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Barbara M. Braunger, Stefan M. Pielmeier, Cora Demmer, Victoria Landstorfer, Daniela Kawall, Herbert Jaegle, Ernst R. Tamm; Deficiency In Tgf Beta Signaling Enhances Apoptosis Of Retinal Neurons During Development. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5417.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of TGF-beta signaling during ontogenetic cell death in the mouse retina. In the spinal cord, TGF-beta does promote neuronal survival and may have similar effects on neurons in the retina.
Floxed TGFbr2 mice in which LoxP sites had been introduced in the coding sequences of the gene encoding for the type 2 TGF-beta receptor (TGFbr2) were crossed with alpha-Cre mice expressing Cre recombinase in retinal neurons under control of the alpha- enhancer of the Pax6 promoter. As control, floxed Smad7 mice were used, in which LoxP sites had been introduced in the coding sequences of Smad7, an endogenous inhibitor of TGF-beta signaling. Western Blot analysis and quantitative real-time PCR were performed to confirm the conditional knockout. Apoptotic cell death of retinal neurons was analyzed at embryonic days (E) 12.4, 14.5, 16.5 and at postnatal days (P) 4, 7, 9 by TUNEL labeling and an ELISA for free nucleosomes.
Western blot analysis for TGFR-2 and Smad7 confirmed the conditional knock out in the retina in both mouse lines. Immunohistochemistry of phosphorylated Smad2/3 (pSmad) showed immunoreactivity in nuclei of retinal ganglion cells in wild-type animals, indicating activated TGF-beta signaling, whereas TGFbr2flox/flox/alpha-Cre mice showed dramatically reduced immunoreactivity. TUNEL analysis showed significantly (p < 0.01 - 0.05) more TUNEL positive cells in the retina of TGFbr2flox/flox/alpha-Cre mice at each time point when compared to their wild-type littermates. The increase in cell death resulted in a significantly thinner inner nuclear layer in adult mice and significantly fewer axons (p < 0.01) in the optic nerve. Standard flash-ERG indicates functional changes. In contrast, Smad7flox/flox/alpha-Cre mice showed significantly (p < 0.01 - 0.02) less apoptotic cell death in the retina when compared to their wild-type littermates.
TGF-beta signaling protects retinal neurons from apoptotic cell death during development and may have comparable effects following retinal injury in adult animals.
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