Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in NBN or ATM result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show in vivo consequences of conditional inactivation of both genes in neural stem/progenitor cells using Nestin-Cre mice. Genetic inactivation of Atm in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with Nbn deletion was prevented by simultaneous inactivation of Atm in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.


Introduction
The central nervous system (CNS) exhibits an acute sensitivity to double-strand breaks (DSBs) during its development [1]. Indeed, the majority of the human diseases associated with mutations in DSB signaling or repair genes present a wide spectrum of neurological abnormalities ranging from microcephaly to neurodegeneration [1].
Hypomorphic mutations in NBN lead to the Nijmegen Breakage Syndrome (NBS, OMIM 251260) a rare autosomal recessive disorder associated with growth retardation, immunodeficiency, neurological defects, radiosensitivity and tumor predisposition, including astrocytomas and medulloblastomas. Notably, CNS malignancies are rarely found in NBS and other related inherited diseases, such as Ataxia-Telangiectasia (A-T, ATM) [2,3]. In fact, these common features are expected, since the nibrin (NBN) protein is a target of DNA damage signaling kinases, such as ATM or ATR, and is a component of the MRN complex (with RAD50 and MRE11) that is involved in DNA damage signaling and repair, telomere maintenance, cell cycle checkpoint activation and processing of stalled replication forks [4]. NBN is a key sensor of the DSBs and is essential for the efficient activation of DNA repair PI-3 like kinases ATM or ATR in response to both exogenous and endogenous DNA damaging agents such as ionizing radiation (IR), ultra-violet (UV) and stalled replication forks [5]. Phosphorylation of NBN at serines 278 and 343 by the same PI-3 kinases is required for the activation of the intra-S phase checkpoint [6][7][8]. Finally, Nbn has also been shown to be required for the DSB repair branching between the Non Homologous End Joining (NHEJ) and Homologous Recombination Repair (HRR) [9].
The inactivation of Nbn leads to early embryonic lethality, while the hypomorphic mutant mice are viable and barely exhibit the NBS-associated neurological defects [10][11][12][13][14]. The specific inactivation of Nbn in mouse neural tissues using Nestin-cre transgenic mice results in a combination of the neurological abnormalities of NBS, A-T and A-TLD, including microcephaly, growth retardation, cerebellar defects and ataxia [15]. Analysis of Nbn conditional knockout mice indicated that the loss of Nbn impairs the proliferation of granule cell progenitors and increased apoptosis of post mitotic neurons in the cerebellum [15]. It was also shown that Nbn inactivation leads to defects in myelin formation, oligodendrocyte development and astrocyte dysfunction [16][17][18]. In addition, Nbn-deficient neural stem cells exhibit proliferation defects, but not increased apoptosis, and contain more chromosomal breaks accompanied by Atm-mediated p53 activation [15]. Importantly, depletion of p53 significantly rescues the neurological defects of Nbn mutant mice, while inactivation of Atm in Nbndeficient neural stem cells seems to worsen the cerebellar defects of Nbn deficient mice [17]. Apart from neurological defects these mice also exhibit severe eye phenotypes, such as micropthalamia, disorganization of the lens, impaired visual function and cataracts [16,19].
Even though it is clear that functional interaction between NBN and ATM is required for a proper DNA damage response and that both are crucial for CNS development, it remains unclear whether the functional relationship between NBN and ATM is identical or equally relevant in all developing tissues. For example, nothing or little is known about their functional relationship during eye and brain development. To study how Nbn and Atm are functionally interconnected in the development of these tissues and to better understand the origins of the developmental defects caused by Nbn and/or Atm-deficiency, we simultaneously inactivated them in various neural and eye tissues using multiple Cre/LoxP systems. We report that Atm inactivation worsens the Nbn-deficient phenotype causing increased genomic instability and increased apoptosis of neural progenitors. Similar results were observed for progenitor cells of the lens anterior epithelia, but not for retinal progenitor cells (RPC). Interestingly, even though both Nbn and Atm are ubiquitously expressed in the CNS and in the eye [20,21], we show that the phenotype severity caused by simultaneous inactivation of Nbn and Atm is restricted to some tissues or to specific stages of development. Therefore, we propose that functional synergy between Nbn and Atm to prevent and repair DSBs and apoptosis during development is highly dependent on the tissue type and its developmental stage.

Results
Atm deficiency promotes premature growth defect and death of Nbn Nes-Cre mice It has been shown that inactivation of Nbn in mouse neural stem cells (Nbn Nes-Cre ) was associated with severe growth defects and premature death around 21 days after birth (P21) [15]. Concomitant deletion of Atm and Nbn in neural progenitor cells (Nbn/ Atm Nes-Cre ) exacerbated this phenotype, albeit Nbn Nes-Cre and Nbn/ Atm Nes-Cre mice were viable and appeared indistinguishable from their littermates at birth. Nbn/Atm Nes-Cre exhibited a stronger growth defect at P9, as compared to Nbn Nes-Cre mice ( Figure 1A) and prematurely died by P14. Nevertheless, both genotypes exhibited a similar tremor and ataxia. The brain weight and size were indistinguishable between Nbn Nes-Cre and Nbn/Atm Nes-Cre mice at P9 (Figure1 B-C).

Nbn and Atm are not essential for Purkinje Cells homeostasis
Microscopic examination of the Nbn Nes-Cre and Nbn/Atm Nes-Cre P9 brains showed identical granule cell loss, cerebellar and cortical developmental defects by P9 ( Figure 1D-F). Although both Nbn Nes-Cre and Nbn/Atm Nes-Cre Purkinje cells presented impaired localization and altered dendritic arborization, their numbers were not different from the control mice ( Figure 1E, 1G). To test whether Nbn and/or Atm play a role in the homeostasis of differentiated Purkinje cells, Nbn PcP2-Cre and Nbn/Atm PcP2-Cre mice were generated. PcP2-Cre/ L7-Cre allows a specific genetic inactivation in post-mitotic Purkinje cells during postnatal development (7 days of age/P7) [22]. Macroscopic analysis of Nbn/Atm PcP2-Cre P21 brains did not reveal abnormalities of the cerebellum structure (data not shown). Histological analysis did not show loss of granule cells, morphological abnormalities or alterations in the number of Purkinje cells ( Figure 1H), suggesting that both Nbn and Atm are dispensable for Purkinje cells homeostasis in mouse.
Atm inactivation does not worsen the Nbn Nes-Cre phenotype in neural stem/progenitor cells Since Nbn/Atm deficient mice exhibited premature growth defects and death as compared to Nbn-deficient ones, we investigated whether Atm inactivation could worsen the Nbn deficiency phenotype in the neural stem cells (NSCs). NSCs from Nbn Ctrl , Nbn Nes-Cre and Nbn/Atm Nes-Cre from E14.5 brains were isolated and cultured in vitro (Figure 2A). At this stage the deletion of both Nbn and Atm is completed using Nestin-Cre [15]. Both neurospheres derived from Nbn Nes-Cre and Nbn/Atm Nes-Cre brains exhibit substantial reduction in size and number ( Figure 2B-C) as compared to those derived from control brains. Interestingly, no significant difference on the number of neurospheres per ml or number of cells per neurosphere was observed between NSCs derived from Nbn Nes-Cre or Nbn/Atm Nes-Cre mice ( Figure 2B-C). These findings suggest that the slight differences arising later in life may occur during development due to the effects of Atm deficiency on specific cells.

Atm prevents premature DSBs accumulation and apoptosis in Nbn-deficient cerebellum and forebrain
To determine whether Atm deficiency would increase accumulation of DSBs in Nbn-deficient granule cells, we measured c-H2AX foci in the cerebellum of E15.5 embryos. We found that the cells of the external germinal layer (EGL) of both Nbn Nes-Cre and Nbn/Atm Nes-Cre presented increased c-H2AX foci compared to those of Nbn Ctrl ( Figure 3A, 3G). Interestingly, the EGL from Nbn/ Atm Nes-Cre exhibit significantly more cells with c-H2AX compared to Nbn Nes-Cre granule cells, suggesting that Atm may play a role in the prevention of DSB accumulation in Nbn-deficient cells. DSBs are frequently associated with an activation of cell cycle checkpoints or apoptosis. Therefore, we first measured BrdU incorporation in EGL cells after a 1 hour BrdU pulse. Consistent with former studies, we found significant reduction of BrdU positive cells in EGL from Nbn-deficient cells. Simultaneous inactivation of Atm did not alter the proportion of BrdU positive cells ( Figure 3D, 3I). In parallel, the number of cells in mitosis was evaluated using the phospho-histone 3 (phospho-H3) marker. A moderate increase in the number of phospho-H3 positive cells was found in Nbn Nes-Cre and Nbn/Atm Nes-Cre EGL ( Figure 3B, 3H), suggesting that G2/M cell cycle checkpoint is activated in Nbn Nes-Cre and Nbn/Atm Nes-Cre deficient EGL cells. To test this hypothesis, we asked whether the DSBs increase would correlate with p53 protein stabilization. Indeed, in EGL of both Nbn Nes-Cre and Nbn/Atm Nes-Cre mice we found a high proportion of p53 positive cells ( Figure 3F) with detectable phosphorylation at serine 18 ( Figure S1). It indicates that in Nbn Nes-Cre and Nbn/Atm Nes-Cre EGL cells, p53 can be stabilized and phosphorylated at serine 18 independently of Nbn and Atm. Since p53 stabilization and apoptosis are often associated in the CNS, we analyzed apoptotic cell death using two independent markers: TUNEL and cleaved caspase-3. As expected, almost no TUNEL or cleaved caspase 3 positive cells were found in the control EGL at E15.5 ( Figure 3C, 3K). In contrast, the proportion of apoptotic cells increased in both Nbn-deficient and Nbn/Atm-deficient cells ( Figure 3E, 3J). Importantly, Nbn/ Atm-deficient EGL exhibit a 4-fold increase of TUNEL and cleaved caspase 3 positive cells as compared to Nbn deficiency alone (p,0,001). Notably, in both genotypes, similar numbers of TUNEL positive cells and cleaved caspase 3 positive cells were observed, suggesting that at E15.5 the apoptosis is always caspase 3dependent. At E17.5, we observed that the apoptosis in Nbndeficient EGL cells reached the same level as in the Nbn/Atmdeficient EGL ( Figure 3J, 3K). Here it is worth to notice that in Nbn Nes-Cre cerebella the apoptotic cells were localized in the external border of the EGL where most of the cells exit cell cycle, while in Nbn/Atm Nes-Cre cerebella, cells undergoing apoptotic cell death were localized throughout the EGL. It suggests that Atm is required to prevent apoptosis in dividing cells as well as in post-mitotic Nbndeficient cells. In conclusion, Nbn deficiency in cerebellum primordium induces DNA DSBs, cell cycle checkpoint activation and stabilization of p53. Concomitant inactivation of Atm induces premature apoptosis, suggesting that Atm may be required to promote DNA repair and prevent apoptosis in Nbn-deficient damaged cells.
After investigating the consequences of Nbn and Atm genetic inactivation in cerebellum, we analyzed whether other areas of the Figure 1. Heterogeneous effects of Atm inactivation on Nbn-deficient brain and eye. (A) Body weight of P9 Nbn/Atm F/F ;Nestin-cre (Nbn/ Atm Nes-Cre ) mice was reduced as compared to control (Nbn Ctrl ) and Nbn F/F ;Nestin-Cre (Nbn Nes-Cre ) littermates. (B-C) Brain weight (B) and size (C) of P9 of Nbn Nes-Cre (n = 11) and Nbn/Atm Nes-Cre (n = 22) mice are significantly reduced as compared to those of Nbn Ctrl (n = 123), without dramatic disruption of the general morphology. (D) H&E staining of Nbn Ctrl , Nbn Nes-Cre and Nbn/Atm Nes-Cre brain sections at P9 reveal reduction of the size of the Nbn Nes-Cre and Nbn/Atm Nes-Cre mice cerebella compared to those of Nbn Ctrl mice (Magnification 610).   Atm inactivation exacerbates the growth impairment of Nbn-deficient lens due to DSBs accumulation and premature apoptosis Previous studies showed that Nbn was crucial for the development of both the retina and the lens [16,19]. Nevertheless, the functional relationship of Nbn and Atm in these tissues has remained elusive. Therefore, we analyzed the development of the eye in both Nbn Nes-Cre and Nbn/Atm Nes-Cre mice. The Nestin-Cre transgenic mice present a high Cre recombinase activity in developing lens and retina [23]. First, we confirmed our previous reports that in vivo inactivation of Nbn impaired eye and lens growth ( Figure 1I To extend the results obtained with Nestin-Cre, we used a lens-specific mouse line (Le-Cre) [24] that allows deletion of the genes in early lens development, specifically in the surface ectoderm. We postulated that earlier deletion in lens progenitor cells population would augment the defects already observed in the Nestin-cre. As expected, at P9 the eye growth of Nbn Le-Cre and Nbn/ Atm Le-Cre mice was already affected (data not shown) and at P21, the volume of the eyes were dramatically reduced ( Figure S2A-B) in both Nbn Le-Cre and Nbn/Atm Le-Cre mice. Similarly to what was observed in Nestin-Cre mice, Atm deficiency potentiated the defects observed in Nbn-deficient lens ( Figure S2A-B). Histological analysis revealed that at P21, the lens is severely reduced in the Nbn Le-Cre mice and completely absent in the Nbn/Atm Le-Cre . As expected, the severe impairment of lens development completely abrogated retinal development ( Figure S2A-B).
Altogether, the analysis of cell proliferation, DNA damage and cell death indicate that both Nbn and Atm are crucial for lens progenitor cells survival during embryogenesis. Importantly, the more severe impairment of lens growth as a result of simultaneous inactivation of Nbn and Atm indicates that these proteins have independent roles in the developing eye.

Nbn-deficiency leads to an Atm-dependent apoptosis in developing retina
Previous findings demonstrated that Nbn loss compromises the development of the visual system and indicated that Nbndeficiency results in neurodegeneration in adult retina [16]. Here, we asked whether Nbn and Atm regulates early stages of retinal development. Initially, we performed real time PCR to analyze the expression of Nbn in various stages of retinal development. In the mouse retina, Nbn is expressed as early as E14.5 ( Figure 6A). To test whether Nbn and/or Atm would regulate genome stability in retinal progenitor cells, we quantified c-H2AX positive cells in Nbn Ctrl , Nbn Nes-Cre and Nbn/Atm Nes-Cre embryonic retinas. At E17.5, a slight increase in the proportion of c-H2AX positive cells was observed in both Nbn-and Nbn/Atm-deficient retinas ( Figure 6B). To verify whether the detected DSBs would affect cell cycle progression, we first quantified the proportion of BrdU positive cells. Nbn-deficiency or Nbn/Atm-deficiency did not significantly altered BrdU incorporation in E15.5 or E17.5 retinas ( Figure 6D). To directly analyze the mitotic subpopulation of retinal progenitors, we scored the proportion of phospho-H3 positive cells. At E17.5, Nbn inactivation increased the proportion of mitotic cells. Surprisingly, in contrast to what was observed in the lens, this effect was completely reverted by Atm inactivation. These findings suggest that Nbn-deficiency induced an Atm-dependent G2/M arrest of the cell cycle ( Figure 6C, 6F). To provide additional evidence of Nbn role in retinal development, we asked whether its inactivation would regulate cell death in embryonic retina. At E17.5, Nbn loss induced a 2-fold increase in the number of TUNEL positive cells in the neuroblastic layer (NBL) of the retina ( Figure 6E, 6G). As already observed for phospho-H3 positive cells accumulation, apoptosis induced by Nbn-deficiency, was completely blocked by inactivation of Atm, suggesting that in developing retina both the G2/M arrest and the apoptotic cell death are mediated by an Atm-dependent pathway.

Nbn and Atm are not required for neuronal differentiation during retinal development
To test whether retinal cell fate specification and differentiation would be compromised in the absence of Nbn and/or Atm function, we analyzed the distribution of various retinal cell types in Nbn Ctrl , Nbn Nes-Cre and Nbn/Atm Nes-Cre postnatal retinas. Bipolar cells (Chx10, Figure 6H), horizontal cells (calbindin, Figure 6I), amacrine cells (Pax6, Figure 6J) and photoreceptor cells (recoverin, Figure 6K) were normally generated and distributed in Nbndeficient or Nbn/Atm-deficient P9 retinas. Similar results were obtained when Nbn and Atm were inactivated at earlier stages of retinal development by the use of Pax6-Cre mice line [25]. At P28, no difference in the lamination or distribution of the analyzed retinal cell types was observed when Nbn Ctrl , Nbn Pax6-Cre and Nbn/ Atm Pax6-Cre were compared ( Figure S2C-E). These findings indicate that, in the mouse retina, the programs of neuronal and glial differentiation are not disturbed by genetic inactivation of Nbn and Atm.

Discussion
In this study, we analyzed how Nbn and Atm cooperate in vivo to prevent DSBs accumulation and apoptosis during CNS and eye development. Understanding the functional interaction between Nbn and Atm in vivo is of prime importance considering that human neurological diseases such as NBS and A-T are associated with mutations in NBN and ATM. We provide evidence that both Nbn and Atm play important roles in the maintenance of genomic integrity in different areas of the developing CNS and in the eye. More importantly, we showed that the functional cooperation between Nbn and Atm is both tissue-and developmental stagespecific. Additionally, our findings support the idea that the neurological malformations observed in Nbn and Nbn/Atmdeficient mice are developmental, rather than resulting from neurodegeneration.
We expected that combined inactivation of Nbn and Atm would augment the developmental defects associated with Nbn deletion during CNS development because of the synthetic lethality already observed in Nbn DB/DB , Atm 2/2 mice [14] or in the acute developmental defects of the Nbn-CNS-D/ Atm 2/2 cerebella [17]. We demonstrate that instead of broadly compromising the entire neurogenesis, inactivation of Nbn or Nbn/Atm affected only few areas of the developing CNS and eye, including the cerebellum, the GE and the lens, as well as the retina and did not compromise survival of differentiated neurons. The cerebellum and in particular the granule cells (GCs) were already shown to be highly sensitive to Nbn inactivation [15,17,26,27]. The sensitivity of cerebellum to DSBs genes deficiency was often attributed to the extremely fast cell proliferation of the GCs at a certain stages of development, which would render them more susceptible to apoptosis associated with replication forks collapse [28]. Besides, increased apoptosis during EGL development in Nbn-and Nbn/ Atm-deficient cerebella was untimely correlated with the increased proliferation of GCs progenitors. Furthermore, we found that the viability of differentiated neurons from the cerebellum, such as Purkinje cells did not require Nbn or Atm and that these cells did not present spontaneous DSBs. Despite numerous studies, the function of the MRN complex in differentiated neurons remains controversial. Indeed, a marked reduction of Purkinje cells in Nbn-CNS-D [26] and Nbn-CNS-D/ Atm 2/2 [17] was reported. In contrast, consistent with the data presented in this study, no defects were observed in Purkinje cells following inactivation of Nbn [27] or Rad50 [29] using Pcp2-cre even in a long term study. Several other findings support the hypothesis that Nbn and Atm are dispensable for the homeostasis of differentiated Purkinje cells. Purkinje cells in mouse exit the cell cycle early during cerebellar development around E12.5 making them less sensitive to the DNA damage associated with long term proliferation and replicative stress [30]. Moreover, the remaining GCs may be sufficient to maintain Purkinje cells viability despite their altered branching as observed in other mouse models [31,32]. It was shown that postmitotic and differentiated neurons repair DSBs resulting from oxidative stress mainly by Non Homologous End Joining (NHEJ) [33]. In addition, Nbn has been shown to be dispensable for classic NHEJ and Nbn inactivation was shown to favor NHEJ in mouse cells [9,34,35]. In conclusion, similarly to what was shown for Rad50, we demonstrated that Nbn is dispensable for the viability of post-mitotic differentiated neural cells.
In our study, we show also that in addition to the cerebellum, the SVZ from the developing forebrain composed of four regions the medial ganglionic eminence (MGE), the lateral ganglionic eminence the caudal ganglionic eminence and the fetal neocortical SVZ presented an acute sensitivity to both Nbn and Nbn/Atm deficiency (Figure 4). Among them the MGE appeared to be the most affected with severe apoptosis as early as E15.5. The MGE has been shown to give rise to a tremendous variety of cell types populating various CNS structures including cortex, striatum, thalamus, hippocampus and olfactory bulbs [36,37]. It is the major source of the GABAergic interneurons of the cortex populating its layers II to V, and representing up to 30% of its neural population [38]. The MGE was also demonstrated to be a source of oligodendrocytes [39]. In human, the lack of cortex interneurons or their defective migration and localization were often associated with diseases such as autism, epilepsy or seizures [40]. This is consistent with some NBS patients suffering severe epilepsy [41] and other presenting various form of neuronal migration disorder including schizencephaly [42], pachygyria [43] and partial lissencephaly [41]. In addition Nbn CNS-del mice exhibit severe disruption of olfactory bulbs morphology and reduced cellularity of hippocampus and of the layers II to V from the cortex [44]. Altogether, we demonstrate that Nbn is essential for the cell survival in ganglionic eminences and the formation of interneurons. We also propose a cause for the cortical defects observed in both in NBS patients [41] and Nbn-deficient mice [44].
We also found that the eye development was severely affected by Nbn and Atm deficiencies. These findings resemble the human syndromes Ataxia-Telangiectasia (A-T), and Nijmegen Breakage Syndrome (NBS), which often exhibit developmental or neurodegenerative defects in ocular structures. Ataxia-Telangiectasia (A-T) patients present conjunctival/scleral telangiectasia [45,46], saccadic abnormalities [47], head thrusts, strabismus and abnormal convergence [48]. Surprisingly, the A-TLD syndrome that results from MRE11 mutations and shares close similarities with A-T, exhibits moderated eye defects [48]. NBS patients exhibit also some ophthalmological abnormalities including reduced visual acuity, smaller eyes, cornea diameter lens opacity and refractive errors [49]. In mice, previous studies demonstrated that Nbn function was required for proper lens development. Yang and colleagues concluded that cell proliferation and altered differentiation were the causes of the defects in Nbn-deficient lens [19]. Complementary analysis performed here suggested that cell cycle arrest and apoptotic cell death are associated with the lens defects resulting from increased DSBs within lens progenitor cells. Indeed, we already detected increased c-H2AX foci in both Nbn-and Nbn/Atm-deficient lens epithelial cells as early as E15.5. Both the increased DNA damage in proliferating cells and the apoptotic cell death started earlier in Nbn/Atm-deficient, indicating that Atm may promote DSBs repair in Nbn-deficient lens progenitor cells and that additional deletion of Atm may lead to broader effects on both dividing and differentiating cells in Nbn-deficient lens. Additionally, we provide evidence that Nbn and Atm are required at very early stages in lens development since inactivation of Nbn in the surface ectoderm (using Le-Cre mice) dramatically impaired eye growth and lens development was completely abrogated in Nbn/ Atm Le-Cre mice.
Surprisingly, in contrast to the lens, the embryonic retinal development was only mildly affected by Nbn inactivation. Our findings add to previous reports that showed decreased electroretinogram amplitudes, demyelination of optic nerve, axonal degeneration in addition to oligodendrocytes and ganglion cells degeneration in adult Nbn-deficient eyes [16]. Indeed, our study indicates that Nbn regulates genomic integrity in retinal progenitor cells (RPC) during embryonic development. Inactivation of Nbn increases the number of RPC with DSBs indicating that, in the absence of MRN complex, DNA damage repair in RPC does not occur properly. Interestingly, the increased proportion of RPC containing DSBs observed in Nbn-deficient retina was not significantly altered by simultaneous Atm inactivation. In contrast, apoptosis associated with Nbn deletion was significantly decreased following Atm loss. This is in agreement with the requirement of Atm for apoptosis in developing retina upon irradiation [50]. Persistence of phosphorylation of H2AX in Nbn/Atm-deficient retina strongly suggest that at least one alternative kinase, is able to phosphorylate H2AX independently of both Nbn and Atm in RPC. It has been proposed that DNA-PKcs may regulate retinal development [51]. In addition, after analyzing the distribution of all retinal cell types right after the vast majority of RPC have become post-mitotic (P9), we provide evidence that cell fate specification or early steps of cell differentiation of retinal neurons and glia are not affected by Nbn and/or Atm deficiency. Therefore, we propose that the visual abnormalities described for mice with Nbn-deficient retina [16] most likely result from neuronal and/or glial degeneration during adulthood, but not from developmental malformations.
The similarity of the cellular defects and their timing of occurrence in the tissues affected by Nbn deficiency imply the existence a common molecular mechanism. We hypothesize that complete disruption of Nbn leads to replication fork collapses and the formation of DSBs as indicated by c-H2AX foci in dividing cells. This is consistent with the role of Nbn in preventing the formation of DSBs by favoring stabilization and restart of stalled replication forks directly through activation and retention of ATR at the damage sites [52] or interaction with TopBP1 an ATR activator [53]. Notably, the phosphorylation of H2AX at Ser139 requires neither Nbn nor Atm since both Nbn and Nbn/Atm deficient cells presented foci. We suggest that ATR may be the kinase responsible for c-H2AX, since Nbn has been shown to be dispensable for ATR mediated H2AX phosphorylation [52]. Nevertheless, it is not possible to exclude DNA-PKcs since it may compensate Atm loss in an efficient way and it was also shown to play a role in the survival of neural cells [51,[54][55][56]. In dividing cells, during the first part of the embryonic development, the DSBs trigger a survival signal, through the activation of cell cycle checkpoint mediated by p53 and others. p53 was already shown to be one of the key effectors to activate cell cycle checkpoint and apoptosis in the CNS [1]. The essential function of p53 was also highlighted by the partial or complete rescue of the neurological defects in several DSBs mouse mutants [28]. Interestingly, in cerebellum, the stabilization and phosphorylation at serine 18 of p53 is independent of both Nbn and Atm in contrast to what happens upon low dose of ionizing radiation. In this regard, the cerebellum and lens may slightly differ since p53 has been shown not to rescue the lens phenotype in Nbn-deficient eye [19]. The increase of apoptosis in Nbn/Atm versus Nbn-deficient cells in early embryonic development can be attributed to the essential requirement of Atm in the repair of DSBs resulting from replication forks collapses and activation of Rad51-dependent HR [57] and also the essential function of Atm in the DNA damage response in post-mitotic cells [58]. In late embryonic development the acceleration of cell division, the DSBs repair switch from canonical NHEJ to micro homology-mediated end joining (MMEJ) which requires the MRN complex [59,60] may favor accumulation of unrepaired DSBs to an unsustainable level for the cells leading irremediably to a p53-dependent apoptosis.
Indeed, It is known that the accumulation of unrepaired persistent lesions leads irremediably to apoptosis in the CNS [28], and that neural cells presented a gradient of tolerance to DSBs inversely correlate with their differentiation status [61].
To conclude, our study reveals new findings about the possible origins of ocular and cortical developmental defects of NBS patients. Besides, we demonstrated tissue-specific and developmental stage-specific requirements of Nbn and Atm acting synergistically to prevent accumulation of DSBs and apoptosis in CNS and eye.

Ethics statement
All animal care and procedures followed German legal regulations and were previously approved by the governmental review board of the state of Baden-Württemberg (Regierungsprä sidium Karlsruhe-Abteilung 3-Landwirtschaft, Lä ndlicher Raum, Veterinä r-und Lebensmittelwesen, Germany, licence G-114/11, experimental projects DKFZ 220, 241, 256). All the aspects of the mouse work were carried out following strict guidelines to insure careful, consistent and ethical handling of mice.

RNA extraction, cDNA synthesis, and real-time RT-PCR analysis
Retinal tissue was obtained from staged embryonic (E14.5, E17.5) and postnatal (P0, P3, P11, adult) C57BL/6 mice. RNA extraction and cDNA synthesis were performed as previously described [63,64]. Nbn primers used were as follows: forward-59-ACCCACCCATTGATGAACCAGCTA-39 and reverse 59-CTGAGTTTCTTGTGCTGCTTGGCA-39. Real time RT-PCR reactions were done with Power SYBR Green PCR Master Mix and in ABI7500 machine (Applied Biosystems). PCR product identity was verified by electrophoresis and by melting-point analysis. Nbn gene (Nbn) expression was normalized to Gapdh (Gapd) [65] and gapdh-normalized amount of Nbn gene expression was plotted relative to the mean of delta Ct of all ages analyzed.

Morphometric and Volume measurements of the eye and lens
Eyes from embryonic, newborn (P9) and adult mice (P30) were processed and measured as previously described [63]. After enucleation, eyes were fixed in 4% phosphate-buffered paraformaldehyde. The axial length and two coronal axes (dorso-ventral and medial-lateral) of each eye were measured with a digital paquimeter and the eye volume was calculated after applying formula (4/3*PI)*x*y*z. After dissection of the retina and lens, the same procedure was used to calculate lens volume.

Statistical analysis
Quantification analysis of cell cycle and cell death, immunopositive cells in the EGL, lens and retina were counted in at least 4 representative sections and mean values were calculated and statistically analyzed. These data were collected from at least 3 different animals per each group. One way ANOVA followed by post hoc Newman-Keuls or T-Test analysis were performed using Prism (v5.0, Graphpad). Differences were considered significant when P value was 0.05. Figure S1 Nbn and Atm inactivation leads to p53 phosphorylation at serine 18 in the EGL of the cerebellum. p53 stabilization is associated with its phosphorylation at serine 18 in Nbn Nes-Cre and Nbn/Atm Nes-Cre EGL (Magnification 6400).