BACKGROUND
eutherians and marsupials  diverged between 125– <dig> million years ago  <cit> .
genomic imprinting, in which the monoallelic expression of certain genes depends on the parent of origin, occurs in both therian mammal lineages.
this is in contrast to all other vertebrate species including monotreme mammals in which imprinting of endogenous genes has not been demonstrated  <cit> .
monoallelic expression negates the advantage of diploidy, namely the masking of deleterious alleles.
the cost of imprinting must, therefore, be outweighed by its potential benefits to the genetic fitness of the individual.
the parental conflict hypothesis proposes that imprinting is the product of asymmetric selection on parental genomes, with selection favouring the expression of paternal genes that increase the amount of maternal nutrient transfer while expression from maternally-inherited genes will be favoured if they reduce nutritional demands on the mother  <cit> .
the placentas of marsupials and eutherians mediate the transfer of nutrients between mother and young and the placentas of both groups of mammal express imprinted genes  <cit> .
however, the marsupial placenta is comparatively short-lived and the majority of support for growth and development of the marsupial young occurs during an extended and complex period of lactation  <cit> .
thus marsupials are ideal models in which to examine the evolution of imprinting and its association with mammalian placentation.
cdkn1c  and igf <dig>  are syntenic, and implicated in growth disorders such as beckwith-wiedemann syndrome  <cit> .
igf <dig> is paternally expressed in mouse and human  <cit>  and stimulates cell cycle progression.
igf <dig> is highly conserved in all vertebrates and is expressed during marsupial placentation, as it in the eutherian placenta  <cit> .
igf <dig> is also imprinted in the tammar placenta, indicating imprinting at this locus evolved before the eutherian-marsupial split  <cit> .
in contrast, progression through the cell cycle is negatively controlled by cyclin-dependent kinase  inhibitors, such as p57kip <dig>  encoded by the gene cdkn1c  <cit> .
cdkn1c is expressed from the maternal allele in many tissues of mouse and human, including the placenta  <cit> , but is bi-alellically expressed  in the tammar wallaby  <cit> .
placental development is disrupted in mice carrying a maternally-derived mutation in cdkn1c  <cit>  and mutations in cdkn1c are associated with trophoblastic disease in humans  <cit> .
unlike igf <dig>  cdkn1c is rapidly evolving in mammals and has low homology between mouse, human, and tammar, with only the cdk-inhibiting domain conserved  <cit> .
divergence in the structure of p57kip <dig> suggests functional specialisation in different species and may explain species-specific patterns of expression, subtle differences in the pathologies of human and mouse cdkn1c mutants  <cit> , and the absence of imprinting of this gene in the tammar  <cit> .
most imprinted genes aggregate within the genome and in each imprinted domain gene order and imprint status show considerable similarity between mouse and human  <cit> .
igf <dig> and cdkn1c are syntenic, and reside in an imprinted cluster on mouse distal chromosome  <dig>  included in this region are the maternally expressed genes ipl/tssc <dig>  slc22a1l/tssc <dig>  mash2/ascl <dig>  and cdkn1c and the paternally expressed genes igf <dig> and ins <dig>  gene order and the imprint status of most genes within this region are conserved with human chromosome 11p <dig>   <cit> .
the entire region is regulated by two differentially methylated imprinting control regions  one of which is the kcnq1ot <dig> antisense-rna transcript, that regulates the expression of cdkn1c, kcnq <dig>  and ascl <dig>  <cit> .
conserved synteny may reflect the co-ordinated transcriptional regulation of several imprinted genes within each domain  <cit> .
the igf2-cdkn1c region is highly conserved even between human and chicken, despite a lack of imprinting in the chicken  <cit> .
nevertheless, synteny may facilitate the co-evolution of maternal and paternal imprints in adjacent regions of the genome  <cit> , possibly by the spread of imprinting mechanisms from one locus to another, as described by the bystander hypothesis  <cit> .
ins and igf <dig> are syntenous in mouse, human and tammar  <cit> .
extension of this synteny from igf <dig> to cdkn1c has recently been described in marsupials  <cit> .
this is of particular importance given that igf <dig> and ins are imprinted in the tammar, as in mouse and human, but cdkn1c is not  <cit> .
differentially methylated regions , often found at cpg islands in or near imprinted genes, clearly contribute to the regulation of imprinted expression in eutherians  <cit> .
however, not all imprinted genes depend on this mechanism.
antisense transcripts and various forms of chromatin modification also regulate imprinted expression in eutherians  <cit> .
kcnq1ot <dig>  is a paternally expressed antisense transcript originating from intron  <dig> of the maternally expressed kcnq <dig>  gene  <cit> .
the human kcnq1ot <dig> promoter starts approximately  <dig> bp upstream of the transcription initiation site and spans  <dig> to  <dig> bp  <cit> , although associated transcription factor binding sites may extend as far as  <dig> bp upstream  <cit> .
the transcription initiation site is located in a cpg island that is methylated in oocytes, but not in sperm.
methylation blocks transcription of the maternal allele of kcnq1ot <dig> and deletion of either the cpg island or kcnq1ot <dig> increases expression of cdkn1c, kcnq <dig>  and ascl <dig>  indicating its role in the coordinated regulation of these loci  <cit> .
dna methylation as a regulator of imprinted expression may have evolved from the molecular systems associated with the silencing of transposable elements.
the host defence hypothesis suggests that dna methylation fortuitously provided a mechanism to regulate parental specific gene expression and similar circumstances may have led to the silencing function of antisense transcripts at imprinted loci  <cit> .
indeed, many imprinted genes are found in association with repeat sequences such as non-ltr elements , dna elements, and endogenous retroviruses  <cit>  and are all known to attract methylation  <cit> .
we determined the expression of cdkn1c and the cellular localisation of its protein, p57kip <dig>  in the placenta of the tammar wallaby because of their evolutionary divergence from eutherian mammals.
we examined whether cdkn1c became imprinted after it acquired a role in placental development, or if its placental expression pre-dated the evolution of its imprinting.
in addition, to gain insight into the evolution of imprinting within this cluster, the structure and repeat distribution of the cdkn1c domain, including kcnq <dig> and the icr, kcnq1ot <dig>  were investigated in the tammar.
RESULTS
p57kip <dig> localises to the tammar placenta
while cdkn1c is imprinted in eutherians and vital for placentation, it is not imprinted in the tammar  <cit> .
the chorio-vitelline placenta of the tammar  consists of two functional regions, a vascular and non-vascular one.
the vascular, trilaminar placenta is thought to be the primary site of gas exchange, while that of the avascular, bilaminar region, the predominant site for nutritional exchange  <cit> .
we used immunohistochemistry to examine p57kip <dig> in both regions of the marsupial placenta.
p57kip <dig> was localised in the cytoplasm and nucleus of all cell types in the bilaminar and trilaminar yolk sac placenta .
however, while the protein was consistently present in many cells of the yolk sac endoderm and trophoblast, staining in the mesenchymal and endothelial cells was sporadic .
no notable difference in staining intensity or in the cross-reactivity of different cell types was evident for the stages examined .
weak non-specific cytoplasmic staining was evident in the yolk sac endoderm and, to a lesser extent, the trophoblast in matched goat igg treated controls , but was clearly distinguishable from the dense staining in both the cytoplasm and nucleus with p57kip <dig> ab- <dig>  there was no cytoplasmic or nuclear staining in no-antibody negative controls .
cdkn1c expression increases in the final days of gestation
cdkn1c was expressed in the tammar placenta, but immunohistochemistry did not indicate if there were changes in the quantity of protein over time.
we used quantitative rt-pcr to examine quantitative changes in the expression of cdkn1c.
between days  <dig> and  <dig>  cdkn1c expression was higher in the trilaminar yolk sac than in the bilaminar yolk sac placenta  .
however, in the two days before birth  there was no longer a significant difference in cdkn1c expression between vascular and avascular regions , but there was a significant increase in cdkn1c expression in both regions of the placenta .
genomic analysis – gene order is conserved in the tammar and eutherians
we examined the region containing igf <dig> and cdkn1c in the tammar and compared this to human and mouse.
a blast-n with tammar cdkn1c sequence identified a single clone of tammar genomic dna .
successive blast-n searches identified five more overlapping clones joining the igf2-contianing clone to the cdkn1c-containing clone  .
each set of overlapping clones showed 100% identity over at least  <dig> bp.
the overlapping clones provided almost  <dig> mb of continuous sequence spanning the domains bordered by igf <dig> and cdkn1c.
several regions were conserved or homologous between human, mouse, and tammar as indicated by a multi-species percent identity plot  .
most of the conserved regions corresponded to genomic locations with a high gene density, such as the gene cluster bordered by ascl <dig> and trpm <dig>  however, the large intergenic region between th and ascl <dig> also had two regions conserved or homologous in all three species .
a pip of the orthologous region of human against the tammar sequence was generated.
regions of high homology to human indicated the location of putative exons in the tammar sequence.
the sequence of each possible exon was entered into a blastn to confirm gene identity and was aligned back to human sequences to confirm the location of exon-intron boundaries .
of the nine genes located between igf <dig> and cdkn1c in human, eight were identified in the tammar with kcnq1ot <dig> the only gene not found by homology .
exons homologous to human tssc <dig>  which lies downstream of cdkn1c, were also identified, but the partial sequence available did not extend to cover the entire gene.
gene order was highly conserved between human, mouse, and tammar , as was gene structure.
a multi-species pip of the kcnq <dig> gene of human against mouse, tammar, and chicken, shows conserved gene structure in all four species, with regions of homology corresponding to exon locations .
however, while the exon-intron structure of kcnq <dig> was generally highly conserved, intron  <dig> was absent in chicken and intron  <dig> was significantly reduced in length in the tammar and in chicken .
in mouse and human the kcnq1ot <dig> transcript starts in intron  <dig> and extends into intron  <dig>  mouse intron  <dig> and  <dig> of kcnq <dig> had significant homology with human introns  <dig> and  <dig>  while no homology was evident for the tammar or chicken .
genomic analysis – tammar sequences lack the kcnq1ot <dig> promoter and cpg island
the cpg island in intron  <dig> of kcnq <dig> is essential for imprinted expression of the kcnq1ot <dig> transcript in mouse and human.
we examined the cpg content of the orthologous region in the tammar.
there were  <dig> cpg islands, grouped into nine clusters, in the sequence spanning igf <dig> to cdkn1c in the tammar, while in human there were  <dig>  and in mouse  <dig> .
six cpg islands in the human sequence were greater than  <dig> bp in length with the longest island  <dig> bp.
in comparison, only one of the islands in the tammar sequence was longer than  <dig> bp .
however, mouse also had only two cpg islands over  <dig> bp .
although both human and mouse had fewer cpg islands in kcnq <dig> compared to the remaining sequence assessed , there were no cpg islands in kcnq <dig> of the tammar .
like human and mouse, chicken had a cpg island in kcnq <dig> .
despite differences in the cpg island content of kcnq <dig> in the human and tammar, the overall percent gc was similar .
in human, mouse and chicken at least one cpg island was located in intron  <dig> of kcnq <dig> .
in human and mouse the position of the cpg island and the kcnq1ot <dig> promoter region were highly conserved .
although a cpg island was also present in the chicken intron  <dig>  it is not clear if this is orthologous, as no significant homology to the kcnq1ot <dig> transcription start site could be found, and the cpg island was located approximately  <dig> and  <dig> kb downstream of the orthologous cpg islands in human and mouse respectively.
expression analysis of kcnq1o1
primers were designed within the tammar kcnq <dig> intron  <dig> to determine if it still encoded a kcnq1ot <dig> antisense rna molecule despite its lack of conservation with human and mouse.
since primers did not span an intron, extracted rna was dnased and an aliquot removed for pcr to ensure there was no genomic dna contamination .
surprisingly, transcription of the putative kcnq1o <dig> gene was detected in the trilaminar, but not the bilaminar placenta and only during the final stages of pregnancy .
the resulting pcr band was sequence verified to ensure amplification of the correct product.
genomic analysis – analysis of repeat distribution in the igf2-cdkn1c region
repeat sequences may contribute to the evolution and or regulation of many imprinted regions and so the distribution of repetitive elements in the tammar igf2-cdknic region was assessed.
two regions of high homology were identified in the intergenic dna between th and ascl <dig>  and represent areas of high line/sine density in all three species .
the percent sequence covered by all repetitive elements in the region from igf2-cdkn1c was not significantly different between species .
when the kcnq <dig> region was assessed separately, the percent covered by all repetitive sequences in introns  <dig>  1b,  <dig>   <dig>  and  <dig>  still did not differ significantly between species.
however, the percentage of sequenced covered by specific classes of repetitive sequence did differ significantly between species .
there were significantly fewer long-terminal repeat  elements  and dna elements  in tammar kcnq <dig> introns  <dig>  1b,  <dig>   <dig>  and  <dig> compared to the same introns in mouse and human, while there were significantly more low complexity regions .
however, the lower percentage of sequence covered by ltr and dna elements in the tammar compared to mouse and human was also evident across the entire region from igf <dig> to cdkn1c .
however, the relative proportion of line/sines and simple repeats was not notably different between species in either kcnq <dig> or the entire igf2-cdkn1c region .
in both human and mouse there was high concentration of repetitive elements in intron  <dig>  largely due to an increase in lines, which are largely absent in tammar intron  <dig> .
this was significantly different in mouse compared to tammar , but not with human .
a decrease in sines is associated with imprinted regions  <cit> .
however, there was no significant difference in the percentage of sequence covered by sines between tammar and human and mouse.
despite similarities in the overall proportions of different types of repetitive elements in mouse and human, no specific repetitive elements in intron  <dig>  intron  <dig>  or at the transcription start site, were clearly identified as homologous .
however, in both human and mouse an l1mb element  was located approximately  <dig> to  <dig> kb upstream of the kcnq1ot <dig> transcription start site .
discussion
we have confirmed the synteny of cdkn1c with igf <dig> in the tammar wallaby and shown it is highly conserved between the human, mouse and tammar.
igf <dig> and p57kip <dig> antagonistically regulate growth of the eutherian placenta.
although cdkn1c is not imprinted in the tammar wallaby, it is expressed in the placenta and so could antagonise the growth promoting effects of igf <dig> on the marsupial placenta.
furthermore, the antisense transcript, kcnq1ot <dig>  known to regulate cdkn1c imprinting in eutherians is also expressed in the marsupial placenta.
however, a cpg island and promoter, orthologous to eutherian kcnq1ot <dig>  were absent in the tammar genome.
these data suggest that imprinting of the cdkn1c gene is not contingent on its synteny with igf <dig>  expression in the placenta or the expression of the kcnq1ot <dig> gene.
the p57kip <dig> protein was present in the trophoblast, yolk sac endoderm, and some mesenchymal cells of the yolk sac placenta.
in eutherians, p57kip <dig> binds cyclin-dependent kinases in the nucleus.
in the tammar, p57kip <dig> was found in the nucleus as well as the cytoplasm of cells in all tissue types, suggesting that this protein is functional in the marsupial placenta, as in eutherians.
however, members of the kip family, including p57kip <dig>  also have roles outside their cdk activity that may account for their cytoplasmic location  <cit> .
cdkn1c mrna expression was higher in the trilaminar placenta compared to the bilaminar, but this difference was only significant when expression was low .
more conspicuous was the significant increase in cdkn1c expression in both regions of the yolk sac placenta in the two days before birth .
the vascular region of the placenta develops rapidly between days 19– <dig> to facilitate transfer of nutrients to the developing fetus, consistent with a low cdkn1c expression.
the increase in cdkn1c expression immediately before birth is consistent with retarded growth of the placenta at this time  <cit> .
the slightly higher expression of cdkn1c in the trilaminar placenta may represent the terminal differentiation of haematopoietic tissue in this region that requires exit from the cell cycle.
cdkn1c expression in the marsupial placenta is therefore consistent with a role for p57kip <dig> in the inhibition of cell cycle progression and regulation of marsupial placental growth.
igf <dig> and cdkn1c are co-expressed in the placentas of human, mouse, and tammar, suggesting these genes were also co-expressed in the placenta of the therian ancestor.
the antagonistic functional relationship between these genes is also likely to have existed in the ancestor of marsupials and eutherians.
however, the cdkn1c imprint must have been acquired later and only in the eutherian lineage  <cit> .
all genes located between igf <dig> and cdkn1c in human were also syntenic in the tammar.
however, homology to kcnq1ot <dig> was lacking.
despite high conservation of kcnqt <dig> exons between mouse, human and tammar, intron  <dig> of the tammar kcnqt <dig> gene showed no homology to the kcnq1ot <dig> eutherian transcript.
the promoter region and associated cpg island, conserved in human and mouse  <cit> , could not be detected in the tammar.
intron  <dig> of kcnq <dig>  which encodes the terminal end of kcnq1ot <dig>  also lacks homology with its eutherian counterpart and is considerably shorter in the tammar  than in human and mouse.
since kcnq1ot is a non-coding rna transcript, its sequence conservation with eutherians is not critical to its function  <cit> .
we therefore examined the expression of kcnqt <dig> intron  <dig> by rt-pcr.
despite the sequence divergence, transcription was detected suggesting that an antisense rna kcnq1ot1-orthologue exists in marsupials as in eutherians.
this is especially interesting, since cdkn1c is not imprinted in marsupials, despite the presence of kcnq1ot <dig>  it will be important to determine whether kcnq <dig> and kcnq1ot <dig> are imprinted in the tammar, as in eutherians.
as none of the promoter elements of eutherian kcnq1ot <dig> were identified, it is possible that marsupial kcnq1ot <dig> is regulated by a completely different means and has not yet been co-opted into regulating imprinting of cdkn1c.
however, it does show that evolution of kcnq1ot <dig> preceded imprinting of cdkn1c.
throughout the rest of the igf2-cdkn1c region, there was little cross-species sequence homology in the intergenic regions.
however, two regions of high homology were identified in mammals between the th and ascl <dig> genes.
both sites of homology corresponded to regions of high line/sine density suggesting conservation of repetitive elements may account for the sequence homology, rather than any regulatory or control regions.
these repeats may be important for establishing a boundary region between two independently regulated domains, leading to their conservation.
there were significantly fewer dna elements and endogenous retroviruses  across the entire region, spanning both the imprinted igf <dig> domain and the non-imprinted cdkn1c domain.
however, in both mouse and human there was a considerable increase in repetitive elements in intron  <dig>  when compared with the tammar.
this was due to the accumulation of lines that appear to coincide with the termination of the kcnq1ot <dig> transcript.
these results suggest that the truncation of kcnq <dig> intron  <dig> in the tammar and the increase in lines in mouse and human may be associated with the evolution of kcnq <dig> imprinting in the region.
CONCLUSIONS
despite its lack of imprinting, marsupial cdkn1c is expressed in the developing placenta where it may antagonise the actions of igf <dig> on cell-cycle progression, as it does in eutherians.
since cdkn1c resides in synteny with igf <dig>  imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta.
the expression of kcnq1ot <dig> in the absence of cdkn1c imprinting suggests that antisense transcription at this locus may have preceded imprinting of this domain.
these findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that cdkn1c imprinting is not due to its synteny with igf <dig> or its placental expression in mammals.

 eutherians and marsupials diverged between 125– million years ago. genomic imprinting, in which the monoallelic expression of certain genes depends on the parent of origin, occurs in both therian mammal lineages. this is in contrast to all other vertebrate species including monotreme mammals in which imprinting of endogenous genes has not been demonstrated. monoallelic expression negates the advantage of diploidy, namely the masking of deleterious alleles. the cost of imprinting must, therefore, be outweighed by its potential benefits to the genetic fitness of the individual. the parental conflict hypothesis proposes that imprinting is the product of asymmetric selection on parental genomes, with selection favouring the expression of paternal genes that increase the amount of maternal nutrient transfer while expression from maternally-inherited genes will be favoured if they reduce nutritional demands on the mother. the placentas of marsupials and eutherians mediate the transfer of nutrients between mother and young and the placentas of both groups of mammal express imprinted genes. however, the marsupial placenta is comparatively short-lived and the majority of support for growth and development of the marsupial young occurs during an extended and complex period of lactation. thus marsupials are ideal models in which to examine the evolution of imprinting and its association with mammalian placentation. cdkn1c and igf are syntenic, and implicated in growth disorders such as beckwith-wiedemann syndrome. igf is paternally expressed in mouse and human and stimulates cell cycle progression. igf is highly conserved in all vertebrates and is expressed during marsupial placentation, as it in the eutherian placenta. igf is also imprinted in the tammar placenta, indicating imprinting at this locus evolved before the eutherian-marsupial split. in contrast, progression through the cell cycle is negatively controlled by cyclin-dependent kinase inhibitors, such as p57kip encoded by the gene cdkn1c. cdkn1c is expressed from the maternal allele in many tissues of mouse and human, including the placenta, but is bi-alellically expressed in the tammar wallaby. placental development is disrupted in mice carrying a maternally-derived mutation in cdkn1c and mutations in cdkn1c are associated with trophoblastic disease in humans. unlike igf cdkn1c is rapidly evolving in mammals and has low homology between mouse, human, and tammar, with only the cdk-inhibiting domain conserved. divergence in the structure of p57kip suggests functional specialisation in different species and may explain species-specific patterns of expression, subtle differences in the pathologies of human and mouse cdkn1c mutants, and the absence of imprinting of this gene in the tammar. most imprinted genes aggregate within the genome and in each imprinted domain gene order and imprint status show considerable similarity between mouse and human. igf and cdkn1c are syntenic, and reside in an imprinted cluster on mouse distal chromosome included in this region are the maternally expressed genes ipl/tssc slc22a1l/tssc mash2/ascl and cdkn1c and the paternally expressed genes igf and ins gene order and the imprint status of most genes within this region are conserved with human chromosome 11p. the entire region is regulated by two differentially methylated imprinting control regions one of which is the kcnq1ot antisense-rna transcript, that regulates the expression of cdkn1c, kcnq and ascl. conserved synteny may reflect the co-ordinated transcriptional regulation of several imprinted genes within each domain. the igf2-cdkn1c region is highly conserved even between human and chicken, despite a lack of imprinting in the chicken. nevertheless, synteny may facilitate the co-evolution of maternal and paternal imprints in adjacent regions of the genome, possibly by the spread of imprinting mechanisms from one locus to another, as described by the bystander hypothesis. ins and igf are syntenous in mouse, human and tammar. extension of this synteny from igf to cdkn1c has recently been described in marsupials. this is of particular importance given that igf and ins are imprinted in the tammar, as in mouse and human, but cdkn1c is not. differentially methylated regions, often found at cpg islands in or near imprinted genes, clearly contribute to the regulation of imprinted expression in eutherians. however, not all imprinted genes depend on this mechanism. antisense transcripts and various forms of chromatin modification also regulate imprinted expression in eutherians. kcnq1ot is a paternally expressed antisense transcript originating from intron of the maternally expressed kcnq gene. the human kcnq1ot promoter starts approximately bp upstream of the transcription initiation site and spans to bp, although associated transcription factor binding sites may extend as far as bp upstream. the transcription initiation site is located in a cpg island that is methylated in oocytes, but not in sperm. methylation blocks transcription of the maternal allele of kcnq1ot and deletion of either the cpg island or kcnq1ot increases expression of cdkn1c, kcnq and ascl indicating its role in the coordinated regulation of these loci. dna methylation as a regulator of imprinted expression may have evolved from the molecular systems associated with the silencing of transposable elements. the host defence hypothesis suggests that dna methylation fortuitously provided a mechanism to regulate parental specific gene expression and similar circumstances may have led to the silencing function of antisense transcripts at imprinted loci. indeed, many imprinted genes are found in association with repeat sequences such as non-ltr elements, dna elements, and endogenous retroviruses and are all known to attract methylation. we determined the expression of cdkn1c and the cellular localisation of its protein, p57kip in the placenta of the tammar wallaby because of their evolutionary divergence from eutherian mammals. we examined whether cdkn1c became imprinted after it acquired a role in placental development, or if its placental expression pre-dated the evolution of its imprinting. in addition, to gain insight into the evolution of imprinting within this cluster, the structure and repeat distribution of the cdkn1c domain, including kcnq and the icr, kcnq1ot were investigated in the tammar.  p57kip localises to the tammar placenta while cdkn1c is imprinted in eutherians and vital for placentation, it is not imprinted in the tammar. the chorio-vitelline placenta of the tammar consists of two functional regions, a vascular and non-vascular one. the vascular, trilaminar placenta is thought to be the primary site of gas exchange, while that of the avascular, bilaminar region, the predominant site for nutritional exchange. we used immunohistochemistry to examine p57kip in both regions of the marsupial placenta. p57kip was localised in the cytoplasm and nucleus of all cell types in the bilaminar and trilaminar yolk sac placenta. however, while the protein was consistently present in many cells of the yolk sac endoderm and trophoblast, staining in the mesenchymal and endothelial cells was sporadic. no notable difference in staining intensity or in the cross-reactivity of different cell types was evident for the stages examined. weak non-specific cytoplasmic staining was evident in the yolk sac endoderm and, to a lesser extent, the trophoblast in matched goat igg treated controls, but was clearly distinguishable from the dense staining in both the cytoplasm and nucleus with p57kip ab- there was no cytoplasmic or nuclear staining in no-antibody negative controls. cdkn1c expression increases in the final days of gestation cdkn1c was expressed in the tammar placenta, but immunohistochemistry did not indicate if there were changes in the quantity of protein over time. we used quantitative rt-pcr to examine quantitative changes in the expression of cdkn1c. between days and cdkn1c expression was higher in the trilaminar yolk sac than in the bilaminar yolk sac placenta. however, in the two days before birth there was no longer a significant difference in cdkn1c expression between vascular and avascular regions, but there was a significant increase in cdkn1c expression in both regions of the placenta. genomic analysis – gene order is conserved in the tammar and eutherians we examined the region containing igf and cdkn1c in the tammar and compared this to human and mouse. a blast-n with tammar cdkn1c sequence identified a single clone of tammar genomic dna. successive blast-n searches identified five more overlapping clones joining the igf2-contianing clone to the cdkn1c-containing clone. each set of overlapping clones showed 100% identity over at least bp. the overlapping clones provided almost mb of continuous sequence spanning the domains bordered by igf and cdkn1c. several regions were conserved or homologous between human, mouse, and tammar as indicated by a multi-species percent identity plot. most of the conserved regions corresponded to genomic locations with a high gene density, such as the gene cluster bordered by ascl and trpm however, the large intergenic region between th and ascl also had two regions conserved or homologous in all three species. a pip of the orthologous region of human against the tammar sequence was generated. regions of high homology to human indicated the location of putative exons in the tammar sequence. the sequence of each possible exon was entered into a blastn to confirm gene identity and was aligned back to human sequences to confirm the location of exon-intron boundaries. of the nine genes located between igf and cdkn1c in human, eight were identified in the tammar with kcnq1ot the only gene not found by homology. exons homologous to human tssc which lies downstream of cdkn1c, were also identified, but the partial sequence available did not extend to cover the entire gene. gene order was highly conserved between human, mouse, and tammar, as was gene structure. a multi-species pip of the kcnq gene of human against mouse, tammar, and chicken, shows conserved gene structure in all four species, with regions of homology corresponding to exon locations. however, while the exon-intron structure of kcnq was generally highly conserved, intron was absent in chicken and intron was significantly reduced in length in the tammar and in chicken. in mouse and human the kcnq1ot transcript starts in intron and extends into intron mouse intron and of kcnq had significant homology with human introns and while no homology was evident for the tammar or chicken. genomic analysis – tammar sequences lack the kcnq1ot promoter and cpg island the cpg island in intron of kcnq is essential for imprinted expression of the kcnq1ot transcript in mouse and human. we examined the cpg content of the orthologous region in the tammar. there were cpg islands, grouped into nine clusters, in the sequence spanning igf to cdkn1c in the tammar, while in human there were and in mouse. six cpg islands in the human sequence were greater than bp in length with the longest island bp. in comparison, only one of the islands in the tammar sequence was longer than bp. however, mouse also had only two cpg islands over bp. although both human and mouse had fewer cpg islands in kcnq compared to the remaining sequence assessed, there were no cpg islands in kcnq of the tammar. like human and mouse, chicken had a cpg island in kcnq. despite differences in the cpg island content of kcnq in the human and tammar, the overall percent gc was similar. in human, mouse and chicken at least one cpg island was located in intron of kcnq. in human and mouse the position of the cpg island and the kcnq1ot promoter region were highly conserved. although a cpg island was also present in the chicken intron it is not clear if this is orthologous, as no significant homology to the kcnq1ot transcription start site could be found, and the cpg island was located approximately and kb downstream of the orthologous cpg islands in human and mouse respectively. expression analysis of kcnq1o1 primers were designed within the tammar kcnq intron to determine if it still encoded a kcnq1ot antisense rna molecule despite its lack of conservation with human and mouse. since primers did not span an intron, extracted rna was dnased and an aliquot removed for pcr to ensure there was no genomic dna contamination. surprisingly, transcription of the putative kcnq1o gene was detected in the trilaminar, but not the bilaminar placenta and only during the final stages of pregnancy. the resulting pcr band was sequence verified to ensure amplification of the correct product. genomic analysis – analysis of repeat distribution in the igf2-cdkn1c region repeat sequences may contribute to the evolution and or regulation of many imprinted regions and so the distribution of repetitive elements in the tammar igf2-cdknic region was assessed. two regions of high homology were identified in the intergenic dna between th and ascl and represent areas of high line/sine density in all three species. the percent sequence covered by all repetitive elements in the region from igf2-cdkn1c was not significantly different between species. when the kcnq region was assessed separately, the percent covered by all repetitive sequences in introns 1b, and still did not differ significantly between species. however, the percentage of sequenced covered by specific classes of repetitive sequence did differ significantly between species. there were significantly fewer long-terminal repeat elements and dna elements in tammar kcnq introns 1b, and compared to the same introns in mouse and human, while there were significantly more low complexity regions. however, the lower percentage of sequence covered by ltr and dna elements in the tammar compared to mouse and human was also evident across the entire region from igf to cdkn1c. however, the relative proportion of line/sines and simple repeats was not notably different between species in either kcnq or the entire igf2-cdkn1c region. in both human and mouse there was high concentration of repetitive elements in intron largely due to an increase in lines, which are largely absent in tammar intron. this was significantly different in mouse compared to tammar, but not with human. a decrease in sines is associated with imprinted regions. however, there was no significant difference in the percentage of sequence covered by sines between tammar and human and mouse. despite similarities in the overall proportions of different types of repetitive elements in mouse and human, no specific repetitive elements in intron intron or at the transcription start site, were clearly identified as homologous. however, in both human and mouse an l1mb element was located approximately to kb upstream of the kcnq1ot transcription start site. discussion we have confirmed the synteny of cdkn1c with igf in the tammar wallaby and shown it is highly conserved between the human, mouse and tammar. igf and p57kip antagonistically regulate growth of the eutherian placenta. although cdkn1c is not imprinted in the tammar wallaby, it is expressed in the placenta and so could antagonise the growth promoting effects of igf on the marsupial placenta. furthermore, the antisense transcript, kcnq1ot known to regulate cdkn1c imprinting in eutherians is also expressed in the marsupial placenta. however, a cpg island and promoter, orthologous to eutherian kcnq1ot were absent in the tammar genome. these data suggest that imprinting of the cdkn1c gene is not contingent on its synteny with igf expression in the placenta or the expression of the kcnq1ot gene. the p57kip protein was present in the trophoblast, yolk sac endoderm, and some mesenchymal cells of the yolk sac placenta. in eutherians, p57kip binds cyclin-dependent kinases in the nucleus. in the tammar, p57kip was found in the nucleus as well as the cytoplasm of cells in all tissue types, suggesting that this protein is functional in the marsupial placenta, as in eutherians. however, members of the kip family, including p57kip also have roles outside their cdk activity that may account for their cytoplasmic location. cdkn1c mrna expression was higher in the trilaminar placenta compared to the bilaminar, but this difference was only significant when expression was low. more conspicuous was the significant increase in cdkn1c expression in both regions of the yolk sac placenta in the two days before birth. the vascular region of the placenta develops rapidly between days 19– to facilitate transfer of nutrients to the developing fetus, consistent with a low cdkn1c expression. the increase in cdkn1c expression immediately before birth is consistent with retarded growth of the placenta at this time. the slightly higher expression of cdkn1c in the trilaminar placenta may represent the terminal differentiation of haematopoietic tissue in this region that requires exit from the cell cycle. cdkn1c expression in the marsupial placenta is therefore consistent with a role for p57kip in the inhibition of cell cycle progression and regulation of marsupial placental growth. igf and cdkn1c are co-expressed in the placentas of human, mouse, and tammar, suggesting these genes were also co-expressed in the placenta of the therian ancestor. the antagonistic functional relationship between these genes is also likely to have existed in the ancestor of marsupials and eutherians. however, the cdkn1c imprint must have been acquired later and only in the eutherian lineage. all genes located between igf and cdkn1c in human were also syntenic in the tammar. however, homology to kcnq1ot was lacking. despite high conservation of kcnqt exons between mouse, human and tammar, intron of the tammar kcnqt gene showed no homology to the kcnq1ot eutherian transcript. the promoter region and associated cpg island, conserved in human and mouse, could not be detected in the tammar. intron of kcnq which encodes the terminal end of kcnq1ot also lacks homology with its eutherian counterpart and is considerably shorter in the tammar than in human and mouse. since kcnq1ot is a non-coding rna transcript, its sequence conservation with eutherians is not critical to its function. we therefore examined the expression of kcnqt intron by rt-pcr. despite the sequence divergence, transcription was detected suggesting that an antisense rna kcnq1ot1-orthologue exists in marsupials as in eutherians. this is especially interesting, since cdkn1c is not imprinted in marsupials, despite the presence of kcnq1ot it will be important to determine whether kcnq and kcnq1ot are imprinted in the tammar, as in eutherians. as none of the promoter elements of eutherian kcnq1ot were identified, it is possible that marsupial kcnq1ot is regulated by a completely different means and has not yet been co-opted into regulating imprinting of cdkn1c. however, it does show that evolution of kcnq1ot preceded imprinting of cdkn1c. throughout the rest of the igf2-cdkn1c region, there was little cross-species sequence homology in the intergenic regions. however, two regions of high homology were identified in mammals between the th and ascl genes. both sites of homology corresponded to regions of high line/sine density suggesting conservation of repetitive elements may account for the sequence homology, rather than any regulatory or control regions. these repeats may be important for establishing a boundary region between two independently regulated domains, leading to their conservation. there were significantly fewer dna elements and endogenous retroviruses across the entire region, spanning both the imprinted igf domain and the non-imprinted cdkn1c domain. however, in both mouse and human there was a considerable increase in repetitive elements in intron when compared with the tammar. this was due to the accumulation of lines that appear to coincide with the termination of the kcnq1ot transcript. these results suggest that the truncation of kcnq intron in the tammar and the increase in lines in mouse and human may be associated with the evolution of kcnq imprinting in the region.  despite its lack of imprinting, marsupial cdkn1c is expressed in the developing placenta where it may antagonise the actions of igf on cell-cycle progression, as it does in eutherians. since cdkn1c resides in synteny with igf imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. the expression of kcnq1ot in the absence of cdkn1c imprinting suggests that antisense transcription at this locus may have preceded imprinting of this domain. these findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that cdkn1c imprinting is not due to its synteny with igf or its placental expression in mammals. 
these findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that cdkn1c imprinting cannot be due to its synteny with igf or with its placental expression in mammals. cdkn1c resides in synteny with igf demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. while the cpg island in intron of kcnq and promoter elements could not be detected, the antisense transcript kcnq1ot that regulates cdkn1c imprinting in human and mouse is still expressed. genomic imprinting occurs in both marsupial and eutherian mammals. genomic analysis of the tammar region confirmed that cdkn1c is syntenic with igf however, there are fewer ltr and dna elements in the region and in intron of kcnq in addition there are fewer lines in the tammar compared with human and mouse. the expression of kcnq1ot in the absence of cdkn1c imprinting suggests that antisense transcription at this locus preceded imprinting of this domain.
 genomic imprinting occurs in both marsupial and eutherian mammals. the cdkn1c and igf genes are both imprinted and syntenic in the mouse and human, but in marsupials only igf is imprinted. this study examines the evolution of features that, in eutherians, regulate cdkn1c imprinting.  despite the absence of imprinting, cdkn1c protein was present in the tammar wallaby placenta. genomic analysis of the tammar region confirmed that cdkn1c is syntenic with igf however, there are fewer ltr and dna elements in the region and in intron of kcnq in addition there are fewer lines in the tammar compared with human and mouse. while the cpg island in intron of kcnq and promoter elements could not be detected, the antisense transcript kcnq1ot that regulates cdkn1c imprinting in human and mouse is still expressed.  cdkn1c has a conserved function, likely antagonistic to igf in the mammalian placenta that preceded its acquisition of imprinting. cdkn1c resides in synteny with igf demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. the expression of kcnq1ot in the absence of cdkn1c imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. these findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that cdkn1c imprinting cannot be due to its synteny with igf or with its placental expression in mammals. 