FGF receptors 1 and 2 control chemically induced injury and compound detoxification in regenerating livers of mice.

BACKGROUND & AIMS: Fibroblast growth factor receptor 4 (FGFR4) controls bile acid metabolism and protects the liver from fibrosis, but the roles of FGFR1 and FGFR2 in the adult liver are largely unknown. We investigated the functions and mechanisms of action of these receptors in liver homeostasis, regeneration, and fibrosis. METHODS: We generated mice with hepatocytes that lack FGFR1 and FGFR2 and subjected them to acute and chronic carbon tetrachloride-induced liver injury and partial hepatectomy; mice were also injected with FGF7. We performed histology, histomorphometry, real-time reverse transcription polymerase chain reaction, and immunoblot analyses. RESULTS: In hepatocytes, loss of FGFR1 and FGFR2 eliminated responsiveness to FGF7 and related FGF family members but did not affect toxin-induced liver injury and fibrosis. However, mortality after partial hepatectomy increased because of severe hepatocyte necrosis. These effects appeared to be mediated by a failure of hepatocytes to induce the expression of the transcriptional regulators Dbp and Tef upon liver surgery; this affected expression of their target genes, which encode detoxifying cytochrome P450 enzymes. We found that Dbp and Tef expression was directly controlled by FGFR signaling in hepatocytes. As a consequence of the reduced expression of genes that control detoxification, the liver tissue that remained after partial hepatectomy failed to efficiently metabolize endogenous compounds and the drugs applied for anesthesia/analgesia. CONCLUSIONS: We identified a new, cytoprotective effect of FGFR1 and FGFR2 in the regenerating liver and suggest the use of recombinant FGF7 to increase survival of patients after surgical resection of large amounts of liver tissue.

Evidence that Fgf10 contributes to the skeletal and visceral defects of an Apert syndrome mouse model.

Apert syndrome (AS) is a severe congenital disease caused by mutations in fibroblast growth factor receptor-2 (FGFR2), and characterised by craniofacial, limb, visceral, and neural abnormalities. AS-type FGFR2 molecules exert a gain-of-function effect in a ligand-dependent manner, but the causative FGFs and their relative contribution to each of the abnormalities observed in AS remains unknown. We have generated mice that harbour an AS mutation but are deficient in or heterozygous for Fgf10. The genetic knockdown of Fgf10 can rescue the skeletal as well as some of the visceral defects observed in this AS model, and restore a near normal level of FgfR2 signaling involving an apparent switch between ERK(p44/p42) and p38 phosphorylation. Surprisingly, it can also yield de novo cleft palate and blind colon in a subset of the compound mutants. These findings strongly suggest that Fgf10 contributes to AS-like pathologies and highlight a complexity of Fgf10 function in different tissues.

Fgfr2b mediated epithelial-mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning.

Dental trigeminal nerve fiber growth and patterning are strictly integrated with tooth morphogenesis, but it is still unknown, how these two developmental processes are coordinated. Here we show that targeted inactivation of the dental epithelium expressed Fgfr2b results in cessation of the mouse mandibular first molar development at the degenerated cap stage and the failure of the trigeminal molar nerve to establish the lingual branch at E13.5 stage while the buccal branch develops properly. This axon patterning defect correlates to the histological absence of the mesenchymal dental follicle and adjacent Semaphorin3A-free dental follicle target field as well as appearance of ectopic Sema3A expression domain in the lingual side of the epithelial bud. Although the mesenchymal ligands for Fgfr2b, Fgf3 and -10 were present in the Fgfr2b(-/)(-) dental mesenchyme, mutant dental epithelium showed dramatically reduced proliferation and the lack of Fgf3. Tgfbeta1, which controls Sema3A was absent from the Fgfr2b(-/-) tooth germ, and Sema3A was specifically downregulated in the dental mesenchyme at the bud and cap stage. In addition, the epithelial primary enamel knot signaling center although being molecularly present neither was histologically detectable nor expressed Bmp4 and Fgf3 as well as Fgf4, which is essential for tooth morphogenesis and stimulates mesenchymal Fgf3 and Tgfbeta1. Fgf4 beads rescued Tgfbeta1 in the Fgfr2b(-/-) dental mesenchyme explants and Tgfbeta1 induced de novo Sema3A expression in the dental mesenchyme. Collectively these results demonstrate that epithelial Fgfr2b controls tooth morphogenesis and dental axon patterning, and suggests that Fgfr2b, by mediating local epithelial-mesenchymal interactions, integrates these two distinct developmental processes during odontogenesis.

The role of fibroblast growth factor receptor 2b in skin homeostasis and cancer development.

The epithelial isoform of fibroblast growth factor receptor 2 (Fgfr2b) is essential for embryogenesis, and Fgfr2b-null mice die at birth. Using Cre-Lox transgenics to delete Fgfr2b in cells expressing keratin 5, we show that mice lacking epidermal Fgfr2b survive into adulthood but display striking abnormalities in hair and sebaceous gland development. Epidermal hyperthickening develops with age, and 10% of mutant mice develop spontaneous papillomas, demonstrating the role of Fgfr2b in post-natal skin development and in adult skin homeostasis. Mice lacking epithelial Fgfr2b show great sensitivity to chemical carcinogenic insult, displaying several oncogenic ha-ras mutations with dramatic development of papillomas and squamous cell carcinomas. Mutant mice have increased inflammation in the skin, with increased numbers of macrophages and gammadeltaT cells with abnormal morphology. Mutant skin shows several changes in gene expression, including enhanced expression of the pro-inflammatory cytokine interleukin 18 and decreased expression of Serpin a3b, a potential tumor suppressor. Thus we describe a novel role of Fgfr2b and provide the first evidence of a tyrosine kinase receptor playing a tumor suppressive role in the skin.

All-trans retinoic acid suppresses exocrine differentiation and branching morphogenesis in the embryonic pancreas.

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.

Congenital heart defects in Fgfr2-IIIb and Fgf10 mutant mice.

OBJECTIVE: Myocardial progenitor cells expressing Fgf10 give rise to the outflow tract and right ventricle of the mammalian heart. In order to define the role of fibroblast growth factor (FGF) signaling in this process we investigated whether Fgf10 or the major Fgf10 receptor Fgfr2-IIIb are required for normal heart development. METHODS: The cardiac phenotype of Fgf10 and Fgfr2-IIIb mutant mice was analysed by histology, scanning electron microscopy and gene and transgene expression studies. RESULTS: Outflow tract formation from Fgf10 expressing progenitor cells occurs normally in Fgf10 mutant embryos and in the majority of Fgfr2-IIIb mutant embryos; a proportion of Fgfr2-IIIb mutant embryos, however, display outflow tract and right ventricular hypoplasia. The predominant cardiac defects in Fgfr2-IIIb mutant embryos are ventricular septal defects associated with overriding aorta or double outlet right ventricle. In addition, loss of Fgfr2-IIIb is associated with ventricular anomalies including a thin myocardial wall, abnormal trabeculation and muscular ventricular septal defects. In contrast, Fgf10 is required to correctly position the heart in the thoracic cavity but not for outflow tract septation. Both Fgf10 and Fgfr2-IIIb mutant embryos lack pulmonary arteries and veins. CONCLUSIONS: Fgfr2-IIIb and Fgf10 mutant mice have distinct roles during cardiac morphogenesis, although neither gene is essential for outflow tract elongation from Fgf10 expressing progenitor cells. Fgfr2-IIIb and Fgf10 mutant mice provide new models for common components of congenital heart disease.

Stomach development is dependent on fibroblast growth factor 10/fibroblast growth factor receptor 2b-mediated signaling.

BACKGROUND & AIMS: Fibroblast growth factors (Fgfs) and their receptors (Fgfrs) are important intercellular signaling molecules that are essential to mammalian embryonic development. The signaling pathways between endoderm-derived gastric epithelium and the surrounding mesenchyme are largely unknown; however, the developmental expression profile of the IIIb isoform of Fgfr2 (Fgfr2b) and its main ligand, Fgf10, suggest that they may be strong candidates. Mice lacking either component (Fgfr2b-/- or Fgf10-/-) were examined to determine the role of Fgfr2b-mediated signaling during gastric organogenesis. METHODS: Stomachs from embryonic day 13.5-18.5 Fgfr2b-/-, Fgf10-/-, and wild-type littermates were collected and analyzed by conventional histology, immunohistochemistry, in situ hybridization, and electron microscopy. RESULTS: Fgfr2b-/- and Fgf10-/- fetuses had stomachs smaller than wild-type, consisting of relatively proportionate forestomach but disproportionately reduced glandular stomach, the mucosa of which has low cytoarchitectural complexity with a spiral arrangement of large mucosal folds. During mid to late fetal stages (embryonic day 15.5-18.5), epithelial differentiation to mucous and chief cell lineages was rudimentary, with no expression of several early cytodifferentiation markers including GATA4, GATA6, and H+/K+-adenosine triphosphatase and abnormal expression of members of the hedgehog family of signaling molecules. CONCLUSIONS: Fgfr2b and Fgf10 are part of a signaling network with Sonic hedgehog and Indian hedgehog that are essential to anterior-posterior and radial patterning in gastric development.

Fibroblast growth factor signaling in tumorigenesis.

Fibroblast growth factors and their signaling receptors have been associated with multiple biological activities, including proliferation, differentiation and motility. Consequently, they have evoked interest as candidate oncogenes with the potential to initiate and/or promote tumorigenesis. This has resulted in a large literature describing the presence of these growth factors and their receptors in cancer cell lines and primary tumors of diverse origin. However, it is only recently that compelling evidence has emerged to implicate the fibroblast growth factors (Fgfs) and their receptors in the genesis of human cancers. Here, we outline the model systems that demonstrate the potential oncogenic nature of Fgf signaling and summarise recent evidence that implicates aberrant Fgf signaling as important in the natural history of some common human cancers.

Development of the mammalian urethra is controlled by Fgfr2-IIIb.

Development of external genitalia in mammalian embryos requires tight coordination of a complex series of morphogenetic events involving outgrowth, proximodistal and dorsoventral patterning, and epithelial tubulogenesis. Hypospadias is a congenital defect of the external genitalia that results from failure of urethral tube closure. Although this is the second most common birth defect in humans, affecting one in every 250 children, the molecular mechanisms that regulate morphogenesis of the mammalian urethra are poorly understood. We report that mice lacking the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2) exhibit severe hypospadias. Urethral signaling regions, as indicated by Shh and Fgf8 expression, are established in Fgfr2-IIIb null mice; however, cell proliferation arrests prematurely and maturation of the urethral epithelium is disrupted. Fgfr2-IIIb-/- mutants fail to maintain the progenitor cell population required for uroepithelial renewal during tubular morphogenesis. In addition, we show that antagonism of the androgen receptor (AR) leads to loss of Fgfr2-IIIb and Fgf10 expression in the urethra, and an associated hypospadias phenotype, suggesting that these genes are downstream targets of AR during external genital development. Genitourinary defects resulting from disruption of AR activity, by either genetic or environmental factors, may therefore involve negative regulation of the Fgfr2 pathway. This represents the first example of how the developing genitourinary system integrates cues from systemically circulating steroid hormones with a locally expressed growth factor pathway.

A crucial role for fibroblast growth factor signaling in embryonic mammary gland development.

The fibroblast growth factors (Fgfs) represent a large group of intercellular signaling molecules that mediate their effects by binding to a class of cell surface enzymes belonging to the receptor tyrosine kinase family (FgfRs). In vitro, Fgf signaling can induce potent mitogenic, motogenic, and angiogenic cellular responses, and has been associated with a multitude of biological processes. The development of gene targeting and transgenic strategies has provided unequivocal evidence for the key involvement of Fgf signaling in mammalian developmental processes. In this review we highlight recent findings that demonstrate a critical requirement for Fgf signaling in the induction and development of the embryonic mammary gland. Furthermore, we briefly discuss the potential of Fgfs to act as oncogenic factors in mammary neoplasia.

Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate.

Classical research has suggested that early palate formation develops via epithelial-mesenchymal interactions, and in this study we reveal which signals control this process. Using Fgf10-/-, FGF receptor 2b-/- (Fgfr2b-/-), and Sonic hedgehog (Shh) mutant mice, which all exhibit cleft palate, we show that Shh is a downstream target of Fgf10/Fgfr2b signaling. Our results demonstrate that mesenchymal Fgf10 regulates the epithelial expression of Shh, which in turn signals back to the mesenchyme. This was confirmed by demonstrating that cell proliferation is decreased not only in the palatal epithelium but also in the mesenchyme of Fgfr2b-/- mice. These results reveal a new role for Fgf signaling in mammalian palate development. We show that coordinated epithelial-mesenchymal interactions are essential during the initial stages of palate development and require an Fgf-Shh signaling network.

SOX7 and GATA-4 are competitive activators of Fgf-3 transcription.

Fgf-3 is expressed in a dynamic and complex spatiotemporal pattern during mouse development. Previous studies identified GATA-4 as a transcription factor that binds the key regulatory element PS4A of the Fgf-3 promoter and stimulates transcription. Here we show that members of the SOX family of transcription factors also bind PS4A and differentially modulate transcription. At least five SOX genes, Sox2, Sox6, Sox7, Sox13, and Sox17, were expressed in F9 cells, and of these, Sox7 and Sox17 were dramatically induced in parallel with Fgf-3 following differentiation into parietal endoderm-like cells with retinoic acid and dibutyryl cAMP. Complexes could be detected on PS4A with SOX2, SOX7, and SOX17 by using nuclear extracts from differentiated F9 cells. However, only Sox7 expression markedly activated the Fgf-3 promoter in these cells. By contrast, SOX2 was a poor activator of Fgf-3 transcription, and when Sox2 was coexpressed with Gata4, it negatively modulated the strong activation mediated by GATA-4. More detailed analyses showed that SOX7 competes with GATA-4 for PS4A occupancy and to activate the Fgf-3 promoter. In situ hybridization analysis showed that Sox7 is co-expressed with Fgf-3 and Gata4 in the parietal endoderm of E7.5 mouse embryos. In culture, GATA-4-deficient embryonal stem cells were shown to express Fgf-3 upon differentiation into embryoid bodies, although at lower levels than were found in wild type embryonal stem cells. This Fgf-3 expression was virtually abolished when Sox7 expression was suppressed by RNA interference. These results show that SOX7 is a potent activator of Fgf-3 transcription.

Regulatory analysis of the mouse Fgf3 gene: control of embryonic expression patterns and dependence upon sonic hedgehog (Shh) signalling.

Fgf3 displays a dynamic and complex expression pattern during mouse embryogenesis. To address the molecular mechanisms underlying Fgf3 expression, we used a transgenic approach to assay genomic regions from the mouse Fgf3 gene for regulatory activity. We identified an enhancer that mediates major components of embryonic expression, governing expression in the midbrain, hindbrain, surface ectoderm, dorsal roots and dorsal root ganglia (DRG), proximal sensory ganglia, and the developing central nervous system (CNS). Deletional analysis of the enhancer further delimited this regulatory activity to a 5.7-kb fragment. We have also revealed sonic hedgehog (Shh) -dependent and Shh-independent aspects of Fgf3 expression through breeding the Fgf3 reporter transgene into Shh mutants. In the absence of Shh signalling, Fgf3 reporter expression is lost in the ventral CNS, DRG, and superior cervical nerves, whereas activation of reporter expression in cranial ganglion cells is Shh independent. Moreover, detailed re-examination of the Shh phenotype revealed that Shh signalling is required for the correct development/maturation of the DRG.

A crucial role for Fgfr2-IIIb signalling in epidermal development and hair follicle patterning.

To understand the role Fgf signalling in skin and hair follicle development, we analysed the phenotype of mice deficient for Fgfr2-IIIb and its main ligand Fgf10. These studies showed that the severe epidermal hypoplasia found in mice null for Fgfr2-IIIb is caused by a lack of the basal cell proliferation that normally results in a stratified epidermis. Although at term the epidermis of Fgfr2-IIIb null mice is only two to three cells thick, it expresses the classical markers of epidermal differentiation and establishes a functional barrier. Mice deficient for Fgf10 display a similar but less severe epidermal hypoplasia. By contrast, Fgfr2-IIIb-/-, but not Fgf10-/-, mice produced significantly fewer hair follicles, and their follicles were developmentally retarded. Following transplantation onto nude mice, grafts of Fgfr2-IIIb-/- skin showed impaired hair formation, with a decrease in hair density and the production of abnormal pelage hairs. Expression of Lef1, Shh and Bmp4 in the developing hair follicles of Fgfr2-IIIb-/- mice was similar to wild type. These results suggest that Fgf signalling positively regulates the number of keratinocytes needed to form a normal stratified epidermis and to initiate hair placode formation. In addition, Fgf signals are required for the growth and patterning of pelage hairs.

Fibroblast growth factor 22 and its potential role during skin development and repair.

We have isolated, using RT-PCR, a cDNA from mouse skin wounds that encodes fibroblast growth factor (FGF) 22, a recently discovered member of the FGF family, which is closely related to FGF-7 and FGF-10. Transient expression of tagged FGF-22 protein in COS-1 and MCF-7 cells revealed that the protein was present within the cell and at the cell surface but was not apparently released from the cell. Analysis of RNA expression revealed that FGF-22 transcripts were not detected in the developing mouse embryo until day E16.5 and in the adult mouse it was expressed in the brain, tongue, and skin, but not in other tissues examined. After skin injury, FGF-22 mRNA levels were slightly down-regulated within the first 5 days after wounding, but expression increased strongly at the later stages of the repair process. In situ hybridization revealed the presence of FGF-22 mRNA throughout the epidermis and hair follicle keratinocytes of E16.5 embryos, as well as in adult skin and keratinocytes of the hyperthickened wound epithelium. This expression pattern suggests a potential role for FGF-22 in cutaneous development and repair.

The IIIb isoform of fibroblast growth factor receptor 2 is required for proper growth and branching of pancreatic ductal epithelium but not for differentiation of exocrine or endocrine cells.

Fibroblast growth factors (Fgfs) and their receptors have been implicated in embryonic pancreas development. Recently it was shown that Fgf10, a major ligand for the IIIb isoform of fibroblast growth factor receptor 2 (Fgfr2b), has an important regulatory role in early pancreas development. The aim of our study was to define the role of Fgfr2b in pancreas development by analyzing the phenotype of Fgfr2b (-/-) mice. Pancreases of Fgfr2b (-/-) embryos were noticeably smaller than the wild type littermates during embryogenesis, and pancreatic ductal branching as well as duct cell proliferation was significantly reduced. However, both exocrine and endocrine pancreatic differentiation occurred relatively normally. Exogenous addition of Fgfr2b ligands (Fgf7 and Fgf10) stimulated duct cell proliferation and inhibited endocrine cell differentiation in the ex vivo embryonic organ cultures of wild type pancreas. Our results thus suggest that Fgfr2b-mediated signaling plays a major role in pancreatic ductal proliferation and branching morphogenesis, but has little effect on endocrine and exocrine differentiation.

Cyclin D1 is not an essential target of beta-catenin signaling during intestinal tumorigenesis, but it may act as a modifier of disease severity in multiple intestinal neoplasia (Min) mice.

Deregulation of beta-catenin activity is an important step in the development of colorectal cancers. One consequence of this is transcriptional activation of cyclin D1, an oncogene known to be overexpressed in colorectal cancers. We tested the hypothesis that cyclin D1 gene activation is important for intestinal tumorigenesis. Multiple intestinal neoplasia mice (a model for human familial adenomatous polyposis) were crossed with cyclin D1 knockout (Ccnd1(-/-)) mice. Despite the absence of cyclin D1, intestinal tumors still developed. However, Ccnd1(-/-) multiple intestinal neoplasia mice developed significantly fewer tumors than Ccnd1(+/-) or Ccnd1(+/+) mice (P = 0.003). We conclude that cyclin D1 is not essential for intestinal tumorigenesis, but it may act as a modifier gene.

Basolateral targeting of ERBB2 is dependent on a novel bipartite juxtamembrane sorting signal but independent of the C-terminal ERBIN-binding domain.

ERBB2 is a receptor tyrosine kinase present on the basolateral membrane of polarized epithelia and has important functions in organ development and tumorigenesis. Using mutagenic analyses and Madin-Darby canine kidney (MDCK) cells, we have investigated the signals that regulate basolateral targeting of ERBB2. We show that basolateral delivery of ERBB2 is dependent on a novel bipartite juxtamembrane sorting signal residing between Gln-692 and Thr-701. The signal shows only limited sequence homology to known basolateral targeting signals and is both necessary and sufficient for correct sorting of ERBB2. In addition we demonstrate that this motif can function as a dominant basolateral targeting signal by its ability to redirect the apically localized P75 neurotrophin receptor to the basolateral membrane domain of polarized epithelial cells. Interestingly, LLC-PK1 cells, which are deficient for the micro 1B subunit of the AP1B adaptor complex, missort a large proportion of ERBB2 to the apical membrane domain. This missorting can be partially corrected by the introduction of micro 1B, suggesting a possible role for AP1B in ERBB2 endosomal trafficking. Furthermore, we find that the C-terminal ERBIN binding domain of ERBB2 is not necessary for its basolateral targeting in MDCK cells.