dna methylation is an epigenetic modification of vertebrate genomes linked with a repressive chromatin state . 

tet ( ten-11 translocation ) proteins ( tet1/2/3 ) can enzymatically oxidise 5-methylcytosine ( 5-mc ) generating another stable cytosine modification , 5-hydroxymethylcytosine ( 5-hmc ) , which differs from 5-mc in its genomic and tissue distribution as well as ( likely ) biological function .
recent reports demonstrated that 5-hmc can by oxidised further forming 5-formylcytosine ( 5-fc ) and 5-carboxylcytosine ( 5-cac ) .
since both 5-fc and 5-cac can be recognized and removed from the dna by the components of base excision repair machinery , a model according to which these oxidised forms of 5-methylcytosine may serve as intermediates in active demethylation process has been suggested but it is still unclear if this model is being employed during vertebrate development and differentiation .
thus , it has been reported that 5-methylcytosine is converted to 5-fc and 5-cac in mouse zygotes and both these modifications exhibit replication - dependent dilution during mouse pre - implantation development , which suggests that they are relatively stable and may play distinct biological roles in early mouse embryos .
although significant levels of both 5-fc and 5-cac have been found in escs , no detectable levels of 5-carboxylcytosine have been reported for mouse somatic tissues .
here we assessed the tissue distribution of 5-carboxylcytosine in adult amphibian ( axolotl , ambystoma mexicanum ) tissues and demonstrate that 5-cac is immunochemically detectable in the somatic cells of axolotl ovary and localized to euchromatin in their nuclei .
although it can be detected in most of the adult axolotl organs , neuronal cells are significantly enriched with 5-hmc compared with the rest of tissues in amphibians .
employing the same highly sensitive immunochemical staining procedure , based on the use of peroxidase - conjugated secondary antibody coupled with a tyramide signal enhancement system , we performed co - immunostaining of 5-cac and 5-hmc on the tissues of young axolotl adults ( fig .  1 ) .
using-5-cac antibody ( active motif ) , which is able to specifically recognize 5-cac but not 5-mc , 5-hmc or 5-fc according to the latest report , we could not detect any 5-cac signal in axolotl neural tube , muscle or connective tissue ( fig . 
1a and b and data not shown ) . on the other hand , we detected specific 5-carboxylcytosine staining evident in mouse escs , and showed that this is eliminated in the presence of 5-cac- but not 5-mc- , 5-hmc - containing or unmodified dna ( fig .
similar to our previous results , the immunostaining using mouse monoclonal anti-5-hmc antibody produced non - negligible signal in all the tested tissues ( fig .  1 ) .
these results are in line with the previously published data on biochemical detection of 5-cac , where this modification was detectable in mouse escs but not in the adult mouse tissues .
in contrast the immunostaining of ovarian tissue revealed relatively strong signal for both 5-hmc and 5-cac ( fig . 
notably , most of the somatic cells in developing follicles and cell nests , but not the surrounding cells of ovarian theca , exhibited strong 5-cac staining ( fig . 
1a and b ) . as we found that 5-cac is detectable in follicular cells of axolotl we decided to examine if the nuclear distribution of this mark matches that of 5-hydroxymethylcytosine in these cells .
unlike 5-methylcytosine , usually enriched in condensed dapi - bright transcriptionally - silent chromatin , 5-hydroxymethylcytosine has been reported to be predominantly associated with euchromatic regions such as gene bodies and enhancer elements . since the anti-5-mc antibody we routinely use is mouse monoclonal ( clone 33d3 ) and , therefore , it is technically impossible to co - localize all three epigenetic marks ( 5-mc , 5-hmc and 5-cac ) on the same sample , we co - stained ovarian tissue with 5-hmc and 5-cac antibodies in mild denaturing conditions ( 2 m hcl ) , allowing us to visualize dense heterochromatic dna regions with dapi .
analysis of these immunostaining experiments , imaged using confocal microscopy , revealed that both 5-hmc and 5-cac are predominantly localized to gene - rich euchromatic regions but not to the dapi - bright regions of condensed chromatin ( fig . 
quantification of the immunostaining showed that whereas the 2.5xd plots for 5-hmc and 5-cac signals exhibit similar profiles ( fig .  2c and d ) , they both are strikingly different from that of dapi staining ( fig
indeed , the signal profiling across the heterochromatic and euchromatic regions of the nucleus demonstrated that 5-carboxylcytosine signal intensity is highest in the regions of low or moderate dapi signal , while the intensities of both 5-hmc and 5-cac signals are very weak in heterochromatic areas with strong dapi staining ( fig . 
, although both 5-hmc and 5-cac are localized to the euchromatin , their signal profiles are not absolutely identical ( fig . 
thus 5-hmc staining seems to be more punctate than that of 5-cac and certain 5-hydroxymethylcytosine enriched regions do not exhibit enrichment similar to the 5-cac content ( fig . 
( a ) 5-hmc and 5-cac immunostaining of young adult axolotl ovary and neural tube tissue ( indicated ) . developing follicle
( b ) immunohistochemical detection of 5-hmc and 5-cac in axolotl ovary and surrounding tissues .
examples of non - specific ( non - nuclear ) staining produced by anti-5-cac antibody in ovarian theca are marked with white arrow .
( a ) confocal image of a representative field of axolotl ovary stained with 5-hmc , 5-cac and dapi .
an inset shows the cells used for the analysis presented in ( b - e ) under higher magnification .
( b - e ) 2.5xd plot of 5-hmc , 5-cac and dapi signal intensities of the inset in ( a ) .
merge view ( b ) and single channel images ( c , d and e ) are shown .
( a ) immunochemical detection of 5-hmc and 5-cac in a single nucleus of axolotl follicular cell co - stained with dapi using confocal microscopy .
the position of the region used for the generation of the signal intensities profile plot shown in ( b ) is marked with a dashed arrow .
( b ) the profile of dapi , 5-hmc and 5-cac signal intensities ( indicated ) across heterochromatic and euchromatic regions of the follicular cell nucleus presented in ( a ) .
our data show that 5-carboxylcytosine is immunochemically detectable in the somatic cells of amphibian ovary , where both 5-hmc and 5-cac are localized mainly to gene - rich euchromatic regions with similar patterns of nuclear distribution .
as the oxidation of 5-hydroxymethylcytosine to 5-carboxylcytosine is taking place in mammalian zygotes where tet3 protein is likely responsible for this conversion , it would be interesting to check if a tet3 homolog is required for the generation of 5-cac in amphibian follicular cells . since , according to our data , most of the somatic cells in the ovary possess similar levels of 5-carboxylcytosine , were this modification to indicate the presence of active demethylation , our results would imply an extremely dynamic demethylation process , with constant cycles of 5-methylcytosine removal from dna followed by re - methylation .
such genome wide demethylation taking place at the actively transcribed euchromatic regions of follicular cells dna would most likely contribute to switching on and off the transcription of a number of genes active at different phases of amphibian oocyte development .
since according to the recent reports , 5-cac reduces the rate and substrate specificity of rna polymerase ii transcription , and 5-formylcytosine distribution is associated with transcription in mouse escs , an alternative explanation of our results would suggest that 5-carboxylcytosine is not always an intermediate in demethylation but may instead play a distinct functional role in transcription regulation in some biological contexts .
mouse hm1 escs were maintained on gelatine - coated dishes in glasgow 's minimum essential medium ( gibco ) , supplemented with 15% fetal bovine serum , 55 m -mercaptoethanol ( gibco ) , 2 mm l - glutamine , 0.1 mm mem non - essential amino acids , 5 000 units ml penicillin / streptomycin and 1 000 units ml of murine lif ( chemicon ) under feeder - free conditions .
tissues were fixed in 4% formaldehyde for 12 h. tissue sections were de - waxed according to standard procedures and permeabilised for 15 min with pbs containing 0.5% triton x-100 . for 5-hmc and 5-mc staining ,
permeabilised tissue sections were incubated in 2 n hcl for 1 h at 37c and then neutralised in 100 mm tris - hcl ( ph 8.5 ) for 10 min , followed by a standard immunostaining protocol .
anti-5-hmc mouse monoclonal ( active motif , 1:5000 dilution ) and anti-5-cac rabbit polyclonal ( active motif , 1:3000 dilution ) primary antibodies were used .
peroxidase - conjugated anti - rabbit secondary antibody ( dako ) and the tyramide signal enhancement system ( perkin elmer ) were employed for 5-cac detection .
immunostaining competition experiment was performed as described using 5-cac dna standard ( active motif ) and pcr - produced 100-bp dna fragments , where all the cytosines were replaced with either 5-hmc or 5-mc .
images ( 400 nm optical sections ) were acquired with a zeiss lsm 700 axioobserver confocal microscope using a plan - apochromat 63/1.40 oil dic m27 objective .
mouse hm1 escs were maintained on gelatine - coated dishes in glasgow 's minimum essential medium ( gibco ) , supplemented with 15% fetal bovine serum , 55 m -mercaptoethanol ( gibco ) , 2 mm l - glutamine , 0.1 mm mem non - essential amino acids , 5 000 units ml penicillin / streptomycin and 1 000 units ml of murine lif ( chemicon ) under feeder - free conditions .
tissues were fixed in 4% formaldehyde for 12 h. tissue sections were de - waxed according to standard procedures and permeabilised for 15 min with pbs containing 0.5% triton x-100 . for 5-hmc and 5-mc staining ,
permeabilised tissue sections were incubated in 2 n hcl for 1 h at 37c and then neutralised in 100 mm tris - hcl ( ph 8.5 ) for 10 min , followed by a standard immunostaining protocol .
anti-5-hmc mouse monoclonal ( active motif , 1:5000 dilution ) and anti-5-cac rabbit polyclonal ( active motif , 1:3000 dilution ) primary antibodies were used .
peroxidase - conjugated anti - rabbit secondary antibody ( dako ) and the tyramide signal enhancement system ( perkin elmer ) were employed for 5-cac detection .
immunostaining competition experiment was performed as described using 5-cac dna standard ( active motif ) and pcr - produced 100-bp dna fragments , where all the cytosines were replaced with either 5-hmc or 5-mc .
images ( 400 nm optical sections ) were acquired with a zeiss lsm 700 axioobserver confocal microscope using a plan - apochromat 63/1.40 oil dic m27 objective .
 5-methylcytosine ( 5-mc ) is an epigenetic modification associated with gene repression . 
 recent studies demonstrated that 5-mc can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine ( 5-fc ) and 5-carboxylcytsine ( 5-cac ) . 
 5-cac has been found in embryonic stem cells and in mouse pre - implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date . whereas it has been suggested that 5-cac can serve as an intermediate in the process of active demethylation , the function of this form of modified cytosine remains obscure . here 
 we show that 5-cac is immunochemically detectable in somatic cells of axolotl ovary . 
 we demonstrate that both 5-hmc and 5-cac are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution . 
 our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts . 
Results and Discussion
Materials and Methods
ES cell culture
Immunohistochemistry and imaging
Immunostaining competition experiment
Confocal microscopy
Supplementary Material