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Growing evidences suggest that the fibroblast growth factor/FGF receptor (FGF/FGFR) signaling has crucial roles in a multitude of processes during embryonic development and adult homeostasis by regulating cellular lineage commitment, differentiation, proliferation, and apoptosis of various types of cells. In this review, we provide a comprehensive overview of the current understanding of FGF signaling and its roles in organ development, injury repair, and the pathophysiology of spectrum of diseases, which is a consequence of FGF signaling dysregulation, including cancers and chronic kidney disease (CKD). In this context, the agonists and antagonists for FGF-FGFRs might have therapeutic benefits in multiple systems.

CATSHL syndrome, characterized by camptodactyly, tall stature and hearing loss, is caused by loss-of-function mutations of fibroblast growth factor receptors 3 (FGFR3) gene. Most manifestations of patients with CATSHL syndrome start to develop in the embryonic stage, such as skeletal overgrowth, craniofacial abnormalities, however, the pathogenesis of these phenotypes especially the early maldevelopment remains incompletely understood. Furthermore, there are no effective therapeutic targets for this skeleton dysplasia. Methods: We generated fgfr3 knockout zebrafish by CRISPR/Cas9 technology to study the developmental mechanisms and therapeutic targets of CATSHL syndrome. Several zebrafish transgenic lines labeling osteoblasts and chondrocytes, and live Alizarin red staining were used to analyze the dynamical skeleton development in fgfr3 mutants. Western blotting, whole mount in situ hybridization, Edu labeling based cell proliferation assay and Wnt/β-catenin signaling antagonist were used to explore the potential mechanisms and therapeutic targets. Results: We found that fgfr3 mutant zebrafish, staring from early development stage, showed craniofacial bone malformation with microcephaly and delayed closure of cranial sutures, chondroma-like lesion and abnormal development of auditory sensory organs, partially resembling the clinical manifestations of patients with CATSHL syndrome. Further studies showed that fgfr3 regulates the patterning and shaping of pharyngeal arches and the timely ossification of craniofacial skeleton. The abnormal development of pharyngeal arch cartilage is related to the augmented hypertrophy and disordered arrangement of chondrocytes, while decreased proliferation, differentiation and mineralization of osteoblasts may be involved in the delayed maturation of skull bones. Furthermore, we revealed that deficiency of fgfr3 leads to enhanced IHH signaling and up-regulated canonical Wnt/β-catenin signaling, and pharmacological inhibition of Wnt/β-catenin could partially alleviate the phenotypes of fgfr3 mutants. Conclusions: Our study further reveals some novel phenotypes and underlying developmental mechanism of CATSHL syndrome, which deepens our understanding of the pathogenesis of CATSHL and the role of fgfr3 in skeleton development. Our findings provide evidence that modulation of Wnt/β-catenin activity could be a potential therapy for CATSHL syndrome and related skeleton diseases.

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway., Congenital scoliosis is a complex genetic disorder characterized by vertebral malformation. Here, the authors demonstrate that loss of dstyk leads to scoliosis in zebrafish due to dysregulated biogenesis of notochord vacuoles and that DSTYK is required for lysosome biogenesis through mTORC1 regulation.

Acquired heterotopic ossification (HO) is the extraskeletal bone formation after trauma. Various mesenchymal progenitors are reported to participate in ectopic bone formation. Here we induce acquired HO in mice by Achilles tenotomy and observe that conditional knockout (cKO) of fibroblast growth factor receptor 3 (FGFR3) in Col2+ cells promote acquired HO development. Lineage tracing studies reveal that Col2+ cells adopt fate of lymphatic endothelial cells (LECs) instead of chondrocytes or osteoblasts during HO development. FGFR3 cKO in Prox1+ LECs causes even more aggravated HO formation. We further demonstrate that FGFR3 deficiency in LECs leads to decreased local lymphatic formation in a BMPR1a-pSmad1/5-dependent manner, which exacerbates inflammatory levels in the repaired tendon. Local administration of FGF9 in Matrigel inhibits heterotopic bone formation, which is dependent on FGFR3 expression in LECs. Here we uncover Col2+ lineage cells as an origin of lymphatic endothelium, which regulates local inflammatory microenvironment after trauma and thus influences HO development via FGFR3-BMPR1a pathway. Activation of FGFR3 in LECs may be a therapeutic strategy to inhibit acquired HO formation via increasing local lymphangiogenesis., Different types of mesenchymal progenitors participate in ectopic bone formation. Here, the authors show Col2+ lineage cells adopt a lymphatic endothelium cell fate, which regulates local inflammatory microenvironment after trauma, thus influencing heterotopic ossification (HO) development via a FGFR3-BMPR1a pathway.

Objective: Scoliosis is a common disease characterized by spinal curvature with variable severities. There is no generally accepted theory about the physical origin of the spinal deformation of scoliosis. The aim of this study was to explore a new hypothesis suggesting that the curvatures in scoliosis may be associated with the imbalance growth between thoracic vertebral column and sternum. Methods: We undertook a comparative computed tomography (CT) based morphology study of thoracic vertebrae and sternum of patients with adolescent idiopathic scoliosis (AIS) and age-gender matched normal subjects. We further measured the ratios between the lengths of the sternum and thoracic vertebra of mice with deficiency of fibroblast growth factor receptor 3 (FGFR3), which exhibit scoliosis. Three-week-old C57BL/6J mice were used to generate bipedal and sternal growth plate injury model. Radiographs and histological images were obtained to observe the presence of sternal and spinal deformity. Results: There was a significant correlation between the severities of scoliosis and the ratios of the sternum to thoracic vertebral lengths. We also found that FGFR3 deficient mice showed smaller ratio of the sternum to thoracic vertebra lengths than that of the wild-type mice, which were similar with that of the AIS patients. Surgery-induced injuries of sternal growth plates can accelerate and aggravate the scoliosis in bipedal mice and imbalanced development of anterior and posterior thoracic occurred before the appearance of scoliosis. Conclusions: Our findings suggest that the imbalanced growth between the thoracic vertebral column and the sternum is an important causative factor for the pathogenesis of scoliosis including AIS. The translational potential of this article: Imbalanced growth between the thoracic vertebral column and the sternum is associated with scoliosis. Surgical or rehabilitation intervention for scoliosis should focus on all components involved in the pathogenesis of curvature to obtain better outcome. Keywords: Growth plate, Imbalanced growth, Scoliosis, Sternum, Vertebrae

Apert syndrome (AS) is a common genetic syndrome in humans characterized with craniosynostosis. Apert patients and mouse models showed abnormalities in sutures, cranial base and brain, that may all be involved in the pathogenesis of skull malformation of Apert syndrome. To distinguish the differential roles of these components of head in the pathogenesis of the abnormal skull morphology of AS, we generated mouse strains specifically expressing mutant FGFR2 in chondrocytes, osteoblasts, and progenitor cells of central nervous system (CNS) by crossing Fgfr2+/P253R-Neo mice with Col2a1-Cre, Osteocalcin-Cre (OC-Cre), and Nestin-Cre mice, respectively. We then quantitatively analyzed the skull and brain morphology of these mutant mice by micro-CT and micro-MRI using Euclidean distance matrix analysis (EDMA). Skulls of Col2a1-Fgfr2+/P253R mice showed Apert syndrome-like dysmorphology, such as shortened skull dimensions along the rostrocaudal axis, shortened nasal bone, and evidently advanced ossification of cranial base synchondroses. The OC-Fgfr2+/P253R mice showed malformation in face at 8-week stage. Nestin-Fgfr2+/P253R mice exhibited increased dorsoventral height and rostrocaudal length on the caudal skull and brain at 8 weeks. Our study indicates that the abnormal skull morphology of AS is caused by the combined effects of the maldevelopment in calvarias, cranial base, and brain tissue. These findings further deepen our knowledge about the pathogenesis of the abnormal skull morphology of AS, and provide new clues for the further analyses of skull phenotypes and clinical management of AS.

Summary: PTH stimulates bone formation in Fgfr3 knockout mice through promotion of proliferation and differentiation in osteoblasts Introduction: Previous studies showed that endogenous fibroblast growth factor 2 (FGF-2) is required for parathyroid hormone (PTH)-stimulated bone anabolic effects, however, the exact mechanisms by which PTH stimulate bone formation and the function of FGF receptors in mediating these actions are not fully defined. FGF receptor 3 (FGFR3) has been characterized as an important regulator of bone metabolism and is confirmed to cross-talk with PTH/PTHrP signal in cartilage and bone development. Methods: Fgfr3 knockout and wild-type mice at 2-month-old and 4-month-old were intraperitoneally injected with PTH intermittently for 4 weeks and then the skeletal responses to PTH were assessed by dual energy X-ray absorptiometry (DEXA), micro-computed tomography (μCT) and bone histomorphometry. Results: Intermittent PTH treatment improved bone mineral density (BMD) and femoral mechanical properties in both Fgfr3-/- and wild-type mice. Histomorphometric analysis showed that bone formation and bone resorption were increased in both genotypes following PTH treatment. PTH treatment increased trabecular bone volume (BV/TV) in WT and Fgfr3-deficient mice. The anabolic response in Fgfr3-deficient and wild-type bone is characterized by an increase of both bone formation and resorption-related genes following PTH treatment. In addition, we found that Fgfr3 null osteoblasts (compared to wild-type controls) maintained normal abilities to response to PTH-stimulated increase of proliferation, differentiation, expression of osteoblastic marker genes (Cbfa1, Osteopontin and Osteocalcin), and phosphorylation of Erk1/2. Conclusions: Bone anabolic effects of PTH were not impaired by the absence of FGFR3, suggesting that the FGFR3 signaling may not be required for osteoanabolic effects of PTH activities. Refereed/Peer-reviewed

Osteoarthritis (OA) in the temporomandibular joint (TMJ) is a common degenerative disease in adult, which is characterized by progressive destruction of the articular cartilage. To investigate the role of FGFR3 in the homeostasis of TMJ cartilage during adult stage, we generated Fgfr3f/f; Col2a1-CreERT2 (Fgfr3 cKO) mice, in which Fgfr3 was deleted in chondrocytes at 2 months of age. OA-like defects were observed in Fgfr3 cKO TMJ cartilage. Immunohistochemical staining and quantitative real-time PCR analyses revealed a significant increase in expressions of COL10, MMP13 and AMAMTS5. In addition, there was a sharp increase in chondrocyte apoptosis at the Fgfr3 cKO articular surface, which was accompanied by a down-regulation of lubricin expression. Importantly, the expressions of RUNX2 and Indian hedgehog (IHH) were up-regulated in Fgfr3 cKO TMJ. Primary Fgfr3 cKO chondrocytes were treated with IHH signaling inhibitor, which significantly reduced expressions of Runx2, Col10, Mmp13 and Adamts5. Furthermore, the IHH signaling inhibitor partially alleviated OA-like defects in the TMJ of Fgfr3 cKO mice, including restoration of lubricin expression and improvement of the integrity of the articular surface. In conclusion, our study proposes that FGFR3/IHH signaling pathway plays a critical role in maintaining the homeostasis of TMJ articular cartilage during adult stage.

Fibroblast growth factor receptor 3 (FGFR3), highly conserved in both humans and murine, is one of key tyrosine kinase receptors for FGF. FGFR3 is expressed in different tissues, including cartilage, brain, kidney, and intestine at different development stages. Conventional knockout of Fgfr3 alleles leads to short life span, and overgrowth of bone. In clinic, human FGFR3 mutations are responsible for three different types of chondrodysplasia syndromes including achondroplasia (ACH), hypochondroplasia (HCH) and thanatophoric dysplasia (TD). For better understanding of the roles of FGFR3 in different tissues at different stages of development and in pathological conditions, we generated Fgfr3 conditional knockout mice in which loxp sites flank exons 9-10 in the Fgfr3 allele. We also demonstrated that Cre-mediated recombination using Col2a1-Cre, a Cre line expressed in chondrocyte during bone development, results in specific deletion of the gene in tissues containing cartilage. This animal model will be useful to study distinct roles of FGFR3 in different tissues at different ages.

Gain-of-function mutations in fibroblast growth factor receptor-3 (FGFR3) lead to several types of human skeletal dysplasia syndromes including achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Currently, there are no effective treatments for these skeletal dysplasia diseases. In this study, we screened, using FGFR3 as a bait, a random 12-peptide phage library and obtained 23 positive clones that share identical amino acid sequences (VSPPLTLGQLLS), named as peptide P3. This peptide had high binding specificity to the extracellular domain of FGFR3. P3 inhibited tyrosine kinase activity of FGFR3 and its typical downstream molecules, extracellular signal-regulated kinase/mitogen-activated protein kinase. P3 also promoted proliferation and chondrogenic differentiation of cultured ATDC5 chondrogenic cells. In addition, P3 alleviated the bone growth retardation in bone rudiments from mice mimicking human thanatophoric dysplasia type II (TDII). Finally, P3 reversed the neonatal lethality of TDII mice. Thus, this study identifies a novel inhibitory peptide for FGFR3 signaling, which may serve as a potential therapeutic agent for the treatment of FGFR3-related skeletal dysplasia. Refereed/Peer-reviewed