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DNA Research Advance Access originally published online on March 21, 2006
DNA Research 2006 13(2):65-75; doi:10.1093/dnares/dsi028
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© The Author 2006. Kazusa DNA Research Institute
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

The Role of Trp53 in the Transcriptional Response to Ionizing Radiation in the Developing Brain

J. Verheyde1,2, L. de Saint-Georges1, L. Leyns2 and M.A. Benotmane1,*

1 Laboratory of Radiobiology, Studiecentrum voor Kernenergie/Centre d'étude de l'Énergie Nucléaire (SCK•CEN) Boeretang 200, Mol B-2400, Belgium
2 Laboratory for Cell Genetics, Vrije Universiteit Brussel Pleinlaan 2, Brussels B-1050, Belgium

Received 22 September 2005; revised 11 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary Data
 Acknowledgements
 References
 
Brain formation results from a series of well-timed consecutive waves of cellular proliferation, migration and differentiation. Acute irradiation during pregnancy selectively interferes with these events to result in malformations such as microcephaly, reduced cortical thickness and mental retardation. In the present study we performed a straight-through cDNA-microarray analysis of the developing mouse brain at embryonic day E13, 3 h after in utero exposure to 50 cGy X-radiation. This dataset was used as an indication of genes involved in different pathways that are activated upon early radiation exposure, and for further evaluation using quantitative PCR (qPCR). Microarray and qPCR data revealed that the main activated pathways in irradiated wild-type embryos are involved in the regulation of a p53-mediated pathway that may lead to cell cycle delay/arrest and increased levels of apoptosis. To define whether the transcriptional radiation response was solely p53 mediated, we analysed the expression of cell cycle regulating genes in a Trp53 null mutant. The modulated expression of cell cycle regulating genes such as cyclins and Cdk genes indicated the induction of a cell cycle arrest, without evidence for the onset of apoptosis. Additional gene-expression studies have shown that various E2F transcription factors may be involved in this event. Together, these results provide a detailed view of the different p53-related mechanisms that are triggered in response to ionizing radiation in the developing brain.

Key words: cDNA microarray; ionizing radiation; brain development; Trp53; p53


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Supplementary Data
 Acknowledgements
 References
 
Brain development can be seen as long interrelated sequences of proliferation, migration and differentiation of neuroblasts and support cells.1Go,2Go Exposure of the developing brain to moderate or high doses of X- or {gamma}-ray ionizing radiation are known to interfere with these events. Epidemiological studies on children from radiation exposed pregnant women of Hiroshima and Nagasaki have emphasized the consequence of radiation exposure.3Go–6Go This exposure resulted in the occurrence of a range of distinct abnormalities such as reduced cortical thickness, abnormal neuronal migration, synaptic disturbance between neural cells, mental retardation and microcephaly and an accumulation of cell death. Ionizing radiation causes direct and indirect cellular effects, partially by targeting the DNA damage structure or signal transduction pathways. These pathways may consequently induce cell cycle arrest, aberrant mitosis and necrotic/apoptotic cell death in a dose-dependent manner.7Go Following radiation exposure, DNA is recognized by the ataxia-telangiectasia mutated (Atm) serine/threonine protein kinase.8Go Atm activates multiple targets that in turn regulate DNA double-strand break repair, induce cell cycle delay/arrest or trigger a pro-apoptotic cascade.9Go Cell cycle arrest induced at different cell cycle checkpoints is important to allow genetic repair or subsequent induction of cell death. Although Atm can activate such a cell cycle checkpoint via Chek2 and Cdc25, a more common system exist that involves p53 activation triggered by an Atm-regulated release of the Mdm2-mediated inhibition on p53 and the downstream activation of p21.9Go

Under normal conditions the oncogene protein p53, encoded by the Trp53 gene, is a short-lived protein that is maintained at low levels in the cell. Radiation exposure-triggered pathways activate four proteins—including Mdm2 and Cyclin G1—leading to the formation of an intricate complex that regulates p53 activity.10Go Active p53 induces the transcription of several downstream genes that in turn can trigger a variety of biological processes such as cell cycle arrest, apoptosis, DNA repair, replicative senescence and differentiation.11Go One of the downstream mechanisms involves the p53-mediated transcriptional activation of p21, which is encoded by the Cdkna1 gene. It was shown that p21 can arrest the cell cycle progression at the S-phase entry either through inhibition of the intra-G1 cell cycle phase cyclins12Go–14Go or by a combined effect of p21 and 14-3-3{sigma}.15Go After the onset of a cell cycle arrest, and depending on the severity of the X-radiation induced damage, p53 may activate DNA repair genes or downstream intermediates, such as Bcl2, Bax and Caspase3, to induce apoptosis.16Go Although the exact mechanisms are not yet fully understood, studies of Trp53 null and Trp53 mutated cells have shown that other X-irradiation triggered mechanisms independently of Trp53 exist, which may also lead to cell cycle arrest and apoptosis.12Go,13Go,17Go–20Go

While prenatal irradiation is a known risk-factor for adult neurological defects, most of the earlier performed global gene-expression studies that analyse ionizing radiation exposure were performed on adult brain tissues or haematopoietic cell populations.20Go–22Go For instance, Yin et al.22Go analysed the effects of both low- (10 cGy) and high-dose (2 Gy) radiation on the adult mouse brain and showed a time- and dose-dependent gene-expression profile. Genes associated with functions such as RNA synthesis and modification, DNA repair, cell cycle, heat shock mechanisms and chromatin structure regulation were primarily modulated 30 min after irradiation. Only 4 h after irradiation, genes involved in protein synthesis, cell growth, migration and maintenance showed a modulated expression. Remarkably, even a low dose of 10 cGy appeared to be sufficient to induce genes involved in stress response, synaptic signalling, DNA repair and cell cycle control.22Go Therefore, evaluating the gene expression in the embryo brain after radiation exposure could provide a better view of the radiation responsive mechanisms that occur in vivo. In this way a wide spectrum of known and unknown radio-induced or radio-repressed genes could be found. In our study we first generated an overview of the modulated mechanisms and signalling pathways involved in the early radiation response of the embryonic brain. For this purpose we performed a microarray gene-expression profiling on 22.680 transcripts 3 h after 50 cGy X-irradiation in wild-type (wt) as well as in Trp53 null mutant embryo brains at development day E13. The role of p53 was evaluated, since p53 activity is clearly related with the molecular radiation response.23Go Among the many candidate genes identified, we selected a subset of genes involved in distinct radiation responsive pathways for further in depth investigation using quantitative PCR (qPCR) on three biological replicates. As indicated by our results, and although the main radiation induced cellular response is considered to be p53 dependent, cell cycle related genes were modulated at the transcriptional level after exposure to radiation in the Trp53 null mutant.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Supplementary Data
 Acknowledgements
 References
 
2.1. Sample preparation
All animal experiments were handled in agreement with the laboratory animal Belgian legislation (ref. 02-012 of the local ethical commission). Balb/cJ@Rj wild type (+/+, Janvier/Bio-services) and Trp53 null mutant (–/–) in Balb/c background (in-house breeding) were maintained in a normal 12:30/11:30 light cycle. Food and water were available ad libitum. Trp53 wild type and Trp53 null mutant female mice were mated with males of the same genotype from 05:30 am to 09:00 am using automatic time-operated cages. The day at which the presence of the copulating plug was detected was set as day E1.

Subsequently, at day E13 pregnant females were whole-body irradiated with 50 cGy at a dose rate of 0.35 Gy/min using a Pantak RX, 250kV–15mA, 1mm Cu filter installation. Two sham-exposed mice were used as controls. Three hours after the irradiation, embryos of both a Trp53 wild type and a Trp53 null mutant genotype were isolated and the brains were microdissected. Both brains and the remaining bodies were snap-freezed in liquid nitrogen. Body tissue samples were used for DNA extraction for Trp53 locus genotyping by PCR using three designed primer sets (Supplementary Table 1 is available at www.dnaresearch.oxfordjournals.org). To eliminate the possible influence of gender on differential gene expression, microarray hybridizations were performed using only female embryo mRNA for all conditions.24Go As previously described by Lambert et al.24Go sex determination was performed by PCR using the Sry-gene, located on Y chromosome. IL-3 was used as the positive control.24Go

2.2. RNA/DNA isolation
Total RNA was isolated using the Trizol method (Invitrogen, USA) according to the manufacturer's recommendations. The quantity and quality of the purified RNA was assessed using spectrometry and electrophoretic gel-assay. For PCR genotyping and sex-determination DNA extraction was performed using the High Pure PCR template preparation kit (Roche Diagnostics, USA). The DNA used for DNA fragmentation analysis was extracted according the guidelines of the Wizard Genomic DNA kit (Promega, USA).

2.3. Microarray experiments and processing
Five micrograms total RNA of each sample were used for hybridization on the microarray. Microarray hybridization and washing steps were processed in the same way as described previously by Mori et al.25Go To correct for non-equilibrated dye incorporation, a straight and colour-flipped hybridization was performed for each pair of samples on in-house spotted cDNA microarrays containing 22 680 different clones, including spikes and internal controls (Microarray Facility, VIB-Vlaams Interuniversitair Instituut voor Biotechnologie, Belgium—www.microarray.be). After dual laser scanning (Generation III scanner, Amersham Biosciences, UK), image analysis was performed using the ArrayVision software (GE Healthcare, UK). For duplicate and colour-flipped clones, ratios of corrected Cy5 intensity over corrected Cy3 intensity were calculated and deviations in the ratio-intensity plot were smoothened using Lowess-normalization.26Go Using a two-sample T-test, significance intervals were calculated based on four intensity values obtained from the two hybridized slides. When multiple clones of the same gene were present, the average intensity and corresponding standard deviation were calculated. Genes with an average value <1.5-fold induction/repression that showed strong differential expression for at least one clone were maintained for selection. Transcripts with a threshold of induction and repression of greater than 1.50- and less than –1.50-fold and ESTs differentially expressed in the original microarray-dataset were screened for annotation based on public databases such as the Unigene database (http://www.ncbi.nlm.nih.gov, 05/2005), the Mouse Genome Institute (MGI, http://www.informatics.jax.org) and Matchminer.27Go Only the annotated genes were included for further analysis and clustering in biological significant groups. To evaluate p53 interactions, the selected genes were matched against two publications, which reported analysed genes that contain p53 binding sites on their promoter region (http://linkage.rockefeller.edu/p53).23Go,28Go

2.4. Two step real-time qPCR
Three additional RNA samples were extracted from both wt and Trp53 null mutant embryos, 50 cGy irradiated at E13. A total of 5 µg mRNA was reverse transcribed to cDNA according to the manufacturer's guidelines (Roche Diagnostics, USA). Gene-specific primers were designed using the Primer Express software (Applied Biosystems, USA) and were checked for gene specificity using NCBI Blast (http://www.ncbi.nlm.nih.gov/blast). In presence of SYBR® Green (Eurogentec, Belgium) the primers were used to amplify the expressed cDNA using the ABI 5700 real time PCR system (Applied Biosystems, USA). The amplification of each gene was corrected against the expression level of the house-keeping gene ß-actin. The relative quantification of gene expression was calculated using the {Delta}{Delta}Ct comparative method in which {Delta}{Delta}Ct = (Ctsample – Ctref)ctrl – (Ctsample – Ctref)irradiated and where the estimated expression ratio is equal to 2{Delta}{Delta}Ct.29Go A one sample T-test was used to statistically analyse the difference of the derived expression ratios of irradiated versus non-irradiated samples.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Supplementary Data
 Acknowledgements
 References
 
3.1. Microarrays
Since DNA ladder analysis showed apoptosis 6 h after exposure in wt developing mouse brains at E14 (Supplementary Figure 1 is available at www.dnaresearch.oxfordjournals.org), we hypothesized that radiation responsive gene-expression changes may be observed as early as 3 h after irradiation. The mRNA microarray expression profile of each irradiated embryonic brain was compared with non-irradiated controls of the same developmental stage. At E13, the total numbers of genes differentially expressed with a threshold of >1.5 and <–1.5 were 2785 transcripts including 1150 annotated genes in the wt and 2115 transcripts including 1236 annotated genes in the Trp53 null mutant (Fig. 1). We have chosen to put the threshold level to ±1.50, since most of the interesting genes were analysed using qPCR and in this way the maximum number of potential candidate genes that are involved in the radiation response were obtained. All annotated genes were combined according to their gene-ontology classification in 14 biological groups (Table 1). Based on the involvement in stress response, only groups AII, AIII, AVII, BI, BII and D, of both wt and Trp53 null mutant embryos, were used for the selection of a series of genes (Table 1). The groups AI, AIV, AV, AVI, AVIII, AIX, C and E were excluded from analysis, since they were related to metabolic, cytoskeleton, small molecule/protein binding or transport functions. From the 125 genes ultimately selected in the irradiated wt model, 60 (48%) were upregulated and 65 (52%) downregulated at E13. From the 130 differentially expressed selected genes in the irradiated Trp53 null mutant, 45 (35%) were upregulated and 85 (65%) downregulated. To further study the modulated mechanism found after radiation exposure, a subset of the previously selected genes was then used for qPCR.


Figure 1
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  		Figure 1. Process of selection for genes differentially expressed in the E13 wild-type (+/+) and Trp53 null mutant (–/–) embryonic brains.

 

View this table:
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  		Table 1. Functional Gene Ontology (GO) classification of up- and downregulated (greater than 1.5- and less than –1.5-fold) transcripts as found in the microarray data from 50 cGy irradiated wild type and Trp53 null mutant embryonic brains at E13.

 
3.2. Differential expression of transcripts in the irradiated wild-type embryos
A first observation of the differentially expressed genes, selected from the 50 cGy X-irradiated wt embryo at developmental stage E13, pointed to transcripts involved in various cellular functions, such as cell proliferation/DNA modification and cell interaction (AII–AIII), stress response (AVII), cell to cell communication (BI–BII) and development (D). Based on the selection of differentially expressed genes, three major mechanisms that were activated or repressed upon irradiation could be observed. A first set of these overexpressed genes belongs to the known p53 pathway involved in stress response. These genes were Cyclin G1 (Ccng1), mouse double minute 2 gene (Mdm2), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1), cyclin-dependent kinase inhibitor 1a (Cdkn1a/p21), transcription factor Max and oncogenes c-Jun and c-Myc. There were 23 differentially expressed transcripts classified as signalling receptors/cytokines/ligands (BI). The two major pathways, which could be identified, involve the insulin growth factor (Igf) family pathway (Igf1, Igf2, Igfbp1, and Igfbp4) and the interferon-gamma (IFN-{gamma}) signalling pathway (Iigp, Ifrd1, Ifi30, Ikbke/IKK-i and Isg20 (Supplementary Table 2 is available at www.dnaresearch.oxfordjournals.org).

We have selected a small set of genes from the microarray that were involved in the p53-mediated pathway. These genes were used for further investigation using qPCR, and included Ccng1, Mdm2, Cdkn1a and Trp53inp1, as well as genes that have been suggested to participate in the same p53-dependent pathway, but which were not present on the microarray (Fig. 2A and B). These included Atm, Atr, the checkpoint kinases Chek1/2, P19Arf/Cdkn2a and protein phosphatase 2 subunits Ppp2r2a/b/c/d, Hipk2/3, Trp73 and Trp63. Average qPCR relative fold induction resulting from three independent brain samples of each treatment showed an upregulation of 4.69-fold for Atm, while Atr was downregulated by –11.20-fold (Fig. 2A). Further downstream in the pathway, Chek1 and Chek2 were downregulated by –8.69 and –6.52, respectively. The PP2A-B subunits and P19Arf were differentially expressed and are partners of the same complex that also involves Ccng1/Mdm2. All the PP2A-B subunits were upregulated with Ppp2r2a: 4.57-fold, Ppp2r2b: 21.14-fold, Ppp2r2c: 15.82-fold and Ppp2r2d: 4.18-fold, while P19Arf was downregulated with –7.51-fold. We also analysed the mRNA expression of the p53 family members Trp63 (+2.11-fold) and Trp73 (+1.31-fold), as their molecular function can be complementary to that of p53 (Fig. 2B).30Go


Figure 2
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  		Figure 2. (A and B). Gene expression of two pathways involving p53 upstream and p53 downstream genes. Real time quantitative PCR showing relative fold expression level (IR/Ctrl) of 15 Trp53-dependent transcripts in three individual wild-type brain embryos after exposure to 50 cGy X-radiation. Analysis was performed based on the {Delta}{Delta}Ct method and corrected on ß-actin expression. Genes suggested to participate in the Trp53-dependent pathway but which were not differentially expressed or not present on the microarray were also included in the analysis. (Figure 2: P < 0.1; *: P < 0.05; **: P < 0.01 and ***: P < 0.001)

 
3.3. Differential expression of transcripts in the irradiated Trp53 mutant embryos
We chose to include a p53 null mutant embryonic brain for the analysis, since the p53 protein is undoubtfully linked with the molecular response to ionizing radiation. Therefore, similar experimental conditions were used for microarray analysis, to identify modulated transcripts in the irradiated Trp53 mutant embryonic brain. To confirm the inactivity of the main mediated p53 pathway, we analysed the mRNA expression level of Cdkn1a using qPCR. Cdkn1a was not differentially expressed in the irradiated Trp53 null mutant embryo and showed even a slight downregulation with –1.62-fold after irradiation (Fig. 3D). Furthermore, no other known p53-mediated transcripts present in the irradiated wt embryos microarray dataset were modulated in the null mutant dataset. The same biological clusters were applied for the selection of differentially expressed transcripts in the irradiated Trp53 null mutant (Table 1). From the biological cluster concerning cell proliferation/DNA modification (AII) there were 32 modulated transcripts, of which 26 were downregulated and directly involved in cell cycle progression. These include the cyclins Ccna2, Ccnb1/2, Ccnd1/3, Ccne1 and the cyclin-dependent kinases Cdc20, Cdc6, Cdca5/8 and Cdk2 (Supplementary Table 3 is available at www.dnaresearch.oxfordjournals.org). The downregulation of numerous cell cycle genes suggests that ionizing radiation has a major effect on cell proliferation, even in Trp53 null mutant mice. In order to better understand this phenomenon we evaluated the expression levels of different cyclins and cyclin-dependent kinases using qPCR. These included the genes present on the array as well as seven additional genes. Our qPCR analysis evidenced the strong downregulation of G2 phase cell cycle genes; however, not all genes involved in G1 phase transition showed a downregulated expression (Fig. 3A and B). To further explore the radiation induced cell cycle regulation effects, we evaluated the expression levels of different members of the E2F family of transcription factors. These genes are known to have cell cycle regulating functions. Analysis using qPCR showed a differential expression for the seven E2F family members, suggesting their involvement in this event. Cdc6, E2F6 and E2F7 were downregulated –1.95-fold, –1.93-fold and –3.08-fold, respectively (Fig. 3C). Remarkably, E2F2 and E2F4 were upregulated, 1.97- and 4.53-fold, respectively (Fig. 3C).


Figure 3
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  		Figure 3. (A–D) qPCR graphs showing the relative fold expression level (IR/Ctrl) of 15 cell cycle and 7 Atm/p53-mediated transcripts analysed in three independent mutant brain embryos after exposure to 50 cGy X-radiation. Analysis was based on the {Delta}{Delta}Ct method and corrected on ß-actin expression. Genes suggested to participate in the same pathway but which were not differentially expressed or not present on the microarray were also included in the analysis. (Figure 3: P < 0.1; *: P < 0.05; **: P < 0.01 and ***: P < 0.001)

 
Finally, to determine whether alternative pathways were triggered by Trp63 and Trp73, and whether they replace p53, we analysed the expression of the two genes using qPCR. Both genes were downregulated, –3.33-fold for Trp63 and –10.00-fold for Trp73 (Fig. 3D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Supplementary Data
 Acknowledgements
 References
 
4.1. Activation of Trp53-dependent transcripts in irradiated wt embryos
The main result from our microarray and qPCR data on irradiated wt embryos was the upregulation of genes involved in the p53-mediated DNA-damage responsive pathway. Although gene-expression analysis of individual or small subsets of these genes has been previously reported in response to radiation, our analysis provides an overview of the different pathways induced by ionizing radiation.31Go,32Go

We observed using qPCR an upregulated expression for Atm 3 h after irradiation, but not for Atr. This confirms the previously published work that suggests that Atm is required for p53 stabilization and activation after ionizing radiation exposure, while Atr is needed for the same function after UV radiation exposure (Fig. 4).10Go It has been proposed that in response to stress, Atm activates in parallel different pathways that function in a p53-dependent and p53-independent manner.33Go Although Atm activation could lead to a G1/S phase arrest through Chek2 and Cdc25a, it does not seem the preferred signalling pathway as indicated by the absence of upregulation of these genes in the microarray or qPCR data.33Go Our data show however that three mechanisms are triggered by ionizing radiation. The first one involves the Atm mediated activation of Trp53inp1, a recently identified gene that encodes two splice variants (SIP27 and SIP18) and that can regulate p53 activity through Hipk2 (Fig. 4). We suggest that Atm could directly or indirectly regulate SIP18/SIP27 activity in a positive manner and stimulate p53 activity as such after exposure to ionizing radiation.34Go–36Go


Figure 4
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  		Figure 4. Combined scheme showing irradiation-induced Trp53 molecular signalling pathway according to Kurz et al. (2004); Fei et al. (2003) and Kimura et al. (2002)9Go,10Go,31Go The genes associated with the Trp53-dependent radiation response that were modulated in our microarray are indicated (closed triangle, upregulated genes; closed inverted triangle, downregulated genes). Solid arrows represent activation, while blocked lines represent inhibition. Dotted arrows depict hypothesized pathways.

 
However, the main part of our data points towards a second and more studied p53-dependent molecular mechanism. This mechanism is known to be activated in response to ionizing radiation and involves the p53-mediated signal transduction genes Ccng1, Mdm2 and Cdkn1a (Fig. 4). As previously described in studies on neural and tumoral tissue, cyclin G1 or the protein encoded by Ccng1 is activated during normal brain development, but it's expression is increased in response to DNA damage.10Go,34Go,35Go,37Go,38Go The interaction of Cyclin G1 with p53 is mediated in an intricate feedback loop-system in which Ccng1 functions as a transcriptional target of p53 and subsequently regulates the p53 activity.39Go Studies using high dose of {gamma}-radiation (10 Gy) treatment showed that cyclin G1 promotes the formation and stabilization of a complex consisting of Mdm2, Cyclin G1 and either P19Arf or the PP2A-B subunits in a time dependent way.10Go Dependent on the time after exposure, P19Arf and the PP2A-B subunits regulate p53 activity in a different way.10Go Although this kind of model has only been described in high-dose-irradiated cells, we observed an upregulation for Ccng1 and all the four PP2A-B subunits 3 h after irradiation. This suggests that even a dose of 50 cGy is high enough to induce the formation of a complex that consists of the PP2A-B subunits in brain cells (Fig. 4).10Go Furthermore, it is known that overexpression of Ccng1-transcripts can induce a delayed S- and G2/M-phase cell cycle arrest by impairing p53 activity, as shown after a 1Gy radiation dose.40Go This may actually be the case in our model since such effect can be further evidenced by the downstream upregulation of Cdkn1a. Cdkn1a can inhibit cell cycle progression at two cell cycle checkpoints. The downregulation of Ccnd2, Ccnd3 and Cdk5 however may indicate that the preferential target is the G1/S phase checkpoint and not the G2/M phase checkpoint.14Go,41Go

A third suggested mechanism that can be activated by Atm in response to radiation in the embryonic brain is mediated by the p53 family members p63 and p73 (Fig. 4).42Go Although, the exact role of these genes in the radiation response is still elusive, they are known to play a role in the processes of cell cycle arrest, apoptosis and development.43Go,44Go The upregulated expression of Trp63 and Trp73 suggests that after irradiation there is a well-coordinated balance of p53, p63 and p73 activation in the wt embryonic brain, which may be essential for eliminating damaged brain cells. Together with the p53-mediated response, the irradiated embryonic brain is also characterized by the induction of a spectrum of inflammatory factors, which may moderate the radiation induced negative effects in neural cells. Although the global effects of these cellular inflammatory pathways are not discussed in detail, it is known that the survival of neurons and possibly also their support cells relies partially on a multifactoral support of autocrine/paracrine functions of such neurotrophic factors by which—even in absence of an individual factor—maximal survival is ensured.45Go,46Go Upregulation of these inflammatory pathway intermediates may relate to a direct radiation induced response and with the invasion of inflammatory cells among damaged neural cells.47Go However, DNA fragmentation analysis showed increased levels of apoptosis in developing brain, suggesting that this combined activation of protective mechanisms seems to be not sufficient against the strong Trp53-dependent pro-apoptotic and IFN-{gamma} pro-inflammation signal in the majority of cells.

4.2. Cell cycle related transcripts are downregulated in irradiated Trp53 mutant embryos
Using our data, we first identified whether the presence of a p53 binding site in the promoter region could be an indication of the difference in radiation response between wt and Trp53 null mutant embryos. Therefore, we compared our selected genes with two lists of putative p53 interacting genes identified with p53 binding capacities based on their molecular sequence using a computational approach.23Go,28Go Analysis of our selected dataset revealed that 41 (33%) genes in the wt embryo and 52 (29%) genes in the Trp53 null mutant embryo had potential p53 binding sites in their promoter region. From this selection, 54% in the wt embryo and only 35% in the Trp53 null mutant embryo were upregulated. The downregulated p53 interacting genes (65%) in the Trp53 null mutant indicate that p53 and p53-mediated mechanisms are the main activation targets after ionizing radiation exposure (Supplementary Tables 2 and 3 are available at www.dnaresearch.oxfordjournals.org).

Despite the absence of apoptosis in the X-irradiated Trp53 null mutant embryos 6 h after exposure to ionizing radiation, our qPCR data showed the induction of several other stress response mechanisms.48Go–50Go First, to confirm the inactivity of p53-mediated mechanisms in our null mutant model, we evaluated the expression levels of seven p53 up- and downstream genes using qPCR. Although the upregulation of Atm indicates the induction of DNA damage by ionizing radiation, downregulation of the p53 downstream gene Cdkn1a (Fig. 3D) suggests that the p53-dependent mechanisms are not activated in the irradiated Trp53 null mutant embryonic brain. Therefore the p53-mediated pathway could not be directly responsible for the downregulation of the observed cell cycle regulating genes. As most of these genes are considered to be post-translationally modified, it still seems that p53 controls their transcription at a basal level.51Go,52Go

In the Trp53 null mutant embryo we also analysed the expression of Trp63 and Trp73, as they can replace Trp53 in stress response. As described above, both genes have distinct developmental functions, and a loss of p73 activity has been related to hippocampus dysgenesis, which in turn is due to a loss of reelin-producing Cajal-Retzius neurons.53Go Downregulation of these two genes in the irradiated Trp53 null mutant brain suggests not only that p53 is essential for their activity in the developing brain, but that they also are regulated in a negative feedback loop-system.4Go,54Go,55Go However, further research should be conducted to clarify the exact role of these genes in the onset of developmental defects.

Although there is no evidence for increased levels of apoptosis after irradiation in the p53 null mutant embryo brain at E13, transcriptional evidence showed the occurrence of increased numbers of cells that exhibit a proliferative arrest in the irradiated tissue. This is indicated by the observed downregulation of various cyclins, Cdks and Cdc genes in the analysed embryos (Fig. 3A and B). Different gene-products, mediating independently of p53, have the ability to break the formed Cyclin/Cdk complexes or inhibit their formation (Fig. 5).12Go,13Go It has been shown that ionizing radiation may trigger this event in multitude of cells and induce a G1 or G2 phase cell cycle arrest.37Go,56Go,57Go The downregulation of cell cycle G1 and G2 phase genes in our microarray and qPCR initially suggests that the cell proliferation arrest is not limited to a specific phase of the cell cycle or is not specifically dependent on p53 activity.58Go However, a further in depth qPCR study showed that mainly cell cycle G2 phase genes were downregulated and not those of the G1 phase.


Figure 5
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  		Figure 5. The combined scheme, according to Vermeulen et al. (2003) and Pawlik and Keyomarsi (2004), showing the genes essential for normal cell cycle progression and the genes that may jeopardize this progression.12Go,13Go,57Go The genes associated with the mammalian cell cycle that were modulated in our microarray are indicated (closed triangle, upregulated genes; closed inverted triangle, downregulated genes). Solid arrows represent activation, while blocked lines represent inhibition. Dotted arrows depict putative pathways.

 
A first group of genes that could be responsible for the induction of the cell cycle arrest are the different members of the E2F transcription factor family (Fig. 5). E2F factors are known to control the cell cycle by regulating transcription of various growth-related genes such as Cdc2, Cdc6, Cdk2, Cyclin E and E2F1.59Go Each of the E2F family members has distinct cell cycle repressing or inducing activities. Using qPCR, we observed the downregulation of Cdc6, E2F6 and E2F7 and the upregulation of E2F2 and E2F4 in the irradiated Trp53 null mutant embryo brain (Fig 3D). However, the exact mechanism of the E2F factors is not completely understood. Robles et al.60Go reported that Cdc6 is an important factor with a dual role in regulating both G1/S and G2/S cell cycle checkpoints and that E2F factors regulate Cdc6 expression to control cell cycle progression. Since E2F4 has been stated responsible for the induction of growth arrest in mammalian cells,61Go we suggest that the elevated expression level of E2F4 after irradiation may be responsible for the downregulation of Cdc6 and possibly of other E2F target genes. Furthermore, as Takahashi et al.62Go reported, pRb together with the E2F4 factor may lead to the downregulation of E2F factors or the repression of E2F target genes to result in the onset of cellular senescence or growth arrest.

Similar to the wt embryonic brain, different members of the pro-inflammatory signalling pathway showed induction in response to ionizing radiation in the Trp53 null mutant. The observed induction of IFN-{gamma} related transcripts that were observed in our microarray data included Icsbp1, Isg20 and Iigp. As such, these mechanisms could function as an additional, though subtle, system for stress response, mediated independently of p53. Also, various development-related transcripts were downregulated, including Hoxa7, Hoxb3, Hoxb6, Hoxc5, Hoxd8 and Fzd4 (frizzled-4). Although the gene expression was not investigated further, their downregulation may relate to the described cognitive defects after radiation exposure of embryos.

In summary, the initial aim of the study was to better understand the basic underlying molecular mechanisms that are activated in vivo by radiation exposure in the developing brain. Previously performed studies on atomic bomb survivors have shown that in utero irradiated children have a higher incidence for late neurological defects, such as mental retardation and decreased IQ levels.4Go To expand the knowledge that is available on the early transcriptional radiation response, and since p53 is indisputably connected with the stress response, we analysed the modulated gene expression of the whole developing brain of wt and Trp53 null mutant embryos exposed to 50 cGy of ionizing radiation. The results on the irradiated wt embryos not only confirmed the importance of a p53-mediated stress response, but also provided a range of other candidate genes that may participate to produce stress response. In the Trp53 null mutant embryo, we observed other pathways leading to a cell cycle arrest without the observed onset of apoptosis. This shows that several ionizing radiation triggered mechanisms may be activated in parallel or may circumvent each other. These mechanisms were analysed in the present study at their transcriptional level on whole embryonic brains. Therefore, further study should be performed on homogeneous neuron or glial cell cultures in order to elucidate the cell type dependent contribution of the tissue reported effect.


   Supplementary Data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary Data
Acknowledgements
 References
 
Supplementary Data is available online at http://dnaresearch.oxfordjournals.org.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary Data
 Acknowledgements
References
 
The authors thank Dr P. Van Hummelen of the Microarray facility (VIB, Belgium) for his help with analysing the microarrays and Prof. M. Mergeay and Dr P. Janssen (SCK•CEN, Belgium) for the excellent discussions and reviewing of the paper. Furthermore, we thank A. Michaux for her intensive help on mouse embryo genotyping and sex determination. Prof. Dr L. Leyns was funded by the Belgian Science Policy agency (grant IAP-V 35) for this research. J. Verheyde is an Assistant Scientific Collaborator (AWM) at the SCK-CEN. This work is dedicated to the memory of P. Govaerts ({dagger}, 25 January 2006) SCK-CEN general manager.


   Footnotes
 
*To whom correspondence should be addressed. Tel. +32-14-33-27-31, Fax. +32-14-31-47-93, E-mail: abenotma{at}sckcen.be

Communicated by Shoji Tsuji


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Supplementary Data
 Acknowledgements
 References
 

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