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TGF-ß modulates programmed cell death in the retina of the developing chick embryo

Nicole Dünker^*, Norbert Schuster^* and Kerstin Krieglstein

University of Saarland, Department of Anatomy, D-66421 Homburg/Saar, Germany
^* These authors contributed equally to this work

View larger version (81K): [in a new window]   Fig. 1. Localization of TGF-ß ligands and receptor in the developing chick retina. (A) An E6 chick eye showing the distribution of apoptotic nuclei (green stars). The main areas of cell death (ellipses) are localized in the vicinity of the optic nerve. The box outlines the region shown at higher magnification in B-F. (B-F) Expression of TGF-ß isoforms and receptor (TßRII) in the E6 chick retina (B-D, indirect fluorescence; E,F, DAB stain). (B) TßRII immunoreactivity is distributed evenly over the entire retinal surface. TGF-ß2 (C,E) and TGF-ß3 (D,F) immunoreactivity was observed mainly in the central retina in the region around the optic nerve head, where apoptosis is most prominent during the early period of PCD. In addition, TGF-ß3 is localized in the pigment epithelium (asterisk in D), in structures that span the neuroepithelium, resembling Müller glia cells or radial Müller fibers (arrowheads in D,F) and in the presumptive ganglion cell layer (arrows in D,F). le, lens; nr, neural retina; on, optic nerve; pe, pigment epithelium; vb, vitreous body; ov, optic ventricle. Scale bar: 50 µm.

View larger version (34K): [in a new window]   Fig. 2. Cell death in the developing chick retina. (A,B) Detection of DNA fragmentation in apoptotic nuclei by TUNEL staining of retinal sections, showing the optic nerve region of E6 chick retinae. TUNEL-positive nuclei (arrowheads) are interspersed in the pseudostratified neuroepithelium. Many more apoptotic nuclei were labeled in control sections (A) compared with sections from anti-TGF-ß-treated embryos (B). (C-E) Quantification of cell death. Apoptotic levels were quantified in retinal sections using the TUNEL assay (C,E) or in retinal extracts using the nucleosome assay (ELISA; D; see Materials and Methods). Apoptosis was reduced in retinae obtained from anti-TGF-ß-, anti-NGF- and anti-TGF-ß/anti-NGF-treated embryos when compared with untreated controls. Co-treatment with TGF-ß neutralizing antibody and recombinant NGF (E) did not block the anti-TGF-ß effect, resulting in an equivalent decrease in apoptosis. Values are mean±s.e.m. *P<0.01; **P<0.005; ***P<0.001; unpaired Student’s t-test. on, optic nerve; ov, optic ventricle. Scale bar, 50 µm.

View larger version (139K): [in a new window]   Fig. 3. Morphological alterations of E6 chick retinae after neutralization of TGF-ß. Hematoxylin and Eosin-stained retinal cross sections of untreated (A,C) or anti-TGF-ß-treated (B,D) E6 chick embryos. The insets in A and B demarcate the region shown at higher magnification in C and D, respectively. Compared with control retinae, retinae of anti-TGF-ß-treated embryos are noticeably thicker and the retinal neuroepithelium exhibits protrusions and indentations (asterisks in B). le, lens; nr, neural retina; on, optic nerve; pe, pigment epithelium. Scale bars: 500 µm in A,B; 50 µm in C,D.

View larger version (84K): [in a new window]   Fig. 4. Cell proliferation in the developing chick retina, as detected by BrdU incorporation. Staining with a monoclonal antibody to BrdU results in a brown reaction product. No increase in cell proliferation was detected in the optic nerve region (A,B) of the central retina, in regions neighboring the optic nerve region (C,D; higher magnification of region resembling area of insets in Fig. 3A,B) or in more peripheral regions (E,F; higher magnification of the proliferation zone) when comparing control (A,C,E) and anti-TGF-ß-treated (B,D,F) retinae. (G) Quantification of cell proliferation. Comparison shown as number of BrdU-positive cells in control versus anti-TGF-ß-treated retinae. nr, neural retina; on, optic nerve; ov optic ventricle; pe, pigment epithelium. Scale bars: in A, 100 µm for A,B; in C, 50 µm for C,D; in E, 50 µm for E,F.

View larger version (73K): [in a new window]   Fig. 5. Acidic phosphatase labeling of microglial cells in the developing E4 (B,C) and E6 (D,E) chick retina. The product of this enzymatic activity can be detected by a histochemical method that yields a red product. Intensely stained acidic phosphatase-positive cells are seen on the vitreal surface of anti-TGF-ß-treated (arrowheads in B,D) and untreated retinae (arrowheads in C,E). No enzyme activity was found in control sections incubated in medium without substrate (naphtol AS BI phosphate; A,C). Scale bar: 20 µm.

View larger version (80K): [in a new window]   Fig. 6. Expression of NGF in the E6 chick retina. RT-PCR with NGF- and GAPDH-specific primers was performed as described (see Materials and Methods). The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. NGF is detectable in both control and anti-TGF-ß-treated retinae. NGF, lane 4 (control) and lane 5 (anti-TGF-ß); GAPDH, lane 9 (control) and lane 10 (anti-TGF-ß). PCR with crude RNA preparations revealed no amplification product (lanes 2,3,7,8), ruling out cross contamination with genomic DNA. M, marker lane.

View larger version (44K): [in a new window]   Fig.7 . Expression of p75NTR in the E6 chick retina. p75NTR immunoreactivity was observed in the prospective ganglion cell layer and optic fiber layer (arrowheads in A-C) of control (A) and anti-TGF-ß-treated (B,C) retinae. Staining was also detectable in the optic nerve (arrows in C). nr, neural retina; on, optic nerve; pe, pigment epithelium. Scale bar: 20 µm.

View larger version (35K): [in a new window]   Fig. 8. p75NTR (A) and TßRII (B) immunoreactivity in consecutive cryosections of an E5 chick retina, as revealed by indirect fluorescent stains (p75NTR, FITC/green; TßRII, CY3/red). TßRII immunoreactivity is distributed evenly, whereas p75NTR staining is mainly observed in the prospective ganglion cell layer and optic fiber layer, as well as in the optic nerve. The main overlap of both receptors can be found in the optic nerve region, where most cells stain positively for both, p75NTR and TßRII. on, optic nerve; ov, optic ventricle. Scale bar: 50 µm.

View larger version (56K): [in a new window]   Fig. 9. Expression of TGF-ß isoforms (B,C) and receptor (TßRII; A) in anti-NGF-treated E6 chick retinae, revealed by indirect immunofluorescence stains. As in control retinae, TßRII immunoreactivity is distributed evenly over the entire retinal surface (A), whereas TGF-ß2 (B) and TGF-ß3 (C) immunoreactivity was observed mainly in the central retina in the region around the optic nerve head. In addition, TGF-ß3 is localized in structures that span the neuroepithelium, resembling Müller glia cells or radial Müller fibers (arrowheads in C), and in the presumptive ganglion cell layer (arrow in C). nr, neural retina; on, optic nerve; ov, optic ventricle; pe, pigment epithelium. Scale bar: 50 µm.