Fibroblast Growth Factor 10 and Vertebrate Limb Development

Early limb development requires fibroblast growth factor (Fgf)-mediated coordination between growth and patterning to ensure the proper formation of a functional organ. The apical ectodermal ridge (AER) is a domain of thickened epithelium located at the distal edge of the limb bud that coordinates outgrowth along the proximodistal axis. Considerable amount of work has been done to elucidate the cellular and molecular mechanisms underlying induction, maintenance and regression of the AER. Fgf10, a paracrine Fgf that elicits its biological responses by activating the fibroblast growth factor receptor 2b (Fgfr2b), is crucial for governing proximal distal outgrowth as well as patterning and acts upstream of the known AER marker Fgf8. A transgenic mouse line allowing doxycycline-based inducible and ubiquitous expression of a soluble form of Fgfr2b has been extensively used to identify the role of Fgfr2b ligands at different time points during development. Overexpression of soluble Fgfr2b (sFgfr2b) post-AER induction leads to irreversible loss of cellular β-catenin organization and decreased Fgf8 expression in the AER. A similar approach has been carried out pre-AER induction. The observed limb phenotype is similar to the severe proximal truncations observed in human babies exposed to thalidomide, which has been proposed to block the Fgf10-AER-Fgf8 feedback loop. Novel insights on the role of Fgf10 signaling in limb formation pre- and post-AER induction are summarized in this review and will be integrated with possible future investigations on the role of Fgf10 throughout limb development

A Comprehensive Analysis of Fibroblast Growth Factor Receptor 2b Signaling on Epithelial Tip Progenitor Cells During Early Mouse Lung Branching Morphogenesis

This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells, via ß-catenin/EP300, controls, through a comprehensive set of developmental genes, morphogenesis, and differentiation. Fibroblast growth factor (FGF) 10 signaling through FGF receptor 2b (FGFR2b) is mandatory during early lung development as the deletion of either the ligand or the receptor leads to lung agenesis. However, this drastic phenotype previously hampered characterization of the primary biological activities, immediate downstream targets and mechanisms of action. Through the use of a dominant negative transgenic mouse model (Rosa26rtTA; tet(o)sFgfr2b), we conditionally inhibited FGF10 signaling in vivo in E12.5 embryonic lungs via doxycycline IP injection to pregnant females, and in vitro by culturing control and experimental lungs with doxycycline. The impact on branching morphogenesis 9 h after doxycycline administration was analyzed by morphometry, fluorescence and electron microscopy. Gene arrays at 6 and 9 h following doxycycline administration were carried out. The relationship between FGF10 and ß-catenin signaling was also analyzed through in vitro experiments using IQ1, a pharmacological inhibitor of ß-catenin/EP300 transcriptional activity. Loss of FGF10 signaling did not impact proliferation or survival, but affected both adherens junctions (up-regulation of E-cadherin), and basement membrane organization (increased laminin). Gene arrays identified multiple direct targets of FGF10, including main transcription factors. Immunofluorescence showed a down-regulation of the distal epithelial marker SOX9 and mis-expression distally of the proximal marker SOX2. Staining for the transcriptionally-active form of ß-catenin showed a reduction in experimental vs. control lungs. In vitro experiments using IQ1 phenocopied the impacts of blocking FGF10. This study demonstrates that FGF10/FGFR2b signaling on distal epithelial progenitor cells via ß-catenin/EP300 controls, through a comprehensive set of developmental genes, cell adhesion, and differentiation

Characterization of Tg(Etv4-GFP) and Etv5RFP Reporter Lines in the Context of Fibroblast Growth Factor 10 Signaling During Mouse Embryonic Lung Development

Members of the PEA3 transcription factors are emerging as bone fide targets for fibroblast growth factor (FGF) signaling. Among them, ETV4 and ETV5 appear to mediate FGF10 signaling during early embryonic lung development. In this paper, recently obtained Tg(Etv4-GFP) and Etv5CreERT2−RFP fluorescent reporter lines were generally characterized during early embryonic development and in the context of FGF10 signaling, in particular. We found that both Tg(Etv4-GFP) and Etv5CreERT2−RFP were primarily expressed in the epithelium of the lung during embryonic development. However, the expression of Etv5CreERT2−RFP was much higher than that of Tg(Etv4-GFP), and continued to increase during development, whereas Tg(Etv4-GFP) decreased. The expression patterns of the surrogate fluorescent protein GFP and RFP for ETV4 and ETV5, respectively, agreed with known regions of FGF10 signaling in various developing organs, including the lung, where ETV4-GFP was seen primarily in the distal epithelium and to a lesser extent in the surrounding mesenchyme. As expected, ETV5-RFP was restricted to the lung epithelium, showing a decreasing expression pattern from distal buds to proximal conducting airways. FGF10 inhibition experiments confirmed that both Etv4 and Etv5 are downstream of FGF10 signaling. Finally, we also validated that both fluorescent reporters responded to FGF10 inhibition in vitro. In conclusion, these two reporter lines appear to be promising tools to monitor FGF10/FGFR2b signaling in early lung development. These tools will have to be further validated at later stages and in other organs of interest

Mesenchymal adenomatous polyposis coli plays critical and diverse roles in regulating lung development.

BackgroundAdenomatous polyposis coli (Apc) is a tumor suppressor that inhibits Wnt/Ctnnb1. Mutations of Apc will not only lead to familial adenomatous polyposis with associated epithelial lesions, but will also cause aggressive fibromatosis in mesenchymal cells. However, the roles of Apc in regulating mesenchymal cell biology and organogenesis during development are unknown.ResultsWe have specifically deleted the Apc gene in lung mesenchymal cells during early lung development in mice. Loss of Apc function resulted in immediate mesenchymal cell hyperproliferation through abnormal activation of Wnt/Ctnnb1, followed by a subsequent inhibition of cell proliferation due to cell cycle arrest at G0/G1, which was caused by a mechanism independent of Wnt/Ctnnb1. Meanwhile, abrogation of Apc also disrupted lung mesenchymal cell differentiation, including decreased airway and vascular smooth muscle cells, the presence of Sox9-positive mesenchymal cells in the peripheral lung, and excessive versican production. Moreover, lung epithelial branching morphogenesis was drastically inhibited due to disrupted Bmp4-Fgf10 morphogen production and regulation in surrounding lung mesenchyme. Lastly, lung mesenchyme-specific Apc conditional knockout also resulted in altered lung vasculogenesis and disrupted pulmonary vascular continuity through a paracrine mechanism, leading to massive pulmonary hemorrhage and lethality at mid-gestation when the pulmonary circulation should have started.ConclusionsOur study suggests that Apc in lung mesenchyme plays central roles in coordinating the proper development of several quite different cellular compartments including lung epithelial branching and pulmonary vascular circulation during lung organogenesis

Characterization of endogenous players in Fibroblast Growth Factor‐regulated functions of hypothalamic tanycytes and energy‐balance nuclei

The mammalian hypothalamus regulates key homeostatic and neuroendocrine functions ranging from circadian rhythm and energy‐balance to growth and reproductive cycles via the hypothalamo‐pituitary and hypothalamo‐thyroid axes. In addition to its neurons, tanycytes are taking centre stage in the short and long term augmentation and integration of diverse hypothalamic functions, but the genetic regulators and mediators of their involvement are poorly understood. Exogenous interventions have implicated Fibroblast growth factor (FGF) signalling, but the focal point of FGF action and any role for putative endogenous players also remains elusive. We carried out a comprehensive high‐resolution screen of FGF signalling pathway mediators and modifiers using a combination of in situ hybridization, immunolabelling and transgenic reporter mice, to map their spatial distribution in the adult hypothalamus. Our findings suggest that beta tanycytes are the likely focal point of exogenous and endogenous FGF action in the third ventricular wall, utilising FGF‐receptors (FGFRs) ‐1 and ‐2 IIIc isoforms, but not FGFR3. Key IIIc‐activating endogenous ligands include FGFs 1, 2, 9 and 18, which are expressed by a subset of ependymal and parenchymal cells. In the parenchymal compartment, FGFRs 1‐3 show divergent patterns, with FGFR1 predominant in neuronal nuclei and FGFR3 expression being associated with glial cell function. Intracrine FGFs are also present, suggestive of multiple modes of FGF function. Our findings provide a testable framework for understanding the complex role of FGFs in regulating the metabolic endocrine and neurogenic functions of hypothalamus in vivo

Growth factor signaling in lung morphogenetic centers: automaticity, stereotypy and symmetry

Lung morphogenesis is stereotypic, both for lobation and for the first several generations of airways, implying mechanistic control by a well conserved, genetically hardwired developmental program. This program is not only directed by transcriptional factors and peptide growth factor signaling, but also co-opts and is modulated by physical forces. Peptide growth factors signal within repeating epithelial-mesenchymal temporospatial patterns that constitute morphogenetic centers, automatically directing millions of repetitive events during both stereotypic branching and nonstereotypic branching as well as alveolar surface expansion phases of lung development. Transduction of peptide growth factor signaling within these centers is finely regulated at multiple levels. These may include ligand expression, proteolytic activation of latent ligand, ligand bioavailability, ligand binding proteins and receptor affinity and presentation, receptor complex assembly and kinase activation, phosphorylation and activation of adapter and messenger protein complexes as well as downstream events and cross-talk both inside and outside the nucleus. Herein we review the critical Sonic Hedgehog, Fibroblast Growth Factor, Bone Morphogenetic Protein, Vascular Endothelial Growth Factor and Transforming Growth Factorβ signaling pathways and propose how they may be functionally coordinated within compound, highly regulated morphogenetic gradients that drive first stereotypic and then non-stereotypic, automatically repetitive, symmetrical as well as asymmetrical branching events in the lung

Mechanisms of TGFβ inhibition of LUNG endodermal morphogenesis: The role of TβRII, Smads, Nkx2.1 and Pten

AbstractTransforming growth factor-beta is a multifunctional growth factor with roles in normal development and disease pathogenesis. One such role is in inhibition of lung branching morphogenesis, although the precise mechanism remains unknown. In an explant model, all three TGFβ isoforms inhibited FGF10-induced morphogenesis of mesenchyme-free embryonic lung endoderm. Inhibition of budding by TGFβ was partially abrogated in endodermal explants from Smad3−/− or conditional endodermal-specific Smad4Δ/Δ embryonic lungs. Endodermal explants from conditional TGFβ receptor II knockout lungs were entirely refractive to TGFβ-induced inhibition. Inhibition of morphogenesis was associated with dedifferentiation of endodermal cells as documented by a decrease in key transcriptional factor, NKX2.1 protein, and its downstream target, surfactant protein C (SpC). TGFβ reduced the proliferation of wild-type endodermal cells within the explants as assessed by BrdU labeling. Gene expression analysis showed increased levels of mRNA for Pten, a key regulator of cell proliferation. Conditional, endodermal-specific deletion of Pten overcame TGFβ's inhibitory effect on cell proliferation, but did not restore morphogenesis. Thus, the mechanisms by which TGFβ inhibits FGF10-induced lung endodermal morphogenesis may entail both inhibition of cell proliferation, through increased Pten, as well as inhibition or interference with morphogenetic mediators such as Nkx2.1. Both of the latter are dependent on signaling through TβRII

Fibroblast growth factor 10 is a negative regulator of postnatal neurogenesis in the mouse hypothalamus

New neurons are generated in the postnatal rodent hypothalamus, with a subset of tanycytes in the third ventricular (3V) wall serving as neural stem/progenitor cells. However, the precise stem cell niche organization, the intermediate steps and the endogenous regulators of postnatal hypothalamic neurogenesis remain elusive. Quantitative lineage-tracing in vivo revealed that conditional deletion of fibroblast growth factor 10 (Fgf10) from Fgf10-expressing beta-tanycytes at postnatal days (P)4-5 results in the generation of significantly more parenchymal cells by P28, composed mostly of ventromedial and dorsomedial neurons and some glial cells, which persist into adulthood. A closer scrutiny in vivo and ex vivo revealed that the 3V wall is not static and is amenable to cell movements. Furthermore, normally beta-tanycytes give rise to parenchymal cells via an intermediate population of alpha-tanycytes with transient amplifying cell characteristics. Loss of Fgf10 temporarily attenuates the amplification of beta-tanycytes but also appears to delay the exit of their alpha-tanycyte descendants from the germinal 3V wall. Our findings suggest that transience of cells through the alpha-tanycyte domain is a key feature, and Fgf10 is a negative regulator of postnatal hypothalamic neurogenesis.Peer reviewe

SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains

Mechanisms of gene repression by transforming growth factor-beta (TGF-beta) are not well understood. TGF-beta represses transcription of pulmonary surfactant protein-B gene in lung epithelial cells. Repression is mediated by SMAD3 through interactions with NKX2.1 and FOXA1, two key transcription factors that are positive regulators of SpB transcription. In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins. The sites of interaction on NKX2.1 are located within the NH2 and COOH domains, known to be involved in transactivation function. In comparison, weaker interaction of FOXA1 winged helix, and the NH2-terminal domains was documented with SMAD3. Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter. Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors

The Potentials and Caveats of Mesenchymal Stromal Cell-Based Therapies in the Preterm Infant

Preponderance of proinflammatory signals is a characteristic feature of all acute and resulting long-term morbidities of the preterm infant. The proinflammatory actions are best characterized for bronchopulmonary dysplasia (BPD) which is the chronic lung disease of the preterm infant with lifelong restrictions of pulmonary function and severe consequences for psychomotor development and quality of life. Besides BPD, the immature brain, eye, and gut are also exposed to inflammatory injuries provoked by infection, mechanical ventilation, and oxygen toxicity. Despite the tremendous progress in the understanding of disease pathologies, therapeutic interventions with proven efficiency remain restricted to a few drug therapies with restricted therapeutic benefit, partially considerable side effects, and missing option of applicability to the inflamed brain. The therapeutic potential of mesenchymal stromal cells (MSCs)-also known as mesenchymal stem cells-has attracted much attention during the recent years due to their anti-inflammatory activities and their secretion of growth and development-promoting factors. Based on a molecular understanding, this review summarizes the positive actions of exogenous umbilical cord-derived MSCs on the immature lung and brain and the therapeutic potential of reprogramming resident MSCs. The pathomechanistic understanding of MSC actions from the animal model is complemented by the promising results from the first phase I clinical trials testing allogenic MSC transplantation from umbilical cord blood. Despite all the enthusiasm towards this new therapeutic option, the caveats and outstanding issues have to be critically evaluated before a broad introduction of MSC-based therapies