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.     

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.     

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.     

Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system

The availability of the first marsupial genome sequence has allowed us to characterize the immunome of the gray short-tailed opossum (Monodelphis domestica). Here we report the identification of key immune genes, including the highly divergent chemokines, defensins, cathelicidins, and Natural Killer cell receptors. It appears that the increase in complexity of the mammalian immune system occurred prior to the divergence of the marsupial and eutherian lineages approximately 180 million years ago. Genomes of ancestral mammals most likely contained all of the key mammalian immune gene families, with evolution on different continents, in the presence of different pathogens leading to lineage specific expansions and contractions, resulting in some minor differences in gene number and composition between different mammalian lineages. Gene expansion and extensive heterogeneity in opossum antimicrobial peptide genes may have evolved as a consequence of the newborn young needing to survive without an adaptive immune system in a pathogen laden environment. Given the similarities in the genomic architecture of the marsupial and eutherian immune systems, we propose that marsupials are ideal model organisms for the study of developmental immunology.     

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.     

Postnatal lung and metabolic development in two marsupial and four eutherian species

Two marsupial species (Monodelphis domestica, Macropus eugenii) and four eutherian species (Mesocricetus auratus, Suncus murinus, Tupaia belangeri and Cavia aperea) were examined to compare and contrast the timing of lung and metabolic development during the postnatal maturation of the mammalian respiratory apparatus. Using light, scanning and transmission electron microscopy, the lung structural changes were correlated with indirect calorimetry to track the metabolic development. Marsupial and eutherian species followed the same pattern of mammalian lung development, but differed in the developmental pace. In the two newborn marsupial species, the lung parenchyma was at the early terminal sac stage, with large terminal air sacs, and the lung developed slowly. In contrast, the newborn eutherian species had more advanced lungs at the late terminal sac stage in altricial species (M. auratus, S. murinus) and at the alveolar stage in precocial species (T. belangeri, C. aperea). Postnatal lung development proceeded rapidly in eutherian species. The marsupial species had a low metabolic rate at birth and achieved adult metabolism late in postnatal development. In contrast, newborn eutherian species had high metabolic rates and reached adult metabolism during the first week of life. The time course of the metabolic development is thus tightly linked to the structural differentiation of the lungs and the timing of postnatal lung development. These differences in the neonatal lung structure and the timing of postnatal lung maturation between marsupial and eutherian species reflect their differing reproductive strategies.     

An analysis of the gene complement of a marsupial, Monodelphis domestica: evolution of lineage-specific genes and giant chromosomes

The newly sequenced genome of Monodelphis domestica not only provides the out-group necessary to better understand our own eutherian lineage, but it enables insights into the innovative biology of metatherians. Here, we compare Monodelphis with Homo sequences from alignments of single nucleotides, genes, and whole chromosomes. Using PhyOP, we have established orthologs in Homo for 82% (15,250) of Monodelphis gene predictions. Those with single orthologs in each species exhibited a high median synonymous substitution rate (d(S) = 1.02), thereby explaining the relative paucity of aligned regions outside of coding sequences. Orthology assignments were used to construct a synteny map that illustrates the considerable fragmentation of Monodelphis and Homo karyotypes since their therian last common ancestor. Fifteen percent of Monodelphis genes are predicted, from their low divergence at synonymous sites, to have been duplicated in the metatherian lineage. The majority of Monodelphis-specific genes possess predicted roles in chemosensation, reproduction, adaptation to specific diets, and immunity. Using alignments of Monodelphis genes to sequences from either Homo or Trichosurus vulpecula (an Australian marsupial), we show that metatherian X chromosomes have elevated silent substitution rates and high G+C contents in comparison with both metatherian autosomes and eutherian chromosomes. Each of these elevations is also a feature of subtelomeric chromosomal regions. We attribute these observations to high rates of female-specific recombination near the chromosomal ends and within the X chromosome, which act to sustain or increase G+C levels by biased gene conversion. In particular, we propose that the higher G+C content of the Monodelphis X chromosome is a direct consequence of its small size relative to the giant autosomes.     

Molecular characterisation of the tammar wallaby (Macropus eugenii) CD3 epsilon chain cDNA.

The cDNA encoding the epsilon chain of the tammar wallaby CD3 complex (CD3epsilon) was isolated by PCR. This is the first CD3 component to be cloned in a marsupial. The tammar wallaby cDNA coding region was 61.7 and 63.0% identical to the human and mouse cDNA coding sequences, respectively. Similarly, the predicted amino acid sequence was 56.5 and 52.9% identical to the human and mouse sequences. When compared with other known CD3epsilon peptide sequences, the most conserved region of the tammar wallaby CD3epsilon chain peptide was the cytoplasmic domain and the least conserved was the extracellular portion. Phylogenetic reconstruction based on the deduced amino acid sequence placed the tammar wallaby sequence in its expected position outside of all the eutherian mammals.     

Interferon alpha/beta genes from a marsupial, Macropus eugenii

This paper describes the cloning of full length marsupial type I interferon (IFN) genes and their flanking regions using a genome walking approach and PCR primers based on previously isolated partial DNA sequences. We confirm that the two major classes of Tammar Wallaby type I IFN genes are homologous with the eutherian IFN-alpha and IFN-beta gene families. The wallaby IFN genes share a number of conserved features with their eutherian counterparts, including codons for cysteines at equivalent positions, implying similar secondary structures for the encoded proteins, and promoter regions with conserved putative regulatory motifs. Moreover, the wallaby genes have AT-rich elements in their flanking sequence corresponding to the mRNA 3'-untranslated regions, also implying that, as in eutherian mammals, rapid mRNA degradation plays a role in regulating expression of these genes. The complex nature of the type I IFN gene families in viviparous mammals (eutherians and marsupials) may reflect their recruitment into nonimmunological processes and this concept is discussed.     

Histology of the pouch epithelium and the mammary glands during chemically induced oestrus in the brushtail possum (Trichosurus vulpecula)

Changes in the epithelium of the maternal pouch and the mammary gland of brushtail possums (Trichosurus vulpecula) were examined after animals were treated to induce ovulation with follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mares' serum gonadotrophin (PMSG) and oestradiol. The mammary glands were similar in appearance to those described in eutherian mammals and in previous studies on other marsupials. Exposure of possums to these compounds, particularly PMSG, appeared to result in changes in the mammary glands that could be associated with milk/secretion production. In contrast, the pouch epithelium had a similar histological appearance to that of epithelium from other parts of the body regardless of whether the animal was exposed to stimulants. These preliminary observations are discussed in the context of the purported role of the pouch epithelium and the mammary gland in production of secretions at oestrus and provision of immunological protection to the neonatal marsupial.     

Histological and immunohistological investigation of the lymphoid tissue in normal and mycobacteria-affected specimens of the Rufous Hare-wallaby (Lagorchestes hirsutus)

The histology of the spleen, lymph nodes, Gut‐associated lymphoid tissue (GALT) and Bronchus‐associated lymphoid tissue (BALT) are described for samples collected opportunistically from healthy and mycobacteria‐affected specimens of the endangered marsupial Lagorchestes hirsutus, the Rufous Hare‐wallaby. The structural elements, organization and distribution of T and B lymphocytes determined by immunohistological techniques using species cross‐reactive antibodies in the lymph nodes, spleen and GALT of this species demonstrated lymphoid cell distributions that were consistent with other marsupial and eutherian mammals. The tissues of animals identified as acid‐fast positive displayed immunopathology consistent with the responses to intracellular bacteria displayed in some eutherian mammals and included the presence of focal lesions, giant cells in the lung and lymphoid aggregations situated adjacent to blood and airway vessels. This is the first study to describe the lymphoid tissue of this rare macropod species and the first to document the tissue bed response to mycobacteria.     

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.     

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.     

Evidence for an early appearance of modern post-switch immunoglobulin isotypes in mammalian evolution (II); cloning of IgE,IgG1 and IgG2 from a monotreme,the duck-billed platypus,Ornithorhynchus anatinus

To trace the emergence of the modern post-switch immunoglobulin (Ig) isotypes in vertebrate evolution we have studied Ig expression in mammals distantly related to eutherians. We here present an analysis of the Ig expression in an egg-laying mammal, a monotreme, the duck-billed platypus (Ornithorhynchus anatinus). Fragments of platypus IgG and IgE cDNA were obtained by a PCR-based screening using degenerate primers. The fragments obtained were used as probes to isolate full-length cDNA clones of three platypus post-switch isotypes, IgG1, IgG2, and IgE. Comparative amino acid sequence analysis against IgY, IgE and IgG from various animal species revealed that platypus IgE and IgG form branches that are clearly separated from those of their eutherian (placental) counterparts. However, the platypus IgE and IgG still conform to the general structure displayed by the respective Ig isotypes of eutherian and marsupial mammals. According to our findings, all of the major evolutionary changes in the expression array and basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the separation of monotremes from marsupial and placental mammals. Hence, our results indicate that the modern post-switch isotypes appeared very early in the mammalian lineage, possibly already 310-330 million years ago.