Abstract
Purpose: :
To analyze activation of the DNA damage response during naturally occurring and induced cell death in the developing retina. The histones that are part of the nucleosome primarily pack the DNA, but recent studies have shown that certain histones have additional functions. A variant of histone H2A - H2AX is part of the cell cycle S/G2-phase DNA-damage checkpoint. H2AX becomes phosphorylated, gamma-H2AX, by the phosphatidylinositon-3 kinase family of kinases including ataxia telangectasia mutated (ATM) and ataxia telangectasia Rad3-rated (ATR) protein kinases, upon double stand breaks. Here we have investigated the presence of gamma-H2AX in the normal developing chick retina at the period of naturally occurring neuronal death and after injection of a DNA damaging substance.
Methods: :
Fertilized White Leghorn eggs were injected at Hamburger & Hamilton stage 25 (E5.5) either with the DNA damaging agent Neocarzinostatin (NCS) or with vehicle. NCS is an antiproliferative and antitumor compound. Injection of 2µl 20ng/µl NCS in to the eye of the chick embryo leads to double strand brakes and neuronal apoptosis in the retina. Embryos were incubated for 2hs before analysis. Gamma-H2AX immunoreactivity, together with TUNEL staining, was analyzed in the injected retinas as well as on different stages of normal chick retina representing the peak of naturally occurring cell death.
Results: :
In the NCS treated retinas massive TUNEL staining was observed already 2 hrs after NCS injection indicating that many cells immediately entered apoptosis. In the vehicle injected retinas a few TUNEL cells were found, close to the site of injection. Strong gamma-H2AX immunoreactivity was seen in TUNEL-negative or weakly TUNEL stained cells of the injected eyes. This suggested that H2AX is phosphorylated prior to DNA fragmentation during apoptosis in the chick retina. In normal retina, from stages with abundant apoptosis, a similar result was observed.
Conclusions: :
We conclude that the DNA damage response system is activated, as shown by the presence of gamma-H2AX immunoreactive cells, prior to the apoptosis induced DNA fragmentation as indicated by TUNEL staining. This suggests that the cell cycle DNA-damage checkpoint is triggered during naturally occurring cell death in the developing chick retina.
Keywords: retinal development • apoptosis/cell death • phosphorylation