South American Opossum Monodelphis Domestica Research Articles

The rod signaling pathway in marsupial retinae.

The retinal rod pathway, featuring dedicated rod bipolar cells (RBCs) and AII amacrine cells, has been intensely studied in placental mammals. Here, we analyzed the rod pathway in a nocturnal marsupial, the South American opossum Monodelphis domestica to elucidate whether marsupials have a similar rod pathway. The retina was dominated by rods with densities of 338,000–413,000/mm². Immunohistochemistry for the RBC-specific marker protein kinase Cα (PKCα) and the AII cell marker calretinin revealed the presence of both cell types with their typical morphology. This is the first demonstration of RBCs in a marsupial and of the integration of RBCs and AII cells in the rod signaling pathway. Electron microscopy showed invaginating synaptic contacts of the PKCα-immunoreactive bipolar cells with rods; light microscopic co-immunolabeling for the synaptic ribbon marker CtBP2 confirmed dominant rod contacts. The RBC axon terminals were mostly located in the innermost stratum S5 of the inner plexiform layer (IPL), but had additional side branches and synaptic varicosities in strata S3 and S4, with S3-S5 belonging to the presumed functional ON sublayer of the IPL, as shown by immunolabeling for the ON bipolar cell marker Gγ13. Triple-immunolabeling for PKCα, calretinin and CtBP2 demonstrated RBC synapses onto AII cells. These features conform to the pattern seen in placental mammals, indicating a basically similar rod pathway in M. domestica. The density range of RBCs was 9,900–16,600/mm2, that of AII cells was 1,500–3,260/mm2. The numerical convergence (density ratio) of 146–156 rods to 4.7–6.0 RBCs to 1 AII cell is within the broad range found among placental mammals. For comparison, we collected data for the Australian nocturnal dunnart Sminthopsis crassicaudata, and found it to be similar to M. domestica, with rod-contacting PKCα-immunoreactive bipolar cells that had axon terminals also stratifying in IPL strata S3-S5.

Genomic imprinting of IGF2 in marsupials is methylation dependent

Background-Parent-specific methylation of specific CpG residues is critical to imprinting in eutherian mammals, but its importance to imprinting in marsupials and, thus, the evolutionary origins of the imprinting mechanism have been the subject of controversy. This has been particularly true for the imprinted Insulin-like Growth Factor II (IGF2), a key regulator of embryonic growth in vertebrates and a focal point of the selective forces leading to genomic imprinting. The presence of the essential imprinting effector, DNMT3L, in marsupial genomes and the demonstration of a differentially methylated region (DMR) in the retrotransposon-derived imprinted gene, PEG10, in tammar wallaby argue for a role for methylation in imprinting, but several studies have found no evidence of parent-specific methylation at other imprinted loci in marsupials.Results-We performed the most extensive search to date for allele-specific patterns of CpG methylation within CpG isochores or CpG enriched segments across a 22 kilobase region surrounding the IGF2 gene in the South American opossum Monodelphis domestica. We identified a previously unknown 5'-untranslated exon for opossum IGF2, which is flanked by sequences defining a putative neonatal promoter, a DMR and an active Matrix Attachment Region (MAR). Demethylation of this DMR in opossum neonatal fibroblasts results in abherrant biallelic expression of IGF2.Conclusion-The demonstration of a DMR and an active MAR in the 5' flank of opossum IGF2 mirrors the regulatory features of the 5' flank of Igf2 in mice. However, demethylation induced activation of the maternal allele of IGF2 in opossum differs from the demethylation induced repression of the paternal Igf2 allele in mice. While it can now be concluded that parent-specific DNA methylation is an epigentic mark common to Marsupialia and Eutheria, the molecular mechanisms of transcriptional silencing at imprinted loci have clearly evolved along independent trajectories.

Primary sensory afferent innervation of the developing superficial dorsal horn in the South American opossumMonodelphis domestica

The development of the primary sensory innervation of the superficial dorsal horn (SDH) was studied in postnatal opossums Monodelphis domestica by using DiI labelling of primary afferents and with GSA-IB(4) lectin binding and calcitonin gene-related peptide (CGRP) immunoreactivity to label primary afferent subpopulations. We also compared the timing of SDH innervation in the cervical and lumbar regions of the spinal cord. The first primary afferent projections to SDH emerge from the most lateral part of the dorsal root entry zone at postnatal day 5 and project around the lateral edge of the SDH toward lamina V. Innervation of the SDH occurs slowly over the second and third postnatal weeks, with the most dorsal aspect becoming populated by mediolaterally oriented varicose fibers before the rest of the dorsoventral thickness of the SDH becomes innervated by fine branching varicose fibers. Labelling with GSA-IB(4) lectin also labelled fibers at the lateral edge of the dorsal horn and SDH at P5, indicating that the GSA-IB(4) is expressed on SDH/lamina V primary afferents at the time when they are making their projections into the spinal cord. In contrast, CGRP-immunoreactive afferents were not evident until postnatal day 7, when a few short projections into the lateral dorsal horn were observed. These afferents then followed a pattern similar to the development of GSA-IB(4) projects but with a latency of several days. The adult pattern of labelling by GSA-IB(4) is achieved by about postnatal day 20, whereas the adult pattern of CGRP labelling was not seen until postnatal day 30. Electron microscopy revealed a few immature synapses in the region of the developing SDH at postnatal day 10, and processes considered to be precursors of glomerular synapses (and thus of primary afferent origin) were first seen at postnatal day 16 and adopted their definitive appearance between postnatal days 28 and 55. Although structural and functional development of forelimbs of neonatal Monodelphis is more advanced than the hindlimbs, we found little evidence of a significant delay in the invasion of the spinal cord by primary afferents in cervical and lumbar regions. These observations, together with the broadly similar maturational appearance of histological sections of rostral and caudal spinal cord, suggest that, unlike the limbs they innervate, the spinal regions do not exhibit a large rostrocaudal gradient in their maturation.

Spinal repair in immature animals: a novel approach using the South American opossum Monodelphis domestica.

1. The adult mammalian central nervous system (CNS) is unable to regenerate following injury and repair has only been seen when implants of peripheral nervous tissue, fetal tissue or Schwann cells are used, or antibodies or trophic molecules applied. However, the immature mammalian CNS has revealed a capacity to repair without extrinsic influence. 2. The marsupial mammal provides a unique opportunity to access the immature CNS without invasive in utero surgery. In particular, the South American opossum Monodelphis domestica is an ideal animal for spinal cord injury studies examining the ability of the immature CNS to repair after injury. 3. The Monodelphis spinal cord may be examined for its response to injury either as an in vitro or in vivo system and, therefore, is a flexible model, allowing many different questions to be addressed by the most suitable approach. 4. The immature Monodelphis CNS was able to support fibre growth that reappeared 4 days after a crush at P3-P8 in vitro. Conduction was also restored at this time, accompanied by synaptic connections. 5. A cut lesion performed in vivo on Monodelphis spinal cords at P7 took longer to repair, with fibres reappearing across the injury site 2 weeks after the lesion; greater disruption to structure was noted both during early stages of repair and in adulthood. 6. Neural pathway tracing with dextran amine from the lumbar cord to the brain in adult Monodelphis, which received spinal lesions at P7, revealed a similar distribution of labelled cells in brainstem and mid-brain nuclei to that of control animals. 7. Studies of the locomotor behaviour of adult Monodelphis that had received either a cut or crush lesion at P7-P8 showed remarkably similar abilities to control animals when performing complex tasks. 8. The results of spinal cord injury studies with the immature Monodelphis CNS may help in the development of treatments for spinal injury patients.

Development of motoneurons and primary sensory afferents in the thoracic and lumbar spinal cord of the South American opossum Monodelphis domestica.

The postnatal development of the primary sensory afferent projection to the thoracic (T4) and lumbar (L4) spinal cord of the marsupial species Monodelphis domestica was studied by using anterograde and retrograde neuronal tracers. Large numbers of primary afferents and motoneurons were labelled by application of the carbocyanine dye DiI into individual dorsal root ganglia (DRG) afferents in short-term organ cultures. Dorsal root axons had entered the cord at birth, but most primary afferent innervation of the grey matter and the establishment of cytoarchitectural lamination occurs postnatally. In addition to ipsilateral projections, some primary afferents that projected to the dorsal horn extended across the midline into the equivalent contralateral regions of the grey matter. Similarly, motoneuron dendrites occasionally extended across midline and into the contralateral grey matter. The first fibres innervating the spinal cord project to the ventral horn and formed increasingly complex terminal arbours in the motor columns between P1 and P7. After P5 many afferents were seen projecting to the dorsal horn, with the superficial dorsal horn being the last region of the spinal grey to be innervated. Histochemical labelling with the lectin Griffonia simplicifolia indicated that C fibre primary afferents had arborised in the superficial dorsal horn by P14. The sequence of primary afferent innervation is thus similar to that described in the rat, but this sequence occurs over a period of several weeks in Monodelphis, compared with several days in the rat.

H-ras Oncogene Activation in Invasive UVR-Induced Corneal Sarcomas of the Opossum Monodelphis domestica

Chronic exposure to ultraviolet radiation (UVR) induces corneal sarcomas in the South American opossum Monodelphis domestica. Cell lines are readily established from these tumors. Northern blotting of mRNA from six such cell lines revealed high expression of the H-ras oncogene. H-ras cDNA from an eye tumor cell line was cloned and characterized; the germline sequence of codons 12, 13, and 61 was confirmed by examination of H-ras sequences amplified from liver DNA by the polymerase chain reaction. The Monodelphis H-ras coding sequence is 84-89% identical to that of other vertebrates at the nucleotide level, and the predicted 189-amino-acid sequence differs by 2-12 amino acids from that of other vertebrates. Analysis of 12 primary invasive corneal sarcomas induced by chronic UVR exposure revealed no evidence of H-ras gene amplification or rearrangement. One tumor was heterozygous for an activating point mutation in codon 61 of the H-ras gene; the tumor was also homozygous for a point mutation at an adjacent site in codon 62. These results provide additional evidence for the functional importance and consequent evolutionary conservation of the ras oncogenes.

S-100 immunoreactivity in melanomas of the South American opossum Monodelphis domestica.

S-100 immunoreactivity was determined 1) in foci of melanocytic hyperplasia, 2) in naturally occurring, ultraviolet radiation-induced, and 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced primary melanomas, and 3) in metastatic melanoma lesions in the South American opossum Monodelphis domestica. Preneoplastic lesions of melanocytic hyperplasia contained scattered cells with S-100-positive nuclei. All primary melanomas, with the exception of a single DMBA-induced tumor, contained cells with S-100-positive nuclei. The pattern of S-100 reactivity in tumors varied from large foci of S-100-positive cells to scattered individual S-100-positive cells. Lymph node metastases were S-100 positive, but metastatic masses in internal organs were usually S-100 negative. Although S-100 reactivity did not distinguish preneoplastic lesions from tumors or benign melanomas from malignant melanomas, identification of metastatic tumor cells clearly demonstrated malignancy.

HPRT activity in embryos of a South American opossum Monodelphis domestica.

Marsupial females show preferential paternal X-inactivation. However, the time at which X-inactivation occurs in early development has not yet been determined. A double microassay which measures the activities of X-linked hypoxanthine phosphoribosyl transferase (HPRT) and the autosomally-coded adenine phosphoribosyl transferase (APRT) from the same sample was performed on a collection of embryos from a South American opossum Monodelphis domestica. The embryos ranged in age from the 2-cell stage to the bilaminar blastocyst stage. The results indicate that their embryonic HPRT and APRT are not expressed until just before the unilaminar blastocyst stage in M. domestica. This is at a later stage of development than that in the mouse where embryonic HPRT and APRT expression first occurs at the 4-8-cell stage. It is concluded that HPRT is an uniformative enzyme for assessing X chromosome activity in cleaving embryos of M. domestica. The widespread distribution of HPRT:APRT ratios after the unilaminar blastocyst stage also makes it difficult to draw conclusions about the state of X chromosome activity in early marsupial development.

Isolation and characterization of a marsupial DNA photolyase.

Abstract— Post UV‐B(280–320 nm) exposure to UV‐A(320–400 nm) reverses pyrimidine dimers in the epidermal DNA of the South American opossum Monodelphis domestica [Ley, R. D. (1984) photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica. Photochem. Photobiol. 40,141–143.] To demonstrate that the observed photorepair is mediated by an enzyme, we have isolated a DNA photolyase from the opossum. DNA photolyase from liver was purified 3000‐fold by ammonium sulfate fractionation and phenylsepharose, hydroxylapatite, DEAE‐cellulose and DNA‐cellulose column chromatography. Heat denaturation (60°C for 4 min) completely eliminated the photoreactivating activity. The enzyme was active in the pH range of 5.5 to 8.5 with a pH optimum of 7.5. The enzyme has an apparent molecular weight of 32 000 under nondenaturing conditions. The activity of the enzyme was not affected by sodium chloride up to 250 mM. The action spectrum for the purified DNA photolyase showed activity in the range of325–475 nm with peak actvity at 375 nm.

Monodelphis domestica: a new animal model for studies in photodermatology.

AbstractThe ability to photoreverse pyrimidine dimers in DNA of the South American opossum Monodelphis domestica provides a powerful tool with which to probe the role of pyrimidine dimers in ultraviolet radiation (UVR)‐induced histopathologic changes of the skin of this mammal. We have observed that post‐UVR exposure to photoreactivation light not only reversed pyrimidine dimers in epidermal DNA, but also suppressed the capacity of UVR to induce macroscopic and microscopic changes in the skin of M. domestica.