Exogenous FGF10 can rescue an eye-open at birth phenotype of Fgf10-null mice by activating activin and TGFalpha-EGFR signaling

Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit an eye-open phenotype at birth. It has previously been shown that FGF10 has a dual role in proliferation and migration during the early and later stages of eyelid development, respectively. To verify the role of FGF10 during eyelid closure, explant culture of Fgf10-null eyelid anlagen was performed, by which it was examined whether or not exogenous FGF10 could rescue the expression of activin betaB and transforming growth factor alpha, known to be required for eyelid closure. We found that the expression of these genes was markedly induced while that of Shh or Ptch1, Ptch2 was not. We also observed the distribution of filamentous actin (F-actin) after FGF10 application in the mutant eyelid explant, finding that the FGF10 protein induced F-actin accumulation. We further examined filopodia of the eyelid leading edge cells, finding the length of the filopodia was significantly reduced in the mutant. These results verify that FGF10 promotes eyelid closure through activating activin and TGFalpha-EGFR signaling.     

Growth arrest specific gene 1 acts as a region-specific mediator of the Fgf10/Fgf8 regulatory loop in the limb

Proximal-to-distal growth of the embryonic limbs requires Fgf10 in the mesenchyme to activate Fgf8 in the apical ectodermal ridge (AER), which in turn promotes mesenchymal outgrowth. We show here that the growth arrest specific gene 1 (Gas1) is required in the mesenchyme for the normal regulation of Fgf10/Fgf8. Gas1 mutant limbs have defects in the proliferation of the AER and the mesenchyme and develop with small autopods, missing phalanges and anterior digit syndactyly. At the molecular level, Fgf10 expression at the distal tip mesenchyme immediately underneath the AER is preferentially affected in the mutant limb, coinciding with the loss of Fgf8 expression in the AER. To test whether FGF10 deficiency is an underlying cause of the Gas1 mutant phenotype, we employed a limb culture system in conjunction with microinjection of recombinant proteins. In this system, FGF10 but not FGF8 protein injected into the mutant distal tip mesenchyme restores Fgf8 expression in the AER. Our data provide evidence that Gas1 acts to maintain high levels of FGF10 at the tip mesenchyme and support the proposal that Fgf10 expression in this region is crucial for maintaining Fgf8 expression in the AER.     

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in α-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.     

Fibroblast growth factor 10 is required for proper development of the mouse whiskers

Fibroblast Growth Factor (FGF) signaling is known to play an important role during cutaneous development. To elucidate the role of FGF10 during whisker formation, we examined the expression of Fgf10 in normal developing whiskers and phenotypes of Fgf10-deficient whiskers. Fgf10 is first expressed in the maxillary process, lateral and medial nasal processes, then in the mesenchymal cells underneath the future whisker placodes, and in the surrounding mesenchyme of developing whiskers. Fgf10-null whiskers exhibit a significant decrease in number and their structure is disorganized as revealed by scanning electron microscopy. Hair follicle marker genes such as Sonic hedgehog, Patched, and Patched 2 are aberrantly expressed in the mutant whiskers. Thus, FGF10 is required for proper whisker development mediated by SHH signaling in the mouse.     

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, we show that the pool of Fgf10-positive cells in the distal lung mesenchyme contains progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed us to create a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease in beta-galactosidase-positive cells around the bronchial epithelium associated with an accumulation of beta-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of alpha smooth muscle actin expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. We provide support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells.     

HB-EGF promotes epithelial cell migration in eyelid development

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and ERBB4. Here, we show that HB-EGF-EGFR signaling is involved in eyelid development. HB-EGF expression is restricted to the tip of the leading edge of the migrating epithelium during eyelid closure in late gestation mouse embryos. Both HB-EGF null (HB(del/del)) and secretion-deficient (HB(uc/uc)) mutant embryos exhibited delayed eyelid closure, owing to slower leading edge extension and reduced actin bundle formation in migrating epithelial cells. No changes in cell proliferation were observed in these embryos. In addition, activation of EGFR and ERK was decreased in HB(del/del) eyelids. Crosses between HB(del/del) mice and waved 2 mice, a hypomorphic EGFR mutant strain, indicate that HB-EGF and EGFR interact genetically in eyelid closure. Together with our data showing that embryos treated with an EGFR-specific kinase inhibitor phenocopy HB(del/del) embryos, these data indicate that EGFR mediates HB-EGF-dependent eyelid closure. Finally, analysis of eyelid closure in TGFalpha-null mice and in HB-EGF and TGFalpha double null mice revealed that HB-EGF and TGFalpha contribute equally to and function synergistically in this process. These results indicate that soluble HB-EGF secreted from the tip of the leading edge activates the EGFR and ERK pathway, and that synergy with TGFalpha is required for leading edge extension in epithelial sheet migration during eyelid closure.     

Fibroblast Growth Factor 10-Fibroblast Growth Factor Receptor 2b Mediated Signaling Is Not Required for Adult Glandular Stomach Homeostasis

The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10''s main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.     

Neuron-derived FGF9 is essential for scaffold formation of Bergmann radial fibers and migration of granule neurons in the cerebellum

Although fibroblast growth factor 9 (FGF9) is widely expressed in the central nervous system (CNS), the function of FGF9 in neural development remains undefined. To address this question, we deleted the Fgf9 gene specifically in the neural tube and demonstrated that FGF9 plays a key role in the postnatal migration of cerebellar granule neurons. Fgf9-null mice showed severe ataxia associated with disrupted Bergmann fiber scaffold formation, impaired granule neuron migration, and upset Purkinje cell maturation. Ex vivo cultured wildtype or Fgf9-null glia displayed a stellate morphology. Coculture with wildtype neurons, but not Fgf9-deficient neurons, or treating with FGF1 or FGF9 induced the cells to adopt a radial glial morphology. In situ hybridization showed that Fgf9 was expressed in neurons and immunostaining revealed that FGF9 was broadly distributed in both neurons and Bergmann glial radial fibers. Genetic analyses revealed that the FGF9 activities in cerebellar development are primarily transduced by FGF receptors 1 and 2. Furthermore, inhibition of the MAP kinase pathway, but not the PI3K/AKT pathway, abrogated the FGF activity to induce glial morphological changes, suggesting that the activity is mediated by the MAP kinase pathway. This work demonstrates that granule neurons secrete FGF9 to control formation of the Bergmann fiber scaffold, which in turn, guides their own inward migration and maturation of Purkinje cells.     

FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for na?ve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.     

Development of the thymus requires signaling through the fibroblast growth factor receptor R2-IIIb

Mice deficient for fibroblast growth factor (Fgf)R2-IIIb show a block in thymic growth after embryonic day 12.5, a stage that just precedes its detection in thymic epithelial cells. Fgf7 and Fgf10, the main ligands for FgfR2-IIIb, are expressed in the mesenchyme surrounding the thymic epithelial primordium, and Fgf10-deficient mice also exhibit impaired thymic growth. Hence, Fgf signaling is essential for thymic epithelial proliferation. In addition to the proliferative block, most thymic epithelial cells fail to progress from an immature cytokeratin 5-positive to a cytokeratin 5-negative phenotype. Nevertheless, sufficient epithelial cell differentiation occurs in the severely hypoplastic thymus to allow the development of CD4/CD8-double-positive thymocytes and a very small number of single-positive thymocytes expressing TCRs.     

An FGF3-BMP Signaling Axis Regulates Caudal Neural Tube Closure,Neural Crest Specification and Anterior-Posterior Axis Extension

During vertebrate axis extension, adjacent tissue layers undergo profound morphological changes: within the neuroepithelium, neural tube closure and neural crest formation are occurring, while within the paraxial mesoderm somites are segmenting from the presomitic mesoderm (PSM). Little is known about the signals between these tissues that regulate their coordinated morphogenesis. Here, we analyze the posterior axis truncation of mouse Fgf3 null homozygotes and demonstrate that the earliest role of PSM-derived FGF3 is to regulate BMP signals in the adjacent neuroepithelium. FGF3 loss causes elevated BMP signals leading to increased neuroepithelium proliferation, delay in neural tube closure and premature neural crest specification. We demonstrate that elevated BMP4 depletes PSM progenitors in vitro, phenocopying the Fgf3 mutant, suggesting that excessive BMP signals cause the Fgf3 axis defect. To test this in vivo we increased BMP signaling in Fgf3 mutants by removing one copy of Noggin, which encodes a BMP antagonist. In such mutants, all parameters of the Fgf3 phenotype were exacerbated: neural tube closure delay, premature neural crest specification, and premature axis termination. Conversely, genetically decreasing BMP signaling in Fgf3 mutants, via loss of BMP receptor activity, alleviates morphological defects. Aberrant apoptosis is observed in the Fgf3 mutant tailbud. However, we demonstrate that cell death does not cause the Fgf3 phenotype: blocking apoptosis via deletion of pro-apoptotic genes surprisingly increases all Fgf3 defects including causing spina bifida. We demonstrate that this counterintuitive consequence of blocking apoptosis is caused by the increased survival of BMP-producing cells in the neuroepithelium. Thus, we show that FGF3 in the caudal vertebrate embryo regulates BMP signaling in the neuroepithelium, which in turn regulates neural tube closure, neural crest specification and axis termination. Uncovering this FGF3-BMP signaling axis is a major advance toward understanding how these tissue layers interact during axis extension with important implications in human disease.     

Dynamic Fgf signaling couples morphogenesis and migration in the zebrafish lateral line primordium

The collective migration of cells in the form of cohesive tissues is a hallmark of both morphogenesis and repair. The extrinsic cues that direct these complex migrations usually act by regulating the dynamics of a specific subset of cells, those at the leading edge. Given that normally the function of tissue migration is to lay down multicellular structures, such as branched epithelial networks or sensory organs, it is surprising how little is known about the mechanisms that organize cells behind the leading edge. Cells of the zebrafish lateral line primordium switch from mesenchyme-like leader cells to epithelial rosettes that develop into mechanosensory organs. Here, we show that this transition is regulated by an Fgf signaling circuit that is active within the migrating primordium. Point sources of Fgf ligand drive surrounding cells towards a ;non-leader' fate by increasing their epithelial character, a prerequisite for rosette formation. We demonstrate that the dynamic expression of Fgf ligands determines the spatiotemporal pattern of epithelialization underlying sensory organ formation in the lateral line. Furthermore, this work uncovers a surprising link between internal tissue organization and collective migration.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

FGF10 maintains stem cell compartment in developing mouse incisors

Mouse incisors are regenerative tissues that grow continuously throughout life. The renewal of dental epithelium-producing enamel matrix and/or induction of dentin formation by mesenchymal cells is performed by stem cells that reside in cervical loop of the incisor apex. However, little is known about the mechanisms of stem cell compartment formation. Recently, a mouse incisor was used as a model to show that fibroblast growth factor (FGF) 10 regulates mitogenesis and fate decision of adult stem cells. To further illustrate the role of FGF10 in the formation of the stem cell compartment during tooth organogenesis, we have analyzed incisor development in Fgf10-deficient mice and have examined the effects of neutralizing anti-FGF10 antibody on the developing incisors in organ cultures. The incisor germs of FGF10-null mice proceeded to cap stage normally. However, at a later stage, the cervical loop was not formed. We found that the absence of the cervical loop was due to a divergence in Fgf10 and Fgf3 expression patterns at E16. Furthermore, we estimated the growth of dental epithelium from incisor explants of FGF10-null mice by organ culture. The dental epithelium of FGF10-null mice showed limited growth, although the epithelium of wild-type mice appeared to grow normally. In other experiments, a functional disorder of FGF10, caused by a neutralizing anti-FGF10 antibody, induced apoptosis in the cervical loop of developing mouse incisor cultures. However, recombinant human FGF10 protein rescued the cervical loop from apoptosis. Taken together, these results suggest that FGF10 is a survival factor that maintains the stem cell population in developing incisor germs.     

Reciprocal epithelial-mesenchymal FGF signaling is required for cecal development

Fibroblast growth factor (FGF) signaling mediates reciprocal mesenchymal-epithelial cell interactions in the developing mouse lung and limb. In the gastrointestinal (GI) tract, FGF10 is expressed in the cecal mesenchyme and signals to an epithelial splice form of FGF receptor (FGFR) 2 to regulate epithelial budding. Here, we identify FGF9 as a reciprocal epithelial-mesenchymal signal required for cecal morphogenesis. Fgf9 null (Fgf9(-/-)) mouse embryos have agenesis of the embryonic cecum, lacking both mesenchymal expansion and an epithelial bud. In the cecal region of Fgf9(-/-) embryos, mesenchymal expression of Fgf10 and Bmp4 is notably absent, whereas the expression of epithelial markers, such as sonic hedgehog, is not affected. Using epithelial and whole explant cultures, we show that FGF9 signals to mesenchymal FGFRs and that FGF10 signals to epithelial FGFRs. Taken together, these data show that an epithelial FGF9 signal is necessary for the expansion of cecal mesenchyme and the expression of mesenchymal genes that are required for epithelial budding. Thus, these data add to our understanding of FGF-mediated reciprocal epithelial-mesenchymal signaling.