Physical mapping of the IGF2 locus in the South American opossum Monodelphis domestica

The South American opossum Monodelphis domestica has been a model organism for marsupials for many years and has recently been the subject of a large-scale genome sequencing effort that will provide the foundation for comparative studies of gene function and regulation. Genomic imprinting is one mechanism of gene regulation that has received increasing attention due to the impact of imprinting defects on development and disease. We have mapped the imprinted insulin-like growth factor II (IGF2) gene of M. domestica as a first step in understanding the regulatory mechanisms involved in genomic imprinting in this marsupial.     

Divergent neuropeptide evolutionary drifts between American and Australian marsupials

Present-day marsupials, which are supposed to have arisen from a single stem diverging from the placental stem some 130 million years ago, exist only in the American and Australian continents. Comparison of the homologous genes and their protein products, which evolved under different environmental conditions, may provide arguments for either selective or neutral evolution. In contrast to Australian Macropodidae, which have pecuIiar neurohypophysial peptides, namely mesotocin and two pressor peptides, lysine vasopressin and phenypressin, the South American oppossum,Didelpbis marsupialis, has oxytocin, lysine vasopressin, and arginine vasopressin. Because placental mammals have oxytocin and usually arginine vasopressin, and nonmammalian tetrapods have mesotocin and arginine vasotocin, it is assumed that (1) selective change of arginine vasotocin into arginine vasopressin occurred in mammalian ancestors and a subsequent gene duplication in the marsupial line gave rise to two pressor peptides with divergent neutral drifts in American and Australian groups, and (2) mesotocin of nonmammalian tetrapods has been preserved in Australian marsupials and reclaimed for milk-ejecting function whereas it has been converted into oxytocin in South American oppossums. The change of mesotocin into oxytocin seems neutral rather than selective.     

Marsupial light chains: IGK with four V families in the opossum Monodelphis domestica

 A full-length and several partial cDNAs encoding IGK light chains from the marsupial South American opossum, Monodelphis domestica, were isolated and characterized. Using these clones as a starting point, the expressed IGKV repertoire was sampled by anchored polymerase chain reaction using an IGKC-specific primer. Based on nucleotide sequences of twenty unique, expressed IGKV-J combinations, there are at least four IGKV families and two J segments. Southern blot analysis revealed each IGK-V family contains multiple gene segments totaling at least thirty-five IGKV in the opossum genome. No evidence for particular, recurrent IGKV-J combinations in the opossum IGK repertoire was seen, rather the V-J combinations appeared random and diverse. Each of the four IGKV families appear more closely related to V segments from placental mammals than to each other, suggesting the duplication of the IGKV families prior to the separation of marsupials and placental mammals more than one-hundred-million years ago. Overall, the complexity of opossum light chain V segments appears greater than that found in the heavy chain, and light chains are likely to contribute significantly to Ig diversity in this species.With this report, the homologues encoding all three classes of eutherian Ig chains, IGH, IGL, and IGK, have been described in a non-placental mammal. Received: 5 April 1999 / Revised: 3 June 1999     

The organization of somatosensory cortex in the short-tailed opossum ( Monodelphis domestica )

The organization of neocortex in the short-tailed opossum ( Monodelphis domestica ) was explored with multiunit microelectrode recordings from middle layers of cortex. Microelectrode maps were subsequently related to the chemoarchitecture of flattened cortical preparations, sectioned parallel to the cortical surface and processed for either cytochrome oxidase (CO) or NADPH-diaphorase (NADPHd) histochemistry. The recordings revealed the presence of at least two systematic representations of the contralateral body surface located in a continuous strip of cortex running from the rhinal sulcus to the medial wall. The primary somatosensory area (S1) was located medially while secondary somatosensory cortex (S2) formed a laterally located mirror image of S1. Auditory cortex was located in lateral cortex at the caudal border of S2, and some electrode penetrations in this area responded to both auditory and somatosensory stimulation. Auditory cortex was outlined by a dark oval visible in flattened brain sections. A large primary visual cortex (V1) was located at the caudal pole of cortex, and also consistently corresponded to a large chemoarchitecturally visible oval. Cortex just rostral and lateral to V1 responded to visual stimulation, while bimodal auditory/visual responses were obtained in an area between V1 and somatosensory cortex. The results are compared with brain organization in other marsupials and with placentals and the evolution of cortical areas in mammals is discussed.     

Reproduction, development and physiology of the gray short-tailed opossum (Monodelphis domestica)

The gray short-tailed opossum (Monodelphis domestica) has become a useful marsupial for biomedical research since it breeds reliably in the laboratory. During a 3 year period (1983-1986), a total of 129 litters were born in the colony at NJMS resulting in five generations of captive-born animals. Litter size ranged from 2 to 13 and the average sex ratio at weaning was 1:1. Mean body temperature of M. domestica was approximately 32.3 degrees C and respiratory rate approximately 54 breaths/minute. Systolic blood pressures of approximately 188 mmHg and heart rates of 345 beats/minute were found. Techniques of blood collection, anesthesia, implanting of hormones, ovariectomies and castrations, and treatment of neonates are described as well as current practices of colony management.     

Mammary glands in male marsupials: I. Primordia in neonatal opossums Didelphis virginiana and Monodelphis domestica

Neonates of the American didelphid marsupials Didelphis virginiana and Monodelphis domestica were sexed by karyotype and histologically examined on the day of birth. Mammary anlagen were found in both sexes of both species, but the neonatal males had less than one-third of the full female complement of mammary glands. Male neonates of both species also had paired scrotal bulges anterior to the genital tubercle but these were never present in females, once again raising the question of whether the pouch and scrotum are homologous structures. Mammary anlagen are not found in male neonates of the Australian marsupial species so far studied, which suggests a dichotomy in the control of some aspects of sexual differentiation in the two marsupial lineages.     

Age-dependent changes in the proteome following complete spinal cord transection in a postnatal South American opossum (Monodelphis domestica)

Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies.     

Timetable of in vivo embryonic development in the grey short-tailed opossum (Monodelphis domestica)

The timing of development was examined in 496 embryos from female Monodelphis domestica, collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three-celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two-cell stage. The four-cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96–100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.© 1994 Wiley-Liss, Inc.     

An evaluation of three anaesthetic regimes in the gray short-tailed opossum (Monodelphis domestica)

The effect of several anaesthetic agents on the gray short-tailed opossum (Monodelphis domestica) was investigated. Pentobarbitone sodium at a dose of 50 mg/kg sedated the animals but did not produce analgesia or anaesthesia. A combination of ketamine hydrochloride and xylazine at 40 mg/kg and 5 mg/kg, respectively, sedated the animals, but anaesthetic levels were not attained. Halothane was most effective in producing anaesthesia in Monodelphis domestica. Hypothermia was a major side effect with all three anaesthetic regimes.     

The hormonal control of birth behavior in the gray short-tailed opossum (Monodelphis domestica)

In all major groups of Australian marsupials, prostaglandin F2alpha (PGF) or oxytocin injection initiates birth behavior in adult females, adult males and pouch young. Because inhibitors of PGF synthesis block this initiation, oxytocin may activate birth behavior via the stimulation of PGF synthesis. In this study, the role of PGF and oxytocin in the activation of birth behavior was examined in an American marsupial, the gray short-tailed opossum (Monodelphis domestica). Adult male and female gray opossums were given PGF, oxytocin, or saline (control) before behavioral observation. On the next day, the animals in the oxytocin group were injected with the PGF inhibitor flunixin meglumide (Finadyne, Schering Corp., U.S.A.) before oxytocin reinjection and behavioral observation. Both males and females showed birth behavior in response to PGF but only females responded to oxytocin. There was no significant difference in the latency of response of females to oxytocin alone versus response to oxytocin after receipt of the PGF inhibitor. These results suggest that, in contrast to Australian species, in this American marsupial, oxytocin initiates birth behavior only in females and does not operate via stimulation of prostaglandin secretion.     

Biochemical genetics of alcohol dehydrogenase isozymes in the gray short-tailed opossum (Monodelphis domestica)

Polyacrylamide gel-isoelectric focusing (PAGE-IEF) methods were used to examine the multiplicity, tissue distribution, and biochemical genetics of alcohol dehydrogenase (ADH) isozymes among gray short-tailed opossums (Monodelphis domestica). Seven ADH isozymes were resolved and distinguished on the basis of their isoelectric points, tissue distributions, and substrate and inhibitor specificities. ADH1 and ADH2 exhibited Class I properties and were observed in liver (and intestine) extracts. ADH3, ADH4, and ADH5 showed “high-K _m ” (possibly Class IV) properties, with ADH3 and ADH4 exhibiting high activity in cornea, ear, stomach, and esophagus extracts. ADH6 and ADH7 exhibited Class III properties, including activities as formaldehyde dehydrogenases, with each showing different tissue distribution characteristics; ADH6 was widely distributed, and ADH7 was restricted to prostate extracts. An additional form of formaldehyde dehydrogenase (FDH) was observed, which was inactive with hexenol and ethanol as substrates. Isoelectric point variants were observed for ADH3 (three forms) and for ADH4 (two forms), and the inheritance of ADH3 was studied in 15 families ofM. domestica. The data were consistent with codominant inheritance of two alleles (ADH3*A andADH3*B) at a single autosomal locus (designatedADH3) and with a model involving a dimeric ADH isozyme: ADH3 (γ₂ isozyme, forming three dimers designated γ ₂ ¹ , γ¹ γ², and γ ₂ ² in heterozygous individuals).     

Proteins in cerebrospinal fluid and plasma of postnatal Monodelphis domestica (grey short-tailed opossum)

1. The protein composition and concentrations of total protein in cerebrospinal fluid (CSF) and plasma of opossum (Monodelphis domestica) from birth until adulthood have been estimated. 2. Total protein in CSF increased from birth to a peak concentration between 5 and 10 days (500 mg/100 ml) after which it declined rapidly. 3. Total protein in plasma started at a low level at birth (below 400 mg/100 ml) and increased progressively to the adult value (8500 mg/100 ml). 4. Proteins identified in CSF and plasma were: albumin, alpha 1 antitrypsin, transferrin, alpha 2 macroglobulin, lipoproteins and immunoglobulin G. A fetal protein, probably alpha-fetoprotein, has been identified. 5. The only plasma proteins found within the brain, even in the very immature newborn of this species, appeared to have an intracellular rather than an extracellular distribution. This indicates that the blood-brain barrier is present very early in development and that there is also a CSF-brain barrier that appears to exclude CSF protein from brain extracellular space.     

Comparative analysis of alphaglobin and carbonic anhydrase I-cDNAs of the opossum Monodelphis domestica with related vertebrate sequences

Alphaglobin and carbonic anhydrase I coding sequences and part of their flanking untranslated regions were isolated from a cDNA library of the opossum Monodelphis domestica. This paper describes their characteristics and presents a brief phylogenetic analysis of these sequences and related ones.     

C-->U editing of apolipoprotein B mRNA in marsupials: identification and characterisation of APOBEC-1 from the American opossum Monodelphus domestica.

The C->U editing of RNA is widely found in plant and animal species. In mammals it is a discrete process confined to the editing of apolipoprotein B (apoB) mRNA in eutherians and the editing of the mitochondrial tRNA for glycine in marsupials. Here we have identified and characterised apoB mRNA editing in the American opossum Monodelphus domestica. The apoB mRNA editing site is highly conserved in the opossum and undergoes complete editing in the small intestine, but not in the liver or other tissues. Opossum APOBEC-1 cDNA was cloned, sequenced and expressed. The encoded protein is similar to APOBEC-1 of eutherians. Motifs previously identified as involved in zinc binding, RNA binding and catalysis, nuclear localisation and a C-terminal leucine-rich domain are all conserved. Opossum APOBEC-1 contains a seven amino acid C-terminal extension also found in humans and rabbits, but not present in rodents. The opossum APOBEC-1 gene has the same intron/exon organisation in the coding sequence as the eutherian gene. Northern blot and RT-PCR analyses and an editing assay indicate that no APOBEC-1 was expressed in the liver. Thus the far upstream promoter responsible for hepatic expression in rodents does not operate in the opossum. An APOBEC-1-like enzyme such as might be involved in C->U RNA editing of tRNA in marsupial mitochondria was not demonstrated. The activity of opossum APOBEC-1 in the presence of both chicken and rodent auxiliary editing proteins was comparable to that of other mammals. These studies extend the origins of APOBEC-1 back 170 000 000 years to marsupials and help bridge the gap in the origins of this RNA editing process between birds and eutherian mammals.     

Increase of annexin 1 immunoreactivity in spinal cord of newborn opossum (Monodelphis domestica) at the time when regeneration after injury stops being possible

Annexins constitute a family of proteins that associate reversibly with cell membranes in a calcium dependent manner. We have studied the distribution of annexin 1, which is known to mediate anti-inflammatory actions of glucocorticoids, and which is upregulated after spinal cord injury, in newborn and adult South American opossum (Monodelphis domestica) spinal cord. We show the increase in the annexin 1 immunoreactivity in spinal cords of neonatal opossums over the critical period when regeneration after injury ceases to be possible. We further show the restricted and specific sites at which it is detected in adult opossum cerebellum and hippocampus. Since the procedures used in immunochemistry of annexin in CNS have in the past yielded conflicting results, different procedures were tested and shown to be reliable. As a control, annexin 1 distribution was surveyed in kidney.