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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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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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.
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ItemDifferential roles of TGIF family genes in mammalian reproduction(BIOMED CENTRAL LTD, 2011-09-29)BACKGROUND: TG-interacting factors (TGIFs) belong to a family of TALE-homeodomain proteins including TGIF1, TGIF2 and TGIFLX/Y in human. Both TGIF1 and TGIF2 act as transcription factors repressing TGF-β signalling. Human TGIFLX and its orthologue, Tex1 in the mouse, are X-linked genes that are only expressed in the adult testis. TGIF2 arose from TGIF1 by duplication, whereas TGIFLX arose by retrotransposition to the X-chromosome. These genes have not been characterised in any non-eutherian mammals. We therefore studied the TGIF family in the tammar wallaby (a marsupial mammal) to investigate their roles in reproduction and how and when these genes may have evolved their functions and chromosomal locations. RESULTS: Both TGIF1 and TGIF2 were present in the tammar genome on autosomes but TGIFLX was absent. Tammar TGIF1 shared a similar expression pattern during embryogenesis, sexual differentiation and in adult tissues to that of TGIF1 in eutherian mammals, suggesting it has been functionally conserved. Tammar TGIF2 was ubiquitously expressed throughout early development as in the human and mouse, but in the adult, it was expressed only in the gonads and spleen, more like the expression pattern of human TGIFLX and mouse Tex1. Tammar TGIF2 mRNA was specifically detected in round and elongated spermatids. There was no mRNA detected in mature spermatozoa. TGIF2 protein was specifically located in the cytoplasm of spermatids, and in the residual body and the mid-piece of the mature sperm tail. These data suggest that tammar TGIF2 may participate in spermiogenesis, like TGIFLX does in eutherians. TGIF2 was detected for the first time in the ovary with mRNA produced in the granulosa and theca cells, suggesting it may also play a role in folliculogenesis. CONCLUSIONS: The restricted and very similar expression of tammar TGIF2 to X-linked paralogues in eutherians suggests that the evolution of TGIF1, TGIF2 and TGIFLX in eutherians was accompanied by a change from ubiquitous to tissue-specific expression. The distribution and localization of TGIF2 in tammar adult gonads suggest that there has been an ultra-conserved function for the TGIF family in fertility and that TGIF2 already functioned in spermatogenesis and potentially folliculogenesis long before its retrotransposition to the X-chromosome of eutherian mammals. These results also provide further evidence that the eutherian X-chromosome has actively recruited sex and reproductive-related genes during mammalian evolution.
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ItemOestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal(BMC, 2010-08-31)BACKGROUND: Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development. RESULTS: We examined the expression of key testicular (SRY, SOX9, AMH and FGF9) and ovarian (WNT4, RSPO1, FOXL2 and FST) markers during gonadal development in the marsupial tammar wallaby (Macropus eugenii) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of AMH and SOX9 as in the mouse and human testis, but this upregulation was initiated before the peak in SRY expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of FOXL2 and WNT4, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of SRY, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad. CONCLUSIONS: We have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein. See commentary: http://www.biomedcentral.com/1741-7007/8/110.
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ItemComparative analysis of the mammalian WNT4 promoter(BMC, 2009-09-06)BACKGROUND: WNT4 is a critical signalling molecule in embryogenesis and homeostasis, but the elements that control its transcriptional regulation are largely unknown. This study uses comparative cross species sequence and functional analyses between humans and a marsupial (the tammar wallaby,Macropus eugenii) to refine the mammalian Wnt4 promoter. RESULTS: We have defined a highly conserved 89 bp minimal promoter region in human WNT4 by comparative analysis with the tammar wallaby. There are many conserved transcription factor binding sites in the proximal promoter region, including SP1, MyoD, NFkappaB and AP2, as well as highly conserved CpG islands within the human, mouse and marsupial promoters, suggesting that DNA methylation may play an important role in WNT4 transcriptional regulation. CONCLUSION: Using a marsupial model, we have been able to provide new information on the transcriptional regulators in the promoter of this essential mammalian developmental gene, WNT4. These transcription factor binding sites and CpG islands are highly conserved in two disparate mammals, and are likely key controlling elements in the regulation of this essential developmental gene.
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ItemEvolution of the CDKN1C-KCNQ1 imprinted domain(BMC, 2008-05-29)BACKGROUND: Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting. RESULTS: 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 IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed. CONCLUSION: CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, 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 KCNQ1OT1 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 IGF2 or with its placental expression in mammals.
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ItemThe evolution of the DLK1-DIO3 imprinted domain in mammals(PUBLIC LIBRARY SCIENCE, 2008-06)A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.
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ItemExpression and protein localisation of IGF2 in the marsupial placenta(BIOMED CENTRAL LTD, 2008-02-20)BACKGROUND: In eutherian mammals, genomic imprinting is critical for normal placentation and embryo survival. Insulin-like growth factor 2 (IGF2) is imprinted in the placenta of both eutherians and marsupials, but its function, or that of any imprinted gene, has not been investigated in any marsupial. This study examines the role of IGF2 in the yolk sac placenta of the tammar wallaby, Macropus eugenii. RESULTS: IGF2 mRNA and protein were produced in the marsupial placenta. Both IGF2 receptors were present in the placenta, and presumably mediate IGF2 mitogenic actions. IGF2 mRNA levels were highest in the vascular region of the yolk sac placenta. IGF2 increased vascular endothelial growth factor expression in placental explant cultures, suggesting that IGF2 promotes vascularisation of the yolk sac. CONCLUSION: This is the first demonstration of a physiological role for any imprinted gene in marsupial placentation. The conserved imprinting of IGF2 in this marsupial and in all eutherian species so far investigated, but not in monotremes, suggests that imprinting of this gene may have originated in the placenta of the therian ancestor.