Characterisation of marsupial PHLDA2 reveals eutherian specific acquisition of imprinting

Abstract

Background: Genomic imprinting causes parent-of-origin specific gene expression by differential epigenetic modifications between two parental genomes. We previously reported that there is no evidence of genomic imprinting of CDKN1C in the KCNQ1 domain in the placenta of an Australian marsupial, the tammar wallaby (Macropus eugenii) whereas tammar IGF2 and H19, located adjacent to the KCNQ1 domain in eutherian mammals, are imprinted. We have now identified and characterised the marsupial orthologue of PHLDA2, another gene in the KCNQ1 domain (also known as IPL or TSSC3) that is imprinted in eutherians. In mice, Phlda2 is a dose-sensitive negative regulator of placental growth, as Cdkn1c is for embryonic growth.

Results: Tammar PHLDA2 is highly expressed in the yolk sac placenta compared to other fetal tissues, confirming a similar expression pattern to that of mouse Phlda2. However, tammar PHLDA2 is biallelically expressed in both the fetus and yolk sac placenta, so it is not imprinted. The lack of imprinting in tammar PHLDA2 suggests that the acquisition of genomic imprinting of the KCNQ1 domain in eutherian mammals, accompanied with gene dosage reduction, occurred after the split of the therian mammals into the marsupials and eutherians.

Conclusions: Our results confirm the idea that acquisition of genomic imprinting in the KCNQ1 domain occurred specifically in the eutherian lineage after the divergence of marsupials, even though imprinting of the adjacent IGF2-H19 domain arose before the marsupial-eutherian split. These data are consistent with the hypothesis that genomic imprinting of the KCNQ1 domain may have contributed to the evolution of more complex placentation in the eutherian lineage by reduction of the gene dosage of negative regulators for both embryonic and placental growth.

Figure 1

Partial genomic structure and amino acid sequence of tammar PHLDA2. (A) Exons are depicted as open boxes. The start and end of the ORF and the poly-A signal sequence are represented as ATG, TGA and AATAAA, respectively. The numbers indicate the nucleotide distance from the start of the ORF. (B) The alignment was created using "CLC Sequence Viewer 6" software. Identical and similar amino acids are indicated by the same color at each location. The bold red line represents the region of the PH domain. The amino acid sequences for human, mouse, tammar, platypus and chicken PHLDA2 were derived from the following accession numbers respectively: Genbank:NM_003311, Genbank:NM_009434, DDBJ:AB537423, GenBank:XM_001507454, GenBank:XM_421020.

Figure 2

Relative expression levels of tammar PHLDA2 mRNA. The vertical axis represents the percentage expression levels when the highest result is regarded as 100%. The numbers on the horizontal axis indicate the day of gestation of each sample. "BYS" represents the bilaminar yolk sac; "TYS": trilaminar yolk sac; "Lm": limb and "Lu": lung.

Figure 3

PHLDA2 immunohistochemistry in the tammar yolk sac placenta. Both bilaminar and trilaminar yolk sac membranes are shown as the cord like structure in the sections (A: bilaminar yolk sac, B: trilaminar yolk sac). "T" indicates trophoblast cells, the large cells with large nuclei, and "En" indicates yolk sac endodermal cells, the flat and thin cells with relatively small nuclei.

Figure 4

Allelic expression analysis of tammar PHLDA2. (A) The difference of genomic structure by the length polymorphism is described. The open boxes represent exonic regions. Each black arrow represents a single 31 bp repeat unit. The longer allele has two units as a tandem repeat while the shorter allele has no repeat. The gel picture shows the PCR results using genomic DNA extracted from three different individuals having each genotype. "S" represents shorter allele; "L": longer allele. (B) Electrophoresis of RT-PCR products. Two different bands correspond to the amplified products from the longer and shorter allele. "gDNA" represents genomic DNA; "mDNA": maternal DNA; "YSP": yolk sac placenta. Three independent RT-PCR results were shown for the fetus #3 and pouch young #1. (C) The results of direct sequencing for tammar IGF2 PCR products amplified the unspliced transcript.

Figure 5

Summary illustration. The branched black arrow represents the evolutionally divergence between marsupials and eutherians which occurred at least 130-148 million years ago. The broken red arrow represents the evolution of eutherian-type gestation including the prolongation of inter-uterine development with a chorioallantoic placenta. The broken green arrow represents the evolution of the advanced complex lactation system as one of the remarkable and specialised features of marsupials. The acquisition of genomic imprinting in the KCNQ1 domain, accompanied with gene dosage reduction of CDKN1C and PHLDA2, occurred only in the evolution of the eutherian linage as well as the SNURF-SNRPN and DLK1-GTL2 domains [29,32]. On the other hand, imprinting of IGF2-H19 domain, IGF2R, PEG1/MEST and PEG10 occurred before the divergence of marsupials [26-28,30,31,33]. This study and others provide evidence that imprinting occurred at two critical time points during the evolution of mammals. For the third time point, whether marsupial-specific imprinting occurred or not, is currently still unknown.