The origin and evolution of genomic imprinting and viviparity in mammals

Genomic imprinting is widespread in eutherian mammals. Marsupial mammals also have genomic imprinting, but in fewer loci. It has long been thought that genomic imprinting is somehow related to placentation and/or viviparity in mammals, although neither is restricted to mammals. Most imprinted genes are expressed in the placenta. There is no evidence for genomic imprinting in the egg-laying monotreme mammals, despite their short-lived placenta that transfers nutrients from mother to embryo. Post natal genomic imprinting also occurs, especially in the brain. However, little attention has been paid to the primary source of nutrition in the neonate in all mammals, the mammary gland. Differentially methylated regions (DMRs) play an important role as imprinting control centres in each imprinted region which usually comprises both paternally and maternally expressed genes (PEGs and MEGs). The DMR is established in the male or female germline (the gDMR). Comprehensive comparative genome studies demonstrated that two imprinted regions, PEG10 and IGF2-H19, are conserved in both marsupials and eutherians and that PEG10 and H19 DMRs emerged in the therian ancestor at least 160 Ma, indicating the ancestral origin of genomic imprinting during therian mammal evolution. Importantly, these regions are known to be deeply involved in placental and embryonic growth. It appears that most maternal gDMRs are always associated with imprinting in eutherian mammals, but emerged at differing times during mammalian evolution. Thus, genomic imprinting could evolve from a defence mechanism against transposable elements that depended on DNA methylation established in germ cells.     

Germ cells, gonads and sex reversal in marsupials.

The formation of the testis or ovary is a critical step in development. Alterations in gonadal development during fetal or postnatal life can lead to intersexuality or infertility. Several model systems have been particularly useful in studying gonadal differentiation, the eutherian mammal and amphibia, fish, and birds. However, marsupials provide a unique opportunity to investigate gonadal development and the interactions of genes and hormones in gonadal differentiation and germ cell development in all mammals. On the one hand the genetic mechanisms appear to be identical to those in eutherian mammals, including the testis-determining SRY gene. On the other hand, marsupials retain in part the plasticity of the amphibian gonad to hormonal manipulation. It is possible to induce female to male and also male to female gonadal sex reversal in marsupials by hormonal manipulation, and oestradiol can induce male germ cells to enter meiosis at the time the oogonia do. In addition, in marsupials the development of the scrotum and mammary glands are independent of testicular androgens and instead are controlled by a gene or genes on the X-chromosome. Thus marsupials provide a number of opportunities for manipulating the sexual differentiation of the gonads that are not possible in eutherian mammals and so provide a unique perspective for understanding the common mechanisms controlling sexual development.     

Foetal age determination and development in elephants

Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation.     

Post-natal imprinting: evidence from marsupials

Genomic imprinting has been identified in therian (eutherian and marsupial) mammals but not in prototherian (monotreme) mammals. Imprinting has an important role in optimising pre-natal nutrition and growth, and most imprinted genes are expressed and imprinted in the placenta and developing fetus. In marsupials, however, the placental attachment is short-lived, and most growth and development occurs post-natally, supported by a changing milk composition tailor-made for each stage of development. Therefore there is a much greater demand on marsupial females during post-natal lactation than during pre-natal placentation, so there may be greater selection for genomic imprinting in the mammary gland than in the short-lived placenta. Recent studies in the tammar wallaby confirm the presence of genomic imprinting in nutrient-regulatory genes in the adult mammary gland. This suggests that imprinting may influence infant post-natal growth via the mammary gland as it does pre-natally via the placenta. Similarly, an increasing number of imprinted genes have been implicated in regulating feeding and nurturing behaviour in both the adult and the developing neonate/offspring in mice. Together these studies provide evidence that genomic imprinting is critical for regulating growth and subsequently the survival of offspring not only pre-natally but also post-natally.     

Sex-linked and autosomal microsatellites provide new insights into island populations of the tammar wallaby

The emerging availability of microsatellite markers from mammalian sex chromosomes provides opportunities to investigate both male- and female-mediated gene flow in wild populations, identifying patterns not apparent from the analysis of autosomal markers alone. Tammar wallabies (Macropus eugenii), once spread over the southern mainland, have been isolated on several islands off the Western Australian and South Australian coastlines for between 10 000 and 13 000 years. Here, we combine analyses of autosomal, Y-linked and X-linked microsatellite loci to investigate genetic variation in populations of this species on two islands (Kangaroo Island, South Australia and Garden Island, Western Australia). All measures of diversity were higher for the larger Kangaroo Island population, in which genetic variation was lowest at Y-linked markers and highest at autosomal markers (θ=3.291, 1.208 and 0.627 for autosomal, X-linked and Y-linked data, respectively). Greater relatedness among females than males provides evidence for male-biased dispersal in this population, while sex-linked markers identified genetic lineages not apparent from autosomal data alone. Overall genetic diversity in the Garden Island population was low, especially on the Y chromosome where most males shared a common haplotype, and we observed high levels of inbreeding and relatedness among individuals. Our findings highlight the utility of this approach for management actions, such as the selection of animals for translocation or captive breeding, and the ecological insights that may be gained by combining analyses of microsatellite markers on sex chromosomes with those derived from autosomes.     

Evolution of coding and non-coding genes in HOX clusters of a marsupial

ABSTRACT: BACKGROUND: The HOX gene clusters are thought to be highly conserved amongst mammals and othervertebrates, but the long non-coding RNAs have only been studied in detail in human andmouse. The sequencing of the kangaroo genome provides an opportunity to use comparativeanalyses to compare the HOX clusters of a mammal with a distinct body plan to those ofother mammals. RESULTS: Here we report a comparative analysis of HOX gene clusters between an Australian marsupialof the kangaroo family and the eutherians. There was a strikingly high level of conservationof HOX gene sequence and structure and non-protein coding genes including the microRNAsmiRNA-196a, miRNA-196b, miRNA-10a and miRNA-10b and the long non-coding RNAsHOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expressionand controlling development. By microRNA deep sequencing and comparative genomicanalyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one newcandidate microRNA with typical hairpin precursor structure that is expressed in bothfibroblasts and testes was found. The prediction of microRNA target analysis showed thatseveral known microRNA targets, such as mir-10, mir-414 and mir-464, were found in thetammar HOX clusters. In addition, several novel and putative miRNAs were identified thatoriginated from elsewhere in the tammar genome and that target the tammar HOXB andHOXD clusters. CONCLUSIONS: This study confirms that the emergence of known long non-coding RNAs in the HOXclusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified anew potentially functional microRNA as well as conserved miRNAs. These non-codingRNAs may participate in the regulation of HOX genes to influence the body plan of thismarsupial.     

3' RACE walking along a large cDNA employing tiered suppression PCR

Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR 5' RACE procedure but includes a specific and limited second-strand cDNA synthesis and a tiered "panhandle" suppression of nonspecific products. The method generated 900 bp of new sequence for the large tammar wallaby ATRY gene in two easy steps, in which standard 3' RACE and PCR-based cDNA library walking proved unsuccessful. This robust approach represents a new tool for isolating unknown sequence under challenging cloning scenarios such as poor library representation, long coding regions, long 3' untranslated regions, and difficult template regions.