The admixed brushtail possum genome reveals invasion history in New Zealand and novel imprinted genes.

Combining genome assembly with population and functional genomics can provide valuable insights to development and evolution, as well as tools for species management. Here, we present a chromosome-level genome assembly of the common brushtail possum (Trichosurus vulpecula), a model marsupial threatened in parts of their native range in Australia, but also a major introduced pest in New Zealand. Functional genomics reveals post-natal activation of chemosensory and metabolic genes, reflecting unique adaptations to altricial birth and delayed weaning, a hallmark of marsupial development. Nuclear and mitochondrial analyses trace New Zealand possums to distinct Australian subspecies, which have subsequently hybridised. This admixture allowed phasing of parental alleles genome-wide, ultimately revealing at least four genes with imprinted, parent-specific expression not yet detected in other species (MLH1, EPM2AIP1, UBP1 and GPX7). We find that reprogramming of possum germline imprints, and the wider epigenome, is similar to eutherian mammals except onset occurs after birth. Together, this work is useful for genetic-based control and conservation of possums, and contributes to understanding of the evolution of novel mammalian epigenetic traits.

Primordial germ cell expression of SSEA1 and DDX4 (VASA) in female Trichosurus vulpecula (Marsupialia) reveals conserved and unique molecular patterns during marsupial germ cell development.

Development of primordial germ cells (PGCs: precursors to adult gametes) is a key process in vertebrate sexual differentiation. Marsupials are ideal to investigate this phenomenon because much of PGC migration and development unusually occurs postnatally in pouch young. However, investigation of the molecular dynamics underpinning PGC development is restricted to one marsupial model species: the tammar wallaby (Macropus eugenii). Given the reproductive diversity among clades, marsupial PGCs likely exhibit diversity in molecular patterns that could help uncover their developmental dynamics. Here we characterise PGC marker expression (SSEA1 and DDX4) in developing ovaries of the brushtail possum, Trichosurus vulpecula. Female germ cells expressed DDX4 from 6 days postpartum (dpp) and almost all germ cells expressed DDX4 by meiosis (40 dpp), consistent with M. eugenii and eutherian mammals. In contrast, PGCs and oogonia expressed SSEA1 from 12 dpp, throughout proliferation and until entry into meiosis (40-63 dpp). SSEA1 expression was temporally distinct from that of M. eugenii, in which SSEA1 expression persists only until 14 dpp, indicating differential expression between marsupial species at equivalent stages of germ cell development. Hence, the molecular characteristics of M. eugenii germ cells cannot be assumed for all marsupials, as at least one key molecule exhibits species-specific expression.

Author Correction: Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Uterine epithelial remodelling during pregnancy in the marsupial Monodelphis domestica (Didelphidae): Implications for mammalian placental evolution.

Mammalian pregnancy involves remodelling of the uterine epithelium to enable placentation. In marsupials, such remodelling has probably played a key role in the transition from ancestral invasive placentation to non-invasive placentation. Identifying uterine alterations that are unique to marsupials with non-invasive placentation can thus elucidate mechanisms of marsupial placental evolution. We identified apical alterations to uterine epithelial cells prior to implantation in Monodelphis domestica, a member of the least derived living marsupial clade (Didelphidae) with invasive (endotheliochorial) placentation. We then compared these traits with those of Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae), both with non-invasive placentation, to identify which alterations to the uterine epithelium are ancestral and which facilitate secondarily evolved non-invasive placentation. In M. domestica, remodelling of the uterine epithelium involves reduced cellular heterogeneity and development of uterodome-like cells, suggesting that similar alterations may also have occurred in the marsupial common ancestor. These alterations also overlap with those of both T. vulpecula and Ma. eugenii, suggesting that the placental shift from invasive to non-invasive placentation in marsupials involves essential, conserved characteristics, irrespective of placental mode. However, unique apical alterations of both T. vulpecula and Ma. eugenii, relative to M. domestica, imply that lineage-specific alterations underpin the evolutionary shift to non-invasive placentation in marsupials.

Facultative oviparity in a viviparous skink ( Saiphos equalis).

Facultative changes in parity mode (oviparity to viviparity and vice versa) are rare in vertebrates, yet offer fascinating opportunities to investigate the role of reproductive lability in parity mode evolution. Here, we report apparent facultative oviparity by a viviparous female of the bimodally reproductive skink Saiphos equalis-the first report of different parity modes within a vertebrate clutch. Eggs oviposited facultatively possess shell characteristics of both viviparous and oviparous S. equalis, demonstrating that egg coverings for viviparous embryos are produced by the same machinery as those for oviparous individuals. Since selection may act in either direction when viviparity has evolved recently, squamate reproductive lability may confer a selective advantage. We suggest that facultative oviparity is a viable reproductive strategy for S. equalis and that squamate reproductive lability is more evolutionarily significant than previously acknowledged.

Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.

Non-invasive placentation in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae) involves redistribution of uterine Desmoglein-2.

In mammalian pregnancy, the uterus is remodeled to become receptive to embryonic implantation. Since non-invasive placentation in marsupials is likely derived from invasive placentation, and is underpinned by intra-uterine conflict between mother and embryo, species with non-invasive placentation may employ a variety of molecular mechanisms to maintain an intact uterine epithelium and to prevent embryonic invasion. Identifying such modifications to the uterine epithelium of marsupial species with non-invasive placentation is key to understanding how conflict is mediated during pregnancy in different mammalian groups. Desmoglein-2, involved in maintaining lateral cell-cell adhesion of the uterine epithelium, is redistributed before implantation to facilitate embryo invasion in mammals with invasive placentation. We identified localization patterns of this cell adhesion molecule throughout pregnancy in two marsupial species with non-invasive placentation, the tammar wallaby (Macropus eugenii; Macropodidae), and the brushtail possum (Trichosurus vulpecula; Phalangeridae). Interestingly, Desmoglein-2 redistribution also occurs in both M. eugenii and T. vulpecula, suggesting that cell adhesion, and thus integrity of the uterine epithelium, is reduced during implantation regardless of placental type, and may be an important component of uterine remodeling. Desmoglein-2 also localizes to the mesenchymal stromal cells of M. eugenii and to epithelial cell nuclei in T. vulpecula, suggesting its involvement in cellular processes that are independent of adhesion and may compensate for reduced lateral adhesion in the uterine epithelium. We conclude that non-invasive placentation in marsupials involves diverse and complementary strategies to maintain an intact epithelial barrier.

Uterine molecular changes for non-invasive embryonic attachment in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae).

Pregnancy in mammals requires remodeling of the uterus to become receptive to the implanting embryo. Remarkably similar morphological changes to the uterine epithelium occur in both eutherian and marsupial mammals, irrespective of placental type. Nevertheless, molecular differences in uterine remodeling indicate that the marsupial uterus employs maternal defences, including molecular reinforcement of the uterine epithelium, to regulate embryonic invasion. Non-invasive (epitheliochorial) embryonic attachment in marsupials likely evolved secondarily from invasive attachment, so uterine defences in these species may prevent embryonic invasion. We tested this hypothesis by identifying localization patterns of Talin, a key basal anchoring molecule, in the uterine epithelium during pregnancy in the tammar wallaby (Macropus eugenii; Macropodidae) and the brush tail possum (Trichosurus vulpecula; Phalangeridae). Embryonic attachment is non-invasive in both species, yet Talin undergoes a clear distributional change during pregnancy in M. eugenii, including recruitment to the base of the uterine epithelium just before attachment, that closely resembles that of invasive implantation in the marsupial species Sminthopsis crassicaudata. Basal localization occurs throughout pregnancy in T. vulpecula, although, as for M. eugenii, this pattern is most specific prior to attachment. Such molecular reinforcement of the uterine epithelium for non-invasive embryonic attachment in marsupials supports the hypothesis that less-invasive and non-invasive embryonic attachment in marsupials may have evolved via accrual of maternal defences. Recruitment of basal molecules, including Talin, to the uterine epithelium may have played a key role in this transition.

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae).

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non-invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non-invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.

Uterine focal adhesion dynamics during pregnancy in a marsupial (Sminthopsis crassicaudata; Dasyuridae).

Alterations to the basal attachment points between the epithelium of the uterus and the underlying tissue in early pregnancy affect how easily the epithelium can be invaded by the implanting embryo. Attachment points- focal adhesions- disassemble to facilitate highly invasive implantation in rats, but species with less invasive implantation, including marsupials, may require different basal alterations for successful pregnancy. Here we used immunofluorescence microscopy and Western blotting to conduct the first study of basal plasma membrane dynamics in the uterus during marsupial pregnancy. We describe localisation patterns of two key anchoring molecules, talin and paxillin, throughout pregnancy in the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuridae). Basal staining of both molecules occurs in early pregnancy, as it does in the rat. However, unlike rats, there is strong basal localisation of talin and paxillin just before implantation in S. crassicaudata, indicating that focal adhesions do not disassemble during pregnancy in this species, and that molecular reinforcement of the epithelium may be a maternal strategy to regulate invasion. Additionally, talin and paxillin do not co-localise at all stages of pregnancy as they do in the rat. Different localisation patterns among mammalian species demonstrate that not all early pregnancy changes are ubiquitous in mammalian pregnancy, as changes to the basal plasma membrane of the epithelium, in particular, may instead be dependent on mode of implantation. Anat Rec, 300:1150-1159, 2017. © 2016 Wiley Periodicals, Inc.

Uterine morphology during diapause and early pregnancy in the tammar wallaby (Macropus eugenii).

In mammals, embryonic diapause, or suspension of embryonic development, occurs when embryos at the blastocyst stage are arrested in growth and metabolism. In the tammar wallaby (Macropus eugenii), there are two separate uteri, only one of which becomes gravid with the single conceptus at a post-partum oestrus, so changes during pregnancy can be compared between the gravid and non-gravid uterus within the same individual. Maintenance of the viable blastocyst and inhibition of further conceptus growth during diapause in the tammar is completely dependent on the uterine environment. Although the specific endocrine and seasonal signals are well established, much less is known about the cellular changes required to create this environment. Here we present the first detailed study of uterine morphology during diapause and early pregnancy of the tammar wallaby. We combined transmission electron microscopy and light microscopy to describe the histological and ultrastructural changes to luminal and glandular epithelial cells. At entry into diapause after the post-partum oestrus and formation of the new conceptus, there was an increase in abundance of organelles associated with respiration in the endometrial cells of the newly gravid uterus, particularly in the endoplasmic reticulum and mitochondria, as well as an increase in secretory activity. Organelle changes and active secretion then ceased in these cells as they became quiescent and remained so for the duration of diapause. In contrast, cells of the non-gravid, post-partum, contralateral uterus underwent sloughing and remodelling during this time and some organelle changes in glandular epithelial cells continued throughout diapause, suggesting these cells are not completely quiescent during diapause, although no active secretion occurred. These findings demonstrate that diapause, like pregnancy, is under unilateral endocrine control in the tammar, and that preparation for and maintenance of diapause requires substantial changes to uterine endometrial cell ultrastructure and activity.

Uterine epithelial cell changes during pregnancy in a marsupial (Sminthopsis crassicaudata; Dasyuridae).

Formation of a placenta requires intimate contact between the embryonic and maternal uterine epithelia in early pregnancy. Contact is accompanied by a characteristic suite of changes to the plasma membranes of uterine epithelial cells, termed the plasma membrane transformation. The plasma membrane transformation occurs in eutherian mammals and in viviparous (live-bearing) squamate reptiles, and may be fundamental to the evolution of viviparity in amniotes. Marsupials provide an excellent opportunity to test the generality of this phenomenon. Here, we present the first detailed study of the plasma membrane transformation in a marsupial. We combine electron microscopy and immunohistochemistry to describe morphological and molecular features of uterine epithelial cells during pregnancy in the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuridae). Cell morphology changes dramatically in S. crassicaudata during pregnancy. Apical microvilli are replaced by irregular blunt projections, then by spiky projections postimplantation. Cell surfaces flatten and ciliated cells are lost. Junctional complexes between adjacent cells increase in depth, then decrease just before implantation, which is consistent with junctional protein localization in this region of the cell membrane. The uterine cellular changes in S. crassicaudata are consistent with a plasma membrane transformation, and support the idea that this phenomenon is fundamental to the evolution of viviparity in amniote vertebrates.