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In eutherian mammals, sex differentiation is initiated by expression of the testis-determining gene on the Y chromosome. Subsequent phenotypic development of the reproductive tract and genitalia depends on the production of hormones by the differentiated testis. In marsupials the mechanisms of phenotypic development may vary from this pattern, as differentiation of the scrotal primordia has been shown to occur before that of the gonad. Thus, the development of the scrotum in the marsupial has been regarded as an androgen-independent process. We have sought to clarify the ontogeny of scrotal development and the appearance of androgen receptor immunoreactivity by examining Monodelphis domesticaembryos/pups from 1 day prior to birth until 2 days after birth. We have also used immunocytochemistry to determine the expression of the key steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase as an indicator of when the developing gonad may be capable of synthesizing androgens. Expression of this enzyme was first detected in the gonads and adrenals of both sexes 1 day prior to birth and before the appearance of scrotal bulges. Androgen receptor immunoreactivity was detected in the scrotal anlagen of male opossum pups as early as 1 day following birth. This finding is significantly earlier than previous reports and coincides with the appearance 1 day after birth of distinct scrotal bulges. Androgen receptor immunoreactivity was also observed in the genital tubercles of male pups, but not female pups, 2 days after birth. These results suggest that androgens may play an important role in the development of the male genitalia at a much earlier stage than that indicated by previously published work and that scrotal development in this species may not be androgen-independent.

We have examined the distribution of immunoreactivity for GAP-43 in the developing and adult brain of a diprotodontid metatherian, the tammar wallaby ( Macropus eugenii). The distribution of GAP-43 immunoreactivity in the neonatal wallaby brain was strikingly heterogeneous, in contrast to that reported for the newborn polyprotodontid opossum. Immunoreactivity for GAP-43 in the developing wallaby brain showed a caudal-to-rostral spatiotemporal gradient, with the brainstem well in advance of the telencephalon throughout the first 100 days of postnatal life. In many regions examined, GAP-43 immunoreactivity passed through the following phases: 1. intense immunoreactivity in developing fiber tracts and occasional somata; 2. diffuse homogeneous immunoreactivity; 3. selective loss of immunoreactivity in particular nuclei or cortical regions. In the isocortex, selective loss of GAP-43 immunoreactivity in the somatosensory and visual cortex (at postnatal day 115) coincided with the maturation of the laminar distribution of terminal thalamocortical axonal fields. Within adult cortical regions, GAP-43 immunoreactivity was highest in layer I of all regions, lower layers (V and VI) of primary somatosensory and visual cortices, layers II/III of motor and cingulate cortex, and layer IV of entorhinal cortex. Our findings suggest that, while patterning of GAP-43 immunoreactivity in the mature brain is similar across meta- and eutheria, there may be early developmental differences in the distribution of GAP-43 immunoreactivity between poly- and diprotodontid metatheria.

Previous light-microscopic studies have shown a unique population of mast cells in lymphatic sinuses of lymph nodes located in the head, neck, axillary fossa and inguinal region of the opossum. In the present work, scanning and transmission electron-microscopic studies in the opossum mandibular and superficial axillary lymph nodes have strengthened the differences between connective-tissue mast cells (CTMC) and the lymphatic-sinus mast cells (LSMC). Further, close appositions of mast cells to other cells were described. At the nodal capsule, CTMC contacted fibroblast and granulocytes. In the lymphatic sinuses a few CTMC contacted LSMC, macrophages and reticular cells. The LSMC contacted macrophages, reticular cells and other LSMC. A few LSMC could be located in the medullary cord in close contact with plasma cells or other lymphoid cells, keeping the same ultrastructural features of those found in the lymphatic sinuses. An important new finding was provided by light-microscopic studies in nine abdominal lymph nodes. Most of them (para-aortic, common iliac, cardial, cecocolic and those of the body and tail of the pancreas) displayed numerous LSMC with the same distribution and histological features described herein. However, the mesenteric, pyloric and head-of-pancreas lymph nodes were virtually devoid of LSMC. Instead, their mast cells occurred mainly at the medullary cords and were very similar to the CTMC. Ultrastructural studies at the mesenteric lymph nodes confirmed the CTMC character of the mast cells located at both medullary cords and sinuses, and disclosed interactions with macrophages and lymphoid cells.

Blood-borne mononuclear cells invade the developing retina via the hyaloid vasculature at the optic nerve head. Following removal of apoptotic cell debris they give rise to the network of resident microglia. The population of cells recently described in the peripheral subretinal space of developing human eyes may represent a further population of macrophages destined to become microglia. The aim of the present study was to confirm the presence of subretinal macrophages in the developing eye in other mammalian species and perform preliminary immunophenotypic analysis in rat tissues. The range of species chosen included eutherian mammals (rat and rabbit) and marsupials (wallaby and opossum). Ocular tissues from a range of developmental stages were studied by scanning electron microscopy and transmission electron microscopy. Distinctive networks of dendriform and pleomorphic macrophages were observed by scanning electron microscopy in the peripheral subretinal space of D2 rabbits, newborn and D2 rats and D75 wallaby. Transmission electron microscopic studies of D2 rabbit, newborn and D2 rat and all ages of North American opossum revealed cells with the ultrastructural features of macrophages in the peripheral subretinal space, cilio-retinal junction and between ciliary epithelial cells. Preliminary immunoperoxidase studies using a panel of anti-leukocyte monoclonal antibodies on frozen sections of rat ocular tissues (newborn, D2 and D4) revealed ED1(+) Ox42(+) ED2(+) but Ox6(-) cells in the peripheral subretinal space, peripheral retina and ciliary body epithelia. The data confirms that subretinal macrophages are a feature of the developing eye in a broad range of mammalian species and immunophenotypic evidence leads the author to postulate that these cells arise from the ciliary body vasculature and may migrate into peripheral neural retina and mature into resident microglia.

Extracellular matrix proteoglycans accumulated in perineuronal nets and in certain neuropil zones have been shown to influence the immediate neuronal microenvironment, and to contribute to the chemoarchitectonic characteristics of neuronal networks. Studies in different placental mammals, including the human, have suggested that the major principles of extracellular matrix distribution remained constant during phylogenesis of the different mammalian strains. However, the comparison of matrix distribution between various species also indicates that striking deviations from the basic pattern may occur, although their functional significance appears unknown as yet. This study examines the extracellular matrix in the forebrain of a basic American marsupial, which has evolved independently of placental mammals for more than 100 million years. Brain sections obtained from adult gray short-tailed opossums (Monodelphis domestica) were stained for extracellular matrix components using the N-acetylgalactosamine-binding lectin Wisteria floribunda agglutinin (WFA), a polyclonal antibody against chondroitin sulfate proteoglycans (CSPG), and biotinylated hyaluronectin for the detection of hyaluronan. In subcortical regions, the distribution patterns of WFA-stained and CSPG-immunoreactive perineuronal nets were similar to those reported previously in placental mammals. In contrast, a unique distribution was found in the neocortex. This distribution was characterized by the presence of perineuronal nets around pyramidal cells and matrix components within the adjacent neuropil that together form a continuously labeled zone in layer V. Weakly stained nets ensheathed less numerous pyramidal cells in the upper layers II/III and a few multipolar cortical neurons. Dual staining experiments showed that cortical net-associated neurons were rarely immunopositive for parvalbumin. This fact, in addition to the predominant association of extracellular matrix components with layer V pyramidal cells, differentiates the neocortex in Monodelphis from that of all placental mammals studied to date. Regarding the basic phylogenetic position of this marsupial species it remains to be shown if these distribution characteristics of extracellular matrix may represent also a basic feature of cortical organization.

The gray short-tailed opossum Monodelphis domestica is born in a very immature state (eyes and ears closed, budlike hindlimbs, etc.) 14 days post-coitum, but it can locomote with its forelimbs from the mother's genital aperture to a nipple to which it attaches. The forelimb movements allowing this behavior may be the expression of central pattern generators in the spinal cord, but sensory clues must guide it. One such system may be the vestibular system, which senses linear and angular acceleration and has a strong influence on posture and balance at rest and during locomotion in adult animals. Using the neuronal tracer DiI, we have looked at the vestibular primary afferents and the vestibular nuclear projections to the cervical spinal cord in newborn and postnatal opossums, as well as in the adult animal. The projections in the adult opossum conform to those described for other mammals. Fibers of the vestibular portion of the eighth nerve distribute to all four vestibular nuclei and toward the cerebellar primordium on the day of birth. In addition, some of the fibers project to the contralateral vestibular ganglion, a projection that is not found in the adult opossum. Projections from the lateral, medial and inferior vestibular nuclei to the cervical cord are also present in the newborn. Although we cannot exclude the possibility that chemical and/or tactile guidance is used for directing the movements of the newborn opossum, our results support the hypothesis that the vestibular system may be directly involved in the control of these movements. However, not all components of the system are equally developed at birth, and the circuit from the utricle to the lateral vestibular nucleus and from the latter to the cervical cord may be better formed than that from the semicircular canals to the medial and inferior vestibular nuclei to the cervical cord.

The distribution of axons by size in the optic tract of the South American opossum, Didelphis marsupialis was studied. Thin and semi-thin sections were examined, and measurements of axonal diameter were made on electron micrographs taken from various locations across the optic tract of normal opossums. In order to determine the contributions of the different axon diameter classes to the crossed and uncrossed retinofugal pathways, measurements were also made from the tracts of opossums in which one eye had been enucleated 5 weeks previously. Within the opossum's optic tract, the axons are partially segregated by their size: the deepest parts of the tract contain only fine and medium-sized axons, whereas coarse axons are also present superficially. In the middle of the tract, all three size classes are present. At increasingly superficial positions, there is a steady reduction in the proportion of medium-diameter axons, and an increase in the number of the finest axons. Medium and coarse axons contribute to both the crossed and uncrossed pathways, and the uncrossed component is displaced superficially relative to the crossed component. The fine axons in the deeper parts of the tract arise from both retinae, while those in the superficial parts of the tract, near the pial surface, are virtually all crossed. The opossum's optic tract thus displays the segregation of axons by size found in placental mammals, and follows a pattern reminiscent of that found in carnivores. Such a common organizational plan, particularly the similarities between the didelphids and carnivores, is suggestive of an early acquisition of parallel visual pathways in mammalian phylogeny. Since the fiber order in the optic tract of eutherians is a chronological map of axonal arrival during development, these results suggest that a conserved developmental mechanism has led to a common organizational plan.

The Harderian gland of the North American opossum (Didelphis virginiana) is large and well developed, despite the absence of a nictitating membrane in the adult of this species. The elongate glands are surrounded by a delicate connective tissue capsule from which thin septae extend, subdividing the gland into numerous lobules. The secretory units of the opossum Harderian gland are drained by a well defined but not extensive intralobular and interlobular duct system. Most of the secretory end pieces consist of tubulo-alveolar units with widely dilated lumina filled with secretory product. Numerous intact lipid vesicles suspended within an amorphous material constitute the luminal contents. Cells lining the tubulo-alveolar secretory end-pieces are usually columnar in shape, and characterized by numerous lipid-containing secretory vesicles and aggregations of poly-tubular complexes 40-60 nm in diameter. In addition, these cells contain numerous large irregularly shaped mitochondria, whose matrix is of considerable electron density. Intralobular and interlobular ducts are lined by electron-lucent epithelial cells that lack both the lipid-containing vesicles and the large mitochondria, although typical smaller mitochondria are found scattered within the cytoplasm. Both secretory end-pieces and ductal elements are invested by an abundance of myoepithelial cells. A second, smaller serous type of secretory unit may occur near the centre of some Harderian gland lobules. In these units secretory tubules and acini are compactly arranged surrounding a narrow lumen. Serous cells are pyramidal in shape and the cytoplasm is characterized by numerous electron-dense secretory granules and scattered profiles of rough endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study, we have examined the genesis of neurons of the retinal ganglion cell layer of the opossum Didelphis marsupialis by [3H]-thymidine autoradiography. Our results suggest that most neurons surviving to adulthood are generated in postnatal life from day 1 to day 23. Cells are generated according to a coarse gradient from the retinal geometric center to the periphery. Regional analysis of soma size distributions in different cohorts suggest that this gradient is actually formed by two partially-overlapping, concentric waves of cell proliferation. Most medium and large ganglion cells are formed during the early wave, whereas most displaced amacrine cells and small ganglion cells are formed during the late wave. Our results confirm the appropriateness of the opossum as a model for studies of development of the mammalian visual system.

Patterns of myelination have been studied in the optic tract and the superior colliculus (SC), with special reference to the optic layer, in the opossum Didelphis marsupialis. Myelination in the optic tract starts far in precedence of eye opening and follows a rostrocaudal gradient. Myelination in the SC presents the following features: it proceeds according to a general inside-out pattern and follows both rostro-caudal and latero-medial gradients in the optic layer, and it accelerates in the SC optic layer soon after systematic exposure to visual input. The data presented here, together with other available information, suggest that myelination in the opossum optic tract starts in parallel with the stabilization in the number of optic fibers, and advances in the rostro-caudal mode common to most eutherian mammals, and also that myelogenesis in the SC neither correlates necessarily with, nor recapitulates, the sequence of acquisition of GFAP-positive astrocytes in a given set of layers. Changes in the rate of myelination in the optic layer after exposure to visual input are regionally-selective, and seem compatible with the recruitment of thin axons into the myelogenetic cycle rather than with the thickening of pre-existing myelin sheaths. It is concluded that the SC is a favorable structure for the study of the differentiation of glial cells, particularly in species with an extended time course of maturation such as the opossum.

We have employed degeneration techniques to study the ontogeny of major projections to the inferior olivary nucleus in the North American opossum, a species which is born 12 days after conception and which enjoys a protracted development in an external pouch. Subsequent to spinal lesions a small amount of axonal degeneration can be produced within the edge of the olive before subnuclei can be distinguished (7 days after birth, 24 mm, snout-rump length). Degenerating axons are present more deeply within the olive in animals operated 12 days after birth (30 mm, snout-rump length) and by at least day 16 (36 mm, snout-rump length), they are found in all of the regions they occupy in the adult animal. Subsequent to lesions which undercut all descending mesencephalic and diencephalic systems, a small amount of axonal degeneration is found at the dorsolateral edge of the olive by day 7 (23 mm, snout-rump length). Degenerating axons fill more of the olive, particularly caudally, after comparable lesions in older animals and by day 17 (38 mm, snout-rump length), degeneration is present in all of the olivary regions innervated by midbrain and thalamic axons in the adult opossum. There is some evidence that spinal, mesencephalic and diencephalic axons follow a caudal to rostral gradient in their intraolivary growth. Lesions which undercut neurites growing out of the cerebellum produce evidence for cerebello-olivary connections by day 17. Axons from the cerebral cortex reach their olivary targets considerably later than those from either the spinal cord, mesencephalon, diencephalon or cerebellum. It is not until approximately postnatal day 30 (55 mm, snout-rump length) that degenerating axons can be traced into the olive after lesions of the cortical mantle. These data indicate that the inferior olive receives major connections early in development and that there is an orderly sequence to their growth.

The North American opossum is born 12 days after conception and is therefore available for experimental manipulation in an immature state. We have used the opossum to study the growth of cortical axons into the brainstem and spinal cord and have obtained evidence that such growth occurs in an orderly fashion. Cortical axons reach the ventral mesencephalon 12 days after birth and some of them have grown into the caudal medulla where they decussate by 23 days. At the latter stage immature cortical axons also distribute to the midbrain tegmentum, the basilar pons, the inferior olive and the hilum of the nucleus cuneatus. Cortical axons first enter the spinal cord about 30 days after birth where they are present in the white matter before growing into the dorsal horn. The forelimb placing reaction does not develop until well after cortical axons have reached cervical levels. Axons from the cerebral cortex grow into the spinal cord before there is evidence for cortical innervation of either the red nucleus or the bulbar reticular formation and well before pyramidal cells of the neocortex are mature. The relatively late development of corticospinal and corticobulbar systems contrasts markedly with the early growth of bulbospinal axons.

The North American opossum does not have a corpus callosum; neocortical commissural axons are contained entirely within the anterior commissure. We have used axonal transport techniques to study the origin and distribution of commissural axons from somatic motor-sensory cortex in developing and adult opossums. Neocortical axons grow into the anterior commissure by postnatal day (PND) 12, the contralateral external capsule by approximately PND 19, the area deep to the contralateral homotypic cortex by approximately PND 26 and the cortex proper by approximately PND 35. Commissural neurons were first demonstrated at about PND 26, when they form a fairly continuous band in the cortical subplate (presumptive layers V-VI). By at least PND 37, commissural neurons are also present in layers II and III, where they form a continuous band, and in layer IV, where they are sparse. In older pouch young and adult opossums the bands of commissural neurons, especially in layers V-VI, are interrupted, and commissural neurons are rare in layer IV. In general, commissural axons in both pouch-young and adult opossums innervate areas containing commissural neurons as well as layer I. In the acallosal opossum as well as in the callosal rat, the development of commissural connections from somatic motor-sensory cortex is characterized by pauses during the growth of axons into the opposite cortex, by a general inside-out-gradient, and by a transition from continuous bands to patchy, radial columns of commissural neurons and axons. This suggests that similar mechanisms govern the formation of commissural connections in the two species.

Evidence is presented for an early appearance of monoaminergic neurites within the spinal cord of the developing opossum. They are present within the marginal zone before hindlimb movements begin (stage I) and they start to grow into the intermediate zone by the time hindlimb movements are first observed (stage II). Monoaminergic neurites grow first into the dorsolateral intermediate zone and the intermediolateral cell column where they can be found by the beginning of stage II. Shortly thereafter, fluorescent varicosities can be traced into the area dorsal to the central canal presumed to become lamina X. Fluorescent processes extend in to the ventral intermediate zone (ventral horn) somewhat later in development. Monoaminergic axons have grown into all of the areas they occupy in the adult animal, except for laminae I and II, by the time immature hindlimb movements can be altered by cutting all brainstem projections to the lumbosacral cord (stage III). Monoaminergic innervation of laminae I and II is the last to develope, but it is present by the time thoracic transection produces complete spinal shock.

Pre- and postsynaptic elements within the developing inferior olive (IO) of both control and experimental opossums were examined via electron microscopy. Electron dense boutons identified di-/mesencephalic, cerebellar and spinal afferents within the IO of 8–71 day old animals, which survived 4–48 hours following either midbrain hemisections or spinal transections.

The development of ascending spinal pathways has been studied in the North American opossum using degeneration methods and the retrograde transport of horseradish peroxidase. Axons from caudal thoracic and/or lumbosacral levels of the spinal cord reach the lateral reticular nucleus, the inferior olivary complex, the reticular formation of the medulla and pons as well as the cerebellum very early in development. Innervation of the nucleus gracilis occurs somewhat later. Spinal axons grow into most of the caudal brain stem areas they occupy in the adult animal, including the nucleus gracilis, before there is convincing evidence that they reach the thalamus. Although spinal axons enter the cerebellum early in development their adult distribution with its characteristic discontinuities appears relatively late.

We have studied the early development of subcortical projections to presumptive somatic sensory-motor areas of neocortex in the North American opossum Didelphis virginiana. The opossum is born in a very immature state, 12-13 days after conception, and climbs into an external pouch where it is available for experimental manipulation. Using the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase, we have obtained evidence that axons from the dorsal raphe and superior central nuclei, the substantia nigra, the locus coeruleus and the parabrachial nuclei reach presumptive somatic sensory-motor areas of neocortex by at least postnatal day (PND) 10. Axons showing serotonin-like immunoreactivity, presumably from the dorsal raphe and/or superior central nuclei, and axons containing tyrosine hydroxylase immunoreactivity, presumably from the substantia nigra and/or locus coeruleus, are present in the same areas at birth or shortly thereafter. Thalamic axons do not grow into comparable areas of neocortex until after PND 10. Such axons reach the subplate region of ventrolateral neocortex first and then proceed dorsomedially; by estimated PD (EPND) 21, they are present in presumptive layers I, V and VI, but they do not innervate an identified layer IV until EPND 48. The developmental sequences suggested by our study are compared with those reported for other species and are discussed in light of their importance in the formation of major sensory and motor circuits.

The development of the neocortex of the marsupial Monodelphis domestica has been studied from birth until adulthood. Monodelphis is born after a gestational period of 14 days, a time when the neocortex is still at a two-layered “embryonic’ stage of development, that is equivalent to a 13–14 day rat embryo or 6 week human embryo. The cortical plate does not begin to appear until 3 to 5 days postnatal. Thus the whole of neocortical development is a postnatal phenomenon in this species, as has been previously described in other marsupials. The general pattern of development of the characteristic layers of the immature neocortex and the subsequent development of a six-layered adult neocortex is similar to that found in eutherian species. However there are some differences. The depth of the immature cortical plate when compared to the thickness of the neocortical wall is less than in eutherians and the subplate zone is much deeper in Monodelphis; this transient subplate zone consists of widely spaced rows of cells that are aligned parallel to the cortical surface. Unlike eutherians there appears to be no secondary proliferative zone in the subventricular zone of the dorso-lateral neocortical wall. Maturation of the neocortex is apparent by 45 days postnatal and by 60 days (around the time of weaning) the characteristic six-layered adult neocortex is clearly present. The neuronal marker PGP 9.5 was used to define neuronal populations in the adult brain. The density of neurons in Monodelphis appears to be considerably less than in eutherians such as the rat. The suitability of postnatal Monodelphis for studies of neocortical development is discussed.