Monodelphis Research Articles

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 ( Monodelphis domestica) immunoglobulin gamma heavy chain constant region (C γ) probe, resulting in the isolation of a 1518 nucleotide cDNA clone. The sequence corresponds to exons 1–3 of C γ. The Australian marsupial ( T. vulpecula) 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.

Developmental analysis of the peripheral olfactory organ of the opossum Monodelphis domestica

The gray, short-tailed opossum, Monodelphis domestica, is born in a very immature state after a brief (14-day) gestation. As a result, the species provides a unique opportunity to examine very early periods of mammalian development. The present study provides the first detailed morphometric analysis of the development of the olfactory mucosa and the nasal cavity in Monodelphis. The extent of the sensory mucosa increases dramatically across development, covering a growing nasal cavity and increasingly elaborate turbinates. Both nasal cavity convolution (a measure of turbinate complexity) and mucosal surface area show extensive growth between birth and adulthood. These measurements are greatest in the central portion of the mucosa (in the caudal portion of the nose) at all ages examined. A developmental BrdU study reveals a robust decrease in cellular proliferation with age; proliferation decreases to near adult-like patterns by postnatal day (P) 40. Results from these studies show that there is dramatic structural and cellular postnatal growth in the opossum peripheral olfactory organ.

Organization of somatosensory cortex in three species of marsupials, Dasyurus hallucatus, Dactylopsila trivirgata, and Monodelphis domestica: neural correlates of morphological specializations.

The organization of somatosensory neocortex was investigated in three species of marsupials, the northern quoll (Dasyurus hallucatus), the striped possum (Dactylopsila trivirgata), and the short-tailed opossum (Monodelphis domestica). In these species, multiunit microelectrode mapping techniques were used to determine the detailed organization of the primary somatosensory area (SI). In the striped possum and quoll, the topography of somatosensory regions rostral (R), and caudal (C) to SI were described as well. Lateral to SI, two fields were identified in the striped possum, the second somatosensory area (SII) and the parietal ventral area (PV); in the quoll, there appeared to be only one additional lateral field which we term SII/PV. Visual and auditory cortices adjacent to somatosensory cortex were also explored, but the details of organization of these regions were not ascertained. In these animals, electrophysiological recording results were related to cortical myeloarchitecture and/or cytochrome oxidase staining. In one additional species, the fat-tailed dunnart (Sminthopsis crassicaudata), an architectonic analysis alone was carried out, and compared with the cortical architecture and electrophysiological recording results in the other three species. We discuss our results on the internal organization of SI in relation to the morphological specializations that each animal possesses. In addition, we discuss the differences in the organization of SI, and how evolutionary processes and developmental and adult neocortical plasticity may contribute to the observed variations in SI.

Isolation of mhc class II DMA and DMB cDNA sequences in a marsupial: the gray short-tailed opossum (Monodelphis domestica).

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the alpha-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified.

Development of thalamocortical projections in the South American gray short-tailed opossum (Monodelphis domestica)

We determined the time-course and general pattern of thalamocortical development of Monodelphis domestica by tracing projections with carbocyanine dye in fixed postnatal brains between postnatal day 2 (P2) and P30. By P2, the first neurons have migrated to form the preplate of the lateral cortex and have sent out axons into the intermediate zone. By P3, fibers from the preplate of more dorsal cortex have entered the intermediate zone, and, by P5, they reach the primitive internal capsule. Crystal placements in the dorsal thalamus at P2-P3 reveal thalamic axons extending down through the diencephalon and growing out through the internal capsule among groups of back-labelled cells that already project into the thalamus. Thalamic axons arrive at the cortex after the arrival of cells of the true cortical plate has split the preplate into marginal zone and subplate. Axons from the ventral part of the dorsal thalamus reach the lateral cortex by P5: Dorsal thalamic fibers arrive at the extreme dorsal cortex by P9. The deeper layers of the cortex appear to mature relatively earlier in Monodelphis than in eutherian mammals, and the subplate becomes less distinct. Thalamic fibers and their side branches proceed into the cortex without an obvious period of waiting in the subplate, but they do not penetrate the dense cortical plate itself. Monodelphis could provide an excellent model species, because the development of its thalamocortical connections is entirely an extrauterine process: The period P0-P15 corresponds to that of E12-P0 in the rat.

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 (epsilon chain), IgG (gamma chain) and IgA (alpha 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.

Heterogeneity in the accessory olfactory system.

The mammalian accessory olfactory bulb (AOB) is chemoarchitecturally heterogeneous in that it stains differentially with a number of markers; the receptor cells that project to the AOB are similarly heterogeneous. What is the significance of this heterogeneity? We have found that the AOB of the gray, short-tailed opossum, Monodelphis domestica, stains differentially with a number of 'markers': antibodies to olfactory marker protein (OMP) and the alpha subunit of the G protein Gi2, the lectin of Vicia villosa and NADPH-diaphorase. These markers stain the rostral AOB more strongly than the caudal AOB whereas, the G protein subunit G(o) alpha is located predominantly in the posterior subdivision of the AOB. This heterogeneity in the chemoarchitecture of the AOB may reflect a fundamental organizational dichotomy within the vomeronasal system that corresponds to a functional dichotomy. The vomeronasal sensory epithelium also exhibits a chemoarchitectural heterogeneity: receptor cells in the basal third are G(o) alpha-immunoreactive whereas the cells in the middle third are Gi2 alpha-immunoreactive. Tracing studies using WGA-HRP demonstrate that the neurons in the middle third of the vomeronasal sensory epithelium project their axons to the anterior AOB whereas those in the basal third appear to project to the posterior AOB.

Transient, high levels of SNAP-25 expression in cholinergic amacrine cells during postnatal development of the mammalian retina.

In the present study, we have examined the development of cholinergic amacrine cells in the retina of the Brazilian opossum, Monodelphis domestica. An antibody directed against choline acetyltransferase (ChAT) revealed that ChAT-like immunoreactivity (ChAT-IR) was first observed at 15 days postnatal (15PN). By 25PN, ChAT-IR identified two matching populations of amacrine cells in the inner nuclear and ganglion cell layer. Bromodeoxyuridine birth-dating analysis coupled with immunolabeling with the anti-ChAT antibody revealed that the cholinergic amacrine cells are born postnatally, between 2PN and 15PN. In addition, we have examined the differentiation of the cholinergic amacrine cells by using an antibody directed against a presynaptic terminal-associated protein, synaptosomal-associated protein of 25 kDa (SNAP-25). Double-labeling analysis revealed that relatively high levels of SNAP-25-IR were selectively present in cholinergic amacrine cells prior to eye opening. However, in the mature retina, high levels of SNAP-25-IR were no longer observed in the ChAT-IR amacrine cells. These results reveal a distinct period in development, prior to eye opening, when high levels of SNAP-25-IR are selectively expressed in cholinergic amacrine cells. The specificity and time course of the high levels of SNAP-25 in cholinergic amacrine cells may be critical in mediating the transient properties of these cells during visual system development.

Gene Maps of Marsupials.

Increased interest in the structural characteristics of mammalian genomes, together with rapid advances in mapping technology, have led to the explosive expansion of gene mapping activity in recent years. Species for which gene mapping data were virtually nonexistent just a decade ago now possess substantial and serviceable gene maps that are enabling the localization of economically and biomedically relevant loci and are contributing to a fuller understanding of genome evolution and the relationships between genome structure and gene function. Recent progress in the compilation and effective application of gene mapping data has been particularly evident in the fascinating group of species that comprise the mammalian infraclass Metatheria, better known as marsupial mammals. As late as 1988, only 22 marsupial genes had been reported as mapped by any method in any species (a gene is considered mapped if it adheres to any of the following criteria: (1) assigned to a specific chromosome; (2) a member of a linkage group; (3) autosomal in marsupials but known to be X or Y linked in eutherians). These few gene assignments were achieved by a variety of methods, and were scattered piecemeal among a dozen different species (this number excludes 14 additional species in which 1 or more ribosomal RNA (RNR) genes were the only loci mapped [Hayman and Rofe 1977; Hayman and Sharp 1981; Young and others 1982]). As a consequence, few species had more than 2 or 3 genes mapped, and only 1 species could boast as many as 9 gene assignments. Currently, at least 142 loci have been assigned to physical locations or linkage groups in marsupials, and more than 15 species have at least 3 gene assignments. Most important, 2 distantly related species, Macropus eugenii (tammar wallaby) with 70 loci mapped and Monodelphis domestica (gray, short-tailed opossum) with 69

VH Repertoire of a Marsupial (Monodelphis domestica)

AbstractWhen contrasted with information available for placental mammals, very little is known about the development of immunocompetence in marsupials. Marsupials, however, provide interesting immunology problems, since most appear to be born at a stage of development much less mature than that of placental mammals. To further understand the marsupial immune system, the Ig repertoire of the short-tailed opossum, Monodelphis domestica, was characterized. The majority of the VH clones were isolated in an unbiased manner by screening a spleen cDNA phage library, using C region probes, or anchored PCR, using C region-specific primers paired with vector specific primers. Analysis of 54 unique VH sequences from this marsupial revealed the presence of two VH families in the expressed Ig repertoire. The larger family, which contributed the majority of the clones identified, appears to be derived from 10 to 12 germline VH segments. The second family of clones is derived from a single germline VH. Both VH families are related to the group III sequences described in other vertebrates. Unusual codon bias differences between the two families may result in very different patterns of somatic mutation within the opossum Ig repertoire.

Features of the auditory development of the short-tailed Brazilian opossum, Monodelphis domestica: evoked responses, neonatal vocalizations and synapses in the inferior colliculus

The onset of hearing in anesthetized South American opossums (Monodelphis domestica) was determined by the measurement of evoked potentials to click stimuli from the vertex of the skull immediately over the inferior colliculus. Evoked potentials were first recorded at postnatal day 24 at a threshold of 83 dB SPL; thresholds declined over subsequent weeks to below 58 dB at 40 days. Isolation calls emitted by the pups had stereotypic spectra with peaks at near 13 kHz and an octave higher. Such calls declined in frequency by day 32 and were not emitted at day 40. The peak frequency of the calls matched very closely the best frequency of hearing of adult Monodelphis. The number of synapses in the inferior colliculus increased at day 26; when plotted in relation to the number of cells, synaptic density increased steeply from day 27 after the animal had begun to hear. This suggests that environmental sound has a potent effect on the development of synapses in the auditory system.

Evolution of shorter and more hydrophilic transthyretin N-termini by stepwise conversion of exon 2 into intron 1 sequences (shifting the 3' splice site of intron 1)

Transthyretin cDNA was cloned from Eastern Grey Kangaroo liver and its nucleotide sequence determined. Analysis of the derived amino acid sequence of kangaroo transthyretin, together with data obtained previously for transthyretins from other vertebrate species [Duan, W., Richardson, S. J., Babon, J. J., Heyes, R. J., Southwell, B. R., Harms, P. J., Wettenhall, R. E. H., Dziegielewska, K. M., Selwood, L., Bradley, A. J., Brack, C. M. & Schreiber, G. (1995) Eur. J. Biochem. 227, 396-406], showed that the N-terminus is the region which changes most distinctly during evolution. It has been shown for human, mouse and rat transthyretins, that this region is encoded by DNA at the border of exon 1 and exon 2. Therefore, this section of transthyretin genomic DNA was amplified by PCR and directly sequenced for the Buffalo Rat, Tammar Wallaby, Eastern Grey Kangaroo, Stripe-faced Dunnart, Short-tailed Grey Opossum and White Leghorn Chicken. The splice sites at both ends of intron 1 were identified by comparison with the cDNA sequences. The obtained data suggest that the N-termini of transthyretin evolved by successive shifts of the 3' splice site of intron 1 in the 3' direction, resulting in successive shortening of the 5' end of exon 2. At the protein level, this resulted in a shorter and more hydrophilic N-terminal region of transthyretin. Successive shifts in splice sites may be an evolutionary mechanism of general importance, since they can lead to stepwise changes in the properties of proteins. This could be a molecular mechanism for positive Darwinian selection.

Sex Differences and the Development of Social Behavior in a Marsupial, the Gray Short-Tailed Opossum ( Monodelphis Domestica)

Fadem, B. H. and A. Corbett. Sex differences and the development of social behavior in a marsupial, the gray short-tailed opossum (Monodelphis domestica). Physiol Behav 61(6) 857–861, 1997.—The effects of sex and age on social behavior were examined in gray short-tailed opossums ( Monodelphis domestica), small didelphid marsupials. Each animal received five behavior test batteries spanning prepubertal to postpubertal ages. Each test battery consisted of two tests with animals of the same age, one with a male and one with a female. Precopulatory behavior toward females, intermale fighting requiring test interruptions as well as scent marking behavior were seen at higher levels in males than in females and were seen more frequently around and after puberty than before puberty. Females showed more threat behavior than males in mixed-sex and in same-sex interactions. This sex difference was apparent after puberty in tests with male partners and prior to as well as around puberty in tests with female partners. Because climbing over and boxing with another animal were seen more frequently prior to than after puberty, these behaviors may be elements of play fighting (i.e. attack and defense without submission and threat). These findings are discussed with respect to the role of gonadal hormones in the organization and activation of behavior and with reference to their comparative significance in mammals.

Male Gray Short-Tailed Opossums (Monodelphis Domestica) Receive Penile Intromissions When Treated with Estrogen and Progesterone in Adulthood

Following treatment with estradiol and progesterone, gonadectomized male as well as female gray opossums received penile intromissions from intact stimulus males. Intromission was possible in male gray opossums because, like marsupials of both sexes, they possess a single cloaca-like anogenital opening. All subjects that allowed intromission showed anogenital dragging just prior to intromission. While intromission latency was similar in tests involving male and female subjects, total intromission duration was longer in tests involving male subjects than in tests involving female subjects, and sex locks were seen only in tests involving female subjects. These findings are discussed with respect to the potential usefulness of gray opossums for studying the effects of peripheral vs central factors on the display of sex differences in behavior.

Ontogeny of Androgen Receptor-Like Immunoreactivity in the Reproductive Tract of Male Monodelphis domestica 1

The distribution of nuclear androgen receptor-like immunoreactivity was studied in the reproductive tract of the developing and adult gray short-tailed Brazilian opossum (Monodelphis domestica), using the well-characterized rabbit polyclonal androgen receptor antibody, PG21. Androgen receptor-like immunoreactivity was first detected on the fifth day of postnatal age, in the mesenchymal tissues of the ductus deferens, gubernaculum testis, inguinal, and scrotal areas; the urogenital sinus; and the genital tubercle. Androgen receptor-like immunoreactivity was first seen in the interstitial cells of the epididymis at 45 days of age; the testes developed androgen receptor-like immunoreactivity at 60 days of age. The epithelium of prostatic glands contained androgen receptor-like immunoreactivity only in the adult. The presence of androgen receptor-like immunoreactivity during development correlated well with the known androgen dependence of the differentiation of most reproductive organs.

Development of the anterior commissure in the opossum: Midline extracellular space and glia coincide with early axon decussation

While the anterior commissure has been shown to be an important route of information transfer in the forebrain, relatively little is known about its anatomical development. Glial substrates and extracellular spaces have been associated with the maturation of other large-fiber tracts, such as the corpus callosum and retinofugal pathway. The present study examined early stages in the maturation of the commissure in the gray short-tailed opossum, Monodelphis domestica. Monodelphis offspring are born after a short 14-day gestation, and, unlike in rats and mice, the anterior commissure develops entirely during the postnatal period. A number of techniques were employed: the carbocyanine dye Dil was used to label early axons in the region, semithin plastic sections were used to examine the extracellular environment of the developing commissure, and immunocytochemistry for glial fibrillary acidic protein (GFAP) was used to characterize glial components. Results suggest that the first commissural fibers that cross the midline pass through a region of large extracellular spaces and may use GFAP-immunoreactive cells and processes as guides during their midline decussation.

Albumin transfer across the choroid plexus of South American opossum (Monodelphis domestica).

1. Blood-cerebrospinal fluid (CSF) transfer of various exogenous albumins has been investigated in developing Monodelphis domestica (South American grey short-tailed opossum) and compared with the steady-state CSF: plasma ratios for endogenous (Monodelphis) albumin. Ratios for Monodelphis albumin and human albumin were similar and were the highest at postnatal day 5 (P5) (48.2 +/- 4.4 and 40.6 +/- 4.5%, respectively). The ratio for bovine albumin was similar to the steady-state ratio for Monodelphis albumin at P7-8 but became consistently lower than the Monodelphis albumin ratio at all other ages until P32-36 when all albumins tested attained a similar low ratio. The CSF:plasma ratio of chemically modified (succinylated) bovine albumin was always significantly lower than that of other albumins, except at the oldest age examined (P32-36). 2. Immunocytochemistry showed that within the brain, albumin was confined to the lumen and endothelial cells of blood vessels. In the choroid plexus only a small proportion (0.2-1.7% of the total cell number) of epithelial cells was positive for albumin, both endogenous and exogenous, at all ages studied (except the 3rd ventricle where cells were only positive from P8). The CSF was strongly positive for all albumins. The peak proportion of positive cells and of albumin concentrations in CSF occurred at P8. These findings suggest that the primary route for penetration of albumin into CSF is directly across the choroid plexus rather than via the brain. 3. Double-labelling immunocytochemistry revealed that the same epithelial cells contained both endogenous (Monodelphis) and exogenous (human) albumin. In contrast, for succinylated albumin, at P7 only about 35% (lateral ventricle) and 50% (4th ventricle) of Monodelphis albumin-positive cells were also positive for succinylated albumin, but by P30 this proportion increased to 90% at both sites. 4. Thus the developing choroid plexus distinguishes between different albumins. Chemical modification of albumin (succinylation) disrupts this mechanism. It is proposed that in older animals (P32-36) all of the albumin in the CSF is derived from plasma by diffusion (as in adult animals). At earlier stages of development, a proportion of the albumin in CSF also appears to be transferred from the plasma by diffusion with an additional component transferred by a mechanism that can distinguish between different species of albumin. The main route of entry of albumin to CSF seems likely to be via the choroid plexus epithelial cells.

Autoradiographic localization of arginine vasopressin binding sites in the brain of adult and developing Brazilian opossums.

We are utilizing the Brazilian short-tailed opossum, Monodelphis domestica, to study the development of the vasopressinergic system. Earlier studies demonstrated that arginine vasopressin-like immunoreactivity was present very early in the Brazilian opossum brain, suggesting a role for vasopressin in the developing central nervous system of mammals. In this study, we have utilized [3H]arginine vasopressin autoradiography to describe the distribution of arginine vasopressin binding sites in adult and developing Brazilian opossum brains. In general, arginine vasopressin binding patterns in adult opossum brains resembled those of other species. However, we found very few labelled areas in neonatal Brazilian opossum brains. At birth, only the ventral tegmental area and the nucleus of the solitary tract were labelled. Binding was not evident in the forebrain until 25 days of postnatal age. The anterior pituitary was heavily labelled from birth onward, but binding in the brain itself remained at low levels until 35 days postnatal. Heavy binding was observed in only a few areas of the brain in adults, including the dorsal part of the lateral septal nucleus, the suprachiasmatic nucleus, the dorsal and median raphe, the nucleus of the solitary tract, and the caudal part of the spinal trigeminal nucleus. Surprisingly, arginine vasopressin binding sites in the Brazilian opossum appeared much later than arginine vasopressin immunoreactivity and, in many cases, after neurogenesis was complete. These findings suggest that the arginine vasopressin binding sites are not playing a developmental role in opossums, although the peptide is present at an early age.

Craniofacial Heterochrony and Sexual Dimorphism in the Short-Tailed Opossum (Monodelphis domestica)

Craniofacial growth of the short-tailed opossum, Monodelphis domestica , was measured to determine differences in growth between bones of the viscerocranium and of the neuro-cranium and between sexes. Initially, the relative lengths of neurocranial to viscerocranial bones were about the same for both sexes. Viscerocranial bones grew at a faster rate in both sexes, while neurocranial bones grew for a longer period of time at a slower rate. Although females exhibited faster rates of growth, they ultimately had smaller skulls than males, because males were initially larger and grew for a longer period of time. Differences in patterns of growth of the two regions of the skull probably are associated with changes in masticatory function due to weaning and development of the brain.

Ultrastructural study of ensheathing cells in early development of olfactory axons

Ultrastructural observations in the grey short-tailed opossum ( Monodelphis domestica) and rat revealed that ensheathing cells were intimately related to the early formation of olfactory axons. Whilst the axons were still in the olfactory epithelium, they were enveloped by ensheathing cell processes which formed a cradle-like structure on the basal side of the epithelium. Continued downgrowth of the axons towards the lamina propria resulted in an evagination with the ensheathing cell process or cell body in direct contact with the basal lamina. Subsequently the basal lamina became fragmented, and the newly formed olfactory nerve emerged from the olfactory epithelium. As the olfactory nerve grew, it was observed that the ensheathing cell process always extended ahead of the axons while axon terminals moving ahead of ensheathing cells were never observed. The findings in this study suggest strongly that ensheathing cells play a role in regulating and promoting olfactory axon growth.