy to treat lung cancer. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The incomplete inactivation of Fgf8 in the limb ectoderm affects the morphogenesis of the anterior autopod through BMP-mediated cell death.

Here we analyze limb development after the conditional inactivation of Fgf8 from the epiblast, using the previously described MORE (Mox2Cre) line. This line drives variable mosaic recombination of a floxed Fgf8 allele, resulting in a small proportion of AER cells that maintain Fgf8 expression. The phenotype of Mox2Cre;Fgf8 limbs is most similar to that of Msx2Cre;Fgf8 forelimbs, indicating that a small but durable expression of FGF8 is equivalent to an early normal, but transitory, expression. This functional equivalence likely relies on the subsequent Fgf4 upregulation that buffers the differences in the pattern of Fgf8 expression between the two conditional mutants. The molecular analysis of Mox2Cre;Fgf8 limbs shows that, despite Fgf4 upregulation, they develop under reduced FGF signaling. These limbs also exhibit an abnormal area of cell death at the anterior forelimb autopod, overlapping with an ectopic domain of Bmp7 expression, which can explain the abnormal morphogenesis of the anterior autopod.     

Mutations in FGFR1 and FGFR2 cause familial and sporadic Pfeiffer syndrome

Pfeifter syndrome (PS) is an autosomal dominant skeletal disorderwhich affects the bones of the skull, hands and feet. Previously,we have mapped PS in a subset of families to chromosome 8cenby linkage analysis and demonstrated a common mutation in thefibroblast growth factor receptor-1 (FGFR1) gene in the linkedfamilies. Here we report a second locus for PS on chromosome10q25, and present evidence that mutations in the fibroblastgrowth factor receptor-2 (FGFR2) gene on 10q25 cause PS in anadditional subset of familial and sporadic cases. Three differentpoint mutations in FGFR2, which alter the same acceptor splicesite of exon B, were observed in both sporadic and familialPS. In addition, a T to C transition in exon B predicting acysteine to arginine substitution was identified in three sporadicPS individuals. Interestingly, this T to C change is identicalto a mutation in FGFR2 previously reported in Crouzon syndrome,a phenotypically similar disorder but one lacking the hand andfoot anomalies seen in PS. Our results highlight the geneticheterogeneity in PS and suggest that the molecular data willbe an important complement to the clinical phenotype in definingcraniosynostosis syndromes.     

FGF8 initiates inner ear induction in chick and mouse

In both chick and mouse, the otic placode, the rudiment of the inner ear, is induced by at least two signals, one from the cephalic paraxial mesoderm and the other from the neural ectoderm. In chick, the mesodermal signal, FGF19, induces neural ectoderm to express additional signals, including WNT8c and FGF3, resulting in induction of the otic placode. In mouse, mesodermal Fgf10 acting redundantly with neural Fgf3 is required for induction of the placode. To determine how the mesodermal inducers of the otic placode are localized, we took advantage of the unique strengths of the two model organisms. We show that endoderm is necessary for otic induction in the chick and that Fgf8, expressed in the chick endoderm subjacent to Fgf19, is both sufficient and necessary for the expression of Fgf19 in the mesoderm. In the mouse, Fgf8 is also expressed in endoderm as well as in other germ layers in the periotic placode region. We show that otic induction fails in embryos null for Fgf3 and hypomorphic for Fgf8 and expression of mesodermal Fgf10 is reduced. Thus, Fgf8 plays a critical upstream role in an FGF signaling cascade required for otic induction in chick and mouse.     

Proline to arginine mutations in FGF receptors 1 and 3 result in Pfeiffer and Muenke craniosynostosis syndromes through enhancement of FGF binding affinity

Identical proline-->arginine gain-of-function mutations in fibroblast growth factor receptor (FGFR) 1 (Pro252Arg), FGFR2 (Pro253Arg) and FGFR3 (Pro250Arg), result in type I Pfeiffer, Apert and Muenke craniosynostosis syndromes, respectively. Here, we characterize the effects of proline-->arginine mutations in FGFR1c and FGFR3c on ligand binding using surface plasmon resonance and X-ray crystallography. Both Pro252Arg FGFR1c and Pro250Arg FGFR3c exhibit an enhancement in ligand binding in comparison to their respective wild-type receptors. Interestingly, binding of both mutant receptors to FGF9 was notably enhanced and implicates FGF9 as a potential pathophysiological ligand for mutant FGFRs in mediating craniosynostosis. The crystal structure, of Pro252Arg FGFR1c in complex with FGF2, demonstrates that the enhanced ligand binding is due to an additional set of receptor-ligand hydrogen bonds, similar to those gain-of-function interactions that occur in the Apert syndrome Pro253Arg FGFR2c-FGF2 crystal structure. However, unlike the Apert syndrome Pro253Arg FGFR2c mutant, neither the Pfeiffer syndrome Pro250Arg FGFR1c mutant nor the Muenke syndrome Pro250Arg FGFR3c mutant bound appreciably to FGF7 or FGF10. This observation provides a potential explanation for why the limb phenotypes, observed in type I Pfeiffer and Muenke syndromes, are less severe than the limb abnormalities observed in Apert syndrome. Hence, although analogous proline-->arginine mutations in FGFR1-3 act through a common structural mechanism to result in gain-of-function, differences in the primary sequence among FGFRs result in varying effects on ligand binding specificity.     

FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis

Fibroblast growth factor (FGF) signaling is involved in skeletal development of the vertebrate. Gain-of-function mutations of FGF receptors (FGFR) cause craniosynostosis, premature fusion of the skull, and dwarfism syndromes. Disruption of Fgfr3 results in prolonged growth of long bones and vertebrae. However, the role that FGFs actually play in skeletal development in the embryo remains unclear. Here we show that Fgf18 is expressed in and required for osteogenesis and chondrogenesis in the mouse embryo. Fgf18 is expressed in both osteogenic mesenchymal cells and differentiating osteoblasts during calvarial bone development. In addition, Fgf18 is expressed in the perichondrium and joints of developing long bones. In calvarial bone development of Fgf18-deficient mice generated by gene targeting, the progress of suture closure is delayed. Furthermore, proliferation of calvarial osteogenic mesenchymal cells is decreased, and terminal differentiation to calvarial osteoblasts is specifically delayed. Delay of osteogenic differentiation is also observed in the developing long bones of this mutant. Conversely, chondrocyte proliferation and the number of differentiated chondrocytes are increased. Therefore, FGF18 appears to regulate cell proliferation and differentiation positively in osteogenesis and negatively in chondrogenesis.     

Wnt8a and Wnt3a cooperate in the axial stem cell niche to promote mammalian body axis extension

Background: Vertebrate body axis extension occurs in a head‐to‐tail direction from a caudal progenitor zone that responds to interacting signals. Wnt/β‐catenin signaling is critical for generation of paraxial mesoderm, somite formation, and maintenance of the axial stem cell pool. Body axis extension requires Wnt8a in lower vertebrates, but in mammals Wnt3a is required, although the anterior trunk develops in the absence of Wnt3a. Results: We examined mouse Wnt8a^–/– and Wnt3a^–/– single and double mutants to explore whether mammalian Wnt8a contributes to body axis extension and to determine whether a posterior growth function for Wnt8a is conserved throughout the vertebrate lineage. We find that caudal Wnt8a is expressed only during early somite stages and is required for normal development of the anterior trunk in the absence of Wnt3a. During this time, we show that Wnt8a and Wnt3a cooperate to maintain Fgf8 expression and prevent premature Sox2 up‐regulation in the axial stem cell niche, critical for posterior growth. Similar to Fgf8, Wnt8a requires retinoic acid (RA) signaling to restrict its caudal expression boundary and possesses an upstream RA response element that binds RA receptors. Conclusions: These findings provide new insight into interaction of caudal Wnt‐FGF‐RA signals required for body axis extension. Developmental Dynamics 244:797–807, 2015. © 2015 Wiley Periodicals, Inc.