Deletion of FGF9 in GABAergic neurons causes epilepsy.

Fibroblast growth factor 9 (FGF9) has long been assumed to modulate multiple biological processes, yet very little is known about the impact of FGF9 on neurodevelopment. Herein, we found that loss of Fgf9 in olig1 progenitor cells induced epilepsy in mice, with pathological changes in the cortex. Then depleting Fgf9 in different neural populations revealed that epilepsy was associated with GABAergic neurons. Fgf9 CKO in GABAergic neuron (CKOVGAT) mice exhibited not only the most severe seizures, but also the most severe growth retardation and highest mortality. Fgf9 deletion in CKOVGAT mice caused neuronal apoptosis and decreased GABA expression, leading to a GABA/Glu imbalance and epilepsy. The adenylate cyclase/cyclic AMP and ERK signaling pathways were activated in this process. Recombinant FGF9 proteoliposomes could significantly decrease the number of seizures. Furthermore, the decrease of FGF9 was commonly observed in serum of epileptic patients, especially those with focal seizures. Thus, FGF9 plays essential roles in GABAergic neuron survival and epilepsy pathology, which could serve as a new target for the treatment of epilepsy.

Mouse fibroblast growth factor 9 N143T mutation leads to wide chondrogenic condensation of long bones.

Long bones of the appendicular skeleton are formed through endochondral ossification. Endochondral bone formation initiates with mesenchymal condensation, followed by the formation of a cartilage template which is replaced by bone. Fibroblast growth factor 9 (FGF9) regulates bone development. Fgf9-/- mice exhibit disproportionate shortening of proximal skeletal elements. Fgf9 missense mutations in mice and humans induce joint synostosis. Thus, FGF9 is critical for regulating bone length and joint formation. Conversely, mechanisms regulating bone width remain unclear. Here, we showed that the homozygous elbow knee synostosis (Eks) mutant mice harboring N143T mutation in Fgf9 have wide long bones at birth. We investigated the cellular and molecular mechanisms underlying the widened prospective humerus in Fgf9Eks/Eks embryos. Increased and expanded FGF signaling in concert with wider expression domain of Fgf receptor 3 (Fgfr3) during chondrogenic condensation of the humerus led to widened cartilage, which resulted in the formation of wider prospective humeri in neonatal Fgf9Eks/Eks mice. Increased and expanded FGF signaling during chondrogenic condensation led to increased density of chondrocytes of the humeri accompanied by increased proliferation of chondrocytes which express inappropriately higher levels of cyclin D1 in Fgf9Eks/Eks embryos. The results suggest that FGF9 regulates the width of prospective long bones by controlling the width of chondrogenic condensation.

Osteoblast-derived FGF9 regulates skeletal homeostasis.

FGF9 has complex and important roles in skeletal development and repair. We have previously observed that Fgf9 expression in osteoblasts (OBs) is regulated by G protein signaling and therefore the present study was done to determine whether OB-derived FGF9 was important in skeletal homeostasis. To directly test this idea, we deleted functional expression of Fgf9 gene in OBs using a 2.3kb collagen type I promoter-driven Cre transgenic mouse line (Fgf9OB-/-). Both Fgf9 knockout (Fgf9OB-/-) and the Fgf9 floxed littermates (Fgf9fl/fl) mice were fully backcrossed and maintained in an FBV/N background. Three month old Fgf9OB-/- mice displayed a significant decrease in cancellous bone and bone formation in the distal femur and a significant decrease in cortical thickness at the TFJ. Strikingly, female Fgf9OB-/- mice did not display altered bone mass. Continuous treatment of mouse BMSCs with exogenous FGF9 inhibited mouse BMSC mineralization while acute treatment increased the proliferation of progenitors, an effect requiring the activation of Akt1. Our results suggest that mature OBs are an important source of FGF9, positively regulating skeletal homeostasis in male mice. Osteoblast-derived FGF9 may serve a paracrine role to maintain the osteogenic progenitor cell population through activation of Akt signaling.

FGF9-Pitx2-FGF10 signaling controls cecal formation in mice.

Fibroblast growth factor (FGF) signaling to the epithelium and mesenchyme mediated by FGF10 and FGF9, respectively, controls cecal formation during embryonic development. In particular, mesenchymal FGF10 signals to the epithelium via FGFR2b to induce epithelial cecal progenitor cell proliferation. Yet the precise upstream mechanisms controlling mesenchymal FGF10 signaling are unknown. Complete deletion of Fgf9 as well as of Pitx2, a gene encoding a homeobox transcription factor, both lead to cecal agenesis. Herein, we used mouse genetic approaches to determine the precise contribution of the epithelium and/or mesenchyme tissue compartments in this process. Using tissue compartment specific Fgf9 versus Pitx2 loss of function approaches in the gut epithelium and/or mesenchyme, we determined that FGF9 signals to the mesenchyme via Pitx2 to induce mesenchymal Fgf10 expression, which in turn leads to epithelial cecal bud formation.

Fgf-9 is required for angiogenesis and osteogenesis in long bone repair.

Bone healing requires a complex interaction of growth factors that establishes an environment for efficient bone regeneration. Among these, FGFs have been considered important for intrinsic bone-healing capacity. In this study, we analyzed the role of Fgf-9 in long bone repair. One-millimeter unicortical defects were created in tibias of Fgf-9(+/-) and wild-type mice. Histomorphometry revealed that half-dose gene of Fgf-9 markedly reduced bone regeneration as compared with wild-type. Both immunohistochemistry and RT-PCR analysis revealed markedly decreased levels of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2), osteocalcin, Vega-a, and platelet endothelial cell adhesion molecule 1 (PECAM-1) in Fgf-9(+/-) defects. muCT angiography indicated dramatic impairment of neovascularization in Fgf-9(+/-) mice as compared with controls. Treatment with FGF-9 protein promoted angiogenesis and successfully rescued the healing capacity of Fgf-9(+/-) mice. Importantly, although other pro-osteogenic factors [Fgf-2, Fgf-18, and bone morphogenic protein 2 (Bmp-2)] still were present in Fgf-9(+/-) mice, they could not compensate for the haploinsufficiency of the Fgf-9 gene. Therefore, endogenous Fgf-9 seems to play an important role in long bone repair. Taken together our data suggest a unique role for Fgf-9 in bone healing, presumably by initiating angiogenesis through Vegf-a. Moreover, this study further supports the embryonic phenotype previously observed in the developing limb, thus promoting the concept that healing processes in adult organisms may recapitulate embryonic skeletal development.

Different endogenous threshold levels of Fibroblast Growth Factor-ligands determine the healing potential of frontal and parietal bones.

In the skull vault, neural crest derived frontal bones have an increased healing capacity and higher expression levels of Fibroblast Growth Factor-ligands as compared to mesoderm-derived parietal bones. Thus, we asked whether Fibroblast Growth Factor-ligands are responsible for the superior healing potential of frontal bones. Parietal defects in juvenile and adult mice treated with Fibroblast Growth Factor-2, -9 and -18 showed increased bone regeneration, comparable to frontal defects. Immunohistochemistry revealed increased recruitment of osteoprogenitors and activation of FGF-signaling pathways in FGF-treated parietal defects. Conversely, calvarial defects in Fgf-9(+/-) and Fgf-18(+/-) mice showed impaired calvarial healing which could be rescued by exogenous Fibroblast Growth Factor-ligands. Moreover, by utilizing Wnt1Cre/R26R mice, the migration and contribution of dura mater and pericranium cells to calvarial healing could be demonstrated. Taken together our results demonstrated that different endogenous threshold levels of Fibroblast Growth Factor-ligands in frontal and parietal bones have a profound impact on calvarial regeneration. The present study thereby opens new avenues for translational medicine.

An FGF-WNT gene regulatory network controls lung mesenchyme development.

Lung mesenchyme is a critical determinant of the shape and size of the lung, the extent and patterning of epithelial branching, and the formation of the pulmonary vasculature and interstitial mesenchymal components of the adult lung. Fibroblast growth factor 9 (FGF9) is a critical regulator of lung mesenchymal growth; however, upstream mechanisms that modulate the FGF mesenchymal signal and the downstream targets of mesenchymal FGF signaling are poorly understood. Here we have identified a robust regulatory network in which mesenchymal FGF signaling regulates beta-Catenin mediated WNT signaling in lung mesenchyme. By conditionally inactivating beta-Catenin in lung mesenchyme, we show that mesenchymal WNT-beta-Catenin signaling is essential for lung development and acts to regulate the cell cycle G1 to S transition and the FGF responsiveness of mesenchyme. Together, both FGF and WNT signaling pathways function to sustain mesenchymal growth and coordinate epithelial morphogenesis during the pseudoglandular stage of lung development.

FGF9 regulates early hypertrophic chondrocyte differentiation and skeletal vascularization in the developing stylopod.

Gain-of-function mutations in fibroblast growth factor (FGF) receptors result in chondrodysplasia and craniosynostosis syndromes, highlighting the critical role for FGF signaling in skeletal development. Although the FGFRs involved in skeletal development have been well characterized, only a single FGF ligand, FGF18, has been identified that regulates skeletal development during embryogenesis. Here we identify Fgf9 as a second FGF ligand that is critical for skeletal development. We show that Fgf9 is expressed in the proximity of developing skeletal elements and that Fgf9-deficient mice exhibit rhizomelia (a disproportionate shortening of proximal skeletal elements), which is a prominent feature of patients with FGFR3-induced chondrodysplasia syndromes. Although Fgf9 is expressed in the apical ectodermal ridge in the limb bud, we demonstrate that the Fgf9-/- limb phenotype results from loss of FGF9 functions after formation of the mesenchymal condensation. In developing stylopod elements, FGF9 promotes chondrocyte hypertrophy at early stages and regulates vascularization of the growth plate and osteogenesis at later stages of skeletal development.