Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation
Teri Louise Belecky-Adams1,2,
Ruben Adler2 and
David C. Beebe3,*
1 Department of Biology, Indiana University-Purdue University Indianapolis, SL306, Indianapolis, IN 46202, USA
2 Wilmer Institute, Department of Ophthalmolology, Johns Hopkins University, Baltimore, MD, USA
3 Department of Ophthalmolology Visual Sciences, Washington University, St. Louis, MO 63110, USA
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Fig. 1. The effect of over expressing noggin in the eye. Eyes were infected with retroviruses expressing alkaline phosphatase or noggin at HH15-18 (E2.5-3). Lenses were fixed, sectioned and stained at E4 (A,B) or E6 (C,D). Phalloidin staining (red) primarily outlines the cell boundaries and TOTO-1 (green) labels nuclei and, to a lesser degree, cytoplasmic RNA. (A) Lens at E4 from an eye infected with control retrovirus. Primary fiber formation is nearly complete and the lumen of the lens vesicle is nearly occluded. (B) Lens at E4 from an eye infected with retrovirus expressing noggin. Primary fiber cell elongation is retarded and the lumen of the lens vesicle remains open. The apparently greater thickness of the presumptive corneal epithelium in A was an artifact of sectioning. The corneal epithelium shown in B is typical of this stage of development. (C) Lens at E6 from an eye infected with retrovirus expressing alkaline phosphatase. Fiber cell elongation appears normal. (D) Lens at E6 from an eye infected with retrovirus expressing noggin. Cells in the initial stage of fiber cell elongation at the lens equator are shorter than normal, leading to the formation of a gap between the earliest fiber cells and the equatorial epithelial cells. Older fiber cells, deeper in the lens, appear to have recovered and are elongating normally. Scale bars: 50 µm.
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Fig. 2. A graph showing the number of TUNEL-positive cells per lens at HH29-31 (E6) in uninjected eyes and eyes injected with virions expressing alkaline phosphatase or noggin. Error bars are ± s.e.m.
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Fig. 3. Antibodies against pSMAD1 provide evidence of BMP signaling throughout lens development. pSMAD1 immunostaining is in red and nucleic acids are stained green with TOTO-1. (A) At HH10, the nuclei of cells in the lens placode (LP) and the portion of the optic vesicle (OV) in contact with the lens placode stain with antibodies against pSMAD1. B. The nuclei of cells throughout the lens vesicle at HH18 (E3) stain for pSMAD1. Staining is weaker in the adjacent prospective corneal epithelium (CE). C. At HH22 (E4) strong nuclear pSMAD1 staining is localized to cells at the equator of the lens and at the margin of the optic cup, the prospective iris and ciliary epithelia. Nuclear pSMAD1 levels are barely detectable in the lens epithelium and in the more mature lens fiber cells. D. At HH33 (E7) pSMAD1 staining localizes to the nuclei of cells at the lens equator (EQ), the earliest lens fibers. More mature fiber cells deeper in the lens have low to undetectable levels of pSMAD1 staining. Inset: nuclei and cytoplasmic vesicles stained for pSMAD1 in elongating lens fiber cells. This section was not stained for nucleic acids. Scale bars: A-D 25 µm, inset 10 µm.
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Fig. 4. Cell elongation in lens epithelial explants. (A) Diagram showing steps in the preparation of an epithelial explant [modified from Piatigorsky et al. (Piatigorsky et al., 1976 )]. (B) Section of a lens epithelial explant treated with FGF1 (50 ng/ml) for 5 hours. Epithelial cells treated in this manner elongated from approximately 11 µm to an average of 12.5 µm. (C) Section of an explant treated with 20% bovine vitreous humor for 5 hours. Epithelial cells treated with vitreous humor elongated to over 18 µm in length. Explants in B and C were labeled with phalloidin (red) to locate cell boundaries and with TOTO-1 (green) to stain nuclei and cytoplasmic RNA. Scale bars in B and C are 10 µm.
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Fig. 5. The effect of noggin-conditioned medium on lens cell elongation stimulated by vitreous humor or IGF-1. (A) Vitreous humor caused lens epithelial cells to elongate to over 18 µm in 5 hours in the presence of conditioned medium from CHO cells transfected with an empty plasmid (P<<0.001). Medium from CHO cells transfected with a plasmid encoding noggin reduced cell elongation stimulated by vitreous humor to approximately 14 µm (P<0.01). (B) Conditioned medium from cells expressing noggin had no effect on lens epithelial cell elongation stimulated by recombinant chicken IGF-1 (16 ng/ml; P=0.36). Error bars are ± s.e.m.
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Fig. 6. The elongation-promoting activity removed by noggin was restored by adding BMPs. Vitreous humor was incubated with recombinant Fc-noggin and the noggin-bound material was removed with Protein A/G beads. Protein A/G beads alone did not remove the elongation-promoting activity. Treatment of vitreous humor with Fc-noggin reduced cell elongation to approximately 17 µm from over 20 µm. Addition of BMP-2, -4, or -7 restored full elongation (P<0.02 for each BMP added to noggin-depleted vitreous humor compared to cells cultured in noggin-depleted vitreous humor alone). Error bars are ± s.e.m.
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Fig. 7. Adding FGFs and BMPs to lens epithelial cells did not promote elongation comparable to that obtained by treatment with 20% vitreous humor. FGF1 or FGF2 (50 ng/ml) stimulated lens epithelial cells to elongate approximately 2 µm to approximately 13 µm (P<0.01). Addition of BMP-2 (50 ng/ml) to FGF1 or FGF2 increased cell elongation by approximately another 2 µm (P<0.01 compared to FGF alone). Vitreous humor typically stimulated lens epithelial cells to elongate to more than 18 µm.
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© The Company of Biologists Ltd 2002
