Recombinant Mouse Fibroblast growth factor 9 (Fgf9) (Active)

What is the molecular structure of recombinant mouse FGF9?

Recombinant mouse FGF9 is a 23.4 kDa monomeric protein consisting of 207 amino acids with a specific sequence that begins with MPLGEVGSYFG and ends with DILSQS . In its native state, FGF9 exists in a monomer-dimer equilibrium, with the dimerization state playing a crucial role in its biological activity . Structurally, FGF9 shares approximately 30% sequence similarity with other members of the fibroblast growth factor family and is most closely related to FGF-16 . The protein contains regions important for receptor binding and interaction with extracellular matrix components, particularly heparan sulfate proteoglycans, which influence its tissue distribution and signaling range .

What are the primary receptors for FGF9 and how does receptor binding affect cellular responses?

FGF9 demonstrates selective binding to specific FGF receptor subtypes with varying affinities. It functions as a high-affinity, heparin-dependent ligand primarily for FGFR3 and FGFR2, while showing limited binding to FGFR1 and FGFR4 . Specifically, FGF9 efficiently activates the FGFR2c splice form of FGFR2 and both the FGFR3b and FGFR3c splice isoforms of FGFR3 . This receptor specificity determines the cellular responses to FGF9 and explains its tissue-specific effects during development and in adult tissues.

The receptor binding triggers intracellular signaling cascades that control cell proliferation, survival, and differentiation. The biological activity of mouse FGF9 can be measured through dose-dependent stimulation of thymidine uptake by BaF3 cells expressing FGF receptors, with an expected ED50 of approximately 2 x 10^6 units/mg . In experimental systems like NR6R 3T3 cells, FGF9 demonstrates proliferative activity with an ED50 ≤10 ng/mL .

What are the key biological functions of FGF9 in developmental and physiological processes?

FGF9 plays critical roles in multiple developmental and physiological processes:

• Neural Development: FGF9 functions as a growth-stimulating factor for glial cells and is produced mainly by neurons in the nervous system . It serves as a mitogen and survival factor for nerve cells, supporting the growth and survival of motor neurons .

• Embryonic Development: FGF9 is involved in various aspects of embryonic development, including limb and skeletal formation. Mutations in FGF9, such as the Eks mutation in mice, can lead to joint synostosis (fusion) and craniosynostosis (premature fusion of cranial sutures) .

• Sex Determination: FGF9 expression in the gonad is necessary for proper sex determination during development .

• Tissue Repair and Homeostasis: As a member of the FGF family, FGF9 possesses broad mitogenic and cell survival activities that contribute to tissue repair mechanisms .

• Mesenchymal Cell Development: FGF9 acts as a mitogen and survival factor for mesenchymal cells, influencing their proliferation and differentiation .

• Autocrine and Paracrine Signaling: FGF9 functions as both an autocrine and paracrine factor, supporting the growth and survival of target cells including motor neurons and prostate tissue .

These diverse functions highlight the importance of FGF9 in normal development and tissue homeostasis, as well as its potential roles in pathological conditions.

How should recombinant mouse FGF9 be reconstituted and stored for optimal activity?

For optimal reconstitution and storage of lyophilized recombinant mouse FGF9:

• Reconstitution Protocol:

  • Use sterile water to achieve a concentration of 0.1 mg/mL

  • Gently pipette the solution down the sides of the vial rather than directly onto the protein

  • Allow several minutes for complete reconstitution without vortexing, which can denature the protein

• Storage Conditions:

  • Prior to reconstitution: Store lyophilized protein at -20°C

  • Short-term storage (up to 1 month): Store reconstituted protein at 4°C

  • Long-term storage (up to 3 months): Prepare working aliquots in 0.1% BSA solution and store at -20 to -80°C

• Stability Considerations:

  • Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

  • For experiments requiring long-term use, prepare single-use aliquots

  • Lyophilized FGF9 is typically stored in a buffer containing 10 mM sodium phosphate at pH 7.5

Following these guidelines will help ensure the protein maintains its biological activity for subsequent experiments and reduces variability in experimental results due to protein degradation.

What assays can be used to validate the biological activity of recombinant mouse FGF9?

Several established assays can be used to validate the biological activity of recombinant mouse FGF9:

• Cell Proliferation Assays:

  • NR6R 3T3 Cell Proliferation: Measure cell proliferation using appropriate detection methods (MTT, BrdU incorporation, cell counting) with an expected ED50 ≤10 ng/mL

  • BaF3 Thymidine Uptake: Measure thymidine incorporation in BaF3 cells expressing FGF receptors with an expected ED50 of approximately 2 x 10^6 units/mg

• Receptor Binding and Activation Assays:

  • Phosphorylation of downstream signaling molecules (e.g., ERK1/2)

  • Receptor-specific binding assays using cells expressing defined FGFR subtypes

• Quality Control Parameters:

  • Purity assessment by SDS-PAGE (both reducing and non-reducing conditions) should show ≥95% purity

  • Endotoxin testing using Kinetic LAL assay should confirm levels ≤1 EU/μg to avoid non-specific cellular responses

• Functional Validation in Specific Cell Types:

  • Glial cell growth assays, reflecting FGF9's natural role as a glia-activating factor

  • Neurite outgrowth assays in neuronal cultures

  • Migration assays for cells known to respond to FGF9

These assays provide complementary information about different aspects of FGF9 activity, from basic receptor binding to complex cellular responses, ensuring comprehensive validation of the recombinant protein.

How does FGF9 dimerization affect its biological activity and experimental applications?

FGF9 dimerization plays a critical regulatory role that significantly impacts its biological activity and experimental applications:

• Molecular Characteristics:

  • Wild-type FGF9 primarily exists as a dimer with an association constant of approximately 10.4 μM^-1

  • Sedimentation equilibrium centrifugation studies show that wild-type FGF9 has an average molecular mass of ~39,264 Da, approximately twice the calculated monomeric mass (20,090 Da)

• Functional Consequences:

  • Dimerization state affects FGF9's affinity for heparin and heparan sulfate proteoglycans

  • Monomeric FGF9 (e.g., the FGF9^Eks mutant with Asn143Thr substitution) binds heparin with lower affinity than dimeric FGF9

  • This difference in binding results in altered diffusion properties through tissues

• Biological Significance:

  • The Eks mutation in mice prevents FGF9 homodimerization, leading to joint synostosis, craniosynostosis, and lung hypoplasia

  • This suggests that dimerization regulates the signaling range of FGF9 in developing tissues

  • Monomeric FGF9 shows increased diffusion, causing ectopic signaling and developmental abnormalities

• Experimental Implications:

  • Researchers should consider the dimerization state as a critical variable affecting experimental outcomes

  • Storage and handling conditions may affect the monomer-dimer equilibrium

  • Heparin or heparan sulfate concentration in experimental systems will differently affect monomeric versus dimeric FGF9

Understanding these molecular characteristics is essential for properly interpreting experimental results and for designing studies that accurately reflect the physiological activity of FGF9.

What experimental approaches can be used to study FGF9's role in developmental processes?

Investigating FGF9's role in developmental processes requires a combination of experimental approaches:

• Genetic Models:

  • Analysis of FGF9 knockout or mutant mice (e.g., Fgf9^Eks/Eks) to understand developmental consequences of altered FGF9 function

  • Conditional knockout models using tissue-specific Cre recombinase expression to study tissue-specific requirements for FGF9

  • Genetic crosses with reporter mice to visualize FGF9-responsive cell populations

• Expression Analysis Techniques:

  • In situ hybridization to localize FGF9 and FGFR expression during development

  • Immunohistochemistry to detect FGF9 protein and activated signaling components

  • RNA sequencing to identify transcriptional changes associated with FGF9 signaling during development

• Ex Vivo and Organ Culture Systems:

  • Explant cultures treated with recombinant FGF9 to observe tissue-specific responses

  • Organ cultures to study the effects of FGF9 on complex developmental processes

  • Co-cultures of different cell types to study paracrine signaling mechanisms

• Molecular and Cellular Techniques:

  • CRISPR-Cas9 gene editing to introduce specific mutations in FGF9 or its receptors

  • Cell lineage tracing to follow the fate of FGF9-expressing or FGF9-responsive cells

  • Live imaging of developing tissues with fluorescently labeled proteins to visualize dynamics

The combination of these approaches provides comprehensive insights into how FGF9 influences developmental processes across different tissues and developmental stages.

How can researchers identify and validate FGF9-specific effects versus general growth factor responses?

Distinguishing FGF9-specific effects from general growth factor responses requires careful experimental design:

• Receptor Specificity Analysis:

  • Compare responses in cells expressing different FGFR subtypes, recognizing that FGF9 preferentially activates FGFR3 and FGFR2 but not FGFR1 and FGFR4

  • Use receptor-specific blocking antibodies or competitive inhibitors

  • Compare cellular responses to FGF9 with responses to other FGF family members

• Signaling Pathway Discrimination:

  • Monitor activation of downstream signaling molecules with temporal resolution

  • Compare signaling patterns induced by FGF9 versus other growth factors

  • Use pathway-specific inhibitors to determine which downstream effects depend on specific signaling cascades

• Molecular Validation Strategies:

  • Perform gene expression profiling to identify FGF9-specific transcriptional signatures

  • Conduct quantitative RT-PCR to validate expression of FGF9-responsive genes

  • Use chromatin immunoprecipitation (ChIP) to identify transcription factors activated specifically by FGF9

• Functional Validation:

  • Use loss-of-function approaches including FGF9-specific neutralizing antibodies or RNA interference

  • Perform dose-response studies to identify concentration thresholds specific to FGF9

  • Analyze the temporal dynamics of responses to distinguish immediate versus delayed effects

These strategies enable researchers to attribute observed biological responses specifically to FGF9 rather than to general growth factor effects.

What are the emerging roles of FGF9 in pathological conditions?

Research has revealed several important roles for FGF9 in pathological conditions:

• Cancer Biology:

  • FGF9 overexpression has been observed in colon cancer cells

  • Translational regulation mechanisms, including elements in the 5' UTR (upstream open reading frames and internal ribosome entry sites), may contribute to FGF9 overexpression in cancer

  • FGF9 possesses properties that promote tumor growth and invasion, potentially functioning as both an autocrine and paracrine factor in tumor microenvironments

• Developmental Disorders:

  • The Eks mutation in mice (Asn143Thr substitution in FGF9) causes joint synostosis, craniosynostosis, and lung hypoplasia

  • This mutation prevents FGF9 homodimerization and reduces its affinity for heparin, leading to ectopic signaling and developmental abnormalities

  • Similar mechanisms may underlie related human developmental disorders

• Neurological Conditions:

  • As a neurotrophic factor highly expressed in the brain, FGF9 may play roles in neurological disorders

  • Its role in supporting neuronal and glial cell development suggests potential implications for neurodegenerative or neurodevelopmental conditions

• Research Approaches:

  • Analysis of FGF9 expression in pathological versus normal tissues

  • Examination of FGF9 secretion using techniques like enzyme-linked immunosorbent assay (ELISA)

  • Investigation of translational regulation using polysome profiling and other molecular techniques

  • Animal models with altered FGF9 expression or signaling to study disease mechanisms

These emerging areas highlight the importance of FGF9 beyond its developmental roles and suggest potential therapeutic applications targeting FGF9 signaling in various pathological conditions.

What factors affect reproducibility when working with recombinant FGF9, and how can they be mitigated?

Several factors can influence the reproducibility of experiments using recombinant mouse FGF9:

• Protein Handling and Storage:

  • Challenge: Protein degradation due to improper storage or repeated freeze-thaw cycles

  • Solution: Store lyophilized protein at -20°C; prepare single-use aliquots of reconstituted protein in 0.1% BSA and store at -80°C

  • Challenge: Denaturation during reconstitution

  • Solution: Avoid vortexing; gently pipette reconstitution buffer down the sides of the vial

• Experimental Design Considerations:

  • Challenge: Variability in heparin/heparan sulfate availability affecting FGF9 activity

  • Solution: Standardize heparin concentrations in experimental systems; consider supplementing with defined amounts of heparin

  • Challenge: Receptor expression variations across cell lines or primary cultures

  • Solution: Characterize receptor expression profiles; use cells with defined receptor expression patterns

• Quality Control Measures:

  • Challenge: Inconsistent protein quality between batches

  • Solution: Verify protein purity (≥95% by SDS-PAGE) and activity before experiments

  • Challenge: Endotoxin contamination causing non-specific cellular responses

  • Solution: Ensure endotoxin levels are ≤1 EU/μg using appropriate testing methods

• Assay-Specific Variables:

  • Challenge: Different detection methods yielding different results

  • Solution: Standardize detection methods; include appropriate controls and standard curves

  • Challenge: Concentration-dependent effects not being properly captured

  • Solution: Use concentration series spanning the expected ED50 (10 ng/mL for NR6R 3T3 proliferation assays)

Implementing these solutions will help ensure consistent, reproducible results when working with recombinant mouse FGF9.

How does FGF9's interaction with the extracellular matrix influence experimental design?

FGF9's interaction with extracellular matrix components has significant implications for experimental design:

• Heparin Dependency Considerations:

  • FGF9 is a heparin-dependent ligand for its receptors, with binding affinity affected by its dimerization state

  • Experimental solution: Include appropriate heparin concentrations in binding and activity assays; consider how the matrix composition of experimental systems affects FGF9 diffusion and activity

• Diffusion and Distribution Effects:

  • The affinity of FGF9 for heparan sulfate proteoglycans affects its tissue distribution

  • Wild-type (dimeric) FGF9 shows more restricted diffusion compared to monomeric forms

  • Experimental solution: Consider diffusion characteristics when designing experiments involving tissue penetration or gradient formation; use appropriate time points to capture distribution dynamics

• Dimerization State Impacts:

  • The monomer-dimer equilibrium affects biological activity and matrix binding

  • The Eks mutation (Asn143Thr) prevents homodimerization, reducing heparin affinity

  • Experimental solution: Characterize the dimerization state of recombinant FGF9 preparations; consider how experimental conditions might affect this equilibrium

• Tissue-Specific Matrix Variations:

  • Different tissues have varying compositions of extracellular matrix components

  • This affects local FGF9 concentration and activity

  • Experimental solution: Use appropriate matrix components when studying tissue-specific effects; consider using tissue-derived matrices or specific ECM proteins in culture systems

Understanding these interactions is crucial for designing physiologically relevant experiments and for interpreting results in the context of FGF9's natural behavior in tissues.

What advanced techniques can provide insights into FGF9 function in complex biological systems?

Several advanced techniques offer powerful approaches for studying FGF9 function in complex biological systems:

• Imaging and Visualization Techniques:

  • Fluorescently tagged FGF9 to track protein distribution and dynamics in real-time

  • Multiphoton microscopy for deep tissue imaging in explants or whole organisms

  • Correlative light and electron microscopy to link functional observations with ultrastructural context

• Molecular and Genetic Tools:

  • CRISPR-Cas9 genome editing to introduce specific mutations or regulatory elements

  • Single-cell RNA sequencing to capture heterogeneous responses to FGF9 in complex tissues

  • Conditional expression systems for spatiotemporal control of FGF9 expression or receptor activation

• Biochemical and Protein Analysis Methods:

  • Surface plasmon resonance to study binding kinetics with receptors or matrix components

  • Analytical ultracentrifugation to characterize protein states (as used in studying FGF9 dimerization)

  • Mass spectrometry-based approaches to identify FGF9 interaction partners

• Translational Research Approaches:

  • Polysome profiling to study translational regulation of FGF9 expression

  • Patient-derived samples or data to correlate experimental findings with human conditions

  • Disease models to validate the relevance of FGF9 mechanisms in pathological states

• Systems Biology Approaches:

  • Multi-omics integration to understand FGF9 in broader cellular and tissue contexts

  • Mathematical modeling of FGF9 diffusion, binding, and signaling dynamics

  • Network analysis to position FGF9 signaling within larger regulatory networks

These advanced techniques provide comprehensive insights into FGF9 function beyond what can be achieved with traditional methods, allowing researchers to address complex questions about FGF9's roles in development, homeostasis, and disease.