Exploring the Origin and Physiological Significance of DNA Double Strand Breaks in the Developing Neuroretina

Abstract

Genetic mosaicism is an intriguing physiological feature of the mammalian brain that generates altered genetic information and provides cellular, and prospectively functional, diversity in a manner similar to that of the immune system. However, both its origin and its physiological significance remain poorly characterized. Most, if not all, cases of somatic mosaicism require prior generation and repair of DNA double strand breaks (DSBs). The relationship between DSB generation, neurogenesis, and early neuronal cell death revealed by our studies in the developing retina provides new perspectives on the different mechanisms that contribute to DNA rearrangements in the developing brain. Here, we speculate on the physiological significance of these findings.

Keywords: DSBs; NHEJ; Rag2; neurogenesis; neuron somatic mosaicism; recombination.

Figure 1

Intrinsic events during early neurogenesis that may contribute to somatic mosaicism and functional diversity in the mature nervous system. In the nervous system, neuronal genetic diversity seems to arise intrinsically during early neuronal differentiation. Diverse genetic alterations have been observed in healthy neurons, most of them involving the generation and repair of DNA double strand breaks. The pool of neurons with cell-unique differences in their DNA could be even larger than the vast numbers of antibodies generated by the V(d)J recombination in the immune system [14]. During neuronal differentiation, more than 60% of the recently generated neurons undergo cell death events [15]. The surviving neurons, many of them carrying genetic alterations, may configure a functional repertoire characterized by the physiologically generated somatic mosaicism.

Figure 2

Impaired retinal development in mutant mice defective in components of DSB generation and repair mechanisms. Comparison of retinal phenotypes in mutant mice defective in DNA polymerase mu (polµ-/-; E-H), DNA-PK (SCID; I-L), and one of the subunits of the RAG-1,2 endonuclease responsible for generating the DSBs that originate the V(d)J recombination in the immune system (rag2-/-; M-P). The phenotype of the WT mouse is displayed in panels (A–D,A,E,I,M) E13.5 dissociated retinal cells were immunostained for γH2AX (cyan) to reveal DSBs and counterstained with DAPI (blue) to visualize the nuclei. Notice that the foci numbers are increased in the repair defective mutants and reduced in the RAG2 defective mutant, with respect to the WT. (B,F,J,N) programmed cell death was detected by TUNEL (green) in whole mount E13.5 retinas. Notice that apoptotic nuclei numbers are increased in all three mutant mice with respect to the WT mouse. (C,G,K,O) E13.5 dissociated retinal cells were cultured on polyornithine/laminin-treated plates. Neurite emission was visualized by TUJ-1 immunostaining (red). Notice the disturbed axonal trajectories in all three mutant mice, with respect to the WT mouse. (D,H,L,P) E13.5 whole-mount retinas were immunostained with TUJ-1 (red) to visualize RGC axonal trajectories. Notice the disturbed axonal trajectories in all three mutant mice, with respect to the WT mouse. Images adapted from [48,49,62,85].