The Role of FGFs and BMPs in Lens Fiber Cell
Differentiation
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Jia-Jan Fu
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During lens development, the epithelial cells of the lens vesicle undergo a rapid change. Although the epithelial cells are genetically the same, the anterior portion continues to proliferate while the posterior epithelial cells stop dividing and elongate. This process&emdash;in which the posterior cells change from epithelial cells to fiber cells&emdash;is called lens fiber cell differentiation.
The agent that causes lens fiber cells to differentiate is unknown but has been previously shown to be a component of the vitreous humor. Vitreous humor is a clear gel-like substance that is composed of collagens and contains many nutrients, growth factors, and other proteins needed for the development of the eye. Specifically, FGFs (fibroblast growth factors) and BMPs (bone morphogenetic proteins) are growth factors present in the vitreous body. Recent studies have shown that high concentrations of FGFs stimulate epithelial-to-fiber cell differentiation in chick lenses. There is also evidence that BMPs may also be involved in lens fiber cell differentiation. Thus, I tested the hypothesis that there are multiple factors, working in concert, which are required for lens fiber cell differentiation. It was my objective to investigate whether FGF2 (basic fibroblast growth factor) stimulated the production of other growth factors, mainly BMPs.
To determine whether or not FGF is causing the production of BMPs, we used an assay to measure the activity of the BMP signaling pathway. When a BMP receptor is activated by the binding of a BMP, the receptors dimerize. This causes the receptor to phosphorylate a SMAD, a protein, which travels to the nucleus to regulate the expression of different genes. However, this process can be inhibited by an extra-cellular protein called noggin. Noggin binds to the BMPs preventing there interaction.
Central epithelium explants were prepared from embryonic day 6 (E6) chick lens. The explants were treated with the additives FGF2 and noggin in medium 199 supplemented with 0.1% bovine albumin. Then the explants were put into an incubator (at 37oC in 5% CO2) for 6 days, with a change of medium 24 hours after preparation.
After the 6 day incubation period, the explants were fixed and immunostained with phospho-SMAD1 as the primary antibody and Alexa 568 goat-anti-rabbit as the secondary antibody. The primary antibody attaches to phosphorolated SMAD1, then the fluorescent secondary antibody binds to and locates the complex. A confocal microscope detects the fluorescence emitted by the sample. The location of the fluorescence reveals whether or not the BMP receptors were activated. Immunostaining in the nucleus signifies that the BMP receptors were stimulated by treatment with FGF.
My results are as follows:
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EXPLANTS |
Differentiated? |
Staining of PO4-SMAD1 |
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At time zero |
No |
Located in cytoplasm |
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With FGF |
Yes |
Located in nuclei |
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With FGF and Noggin |
Partially inhibited |
Partly in the cytoplasm and weak staining in some nuclei |
The results from my experiment were positive, however, inconclusive without further research. It's possible that FGFs could be directly or indirectly stimulating the production of BMPs that, in turn, causes lens fiber cell differentiation. Or FGFs might be, by some unknown pathway, producing phospho-SMAD1 and bypassing the BMP pathway. Thus, further studies are needed to explore these possibilities. These include western blotting to determine the levels of phospho-SMAD1 in FGF treated lens epithelia and using gene chips and western blotting to identify the BMPs produced in lens epithelia in response to FGF.
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