Insights into Craniofacial Development and Anomalies: Exploring Fgf Signaling in Zebrafish Models.

PURPOSE OF REVIEW: To illustrate the value of using zebrafish to understand the role of the Fgf signaling pathway during craniofacial skeletal development under normal and pathological conditions. RECENT FINDINGS: Recent data obtained from studies on zebrafish have demonstrated the genetic redundancy of Fgf signaling pathway and have identified new molecular partners of this signaling during the early stages of craniofacial skeletal development. Studies on zebrafish models demonstrate the involvement of the Fgf signaling pathway at every stage of craniofacial development. They particularly emphasize the central role of Fgf signaling pathway during the early stages of the development, which significantly impacts the formation of the various structures making up the craniofacial skeleton. This partly explains the craniofacial abnormalities observed in disorders associated with FGF signaling. Future research efforts should focus on investigating zebrafish Fgf signaling during more advanced stages, notably by establishing zebrafish models expressing mutations responsible for diseases such as craniosynostoses.

Constitutively-active FGFR3 disrupts primary cilium length and IFT20 trafficking in various chondrocyte models of achondroplasia.

Fibroblast growth factor receptor 3 (FGFR3) gain-of-function mutations cause dwarfisms, including achondroplasia (ACH) and thanatophoric dysplasia (TD). The constitutive activation of FGFR3 disrupts the normal process of skeletal growth. Bone-growth anomalies have been identified in skeletal ciliopathies, in which primary cilia (PC) function is disrupted. In human ACH and TD, the impact of FGFR3 mutations on PC in growth plate cartilage remains unknown. Here we showed that in chondrocytes from human (ACH, TD) and mouse Fgfr3Y367C/+ cartilage, the constitutively active FGFR3 perturbed PC length and the sorting and trafficking of intraflagellar transport (IFT) 20 to the PC. We demonstrated that inhibiting FGFR3 with FGFR inhibitor, PD173074, rescued both PC length and IFT20 trafficking. We also studied the impact of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) pathway. Interestingly, mTOR inhibition also rescued PC length and IFT20 trafficking. Together, we provide evidence that the growth plate defects ascribed to FGFR3-related dwarfisms are potentially due to loss of PC function, and these dwarfisms may represent a novel type of skeletal disorders with defective ciliogenesis.

Fibrillarin is essential for S-phase progression and neuronal differentiation in zebrafish dorsal midbrain and retina.

Fibrillarin (Fbl) is a highly conserved protein that plays an essential role in ribosome biogenesis and more particularly in the methylation of ribosomal RNAs and rDNA histones. In cellular models, FBL was shown to play an important role in tumorigenesis and stem cell differentiation. We used the zebrafish as an in vivo model to study Fbl function during embryonic development. We show here that the optic tectum and the eye are severely affected by Fbl depletion whereas ventral regions of the brain are less impacted. The morphogenesis defects are associated with impaired neural differentiation and massive apoptosis. Polysome gradient experiments show that fbl mutant larvae display defects in ribosome biogenesis and activity. Strikingly, flow cytometry analyses revealed different S-phase profiles between wild-type and mutant cells, suggesting a defect in S-phase progression.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Theobroma cacao improves bone growth by modulating defective ciliogenesis in a mouse model of achondroplasia.

A gain-of-function mutation in the fibroblast growth factor receptor 3 gene (FGFR3) results in achondroplasia (ACH), the most frequent form of dwarfism. Constitutive activation of FGFR3 impairs bone formation and elongation and many signal transduction pathways. Identification of new and relevant compounds targeting the FGFR3 signaling pathway is of broad importance for the treatment of ACH, and natural plant compounds are prime drug candidate sources. Here, we found that the phenolic compound (-)-epicatechin, isolated from Theobroma cacao, effectively inhibited FGFR3's downstream signaling pathways. Transcriptomic analysis in an Fgfr3 mouse model showed that ciliary mRNA expression was modified and influenced significantly by the Indian hedgehog and PKA pathways. (-)-Epicatechin is able to rescue mRNA expression impairments that control both the structural organization of the primary cilium and ciliogenesis-related genes. In femurs isolated from a mouse model (Fgfr3Y367C/+) of ACH, we showed that (-)-epicatechin eliminated bone growth impairment during 6 days of ex vivo culture. In vivo, we confirmed that daily subcutaneous injections of (-)-epicatechin to Fgfr3Y367C/+ mice increased bone elongation and rescued the primary cilium defects observed in chondrocytes. This modification to the primary cilia promoted the typical columnar arrangement of flat proliferative chondrocytes and thus enhanced bone elongation. The results of the present proof-of-principle study support (-)-epicatechin as a potential drug for the treatment of ACH.

An Fgfr3-activating mutation in immature murine osteoblasts affects the appendicular and craniofacial skeleton.

Achondroplasia (ACH), the most common form of dwarfism, is caused by a missense mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3). The resulting increase in FGFR3 signaling perturbs the proliferation and differentiation of chondrocytes (CCs), alters the process of endochondral ossification and thus reduces bone elongation. Increased FGFR3 signaling in osteoblasts (OBs) might also contribute to bone anomalies in ACH. In the present study of a mouse model of ACH, we sought to determine whether FGFR3 overactivation in OBs leads to bone modifications. The model carries an Fgfr3-activating mutation (Fgfr3Y367C/+) that accurately mimics ACH; we targeted the mutation to either immature OBs and hypertrophic CCs or to mature OBs by using the Osx-cre and collagen 1α1 (2.3 kb Col1a1)-cre mouse strains, respectively. We observed that Fgfr3 activation in immature OBs and hypertrophic CCs (Osx-Fgfr3) not only perturbed the hypertrophic cells of the growth plate (thus affecting long bone growth) but also led to osteopenia and low cortical thickness in long bones in adult (3-month-old) mice but not growing (3-week-old) mice. Importantly, craniofacial membranous bone defects were present in the adult mice. In contrast, activation of Fgfr3 in mature OBs (Col1-Fgfr3) had very limited effects on skeletal shape, size and micro-architecture. In vitro, we observed that Fgfr3 activation in immature OBs was associated with low mineralization activity. In conclusion, immature OBs appear to be affected by Fgfr3 overactivation, which might contribute to the bone modifications observed in ACH independently of CCs.

Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

Fgfr3 Is a Positive Regulator of Osteoblast Expansion and Differentiation During Zebrafish Skull Vault Development.

Gain or loss-of-function mutations in fibroblast growth factor receptor 3 (FGFR3) result in cranial vault defects highlighting the protein's role in membranous ossification. Zebrafish express high levels of fgfr3 during skull development; in order to study FGFR3's role in cranial vault development, we generated the first fgfr3 loss-of-function zebrafish (fgfr3lof/lof ). The mutant fish exhibited major changes in the craniofacial skeleton, with a lack of sutures, abnormal frontal and parietal bones, and the presence of ectopic bones. Integrated analyses (in vivo imaging and single-cell RNA sequencing of the osteoblast lineage) of zebrafish fgfr3lof/lof revealed a delay in osteoblast expansion and differentiation, together with changes in the extracellular matrix. These findings demonstrate that fgfr3 is a positive regulator of osteogenesis. We conclude that changes in the extracellular matrix within growing bone might impair cell-cell communication, mineralization, and new osteoblast recruitment. © 2020 American Society for Bone and Mineral Research.

Tyrosine kinase inhibitor NVP-BGJ398 functionally improves FGFR3-related dwarfism in mouse model.

Achondroplasia (ACH) is the most frequent form of dwarfism and is caused by gain-of-function mutations in the fibroblast growth factor receptor 3-encoding (FGFR3-encoding) gene. Although potential therapeutic strategies for ACH, which aim to reduce excessive FGFR3 activation, have emerged over many years, the use of tyrosine kinase inhibitor (TKI) to counteract FGFR3 hyperactivity has yet to be evaluated. Here, we have reported that the pan-FGFR TKI, NVP-BGJ398, reduces FGFR3 phosphorylation and corrects the abnormal femoral growth plate and calvaria in organ cultures from embryos of the Fgfr3Y367C/+ mouse model of ACH. Moreover, we demonstrated that a low dose of NVP-BGJ398, injected subcutaneously, was able to penetrate into the growth plate of Fgfr3Y367C/+ mice and modify its organization. Improvements to the axial and appendicular skeletons were noticeable after 10 days of treatment and were more extensive after 15 days of treatment that started from postnatal day 1. Low-dose NVP-BGJ398 treatment reduced intervertebral disc defects of lumbar vertebrae, loss of synchondroses, and foramen-magnum shape anomalies. NVP-BGJ398 inhibited FGFR3 downstream signaling pathways, including MAPK, SOX9, STAT1, and PLCγ, in the growth plates of Fgfr3Y367C/+ mice and in cultured chondrocyte models of ACH. Together, our data demonstrate that NVP-BGJ398 corrects pathological hallmarks of ACH and support TKIs as a potential therapeutic approach for ACH.

Contribution of annexin 2 to the architecture of mature endothelial adherens junctions.

The vascular endothelial cadherin (VE-cad)-based complex is involved in the maintenance of vascular endothelium integrity. Using immunoprecipitation experiments, we have demonstrated that, in confluent human umbilical vein endothelial cells, the VE-cad-based complex interacts with annexin 2 and that annexin 2 translocates from the cytoplasm to the cell-cell contact sites as cell confluence is established. Annexin 2, located in cholesterol rafts, binds to both the actin cytoskeleton and the VE-cad-based complex so the complex is docked to cholesterol rafts. These multiple connections prevent the lateral diffusion of the VE-cad-based complex, thus strengthening adherens junctions in the ultimate steps of maturation. Moreover, we observed that the down-regulation of annexin 2 by small interfering RNA induces a delocalization of VE-cad from adherens junctions and consequently a destabilization of these junctions. Furthermore, our data indicate that the decoupling of the annexin 2/p11 complex from the VE-cad-based junction, triggered by vascular endothelial growth factor treatment, facilitates the switch from a quiescent to an immature state.