Isoforms of the CD79 signal transduction component of the macropod B-cell receptor

B cell responses and their concomitant signal transduction pathways are not well understood in marsupial mammals, despite the availability of gene expression data for key immunoglobulin genes and for elements of the CD79a/CD79b heterodimer signalling complex for two model marsupials. Broader studies of factors that influence B cell responses are still hampered by a lack of species-specific reagents and there are few reports of other factors that influence gene expression such as the potential for splice variants in BCR components, which may influence immune signalling pathways. In this study, we characterise CD79a and CD79b genes in the endangered macropod marsupial, Onychogalea fraenata (the bridled nailtail wallaby) and show that domains and residues important for the structural and functional integrity of both monomers are conserved in this species, consistent with results previously reported for the closely-related macropod, Macropus eugenii (the tammar wallaby). We extend this work to report the detection of splice variants for CD79a and CD79b in wallaby species; three CD79a isoforms and one CD79b isoform. Of these, two CD79a isoforms and the CD79b isoform have not been reported in any other mammalian species.     

Isoforms of the CD79 signal transduction component of the macropod B-cell receptor

B cell responses and their concomitant signal transduction pathways are not well understood in marsupial mammals, despite the availability of gene expression data for key immunoglobulin genes and for elements of the CD79a/CD79b heterodimer signalling complex for two model marsupials. Broader studies of factors that influence B cell responses are still hampered by a lack of species-specific reagents and there are few reports of other factors that influence gene expression such as the potential for splice variants in BCR components, which may influence immune signalling pathways. In this study, we characterise CD79a and CD79b genes in the endangered macropod marsupial, Onychogalea fraenata (the bridled nailtail wallaby) and show that domains and residues important for the structural and functional integrity of both monomers are conserved in this species, consistent with results previously reported for the closely-related macropod, Macropus eugenii (the tammar wallaby). We extend this work to report the detection of splice variants for CD79a and CD79b in wallaby species; three CD79a isoforms and one CD79b isoform. Of these, two CD79a isoforms and the CD79b isoform have not been reported in any other mammalian species.     

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.     

Physical map of two tammar wallaby chromosomes: A strategy for mapping in non-model mammals

Marsupials are especially valuable for comparative genomic studies of mammals. Two distantly related model marsupials have been sequenced: the South American opossum (Monodelphis domestica) and the tammar wallaby (Macropus eugenii), which last shared a common ancestor about 70 Mya. The six-fold opossum genome sequence has been assembled and assigned to chromosomes with the help of a cytogenetic map. A good cytogenetic map will be even more essential for assembly and anchoring of the two-fold wallaby genome. As a start to generating a physical map of gene locations on wallaby chromosomes, we focused on two chromosomes sharing homology with the human X, wallaby chromosomes X and 5. We devised an efficient strategy for mapping large conserved synteny blocks in non-model mammals, and applied this to generate dense maps of the X and ‘neo-X’ regions and to determine the arrangement of large conserved synteny blocks on chromosome 5. Comparisons between the wallaby and opossum chromosome maps revealed many rearrangements, highlighting the need for comparative gene mapping between South American and Australian marsupials. Frequent rearrangement of the X, along with the absence of a marsupial XIST gene, suggests that inactivation of the marsupial X chromosome does not depend on a whole-chromosome repression by a control locus. Electronic supplementary material  The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

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.     

Generation of reactive oxygen responses by monotreme and marsupial granulocytes

The granules of circulating leukocytes contain reactive oxygen species that are important components of host defence against bacterial invasion. We report the capacity of marsupials and monotremes to mount such a defence in a manner similar to their eutherian relatives. Using the nitroblue tetrazolium (NBT) test, reactive oxygen species were detected in the peripheral blood cells of five captive marsupial species (the tammar wallaby, Macropus eugenii, the Rufous hare wallaby, Lagorchestes hirsutus, the Brush-tailed bettong, Bettongia penicillata, the Long-footed potoroo, Potorous longipes, and the Long-nosed potoroo, Potorous tridactylus). The study included animals that were clinically healthy and those that were affected by mycobacterial disease. Animals in poor health elicited weak responses, consistent with the NBT test being used as a diagnostic assay for immunodeficiency. The NBT slide assay was also applied to platypus (Ornithorhyncus anatinus) and short-beaked echidna (Tachyglossus aculeatus) granulocytes to confirm the applicability of the test to this unique group of mammals. Presented in part in poster form at the 8th Congress of the International Society of Developmental and Comparative Immunology (Cairns, Australia, 2000).     

Immunocytochemical identification of CD5 positive peripheral blood lymphocytes in four marsupial species

 Immunocytochemical analysis of peripheral blood mononuclear cells was undertaken using a streptavidin biotin–horseradish peroxidase method to detect CD5 positive lymphocytes from the blood of several marsupial species. A monoclonal antibody raised to a conserved peptide sequence of the human CD5 antigen positively labelled lymphocytes in freshly isolated peripheral blood mononuclear cells of the tammar wallaby (Macropus eugenii), the long-footed potoroo (Potorous longipes), the long-nosed potoroo (Potorous tridactylus) and the rufous hare-wallaby (Lagorchestes hirsutus). A polyclonal anti-CD3 antibody also positively labelled circulating lymphocytes from the tammar wallaby. Whereas previous studies using flow cytometry reported labelling of T cells in koala lymphocyte preparations using a polyclonal anti-CD3 antibody, there have been no other reports of marsupial blood immunophenotyping. The current study extends the known applications of monoclonal anti-CD5 and polyclonal anti-CD3 antibodies to blood lymphocytes of small wallaby species using an immunocytochemical slide technique that is simple, can be processed within a day and requires no dedicated large equipment. Received: 2 July 2002 / Accepted: 23 August 2002 Acknowledgements We thank Ron Claassens of Macquarie University Fauna Park (New South Wales, Australia) for assistance with Tammar wallabies; Veterinary Staff at Healesville Sanctuary (Victoria, Australia) for potoroo blood samples; Ro McFarlane of Alice Springs Veterinary Clinic (Northern Territory, Australia) for Mala samples and Margaret Jones of the Leukaemia Research Foundation (Oxford, UK) for the donation of the monoclonal CD3 and CD5 antibodies. Lauren Young was supported by an Australian Postgraduate Award during the period of this study.     

Evolutionary history of novel genes on the tammar wallaby Y chromosome: Implications for sex chromosome evolution

We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis-brain expressed genes on the X.     

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

The inactivation of one of the two X chromosomes in female placental mammals represents a remarkable example of epigenetic silencing. X inactivation occurs also in marsupial mammals, but is phenotypically different, being incomplete, tissue-specific and paternal. Paternal X inactivation occurs also in the extraembryonic cells of rodents, suggesting that imprinted X inactivation represents a simpler ancestral mechanism. This evolved into a complex and random process in placental mammals under the control of the XIST gene, involving notably variant and modified histones. Molecular mechanisms of X inactivation in marsupials are poorly known, but occur in the absence of an XIST homologue. We analysed the specific pattern of histone modifications using immunofluorescence on metaphasic chromosomes of a model kangaroo, the tammar wallaby. We found that all active marks are excluded from the inactive X in marsupials, as in placental mammals, so this represents a common feature of X inactivation throughout mammals. However, we were unable to demonstrate the accumulation of inactive histone marks, suggesting some fundamental differences in the molecular mechanism of X inactivation between marsupial and placental mammals. A better understanding of the epigenetic mechanisms underlying X inactivation in marsupials will provide important insights into the evolution of this complex process. Edda Koina and Julie Chaumeil contributed equally to this work.     

Primary structure and variation of the T-cell receptor delta-chain from a marsupial,Macropus eugenii

Although gammadelta T-cells form only a small portion of circulating T-cells in mice and humans, they are more frequent in many other types of mammals and this has lead to speculation regarding their roles and the evolutionary significance of their relative abundance. Moreover, whilst clear homologues of four types of T-cell receptor (TCR) chains (alpha, beta, delta and gamma) have been identified in vertebrates as distantly related as eutherian mammals and cartilaginous fish, there are still many gaps in our knowledge of these TCR components from various taxa. Such knowledge would further illuminate the evolution and function of these receptors and of gammadelta T-cells. Here, we report the molecular cloning of a TCR-delta chain cDNA from the tammar wallaby (Macropus eugenii) which represents the first component of the gammadelta TCR to be characterised from a marsupial. A PCR-based survey of variable (V) segment usage in tammar wallaby mammary-associated lymph node indicated that, although gammadelta T-cells may be sparse in this type of tissue, this species has at least three subfamilies of V genes that have been broadly conserved across vertebrate evolution. Two V subfamilies found in the tammar wallaby were relatively similar and may have diverged more recently, an event that probably occurred at some point in the marsupial lineage.     

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.     

Chromosome Painting in Marsupials: Genome Conservation in the Kangaroo Family

In order to deduce the ancestral genome arrangement in the karyotypically diverse marsupial family Macropodidae, and to assess chromosome change in this family, chromosome-specific paints from the tammar wallaby (2n = 16) were hybridized to metaphase spreads from the two species proposed to represent the 2n = 22 ancestral karyotype, as well as species with derived 2n = 20 and 2n = 14 karyotypes. Identical patterns were observed in the two 2n = 22 species, from which the rearrangements to form the three derived karyotypes may be easily deduced to be 1, 3 and 4 different fusions, respectively. The identical Thylogale and Dorcopsis genomes may both be used to represent the pleisiomorphic macropodid chromosome complement. Variation in the X chromosome was also investigated by hybridizing an X-Y shared tammar wallaby 12-kb repeat element to chromosomes from the other four macropodid species, finding that it hybridized only to the most closely related species, and therefore is of recent origin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2–3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.     

Spermiogenesis and spermiation in a marsupial, the tammar wallaby (Macropus eugenii)

Fourteen steps of spermatid development in the tammar wallaby (Macropus eugenii), from the newly formed spermatid to the release of the spermatozoon into the lumen of the seminiferous tubules, were recognised at the ultrastructural level using transmission and scanning electron microscopy. This study confirmed that although the main events are generally similar, the process of the differentiation of the spermatid in marsupials is notably different and relatively more complex than that in most studied eutherian mammals and birds. For example, the sperm head rotated twice in the late stage of spermiogenesis: the shape of the spermatid changed from a T-shape at step 10 into a streamlined shape in step 14, and then back to T-shape in the testicular spermatozoa. Some unique figures occurring during the spermiogenesis in other marsupial species, such as the presence of Sertoli cell spurs, the nuclear ring and the subacrosomal space, were also found in the tammar wallaby. However, an important new finding of this study was the development of the postacrosome complex (PAC), a special structure that was first evident as a line of electron dense material on the nuclear membrane of the step 7 spermatid. Subsequently it became a discontinuous line of electron particles, and migrated from the ventral side of the nucleus to the area just behind the posterior end of the acrosome, which was closely located to the sperm–egg fusion site proposed for Monodelphis domestica (Taggart et al. 1993). The PAC and its possible role in both American and Australian marsupials requires detailed examination. Distinct immature features were discovered in the wallaby testicular spermatozoa. A scoop shape of the acrosome was found on the testicular spermatozoa of the tammar wallaby, which was completely different to the compact button shape of acrosome in ejaculated spermatozoa. The fibre network found beneath the cytoplasm membrane of the midpiece of the ejaculated sperm also did not occur in the testicular spermatozoa, although the structure of the principal piece was fully formed and had no obvious morphological difference from that of the epididymal and ejaculated spermatozoa. The time frame of the formation of morphologically mature spermatozoa in the epididymis of the tammar wallaby needs to be determined by further studies.     

Culture and characterisation of peripheral blood monocytes and monocyte-derived adherent cells of the tammar wallaby, Macropus eugenii

Monocytes, monocyte-derived adherent cells and dendritic cells all play a role in cellular immunity. In this study, we describe the isolation of monocyte-derived adherent cells and dendritic cells from a model marsupial, the tammar wallaby, Macropus eugenii, and report that in vitro, these cells appear morphologically similar to these cells found in other mammals. The successful culture of marsupial monocyte and dendritic cells was undertaken in serum-free medium which contained lymphocyte conditioned medium as an absolute requirement. This supports the view that similar to cultured dendritic cells from other species reported to date, specific growth factors are required to promote the maturation and differentiation of these cells.