Increased neuronal death and disturbed axonal growth in the Polμ-deficient mouse embryonic retina

Abstract

Programmed cell death occurs naturally at different stages of neural development, including neurogenesis. The functional role of this early phase of neural cell death, which affects recently differentiated neurons among other cell types, remains undefined. Some mouse models defective in DNA double-strand break (DSB) repair present massive cell death during neural development, occasionally provoking embryonic lethality, while other organs and tissues remain unaffected. This suggests that DSBs occur frequently and selectively in the developing nervous system. We analyzed the embryonic retina of a mouse model deficient in the error-prone DNA polymerase μ (Polμ), a key component of the non-homologous end-joining (NHEJ) repair system. DNA DSBs were increased in the mutant mouse at embryonic day 13.5 (E13.5), as well as the incidence of cell death that affected young neurons, including retinal ganglion cells (RGCs). Polμ−/− mice also showed disturbed RGC axonal growth and navigation, and altered distribution of the axonal guidance molecules L1-CAM and Bravo (also known as Nr-CAM). These findings demonstrate that Polμ is necessary for proper retinal development, and support that the generation of DSBs and their repair via the NHEJ pathway are genuine processes involved in neural development.

RAG-2 deficiency results in fewer phosphorylated histone H2AX foci, but increased retinal ganglion cell death and altered axonal growth

“…Contrary to expectations, the neuronal cell death phenotype observed in RAG-2-deficient mice, in which the number of γ-H2AX + foci is reduced versus WT (Fig. 2), was in line with that described in DNA-PK ( SCID ) and DNA polymerase μ ( polμ −/− ) DSB repair mutants, which accumulate γ-H2AX + foci 18,19 . Based on this observation, we next sought to characterize other phenotypic consequences of RAG-2 deficiency.…”

“…Fig. 1), but the distribution of cell adhesion molecules involved in axonal guidance, namely L1-CAM and Bravo, that were altered in the polμ −/− mutant mice 19 , were not affected (data not shown).

Figure 4 In vivo axonal navigation is altered in E13.5 rag2 −/− mice. RGC axonal trajectories were visualized by TUJ-1 immunostaining in whole-mount retinas from E13.5 WT ( a , c ) and rag2 −/− mice ( b,d , f ).

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“…RGC axonal growth is impaired in mice that lack repair DNA polymerase μ (Pol μ) 19 . We therefore investigated whether RAG-2 deficiency gives rise to a similar phenotype.…”

“…Axonal growth and navigation is a complex process mediated by interactions between environmental cues and cell autonomous determinants 40 . Based on our previous observation of abnormal neurite outgrowth in dissociated polμ −/− retinal cells 19 , we examined neurite phenotype in rag2 −/− retinal cells (Fig. 5).…”

“…Our previous studies have demonstrated that DSBs, as visualized by γ-H2AX immunostaining, are generated during the early stages of retinal development, and have characterized the impact of deficient DNA repair mechanisms on RGC survival and axonal growth in SCID and pol μ −/− mutant mice 18,19 . In this study, we sought to characterize sources of DSBs and determine their physiological relevance.…”