Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9(-/-) lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9(-/-) lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.     

FGF9 and SHH signaling coordinate lung growth and development through regulation of distinct mesenchymal domains

Morphogenesis of the lung is regulated by reciprocal signaling between epithelium and mesenchyme. In previous studies, we have shown that FGF9 signals are essential for lung mesenchyme development. Using Fgf9 loss-of-function and inducible gain-of-function mouse models, we show that lung mesenchyme can be divided into two distinct regions: the sub-mesothelial and sub-epithelial compartments, which proliferate in response to unique growth factor signals. Fibroblast growth factor (FGF) 9 signals from the mesothelium (the future pleura) to sub-mesothelial mesenchyme through both FGF receptor (FGFR) 1 and FGFR2 to induce proliferation. FGF9 also signals from the epithelium to the sub-epithelial mesenchyme to maintain SHH signaling, which regulates cell proliferation, survival and the expression of mesenchymal to epithelial signals. We further show that FGF9 represses peribronchiolar smooth muscle differentiation and stimulates vascular development in vivo. We propose a model in which FGF9 and SHH signals cooperate to regulate mesenchymal proliferation in distinct submesothelial and subepithelial regions. These data provide a molecular mechanism by which mesothelial and epithelial FGF9 directs lung development by regulating mesenchymal growth, and the pattern and expression levels of mesenchymal growth factors that signal back to the epithelium.     

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.     

Expression of Fgf10 and Fgf receptors during development of the embryonic chicken stomach

Fibroblast growth factor 10 (FGF10) is involved in numerous different aspects of embryonic development and especially in active epithelial-mesenchymal interactions during morphogenesis of many organs as a mesenchymal regulator by activating its receptors (FGFR1b and FGFR2b) expressed in the epithelial tissue. FGFR2b is also activated by FGF7 although FGF7 does not bind to FGFR1b. To provide basic data to analyze function of FGFs in the developing gut, here we cloned Fgf7 and studied expression patterns of Fgf7, Fgf10 and Fgfr1-4 during the development of chicken stomach (glandular stomach; proventriculus and muscular stomach; gizzard). Fgf10 is expressed both in the proventricular and gizzard mesenchyme while Fgf7 is expressed only in gizzard mesenchyme. Fgfr1-4 are expressed both in the epithelium and mesenchyme with a different spatial expression patterns. Furthermore, RT-PCR analysis reveals that Fgfr1b and Fgfr2b are expressed only in epithelia of both organs.     

FGF signaling regulates mesenchymal differentiation and skeletal patterning along the limb bud proximodistal axis

Fibroblast growth factors (FGFs) are signals from the apical ectodermal ridge (AER) that are essential for limb pattern formation along the proximodistal (PD) axis. However, how patterning along the PD axis is regulated by AER-FGF signals remains controversial. To further explore the molecular mechanism of FGF functions during limb development, we conditionally inactivated fgf receptor 2 (Fgfr2) in the mouse AER to terminate all AER functions; for comparison, we inactivated both Fgfr1 and Fgfr2 in limb mesenchyme to block mesenchymal AER-FGF signaling. We also re-examined published data in which Fgf4 and Fgf8 were inactivated in the AER. We conclude that limb skeletal phenotypes resulting from loss of AER-FGF signals cannot simply be a consequence of excessive mesenchymal cell death, as suggested by previous studies, but also must be a consequence of reduced mesenchymal proliferation and a failure of mesenchymal differentiation, which occur following loss of both Fgf4 and Fgf8. We further conclude that chondrogenic primordia formation, marked by initial Sox9 expression in limb mesenchyme, is an essential component of the PD patterning process and that a key role for AER-FGF signaling is to facilitate SOX9 function and to ensure progressive establishment of chondrogenic primordia along the PD axis.     

Mesothelial- and epithelial-derived FGF9 have distinct functions in the regulation of lung development

Fibroblast growth factor (FGF) 9 is a secreted signaling molecule that is expressed in lung mesothelium and epithelium and is required for lung development. Embryos lacking FGF9 show mesenchymal hypoplasia, decreased epithelial branching and, by the end of gestation, hypoplastic lungs that cannot support life. Mesenchymal FGF signaling interacts with β-catenin-mediated WNT signaling in a feed-forward loop that functions to sustain mesenchymal FGF responsiveness and mesenchymal WNT/β-catenin signaling. During pseudoglandular stages of lung development, Wnt2a and Wnt7b are the canonical WNT ligands that activate mesenchymal WNT/β-catenin signaling, whereas FGF9 is the only known ligand that signals to mesenchymal FGF receptors (FGFRs). Here, we demonstrate that mesothelial- and epithelial-derived FGF9, mesenchymal Wnt2a and epithelial Wnt7b have unique functions in lung development in mouse. Mesothelial FGF9 and mesenchymal WNT2A are principally responsible for maintaining mesenchymal FGF-WNT/β-catenin signaling, whereas epithelial FGF9 primarily affects epithelial branching. We show that FGF signaling is primarily responsible for regulating mesenchymal proliferation, whereas β-catenin signaling is a required permissive factor for mesenchymal FGF signaling.     

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.     

FGF10 is required for cell proliferation and gland formation in the stomach epithelium of the chicken embryo

The development of digestive organs in vertebrates involves active epithelial-mesenchymal interactions. In the chicken proventriculus (glandular stomach), the morphogenesis and cytodifferentiation of the epithelium are controlled by the inductive signaling factors that are secreted from the underlying mesenchyme. Previous studies have shown that Fgf10 is expressed in the developing chicken proventricular mesenchyme, whereas its receptors are present in the epithelium. In our present study, we show that FGF10 is an early mesenchymal signal that is critically associated with the developmental processes in the proventricular epithelium. Furthermore, virus-mediated Fgf10 overexpression in ovo results in a hypermorphic epithelial structure and an increase in epithelial cell number. In contrast, the overexpression of a secreted FGFR2b (sFGFR2b), an FGF10 antagonist, blocks cell proliferation and gland formation in the proventricular epithelium in ovo. This downregulation of proliferative activity was subsequently found to retard gland formation and also to delay differentiation of the epithelium. These results demonstrate that FGF10 signaling, mediated by FGFR1b and/or FGFR2b, is required for proliferation and gland formation in the epithelium in the developing chick embryo.     

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.     

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.     

Fgf3 and Fgf10 are required for mouse otic placode induction

The inner ear, which contains the sensory organs specialised for audition and balance, develops from an ectodermal placode adjacent to the developing hindbrain. Tissue grafting and recombination experiments suggest that placodal development is directed by signals arising from the underlying mesoderm and adjacent neurectoderm. In mice, Fgf3 is expressed in the neurectoderm prior to and concomitant with placode induction and otic vesicle formation, but its absence affects only the later stages of otic vesicle morphogenesis. We show here that mouse Fgf10 is expressed in the mesenchyme underlying the prospective otic placode. Embryos lacking both Fgf3 and Fgf10 fail to form otic vesicles and have aberrant patterns of otic marker gene expression, suggesting that FGF signals are required for otic placode induction and that these signals emanate from both the hindbrain and mesenchyme. These signals are likely to act directly on the ectoderm, as double mutant embryos showed normal patterns of gene expression in the hindbrain. Cell proliferation and survival were not markedly affected in double mutant embryos, suggesting that the major role of FGF signals in otic induction is to establish normal patterns of gene expression in the prospective placode. Finally, examination of embryos carrying three out of the four mutant Fgf alleles revealed intermediate phenotypes, suggesting a quantitative requirement for FGF signalling in otic vesicle formation.     

Fgf9 signaling regulates inner ear morphogenesis through epithelial-mesenchymal interactions

The mammalian inner ear comprises the cochleovestibular labyrinth, derived from the ectodermal otic placode, and the encasing bony labyrinth of the temporal bone. Epithelial-mesenchymal interactions are thought to control inner ear development, but the modes and the molecules involved are largely unresolved. We show here that, during the precartilage and cartilage stages, Fgf9 is expressed in specific nonsensory domains of the otic epithelium and its receptors, Fgfr1(IIIc) and Fgfr2(IIIc), widely in the surrounding mesenchyme. To address the role of Fgf9 signaling, we analyzed the inner ears of mice homozygous for Fgf9 null alleles. Fgf9 inactivation leads to a hypoplastic vestibular component of the otic capsule and to the absence of the epithelial semicircular ducts. Reduced proliferation of the prechondrogenic mesenchyme was found to underlie capsular hypoplasticity. Semicircular duct development is blocked at the initial stages, since fusion plates do not form. Our results show that the mesenchyme directs fusion plate formation and they give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. In addition to the vestibule, in the cochlea, Fgf9 mutation caused defects in the interactions between the Reissner's membrane and the mesenchymal cells, leading to a malformed scala vestibuli. Together, these data show that Fgf9 signaling is required for inner ear morphogenesis.     

Levels of mesenchymal FGFR2 signaling modulate smooth muscle progenitor cell commitment in the lung

Fibroblast growth factor (FGF) signaling has been shown to regulate lung epithelial development but its influence on mesenchymal differentiation has been poorly investigated. To study the role of mesenchymal FGF signaling in the differentiation of the mesenchyme and its impact on epithelial morphogenesis, we took advantage of Fgfr2c(+/Delta) mice, which due to a splicing switch express Fgfr2b in mesenchymal tissues and manifest Apert syndrome-like phenotypes. Using a set of in vivo and in vitro studies, we show that an autocrine FGF10-FGFR2b signaling loop is established in the mutant lung mesenchyme, which has several consequences. It prevents the entry of the smooth muscle progenitors into the smooth muscle cell (SMC) lineage and results in reduced fibronectin and elastin deposition. Levels of Fgf10 expression are raised within the mutant mesenchyme itself. Epithelial branching as well as epithelial levels of FGF and canonical Wnt signaling is dramatically reduced. These defects result in arrested development of terminal airways and an "emphysema like" phenotype in postnatal lungs. Our work unravels part of the complex interactions that govern normal lung development and may be pertinent to understanding the basis of respiratory defects in Apert syndrome.     

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.     

The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs.

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.