Abstract
DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of γH2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells γH2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of γH2AX-only cells increases after caspase inhibition while the relative number of TUNEL-only cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration.
Highlights
8, and 9) and executioner caspases in addition to pro-apoptotic factors of the intrinsic apoptotic pathway have been implicated in the regression of interdigital tissue[8]
Results γH2AX-postive DNA damage is a precocious event of interdigital cell death
The initial response of most eucaryotic cells to repair DNA damage is the phosphorylation at the break region of substrates, such as the histone variant H2AX that forms γH2AX (H2AXS139PO4; ref. 18) that facilitates the recruitment of additional DNA damage response (DDR) mediators
Summary
8, and 9) and executioner caspases (caspase-3, 6 and −7) in addition to pro-apoptotic factors of the intrinsic (mitochondrial) apoptotic pathway have been implicated in the regression of interdigital tissue[8]. The occurrence of an associated cell senescence process in the regression of interdigits is a striking feature that is difficult to explain[12]. These findings have been largely interpreted as evidence for a functional redundancy between the biological mechanisms responsible for tissue degeneration, allowing each to replace the other should one pathway fail[13]. The DNA damage response (DDR) is destined to repair DNA and results in cell cycle arrest, cell senescence, and apoptosis to ensure genomic stability[15,16]. The aim of this study was to determine whether DNA damage precedes the degradative routes observed in the remodeling of interdigits termed the Interdigital Necrotic Zones (INZs)
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