School of BioSciences - Research Publications
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ItemPromoter-Specific Expression and Imprint Status of Marsupial IGF2(PUBLIC LIBRARY SCIENCE, 2012-07-25)In mice and humans, IGF2 has multiple promoters to maintain its complex tissue- and developmental stage-specific imprinting and expression. IGF2 is also imprinted in marsupials, but little is known about its promoter region. In this study, three IGF2 transcripts were isolated from placental and liver samples of the tammar wallaby, Macropus eugenii. Each transcript contained a unique 5' untranslated region, orthologous to the non-coding exons derived from promoters P1-P3 in the human and mouse IGF2 locus. The expression of tammar IGF2 was predominantly from the P2 promoter, similar to humans. Expression of IGF2 was higher in pouch young than in the adult and imprinting was highly tissue and developmental-stage specific. Interestingly, while IGF2 was expressed throughout the placenta, imprinting seemed to be restricted to the vascular, trilaminar region. In addition, IGF2 was monoallelically expressed in the adult mammary gland while in the liver it switched from monoalleleic expression in the pouch young to biallelic in the adult. These data suggest a complex mode of IGF2 regulation in marsupials as seen in eutherian mammals. The conservation of the IGF2 promoters suggests they originated before the divergence of marsupials and eutherians, and have been selectively maintained for at least 160 million years.
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ItemEvolution of coding and non-coding genes in HOX clusters of a marsupial(BMC, 2012-06-18)BACKGROUND: The HOX gene clusters are thought to be highly conserved amongst mammals and other vertebrates, but the long non-coding RNAs have only been studied in detail in human and mouse. The sequencing of the kangaroo genome provides an opportunity to use comparative analyses to compare the HOX clusters of a mammal with a distinct body plan to those of other mammals. RESULTS: Here we report a comparative analysis of HOX gene clusters between an Australian marsupial of the kangaroo family and the eutherians. There was a strikingly high level of conservation of HOX gene sequence and structure and non-protein coding genes including the microRNAs miR-196a, miR-196b, miR-10a and miR-10b and the long non-coding RNAs HOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expression and controlling development. By microRNA deep sequencing and comparative genomic analyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one new candidate microRNA with typical hairpin precursor structure that is expressed in both fibroblasts and testes was found. The prediction of microRNA target analysis showed that several known microRNA targets, such as miR-10, miR-414 and miR-464, were found in the tammar HOX clusters. In addition, several novel and putative miRNAs were identified that originated from elsewhere in the tammar genome and that target the tammar HOXB and HOXD clusters. CONCLUSIONS: This study confirms that the emergence of known long non-coding RNAs in the HOX clusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified a new potentially functional microRNA as well as conserved miRNAs. These non-coding RNAs may participate in the regulation of HOX genes to influence the body plan of this marsupial.
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ItemEvolution of vertebrate interferon inducible transmembrane proteins(BMC, 2012-04-26)BACKGROUND: Interferon inducible transmembrane proteins (IFITMs) have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. RESULTS: Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. CONCLUSIONS: Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.
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ItemThe Evolution of Mammalian Genomic Imprinting Was Accompanied by the Acquisition of Novel CpG Islands(OXFORD UNIV PRESS, 2011)Parent-of-origin-dependent expression of imprinted genes is mostly associated with allele-specific DNA methylation of the CpG islands (CGIs) called germ line differentially methylated regions (gDMRs). Although the essential role of gDMRs for genomic imprinting has been well established, little is known about how they evolved. In several imprinted loci, the CGIs forming gDMRs may have emerged with the insertion of a retrotransposon or retrogene. To examine the generality of the hypothesis that the CGIs forming gDMRs were novel CGIs recently acquired during mammalian evolution, we reviewed the time of novel CGI emergence for all the maternal gDMR loci using the novel data analyzed in this study combined with the data from previous reports. The comparative sequence analyses using mouse, human, dog, cow, elephant, tammar, opossum, platypus, and chicken genomic sequences were carried out for Peg13, Meg1/Grb10, Plagl1/Zac1, Gnas, and Slc38a4 imprinted loci to obtain comprehensive results. The combined data showed that emergence of novel CGIs occurred universally in the maternal gDMR loci at various time points during mammalian evolution. Furthermore, the analysis of Meg1/Grb10 locus provided evidence that gradual base pair-wise sequence change was involved in the accumulation of CpG sequence, suggesting the mechanism of novel CGI emergence is more complex than the suggestion that CpG sequences originated solely by insertion of CpG-rich transposable elements. We propose that acquisition of novel CGIs was a key genomic change for the evolution of imprinting and that it usually occurred in the maternal gDMR loci.
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ItemSelected imprinting of INS in the marsupial(BMC, 2012-08-28)BACKGROUND: In marsupials, growth and development of the young occur postnatally, regulated by milk that changes in composition throughout the long lactation. To initiate lactation in mammals, there is an absolute requirement for insulin (INS), a gene known to be imprinted in the placenta. We therefore examined whether INS is imprinted in the mammary gland of the marsupial tammar wallaby (Macropus eugenii) and compared its expression with that of insulin-like growth factor 2 (IGF2). RESULTS: INS was expressed in the mammary gland and significantly increased, while IGF2 decreased, during established milk production. Insulin and IGF2 were both detected in the mammary gland macrophage cells during early lactation and in the alveolar cells later in lactation. Surprisingly, INS, which was thought only to be imprinted in the therian yolk sac, was imprinted and paternally expressed in the liver of the developing young, monoallelically expressed in the tammar mammary gland and biallelic in the stomach and intestine. The INS transcription start site used in the liver and mammary gland was differentially methylated. CONCLUSIONS: This is the first study to identify tissue-specific INS imprinting outside the yolk sac. These data suggest that there may be an advantage of selective monoallelic expression in the mammary gland and that this may influence the growth of the postnatal young. These results are not consistent with the parental conflict hypothesis, but instead provide support for the maternal-infant co-adaptation hypothesis. Thus, imprinting in the mammary gland maybe as critical for postnatal growth and development in mammals as genomic imprinting in the placenta is prenatally.
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ItemA novel MSMB-related microprotein in the postovulatory egg coats of marsupials(BMC, 2011-12-30)BACKGROUND: Early marsupial conceptuses differ markedly from those of eutherian mammals, especially during cleavage and early blastocyst stages of development. Additionally, in marsupials the zona pellucida is surrounded by two acellular layers, the mucoid coat and shell, which are formed from secretions from the reproductive tract. RESULTS: We report the identification of a novel postovulatory coat component in marsupials, which we call uterinesecreted microprotein (USM). USM belongs to a family of disulfide-rich microproteins of unconfirmed function that is found throughout deuterostomes and in some protostomes, and includes β-microseminoprotein (MSMB) and prostate-associated microseminoprotein (MSMP). We describe the evolution of this family in detail, including USM-related sequences in other vertebrates. The orthologue of USM in the tammar wallaby, USM1, is expressed by the endometrium with a dynamic temporal profile, possibly under the control of progesterone. CONCLUSIONS: USM appears to have evolved in a mammalian ancestor specifically as a component of the postovulatory coats. By analogy with the known properties of MSMB, it may have roles in regulating sperm motility/survival or in the immune system. However, its C-terminal domain is greatly truncated compared with MSMB, suggesting a divergent function.
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ItemIdentification of two distinct genes at the vertebrate TRPC2 locus and their characterisation in a marsupial and a monotreme(BIOMED CENTRAL LTD, 2011-08-19)BACKGROUND: The vomeronasal organ (VNO) detects pheromones via two large families of vomeronasal receptors: vomeronasal receptor 1 (V1R) and vomeronasal receptor 2 (V2R). Both VRs have a common receptor activation cascade involving transient receptor potential channel, subfamily C, member 2 (TRPC2). RESULTS: We characterised the TRPC2 locus in a marsupial, the tammar wallaby (Macropus eugenii), and identified two independently regulated genes not previously recognised as distinct. 3'-located exons comprise bona fide TRPC2 whilst 5'-located exons, previously identified as part of TRPC2, comprise a distinct gene, which we term XNDR (XRCC1 N-terminal domain-related). The two genes show contrasting expression patterns in the tammar: TRPC2 is specifically expressed in adult and developing VNO, whereas XNDR is widely expressed in many tissues suggesting a non-VNO-specific role. Strong expression of TRPC2 was detected only after about day 30 post-partum, suggesting that the VNO may not be functional during early pouch life of the tammar. Similarly restricted expression of TRPC2 and widespread expression of XNDR was also detected in the platypus. Bioinformatic analysis of the genomes of a wide range of species suggests that the identity of XNDR and TRPC2 as distinct genes is conserved among vertebrates. Finally, we analysed the promoter of mammalian TRPC2 and identified a conserved binding site for NHLH1, a transcription factor previously implicated in VNO receptor neuron development. CONCLUSIONS: Two functionally distinct vertebrate genes-XNDR and TRPC2 - occupy a genomic locus that was previously defined as a single gene in the mouse. The former is widely expressed with a putative role in DNA repair, while the latter shows VNO-specific expression under the probable regulation of NHLH1.
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ItemCharacterisation of marsupial PHLDA2 reveals eutherian specific acquisition of imprinting(BIOMED CENTRAL LTD, 2011-08-19)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.
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ItemIdentification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event(OXFORD UNIV PRESS, 2011-08)In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.
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ItemHOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii(BMC, 2012-01-11)BACKGROUND: Kangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby. RESULTS: We describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken. CONCLUSIONS: This is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.