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Sameer D. Pant, Lindsey D. March, Curtis R. French, Andrew J. Waskiewicz, Ordan J. Lehmann; Molecular Mechanisms Underlying Increased Ocular Apoptosis Observed In gdf6a-/- Zebrafish During Development. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4529.
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© ARVO (1962-2015); The Authors (2016-present)
Gdf6 is a TGFβ family ligand essential for normal development, whose deficiency results in pediatric anomalies including coloboma and microphthalmia. Loss of Gdf6/gdf6 also results in increased retinal apoptosis in animal models, with zebrafish (gdf6a-/-) mutants exhibiting significantly increased apoptosis at 28 hours post fertilization (hpf). The goal of this study was to identify mediators that drive this increase and also to test if retinal apoptosis was responsive to neuroprotective modulators like P7C3.
Embryos obtained from crossing gdf6a+/- heterozygotes were staged to 28 hpf, euthanized, fixed and genotyped using PCR and restriction digestion. Immunohistochemistry (IHC) was performed using antibodies (Ab) against activated Caspase-3 and phospho-p38 MAPK. For inhibiting retinal apoptosis, dechorionated embryos were incubated in embryo media containing either DMSO, a p38 MAPK inhibitor (SB203580), or P7C3 (a novel anti-apoptotic compound). To investigate the role of BCL2 proteins, antisense morpholino oligonucleotides were used to knock down Bax1 and separately Bax2.
IHC with phospho-p38 MAPK Abs demonstrated increased phosphorylated p38 in mutants suggesting that p38 MAPK may mediate retinal apoptosis. The MAPK inhibitor SB203580, was next used to validate these results with data from 3 replicate experiments demonstrating reduced apoptosis [SB203580: 76.0 ± 38.8 signals; DMSO: 137.9 ± 58.9 signals] (p=0.00002). P7C3 treatment also significantly reduced apoptosis compared to DMSO treatment [P7C3 treatment: Mean = 16.1 signals; DMSO treatment: Mean = 76.1 signals] (p<0.00001). Finally, Morpholino (MO) knockdown of Bax1 and Bax2 similarly demonstrated reduced ocular apoptosis in mutants compared to p53 MO controls [p = 0.09 and 0.005 respectively].
These data demonstrate that Gdf6 induced retinal apoptosis is amenable to pharmacological inhibition, with a number of tractable targets available. This apoptosis is mediated in part by p38 MAPK with some involvement of the intrinsic apoptotic pathway evident from the BCL2 data. Since TGFβ signaling is highly conserved, the mechanisms underlying apoptosis in gdf6a-/- mutants are likely common to a broader spectrum of disorders attributable to other TGFβ ligands.
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