Organization of the Olfactory and Respiratory Skeleton in the Nose of the Gray Short-Tailed Opossum Monodelphis domestica

The internal nasal skeleton in Monodelphis domestica, the gray short-tailed opossum, primarily supports olfactory and respiratory epithelia, the vomeronasal organ, and the nasal gland. This scaffold is built by the median mesethmoid, and the paired vomer and ethmoid bones. The mesethmoid ossifies within the nasal septum cartilage. The bilateral ethmoid segregates respiratory and olfactory regions, and its geometry offers insight into the functional, developmental, and genomic organization of the nose. It forms through partial coalescence of separate elements known as turbinals, which in Monodelphis comprise the maxilloturbinal, nasoturbinal, five endoturbinals, and two ectoturbinals. Geometry of the ethmoid increases respiratory mucosal surface area by a factor of six and olfactory mucosal surface by nearly an order of magnitude. Respiratory epithelium warms and humidifies inspired air, recovers moisture as air is exhaled, and may help mediate brain temperature. In contrast, the olfactory skeleton functions as a series of small funnels that support growth of new olfactory neurons throughout life. Olfactory mucosa lines the mouth of each funnel, forming blind olfactory recesses known as the ethmoid cells, and neuronal axons are funneled from the epithelium through tiny olfactory foramina in the cribriform plate, into close proximity with target glomeruli in the olfactory bulb of the brain where each axon makes its first synapse. The skeleton may thus mediate topological correspondence between odorant receptor areas in the nose with particular glomeruli in the olfactory bulb, enabling growth throughout life of new olfactory neurons and proper targeting by their axons. The geometric arrangement of odorant receptors suggests that a measure of volatility may be a component in the peripheral olfactory code, and that corresponding glomeruli may function in temporal signal processing. Supporting visualizations for this study are available online at www.DigiMorph.org.     

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 α-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. Received: 2 March 1998 / Accepted: 2 June 1998     

Effects of unilateral olfactory deprivation in the developing opossum,Monodelphis domestica

Unilateral naris closure in young rodents leads to striking alterations in the development of the ipsilateral olfactory system. One of the most pronounced effects is a 25% reduction in the size of the experimental olfactory bulb, a change that stems in part from decreased cell survival. Since naris occlusion in rodents alters the system more during development than in adulthood, we investigated the consequences of olfactory deprivation in a species that is born in a very immature state, Monodelphis domestica. In this pouchless marsupial, offspring are born after a short 14-day gestation. In the present study, the thymidine analogue bromodeoxyuridine was used to examine early postnatal neurogenesis in the olfactory bulb. Unlike rats and mice, neurogenesis of the main output neurons (the mitral cells) continues into postnatal life. Unilateral naris closure was begun on postnatal day 4 (P4) or P5 in Monodelphis and continued for 30 or 60 days. Laminar volume measurements revealed a significant reduction in the size of the experimental bulb following 60, but not 30, days of early olfactory deprivation. Mitral cell number estimates indicated a significant reduction after both 30 and 60 days of naris closure. The immaturity of Monodelphis offspring may render the population of mitral cells susceptible to the effects of olfactory deprivation. These findings suggest that afferent activity plays a role in the survival of all bulb neurons, irrespective of cell class. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 429–438, 1997     

Dissecting the Factors Involved in the Locomotion Mode of Neuronal Migration in the Developing Cerebral Cortex

Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cδ (PKCδ), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCδ hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCδ in the early and final phases of migration, respectively.     

Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.     

The developmental reduction of the marsupial coracoid: A case study in Monodelphis domestica

During their embryogenesis, marsupials develop a unique structure, the shoulder arch, which provides the structural and muscle‐attachment support necessary for the newborn's crawl to the teat. One of the most pronounced and important aspects of the shoulder arch is an enlarged coracoid. After marsupial newborns reach the teat, the shoulder arch is remodeled and the coracoid is reduced to a small process on the scapula. Although an understanding of marsupial coracoid reduction has the potential to provide insights into both, marsupial evolution and the origin of mammals, little is known about the morphological and cellular processes controlling this process. To remedy this situation, this study examined the morphological and cellular mechanisms behind coracoid reduction in the gray short‐tailed opossum, Monodelphis domestica. A quantitative, morphometric study of shoulder girdle development revealed that the coracoid is reduced in size relative to other aspects of the shoulder girdle by growing at a slower rate. Using a series of molecular assays for cell death, no evidence was found for programmed cell death playing a role in the reduction of coracoid size in marsupials (in contrast to hypotheses of previous researchers). Although it is likely the case that coracoid growth is reduced through a relatively lower rate of cellular proliferation, differences in proliferative rates in the coracoid and scapula were not great enough to be quantified using standard molecular assays. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Response to novelty in the laboratory Wistar rat, wild-captive WWCPS rat, and the gray short-tailed opossum (Monodelphis domestica)

Behavior of the laboratory gray short-tailed opossums (Monodelphis domestica), Warsaw Wild Captive Pisula Stryjek rats (WWCPS) and laboratory rats (Wistar) has been registered in the period of familiarization with a new environment and consecutive confrontation with a novel, innocuous object placed in that familiarized environment. In the new environment the sequence of anxiety, investigation, and habituation was shortest in the opossum, longer in the laboratory rat and longest in the WWCPS rat. When placed in it, gray short-tailed opossums investigated the new environment with the shortest delay and most intensity. In reaction to novel objects, opossums and laboratory rats prolonged the time spent in the proximity of the new object, while the WWCPS rat did not show that reaction. Both opossums and laboratory rats increased the number of contacts with the new object, whereas WWCPS rats reduced those contacts. Behavior of all three species and lines grouped in different clusters. Some other quantitative and qualitative differences in behavior of the investigated animals are also described, showing a higher level of anxiety in both lines of rats than in the opossum. Behavioral differences between species and lines of animals used in this study may be attributed to different ecological adaptations of rats and opossums and to the effect of domestication in the laboratory rats. These behavioral differences make comparisons of opossums vs rat, and wild rat vs laboratory rat interesting models for studying the brain mechanisms of anxiety and neotic motivations.     

Development of olfactory marker protein and N-CAM expression in chemosensory systems of the opossum,Monodelphis domestica

Using olfactory marker protein (OMP) and neural cell adhesion molecule (N-CAM) immunohistochemistry, the present study describes development of olfactory and vomeronasal systems in pre- and postnatal opossums, Monodelphis domestica. As revealed by OMP expression, development of the main olfactory system precedes that of the vomeronasal system by 1–2 weeks. OMP is expressed throughout (homogeneously) the nerve and glomerular layers of the main (MOB) but is expressed more strongly (heterogeneously) in the anterior as compared to the posterior accessory (AOB) olfactory bulb. N-CAM expression is homogeneous in both MOB and AOB. The heterogeneity in the AOB is developmentally regulated, since in the 30-day-old AOB the expression of OMP is homogeneous, becoming heterogeneous during the second month of life. Maximal expression of N-CAM precedes maximal expression of OMP in the VNS by about 2 weeks. From 7 to 21 days of age only, there is a small population of OMP-positive, N-CAM-negative olfactory and vomeronasal axon terminals that penetrate deep into the brain parenchyma, overgrowing their normal targets in the MOB and AOB, respectively. J. Morphol. 234:109–129, 1997. © 1997 Wiley-Liss, Inc.     

First-generation linkage map of the gray, short-tailed opossum, Monodelphis domestica, reveals genome-wide reduction in female recombination rates

The gray, short-tailed opossum, Monodelphis domestica, is the most extensively used, laboratory-bred marsupial resource for basic biologic and biomedical research worldwide. To enhance the research utility of this species, we are building a linkage map, using both anonymous markers and functional gene loci, that will enable the localization of quantitative trait loci (QTL) and provide comparative information regarding the evolution of mammalian and other vertebrate genomes. The current map is composed of 83 loci distributed among eight autosomal linkage groups and the X chromosome. The autosomal linkage groups appear to encompass a very large portion of the genome, yet span a sex-average distance of only 633.0 cM, making this the most compact linkage map known among vertebrates. Most surprising, the male map is much larger than the female map (884.6 cM vs. 443.1 cM), a pattern contrary to that in eutherian mammals and other vertebrates. The finding of genome-wide reduction in female recombination in M. domestica, coupled with recombination data from two other, distantly related marsupial species, suggests that reduced female recombination might be a widespread metatherian attribute. We discuss possible explanations for reduced female recombination in marsupials as a consequence of the metatherian characteristic of determinate paternal X chromosome inactivation.     

The oocyte of a new world marsupial, Monodelphis domestica: structure, formation, and function of the enveloping mucoid layer

Ovulated oocytes of the gray short-tailed opossum Monodelphis domestica are surrounded by a thin zona pellucida and are devoid of a cumulus oophorus. In the ampulla of the oviduct, oocytes acquire a thick mucoid layer composed of concentrically arranged fibrillar material. Exocytosis by the secretory cells of the oviductal epithelium occurs in the region of the oviduct adjacent to the egg. This suggests that the oocyte-zona-mucus layer complex may influence the oviductal epithelium to secrete. During secretion, fibrillar contents of the secretion granules appear to be transformed into membranous material which presumably becomes fibrillar again as it is incorporated into the forming mucoid layer. Spermatozoa (which are known to pair in the cauda epididymis) are found in pairs and with intact acrosomes in the mucoid layer of fertilized eggs. This suggests that spermatozoa of Mondelphis remain paired until they reach the zona pellucida and that the acrosome functions in zona binding and/or penetration.     

Injection of a boar sperm factor causes calcium oscillations in oocytes of the marsupial opossum, Monodelphis domestica

At fertilisation, the sperm triggers an abrupt and transient increase in intracellular calcium ([Ca2+]i) in the oocyte cytoplasm. In eutherian mammals, oocytes exhibit multiple [Ca2+]i transients which are necessary for egg activation. We investigated [Ca2+]i in the marsupial opossum, Monodelphis domestica. Embryo development in this therian mammal is quite distinct from that in most Eutheria. Oestrus was induced in an adult female opossum by introduction of a male into her cage. Injection of a boar sperm extract induced repetitive increases in [Ca2+]i. Each oscillation travelled across and around the periphery of the egg in a wave-like manner. A control injection of KCl elicited no change in [Ca2+]i. This is the first observation of [Ca2+]i oscillations in the oocyte of a marsupial. The repetitive nature of the [Ca2+]i changes were more similar to those in oocytes of Eutheria than those in oocytes of non-mammalian vertebrates.     

Spontaneous development of full weight-supported stepping after complete spinal cord transection in the neonatal opossum, Monodelphis domestica

Spinal cord trauma in the adult nervous system usually results in permanent loss of function below the injury level. The immature spinal cord has greater capacity for repair and can develop considerable functionality by adulthood. This study used the marsupial laboratory opossum Monodelphis domestica, which is born at a very early stage of neural development. Complete spinal cord transection was made in the lower-thoracic region of pups at postnatal-day 7 (P7) or P28, and the animals grew to adulthood. Injury at P7 resulted in a dense neuronal tissue bridge that connected the two ends of the cord; retrograde neuronal labelling indicated that supraspinal and propriospinal innervation spanned the injury site. This repair was associated with pronounced behavioural recovery, coordinated gait and an ability to use hindlimbs when swimming. Injury at P28 resulted in a cyst-like cavity encased in scar tissue forming at the injury site. Using retrograde labelling, no labelled brainstem or propriospinal neurons were found above the lesion, indicating that detectable neuronal connectivity had not spanned the injury site. However, these animals could use their hindlimbs to take weight-supporting steps but could not use their hindlimbs when swimming. White matter, demonstrated by Luxol Fast Blue staining, was present in the injury site of P7- but not P28-injured animals. Overall, these studies demonstrated that provided spinal injury occurs early in development, regrowth of supraspinal innervation is possible. This repair appears to lead to improved functional outcomes. At older ages, even without detectable axonal growth spanning the injury site, substantial development of locomotion was still possible. This outcome is discussed in conjunction with preliminary findings of differences in the local propriospinal circuits following spinal cord injury (demonstrated with fluororuby labelling), which may underlie the weight bearing locomotion observed in the apparent absence of axons bridging the lesion site in P28-injured Monodelphis.     

A new DNA marker, U15557, is linked to protease inhibitor and adenylate kinase-1 in the laboratory opossum, Monodelphis domestica

A 323-bp DNA fragment (U15557) was isolated, cloned, and sequenced after polymerase chain reaction (PCR) amplification from Monodelphis domestica genomic DNA. AHindIII restriction fragment length polymorphism was identified in this species using the U15557 PCR. fragment as a hybridization probe. DNA samples exhibited either a 6.4kb band, a 7.2 kb band, or both bands simultaneously. Behaviour of these two variants in family studies was consistent with codominant autosomal inheritance. Linkage between this marker and the loci encoding protease inhibitor (PI) and adenylate kinase 1 (AK1) was found in M. domestica.