RESUMO
Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.
Assuntos
Fator 8 de Crescimento de Fibroblasto , Incisivo , Mesoderma , Dente Molar , Animais , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Camundongos , Incisivo/anormalidades , Incisivo/metabolismo , Mesoderma/metabolismo , Mesoderma/patologia , Dente Molar/anormalidades , Dente Molar/metabolismo , Anodontia/genética , Anodontia/metabolismo , Anodontia/patologia , Apoptose , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Transdução de Sinais , Regulação da Expressão Gênica no Desenvolvimento , Odontogênese/genética , Camundongos TransgênicosRESUMO
The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time. The radial dimension of the ganglion cell layer (GCL) was seen to be the greatest at the HAA throughout development, beginning during the period of neurogenesis, suggesting that genesis, rather than cell death, creates a higher level of retinal ganglion cells (RGCs) in this area. In contrast, the HAA acquired its characteristic high density of cone photoreceptors post-hatching, which is well after the period of neurogenesis. We also confirmed that rod photoreceptors are not present in the HAA. Analyses of cell death in the developing photoreceptor layer, where rods would reside, did not show apoptotic cells, suggesting that lack of genesis, rather than death, created the "rod-free zone" (RFZ). Quantification of each cone photoreceptor subtype showed an ordered mosaic of most cone subtypes. The changes in cellular densities and cell subtypes between the developing and mature HAA provide some answers to the overarching strategy used by the retina to create this area and provide a framework for future studies of the mechanisms underlying its formation.
Assuntos
Retina , Células Ganglionares da Retina , Animais , Embrião de Galinha , Células Ganglionares da Retina/citologia , Retina/embriologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Galinhas , Neurogênese/fisiologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Hibridização in Situ Fluorescente , Fóvea Central/embriologia , Acuidade Visual , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/citologia , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
The asymptomatic nature, high rate of disease recurrence, and resistance to platinum-based chemotherapy highlight the need to identify and characterize novel target molecules for ovarian cancer. Fibroblast growth factor 8 (FGF8) aids in the development and metastasis of ovarian cancer; however, its definite role is not clear. We employed ELISA and IHC to examine the expression of FGF8 in the saliva and tissue samples of epithelial ovarian cancer (EOC) patients and controls. Furthermore, various cell assays were conducted to determine how FGF8 silencing influences ovarian cancer cell survival, adhesion, migration, and invasion to learn more about the functions of FGF8. In saliva samples, from controls through low-grade to high-grade EOC, a stepped overexpression of FGF8 was observed. Similar expression trends were seen in tissue samples, both at protein and mRNA levels. FGF8 gene silencing in SKOV3 cells adversely affected various cell properties essential for cancer cell survival and metastasis. A substantial reduction was observed in the cell survival, cell adhesion to the extracellular matrix, migration, and adhesion properties of SKOV3 cells, suggesting that FGF8 plays a crucial role in the development of EOC. Conclusively, this study suggests a pro-metastatic function of FGF8 in EOC.
Assuntos
Recidiva Local de Neoplasia , Neoplasias Ovarianas , Humanos , Feminino , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/genética , Neoplasias Ovarianas/patologia , Carcinoma Epitelial do Ovário/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de CélulasRESUMO
Gap junction intercellular communication (GJIC) is essential for regulating the development of the organism and sustaining the internal environmental homeostasis of multi-cellular tissue. Fibroblast growth factor 8 (FGF8), an indispensable regulator of the skeletal system, is implicated in regulating chondrocyte growth, differentiation, and disease occurrence. However, the influence of FGF8 on GJIC in chondrocytes is not yet known. The study aims to investigate the role of FGF8 on cell-cell communication in chondrocytes and its underlying biomechanism. We found that FGF8 facilitated cell-cell communication in living chondrocytes by the up-regulation of connexin43 (Cx43), the major fundamental component unit of gap junction channels in chondrocytes. FGF8 activated p38-MAPK signaling to increase the expression of Cx43 and promote the cell-cell communication. Inhibition of p38-MAPK signaling impaired the increase of Cx43 expression and cell-cell communication induced by FGF8, indicating the importance of p38-MAPK signaling. These results help to understand the role of FGF8 on cell communication and provide a potential cue for the treatment of cartilage diseases.
Assuntos
Condrócitos , Conexina 43 , Conexina 43/genética , Conexina 43/metabolismo , Condrócitos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Comunicação Celular/fisiologia , Sistema de Sinalização das MAP Quinases , Junções Comunicantes/metabolismoRESUMO
The cochlear auditory epithelium contains two types of sound receptors, inner hair cells (IHCs) and outer hair cells (OHCs). Mouse models for labelling juvenile and adult IHCs or OHCs exist; however, labelling for embryonic and perinatal IHCs or OHCs are lacking. Here, we generated a new knock-in Fgf8P2A-3×GFP/+ (Fgf8GFP/+) strain, in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements. After confirming that GFP expression accurately reflects the expression of Fgf8, we successfully obtained both embryonic and neonatal IHCs with high purity, highlighting the power of Fgf8GFP/+. Furthermore, our fate-mapping analysis revealed, unexpectedly, that IHCs are also derived from inner ear progenitors expressing Insm1, which is currently regarded as an OHC marker. Thus, besides serving as a highly favorable tool for sorting early IHCs, Fgf8GFP/+ will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.
Assuntos
Cóclea , Células Ciliadas Auditivas Internas , Animais , Camundongos , Cóclea/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Modelos Animais de Doenças , Fator 8 de Crescimento de Fibroblasto/metabolismoRESUMO
Development of the outflow tract of the heart requires specification, proliferation and deployment of a progenitor cell population from the second heart field to generate the myocardium at the arterial pole of the heart. Disruption of these processes leads to lethal defects in rotation and septation of the outflow tract. We previously showed that Fibroblast Growth Factor 8 (FGF8) directs a signaling cascade in the second heart field that regulates critical aspects of OFT morphogenesis. Here we show that in addition to the survival and proliferation cues previously described, FGF8 provides instructive and patterning information to OFT myocardial cells and their progenitors that prevents their aberrant differentiation along a working myocardial program.
Assuntos
Coração , Miocárdio , Diferenciação Celular/fisiologia , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Mesoderma/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos , Animais , CamundongosRESUMO
BACKGROUND: microRNAs (miRNAs) are involved in the progression of Lung adenocarcinoma (LUAD), however, the functions of miR-6742-5p in LUAD remains unknown, thereby this study was carried on. METHODS: The mRNA and miRNA expression data from the LUAD and normal control were obtained from Gene Expression Omnibus (GEO) database, TargetScan and mirDIP were applied to predict the relationship between miR-6742-5p and FGF8.Q-PCR, western blot, dual-luciferase, wound Healing and transwell assays were performed to test the functions of miR-6742-5p in LUAD. RESULTS: Bioinformatics analysis and dual-luciferase identified FGF8 is the target-gene of miR-6742-5p, which is declined in LUAD of human tissues and cell lines, and miR-6742-5P OE suppressed the progression of LUAD in nude mice. MiR-6742-5p OE and KD suppressed or increased the abilities of LUAD' metastasis tested by wound healing and transwell assays H522 and PC-9 cells, these effects about miR-6742-5p OE were reversed by FGF8; miR-6742-5p OE, KD inhibited and increased the expression of FGF8 as its downstream p-ERK1/2, MMP-2/-9, these results were corrected by ERK1/2 inhibitor: Ro 67-7476; the miR-6742-5p KD increased the migrated and invaded cells and suppressed by MMPs inhibitor: S3304. These results identified the negative correlation of miR-6742-5p with FGF8-ERK1/2 signal pathway in LUAD progression. CONCLUSIONS: We conclude that miR-6742-5p might be a regulator of LUAD progression by targeting FGF8/ERK1/2/MMPs signaling pathway, which provides a novel therapeutic target for LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , MicroRNAs , Animais , Camundongos , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Nus , Linhagem Celular Tumoral , Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismoRESUMO
Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK/BAX and for subsequent steps involving, as yet, undefined roles in kidney development.
Assuntos
Rim , Néfrons , Camundongos , Animais , Feminino , Gravidez , Proteína X Associada a bcl-2/metabolismo , Néfrons/metabolismo , Apoptose , Diferenciação Celular , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismoRESUMO
INTRODUCTION: Gelatinases, namely MMP2 and MMP9, are involved in the natural turnover of articular cartilage, as well as the loss of the cartilage matrix in osteoarthritis (OA). Studies have reported that fibroblast growth factor 8 (FGF8) promoted the degradation of cartilage in OA. In the present study, we predicted that FGF8 promoted chondrocyte expression and secretion of gelatinases by activating NF-κB p65 signaling. MATERIALS AND METHODS: Primary chondrocytes from C57 mice were cultured with recombinant FGF8. RNA sequencing was employed to explore the gene expression changes of gelatinases. Gelatin zymography was used to determine the activation of gelatinases. Western blot was used to investigate the expression of the gelatinases and NF-κB p65 signaling pathways, and immunofluorescence staining and NF-κB inhibitor assays were performed to confirm the activation of NF-κB p65 signaling. RESULTS: FGF8 could increase the expression and activity of gelatinases in primary chondrocytes. And FGF8-induced expression of gelatinases was regulated through activation of NF-κB signaling with acetylated p65 accumulating in the cell nucleus. We further found that the NF-κB inhibitor, BAY 11-7082, could suppress up-regulation of gelatinase induced by FGF8. CONCLUSION: FGF8 enhanced the expression and activity of MMP2 and MMP9 in chondrocytes via NF-κB p65 signaling.
Assuntos
Cartilagem Articular , Osteoartrite , Camundongos , Animais , NF-kappa B/metabolismo , Condrócitos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Gelatinases/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Osteoartrite/metabolismo , Cartilagem Articular/metabolismo , Células CultivadasRESUMO
Kidneys develop via iterative branching of the ureteric epithelial tree and subsequent nephrogenesis at the branch points. Nephrons form in the cap mesenchyme as the metanephric mesenchyme (MM) condenses around the epithelial ureteric buds (UBs). Previous work has demonstrated that FGF8 is important for the survival of nephron progenitor cells (NPCs), and early deletion of Fgf8 leads to the cessation of nephron formation, which results in post-natal lethality. We now reveal a previously unreported function of FGF8. By combining transgenic mouse models, quantitative imaging assays and data-driven computational modelling, we show that FGF8 has a strong chemokinetic effect and that this chemokinetic effect is important for the condensation of NPCs to the UB. The computational model shows that the motility must be lower close to the UB to achieve NPC attachment. We conclude that the FGF8 signalling pathway is crucial for the coordination of NPC condensation at the UB. Chemokinetic effects have also been described for other FGFs and may be generally important for the formation of mesenchymal condensates.
Assuntos
Rim , Néfrons , Camundongos , Animais , Néfrons/metabolismo , Rim/metabolismo , Organogênese , Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco/metabolismo , Camundongos Transgênicos , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismoRESUMO
Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time- and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10â days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25â ng/ml, 50â ng/ml or 100â ng/ml) for 10â days or by treatment with 100â ng/ml FGF8 for different durations (2, 4, 6 or 10â days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5â days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (P<0.05, Rs=0.49). However, cells treated with 100â ng/ml FGF8 for 2â days induced the expression of genes, such as FOXG1, ETV5 and SPRY2, and continued FGF8 treatment induced the dynamic expression of several other genes. Moreover, during exposure to FGF8, FGFR1 localized to the cell surface and its specific inhibition with the FGFR1 inhibitor PD166866 reduced expression of GNRH1 (P<0.05). In neurons, FGFR1 also localized to the nucleus. Our results suggest that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny. This article has an associated First Person interview with the first author of the paper.
Assuntos
Hormônio Liberador de Gonadotropina , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fator 8 de Crescimento de Fibroblasto/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Neurônios/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
The expansion of cumulus cells associated with oocytes is an essential phenomenon in normal mammalian ovulation. Indeed, attenuated expression of cumulus expansion-related genes, including Has2, Ptgs2, Ptx3, and Tnfaip6, results in ovulation failure, leading to female subfertility or infertility. Moreover, emerging evidence suggests that proteins of the fibroblast growth factor (FGF) family, produced within ovarian follicles, regulate the development and function of cumulus cells; however, the effects of FGF signaling on cumulus expansion have not been investigated extensively. Herein, we investigate the effects of FGF signaling, particularly those of FGF8 secreted by oocytes, on epidermal growth factor-induced cumulus expansion in mice. The phosphorylation level of MAPK3/1, an intracellular mediator of FGF signaling, was significantly decreased in cumulus-oocyte complexes (COCs) following treatment with NVP-BGJ398, an FGF receptor inhibitor. Moreover, even though NVP-BGJ398 treatment did not affect cumulus cell expansion, it significantly upregulated the expression of Ptgs2 and Ptx3. In contrast, treatment with recombinant FGF8 did not affect the degree of cumulus expansion or the expression of expansion-related genes in COCs or oocytectomized cumulus cell complexes. Collectively, these results suggest that FGFs, other than FGF8, exert suppressive effects on the cumulus expansion process in mice.
Assuntos
Células do Cúmulo , Fatores de Crescimento de Fibroblastos , Animais , Células do Cúmulo/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Mamíferos , Camundongos , Oócitos/metabolismo , Folículo Ovariano/fisiologiaRESUMO
The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.
Assuntos
Ambystoma mexicanum , Via de Sinalização Wnt , Ambystoma mexicanum/genética , Animais , Galinhas/genética , Extremidades/fisiologia , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , Ligantes , Vertebrados/genéticaRESUMO
Somitogenesis, the segmentation of the antero-posterior axis in vertebrates, is thought to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. The segment (somite) size is set by the product of the oscillator period and the velocity of the determination wavefront. Surprisingly, while the segmentation period can vary by a factor three between 20 °C and 32 °C, the somite size is constant. How this temperature independence is achieved is a mystery that we address in this study. Using RT-qPCR we show that the endogenous fgf8 mRNA concentration decreases during somitogenesis and correlates with the exponent of the shrinking pre-somitic mesoderm (PSM) size. As the temperature decreases, the dynamics of fgf8 and many other gene transcripts, as well as the segmentation frequency and the PSM shortening and tail growth rates slows down as T-Tc (with Tc = 14.4 °C). This behavior characteristic of a system near a critical point may account for the temperature independence of somitogenesis in zebrafish.
Assuntos
Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Desenvolvimento Embrionário/genética , Fator 8 de Crescimento de Fibroblasto/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra , p-Aminoazobenzeno/análogos & derivados , p-Aminoazobenzeno/farmacologiaRESUMO
Fibroblast growth factor 8 (FGF-8), also known as androgen-induced growth factor (AIGF), is presumed to be a potent mitogenic cytokine that plays important roles in early embryonic development, brain formation and limb development. In the bone environment, FGF-8 produced or received by chondrocyte precursor cells binds to fibroblast growth factor receptor (FGFR), causing different levels of activation of downstream signalling pathways, such as phospholipase C gamma (PLCγ)/Ca2+ , RAS/mitogen-activated protein kinase-extracellular regulated protein kinases (RAS/MAPK-MEK-ERK), and Wnt-ß-catenin-Axin2 signalling, and ultimately controlling chondrocyte proliferation, differentiation, cell survival and migration. However, the molecular mechanism of FGF-8 in normal or pathological cartilage remains unclear, and thus, FGF-8 represents a novel exploratory target for studies of chondrocyte development and cartilage disease progression. In this review, studies assessing the relationship between FGF-8 and chondrocytes that have been published in the past 5 years are systematically summarized to determine the probable mechanism and physiological effect of FGF-8 on chondrocytes. Based on the existing research results, a therapeutic regimen targeting FGF-8 is proposed to explore the possibility of treating chondrocyte-related diseases.
Assuntos
Condrogênese , Fatores de Crescimento de Fibroblastos , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
The MAPK/ERK pathway regulates a variety of physiological cellular functions, including cell proliferation and survival. It is abnormally activated in many types of human cancers in response to driver mutations in regulators of this pathway that trigger tumor initiation. The early steps of oncogenic progression downstream of ERK overactivation are poorly understood due to a lack of appropriate models. We show here that ERK1/2 overactivation in the trunk neural tube of the chicken embryo through expression of a constitutively active form of the upstream kinase MEK1 (MEK1ca), rapidly provokes a profound change in the transcriptional signature of developing spinal cord cells. These changes are concordant with a previously established role of the tyrosine kinase receptor ligand FGF8 acting via the ERK1/2 effectors to maintain an undifferentiated state. Furthermore, we show that MEK1ca-transfected spinal cord cells lose neuronal identity, retain caudal markers, and ectopically express potential effector oncogenes, such as AQP1. MEK1ca expression in the developing spinal cord from the chicken embryo is thus a tractable in vivo model to identify the mechanisms fostering neoplasia and malignancy in ERK-induced tumorigenesis of neural origins.
Assuntos
Transformação Celular Neoplásica/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Animais , Galinhas , Modelos Animais de Doenças , Humanos , Medula Espinal/patologiaRESUMO
FGF8, which is specifically expressed in the dental epithelium prior to the E12.5 bud stage, is a key player during odontogenesis, being responsible for the initiation of tooth development. Here, to investigate the impact of persistent FGF8 signaling on tooth development, we forcibly activated FGF8 signaling in the dental epithelium after the bud stage by generating K14-Cre;R26R-Fg8 mice. We found that a unique type of fused supernumerary incisors is formed, although morphologically resembling the features of type II dens invaginatus in humans. Further analysis revealed that ectopically activated epithelial FGF8 alters the cell fate of the incisor lingual outer enamel epithelium, endowing it with odontogenic potential by the activation of several key tooth genes, including Pitx2, Sox2, Lef-1, p38, and Erk1/2, and induces de novo formation of an extra incisor crown lingually in parallel to the original one, leading to the formation of an extra incisor crown and fused with the original incisor eventually. Meanwhile, the overdosed epithelial FGF8 signaling dramatically downregulates the expression of mesenchymal Bmp4, leading to severely impaired enamel mineralization. Based on the location of the extra incisors, we propose that they are likely to be rescued replacement teeth. Our results further demonstrate the essential role of FGF8 signaling for tooth initiation and the establishment of progenitor cells of dental epithelial stem cells during development.
Assuntos
Incisivo , Odontogênese , Amelogênese/genética , Animais , Anodontia , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Incisivo/anormalidades , Camundongos , Odontogênese/genética , Germe de DenteRESUMO
Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/ß-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3ß inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/ß-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice.
Assuntos
Células-Tronco Mesenquimais , Odontoblastos , Animais , Diferenciação Celular , Fator 8 de Crescimento de Fibroblasto/metabolismo , Camundongos , Odontoblastos/metabolismo , OsteoblastosRESUMO
Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The â¼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.
Assuntos
Duplicação Cromossômica , Cromossomos Humanos Par 10/química , Variações do Número de Cópias de DNA , Fator 8 de Crescimento de Fibroblasto/genética , Deformidades Congênitas das Extremidades Inferiores/genética , Adolescente , Alelos , Animais , Sistemas CRISPR-Cas , Pré-Escolar , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos Par 10/metabolismo , Elementos Facilitadores Genéticos , Família , Feminino , Fêmur/anormalidades , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Edição de Genes , Heterozigoto , Humanos , Lactente , Deformidades Congênitas das Extremidades Inferiores/diagnóstico por imagem , Deformidades Congênitas das Extremidades Inferiores/metabolismo , Deformidades Congênitas das Extremidades Inferiores/patologia , Masculino , Camundongos , Camundongos Transgênicos , Linhagem , FenótipoRESUMO
High uncoupling protein 1 (Ucp1) expression is a characteristic of differentiated brown adipocytes and is linked to adipogenic differentiation. Paracrine fibroblast growth factor 8b (FGF8b) strongly induces Ucp1 transcription in white adipocytes independent of adipogenesis. Here, we report that FGF8b and other paracrine FGFs act on brown and white preadipocytes to upregulate Ucp1 expression via a FGFR1-MEK1/2-ERK1/2 axis, independent of adipogenesis. Transcriptomic analysis revealed an upregulation of prostaglandin biosynthesis and glycolysis upon Fgf8b treatment of preadipocytes. Oxylipin measurement by LC-MS/MS in FGF8b conditioned media identified prostaglandin E2 as a putative mediator of FGF8b induced Ucp1 transcription. RNA interference and pharmacological inhibition of the prostaglandin E2 biosynthetic pathway confirmed that PGE2 is causally involved in the control over Ucp1 transcription. Importantly, impairment of or failure to induce glycolytic flux blunted the induction of Ucp1, even in the presence of PGE2 . Lastly, a screening of transcription factors identified Nrf1 and Hes1 as required regulators of FGF8b induced Ucp1 expression. Thus, we conclude that paracrine FGFs co-regulate prostaglandin and glucose metabolism to induce Ucp1 expression in a Nrf1/Hes1-dependent manner in preadipocytes, revealing a novel regulatory network in control of Ucp1 expression in a formerly unrecognized cell type.