Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE,IgG and IgA from the marsupial Monodelphis domestica

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.     

The region homologous to the X-chromosome inactivation centre has been disrupted in marsupial and monotreme mammals

Summary Marsupial, as well as eutherian, mammals are subject to X chromosome inactivation in the somatic cells of females, although the phenotype and the molecular mechanism differ in important respects. Monotreme mammals appear to subscribe at least to a form of dosage compensation of X-borne genes. An important question is whether inactivation in these non-eutherian mammals involves co-ordination by a control locus homologous to the XIST gene and neighbouring genes, which play a key regulatory role in human and mouse X inactivation. We mapped BACs containing several orthologues of protein-coding genes that flank human and mouse XIST and genes that lie in the homologous region in chicken and frog. We found that these genes map to two distant locations on the opossum X, and also to different locations on a platypus autosome. We failed to find any trace of an XIST orthologue in any marsupial or monotreme or on any flanking BAC, confirming the conclusion from recent work that non-eutherian mammals lack XIST. We propose the region homologous to the human and mouse X-inactivation centre expanded in early mammals, and this unstable region was disrupted independently in marsupial and monotreme lineages. In the eutherian lineage, inserted and existing sequences provided the starting material for the non-translated RNAs of the X-inactivation centre, including XIST. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.     

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.     

Cellular immune response of a marsupial, Monodelphis domestica.

Marsupials are interesting subjects for studies of comparative and developmental immunology because they separated from eutherian mammals over 100 million years ago and because the newborns are still in a fetal state. We studied cellular immunity in a fully pedigreed colony of the marsupial, M. domestica (commonly called the gray short-tailed opossum). Peripheral blood lymphocytes were separated on nylon wool columns into adherent cells bearing surface immunoglobulin (B cells) and nonadherent cells (T cells) recovered in the ratio of 1:3. Peripheral blood lymphocytes responded by proliferation to Con A and other mitogens. Nonadherent cells were responsive to Con A, but adherent cells were not. Peripheral blood lymphocytes were stimulated weakly or not at all by allogeneic or xenogeneic (mouse) cells in mixed lymphocyte culture. Despite the weak MLC response, which was not due to genetic homogeneity, allogeneic and xenogeneic tail skin grafts were rejected promptly. These data suggest that the cellular immune response of M. domestica is similar to that of eutherian mammals with the notable exception of weak MLC responses.     

The origin and evolution of the pseudoautosomal regions of human sex chromosomes

The human X and Y chromosomes share two homologous pseudoautosomal regions (PARs) which pair and recombine at meiosis. PAR1 lies at the tips of the short arms, and the smaller PAR2 at the tips of the long arms. PAR1 contains several active genes, and has been thought to be critical for pairing and fertility. The inconsistent gene content of the PARs between different species of eutherian ('placental') mammals suggests that gene content is immaterial to function, and the failure to detect a PAR at all in some rodents and all marsupials implies that homologous pairing is not universally essential for fertility. The autosomal localization of marsupial homologues of human PAR1 genes and their co-localization with human Xp22 genes implies that the human PAR1 represents a relic of part of an autosomal region added to both X and Y chromosomes between 80 and 130 MYrBP. The same argument may be made for part of PAR2. Independent additions to the sex chromosomes of macropodid marsupials and monotremes can also be inferred from comparative mapping. We conclude that the PARs are relics of differential additions, loss, rearrangement and degradation of the Y chromosome in different mammalian lineages.      

The search for a marsupial XIC reveals a break with vertebrate synteny

X-chromosome inactivation (XCI) evolved in mammals to deal with X-chromosome dosage imbalance between the XX female and the XY male. In eutherian mammals, random XCI of the soma requires a master regulatory locus known as the ‘X-inactivation center’ (XIC/Xic), wherein lies the noncoding XIST/Xist silencer RNA and its regulatory antisense Tsix gene. By contrast, marsupial XCI is imprinted to occur on the paternal X chromosome. To determine whether marsupials and eutherians share the XIC-driven mechanism, we search for the sequence equivalents in the genome of the South American opossum, Monodelphis domestica. Positional cloning and bioinformatic analysis reveal several interesting findings. First, protein-coding genes that flank the eutherian XIC are well-conserved in M. domestica, as well as in chicken, frog, and pufferfish. However, in M. domestica we fail to identify any recognizable XIST or TSIX equivalents. Moreover, cytogenetic mapping shows a surprising break in synteny with eutherian mammals and other vertebrates. Therefore, during the evolution of the marsupial X chromosome, one or more rearrangements broke up an otherwise evolutionarily conserved block of vertebrate genes. The failure to find XIST/TSIX in M. domestica may suggest that the ancestral XIC is too divergent to allow for detection by current methods. Alternatively, the XIC may have arisen relatively late in mammalian evolution, possibly in eutherians with the emergence of random XCI. The latter argues that marsupial XCI does not require XIST and opens the search for alternative mechanisms of dosage compensation.     

Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs.     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula)

The cell-surface glycoproteins encoded by the major histocompatibility complex (MHC) bind to processed foreign antigens and present them to T lymphocytes. Two classes of MHC molecules and their corresponding gene sequences have been extensively studied in eutherian mammals and birds, but data on the marsupial MHC are limited. Marsupials split from eutherian mammals about 125 million years ago and represent a distinct branch in mammalian evolution. Here the cDNA cloning of MHC class II genes of the brushtail possums (Trichosurus vulpecula) is reported. The sequences obtained were found to be relatively conserved when compared to the red-necked wallaby (Macropus rufogriseus) and an South American marsupial, Monodelphis domestica. The T. vulpecula sequence shared an average overall sequence identity of 75.4% at the deduced amino acid level with M. rufogriseus and M. domestica, respectively.     

Morphogenesis of the fibrous sheath in the marsupial spermatozoon

The spermatozoon fibrous sheath contains longitudinal columns and circumferential ribs. It surrounds the axoneme of the principal piece of the mammalian sperm tail, and may be important in sperm stability and motility. Here we describe its assembly during spermiogenesis in a marsupial, the brush-tail possum, and compare its structural organization with that of eutherian mammals, birds and reptiles. Transmission electron microscopy showed that possum fibrous sheath assembly is a multistep process extending in a distal-to-proximal direction along the axoneme from steps 4 to 14 of spermiogenesis. For the most part, assembly of the longitudinal columns occurs before that of the circumferential ribs. Immunohistochemical and immunogold labelling showed that fibrous sheath proteins are first present in the spermatid cytoplasm; at least some of the proteins of the sheath precursors differ from those in the mature fibrous sheath. That immunoreactivity develops after initiation of chromatin condensation suggests that fibrous sheath proteins, or their mRNAs, are stored within the spermatid cytoplasmic lobule prior to their assembly along the axoneme. These findings are similar to those in laboratory rats, and thus suggests that the mode of fibrous sheath assembly evolved in a common ancestor over 125 million years ago, prior to the divergence of marsupial and eutherian lineages.     

The Unique Paired Retinal Vessels of the Gray Short‐Tailed Opossum (Monodelphis domestica) and Their Relationship to Astrocytes and Microglial Cells

In marsupials that possess a retinal vasculature, the arterial and venous segments, down to the smallest calibre capillaries, have been shown to occur in pairs. This pattern is seen in the marsupial central nervous system (CNS) but not in other tissues in this group or in any tissues in eutherian mammals. The present study aimed to determine if the gray short‐tailed opossum (Monodelphis domestica), a south American marsupial, possesses double retinal vessels. Secondly, we investigated the relationship between vessels and astrocytes and microglia, which are known to play pivotal roles in the blood retinal barrier and immune surveillance respectively. Eyes from M. domestica between 2 months and 33 months of age were examined by bright field and fluorescein angiography, resin histology, and wholemount immunostaining. Retinal vessels in this marsupial always occur in closely related pairs with the arteriolar limb usually on the vitread aspect. Branches penetrate the retina to form layers of paired capillaries as far as the outer nuclear layer. Dense networks of GFAP⁺ astrocytes enveloped the vitread aspect of vessels. No particularly strong association with blood vessels and ramified Iba1⁺ and Ib4⁺ microglia was noted. M. domestica possessed the unusual paired vasculature and capillary loops arrangement previously described in the marsupial CNS. These observations in a small laboratory‐friendly marsupial open up new frontiers to investigate the factors that regulate paired blood vessel development and the functional significance of this arrangement when compared to the anastomotic pattern observed in the retina of eutherian mammals. Anat Rec, 300:1391–1400, 2017. © 2017 Wiley Periodicals, Inc.     

Those other mammals: The immunoglobulins and T cell receptors of marsupials and monotremes

This review summarizes analyses of marsupial and monotreme immunoglobulin and T cell receptor genetics and expression published over the past decade. Analyses of recently completed whole genome sequences from the opossum and the platypus have yielded insight into the evolution of the common antigen receptor systems, as well as discovery of novel receptors that appear to have been lost in eutherian mammals. These species are also useful for investigation of the development of the immune system in organisms notable for giving birth to highly altricial young, as well as the evolution of maternal immunity through comparison of oviparous and viviparous mammals.     

Conserved and divergent expression patterns of markers of axial development in the laboratory opossum,Monodelphis domestica

Background: Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short‐tailed opossum, a metatherian. Results: Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post‐coitus. Conclusions: Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176–1188, 2016. © 2016 Wiley Periodicals, Inc.     

Isolation and sequence of a cDNA coding for the heavy chain constant region of IgG from the Australian brushtail possum, Trichosurus vulpecula.

A brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened with a South American short-tailed opossum (Monodlelphis domestica) immunoglobulin gamma heavy chain constant region (Cgamma) probe, resulting in the isolation of a 1518 nucleotide cDNA clone. The sequence corresponds to exons 1-3 of Cgamma. The Australian marsupial (T. vulpeculla) sequence is 70% identical at the amino acid level with the American marsupial (M. domestica) sequence, but less similar to the eutherian mammals (45-50%). These data provide the opportunity to compare the evolution of IgG between orders of marsupials separated by at least 75 million years and confirm the appearance of IgG prior to the metatherian/eutherian divergence.     

On the prenatal initiation of T cell development in the opossum Monodelphis domestica

Thymus‐dependent lymphocytes (T cells) are a critical cell lineage in the adaptive immune system of all jawed vertebrates. In eutherian mammals the initiation of T cell development takes place prenatally and the offspring of many species are born relatively immuno‐competent. Marsupials, in contrast, are born in a comparatively altricial state and with a less well developed immune system. As such, marsupials are valuable models for studying the peri‐ and postnatal initiation of immune system development in mammals. Previous results supported a lack of prenatal T cell development in a variety of marsupial species. In the gray short‐tailed opossum, Monodelphis domestica, however, there was evidence that αβT cells were present on postnatal day 1 and likely initiated development prenatally. Demonstrated here is the presence of CD3ε⁺ lymphocytes in late‐stage embryos at a site in the upper thoracic cavity, the site of an early developing thymus. CD3ε⁺ cells were evident as early as 48 h prior to parturition. In day 14 embryos, where there is clear organogenesis, CD3ε⁺ cells were only found at the site of the early thymus, consistent with no extra‐thymic sites of T cell development in the opossum. These observations are the first evidence of prenatal T cell lineage commitment in any marsupial.     

The opossum genome: insights and opportunities from an alternative mammal

The strategic importance of the genome sequence of the gray, short-tailed opossum, Monodelphis domestica, accrues from both the unique phylogenetic position of metatherian (marsupial) mammals and the fundamental biologic characteristics of metatherians that distinguish them from other mammalian species. Metatherian and eutherian (placental) mammals are more closely related to one another than to other vertebrate groups, and owing to this close relationship they share fundamentally similar genetic structures and molecular processes. However, during their long evolutionary separation these alternative mammals have developed distinctive anatomical, physiologic, and genetic features that hold tremendous potential for examining relationships between the molecular structures of mammalian genomes and the functional attributes of their components. Comparative analyses using the opossum genome have already provided a wealth of new evidence regarding the importance of noncoding elements in the evolution of mammalian genomes, the role of transposable elements in driving genomic innovation, and the relationships between recombination rate, nucleotide composition, and the genomic distributions of repetitive elements. The genome sequence is also beginning to enlarge our understanding of the evolution and function of the vertebrate immune system, and it provides an alternative model for investigating mechanisms of genomic imprinting. Equally important, availability of the genome sequence is fostering the development of new research tools for physical and functional genomic analyses of M. domestica that are expanding its versatility as an experimental system for a broad range of research applications in basic biology and biomedically oriented research.     

Further characterization of T cell receptor chains of marsupials

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.