the insulin - like growth factor 2 gene ( igf2 ) is an imprinted gene , paternally expressed and encodes for the insulin - like growth factor ii peptide [ 1 , 2 ] . in mice the igf2 gene has five promoters , from which three main transcripts , a placenta specific transcript and a newly described mesoderm - specific transcript , originate .
described multiple imprinted sense and antisense transcripts from the igf2 locus . the igf2as gene located within igf2
we recently found that igf2as transcripts are located in the cytoplasm and associated with polysomes indicating a protein coding function . to further elucidate the function of igf2as
this mouse has a 5  kb deletion within the igf2 gene comprising dmr1 , an adjacent repeat sequence mostly embedded in exon u2 of the placenta specific igf2 p0 transcript and igf2as transcripts .
the maternal transmission of the dmr1-u2 deletion results in loss of igf2 imprinting in heart , kidney , and lung without affecting h19 gene expression .
the authors conclude that their result demonstrates that dmr1 plays a role in igf2 imprinting regulation and gene expression independent of h19 . in that study ,
paternal transmission of the dmr1-u2 deletion was associated with intrauterine growth restriction ( iugr ) manifested by mutants birth weight being 71% that of normal mice .
later , constncia et al .   reported that the 5  kb dmr1-u2 deletion abolishes expression of the igf2 p0 transcript in the labyrinthine trophoblast of the placenta where it is specifically expressed .
noteworthy , placental growth deficiency in dmr1-u2 placentas resembled that of igf2-null mice lacking igf - ii peptide in all placental layers and the fetus which was unexpected since p0 transcripts constitute about 10% of total igf2 transcripts in placenta .
however , the lower birth weight of mutant dmr1-u2 pups was compensated during the first three months of life .
they could also demonstrate with inert hydrophilic molecules that passive permeability of the mutant placenta was reduced compared to the wild type .
however , they showed that the mutant placenta actively transferred more than wild - type placenta indicating compensatory effects to the passive permeability and the smaller placenta size .
the permeability of the mouse placenta to hydrophilic solutes was further studied in dmr1-u2 mice versus wild - type mice . in their study
they found that the permeability for hydrophilic solutes was significantly reduced in the dmr1-u2 knockout placenta at e19 .
stereological analysis showed a reduction in surface area and an increase in thickness of the exchange barrier in the dmr1-u2 knockout labyrinthine layer of the placenta .
this suggests that labyrinthine p0 igf2 expression has a function in the development of normal diffusional exchange characteristics of the mouse placenta influencing fetal growth .
further investigated the placental nutrient supply and the fetal demand in the dmr1-u2 deletion mouse model ( igf2 p0 ) and an igf2-null mouse model .
could further show by using igf2 p0 and igf2-null mice models that placental nutrient transfer occurs in response to fetal nutrient demands and involves igf2 mediation .
additional evidence for the placental adaption was provided by showing lower expression of the transplacental calcium transfer protein calbindin - d9k in igf2 p0 at e17 but not at e19 compared to that of wild types .
more recently , it was shown that adult igf2 p0 mice but not igf2-null mice exhibited behavioral phenotypes .
these mice were exposed to a wide range of emotion - related behaviors tests and igf2 p0 mice showed increased reactivity to acute anxiety - including stimuli . it was further found that anxiety associated genes in the hippocampus of these mice were altered .
these long - term behavioral effects were attributed to the imbalance between fetal demand and placental supply of nutrients during gestation . in this study
, we performed an expression analysis of igf2 and igf2as transcripts in dmr1-u2 and wild - type placentas from different development stages .
the expression of these genes is studied in wild type placentas and in dmr1-u2 placentas which are lacking igf2 p0 and igf2as expression .
we also followed the expression patterns of this sense / antisense pair in different tissues during development and measured igf2/igf2as expression in differentiating c2c12 cells for functional characterization of the putative protein coding igf2as gene .
the cells were grown to 100% confluence and changed into a differentiation medium ( dulbecco 's modified eagle 's medium supplemented with 2% horse serum and 1% penicillin - streptomycin solution ) .
the differentiation media were replaced daily during a period of 6 days . at each time point , the cells of three culture flasks were harvested separately , rna isolated , and used for quantification by real - time pcr .
the experiments were carried out in strict accordance with swiss federal law on animal protection of 16 december 2005 ( tierschutzgesetz tschg , sr 455 ) , art .
32 , absatz 1 ; ordinance on animal protection of 23 april 2008 ( tierschutzverordnung tschv , sr 455.1 ) .
brain , muscle and liver tissues were collected from wild - type c57bl/6 fetuses at e18 ( embryonic day 1 ( e1 ) = day of plug ) , from newborn and adult mice .
dmr1-u2 transgenic male mice carrying the mutation on the paternal allele were donated from m.r .
mice resulting from these crossings were sacrificed at e12 , e14 , e16 , and e19 and placentas were collected .
the primers used for this pcr test are shown in table 1 . from the crosses between male carriers of the dmr1-u2 deletion and c57bl/6 females , three mutant placentas and three wild - type littermates
rna extraction was performed using trizol reagent according to manufacturer 's protocol ( invitrogen ) .
the cdna was synthesized by reverse transcription of 1  g total rna using the quantitect reverse transcription kit according to the manufacturer 's protocol ( qiagen ) .
each sample was quantified in triplicates for all igf2 variants ( v1 , v2 , v3 , and pm ) and the igf2as transcript by qpcr using taqman probes or conjugated minor groove binder ( mgb ) probes to measure igf2as transcription ( table 2 ) ( figure 1 ) .
the ct values of the target were normalized by subtracting the mean of the ct values from actb and gapdh .
the relative quantifications were calculated relative to the first value which was set to 1 in each respective figure .
the calculations were performed for each of three samples measured in triplicate , averaged , and had the standard deviations calculated .
the normalized data was analyzed by using two - tailed student 's t - test .
we found relatively high igf2 and igf2as gene expression in fetal and newborn liver and muscle tissue samples and a significant downregulation of these genes in adult tissues ( figures 2(a ) and 2(b ) ) .
all three igf2 variants followed similar pattern of expression at fetal , newborn , and adult stages of development . in muscle
, we found comparable expression levels of igf2as transcripts in fetus and newborn whereas the expression of igf2 variants increased in newborn ( figure 2(a ) ) . igf2as and
igf2 variants were significantly downregulated in adult tissues ( figures 2(a ) and 2(b ) ) .
igf2as levels were not detectable after 40  qpcr cycles in adult muscle tissue . in liver ,
the level of igf2as and igf2 expressions is similar in fetus and newborn , except for variant 3 which significantly increased in newborn ( figure 2(b ) ) .
transcripts originating from the pm promoter were expressed at very low levels ( data not shown ) . in brain
we found significantly higher expression of igf2 variant 2 and igf2as in embryos compared to fetus with a continuous down - regulation towards the adult stage ( figure 2(c ) ) .
igf2 variants and igf2as followed this pattern , except variant 3 ( figure 2(c ) ) .
we used c2c12 myoblast cells differentiation to monitor the sense / antisense igf2/igf2as gene pair for potential interactions between the two partially complementary transcripts .
cells were differentiated by adding horse serum and the gene pair quantified at 0  h up to 144  h corresponding to 6 days ( figure 3 ) .
igf2 variants 1 and 2 as well as pm transcripts were expressed at very low levels ( data not shown ) . however , at 144  h igf2 variant 1 peaked for unknown reason ( data not shown ) .
interestingly , igf2as gene expression did not follow the increasing expressions trend of igf2 variant 3 during differentiation of c2c12 cells .
igf2as and igf2 expression increased significantly between 24  h and 48  h coinciding with the start of myotube formation .
we measured igf2 and igf2as expressions in placentas of dmr1-u2 mice and their wild - type litter mates ( figure 4 ) .
we found no differences in the expression of igf2 variants 13 between mutant and wild - type placentas except for igf2 variant 3 at e16 , which was more highly expressed in the knockout placentas ( figure 4(c ) ) .
igf2as expression levels in wild - type placenta significantly increased from e16 to e19 ( figure 5 ) .
the expression levels of igf2 p0 in wild - type placenta were constant during e12 to e16 and augmented significantly from e16 to e19 ( figure 5 ) . in dmr1-u2
the aim of this study was to further contribute to ascertaining the function of igf2as .
we studied igf2 and igf2as gene expression in different tissues during development , in differentiating c2c12 cells , and in placenta of a dmr1-u2 deletion mouse model .
all igf2 variants and igf2as followed a similar expression pattern in different tissues during development indicating a common regulation . in brain , we found a more continuous downregulation of igf2 and igf2as transcription compared to distinct downregulation of these genes in adult liver and muscle .
most relevant is , however , that we did not find any consistent evidence for an interaction between igf2 and igf2as transcripts although they are both present in the cytoplasm and contain complementary nucleotide sequences .
additionally , we observed that the lack of igf2as transcripts in placenta did not produce significant changes in igf2 variants expression levels compared to wild type with the exception for igf2 variant 3 at e16 .
igf2 variant 3 does not contain complementary sequences which could directly interact with igf2as transcripts to form double - stranded rna molecules .
in addition variant 3 expression at e12 , e14 , and e19 was not different compared to wild - type mice and thus the interpretation of this event remains elusive .
the comparison of igf2 and igf2as expression between dmr1-u2 and wild - type mice argues against the hypothesis that igf2as transcripts have a role in igf2 gene regulation .
the increase of igf2 variant 3 at e16 in mutant mice may have compensatory effect on igf2 protein content in labyrinthine trophoblast challenging the notion that the dmr1-u2 phenotype is solely caused by the lack of p0 transcripts [ 7 , 10 ] . during c2c12 differentiation
, we observed different expression patterns for igf2 variant 3 and igf2as expressions arguing against an interaction between the two genes .
noteworthy is the increased expression of igf2 variant 3 and igf2as transcripts between 24  h and 48  h of differentiation when myotubes start to form .
the knockout mouse carrying the dmr1-u2 deletion was extensively studied in regard to placental nutrient supply and fetal demand [ 6 , 7 , 911 ] . here , we argue that the previously observed effects of the placenta specific igf2 p0 transcript may be confounded with the effects of the putative protein - coding igf2as gene .
our alternative or additional explanation for the observed dmr1-u2 deletion effects is therefore the lack of a functional igf2as gene .
igf2as is an imprinted and paternally expressed gene and therefore the iugr phenotype described in dmr1-u2 mice agrees with common functions of paternally expressed genes .
assuming that the lack of a functional igf2as gene contributes to the phenotype of iugr , expressed as smaller placentas and lower birth weight , it might have similar functions as the igf2 gene .
interestingly , the recently described long - term behavioral effects found in dmr1-u2 mice could also be , at least in part , due to the absence of igf2as function .
we think that the presented results indicate that previous studies have to be interpreted in the light of potential igf2as gene effects . in summary , igf2as is most expressed during prenatal development followed by a pronounced downregulation after birth in most tissues similar to the igf2 gene . in brain
, we found also highest igf2as expression in early development with a gradual downregulation from embryo brains to adult brains .
 insulin - like growth factor antisense gene ( igf2as ) expression was investigated in different mouse tissues during development , in differentiating c2c12 cells and in a dmr1-u2 knockout mouse model . 
 the expression levels of igf2as were high in fetal and newborn liver and muscle tissues compared to adults . 
 the igf2as gene was also expressed in placenta and in brain . 
 the expression data suggests that the igf2as gene plays a role in early development of the mouse and in placenta . 
 there was no consistent evidence for an interaction between igf2 and igf2as transcripts . 
 furthermore , in knockout placentas lacking igf2as transcription , igf2 expression was comparable to that in wild type . 
 these results indicate that igf2as does not regulate igf2 sense transcripts . in previous studies 
 , it was suggested that the dmr1-u2 knockout mouse showing intrauterine growth restriction was caused by the absence of placenta - specific igf2 p0 transcription . 
 we conclude that the dmr1-u2 deletion phenotype should be reconsidered in the light of a functional igf2as gene . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion