BACKGROUND
the base excision repair  pathway, one of the major dna repair pathways, is dedicated to the repair of damaged dna bases arising from endogenous and exogenous insults  <cit> .
the dna bases may be subjected to oxidation, alkylation and deamination and oxidative dna damage, in particular, has been implicated in the pathogenesis of many diseases including cancer, atherosclerosis, neurodegenerative diseases such as parkinson's and alzheimer's, and even aging  <cit> .
dna glycosylases are the key enzymes of ber; they initiate repair by catalysing the hydrolysis of the n-glycosylic bond between modified bases and the sugar-phosphate backbone  <cit> .
to date, four mammalian dna glycosylases that recognize and excise oxidized bases have been thoroughly characterized: ogg <dig>  nth <dig>  neil <dig> and neil <dig>  <cit> .
neil <dig> was identified together with neil <dig> and neil <dig> as a gene product with significant structural similarities to the e. coli fpg and nei dna glycosylases  <cit> .
however, in contrast to neil <dig> and neil <dig> no substantial dna glycosylase activity has been detected  <cit> .
expression of human neil <dig> has been reported in thymus and testis, while mouse neil <dig> has been demonstrated to be highly expressed in spleen, bone marrow, thymus, blood cells and brain regions that harbour progenitor cells  <cit> .
neil <dig> has also been shown to be highly expressed in primary malignant melanomas associated with metastasis  <cit> .
most studies have been performed on adult organs and a recent paper described neil <dig> expression in the developing mouse  <cit> .
here, we describe the expression pattern of neil <dig> during embryonic development in mice with focus on the expression of neil <dig> during brain development.
the expression of neil <dig> in multiple human cancers was also examined.
RESULTS
neil <dig> is expressed during embryogenesis
the temporal expression pattern of neil <dig> during embryonic development was examined by in silico electronic northern blot  and northern blot analysis.
we used the unigene collection  to generate in silico expression profiles for neil <dig> at different stages during embryonic development.
as seen in figure 1a, there is a very high expression in the oocyte, unfertilised oocyte and zygote and this expression dramatically falls after the zygote stadium.
during embryogenesis, neil <dig> transcription is not detected until e <dig> – <dig> .
northern blot analysis was performed by using a northern blot containing poly+ rna isolated from whole mouse embryos at days  <dig>   <dig>   <dig> and  <dig> of gestation.
expression of neil <dig> was not observed until e <dig> .
neil <dig> is highly expressed in developing brain
next, we examined the expression of neil <dig> in developing mouse brain.
messenger rna was isolated from brains at days  <dig> ,  <dig>  and  <dig>  of gestation and at p <dig>  and subjected to quantitative real-time pcr.
figure 2a shows the expression levels of neil <dig> relative to gapdh.
the highest expression level of neil <dig> was observed at e <dig> .
to dissect the sites of neil <dig> expression, we evaluated the distribution of neil <dig> by transcript profiling in various brain regions at five different stages of development: e <dig>  e <dig> and e <dig> , p <dig>  and  <dig> weeks .
as seen in figure 2b, neil <dig> expression was highest at e <dig> in the fore- and midbrain and the expression decreased during development.
in neonatal mice the expression was relatively low in all areas examined, except for in the cerebellum and the spinal cord.
in adult mice, neil <dig> expression was below the detection limit .
neil  <dig> is highly expressed in regions of active neurogenesis in the developing brain
in order to characterize in more detail the expression of neil <dig> mrna in developing brain, in situ hybridization was performed on foetal mouse forebrains at e <dig>  e <dig>  and p <dig> .
at e <dig> abundant neil <dig> mrna was detected in the subventricular zone  of the lateral ventricles .
the expression of neil <dig> decreased during development and at e <dig>  and p <dig>  expression was limited to distinct cells in the cortical svz, in cells of the secondary matrix , the dentate gyrus migratory route  and the dentate gyrus .
thus, neil <dig> was expressed in regions that are rich in progenitor cells.
no signals were detected for the sense dig-rna probe used as a control .
neil <dig> is expressed in various tumor samples
neil <dig> has recently been shown to be highly expressed in melanomas  <cit> .
we therefore investigated the expression of neil <dig> in several tumor types and their normal counterparts by using qpcr arrays containing cdna from diseased and normal tissues.
data shown in figure  <dig> is from one of the two experiments performed and represents relative neil <dig> mrna levels in normal and tumor tissues.
raw data and information about the samples examined are shown in additional file  <dig>  results revealed that although we observed high variation within the different cancer samples from the same tissue, tumor samples in general displayed a higher expression of neil <dig> than the corresponding normal tissues, except for testis and pancreas.
furthermore, neil <dig> was highly abundant in normal testis tissue, as has been described before  <cit> .
discussion
in this study the expression of neil <dig> during embryonic brain development was investigated.
our results showed that neil <dig> was not uniformly expressed in the brain, but limited to specific cells in certain regions.
the expression appeared to be tightly regulated both spatially and temporally during brain development.
results from northern blot analysis of total embryos revealed the presence of neil <dig> after e <dig>  while rt-pcr experiments showed the presence of neil <dig> in the developing brain from e <dig>  thus, the expression of neil <dig> appears to coincide with organogenesis.
this is also supported by the in silico analysis, where neil <dig> transcripts were not detected until e <dig> –e <dig> .
intriguingly, the in silico analysis revealed high expression of neil <dig> during preimplantation.
mammalian embryos undergo major changes in their gene expression patterns throughout most stages of preimplantation development and massive maternal degradation of mrna characterizes the transition from oocyte to early embryo before the onset of new transcription  <cit> .
genes that follow this expression pattern are suggested to have specific functions either in oogenesis, oocyte maturation, fertilization, and/or early phases of preimplantation development  <cit> .
our results suggest that neil <dig> can be classified with this group of genes since neil <dig> mrna is highly expressed in unfertilised eggs and is abundant up to the zygote stadium before it is apparently degraded.
in mice, embryonic neurogenesis occurs from e <dig> to e <dig>  <cit> .
there are two major proliferative populations in the developing brain  <cit> .
the first to appear is a region called the ventricular zone  in the lateral ventricle.
the vz is lined by a population of cells that contains multipotent neural stem cells that give rise to most neurons and glial cells of the cortex.
these cortical precursor cells constitute a heterogeneous population and there is increasing evidence that different precursor cells generate distinct differentiated cell types  <cit> .
another proliferative population is found in the subventricular zone , located adjacent to the vz along the lateral ventricle.
notably, neurogenesis continues in this region throughout adulthood, and in the adult mammalian brain, new neurons born in the svz migrate anteriorly into the olfactory bulb , where they mature  <cit> .
in post-natal mice, a second germinal zone exists; the subgranular zone  of dentate gyrus in the hippocampus  <cit> .
our in situ results showed that during brain development neil <dig> was highly expressed in cells found in places rich in neural progenitors such as the vz and the svz.
the expression decreased during embryonic development and at p <dig>  only a few cells around the lateral ventricles and in the dentate gyrus of the hippocampus were positive for neil <dig> expression.
we have recently described the distribution of neil <dig> during postnatal development and found neil <dig> expression in the svz, the rostral migratory stream , the dentate gyrus of the hippocampal formation and the purkinje cells of the cerebellum in p <dig> mice.
the expression of neil <dig> decreased dramatically in postnatal mice so that in 1-month-old mice only a few cells in the svz and in layer v of neocortex were detected.
in 1-year-old mice neil <dig> was detected in layer v of neocortex only  <cit> .
in the present study no neil <dig> expression was detected in brains from 5-week-old mice when using quantitative real-time pcr.
the discrepancy between our results and the previously reported data could be due to a lower sensitivity of real-time pcr compared to in situ hybridization.
thus, the limited number of neil3-positive cells detected in 1-month-old mice by in situ hybridization is probably below the detection limit of the real-time pcr.
altogether, previous data and results presented here suggest that neil <dig> is highly expressed in regions where neurogenesis occurs during embryogenesis and to a lesser extent in neonatal animals  <cit> .
hippocampal neil <dig> expression disappeared in 1-month-old mice but could still be observed in the svz  <cit> .
this may suggest the existence of different progenitors in the two germinal areas.
since neil <dig> appears to be enhanced in proliferating cells, the decline in neil <dig> expression during development could be due to a decline in cell proliferation.
neil <dig> knockout mice were generated a few years ago and these mice are viable and remain apparently healthy into adulthood with no overt phenotype  <cit> .
a putative role in lymphocytes and/or other immune cells has been proposed since neil <dig> is highly expressed in lymphatic cells and tissues and the knockout mice have a slightly reduced number of blood cells  <cit> .
the expression pattern described in this paper warrants a closer examination of the distribution/phenotype of neural progenitor cells in the embryo and adult neil <dig> knockout mice.
studies using the neil <dig> knockout mice model that involve insults to the brain such as ischemic stroke would be central in revealing whether neil <dig> has a role in the regeneration of damaged tissue.
in a recent study, neil <dig> was shown to be overexpressed in primary melanomas giving rise to metastasis  <cit> .
neil <dig> was therefore suggested to be associated with the progression of primary melanoma to distant metastasis  <cit> .
interestingly, we found increased neil <dig> expression in  <dig> of  <dig> cancer tissues compared to normal tissues.
intriguingly, neil <dig> has also recently been suggested to be a host factor for hiv and thus, a target for antiviral medicines  <cit> .
taken together all these observations and the fact that no robust dna glycosylase activity has been detected  <cit> , indicate that neil <dig> is not a typical dna glycosylase but rather a protein involved in processes where extensive proliferation is a hallmark such as brain development and possibly tumor development/progression.
CONCLUSIONS
we have characterized the expression pattern of neil  <dig> during embryonic development.
our results clearly showed that the expression of neil <dig> appeared to be tightly regulated both temporally and spatially.
high expression was detected in the oocytes and preimplantation stages of development and later during organogenesis.
in the embryonic brain, neil <dig> was detected in sites rich in neural stem/progenitor cells.
finally, tumor tissues were shown to express higher amounts of neil <dig> compared to their normal counterparts, except for testis and pancreas.
these results call for a closer examination of the brains of neil <dig> knock-out mice not only during development and aging, but also under detrimental conditions such as ischemic stress.

 the base excision repair pathway, one of the major dna repair pathways, is dedicated to the repair of damaged dna bases arising from endogenous and exogenous insults. the dna bases may be subjected to oxidation, alkylation and deamination and oxidative dna damage, in particular, has been implicated in the pathogenesis of many diseases including cancer, atherosclerosis, neurodegenerative diseases such as parkinson's and alzheimer's, and even aging. dna glycosylases are the key enzymes of ber; they initiate repair by catalysing the hydrolysis of the n-glycosylic bond between modified bases and the sugar-phosphate backbone. to date, four mammalian dna glycosylases that recognize and excise oxidized bases have been thoroughly characterized: ogg nth neil and neil. neil was identified together with neil and neil as a gene product with significant structural similarities to the e. coli fpg and nei dna glycosylases. however, in contrast to neil and neil no substantial dna glycosylase activity has been detected. expression of human neil has been reported in thymus and testis, while mouse neil has been demonstrated to be highly expressed in spleen, bone marrow, thymus, blood cells and brain regions that harbour progenitor cells. neil has also been shown to be highly expressed in primary malignant melanomas associated with metastasis. most studies have been performed on adult organs and a recent paper described neil expression in the developing mouse. here, we describe the expression pattern of neil during embryonic development in mice with focus on the expression of neil during brain development. the expression of neil in multiple human cancers was also examined.  neil is expressed during embryogenesis the temporal expression pattern of neil during embryonic development was examined by in silico electronic northern blot and northern blot analysis. we used the unigene collection to generate in silico expression profiles for neil at different stages during embryonic development. as seen in figure 1a, there is a very high expression in the oocyte, unfertilised oocyte and zygote and this expression dramatically falls after the zygote stadium. during embryogenesis, neil transcription is not detected until e –. northern blot analysis was performed by using a northern blot containing poly+ rna isolated from whole mouse embryos at days and of gestation. expression of neil was not observed until e. neil is highly expressed in developing brain next, we examined the expression of neil in developing mouse brain. messenger rna was isolated from brains at days, and of gestation and at p and subjected to quantitative real-time pcr. figure 2a shows the expression levels of neil relative to gapdh. the highest expression level of neil was observed at e. to dissect the sites of neil expression, we evaluated the distribution of neil by transcript profiling in various brain regions at five different stages of development: e e and e, p and weeks. as seen in figure 2b, neil expression was highest at e in the fore- and midbrain and the expression decreased during development. in neonatal mice the expression was relatively low in all areas examined, except for in the cerebellum and the spinal cord. in adult mice, neil expression was below the detection limit. neil is highly expressed in regions of active neurogenesis in the developing brain in order to characterize in more detail the expression of neil mrna in developing brain, in situ hybridization was performed on foetal mouse forebrains at e e and p. at e abundant neil mrna was detected in the subventricular zone of the lateral ventricles. the expression of neil decreased during development and at e and p expression was limited to distinct cells in the cortical svz, in cells of the secondary matrix, the dentate gyrus migratory route and the dentate gyrus. thus, neil was expressed in regions that are rich in progenitor cells. no signals were detected for the sense dig-rna probe used as a control. neil is expressed in various tumor samples neil has recently been shown to be highly expressed in melanomas. we therefore investigated the expression of neil in several tumor types and their normal counterparts by using qpcr arrays containing cdna from diseased and normal tissues. data shown in figure is from one of the two experiments performed and represents relative neil mrna levels in normal and tumor tissues. raw data and information about the samples examined are shown in additional file results revealed that although we observed high variation within the different cancer samples from the same tissue, tumor samples in general displayed a higher expression of neil than the corresponding normal tissues, except for testis and pancreas. furthermore, neil was highly abundant in normal testis tissue, as has been described before. discussion in this study the expression of neil during embryonic brain development was investigated. our results showed that neil was not uniformly expressed in the brain, but limited to specific cells in certain regions. the expression appeared to be tightly regulated both spatially and temporally during brain development. results from northern blot analysis of total embryos revealed the presence of neil after e while rt-pcr experiments showed the presence of neil in the developing brain from e thus, the expression of neil appears to coincide with organogenesis. this is also supported by the in silico analysis, where neil transcripts were not detected until e –e. intriguingly, the in silico analysis revealed high expression of neil during preimplantation. mammalian embryos undergo major changes in their gene expression patterns throughout most stages of preimplantation development and massive maternal degradation of mrna characterizes the transition from oocyte to early embryo before the onset of new transcription. genes that follow this expression pattern are suggested to have specific functions either in oogenesis, oocyte maturation, fertilization, and/or early phases of preimplantation development. our results suggest that neil can be classified with this group of genes since neil mrna is highly expressed in unfertilised eggs and is abundant up to the zygote stadium before it is apparently degraded. in mice, embryonic neurogenesis occurs from e to e. there are two major proliferative populations in the developing brain. the first to appear is a region called the ventricular zone in the lateral ventricle. the vz is lined by a population of cells that contains multipotent neural stem cells that give rise to most neurons and glial cells of the cortex. these cortical precursor cells constitute a heterogeneous population and there is increasing evidence that different precursor cells generate distinct differentiated cell types. another proliferative population is found in the subventricular zone, located adjacent to the vz along the lateral ventricle. notably, neurogenesis continues in this region throughout adulthood, and in the adult mammalian brain, new neurons born in the svz migrate anteriorly into the olfactory bulb, where they mature. in post-natal mice, a second germinal zone exists; the subgranular zone of dentate gyrus in the hippocampus. our in situ results showed that during brain development neil was highly expressed in cells found in places rich in neural progenitors such as the vz and the svz. the expression decreased during embryonic development and at p only a few cells around the lateral ventricles and in the dentate gyrus of the hippocampus were positive for neil expression. we have recently described the distribution of neil during postnatal development and found neil expression in the svz, the rostral migratory stream, the dentate gyrus of the hippocampal formation and the purkinje cells of the cerebellum in p mice. the expression of neil decreased dramatically in postnatal mice so that in 1-month-old mice only a few cells in the svz and in layer v of neocortex were detected. in 1-year-old mice neil was detected in layer v of neocortex only. in the present study no neil expression was detected in brains from 5-week-old mice when using quantitative real-time pcr. the discrepancy between our results and the previously reported data could be due to a lower sensitivity of real-time pcr compared to in situ hybridization. thus, the limited number of neil3-positive cells detected in 1-month-old mice by in situ hybridization is probably below the detection limit of the real-time pcr. altogether, previous data and results presented here suggest that neil is highly expressed in regions where neurogenesis occurs during embryogenesis and to a lesser extent in neonatal animals. hippocampal neil expression disappeared in 1-month-old mice but could still be observed in the svz. this may suggest the existence of different progenitors in the two germinal areas. since neil appears to be enhanced in proliferating cells, the decline in neil expression during development could be due to a decline in cell proliferation. neil knockout mice were generated a few years ago and these mice are viable and remain apparently healthy into adulthood with no overt phenotype. a putative role in lymphocytes and/or other immune cells has been proposed since neil is highly expressed in lymphatic cells and tissues and the knockout mice have a slightly reduced number of blood cells. the expression pattern described in this paper warrants a closer examination of the distribution/phenotype of neural progenitor cells in the embryo and adult neil knockout mice. studies using the neil knockout mice model that involve insults to the brain such as ischemic stroke would be central in revealing whether neil has a role in the regeneration of damaged tissue. in a recent study, neil was shown to be overexpressed in primary melanomas giving rise to metastasis. neil was therefore suggested to be associated with the progression of primary melanoma to distant metastasis. interestingly, we found increased neil expression in of cancer tissues compared to normal tissues. intriguingly, neil has also recently been suggested to be a host factor for hiv and thus, a target for antiviral medicines. taken together all these observations and the fact that no robust dna glycosylase activity has been detected, indicate that neil is not a typical dna glycosylase but rather a protein involved in processes where extensive proliferation is a hallmark such as brain development and possibly tumor development/progression.  we have characterized the expression pattern of neil during embryonic development. our results clearly showed that the expression of neil appeared to be tightly regulated both temporally and spatially. high expression was detected in the oocytes and preimplantation stages of development and later during organogenesis. in the embryonic brain, neil was detected in sites rich in neural stem/progenitor cells. finally, tumor tissues were shown to express higher amounts of neil compared to their normal counterparts, except for testis and pancreas. these results call for a closer examination of the brains of neil knock-out mice not only during development and aging, but also under detrimental conditions such as ischemic stress. 
the base excision repair pathway is responsible for repairing small dna base lesions caused by endogenous and exogenous damaging agents. during embryogenesis and in newborn mice specific expression was observed in areas known to harbour neural stem and progenitor cells such as the subventricular zone and the dentate gyrus. quantitative real-time pcr and in situ hybridization revealed that neil was highly expressed at embryonic days 12– when neurogenesis starts. the expression decreased during development and in the adult brain,neil could not be detected in any of the brain areas examined by quantitative real-time pcr. we have also looked at the expression of neil in several normal and tumor tissues. in addition, neil seems to be upregulated in tumor tissues compared to normal tissues. in this study we describe the expression pattern of neil during mouse embryonic development with special focus on brain development. finally, neil expression was higher in tumors compared to normal tissues, except for testis and pancreas. repair is initiated by dna glycosylases that recognize and remove the lesions. neil is one of mammalian dna glycosylases identified to date and it was discovered on the basis of sequence homology to the e. coli fpg and nei glycosylases.
 the base excision repair pathway is responsible for repairing small dna base lesions caused by endogenous and exogenous damaging agents. repair is initiated by dna glycosylases that recognize and remove the lesions. neil is one of mammalian dna glycosylases identified to date and it was discovered on the basis of sequence homology to the e. coli fpg and nei glycosylases. difficulties in purifying the protein have limited its biochemical characterization and in contrast to the other glycosylases, its function remains unclear.  in this study we describe the expression pattern of neil during mouse embryonic development with special focus on brain development. we have also looked at the expression of neil in several normal and tumor tissues. quantitative real-time pcr and in situ hybridization revealed that neil was highly expressed at embryonic days 12– when neurogenesis starts. the expression decreased during development and in the adult brain,neil could not be detected in any of the brain areas examined by quantitative real-time pcr. during embryogenesis and in newborn mice specific expression was observed in areas known to harbour neural stem and progenitor cells such as the subventricular zone and the dentate gyrus. finally, neil expression was higher in tumors compared to normal tissues, except for testis and pancreas.  our findings indicate that mammalian neil is specifically expressed in brain areas where neurogenesis takes place during development and that its expression is tightly regulated both temporally and spatially. in addition, neil seems to be upregulated in tumor tissues compared to normal tissues. altogether, mammalian neil seems to be highly expressed in cells with high proliferative potential. 