FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells

Signaling by fibroblast growth factor (FGF) is essential is for trophoblast stem (TS) cells and preimplantation embryos. FGF4 provides essential signaling, but the expression of the complete set of 23 FGF family members has not been analyzed. Here, semi-quantitative RT-PCR and microarray analyses were used to define expression of all FGF ligand mRNA. RT-PCR was done for developmentally important FGF subfamilies, FGF10/FGF22 and FGF8/FGF17/FGF18 as well as FGF11. FGF4 and FGF18 are detected at highest levels by RT-PCR and microarrays. FGF10 was detected at low levels in both assays. FGF11 was detected at moderate levels by microarray, but not by RT-PCR. FGF17 was detected at low levels by array and moderate levels by RT-PCR. FGF8 and FGF22 were detected by RT-PCR, but not by microarrays during late cleavage divisions. FGF8, FGF5, and FGF9 were detected in the oocyte by microarray. FGF2, FGF3, and FGF7 were not detected by RT-PCR or microarrays and FGF13, FGF14, and FGF23 were not detected by microarray. Since a major role of FGF is to maintain TS cells, we tested human and mouse placental cell lines and early gestation human placenta for expression of FGF ligands. Expression in mouse TS cells was compared with preimplantation embryos, and human placental cell line expression was compared with human placenta, to infer which ligands are expressed in placental lineage vs. other cell lineages. The data suggest that human and mouse placenta share FGF18 and its high expression suggests preimplantation and early placental function.     

FGF23 and disorders of phosphate homeostasis

It is well known that fibroblast growth factor (FGF) family members are associated with embryonic development and are critical for basic metabolic functions. This review will focus upon fibroblast growth factor-23 (FGF23) and its roles in disorders associated with phosphate handling. The discovery that mutations in FGF23 were responsible for the isolated renal phosphate wasting disorder autosomal dominant hypophosphatemic rickets (ADHR) has ascribed novel functions to the FGF family. FGF23 circulates in the bloodstream, and animal models demonstrate that FGF23 controls phosphate and Vitamin D homeostasis through the regulation of specific renal proteins. The ADHR mutations in FGF23 produce a protein species less susceptible to proteolytic processing. X-linked hypophosphatemic rickets (XLH), tumor-induced osteomalacia (TIO), and fibrous dysplasia of bone (FD) are disorders involving phosphate homeostasis that share phenotypes with ADHR, indicating that FGF23 may be a common denominator for the pathophysiology of these syndromes. Our understanding of FGF23 will help to develop novel therapies for phosphate wasting disorders, as well as for disorders of increased serum phosphate, such as tumoral calcinosis, a rare disorder, and renal failure, a common disorder.     

Evolutionarily conserved and divergent expression of members of the FGF receptor family among vertebrate embryos, as revealed by FGFR expression patterns in Xenopus

Fibroblast growth factors (FGFs) mediate many cell-cell signaling events during early development. While the actions of FGFs have been well-studied, the roles played by specific members of the FGF receptor (FGFR) family are poorly understood. To characterize the roles played by individual FGFRs we compared the regulation and expression of the three Xenopus FGFRs described to date (XFGFR-1, XFGFR-2, and XFGFR-4). First, we describe the expression of Xenopus FGFR-4; XFGFR-4 is present as a maternal mRNA and is found in the embryo through at least the tadpole stage. XFGFR-4 and XFGFR-1 mRNAs are present at comparable levels, arguing that both mediate FGF signaling during early development. Second, the expression of XFGFR-4 in animal caps differs from the expression of XFGFR-1 and XFGFR-2, suggesting that the FGFRs are independently regulated in ectoderm. Third, using whole-mount in situ hybridization, we show that XFGFR-1, XFGFR-2, and XFGFR-4 are expressed in dramatically different patterns, arguing that specific FGF signaling events are mediated by different members of the FGFR family. Among these, FGF signaling during the induction of neural crest cells is likely to be mediated by XFGFR-4. Comparison of our results with previously reported FGFR expression patterns reveals that FGFR-1 expression is highly conserved among vertebrate embryos, and FGFR-2 expression shows many features that are conserved and some that are divergent. In contrast, the expression pattern of FGFR-4 is highly divergent among vertebrate embryos. Received: 5 August 1999 / Accepted: 18 January 2000     

Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.     

Roles of intracellular fibroblast growth factors in neural development and functions

Fibroblast growth factors (FGFs) can be classified as secretory (FGF1-10 and FGF15-23) or intracellular non-secretory forms (FGF11-14). Secretory forms of FGF and their receptors are best known for their regulatory roles in cell growth, differentiation and morphogenesis in the early stages of neural development. However, the functions of intracellular FGFs remain to be explored. FGF12 and FGF14 are found to interact with voltage-gated sodium channels, and regulate the channel activity in neurons. FGF13 is expressed in primary sensory neurons, and is colocalized with sodium channels at the nodes of Ranvier along the myelinated afferent fibers. FGF13 is also expressed in cerebral cortical neurons during the late developmental stage. A recent study showed that FGF13 is a microtubule-stabilizing protein required for regulating the neuronal development in the cerebral cortex. Thus, non-secretory forms of FGF appear to have important roles in the brain, and it would be interesting to further investigate the functions of intracellular FGFs in the nervous system and in neural diseases.     

Involvement of fibroblast growth factor (FGF)18-FGF8 signaling in specification of left-right asymmetry and brain and limb development of the chick embryo

To elucidate roles of fibroblast growth factors (FGF)18 during vertebrate development, we examined expression patterns of Fgf18 in chick embryos and observed effects of FGF18 protein on the Hensen's node, isthmus, and limb buds. Fgf18 is expressed on the right side of the node before the expression of Fgf8 starts. FGF18 protein can induce expression of Fgf8 on the left side of the node, indicating involvement of both FGFs in specification of left-right asymmetry. In the developing brain, Fgf18 is expressed in the isthmus, following the Fgf8 expression. Since Fgf18 is induced ectopically during formation of the second midbrain by FGF8 protein, both FGFs also elaborate midbrain development. In the limb bud, Fgf18 is expressed in the mesenchyme and ectopic application of FGF18 protein inhibits bone growth in the limb. FGF18 is thus likely an endogenous ligand of FGF receptor 3, whose mutation causes bone dysplasia in humans. These results demonstrate that the FGF18-FGF8 signaling is involved in various organizing activities and the signaling hierarchies between FGF18 and FGF8 seem to change during patterning of different structures.     

Requirements for FGF3 and FGF10 during inner ear formation

Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.     

Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes

Objective:  Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.
Materials and methods:  We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA).
Results:  FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-β), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-β expression, suggesting a decrease in responsiveness of TGF-β to FGF18 stimulation in the dedifferentiated chondrocytes.
Conclusion:  FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.     

FGF23 Suppresses Chondrocyte Proliferation in the Presence of Soluble α-Klotho both in Vitro and in Vivo

Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH.     

AUF1 p42 isoform selectively controls both steady-state and PGE2-induced FGF9 mRNA decay

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays vital roles in many physiologic processes including embryonic development. Aberrant expression of FGF9 causes human diseases and thus it highlights the importance of controlling FGF9 expression; however, the mechanism responsible for regulation of FGF9 expression is largely unknown. Here, we show the crucial role of an AU-rich element (ARE) in FGF9 3′-untranslated region (UTR) on controlling FGF9 expression. Our data demonstrated that AUF1 binds to this ARE to regulate FGF9 mRNA stability. Overexpression of each isoform of AUF1 (p37, p40, p42 and p45) showed that only the p42 isoform reduced the steady-state FGF9 mRNA. Also, knockdown of p42^AUF1 prolonged the half-life of FGF9 mRNA. The induction of FGF9 mRNA in prostaglandin (PG) E₂-treated human endometrial stromal cells was accompanied with declined cytoplasmic AUF1. Nevertheless, ablation of AUF1 led to sustained elevation of FGF9 expression in these cells. Our study demonstrated that p42^AUF1 regulates both steady-state and PGE₂-induced FGF9 mRNA stability through ARE-mediated mRNA degradation. Since almost half of the FGF family members are ARE-containing genes, our findings also suggest that ARE-mediated mRNA decay is a common pathway to control FGFs expression, and it represents a novel RNA regulon to coordinate FGFs homeostasis in various physiological conditions.     

RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.     

Hormone-like fibroblast growth factors and metabolic regulation

The family of fibroblast growth factors (FGFs) consisting now of 22 members is generally considered to control a wide range of biological functions such as development, differentiation and survival. However, research during the past decade provided substantial evidence that a so called “hormone-like” subgroup of FGFs, comprised of FGF19, FGF21 and FGF23, is involved in the regulation of diverse metabolic pathways to control glucose, lipid, bile acid, phosphate and vitamin D metabolism. The unique properties of these FGFs include predominant production of the factors in selective tissues, their abundance in the blood due to the lack of extracellular heparin-mediated sequestration, and highly specific tissue-targeted action via engagement of their respective co-receptors. The important metabolic context of FGF19, FGF21, and FGF23 actions has revealed important novel roles for FGFs and provided significant means to explore an opportunity for therapeutic targeting of these factors and their corresponding pathways.     

Expression of members of the Fgf family and their receptors during midfacial development

Members of the FGF family play diverse roles in patterning, cell proliferation and differentiation during embryogenesis. To begin to address their function during craniofacial development we have analyzed the expression of 18 members of the Fgf family (Fgf1-15, -17, -18 and -20) and the four members of the FGF-receptor family in the prospective midfacial region between E9.5 and E11.5 by whole-mount in situ hybridization. We show that at E9.5, Fgf3, -8, -9, -10 and -17 are broadly expressed in midfacial ectoderm. Concomitant with the outgrowth of the nasal processes at E10.5, expression of Fgf3, -8, -9, -10, -15, -17 and -18 was detected in spatially restricted regions of ectoderm at the edge of the nasal pit and at the oral edge of the medial nasal process. Expression of Fgf8, Fgf9, Fgf10 and Fgf17 was still observed in these domains at E11.5. In contrast to the restricted expression patterns of the ligands, FgfR1 and FgfR2 were broadly expressed in facial mesenchyme and ectoderm, respectively, indicating a wide competence of midfacial tissue to respond to FGF signaling.     

Plasma Fibroblast Growth Factor 23 Concentration Is Increased and Predicts Mortality in Patients on the Liver-Transplant Waiting List

High plasma fibroblast growth factor-23 (FGF23) concentration predicts the risk of death and poor outcomes in patients with chronic kidney disease or chronic heart failure. We checked if FGF23 concentration could be modified in patients with end stage liver disease (ESLD) and predict mortality. We measured plasma FGF23 in 200 patients with ESLD registered on a liver transplant waiting list between January 2005 and October 2008. We found that median plasma FGF23 concentration was above normal values in 63% of the patients. Increased FGF23 concentration was not explained by its classical determinants: hyperphosphataemia, increased calcitriol concentration or decreased renal function. FGF23 concentration correlated with the MELD score, serum sodium concentration, and GFR. Forty-six patients died before being transplanted and 135 underwent liver transplantation. We analyzed the prognostic value of FGF23 levels. Mortality was significantly associated with FGF23 levels, the MELD score, serum sodium concentration and glomerular filtration rate. On multivariate analyses only FGF23 concentration was associated with mortality. FGF23 levels were independent of the cause of the liver disease. To determine if the damaged liver can produce FGF23 we measured plasma FGF23 concentration and liver FGF23 mRNA expression in control and diethyl-nitrosamine (DEN)-treated mice. FGF23 plasma levels increased with the apparition of liver lesions in DEN-treated mice and that FGF23 mRNA expression, which was undetectable in the liver of control mice, markedly increased with the development of liver lesions. The correlation between FGF23 plasma concentration and FGF23 mRNA expression in DEN-treated mice suggests that FGF23 production by the liver accounts for the increased plasma FGF23 concentration. In conclusion chronic liver lesions can induce expression of FGF23 mRNA leading to increased FGF23 concentration, which is associated with a higher mortality in patients on a liver-transplant waiting list. In these patients FGF23 concentration was the best predictor of mortality.     

Fibroblast growth factor control of cartilage homeostasis

Osteoarthritis (OA) and degenerative disc disease (DDD) are similar diseases involving the breakdown of cartilage tissue, and a better understanding of the underlying biochemical processes involved in cartilage degeneration may allow for the development of novel biologic therapies aimed at slowing the disease process. Three members of the fibroblast growth factor (FGF) family, FGF‐2, FGF‐18, and FGF‐8, have been implicated as contributing factors in cartilage homeostasis. The role of FGF‐2 is controversial in both articular and intervertebral disc (IVD) cartilage as it has been associated with species‐ and age‐dependent anabolic or catabolic events. Recent evidence suggests that FGF‐2 selectively activates FGF receptor 1 (FGFR1) to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix‐degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF‐18, on the other hand, most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF‐8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage, but its precise biological impact on human adult articular cartilage or IVD tissue remains unknown. The available evidence reveals the promise of FGF‐2/FGFR1 antagonists, FGF‐18/FGFR3 agonists, and FGF‐8 antagonists (i.e., anti‐FGF‐8 antibody) as potential therapies to prevent cartilage degeneration and/or promote cartilage regeneration and repair in the future. J. Cell. Biochem. 114: 735–742, 2013. © 2012 Wiley Periodicals, Inc.     

FGF-2 deficiency does not influence FGF ligand and receptor expression during development of the nigrostriatal system

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.     

Cloning and expression analysis of Fgf5, 6 and 7 during early chick development

FGFs with similar sequences can play different roles depending on the model organisms examined. Determining these roles requires knowledge of spatio-temporal Fgf gene expression patterns. In this study, we report the cloning of chick Fgf5, 6 and 7, and examine their gene expression patterns by whole mount in situ hybridization. We show that Fgf5's spatio-temporally restricted expression pattern indicates a potentially novel role during inner ear development. Fgf6 and Fgf7, although belonging to different subfamilies with diverged sequences, are expressed in similar patterns within the mesoderm. Alignment of protein sequences and phylogenetic analysis demonstrate that FGF5 and FGF6 are highly conserved between chick, human, mouse and zebrafish. FGF7 is similarly conserved except for the zebrafish, which has considerably diverged.