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.     

Developmental profile of a fetuin-like glycoprotein in neocortex, cerebrospinal fluid and plasma of post-natal tammar wallaby (Macropus eugenii).

Summary A fetuin-like glycoprotein (FLG) has been shown to be present in early cortical plate cells in the developing brain of the tammar wallaby (Macropus eugenii). The developmental sequence of the occurrence of glycoprotein-positive fibres and cells in the dorsolateral telencephalic wall from newborn to day 40 is described. The level of FLG in CSF (cerebrospinal fluid) and plasma of the tammar wallaby has also been measured during pouch life. The presence of FLG in early postnatal fibre systems and in some cells in the primordial plexiform layer, as well as in early cortical plate cells of the tammar is similar to that of fetuin in fetal brain in sheep, pig and cow, and ₂HS glycoprotein in human fetal brain. The sequence of appearance of FLG-positive cells during neocortical development in the tammar is strikingly similar to that of a transient population of early cortical plate cells previously described in fetal cat and sheep cortex. During postnatal development, levels of FLG in tammar plasma and CSF follow a pattern different from that of other species. The developmental expression of all three related glycoproteins in their respective species is discussed.     

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.     

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.     

The common gamma chain cytokine interleukin‐21 is expressed by activated lymphocytes from two macropod marsupials,Macropus eugenii and Onychogalea fraenata

In mammals, interleukin‐21 is a member of the common gamma chain cytokine family that also includes IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. IL‐21 has pleiotropic effects on both myeloid and lymphoid immune cells and as a consequence, the biological actions of IL‐21 are broad: regulating both innate and adaptive immune responses and playing a pivotal role in antiviral, inflammatory and antitumour cellular responses. While IL‐21 genes have been characterized in mammals, birds, fish and amphibians, there are no reports for any marsupial species to date. We characterized the expressed IL‐21 gene from immune tissues of two macropod species, the tammar wallaby (Macropus eugenii), a model macropod, and the closely related endangered bridled nailtail wallaby (Onychogalea fraenata). The open reading frame of macropod IL‐21 is 462 nucleotides in length and encodes a 153‐mer putative protein that has 46% identity with human IL‐21. Despite the somewhat low amino acid conservation with other mammals, structural elements and residues essential for IL‐21 conformation and receptor association were conserved in the macropod IL‐21 predicted peptides. The detection of IL‐21 gene expression in T‐cell‐enriched tissues, combined with analysis of the promotor region of the tammar wallaby gene, suggests that macropod IL‐21 is expressed in stimulated T cells but is not readily detected in other cells and tissues. The similarity of gene expression profile and functionally important amino acid residues to eutherian IL‐21 makes it unlikely that the differences in B‐ and T‐cell responses that are reported for some marsupial species are due to a lack of important functional residues or IL‐21 gene expression in this group of mammals.     

Cytochemistry of leukocytes from the family Macropodidae

The cytochemical staining characteristics of leukocytes from four species of Macropodidae and one species of Potoroidae were studied to investigate the cellular composition of leukocytes within the Macropodidae family and determine markers that may be useful for identifying cell lineage. Blood smears from the tammar wallaby (Macropus eugenii), the western grey kangaroo (Macropus fuliginosis), the red kangaroo (Macropus rufus), quokka (Setonix brachyurus) and a potoroid species, the woylie (Bettongia pencillata) were examined following reaction for Sudan Black B (SBB), peroxidase (PER), chloracetate esterase (CAE), α-naphthyl butyrate esterase (NBE) and alkaline phosphatase (ALP). Similar to domestic animal species, the neutrophils and eosinophils of macropodid species stained for both SBB and PER while monocytes and lymphocytes showed little or no reaction. CAE and NBE, however, were not useful as markers for macropodid neutrophils and monocytes, respectively. Significant variation between species was seen in ALP content. Tammar wallabies and quokkas demonstrated strong ALP activity within their neutrophils and eosinophils. In contrast, western grey kangaroos and red kangaroos contained no ALP activity in any of their leukocytes and staining in woylies was limited to weak reaction within some eosinophils.     

Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes

The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species.     

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.     

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).     

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.     

Posttesticular development of spermatozoa of the tammar wallaby (Macropus eugenii)

Tammar wallaby spermatozoa undergo maturation during transit through the epididymis. This maturation differs from that seen in eutherian mammals because in addition to biochemical and functional maturation there are also major changes in morphology, in particular formation of the condensed acrosome and reorientation of the sperm head and tail. Of spermatozoa released from the testes, 83% had a large immature acrosome. By the time spermatozoa reached the proximal cauda epididymis 100% of sperm had condensed acrosomes. Similarly 86% of testicular spermatozoa had immature thumb tack or T shape head-tail orientation while only 2% retained this immature morphology in the corpus epididymis. This maturation is very similar to that reported for the common brush tail possum, Trichosurus vulpecula. However, morphological maturation occurred earlier in epididymal transit in the tammar wallaby. By the time spermatozoa had reached the proximal cauda epididymis no spermatozoa had an immature acrosome and thumbtack orientation. Associated with acrosomal maturation was an increase in acrosomal thiols and the formation of disulphides which presumably account for the unusual stability of the wallaby sperm acrosome. The development of motility and progressive motility of tammar wallaby spermatozoa is similar to that of other marsupials and eutherian mammals. Spermatozoa are immotile in the testes and the percentage of motile spermatozoa and the strength of their motility increases during epididymal transit. During passage through the caput and corpus epididymis, spermatozoa first became weakly motile in the proximal caput and then increasingly progressively motile through the corpus epididymis. Tammar wallaby spermatozoa collected from the proximal cauda epididymis had motility not different from ejaculated spermatozoa. Ultrastructural studies indicated that acrosomal condensation involved a complex infolding of the immature acrosome. At spermiation the acrosome of tammar wallaby spermatozoa was a relatively large flat or concave disc which projected laterally and anteriorly beyond the limits of the nucleus. During transit of the epididymal caput and proximal corpus the lateral projections folded inwards to form a cup like structure the sides of which eventually met and fused. The cavity produced by this fusion was lost as the acrosome condensed to its mature form as a small button-like structure contained within the depression on the anterior end of the nucleus. During this process the dorsal surface of the immature acrosome and its outer acrosomal membrane and overlying plasma membrane were engulfed into the acrosomal matrix. This means that the dorsal surface of the acrosomal region of the testicular tammar wallaby sperm head is a transient structure. The dorsal acrosomal surface of the mature spermatozoon appears ultrastructurally to be the relocated ventral surface of the acrosomal projections which previously extended out beyond the acrosomal depression on the dorsal surface of the nucleus of the immature spermatozoon.     

The role of oxytocin and regulation of uterine oxytocin receptors in pregnant marsupials

The oxytocin-like peptide of most Australian marsupials is mesotocin, which differs from oxytocin by a single amino acid. This substitution has no functional significance as both peptides have equivalent affinity for and biological activity on the marsupial oxytocin-like receptor. A role for mesotocin in marsupial parturition has been demonstrated in the tammar wallaby where plasma mesotocin concentrations increase less than one minute before birth. Infusion of an oxytocin receptor antagonist at the end of gestation disrupts normal parturition, probably by preventing mesotocin from stimulating uterine contractions. In the absence of mesotocin receptor activation, a peripartum surge in prostaglandins is delayed which suggests a functional relationship between mesotocin, prostaglandin release and luteolysis. Female marsupials have anatomically separate uteri and in monovular species, such as the tammar wallaby, only one uterus is gravid with a single fetus whereas the contralateral uterus remains non-gravid. We have used this unique animal model to differentiate systemic and fetal-specific factors in the regulation of uterine function during pregnancy. The gravid uterus in the tammar wallaby becomes increasingly sensitive to mesotocin as gestation proceeds, with the maximum contractile response observed at term. This is reflected in a large increase in mesotocin receptor concentrations in the gravid uterus, and a downregulation in the non-gravid uterus in late pregnancy. The upregulation in myometrial mesotocin receptors is pregnancy-specific and independent of systemic steroids. One factor that may influence mesotocin receptor upregulation in the gravid uterus in late pregnancy is mechanical stretch of the uterus caused by the growing fetus. Our data highlight that a local fetal influence is more important than systemic factors in the regulation of mesotocin receptors in the tammar wallaby.     

The human/mouse imprinted genesIGF2, H19, SNRPN andZNF127 map to two conserved autosomal clusters in a marsupial

The four genesIGF2, H19, SNRPN andZNF127 are imprinted in mouse and human.IGF2 andH19 form one conserved cluster on the distal part of mouse chromosome 7 and human chromosome 11p15.5, whereasSNRPN andZNF127 form another on the middle of mouse chromosome 7 and on human chromosome 15q11-13. We have explored the evolution of these imprinted regions by cloning and mappingIGF2, H19, SNRPN andZNF127 homeologues in marsupials. Specifically, we wished to determine whether the arrangements were shared in eutherian and marsupial mammals, and to determine whether they lay on autosomes, or on the X, as might be predicted by the hypothesis that imprinting evolved from X inactivation. Using fluorescencein situ hybridization, we localized the marsupial homeologues ofIGF2 andH19 to the distal part of tammar wallaby chromosome 2p and the marsupial homeologues ofSNRPN andZNF127 to the middle of chromosome 1q. Thus, these genes were originally organized in two separate autosomal clusters in the therian ancestor 180 million years ago, the conservation of which may suggest a functional relationship. The autosomal location of these clusters does not suggest a recent evolutionary relationship between imprinting and X chromosome inactivation.accepted for publication by M. Schmid     

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.