Cellular development and evolution of the mammalian cerebellum.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

Anatomical comparison across heads, fore- and hindlimbs in mammals using network models.

Animal body parts evolve with variable degrees of integration that nonetheless yield functional adult phenotypes: but, how? The analysis of modularity with Anatomical Network Analysis (AnNA) is used to quantitatively determine phenotypic modules based on the physical connection among anatomical elements, an approach that is valuable to understand developmental and evolutionary constraints. We created anatomical network models of the head, forelimb, and hindlimb of two taxa considered to represent a 'generalized' eutherian (placental: mouse) and metatherian (marsupial: opossum) anatomical configuration and compared them with our species, which has a derived eutherian configuration. In these models, nodes represent anatomical units and links represent their physical connection. Here, we aimed to identify: (1) the commonalities and differences in modularity between species, (2) whether modules present a potential phylogenetic character, and (3) whether modules preferentially reflect either developmental or functional aspects of anatomy, or a mix of both. We predicted differences between networks of metatherian and eutherian mammals that would best be explained by functional constraints, versus by constraints of development and/or phylogeny. The topology of contacts between bones, muscles, and bones + muscles showed that, among all three species, skeletal networks were more similar than musculoskeletal networks. There was no clear indication that humans and mice are more alike when compared to the opossum overall, even though their musculoskeletal and skeletal networks of fore- and hindlimbs are slightly more similar. Differences were greatest among musculoskeletal networks of heads and next of forelimbs, which showed more variation than hindlimbs, supporting previous anatomical studies indicating that in general the configuration of the hindlimbs changes less across evolutionary history. Most observations regarding the anatomical networks seem to be best explained by function, but an exception is the adult opossum ear ossicles. These ear bones might form an independent module because the incus and malleus are involved in forming a functional primary jaw that enables the neonate to attach to the teat, where this newborn will complete its development. Additionally, the human data show a specialized digit 1 module (thumb/big toe) in both limb types, likely the result of functional and evolutionary pressures, as our ape ancestors had highly movable big toes and thumbs.

Didelphis albiventris: an overview of unprecedented transcriptome sequencing of the white-eared opossum.

BACKGROUND: The white-eared opossum (Didelphis albiventris) is widely distributed throughout Brazil and South America. It has been used as an animal model for studying different scientific questions ranging from the restoration of degraded green areas to medical aspects of Chagas disease, leishmaniasis and resistance against snake venom. As a marsupial, D. albiventris can also contribute to the understanding of the molecular mechanisms that govern the different stages of organogenesis. Opossum joeys are born after only 13 days, and the final stages of organogenesis occur when the neonates are inside the pouch, depending on lactation. As neither the genome of this opossum species nor its transcriptome has been completely sequenced, the use of D. albiventris as an animal model is limited. In this work, we sequenced the D. albiventris transcriptome by RNA-seq to obtain the first catalogue of differentially expressed (DE) genes and gene ontology (GO) annotations during the neonatal stages of marsupial development. RESULTS: The D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at 5 days of age (P5) and at 10 days of age (P10). The de novo assembly of these transcripts generated 85,338 transcripts. Approximately 30% of these transcripts could be mapped against the amino acid sequences of M. domestica, the evolutionarily closest relative of D. albiventris to be sequenced thus far. Among the expressed transcripts, 2077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1453 between P5 and P10. The enriched GO terms were mainly related to the immune system, blood tissue development and differentiation, vision, hearing, digestion, the CNS and limb development. CONCLUSIONS: The elucidation of opossum transcriptomes provides an out-group for better understanding the distinct characteristics associated with the evolution of mammalian species. This study provides the first transcriptome sequences and catalogue of genes for a marsupial species at different neonatal stages, allowing the study of the mechanisms involved in organogenesis.

Embryonal neoplasms in the opossum: a new model for solid tumors of infancy and childhood.

Opossums fed the chemical carcinogen ethyl nitrosourea early in postnatal life developed a variety of epithelial and mesenchymal embryonal neoplasms that were closely analogous, in morphology and biological behavior, to tumors of human infancy and childhood for which experimental models in laboratory animals are either imprecise or nonexistent. The embryonal tumors were found in association with, and occasionally at the same sites as, a limited number of malformations.

Cranial endocasts from a growth series of Monodelphis domestica (Didelphidae, Marsupialia): A study of individual and ontogenetic variation.

Intraspecific variation (e.g., ontogenetic, individual, sexual dimorphic) is rarely examined among cranial endocasts (infillings of the braincase cavity) because of the difficulty in obtaining multiple specimens of a species, particularly fossil taxa. We extracted digital cranial endocasts from CT scans of a growth series of skulls of Monodelphis domestica, the gray short-tailed opossum, as a preliminary assessment of the amount of intraspecific variation in mammalian endocranial morphology. The goals of this study were 1) to provide an anatomical description to document developmental changes in endocranial morphology of M. domestica and 2) to examine ontogenetic and individual variation with respect to phylogenetic characters of endocranial cavities that are known to be variable between different mammalian taxa. In this study, "ontogenetic variation" refers to variation between specimens of different ages whereas "individual variation" (i.e., polymorphism) is restricted to variation between specimens of comparable age. Aside from size, changes in shape account for the greatest amount of morphological variation between the endocasts of different ages. Endocast length, width, and volume increase with age for the growth series. Relative olfactory bulb cast size increases with age in the growth series, but the relative size of the parafloccular casts shows a slight negative allometric trend through ontogeny. More than one-third of the phylogenetic characters of the endocranial cavity we examined showed some sort of variation (ontogenetic, individual, or both). This suggests that although endocasts are potentially informative for systematics, both ontogenetic and individual variation affect how endocranial characters are scored for phylogenetic analysis. Further studies such as this are necessary to determine the taxonomic extent of significant intraspecific variation of these endocranial characters.

Transcriptomic Changes Associated with Pregnancy in a Marsupial, the Gray Short-Tailed Opossum Monodelphis domestica.

Live birth has emerged as a reproductive strategy many times across vertebrate evolution; however, mammals account for the majority of viviparous vertebrates. Marsupials are a mammalian lineage that last shared a common ancestor with eutherians (placental mammals) over 148 million years ago. Marsupials are noted for giving birth to highly altricial young after a short gestation, and represent humans' most distant viviparous mammalian relatives. Here we ask what insight can be gained into the evolution of viviparity in mammals specifically and vertebrates in general by analyzing the global uterine transcriptome in a marsupial. Transcriptome analyses were performed using NextGen sequencing of uterine RNA samples from the gray short-tailed opossum, Monodelphis domestica. Samples were collected from late stage pregnant, virgin, and non-pregnant experienced breeders. Three different algorithms were used to determine differential expression, and results were confirmed by quantitative PCR. Over 900 opossum gene transcripts were found to be significantly more abundant in the pregnant uterus than non-pregnant, and over 1400 less so. Most with increased abundance were genes related to metabolism, immune systems processes, and transport. This is the first study to characterize the transcriptomic differences between pregnant, non-pregnant breeders, and virgin marsupial uteruses and helps to establish a set of pregnancy-associated genes in the opossum. These observations allowed for comparative analyses of the differentially transcribed genes with other mammalian and non-mammalian viviparous species, revealing similarities in pregnancy related gene expression over 300 million years of amniote evolution.

Phylogenetic evaluation of taxonomic definition of didelphid mouse opossum of the genus Thylamys from valleys of Coquimbo region, Chile.

Only two species of Didelphidae are currently recognized in Chile, the sister species Thylamys elegans, endemic of Mediterranean ecorregion and Thylamys pallidior, the inhabitant of the Puna and desert canyons. Three subspecies have been described for T. elegans: T. e. elegans, T. e. coquimbensis and T. e. soricinus. However, a recent study based on morphological analyses, synonymized T. elegans coquimbensis from the Coquimbo valleys (30-31° S) with T. pallidior and proposed that T. elegans and T. pallidior could be in sympatry at Coquimbo valleys between Fray Jorge (30°40'S) and Paiguano (30°02' S). We assess the current definition of T. e. coquimbensis and T. e. elegans, as well as this taxonomical conflict among the mouse opossums from the Coquimbo valleys through phylogenetic analyses of cytochrome b mitochondrial gene sequences. In this study, for the first time, we used specimens from the type localities of T. e. coquimbensis and T. e. elegans. In addition, we analyzed diagnostic cranial structures for this taxonomic revision. The results supported two allopatric clades, allowing us to keep the taxonomic definition of T. e. elegans and T. e. coquimbensis as phylogenetic reciprocal monophyletic clades and polyphyletic with T. pallidior. This result corroborates previous morphological analyses, which support that mouse opossums from the Coquimbo valleys are T. e. coquimbensis, thus extending its geographic distribution to the coast of Coquimbo and Atacama regions. We don´t have evidence for sympatric distribution between T. elegans and T. pallidior in the Coquimbo region.

Formation of cortical fields on a reduced cortical sheet.

Theories of both cortical field development and cortical evolution propose that thalamocortical projections play a critical role in the differentiation of cortical fields (; ). In the present study, we examined how changing the size of the immature neocortex before the establishment of thalamocortical connections affects the subsequent development and organization of the adult neocortex. This alteration in cortex is consistent with one of the most profound changes made to the mammalian neocortex throughout evolution: cortical size. Removing the caudal one-third to three-fourths of the cortical neuroepithelial sheet unilaterally at an early stage of development in marsupials resulted in normal spatial relationships between visual, somatosensory, and auditory cortical fields on the remaining cortical sheet. Injections of neuroanatomical tracers into the reduced cortex revealed in an altered distribution of thalamocortical axons; this alteration allowed the maintenance of their original anteroposterior distribution. These results demonstrate the capacity of the cortical neuroepithelium to accommodate different cortical fields at early stages of development, although the anteroposterior and mediolateral relationships between cortical fields appear to be invariant. The shifting of afferents and efferents with cortical reduction or expansion at very early stages of development may have occurred naturally in different lineages over time and may be sufficient to explain much of the phenotypic variation in cortical field number and organization in different mammals.

Structural characteristics and barrier properties of the choroid plexuses in developing brain of the opossum (Monodelphis Domestica).

The structural and functional development of the choroid plexuses, the site of the blood-cerebrospinal fluid (CSF) barrier, in an opossum (Monodelphis domestica) was studied. Marsupial species are extremely immature at birth compared with more conventional eutherian species. Choroid plexus tissue of each brain ventricle, from early stages of development, was collected for light and electron microscopy. During development, the choroidal epithelium changes from a pseudostratified to a cuboidal layer. Individual epithelial cells appear to go through a similar maturation process even though the timing is different between and within each plexus. The ultrastructural changes during development in the choroidal epithelial cells consist of an increase in the number of mitochondria and microvilli, and changes in structure of endoplasmic reticulum. There are also changes in the core of plexuses with age. In contrast, the structure of the tight junctions between epithelial cells does not appear to change with maturation. In addition, the route of penetration for lipid insoluble molecules from blood to CSF across the choroid plexuses was examined using a small biotin-dextran. This showed that the tight junctions already form a functional barrier in early development by preventing the paracellular movement of the tracer. Intracellular staining shows that there may be a transcellular route for these molecules through the epithelial cells from blood to CSF. Apart from lacking a glycogen-rich stage, cellular changes in the developing opossum plexus seem to be similar to those in other species, demonstrating that this is a good model for studies of mammalian choroid plexus development.

Observations on the development of brainstem-spinal systems in the North American oppossum.

The North American oppossum is born 12 to 13 days after conception and and is available for 90 days or more in an external pouch where it can be observed and experimentally manipulated. It is of particular interest that the hindlimbs of the newborn opossum are very immature and remain immobile for a week or more after birth. Degeneration techniques reveal that immature brainstem axons are present within the marginal zone of the lumbosacral cord before hindlimb movements begin (our state I) and material processed for formaldehyde induced fluorescence shows that some of them transport monoamines. Several lines of evidence suggest that part of the fluorescent axons arise within the nucleus locus coeruleus. At this early stage the electron microscope reveals that all brainstem-spinal axons are small (0.1--0.4 micrometer in diameter) and unmyelinated. By the time random hindlimb movements can be observed (stage II), brainstem axons, including those transporting monoamines, can be demonstrated to have grown into limited areas of the intermediate zone of the lumbosacral cord and to arise from most of the areas contributing to them in the adult animal (horseradish peroxidase technique). Such axons are still immature and it is not yet clear that they have formed synaptic terminals. Brainstem axons continue to grow into the intermediate zone of the lumbosacral cord for some time and come to occupy all of their adult territories before thoracic transection produces obvious change in hindlimb motility (beginning of stage III). It is still another 20 days or so before thoracic transection produces spinal shock comparable to that in the adult animal. The relatively mature use of the hindlimbs and the full expression of spinal shock correlate with changes in the technique and survival time needed to demonstrate degenerating brainstem axons in experimental material.

Organization and postnatal development of zebrin II antigenic compartmentation in the cerebellar vermis of the grey opossum, Monodelphis domestica.

The mammalian cerebellar cortex consists of a number of parasagittal Purkinje cell compartments that can be demonstrated cytochemically. The afferent inputs to the cerebellum are also compartmentalized, and a complex but reproducible relationship exists between the afferents and the intrinsic maps. Developmental studies in the rat have shown that many of the main features of compartmentation are already established at birth, and are therefore not easily manipulated experimentally. The compartmentation antigen zebrin II is expressed selectively by Purkinje cell subsets in a range of species, including fish and primates. In this study, zebrin II immunoreactivity has been studied in the grey opossum, Monodelphis domestica, in order to develop a marsupial model of compartment formation in which the early developmental events are more readily accessible. A monoclonal antibody to zebrin II from the weakly electric fish Apteronotus recognizes a 36 kD polypeptide in homogenates of Monodelphis cerebellum that appears to be identical to the antigen in the rat. Immunocytochemistry reveals that zebrin II in adult Monodelphis is confined exclusively to the cerebellum, where it is expressed by a subset of Purkinje cells. All regions of the cell, except the nucleus, are stained. The zebrin II+ Purkinje cells are arranged in a set of parasagittal compartments interposed by similar zebrin II- compartments. In each hemicerebellum there is one zebrin II+ band abutting the midline (P1+), and two others laterally in the vermis (P2+, P3+). A fourth zebrin II+ compartment straddles the paravermian region (P4+). Three other compartments have been identified in the hemisphere (P5+, P6+, P7+). This arrangement is very similar to that found in the rat. During postnatal development, zebrin II is first expressed between P14 and P21 in Purkinje cells of the posterior lobe vermis, and spreads throughout the cerebellar cortex by P28. As in rat, there is a stage at which all Purkinje cells are zebrin II+, including those destined to be zebrin II- in the adult. The mature pattern of expression emerges after P35 as immunoreactivity gradually disappears from the cells destined to become zebrin II-. The adult appearance is attained only after P56. The developmental timetable is therefore similar to that in rat, but is rather more protracted. Monodelphis should prove to be a valuable experimental model in which to study the early events leading to the formation of cerebellar compartments.

Localization of cholecystokinin binding sites in the adult and developing Brazilian opossum brain.

Cholecystokinin (CCK) is now recognized as one of the most abundant peptides in the mammalian central nervous system. We have previously used immunohistochemistry to localize CCK in the adult and developing Brazilian opossum brain. However, little is known about the distribution of CCK binding sites in the developing mammalian brain. Therefore, to further our knowledge of the sites of action for CCK during development, we initiated a series of studies to localize CCK binding sites in the adult and developing Brazilian opossum. This species was chosen because pups are born in a fetus-like state. Receptor autoradiography was performed on coronally sectioned brains of 1 to 60 day postnatal (PN) animals and adults with 125I-Bolton Hunter-CCK-8 as the radioligand. Binding is evident in the 1PN opossum brainstem and is observed in the developing forebrain by 5PN. Region-specific binding increases during development, and binding in the 35PN brain resembles the adult pattern. Binding is evident prior to the detection of CCK-like immunoreactivity in many areas. The facial motor nucleus is identifiable and exhibits high levels of binding in Brazilian opossum pups of 10 to 35 days of age. However, binding is undetectable in the facial motor nucleus of 45 and 60PN pups. In general, the binding patterns for CCK in the adult opossum resemble those of other mammals and likely mediate similar physiological functions. However, some cholecystokininergic pathways appear to be unique to neonatal mammals.

Developmental plasticity of reticulospinal and vestibulospinal axons in the north American opossum, Didelphis virginiana.

We have shown previously that rubral axons grow around a lesion of their spinal pathway in the North American opossum if it is made at early stages of development. In the present experiments, we have asked whether reticular and vestibular axons have the same ability. The spinal cord was hemisected at postnatal day 20, 12, or 5, well within the critical period for rubrospinal plasticity, and, approximately 30 days later, bilateral injections of fast blue were made about four segments caudal to the lesion. The pups were killed 4 or 5 days after the injections. In most of the animals lesioned on postnatal day 20, labeled neurons were not found in the medial part of the pontine reticular nucleus or the dorsal part of the lateral vestibular nucleus ipsilateral to the lesion. The spinal projections from both areas are exclusively ipsilateral. When the lesions were made at postnatal day 12 or 5, however, labeled neurons were present in both areas, suggesting that they supported axons that had grown caudal to the lesion. As was expected from previous studies, rubral neurons were labeled contralateral to the lesion in all three groups. In the opossum, as in other species, the red nucleus projects contralaterally. We conclude that reticular and vestibular axons, like axons from the red nucleus, grow around a lesion of their pathway during development and that the critical period for their plasticity ends earlier than that for rubrospinal axons.

Early development of the inferior olivary complex in pouch young opossums. II. An electron microscopic study.

At birth the inferior olivary complex (IOC) is not present in the caudal ventro-medial brainstem of the opossum. In the 3-7-day-old animal (15-19 days post-conception), this same region does contain neurons of the developing IOC. The immature neurons are characterized by large, centrally placed nuclei surrounded by a thin rim of cytoplasm. The neuropil contains numerous small-diameter profiles which contain bundles of filaments and scattered microtubules. Occasional synaptic endings, containing round clear vesicles, contact large, flocculent profiles. By 10-14 days of age, the olivary complex begins to separate into individual nuclei; however, the olivary cell bodies and the surrounding neuropil exhibit many of the same features as in the 3-7-day-old opossums. In opossums 21-25 days old, there is an increase in varicosities and irregular contours along many of the dendritic shafts. Furthermore, synaptic terminals, possessing round clear vesicles, now contact the soma, dendritic shafts, dendritic varicosities, spines, and large, flocculent profiles. Terminals containing pleomorphic vesicles or a mixture of clear and large granular vesicles are presynaptic only to dendritic spines or large, flocculent profiles. Neuroglial cell bodies have been identified at all ages examined. It is not until days 65-68 that pre- and postsynaptic elements are organized into synaptic clusters (glomeruli), which are typical of the adult. Spiny appendages and small-diameter dendrites comprise the central core of the clusters which are surrounded by synaptic endings containing a variety of vesicle types. Thus it would appear that subsequent to their initial arrival (day 16-17), the synaptic relationships of cerebellar and midbrain afferents are modified to reflect their adult configuration by days 65-68. This extended period of development (postnatal days 3-68) for the olivary complex provides a good model for assessing the effects of experimental manipulations.

The development and migration of large multipolar neurons into the cochlear nucleus of the North American opossum.

We have studied the maturation of the inferior colliculus and cochlear nuclei of the North American opossum with particular emphasis on the large multipolar neurons of the cochlear nucleus. These neurons include the principal and giant cells of the dorsal cochlear nucleus (DCN) and the large neurons of the ventral cochlear nucleus (VCN), all of which can be labelled by horseradish peroxidase (HRP) injections into the contralateral inferior colliculus (IC). The size of these neurons, their characteristic Nissl patterns, and their labelling density after injections into the IC render them distinguishable from other neurons in this nuclei, even in young animals. In Nissl-stained sections of newborn opossums, a band of horizontally oriented neurons can be identified dorsomedial to the vestibular nerve root. This band extends from an apparent cytogenetic zone close to the sulcus limitans, to, but not within, the presumptive cochlear nucleus. Between birth and estimated postnatal day 22 (EPND 22) the band shifts laterally, eventually becoming incorporated into the cochlear nucleus. Many neurons in this band have perinuclear caps of Nissl substance similar to those present in the principal cells of the adult DCN. Injections of HRP into the IC as early as EPND 5 (17 days after conception) labelled neurons in the band referred to above but not in the presumptive cochlear nucleus. By EPND 15, labelled cells were clustered mainly within the nucleus proper. Most of these cells were located in the DCN, but a few were scattered in the dorsocentral VCN. Consistent labelling of small neurons in VCN was not obtained until sometime later. From EPND 15 to EPND 20 most of the labelled cells in DCN reoriented in the vertical plane, aligned in layer II, and differentiated into principal neurons. Some, however, remained deep to layer II and differentiated into giant neurons. The heavily labelled cells in VCN differentiated into large neurons. Our results suggest that the large multipolar neurons of the nucleus are generated in a cytogenetic zone medial to the rhombic lip and that they subsequently migrate laterally, in a band, to reach their adult locations in the nucleus. It is during this migratory stage that their axons reach the inferior colliculus.

Evidence for growth of supraspinal axons through the lesion after transection of the thoracic spinal cord in the developing opossum Didelphis virginiana.

In the present study, we asked whether supraspinal axons grow through a complete transection of the spinal cord in the developing opossum Didelphis virginiana. When the thoracic cord was transected at postnatal day (PD) 5 and bilateral injections of Fast Blue (FB) were made four segments caudal to the lesion 30-40 days later, FB-containing neurons were found in each of the supraspinal nuclei labeled by comparable injections in age-matched unlesioned controls. Continuity between the cut ends of the cord was obviously gross when the animals were killed, and histologically recognizable spinal cord was present at the lesion site. When the same procedure was followed on pups subjected to transection at PD12, FB-containing neurons were still present at supraspinal levels, but they appeared to be fewer in number than in the PD5 cases or the age-matched controls, and none were found within the medial pontine reticular and lateral vestibular nuclei. When the lesion was made at PD20, labeled neurons were even fewer in number, and when it was made at PD26, they were restricted to the medullary raphe and the red nuclei. There was no evidence for growth of supraspinal axons across lesions made at PD33. We conclude that supraspinal axons grow through the lesion after transection of the spinal cord in neonatal opossums and that the critical period for growth of reticulospinal and vestibulospinal axons through the lesion ends earlier than that for comparable growth of raphespinal and rubrospinal axons.

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.

Early development of the optic chiasm in the gray short-tailed opossum, Monodelphis domestica.

We have studied the early development of the uncrossed retinofugal projection in the gray short-tailed opossum. Axons that form the adult uncrossed retinofugal projection arise from the temporal crescent of the retina and reach the optic chiasm on postnatal day 7. The sites at which the uncrossed fibres segregate from the crossed fibres and the pattern of this segregation are very different from those seen in eutherian mammals. In the opossum, the uncrossed fibres segregate from the crossed fibres within the juxtachiasmatic part of the optic nerve before they have encountered either the fibres of the other eye or midline structures of the ventral diencephalon. The uncrossed fibres turn perpendicular to the axis of the nerve and grow dorsoventrally through the crossed projection to gather as a discrete bundle at the ventral edge of the nerve. The abrupt divergence of the uncrossed fibres occurs at a border between two glial cell types: the interfascicular glia that characterise the main part of the optic nerve and the radial glia of the juxtachiasmatic part of the nerve. At the ventral part of the nerve, the bundle of uncrossed fibres turns caudally across the axis of the nerve and enters the ipsilateral optic tract. When retinofugal fibres encounter the border between the interfascicular and radial glia, a very specific axonal reorganisation occurs in marsupials, and this is strikingly different from the axonal reorganisation that occurs at the same site in eutherians, where essentially all retinofugal fibres reorganise, not just the uncrossed component. We believe this to be an important example of an identified cellular element that has quite distinct axon-guidance properties in different species.

Development of projections from somatic motor-sensory areas of neocortex to the diencephalon and brainstem in the North American opossum.

The development of projections from somatic motor-sensory areas of neocortex to the diencephalon and brainstem was studied by using the orthograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in a series of pouch-young opossums. The opossum was chosen for study because it is born in a very immature state, 12 days after conception, and has a protracted postnatal development. Cortical axons form a cerebral peduncle by at least postnatal day (PD) 10, a medullary pyramid by estimated PD (EPD) 17, a pyramidal decussation by EPD 26, and reach the first cervical segment of the spinal cord by EPD 29. Cortical axons innervate diencephalic nuclei and perhaps the substantia nigra by EPD 17, but do not grow into more caudal brainstem nuclei until EPD 26. The first brainstem areas innervated by cortical axons are the mesencephalic and rostral pontine tegmentum and parts of the pontine gray adjacent to the pyramidal tract (EPD 29). By EPD 31, cortical axons project to additional areas of the pontine gray, the gigantocellular reticular formation, the medial accessory olive, and the cuneate nucleus. Cortical innervation of the red nucleus and superior colliculus begins at EPD 31 but is not well developed until EPD 35. Cortical axons do not innervate the parvicellular reticular formation or the sensory trigeminal nuclei until EPD 35. Evidence for transient cerebrocerebellar axons was also found.

Olfactory bulb organization and development in Monodelphis domestica (grey short-tailed opossum).

The olfactory bulbs of adult and developing Monodelphis domestica were examined with a number of techniques. Golgi, Nissl, and Timm stains as well as acetylcholinesterase histochemistry revealed a high degree of order within the adult bulb. All major cell classes characteristic of most mammalian species were observed. Tufted cells appeared to be restricted to the superficial portion of the external plexiform layer. Developing Monodelphis pups were examined with Nissl-stained semithin sections and with immunocytochemistry for tyrosine hydroxylase, microtubule-associated protein 2, vimentin, and glial fibrillary acidic protein. Newborn pups are extremely immature, with few postmitotic cells present in the forebrain. Considerable maturation occurs over the first four postnatal weeks, and by postnatal day 30, the bulb assumes an adult-like organization. The extreme immaturity of the bulb at birth, coupled with its strict organization, suggest that Monodelphis is a particularly appropriate species for experimental examinations of olfactory system development.