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[Cancer Research 63, 5335-5343, September 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Atm-, p53-, and Gadd45a-Deficient Mice Show an Increased Frequency of Homologous Recombination at Different Stages during Development1

Alexander J. R. Bishop2, M. Christine Hollander, Bela Kosaras, Richard L. Sidman, Albert J. Fornace, Jr. and Robert H. Schiestl3

Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115 [A. J. R. B.]; National Institutes of Health, National Cancer Institute, Bethesda, Maryland 20892 [M. C. H., A. F.]; Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02115 [B. K., R. L. S.]; and Department of Pathology, University of California at Los Angeles School of Medicine, Los Angeles, California 90095 [R. H. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
%Atm, p53, and Gadd45a form part of a DNA-damage cellular response pathway; the absence of any one of these components results in increased genomic instability. We conducted an in vivo examination of the frequency of spontaneous homologous recombination in Atm-, p53-, or Gadd45a-deficient mice. In the absence of p53, we observed the greatest increase in events, a lesser increase in the absence of Atm, and only a modest increase in the absence of Gadd45a. The striking observation was the difference in the time at which the spontaneous events occurred in atm and trp53 mutant mice. The frequency of homologous recombination in atm mutant mice was increased later during development. In contrast, p53 appears to have a role in suppressing homologous recombination early during development, when p53 is known to spontaneously promote p21 activity. The timing of the increased spontaneous recombination was similar in the Gadd45a- and p53-deficient mice. This temporal resolution suggests that Atm and p53 can act to maintain genomic integrity by different mechanisms in certain in vivo contexts.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After cellular DNA damage, cells respond to and often repair the damage in an orchestrated manner. One signaling pathway that has been demonstrated to play a key role is the Trp53 (p53) damage response pathway (1, 2, 3, 4, 5) . After the activation and stabilization of p53, p53 plays a central part in the type of responses that cells mount to a variety of damages. These responses include apoptosis, cell cycle arrest and DNA repair (reviewed in Ref. 6 ). It is now understood that p53 is normally maintained at a level within the cell that does not result in any significant transcriptional activation of its downstream effectors.

Atm is a protein kinase (7, 8, 9) that is activated in the cell after ionizing radiation exposure (10 , 11) . It is now clear that Atm has many targets (for a review see Ref. 12 ), among them is p53, which is effected both directly (2 , 13, 14, 15, 16, 17) and indirectly (18, 19, 20) . p53 can subsequently transcriptionally up-regulate effectors such as Cdkn1a (p21; see Ref. 21 ) and Gadd45a (2) to produce a coordinated cellular response. The up-regulation of p21 results in a cell cycle arrest at the G1-S border (21, 22, 23, 24, 25, 26, 27) , which is thought to allow time for repair reactions to be enacted. In what manner any repair reaction is controlled by p53 is not fully understood, although the absence of p53 or Gadd45a has been related to a decreased level of nucleotide excision repair (28) . From the point of view of this report, it is interesting to note that the absence of the p53 damage response pathway components Atm (29, 30, 31, 32, 33, 34) , p53 (35 , 36) , or Gadd45a (37) has been observed to confer an increased level of genomic instability.

Our understanding of recombination is that there are a number of different types of recombination mechanisms, broadly divided into HR4 and nonhomologous endjoining. HR is mediated by regions of homologous DNA and is the basis of the assay used in this study.

To determine the frequency of HR in vivo, we identified the number of cells or clones of cells that had deleted a 65-kb DNA duplication to a single copy (see Fig. 1Citation ). The duplication allele, called pink-eyed unstable (pun; see Refs. 38 , 39 ), interrupts the murine pigmentation gene, pink-eyed dilution (p). In the absence of a functional p gene, mice have pink eyes and a dilute coat color (40) . Only a HR event will result in the correct reconstitution of the p gene and the phenotypic pigmentation that is assayed (38 , 39) . Molecular analysis has confirmed that these events are the result of pun reversion (38 , 39 , 41) . The p gene is normally transcribed in melanocytes and cells of the RPE, so that when a deletion/reversion event of pun occurs somatically in a precursor of a melanocyte or RPE cell, this cell will proliferate and differentiate into a clone of pigmented cells. Such patches or spots have been observed in both the fur (38 , 41) and eyes (42, 43, 44) of pun mice. On the C57BL/6J inbred background, approximately 5–10% of pun mice spontaneously display visible fur-spots (38 , 41) and 4 to 5 eyes-pots are observed per RPE (44 , 45) .



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  		Fig. 1. A schematic representation of the pink-eyed unstable mutation (pun; A) and the wild-type gene pink-eyed dilution (p; B). The publicly available murine sequences for the wild-type gene were obtained from the NIH (Internet address: www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_srchdb?chr = mouse_chr.inf). The duplication junction sequenced identified previously (39) were identified within the genomic region to generate the schematic. The duplication was found to include sequences from within intron 7 to within intron 19, spanning ~65 kb. The sequence representations are drawn to scale, a 10-kb scale bar is given. The translation start is in exon 3 and, assuming correct splicing of all exons, the duplication will result in the insertion of a novel alanine created at the junction of exons 19 and 8, followed by 433 amino acids of the duplicated exons 8 to 19. After deletion of one of the internal repeats by HR, a functional protein can be produced and will result in pigmentation in either melanocytes or cells of the RPE.

 
Deletions reverting the pun allele to p+ resulting in fur spots (fur-spot assay) are increased in frequency after exposure to different DNA-damaging agents (41 , 46 , 47) , as well as in different cancer predisposing genetic backgrounds (34 , 48) . In those in vivo studies, atm mutant mice had an increased frequency of recombination (34) , whereas the trp53 mutant mice did not (49) . This was surprising because a number of tissue culture studies with p53-deficient cells have demonstrated an increased frequency of recombination (50, 51, 52, 53, 54) . To address this issue, we developed the pun eye-spot assay. This assay has already proven to be more sensitive in detecting the frequency of recombination after exposure to DNA-damaging agents than the fur-spot assay (45) . In addition, we demonstrated that because of the well-defined developmental pattern of the RPE (55) , we were able to define the time at which a recombination event occurred by its position within the RPE (56) . Here we use the pun eye-spot assay to examine the frequency of spontaneous recombination in mice deficient for Atm, p53, or Gadd45a. The results demonstrated an increased frequency of HR in all three genetic backgrounds, but the timing of the instability was different between Atm-deficient and p53- or Gadd45a-deficient mice. This study confirms our earlier suggestion of an increased frequency of HR in atm mutant mice, while demonstrating that trp53 and gadd45a mutant mice also display an increased frequency of HR early during development.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
C57BL/6J- pun/un mice were obtained from the Jackson Laboratory (Bar Harbor, ME). C57BL/6J pun/un Atm +/- and C57BL/6J pun/un Trp53+/- have been described previously (34 , 49) . Mice heterozygous for Atm or Trp53 and homozygous for pun were bred to generate mice of all three Atm or Trp53 genotypes. Gadd45a mutant mice (37) were bred into the C57BL/6J pun/un genetic background with three back-crosses to produce C57BL/6J-pun/un Gadd45a-/- mice that were maintained. In the generation of this colony, C57BL/6J-pun/un Gadd45a+/+ mice were also produced and maintained as a control. All mice were bred in the institutional animal facility under standard conditions with a 12-h light/dark cycle and were fed standard diet and water ad libitum. The pun/un genotype was observed phenotypically in the progeny of the second back-cross as mice with a dilute (gray) coat color.

PCR Genotype.
The Atm, Trp53, and Gadd45 genotypes were determined by PCR amplification as described previously (34 , 37 , 49) . DNA was prepared from tail biopsies by standard protocols.

Dissection of the RPE.
Eyes from sacrificed 20-day-old mice were removed, fixed, and dissected to expose the RPE layer, as described previously (45 , 55) . The RPE adjacent to the neural retina was isolated for analysis by removing the eye from its orbit, immersing it in fixative [4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4)] for 1 h and then in PBS until dissection. An incision was made at the upper corneo-scleral border to allow removal of the cornea and lens. Six to eight incisions were made into the eyecup from the corneo-scleral margin toward the centrally positioned optic nerve. The dissected eyecup was placed on a glass slide with the retina facing up. The retina was then gently removed and the flattened eyecup, with RPE facing up, was mounted in 90% glycerol for analysis.

Scoring a Single-Reversion Event, Visualized As an Eye-Spot.
We defined two or more adjacent pigmented cells, or pigmented cells separated from each other by no more than one unpigmented cell, as an eye-spot that resulted from one reversion event (55) . The number of eye-spots in each RPE and the number of cells that comprised each eye-spot were counted. Positions of eye-spots were mapped.

Distance Analysis of Eye-Spots from the Optic Nerve.
Spots were identified under the microscope and compared with their scanned digital images. Distances were measured with the Adobe PhotoShop 5.5 Measurement Tool. Distances were converted from pixels to millimeters by counting the number of pixels per millimeter on the image of a micron scale reticule scanned at the same optical settings as the RPE. Two distances were measured for each eye-spot: the "eye-spot distance," the distance from the center of the optic nerve head to the most proximal edge of the eye-spot; and the "RPE distance" of an eye-spot, the distance from the optic nerve through the eye-spot to the outer edge of the RPE. Dividing the eye-spot distance by the RPE distance gave the proportional distance of each eye-spot from the outer edge of the RPE, or its "position." The position of each eye-spot was determined in this manner to compensate for differences in the size of the eyes.

Microscope, Digital Camera, and Software.
Whole-mount RPE were scanned by a DC120 digital camera (Eastman Kodak Company) mounted on a DMLB microscope (Leica Microsystems, Inc., Wetzlar, Germany) using a x2.5 N-plan objective. Embryonic sections were scanned by a RT Slider Spot camera (Diagnostic Instruments, Inc., MI) mounted on a Axioskop microscope (Zeiss, Göttingen, Germany). The images were assembled and examined in Adobe PhotoShop 5.5 on a Macintosh Power Computer. All data were stored and processed with Microsoft Excel 2001.

Statistical Analysis.
Comparison between numbers of events was performed by a standard G test (57) . The G test is equivalent to a contingency {chi}2 test, but allows for classes with zero events. Comparison of the population of events per RPE between genotypes was performed by Wilcoxon rank-sum analysis (58 , 59) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eye-Spot Frequency.
An examination was conducted on the pun reversion frequency in the RPE. The C57BL/6J- pun/un Atm+/+, C57BL/6J- pun/un Trp53+/+ and C57BL/6J- pun/un colonies described in previously reported concurrently performed studies (45 , 56) were not maintained separately, and comparison of the wild-type retinal pigment epithelia revealed no significant difference in pun reversion frequency or pattern (data not shown). The Gadd45a mouse colony was maintained separately; therefore, an extensive analysis was performed to compare the RPE of that colony with the rest of the wild-type controls. Of all of the analyses performed, a significant difference was only found by the relative positional distribution of events (Z = 2.4; P(z) >= 0.015). Taking this minor difference into account, we performed all further analyses for the Gadd45a colony separately from the other colonies and their wild-type controls.

The reversion frequency of the pun locus was determined for 20 C57BL/6J- pun/un Atm-/-, 16 C57BL/6J- pun/un Trp53-/-, 40 C57BL/6J- pun/un combined wild-type control, 32 C57BL/6J- pun/un Gadd45a-/-, and 26 C57BL/6J- pun/un Gadd45a+/+ control RPE (see Table 1Citation ). One unexpected observation that came from this analysis was that 2 of the 16 RPE obtained from C57BL/6J- pun/un Trp53-/- mice and 1 of 32 RPE obtained from C57BL/6J- pun/un Gadd45a-/- mice had what appeared to be a very early reversion events, possibly in progenitor RPE cells, resulting in many eye-spots. No such event has been observed in the 40 combined control RPE examined to date, nor the 26 C57BL/6J- pun/un Gadd45a+/+, the additional 20 Atm-/- derived RPE reported here, or the 45 RPE obtained after either X-ray or benzo(a)pyrene exposure previously reported (45 , 56) . The frequency of such an early event in the Trp53-/- was significantly different from the combined control (G = 5.199878; P(G) >= 0.023), although the frequency observed for the Gadd45a-/- was not significantly different from the Gadd45a control (G = 1.203654; P(G) >= 0.27). The observation of these types of event is already indicative of an increased level of HR early in the development of the RPE in these genetic backgrounds.


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  		Table 1 Summary of eye-spot and pigmented cell frequency

 
The average number of reverted (pigmented) cells per RPE, eye-spots per RPE, and eye-spot size for all genotypes were determined (see Table 1Citation ). On average, all three mutant backgrounds had more reverted RPE cells and eye-spots compared with their controls, but only trp53 and gadd45a mutant mice had an increased average eye-spot size. Because the SD in these analyses was similar to the average, a more correct method to examine the distribution of reversion frequency between the control and the mutant genotypes was to compare the distribution of RPE with a particular number of events, as shown graphically in Fig. 2Citation . Performing a rank-sum analysis of these distributions demonstrated a significant difference between the pooled wild-type control and both atm and trp53 mutant mice (see Fig. 2ACitation ; atm mutant mice: Z = -3.2; P(Z) >= 0.0013; trp53 mutant mice: Z = -3.6; P(Z) >= 0.0003). The difference between Gadd45a wild-type and mutant mice was not significant (see Fig. 2BCitation ; gadd45a mutant mice: Z = 0.9; P(Z) >= 0.37). Therefore, after examining the overall frequency of spontaneous HR events, it was apparent that the atm and trp53 mutant backgrounds displayed a greatly increased level of HR that was not observed in the gadd45a mutant background.



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  		Fig. 2. Comparison of the number of eye-spots found per RPE in the each mutant genotype with their relevant control. A, a graphical representation of eye-spot frequency per RPE of atm and trp53 mutant mice compared with the wild-type control. The majority of RPE obtained from the atm and trp53 mutant mice have more than nine eye-spots, where the majority of wild-type control RPE have fewer than seven eye-spots. This difference is highly significant for both genotypes. B, a graphical representation of eye-spot frequency per RPE of gadd45a mutant mice compared with the Gadd45a control. There is no significant difference in the pattern of eye-spot frequency between these genotypes, although Gadd45a control RPE have six eye-spots on average whereas gadd45a mutant RPE have nine.

 
Examining the Frequency of Different Sized Eye-Spots.
Eye-spots were categorized by size, that is, the number of reverted cells that constitute them. Previous reports have noted that the majority of eye-spots consisted of a single pigmented cell, with fewer two-cell eye-spots, even fewer three-cell eye-spots, and so on (44 , 45 , 56) . We observed no difference in these ratios for either the atm mutant mice (Z = 1.5; P(Z) >= 0.15) or the gadd45a mutant mice (Z = -0.2; P(Z) >= 0.85) and their relevant controls (see Fig. 3, B and DCitation , respectively). In contrast, trp53 mutant mice had a significantly different eye-spot size distribution from both the pooled control (Z = -4.1; P(Z) >= 4.2 x 10-5) and even more so from the atm mutant mice (Z = -5.1; P(Z) >= 3.3 x 10-7). This size redistribution indicates that in the trp53 mutant mice, more HR events occurred in cells that were likely to continue proliferating than was observed with either Atm-deficient or control mice. It is clear, however, that all three mutant backgrounds displayed an increased frequency of each sized eye-spot compared with their control (see Fig. 3, A and CCitation ).



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  		Fig. 3. Frequency and relative frequency of different size eye-spots in the RPE of the mutant mouse genotypes and their relevant controls. Eye-spot size is determined by the number of pigmented cells in the eye-spot. A, the frequency of different size eye-spots per RPE in atm and trp53 mutant mice and the wild-type control. For all sizes of eye-spot, there is an increased frequency per RPE for both genotypes over the control. B, the relative frequency of each size eye-spot per RPE as a proportion of all of the other eye-spots found in atm and trp53 mutant mice and the wild-type control. In both the control wild-type and atm mutant RPE >50% of the eye-spots consisted of only a single cell. Although trp53 mutant RPE also displayed a majority of singlet eye-spots, there were proportionally more larger eye-spots. C, the frequency of different size eye-spots per RPE in gadd45a mutant mice and the Gadd45a control. The gadd45a mutant RPE had more of each size eye-spot per RPE than the control. D, the relative frequency of each size eye-spot per RPE as a proportion of all of the other eye-spots found in gadd45a mutant mice and the Gadd45a control. The distribution of the eye-spot sizes appear to be the same between this mutant genotype and its control.

 
Examining the Positional Distribution of Eye-Spots in the RPE.
We examined the frequency of eye-spots located in different position intervals for the different mutant backgrounds and their relevant controls (see Figs. 4ACitation and 5ACitation ). The data obtained are displayed either as a frequency per RPE over positional intervals or as a relative frequency distribution that more clearly illustrates the pattern of recombination frequency by position. To determine whether there was any significant difference between the positional distributions of these eye-spots in different genotypes, rank-sum analyses were performed. Because we determined previously that the pattern of eye-spots can be resolved into two populations, eye-spots consisting of a single pigmented cell (singlets) and larger eye-spots (56) , with distinguishable distribution patterns, larger eye-spots generally lying more distal to the optic nerve head than the singlets (56) , any positional analysis benefits from the individual consideration of each type of eye-spot population. The distributions of singlet (see Figs. 4BCitation and 5BCitation ) and larger eye-spots (see Figs. 4CCitation and 5CCitation ) are also given.



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  		Fig. 4. An examination of the frequency of eye-spots in different regions of the RPE of atm and trp53 mutant mice compared with the wild-type mice. Results are presented as either the absolute frequency per RPE (A, C, and E) or as a relative positional distribution (B, D, and F). A position of 0.0 is equivalent to the optic nerve head, whereas a position of 1.0 is at the edge of the RPE. Examination was conducted on all sized eye-spots combined (A and B), singlet eye-spots (C and D), or only the larger eye-spots (E and F). Examination of the absolute frequency of events for the 20 atm mutant and 18 of the wild-type RPE (A, C, and E) demonstrates that the frequency of eye-spots of all classes increase from the optic nerve to the periphery of the RPE. This is represented in the positional distributions (B, D, and F) by the increasing proportion of events from 0% at positions 0.0–0.1 to 25% at positions 0.9–1.0, the edge of the RPE. The eye-spot distribution in the RPE derived from the 16 trp53 mutant mice do not display this gradient effect, with the most obvious difference seen with the larger eye-spots (E and F).

 


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  		Fig. 5. An examination of the frequency of eye-spots in different regions of the RPE of gadd45a mutant mice compared with the Gadd45a control mice. Results are presented as either the absolute frequency per RPE (A, C, and E) or as a relative positional distribution (B, D, and F). A position of 0.0 is equivalent to the optic nerve head, whereas a position of 1.0 is at the edge of the RPE. Examination was conducted on all sized eye-spots combined (A and B), singlet eye-spots (C and D) or only the larger eye-spots (E and F). The distribution of eye-spots in the 32 gadd45a mutant RPE examined is significantly different from the eye-spot distribution of the 26 control RPE. The most significant difference between gadd45a mutant and the control eye-spot distribution can be observed for the larger eye-spots (E and F), with an increased proportion of eye-spots located proximal to the optic nerve in the mutant background.

 
The atm mutant mice had an ~30% increase in the frequency of events compared with the wild-type control for each position examined (see Fig. 4ACitation ). The greatest increase in frequency of events was found in the singlet eye-spot class, as compared with the control (compare eye-spot frequency for atm mutant mice in Fig. 4, C and ECitation ). The most interesting aspect of these results, however, is that the overall distribution of these HR events was the same as the distribution found in the wild-type control (Fig. 4BCitation ; Z = 0.4; P(Z) >= 0.66). The same was found to be true with both the singlet (Fig. 4, C and DCitation ; Z = -0.2; P(Z) >= 0.86) and larger (Fig. 4, E and FCitation ; Z = 0.7; P(Z) >= 0.48) eye-spot populations. These results suggest that although there is an increased amount of HR in the atm mutant mouse, the timing and types of event is directly comparable with the pattern observed for wild-type mice.

The trp53 mutant mice had an increased frequency of events at all positions compared with the wild-type control, irrespective of whether examining all eye-spots, the singlets or the larger eye-spots (see Fig. 4, A, C, and ECitation , respectively). The largest increase in events was observed in the optical nerve head proximal region, leading to a significant redistribution in the relative positional pattern of HR events (all eye-spots, Fig. 4BCitation : Z = 6.0, P(Z) >= 2.4 x 10-9; singlets, Fig. 4DCitation : Z = 3.1, P(Z) >= 0.0017; larger eye-spots, Fig. 4FCitation : Z = 5.4, P(Z) >= 7.6 x 10-8), most dramatically in the larger eye-spot population. The distributions of eye-spots in the atm and trp53 mutant mice were also compared and gave similar results, demonstrating a significant difference in the pattern of events for these two genotypes (all eye-spots, Fig. 4BCitation : Z = 5.9, P(Z) >= 4.8 x 10-9; singlets, Fig. 4DCitation : Z = 3.5, P(Z) >= 0.00050; larger eye-spots, Fig. 4FCitation : Z = 4.9, P(Z) >= 8.8 x 10-7).

The positional distribution of events in the gadd45a mutant was compared with the Gadd45a control. An overall increased frequency of events was observed for most positions (Fig. 5A)Citation , although this increase seemed to be mostly attributable to the increase in larger eye-spots (compare Fig. 5, C and ECitation ). Similar to the results with the trp53 mutant mice, the greatest increase in events were found proximal to the optic nerve head, although a significant difference was only found for the total events and more so for the larger eye-spots compared with the control distribution (all eye-spots, Fig. 5BCitation : Z = 2.8, P(Z) >= 0.0052; singlets, Fig. 5DCitation : Z = 0.7, P(Z) >= 0.47; larger eye-spots, Fig. 5FCitation : Z = 3.2, P(Z) >= 0.0016).

An alternative method to examining the overall frequency distribution of eye-spots is to compare the frequency of events within a defined region versus the frequency of events outside of that region. Previously, we have used this method to identify specific regions into which there has been a redistribution of recombination events compared with control (56) . The results of these analyses are given in Table 2Citation and correlate very well with the rank-sum analyses performed on the overall eye-spot distribution patterns (compare Table 2Citation regions with Fig. 4, B, D, and FCitation , and Fig. 5, B, D, and FCitation ). Comparison between the atm mutant mice and the wild-type control revealed no region of significant difference and was, therefore, not included. Trp53 mutant mice demonstrated regions of significant increase and a corresponding region of decrease, compared with the pooled wild-type control for every class of eye-spot. Gadd45a mutant mice demonstrated a very similar pattern to the trp53 mutant mice with the exception that no region of significantly increased singlet eye-spots was identified, only a region of decrease distal to the optic nerve head. These results strongly suggest that the profile of events found in the trp53 and gadd45a mutant backgrounds are very similar, and yet both are very different from the profiles observed for the atm mutant mice. In addition, examining either the total population distribution or limiting the analysis to specific regions demonstrates that the greatest effect of trp53 and gadd45a mutations is an increased frequency of larger eye-spots. Region analysis locates these events proximal to the optic nerve head. Together, these observations suggest that there is an increased frequency of HR events initiated early during development in the trp53 and gadd45a mutant mice, where the atm mutant mice have an increased frequency of events later in development in a pattern that recapitulates the pattern observed for wild-type mice.


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  		Table 2 Comparing the frequencies of eye-spots of different classes in specific regions between mutant and control genotypes

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this study was to examine the roles of Atm, p53, and Gadd45a in controlling the frequency of HR in vivo. Using a similar in vivo system (the pun fur-spot assay), we previously reported that atm mutant mice have an increased frequency of HR and suggested that this increase came later during development (34) . The increased level of recombination may not have been surprising, considering that cells from AT patients display a higher than normal frequency of genomic instability, but the timing aspect was unexpected. In addition, we also reported previously the spontaneous effect of trp53 deficiency on the pun fur-spot assay (49) . In that study, we had the surprising finding that there was no observable increase in spontaneous HR frequency. Since that time, we developed and characterized the pun eye-spot assay (45) . With this assay, we defined a relationship between the regions in the adult mouse RPE where an increased level of HR occurred at the time of exposure to an HR-inducing agent during development and the region of proliferation of the RPE at that time of development (56) . Thus, the pun eye-spot assay provides both a more sensitive assay to determine the frequency of HR events in vivo and the ability to understand the timing of such events during development. With these tools, we have reexamined the roles of Atm and p53 in controlling the level of spontaneous HR during the development of the mouse embryo. In addition, we have included the gadd45a mutant mouse.

The study presented here clearly demonstrates that atm and trp53 mutant mice have an increased frequency of spontaneous HR events compared with control mice. In addition, we have observed that the gadd45a mutant mice also have an increased frequency of HR, although only observable at a significant level by the positional distribution of events in the RPE. The striking result is that the profile of events in the atm mutant background is clearly different from the profile observed in the trp53 and gadd45a mutant backgrounds, which are very similar to each other. In particular, the major spontaneous effect of the trp53 and gadd45 mutant backgrounds appears to be early in development, whereas the atm mutant background seems to affect spontaneous HR at later times of development, increasing at the same rate as wild-type spontaneous events.

The RPE of the mouse is a monolayer of pigmented cells derived from the neural epithelium. The development of this tissue in the mouse follows a well-defined pattern. RPE precursors enter the embryonic eyecup around the optic nerve head at about 8.5 dpc (60) ; the RPE then develops radially away from the optic nerve, with an outer edge-biased pattern of proliferation (55) . In a previous study, we examined with the pun eye-spot assay the effect of exposure to recombination-inducing agents at different times during development (56) . We demonstrated that because of the well-defined developmental pattern of the RPE (55) , we were able to define the time at which a recombination event occurred by its position within the RPE (56) . The earliest exposure was at 8.5 dpc and resulted in a significant increase in events in almost the exact same position as observed here for the trp53 and gadd45a mutant mice, if not slightly more distal from the optic nerve head. These results would suggest that the both trp53 and gadd45a are playing a role in maintaining genomic stability early during development, before 8 dpc.

In our previous report on the effect of trp53 on spontaneous HR frequency using the pun fur-spot assay, we observed no difference in the frequency of events compared with wild-type control (49) . In our efforts over the last decade to characterize the pun fur-spot assay for the most responsive time during development to conduct an exposure to a DNA-damaging agent to demonstrate induction of HR, we found that exposure on 10.5 dpc had the best result. Exposure at 8.5 dpc resulted in little induction, presumably because of the low number of target melanoblasts, whereas exposure at 12.5 dpc resulted in fur-spots that were extremely difficult to detect, probably because of the number of cell divisions left was too limited to make an easily recognizable pigmented spot (data not shown). This would suggest that the pun fur-spot assay would be insensitive to genomic instability at or before 8.5 dpc. Because we suggest that the time before 8 dpc is when an increased level of HR is seen in the trp53 mutant mice, our previous study would have missed that effect.

There have been three reports in the literature on the spontaneous activity of p53 during mouse embryonic development (61, 62, 63) , that is, the p53 dependent transcriptional activation of its downstream effector p21. All three studies reported that p53 is spontaneously active in the embryo up to approximately 8 dpc, when, depending on the tissue and level of differentiation, the spontaneous activity ceases. The lack of spontaneous activity later in development does not preclude the inducibility of p53 after exposure to DNA-damaging agents. These reports have been substantiated by the early embryonic lethality of Mdm2-deficient mice and their rescue by a p53-null genetic background (64 , 65) , suggesting that p53 is highly active over this period and must be negatively regulated. In addition, the hypersensitivity of embryos to ionizing radiation over this same period (66 , 67) , before 8 dpc, also suggests an already potentiated damage response system that is easily triggered to promote an apoptotic response.

The timing of the increased HR frequency in the absence of p53 that we report here correlates surprisingly well with the spontaneous activity of p53 during embryonic development. The majority of events in the trp53 and gadd45a mutant backgrounds appear to occur early, which, as a consequence, results in not only an increased number of events, but also an increase in larger eye-spots compared with singlets. These observations suggest a role for p53 and Gadd45a in maintaining genomic stability early in the developing embryo, similar to that seen in proliferating tissue culture studies (50, 51, 52, 53, 54) . In addition, the results presented here strongly suggest that p53 and Gadd45a are acting in a similar fashion to control spontaneous HR, perhaps acting epistatically, as has already been established by the transcriptional regulation of Gadd45a by p53 in response to exogenous exposure to DNA-damaging agents (2) . Considering that nucleotide excision repair is compromised in the p53 and Gadd45a-deficient backgrounds (28) , it is possible that the HR machinery, in the absence of p53 or Gadd45a, has more opportunity to act on lesions that are normally repaired by nucleotide-excision repair. The result would be the observed increase in HR frequency in these mutant backgrounds. When p53 or Gadd45a are not spontaneously active, we would not expect to see a substantial increase in HR frequency above the background, correspondingly, the fold increase of HR over control is less later in development as determined by the eye-spot assay.

The results from the atm mutant mice directly correlates with our previous findings with the pun fur-spot assay, an increased frequency of spontaneous recombination that increases more in later embryonic development. In addition, the majority of events, leading to greatest increase, were observed in the singlet eye-spots, unlike the observed profile of the trp53 and gadd45 mutant mice, where the greatest increase was in the frequency of larger eye-spots. This suggests that the role of Atm is very different from p53 in regulating spontaneous HR during development.

In the last few years, it has been demonstrated that Atm is involved in adult neurogenesis (68) . To address the possibility that the observed HR patterning was attributable to a defect in RPE proliferation or development we examined the proliferation of RPE cells in 12.5 dpc embryos by BrdUrd incorporation (data not shown). Although markedly smaller and, in fact, 1-day developmentally delayed, determined by the lack of postmitotic ganglion cells, the RPE of atm mutant mice displayed no gross abnormality or any mispatterning of proliferation. We would, therefore, suggest that Atm does not have a tissue-specific role in RPE differentiation or development and is unlikely to be affecting the HR pattern in the RPE by perturbing its genesis.

In summary, we have demonstrated that p53 and Gadd45a play a role in suppressing HR during in vivo development similar to reports from tissue-culture studies. Unlike the tissue-culture studies, we have seen, both directly in this study and indirectly in our previous study (49) , that this effect does not play a role later during the development of the embryo in either a neural epithelial or a neural crest derived tissue. This points to the necessity of relating tissue-culture studies on genomic stability with an in vivo model. In addition, this study also raises the question of the function of p53 and Gadd45a later in embryo development and how this function changes. Finally, we clearly demonstrate that Atm is functioning very differently from p53 and Gadd45a in suppressing HR, acting later in development. This synergy is supported by the increased prenatal death, severe runting, and increased rate of lymphoma reported in the generation of atm trp53 double-mutant mice (69) . Both Atm and p53 act as guardians for genomic stability, yet the differences in their time of action may offer some explanation as to the timing of carcinogenesis in these mouse models, with Atm-deficient mice suffering a more rapid rate of lymphoma onset (32 , 33 , 70) than p53-deficient mice (71 , 72) . If the level of HR stays abnormally high level in Atm-deficient mice, there is a great likelihood that HR events might directly cause oncogenic mutations or might more rapidly result in exposure of mutations in tumor suppressors. In p53-deficient mice, if the initial high burst of HR during development does not have a deleterious effect, the absence of p53 may have an effect later in the life when a cell is presented with a genomic insult and, in the absence of p53, will not be able to suppress an inappropriate HR event.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the National Institute of Environmental Health Sciences, NIH, RO1 Grant ES09519 and KO2 Award ES00299 (to R. H. S.), NIH RCDA Award F32GM19147 (to A. J. R. B.), and an A-T Children’s Fund award (to B. K. and R. L. S.). Back

2 To whom requests for reprints should be addressed, at Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Phone: (617) 432-7553; Fax: (617) 432-7663; E-mail: abishop{at}genetics.med.harvard.edu Back

3 To whom requests for reprints should be addressed, at Department of Pathology, UCLA School of Medicine, 650 Charles E. Young Drive South, Los Angeles, CA 90095. Phone: (310) 267-2087; Fax: (310) 267-2578; E-mail: rschiestl{at}mednet.ucla.edu Back

4 The abbreviations used are: HR, homologous recombination; RPE, retinal pigment epithelium; dpc, days post coitum. Back

Received 12/18/02. Revised 5/22/03. Accepted 6/26/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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