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In the early part of the 20th century, J. P. Hill and K. P. Watson embarked on a comprehensive study of the development of the brain in Australian marsupials. Their work included series from three major groups: dasyurids, peramelids, and diprotodonts, covering early primitive streak through brain closure and folding stages. While the major part of the work was on the development of the brain, in the course of this work they documented that cellular proliferations from the neural plate provided much of the mesenchyme of the branchial arches. These proliferations are now known to be the neural crest. However, except for a very brief note, published shortly after Hill's death, this work was never published. In this study, I present Hill and Watson's work on the development of the early neural plate and the neural crest in marsupials. I compare their findings with published work on the South American marsupial, Monodelphis domestica and demonstrate that patterns reported in Monodelphis are general for marsupials. Further, using their data I demonstrate that in dasyurids, which are ultra-altricial at birth, the neural crest migrates early and in massive quantities, even relative to other marsupials. Finally, I discuss the historical context and speculate on reasons for why this work was unpublished. I find little support for ideas that Hill blocked publication because of loyalty to the germ layer theory. Instead, it appears primarily to have been a very large project that was simply orphaned as Watson and Hill pursued other activities., (© 2020 Wiley Periodicals LLC.)

Embryo implantation is a hallmark of the female reproductive biology of eutherian (placental) mammals and does not exist in a sustainable form in any other vertebrate group. Implantation is the initial process that leads to a sustained fetal-maternal unit engendering a complex functional relationship between the mother and the embryo/fetus. The nature of this relationship is often portrayed as one of conflict between an aggressive embryo and a passive or defensive maternal organism. Recent progress in elucidating the evolutionary origin of eutherian pregnancy leads to a different picture. The emerging scenario suggests that the very initial stages in the evolution of embryo implantation required evolutionary changes to the maternal physiology which modified an ancestral generic mucosal inflammation in response to the presence of the embryo into an active embedding process. This "female-first" evolutionary scenario also explains the role of endometrial receptivity in human pregnancy. On the marsupial side, where in most animals the fetal-maternal interaction is short and does not lead to a long term sustainable placentation, the relationship is mutual. In these mammals, uterine inflammation is followed by parturition in short order. The inflammatory signaling pathways, however, are cooperative, i.e., they are performed by both the fetus and the mother and therefore we call this relationship "cooperative inflammation." Based on these discoveries we reconceive the narrative of the maternal-fetal relationship., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Neonates of marsupial mammals are altricial at birth, because their gestation period is relatively short compared to placental mammals. Yet, as they need to travel to the teat from the birth canal, and suckle on the mother's milk, forelimbs and jaws develop significantly early. Previous studies in opossum (Monodelphis domestica), an experimental marsupial model, have revealed that cranial neural crest cells are generated significantly early compared to those in placental mammals, such as mouse, leading to an early development of jaw primordia. We have previously found that Sox9, an important neural crest-specifier gene, is expressed in the future cranial neural crest of the opossum embryonic ectoderm significantly earlier than that in mouse or quail embryos. As Sox9 is essential for neural crest formation in various vertebrates, it seems likely that the heterochronic expression of Sox9 is critical for the early cranial neural crest formation in the marsupial embryos. In this study, we show a marsupial-specific sequence in the Sox9 neural crest enhancer E3. We also reveal that the mouse E3 enhancer is activated in the cranial neural crest cells of quail embryos, that the E3 enhancer with marsupial-specific sequence is activated earlier in the Pax7-expressing neural border prior to the onset of endogenous Sox9 expression, and that a misexpression of cMyb, which is also a transcriptional activator of Pax7, in the neural border can ectopically activate the "marsupialized" enhancer. Thus, we suggest that the modification of the E3 enhancer sequence in the marsupial ancestor would have promoted the early expression of Sox9 in the neural border, facilitating the early formation of the cranial neural crest cells and the subsequent heterochronic development of the jaw primordia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Marsupial oocytes and conceptuses provide special challenges to scientists wanting to develop reliable in vitro techniques. Yet these techniques are essential to the study of development. Such techniques also provide tools to help prevent further decline in marsupial biodiversity using assisted reproductive technology. Specific marsupial characters have made development of in vitro techniques difficult. Some of these are the high-energy requirements of cleavage and blastocyst formation and maintenance because of cell-zona adhesion; the essential nature of the shell coat for most of development; the prevalence of embryonic arrests in vivo and in vitro during cleavage and unilaminar blastocyst stages; and the fragility of blastocysts leading to precocious shell loss and developmental failure in vitro. The advantages of marsupial culture during development are that the gestation period is very short, that the implantation is superficial in many, and that the neonates are altricial. This chapter outlines solutions to some of these problems in a representative, polytocous, dasyurid marsupial, Sminthopsis macroura. It is a natural superovulator with the shortest gestation period of any mammal, which has been cultured to within 5 h of birth. The other representative marsupial, the monovular phalangerid Trichosurus vulpecula, has a very superficial implantation that allows vesicles to readily detach from the endometrium.

Marsupial embryos of Armadillidium vulgare (Isopoda: Oniscidea) were collected at different stages of development and assayed for products of nitrogen excretion. Stages were classified as early stage one, late stage one (clear embryo and somite differentiation), early stage two (chorion shed, prior to blastokinesis), late stage two (following blastokinesis), and mancae (vitelline membrane shed; second embryonic molt). Stage one and stage two embryos were primarily ammonotelic. Mancae showed a significant increase in stored uric acid and decrease in ammonia production, in most cases to undetectable levels. The increased metabolic rate of mancae, and the fact that they imbibe marsupial fluid prior to exiting the marsupium, may have favored a switch from ammonotely to uricotely to avoid ammonia toxicity. Protein metabolism, estimated from ammonia production, accounted for 7% of the measured catabolic rate in Stage 2 embryos. Newly emerged juveniles showed a >2-fold increase in metabolism relative to mancae, accompanying the transition from aquatic to aerial respiration. Following 48 h post-emergence, juveniles resumed ammonia excretion, volatilizing the base (NH 3 ) as in later instars. Elevated ammonia excretion in early juveniles may derive from the catabolism of remaining yolk protein. A sharp increase in whole-animal glutamine in juveniles is consistent with its role as an intermediary nitrogen store during periodic ammonia excretion. Total ammonia concentration in the marsupial fluid fluctuated but did not increase significantly over time and ammonia was not volatilized across the oostegites, indicating that embryo ammonia is transported into the maternal hemolymph for excretion., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The study of oogenesis and early development of frogs belonging to the family Hemiphractidae provide important comparison to the aquatic development of other frogs, such as Xenopus laevis, because reproduction on land characterizes the Hemiphractidae. In this review, the multinucleated oogenesis of the marsupial frog Flectonotus pygmaeus (Hemiphractidae) is analyzed and interpreted. In addition, the adaptations associated with the incubation of embryos in the pouch of the female marsupial frog Gastrotheca riobambae (Hemiphractidae) and the embryonic development of this frog are summarized. Moreover, G. riobambae gastrulation is compared with the gastrulation modes of Engystomops randi and Engystomops coloradorum (Leptodactylidae); Ceratophrys stolzmanni (Ceratophryidae); Hyalinobatrachium fleischmanni and Espadarana callistomma (Centrolenidae); Ameerega bilinguis, Dendrobates auratus, Epipedobates anthonyi, Epipedobates machalilla, Epipedobates tricolor, and Hyloxalus vertebralis (Dendrobatidae); Eleutherodactylus coqui (Terrarana: Eleutherodactylidae), and X. laevis (Pipidae). The comparison indicated two modes of frog gastrulation. In X. laevis and in frogs with aquatic reproduction, convergent extension begins during gastrulation. In contrast, convergent extension occurs in the post-gastrula of frogs with terrestrial reproduction. These two modes of gastrulation resemble the transitions toward meroblastic cleavage found in ray-finned fishes (Actinopterygii). In spite of this difference, the genes that guide early development seem to be highly conserved in frogs. I conclude that the shift of convergent extension to the post-gastrula accompanied the diversification of frog egg size and terrestrial reproductive modes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mammals evolved from oviparous reptiles that laid eggs in a dry, terrestrial environment, thus requiring large amounts of yolk to support development and tough, outer coats to protect them. Eutherian mammals such as humans and mice exhibit an "extreme" form of viviparity in which yolk and conceptus coats have become largely redundant. However, the "other" mammals-monotremes and marsupials-have retained and modified some features of reptilian development that provide valuable insights into the evolution of viviparity in mammals. Most striking of these are the conceptus coats, which include the zona pellucida, the mucoid coat, and the shell coat. We discuss current knowledge of these coats in monotremes and marsupials, their possible roles, and recently identified components such as the zona pellucida protein ZPAX, conceptus coat mucin (CCM), and nephronectin (NPNT)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Most of our understanding of forebrain development comes from research of eutherian mammals, such as rodents, primates, and carnivores. However, as the cerebral cortex forms largely prenatally, observation and manipulation of its development has required invasive and/or ex vivo procedures. Marsupials, on the other hand, are born at comparatively earlier stages of development and most events of forebrain formation occur once attached to the teat, thereby permitting continuous and non-invasive experimental access. Here, we take advantage of this aspect of marsupial biology to establish and characterise a resourceful laboratory model of forebrain development: the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-sized carnivorous Australian marsupial. We present an anatomical description of the postnatal development of the body, head and brain in dunnarts, and provide a staging system compatible with human and mouse developmental stages. As compared to eutherians, the orofacial region develops earlier in dunnarts, while forebrain development is largely protracted, extending for more than 40 days versus ca. 15 days in mice. We discuss the benefits of fat-tailed dunnarts as laboratory animals in studies of developmental biology, with an emphasis on how their accessibility in the pouch can help address new experimental questions, especially regarding mechanisms of brain development and evolution.

A central question in the evolution of brain development is whether species differ in rates of brain growth during fetal neurogenesis. Studies of neonatal data have found allometric evidence for brain growth rate differences according to physiological variables such as relative metabolism and placental invasiveness, but these findings have not been tested against fetal data directly. Here, we measure rates of exponential brain growth acceleration in 10 eutherian mammals, two marsupials, and two birds. Eutherian brain acceleration exhibits minimal variation relative to body and visceral organ growth, varies independently of correlated growth patterns in other organs, and is unrelated to proposed physiological constraints such as metabolic rate or placental invasiveness. Brain growth rates in two birds overlap with eutherian variation, while marsupial brain growth is exceptionally slow. Peak brain growth velocity is linked in time with forebrain myelination and eye opening, reliably separates altricial species born before it from precocial species born afterwards, and is an excellent predictor of adult brain size ( r 2 = 0.98). Species with faster body growth exhibit larger relative brain size in early ontogeny, while brain growth is unrelated to allometric measures. These findings indicate a surprising conservation of brain growth rates during fetal neurogenesis in eutherian mammals, clarify sources of variation in neonatal brain size, and suggest that slow body growth rates cause species to be more encephalized at birth., (© 2017 The Author(s).)

The first lineage allocation during mouse development forms the trophectoderm and inner cell mass, in which Cdx2 and Pou5f1 display reciprocal expression. Yet Cdx2 is not required for trophectoderm specification in other mammals, such as the human, cow, pig, or in two marsupials, the tammar and opossum. The role of Cdx2 and Pou5f1 in the first lineage allocation of Sminthopsis macroura, the stripe-faced dunnart, is unknown. In this study, expression of Cdx2 and Pou5f1 during oogenesis, development from cleavage to blastocyst stages, and in the allocation of the first three lineages was analyzed for this dunnart. Cdx2 mRNA was present in late antral-stage oocytes, but not present again until Day 5.5. Pou5f1 mRNA was present from primary follicles to zygotes, and then expression resumed starting at the early unilaminar blastocyst stage. All cleavage stages and the pluriblast and trophoblast cells co-expressed CDX2 and POU5F1 proteins, which persisted until early stages of hypoblast formation. Hypoblast cells also show co-localisation of POU5F1 and CDX2 once they were allocated, and this persisted during their division and migration. Our studies suggest that CDX2, and possibly POU5F1, are maternal proteins, and that the first lineage to differentiate is the trophoblast, which differentiates to trophectoderm after shell loss one day before implantation. In the stripe-faced dunnart, cleavage cells, as well as trophoblast and pluriblast cells, are polarized, suggesting the continued presence of CDX2 in both lineages until late blastocyst stages may play a role in the formation and maintenance of polarity., (© 2015 Wiley Periodicals, Inc.)

Metatherians and monotremes are born in an immature state, followed by prolonged nurturing by maternal lactation. Quantitative analysis of isocortical sections held in the collections at the Museum für Naturkunde, Berlin was used to compare the pace of somatosensory cortex development relative to body size and pallial thickness between metatherian groups, monotremes, and the laboratory rat. Analysis indicated that the pace of pallial growth in the monotremes is much lower than that in the metatherians or laboratory rat, with an estimated 8.6-fold increase in parietal cortex thickness between 10 and 100 mm body length, compared to a 10- to 20-fold increase among the metatherians and the rat. It was found that aggregation of cortical plate neurons occurs at similar embryo size in the mammals studied (around 8-14 mm body length) and a similar pallial thickness (around 200 µm), but that proliferative zone involution occurs at a much higher body size and pallial thickness in the monotremes compared to the metatherians and the laboratory rat. The observations suggest that cortical development in the monotremes is slower and subject to different regulatory signals to the therians studied. The slow pace may be related to either generally slower metabolism in monotremes or less efficient nutrient supply to the offspring due to the lack of teats.

A primary goal of evolutionary biology is to identify the factors that shape phenotypic evolution. According to the theory of natural selection, phenotypic evolution occurs through the differential survival and reproduction of individuals whose traits are selectively advantageous relative to other individuals in the population. This implies that evolution by natural selection is contingent upon the distribution and magnitude of phenotypic variation among individuals, which are in turn the products of developmental processes. Development therefore has the potential to affect the trajectory and rate of phenotypic evolution. Recent research in diverse systems (e.g., mammalian teeth, cichlid skulls, butterfly wings, and marsupial limbs) supports the hypothesis that development biases phenotypic variation and evolution, but suggests that these biases might be system-specific., (© 2014 Wiley Periodicals, Inc.)

The young of marsupials and monotremes are all born in an immature state, followed by prolonged nurturing by maternal lactation in either a pouch or nest. Nevertheless, the level of locomotor ability required for newborn marsupials and monotremes to reach the safety of the pouch or nest varies considerably: some are transferred to the pouch or nest in an egg (monotremes); others are transferred passively by gravity (e.g. dasyurid marsupials); some have only a horizontal wriggle to make (e.g. peramelid and didelphid marsupials); and others must climb vertically for a long distance to reach the maternal pouch (e.g. diprotodontid marsupials). In the present study, archived sections of the inner ear and hindbrain held in the Bolk, Hill and Hubrecht collections at the Museum für Naturkunde, Berlin, were used to test the relationship between structural maturity of the vestibular apparatus and the locomotor challenges that face the young of these different mammalian groups. A system for staging different levels of structural maturity of the vestibular apparatus was applied to the embryos, pouch young and hatchlings, and correlated with somatic size as indicated by greatest body length. Dasyurids are born at the most immature state, with the vestibular apparatus at little more than the otocyst stage. Peramelids are born with the vestibular apparatus at a more mature state (fully developed semicircular ducts and a ductus reuniens forming between the cochlear duct and saccule, but no semicircular canals). Diprotodontids and monotremes are born with the vestibular apparatus at the most mature state for the non-eutherians (semicircular canals formed, maculae present, but vestibular nuclei in the brainstem not yet differentiated). Monotremes and marsupials reach the later stages of vestibular apparatus development at mean body lengths that lie within the range of those found for laboratory rodents (mouse and rat) reaching the same vestibular stage., (© 2013 Anatomical Society.)

The mammalian phallus arises from identical primordia in both sexes and is patterned in part by the key morphogen Sonic hedgehog (SHH). We have investigated SHH and other morphogens during phallus development in the tammar wallaby. In this marsupial, testis differentiation and androgen production occurs just after birth, but it takes a further 50-60 days before the phallus becomes sexually dimorphic. One day before birth, SHH was expressed in both sexes in the urethral epithelium. In males, there was a marked upregulation of SHH, GLI2, and AR at day 50 postpartum, a time when testicular androgen production falls. SHH, GLI2, and AR were downregulated in female pouch young treated with androstanediol from days 24-50, but not when treatments were begun at day 29, suggesting an early window of androgen sensitivity. SHH, GLI2, and AR expression in the phallus of males castrated at day 23 did not differ from controls, but there was an increase in SHH and GLI2 and a decrease in FGF8 and BMP4 expression when the animals were castrated at day 29. These results suggest that the early patterning by SHH is androgen-independent followed by an androgen-dependent window of sensitivity and a sharp rise in SHH expression after androgen withdrawal at day 50., (© 2014 S. Karger AG, Basel.)

Background: The classic model of sex determination in mammals states that the sex of the individual is determined by the type of gonad that develops, which in turn determines the gonadal hormonal milieu that creates sex differences outside of the gonads. However, XX and XY cells are intrinsically different because of the cell-autonomous sex-biasing action of X and Y genes., Results: Recent studies of mice, in which sex chromosome complement is independent of gonadal sex, reveal that sex chromosome complement has strong effects contributing to sex differences in phenotypes such as metabolism. Adult mice with two X chromosomes (relative to mice with one X chromosome) show dramatically greater increases in body weight and adiposity after gonadectomy, irrespective of their gonadal sex. When fed a high-fat diet, XX mice develop striking hyperinsulinemia and fatty liver, relative to XY mice. The sex chromosome effects are modulated by the presence of gonadal hormones, indicating an interaction of the sex-biasing effects of gonadal hormones and sex chromosome genes., Conclusions: Other cell-autonomous sex chromosome effects are detected in mice in many phenotypes. Birds (relative to eutherian mammals) are expected to show more widespread cell-autonomous sex determination in non-gonadal tissues, because of ineffective sex chromosome dosage compensation mechanisms., (Copyright © 2013 Wiley Periodicals, Inc.)

Early cell lineage specification in eutherian mammals results in the formation of a pluripotent inner cell mass (ICM) and trophoblast. By contrast, marsupials have no ICM. Here, we present the first molecular analysis of mechanisms of early cell lineage specification in a marsupial, the tammar wallaby. There was no overt differential localisation of key lineage-specific transcription factors in cleavage and early unilaminar blastocyst stages. Pluriblast cells (equivalent to the ICM) became distinguishable from trophoblast cells by differential expression of POU5F1 and, to a greater extent, POU2, a paralogue of POU5F1. Unlike in the mouse, pluriblast-trophoblast differentiation coincided with a global nuclear-to-cytoplasmic transition of CDX2 localisation. Also unlike in the mouse, Hippo pathway factors YAP and WWTR1 showed mutually distinct localisation patterns that suggest non-redundant roles. NANOG and GATA6 were conserved as markers of epiblast and hypoblast, respectively, but some differences to the mouse were found in their mode of differentiation. Our results suggest that there is considerable evolutionary plasticity in the mechanisms regulating early lineage specification in mammals.

The signaling molecule DHH, secreted by Sertoli cells, has essential regulatory functions in testicular differentiation. DHH is required for the differentiation of peritubular myoid cells that line the seminiferous cords and steroidogenic Leydig cells. The testicular cords in Dhh-null male mice lack a basal lamina and develop abnormally. To date, the DHH-signaling pathway has never been examined outside of any eutherian mammals. This study examined the effects of inhibition of DHH signaling in a marsupial mammal, the tammar wallaby, by culturing gonads in vitro in the presence of the hedgehog-signaling inhibitors cyclopamine and forskolin. Disruption of hedgehog signaling in the tammar testes caused highly disorganized cord formation. SOX9 protein remained strongly expressed in Sertoli cells, laminin distribution was highly fragmented, and germ cells were distributed around the cortical regions of treated testes in an ovarianlike morphology. This suggests that hedgehog signaling regulates cord formation in the tammar wallaby testis as it does in eutherian mammals. These data demonstrate that the hedgehog pathway has been highly conserved in mammals for at least 160 million years.

Kallmann syndrome is characterized by hypogonadotrophic hypogonadism and anosmia. The syndrome can be caused by mutations in several genes, but the X-linked form is caused by mutation in the Kallmann syndrome 1 (KAL1). KAL1 plays a critical role in gonadotropin-releasing hormone (GnRH) neuronal migration that is essential for the normal development of the hypothalamic-pituitary-gonadal axis. Interestingly, KAL1 appears to be missing from the rodent X, and no orthologue has been detected as yet. We investigated KAL1 during development and in adults of an Australian marsupial, the tammar wallaby, Macropus eugenii. Marsupial KAL1 maps to an autosome within a group of genes that was added as a block to the X chromosome in eutherian evolution. KAL1 expression was widespread in embryonic and adult tissues. In the adult testis, tammar KAL1 mRNA and protein were detected in the germ cells at specific stages of differentiation. In the adult testis, the protein encoded by KAL1, anosmin-1, was restricted to the round spermatids and elongated spermatids. In the adult ovary, anosmin-1 was not only detected in the oocytes but was also localized in the granulosa cells throughout folliculogenesis. This is the first examination of KAL1 mRNA and protein localization in adult mammalian gonads. The protein localization suggests that KAL1 participates in gametogenesis not only through the development of the hypothalamic-pituitary-gonadal axis by activation of GnRH neuronal migration, but also directly within the gonads themselves. Because KAL1 is autosomal in marsupials but is X-linked in eutherians, its conserved involvement in gametogenesis supports the hypothesis that reproduction-related genes were actively recruited to the eutherian X chromosome.

To understand developmental mechanisms of evolutionary change, we must first know how different morphologies form. The vast majority of our knowledge on the developmental genetics of tooth formation derives from studies in mice, which have relatively derived mammalian dentitions. The marsupial Monodelphis domestica has a more plesiomorphic heterodont dentition with incisors, canines, premolars, and molars on both the upper and the lower jaws, and a deciduous premolar. The complexity of the M. domestica dentition ranges from simple, unicusped incisors to conical, sharp canines to multicusped molars. We examine the development of the teeth in M. domestica, with a specific focus on the enamel knot, a signaling center in the embryonic tooth that controls shape. We show that the tooth germs of M. domestica express fibroblast growth factor (FGF) genes and Sprouty genes in a manner similar to wild-type mouse molar germs, but with a few key differences., (© 2010 Wiley-Liss, Inc.)

Marsupial mammals are born in an embryonic state, as compared with their eutherian counterparts, yet certain features are accelerated. The most conspicuous of these features are the precocial forelimbs, which the newborns use to climb unaided from the opening of the birth canal to the teat. The developmental mechanisms that produce this acceleration are unknown. Here we show that heterochronic and heterotopic changes early in limb development contribute to forelimb acceleration. Using Tbx5 and Tbx4 as fore- and hindlimb field markers, respectively, we have found that, compared with mouse, both limb fields arise notably early during opossum development. Patterning of the forelimb buds is also accelerated, as Shh expression appears early relative to the outgrowth of the bud itself. In addition, the forelimb fields and forelimb myocyte allocation are increased in size and number, respectively, and migration of the spinal nerves into the forelimb bud has been modified. This shift in the extent of the forelimb field is accompanied by shifts in Hox gene expression along the anterior-posterior axis. Furthermore, we found that both fore- and hindlimb fields arise gradually during gastrulation and extension of the embryonic axis, in contrast to the appearance of the limb fields in their entirety in all other known cases. Our results show a surprising evolutionary flexibility in the early limb development program of amniotes and rule out the induction of the limb fields by mature structures such as the somites or mesonephros.

Genomic imprinting imposes an obligate mode of biparental reproduction in mammals. This phenomenon results from the monoparental expression of a subset of genes. This specific gene regulation mechanism affects viviparous mammals, especially eutherians, but also marsupials to a lesser extent. Oviparous mammals, or monotremes, do not seem to demonstrate monoparental allele expression. This phylogenic confinement suggests that the evolution of the placenta imposed a selective pressure for the emergence of genomic imprinting. This physiological argument is now complemented by recent genomic evidence facilitated by the sequencing of the platypus genome, a rare modern day case of a monotreme. Analysis of the platypus genome in comparison to eutherian genomes shows a chronological and functional coincidence between the appearance of genomic imprinting and transposable element accumulation. The systematic comparative analyses of genomic sequences in different species is essential for the further understanding of genomic imprinting emergence and divergent evolution along mammalian speciation.

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT-PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two- and four-cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two- to eight-cell stage. Lipid droplets largely segregate to pluriblast cells at the 16-cell stage, suggesting a role in pluriblast lineage allocation.

POU5F1 (OCT4) encodes a master regulator of pluripotency that is present in all mammals. A paralogue, POU2, is also present in the genomes of marsupials and monotremes and is an orthologue of zebrafish pou2 and chicken POUV. We explored the evolution of class V POU domain transcription factors and show that POU5F1 arose by gene duplication of pou2 early in the evolution of tetrapods and is not mammal-specific, as previously thought. Instead, either POU5F1 or POU2/POUV has become extinct independently in various lineages, although all gnathostomes appear to possess at least one or the other. In the tammar wallaby, POU5F1 expression is limited to pluripotent cell types (embryonic tissues and germ cells). POU2 is similarly expressed in pluripotent tissues but is also expressed in a broad range of adult tissues. Thus, unlike POU5F1, the role of POU2 may not be restricted to pluripotent cell types but could have a related function such as maintaining multipotency in adult stem cells.

There are three fetal membranes in mammals, namely the yolk sac, allantois and amnion, but only the first two form a placenta. In Monotremata (monotremes), Marsupialia (marsupials) and Placentalia (eutherians), the yolk sac transfers nutrients originating from uterine glandular secretion or the maternal blood. Ontogenetically, the yolk sac of most eutherians loses contact with the peripheral chorion and forms a free splanchnopleuric yolk sac that transfers substances from the exocoelomic cavity, not directly from the endometrium as in marsupials and rodents. This free yolk sac has been preserved in humans and substances originating from glandular secretions are transferred from the exocoelomic cavity to the embryo via this route. The therian yolk sac expresses numerous growth and transcription factors, associated binding proteins and receptors that control its differentiation and function, including hematopoiesis and angiogenesis. Errors in yolk sac development and function could contribute to embryonic malformation, miscarriage and growth diseases. Factors regulating transfer and metabolism, notably insulin and IGF2, are imprinted in the human yolk sac, as in all therians so far studied. This suggests persistence of a strong selective pressure for parentally controlled allocation of resources to the growing embryo via this fetal membrane. The metabolic and biosynthetic functions of the yolk sac of the ancestral therian stem species, as well as hematopoiesis in the eutherian ancestor, appear to have been retained by the human yolk sac. Thus, the yolk sac of humans, like that of all viviparous mammals, is a true placenta crucial for early embryonic development and survival.

Marsupials represent only 6% of all living mammals. Marsupialia and Placentalia are distinguished mainly by their modes of reproduction. In particular, the differences in the stage of development of the neonates may be one explanation for the divergent evolutionary success. In this respect one important question is whether the survivability of the neonate depends on the degree of maturation of the respiratory system relative to the metabolic capacity at the time of birth. Therefore, this review highlights the differences in lung morphology and metabolic development of extant Marsupialia and Placentalia. The Marsupial neonate is born with a low birth weight and is highly immature. The neonatal lung is characterized by large terminal sacs, a poorly developed bronchial system and late formation of alveoli. Marsupialia have a low metabolic rate at birth and attain adult metabolic rate and thermoregulatory capacity late in postnatal development. In contrast, the eutherian neonate is born with a relative high birth weight and is always more mature than marsupial neonates. The neonatal lung has small terminal sacs, the bronchial system is well developed and the formation of alveoli begins few days after birth. Placentalia have a high metabolic rate at birth and attain adult metabolic rate and thermoregulatory capacity early in postnatal development. The differences in the developmental degree of the newborn lung between Marsupialia and Placentalia have consequences for their metabolic and thermoregulatory capacity. These differences could be advantageous for Placentalia in the changing environments in which they evolved.

The marsupial conceptus is surrounded by a uterine-secreted shell coat for 60-80% of gestation. Coat protein 4 (CP4) is the only marsupial shell coat protein characterized and it has only been identified in one species, the Common Brushtail Possum. In this possum, uterine transcription and secretion of cp4 during the oestrous cycle is biphasic and associated with the stage of conceptus development. Here we cloned cp4 (sm-cp4) from a distantly related species, the Stripe-faced Dunnart (Sminthopsis macroura). Transcription of sm-cp4 and secretion of smCP4 were identified by semi-quantitative RT-PCR and immunohistochemistry, respectively. The effect of reproductive hormones on sm-cp4 transcription was investigated in vitro by treatment of uterine explant cultures with oestrogen and/or progesterone. In vivo, uterine expression of smCP4 was biphasic and associated with conceptus development. Uterine smCP4 expression (transcription and secretion) began during the pre-ovulatory period and continued post-ovulation during cleavage stages. Transcription of sm-cp4 continued during the unilaminar blastocyst stage, but smCP4 secretion was reduced during this stage. During the bilaminar blastocyst stage, both transcription of sm-cp4 and secretion of smCP4 were low before they both resumed during the trilaminar blastocyst stage, and continued during the embryo and fetal stages. In vitro uterine transcription of sm-cp4 increased after incubation with progesterone.

In contrast to placentals, marsupials are born with forelimbs that are greatly developmentally advanced relative to their hind limbs. Despite significant interest, we still do not know why this is the case, or how this difference is achieved developmentally. Studies of prechondrogenic and chondrogenic limbs have supported the traditional hypothesis that marsupial forelimb development is accelerated in response to the functional requirements of the newborn's crawl to the teat. However, limb ossification studies have concluded that, rather than the forelimb being accelerated, hind limb development is delayed. By increasing the taxonomic coverage and number of prechondrogenic events relative to previous studies, and combining traditional phylogenetic analyses of event sequences with novel analyses of relative developmental rates, this study demonstrates that the timing of limb development in marsupials is more complex than commonly thought. The marsupial phenotype was derived through two independent evolutionary changes in developmental rate: (1) an acceleration of the forelimb's first appearance and (2) a delay of hind limb development from the bud stage onward. Surprisingly, this study also provides some support for an evolutionary acceleration of the marsupial hind limb's first appearance. Further study is needed on the developmental and genetic mechanisms driving these major evolutionary transitions.

Close examination of hormonal profiles and uterine morphology in the marsupial reproductive cycle highlights significant differences between pregnant and non-pregnant cycles. In the polyovular dasyurid marsupial Sminthopsis macroura, we identified changes associated with gestation by comparing ovarian and plasma progesterone concentrations, uterine weights, uterine epithelial mitoses, body weights and gestation lengths between pregnant and non-pregnant luteal phases. The plasma progesterone profile of S. macroura was biphasic, peaking during unilaminar blastocyst expansion and on the day of implantation. Periods of rapid embryonic development were associated with increasing plasma progesterone concentrations and animal body weight. For the first time in a polyovular marsupial, we identified 1) a correlation between ovarian progesterone concentration and conceptus number during the luteal phase just prior to implantation (total ovarian progesterone), indicating a conceptus influence on progesterone concentration; 2) a pulse of uterine epithelial mitotic activity at the time of implantation and 3) increased mitotic activity in pregnant animals during unilaminar blastocyst formation compared with non-pregnant animals. Gestation length was reduced by up to 15%, due to the loss of, or reduction in, the four-cell arrest and more rapid definitive blastocyst expansion. This is the first time a conceptus influence on gestation length has been identified in a dasyurid. This study provides further evidence for the modification of the luteal phase by pregnancy in S. macroura.

Genomic imprinting is a widespread epigenetic phenomenon in eutherian mammals, which regulates many aspects of growth and development. Parental conflict over the degree of maternal nutrient transfer is the favoured hypothesis for the evolution of imprinting. Marsupials, like eutherian mammals, are viviparous but deliver an altricial young after a short gestation supported by a fully functional placenta, so can shed light on the evolution and time of acquisition of genomic imprinting. All orthologues of eutherian imprinted genes examined have a conserved expression in the marsupial placenta regardless of their imprint status. Differentially methylated regions (DMRs) are the most common mechanism controlling genomic imprinting in eutherian mammals, but none were found in the marsupial imprinted orthologues of IGF2 receptor (IGF2R), INS or mesoderm-specific transcript (MEST). Instead, histone modification appears to be the mechanism used to silence these genes. At least three genes in marsupials have DMRs: H19, IGF2 and PEG10. PEG10 is particularly interesting as it is derived from a retrotransposon, providing the first direct evidence that retrotransposon insertion can drive the evolution of an imprinted region and of a DMR in mammals. The insertion occurred after the prototherian-therian mammal divergence, suggesting that there may have been strong selection for the retention of imprinted regions that arose during the evolution of placentation. There is currently no evidence for genomic imprinting in the egg-laying monotreme mammals. However, since these mammals do have a short-lived placenta, imprinting appears to be correlated with viviparity but not placentation.

Brown adipose tissue expressing uncoupling protein 1 (UCP1) is responsible for adaptive nonshivering thermogenesis giving eutherian mammals crucial advantage to survive the cold. The emergence of this thermogenic organ during mammalian evolution remained unknown as the identification of UCP1 in marsupials failed so far. Here, we unequivocally identify the marsupial UCP1 ortholog in a genomic library of Monodelphis domestica. In South American and Australian marsupials, UCP1 is exclusively expressed in distinct adipose tissue sites and appears to be recruited by cold exposure in the smallest species under investigation (Sminthopsis crassicaudata). Our data suggest that an archetypal brown adipose tissue was present at least 150 million yr ago allowing early mammals to produce endogenous heat in the cold, without dependence on shivering and locomotor activity.

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.

Biologists have long studied the evolutionary consequences of the differences in reproductive and life history strategies of marsupial and eutherian mammals. Over the past few decades, the impact of these strategies on the development of the marsupial embryo and neonate has received attention. In this review, the differences in development in the craniofacial region in marsupial and eutherian mammals will be discussed. The review will highlight differences at the organogenic and cellular levels, and discuss hypotheses for shifts in the expression of important regulatory genes. The major difference in the organogenic period is a whole-scale shift in the relative timing of central nervous system structures, in particular those of the forebrain, which are delayed in marsupials, relative to the structures of the oral-facial apparatus. Correlated with the delay in development of nervous system structures, the ossification of the bones of the neurocranium are delayed, while those of the face are accelerated. This study will also review work showing that the neural crest, which provides much of the cellular material to the facial skeleton and may also carry important patterning information, is notably accelerated in its development in marsupials. Potential consequences of these observations for hypotheses on constraint, evolutionary integration, and the existence of developmental modules is discussed. Finally, the implications of these results for hypotheses on the genetic modulation of craniofacial patterning are presented., ((c) 2006 Wiley-Liss, Inc.)

Hypoblast formation in Sminthopsis macroura starts in blastocysts with a size between 1.0 and 1.4 mm, in which cells appear to be similar to each other, and finishes at the complete 2.6- or 2.7-mm bilaminar blastocyst, which is fully lined with hypoblast cells. When hypoblast cells begin allocation, the pluriblast region progressively differentiates from the trophoblast. Some pluriblast cells, which are otherwise undistinguished, lying on one side near the boundary of the circular pluriblast, move to the inside as hypoblast cells by mitosis or migration. They initially line the pluriblast and then the trophoblast. Hypoblast cells continue to leave the pluriblast/epiblast and intercalate into the underlying hypoblast layer until the advanced stages of bilaminar blastocysts. Associated with the origin of the hypoblast cells, the residual surface epiblast cells become less flatted and more cuboidal or rounded in shape. Characteristics are increased density of ribosomes, granular endoplasmic reticulum and a marked apical-basal polarity related to apical microvilli and endocytosis and more vesicles with flocculent content and a loss of the crystalloid deposits that were typical for earlier stages. Trophoblast cells become flat and elongated with only few vesicles, and they transform into extra-embryonic ectoderm cells, which are broader, rather square and with a higher density of ribosomes. Hypoblast cells are characterized by a relatively high level of ribosomes and endoplasmic reticulum, fewer small vesicles and no noticeable endocytotic processes and initially form a reticulum because the cells preferentially migrate along cell-cell boundaries by extension of long filopodia. Once hypoblast cells reach the boundary of the embryonic area and extend to line the trophoblast, they progressively consolidate into a squamous epithelium. It is suggested that the origin of the hypoblast from one side of the pluriblast and its invasion under the trophoblast from proliferating centres at the edge of the embryonic area provide mechanisms for patterning epiblast, hypoblast, trophoblast and extra-embryonic ectoderm.

Throughout their evolutionary histories, marsupial mammals have been taxonomically and morphologically less diverse than their sister taxa the placentals. Because of this, it has been proposed that the evolution of marsupials has been constrained by the functional requirements of their mode of reproduction. Marsupials give birth after short gestation times to immature neonates that immediately crawl, under the power of their precociously developed shoulder girdles, to the teat where they attach and complete their early development. Using a novel approach incorporating adult and embryological morphological data, this study is the first to both: (1) statistically support adult patterns of morphological divergence consistent with the constraint hypothesis, and (2) identify ontogenetic patterns of morphological change that demonstrate that the constraint was responsible, at least in part, for their formation. As predicted by the marsupial constraint, the shoulder girdles of adult marsupials are less diverse than those of adult placentals, and adult marsupial scapulae are less morphologically diverse than adult marsupial pelves. Furthermore, marsupials that complete an extensive crawl to the teat are restricted to a common pattern of ontogenetic scapular shape change, strongly supporting the hypothesis that the morphological development of the marsupial scapula has been limited evolutionarily by its obligate role in the crawl to the teat. Because this study establishes that ontogenetic and evolutionary morphological change is correlated within mammalian scapulae, it is probable that the marsupial constraint also restricted the morphological divergence of the scapula over evolutionary time by limiting ontogenetic change in the scapula. These findings, coupled with the importance of the shoulder girdle in mammalian locomotor specialization, support the conclusion that the low morphological diversity of marsupial forms over evolutionary time could be directly due to the constraint on marsupial morphological evolution caused by the functional requirements of the crawl to the teat.

This study, based on 38 samples taken between the 16-cell stage on day 2.5 of gestation and the expanded 1.0-mm-diameter unilaminar blastocyst on day 6, describes the ultrastructural changes that occur in the conceptus of the marsupial Sminthopsis macroura in relation to cell-zonal adhesion initiated at the zygote stage and cell-cell adhesion initiated at the 16-cell stage, lineage allocation, extracellular matrix (ECM) secretion and embryo coat changes. In S. macroura, rather flattened pluriblast and rounded trophoblast cells appear as different cell types during the fourth division when nucleolar reticulation suggests activation of the zygotic genome in both cell types. The differences disappear nearly completely in blastocysts of 0.6-0.8 mm in diameter, but the two cell types then reappear as two distinct populations. The ECM varies depending on its location within the conceptus up to the stage of the expanding blastocyst. It is of rather granular appearance between the cell lining and zona pellucida and consists of patches of homogeneous material embedded in an electron-lucent substance in the cleavage cavity. Homogeneous ECM coats trophoblast but not pluriblast cells on blastocoelic surfaces. Transient structures such as 'myosin-like' fibrillar arrays, probably associated with exocytosis of ECM, and pearl string-like whorls are still present, but both disappear during further expansion of the 0.6- to 0.8-mm blastocyst. During blastocyst expansion, the patchy homogeneous ECM in the blastocoel changes structure and appears flocculent, while the continuous ECM coating trophoblast cells disappears. Pluriblast cells and yolk mass identify the embryonic pole and hemisphere, and the opposite hemisphere becomes abembryonic and is eventually fully lined by trophoblast cells. An increase in endocytotic, mainly coated vesicles at the apical, zona-orientated surface of both cell types is noticed and is probably responsible for uptake of the mucoid coat. In 1-mm blastocysts, numerous vesicles contain rod-shaped crystalline inclusions.

The development of the scapula was studied in embryonic and postnatal specimens of Monodelphis domestica and perinatal specimens of Philander opossum, Caluromys philander, and Sminthopsis virginiae using histological sections and 3D reconstructions. Additionally, macerated skeletons of postnatal M. domestica were examined. This study focused on the detachment of the scapulocoracoid from the sternum and on the acquisition of a supraspinous fossa, a supraspinatus muscle, and a scapular spine, all these events associated with the origin of the therian shoulder girdle. In none of the specimens is there a continuity of the cartilaginous scapulocoracoid with the sternum, even though the structures are in close proximity, especially in S. virginiae. At birth, the first rib laterally presents a pronounced boss that probably contacts the humerus during certain movements. Only the acromial portion of the scapular spine, which originates from the anterior margin of the scapular blade, is preformed in cartilage. The other portion is formed by appositional bone ("Zuwachsknochen"), which expands from the perichondral ossification of the scapula into an intermuscular aponeurosis between the supra- and infraspinous muscles. This intermuscular aponeurosis inserts more or less in the middle of the lateral surface of the developing scapula. Thus, the floor of the supraspinous fossa is present from the beginning of scapular development, simultaneously with the infraspinous fossa. The homology of the therian spine with the anterior border of the sauropsid and monotreme scapula is questioned. We consider the dorsal portion (as opposed to the ventral or acromial portion) of the scapular spine a neomorphic structure of therian mammals., (Copyright 2003 Wiley-Liss, Inc.)

Marsupials are potentially excellent models for the study of gastrulation because of their superficial embryonic area (EA), post-gastrulation implantation and their potential to provide information about the evolution of gastrulation. Very few studies have examined this developmental period in marsupials. Using an established developmental timetable, peri-gastrula stage Sminthopsis macroura blastocysts were collected and described in detail by observations on live blastocysts and by the use of histological and immunohistochemical techniques on fixed blastocysts. Gastrulation in S. macroura shares several aspects common to that of both eutherian mammals and birds, but in terms of tissue arrangement and conceptus form, is more similar to the chick than other mammals. Two methods of culturing EA explants flat without their shell were devised. The techniques will markedly increase the number of possible experimental manipulations, which previously were limited by the presence of blastocyst investments. Exposure of fractions of explants of round, morphologically uniform pre-gastrula stage EA to growth factors or signaling molecules implicated in vertebrate gastrulation suggests that like the chick and mouse, the marsupial epiblast is patterned prior to gastrulation. Of all factors tested, basic fibroblast growth factor (bFGF) had the most prominent effect, promoting cell differentiation, and possible mesoderm formation. Data from explant culture suggests that similar to the chick and mouse, limited specification precedes the onset of gastrulation., (Copyright 2003 Wiley-Liss, Inc.)

This study outlines the ultrastructural changes that occur in Sminthopsis macroura tubal zygotes to the 8-cell stage in relation to observations of development in vitro, oocyte polarity and cell-zona adhesion. The extremely polarized mature oocytes and zygotes have nuclear material at one pole and accumulated vesicular bodies at the other. The first division is associated with extrusion of vesicular bodies and some cytoplasm as a membrane-bound yolk mass into the perivitelline space. Early cleavage is accompanied by the appearance of an extensive, highly structured extracellular matrix (ECM) comprised of amorphous substance, granules and filaments. At the 2- and 4-cell stage the decrease in density of the ECM in the vicinity of the blastomeres may facilitate cell-zona contact. At the 8-cell stage, discharge of vesicular bodies, which mostly appear to be empty, may contribute to the ECM by increasing the area of plasma membrane for synthesis of a hyaluronan-like ECM. As in other marsupials, the precedence of cell-zona adhesion over cell-cell contacts prevents morula formation. The earliest cell-zona contacts appear when microvilli contact the zona in the uterine zygote 12-16 h after uterine entry and continue at later stages. This early contact is possible because of the absence of a dense subzonal ECM in this species. Between late zygote and late 4-cell stage the cytoplasm also contains, beside a large amount of vesicular bodies, demarcated areas where smooth endoplasmic reticulum encloses mitochondria, vesicles, granular material and fibrillar arrays. The latter develop in the late zygote stage and are found outside demarcated areas as well, often closely surrounding large vesicles, probably helping vesicle extrusion. A putative germ plasm was identified at the 4-cell stage., (Copyright 2003 S. Karger AG, Basel)

We report a possible case of extended gestation in the koala, Phascolarctos cinereus. Birth of a pouch young was first observed 127 days after the removal of the male from a multi-female colony at Taronga Zoo. No other males were present at that time or had access to the facility. Head measurements and other growth data collected at the time of detection and over the period of pouch life indicates the time from removal of the male and the date of birth to be between 50 and 77 days. DNA fingerprinting using microsatellite loci unambiguously assigned paternity of the pouch young to this male. These observations suggest either an extended period of gestation of at least 50 days, or activation of a dormant blastocyst from the previous breeding season, as the female entered the period of seasonal oestrus.

This study is based on the examination of histological sections of specimens of different ages and of adult ossicles from macerated skulls representing a wide range of taxa and aims at addressing several issues concerning the evolution of the ear ossicles in marsupials. Three-dimensional reconstructions of the ear ossicles based on histological series were done for one or more stages of Monodelphis domestica, Caluromys philander, Sminthopsis virginiae, Trichosurus vulpecula, and Macropus rufogriseus. Several common trends were found. Portions of the ossicles that are phylogenetically older develop earlier than portions representing more recent evolutionary inventions (manubrium of the malleus, crus longum of the incus). The onset of endochondral ossification in the taxa in which this was examined followed the sequence; first malleus, then incus, and finally stapes. In M. domestica and C. philander at birth the yet precartilaginous ossicles form a supportive strut between the lower jaw and the braincase. The cartilage of Paauw develops relatively late in comparison with the ear ossicles and in close association to the tendon of the stapedial muscle. A feeble artery traverses the stapedial foramen of the stapes in the youngest stages of M. domestica, C. philander, and Sminthopsis virginiae examined. Presence of a large stapedial foramen is reconstructed in the groundplan of the Didelphidae and of Marsupialia. The stapedial foramen is absent in all adult caenolestids, dasyurids, Myrmecobius, Notoryctes, peramelids, vombatids, and phascolarctids. Pouch young of Perameles sp. and Dasyurus viverrinus show a bicrurate stapes with a sizeable stapedial foramen. Some didelphids examined to date show a double insertion of the Tensor tympani muscle. Some differences exist between M. domestica and C. philander in adult ossicle form, including the relative length of the incudal crus breve and of the stapes. Several differences exist between the malleus of didelphids and that of some phalangeriforms, the latter showing a short neck, absence of the lamina, and a ventrally directed manubrium. Hearing starts in M. domestica at an age in which the external auditory meatus has not yet fully developed, the ossicles are not fully ossified, and the middle ear space is partially filled with loose mesenchyme. The ontogenetic changes in hearing abilities in M. domestica between postnatal days 30 and 40 may be at least partially related to changes in middle ear structures., (Copyright 2002 Wiley-Liss, Inc.)

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.

Developing patterns in early embryogenesis are analysed in conceptuses from several families, including Dasyuridae, Phalangeridae, Macropodidae and Didelphidae, in which cleavage has been examined in some detail. Features common to cleavage and blastocyst formation, and in some cases to hypoblast formation, are used to develop an outline of possible mechanisms leading to axis formation and lineage allocation. Relevant features that have been described only in some species are also included. It is suggested that certain features of marsupial cleavage establish patterns in the developing blastocyst epithelia, pluriblast, trophoblast and hypoblast that contribute to axis formation and lineage allocation. All marsupials examined had a polarized oocyte or conceptus, the polarity of which was related to the conceptus embryonic-abembryonic axis and, eventually, the conceptus dorsal-ventral axis and the formation of the pluriblast (future embryo) and trophoblast. The embryonic dorsal-ventral and anterior-posterior axes were established after the allocation of hypoblast and epiblast. Mechanisms that appear to result in patterning of the developing epithelia leading to axis formation and lineage allocation are discussed, and include sperm entry point, gravity, conceptus polarity, differentials in cell-zona, cell-cell and cell-type (boundary effects) contacts, cell division order during cleavage and signals external to the conceptus. A model of the patterning effects is included. The applicability of these mechanisms to other amniotes, including eutherian mammals, is also examined.

Several morphological features of early development differ between marsupials and eutherians. These include early overt conceptus polarity, the mode of segregation of pluriblast and trophoblast lineages, and the superficial position of the pluriblast within the unilaminar blastocyst epithelium. Knowledge of the molecular mechanisms of early development is lacking in marsupials. We have cloned from the brushtail possum, Trichosurus vulpecula, the partial cDNA of a marsupial homologue of mammalian POU5F1, which encodes octamer-binding transcription factor-4 (Oct-4). The 579 bp cloned coding sequence of possum POU5F1 (tvPOU5F1) shares 74, 78, and 79% identity with murine Pou5f1, human POU5F1, and bovine bPOU5F1, respectively, at the protein level. The mRNA for tvPOU5F1 was detected by in situ hybridization in oogonia and in oocytes of growing follicles, but not in early meiotic oocytes. In a gastrulation-stage conceptus, high tvPOU5F1 expression was found in the embryonic ectoderm, with low or undetectable expression in other cell types. These results are comparable with those of murine and bovine expression studies, and suggest that the putative role of POU5F1 in totipotent and pluripotent cells is conserved among mammals., (Copyright 2001 Wiley-Liss, Inc.)

Marsupials have many characteristic features that make them ideal models to study the control of sexual differentiation and development. They are distinguished from eutherian mammals in their mode of reproduction and their greater dependence on the teat and mammary gland than on the placenta for development. They give birth to a highly altricial young which completes its development while firmly attached to a teat, usually within the confines of a pouch. At birth, the marsupial neonate has a well-developed digestive, respiratory and circulatory system, but retains its fetal excretory system with a fully functional mesonephric kidney and undifferentiated gonads and genitalia.

The formation of the testis or ovary is a critical step in development. Alterations in gonadal development during fetal or postnatal life can lead to intersexuality or infertility. Several model systems have been particularly useful in studying gonadal differentiation, the eutherian mammal and amphibia, fish, and birds. However, marsupials provide a unique opportunity to investigate gonadal development and the interactions of genes and hormones in gonadal differentiation and germ cell development in all mammals. On the one hand the genetic mechanisms appear to be identical to those in eutherian mammals, including the testis-determining SRY gene. On the other hand, marsupials retain in part the plasticity of the amphibian gonad to hormonal manipulation. It is possible to induce female to male and also male to female gonadal sex reversal in marsupials by hormonal manipulation, and oestradiol can induce male germ cells to enter meiosis at the time the oogonia do. In addition, in marsupials the development of the scrotum and mammary glands are independent of testicular androgens and instead are controlled by a gene or genes on the X-chromosome. Thus marsupials provide a number of opportunities for manipulating the sexual differentiation of the gonads that are not possible in eutherian mammals and so provide a unique perspective for understanding the common mechanisms controlling sexual development.

We report for the first time the development and morphological characteristics of the spleen thymus, lymph nodes and liver of the northern brown bandicoot, Isoodon macrourus. To date few marsupial species have been studied. The development and morphological characteristics of the organs shared the typical features of those few other marsupials studied as well as those of eutherians. These suggest comparative functional properties with the eutherian immune system. The thymus differentiated within the first week of pouch life and showed evidence of immunolymphopoiesis up to juvenile stages. The spleen, though seeded by lymphocytes within the first week of pouch life, was slower to mature, but differentiated and showed signs of immunocompetency by the time young left the pouch. The mature spleen displayed the same anatomical blood filtering and immunosurveillance properties as that of the eutherian spleen, with evidence of erythrocyte destruction, thrombopoietic activity, activation and differentiation of immunocompetent lymphocytes. However, the absence of sheathed capillary structures in the spleen may indicate differences in the humoral response to circulating antigens. Similarly, lymph nodes also mature by this stage with anterior nodes appearing before posterior nodes. The mature lymph nodes displayed structural features of secondary immuno-lymphoid organs consistent with production of immune responses. Finally, the liver displayed haemopoietic activity for the first four weeks of pouch life. The pattern of development in the bandicoot appears to parallel the pattern reported for other marsupials, yet the thymus matures considerably earlier than previously reported and may be of significance in the development and onset of cell-mediated immunity. Current studies to characterise cellular components, such as T/B lymphocyes and accessory cells of these organs will help to define the mechanisms of immune recognition, activation and hence outline the basis of the marsupial humoral and cellular immunity.

This paper described several developmental stages of the male urogenital system in the koala Phascolarctos cinereus, employing both light and scanning electron microscopy. There are few studies of the development of the urogenital system in male marsupials. Findings by White and Timms (1994) that male koalas can be infected with Chlamydia psittaci emphasise the importance of studies on male animals and in particular their reproductive system. Specimens in our study ranged in age from 15 days postnatal to adults. Due to the rarity of such specimens, details of each specimen are linked to the changes of the structures at each available stage. Light microscopy revealed that differentiation of the gonads had commenced by 15 days postnatal and that the cytological arrangements of the urogenital system are essentially the same as those of other mammals. Scanning electron microscopy revealed stereocilia and microvilli along the lumen of each ductus epididymis and cilia and microvilli along the lumina of the vasa deferentia and urethra. The development of these structures coincided with the onset of sexual maturation, sperm production and differentiation at about three years of age.

The development of marsupial oocytes and embryos in vitro is reviewed. Most stages of development have been cultured successfully, usually in a complex medium with added fetal calf serum. Simpler media without added serum have been developed for fertilization and cleavage in vitro. Culture systems have been established for oocyte maturation and fertilization in the grey short-tailed opossum and for cleavage from the zygote to the early expanding unilaminar blastocyst in a number of other marsupials. Survival in vitro of the unilaminar and early bilaminar blastocyst stages is limited in all species examined. In contrast, late bilaminar, trilaminar, embryonic and fetal stages develop at rates approximating those in vivo. More stages have been cultured successfully in Sminthopsis macroura than in any other species. It has been cultured from the late bilaminar blastocyst to within 18 h of birth. Stages of cleavage and unilaminar blastocyst formation of Monodelphis domestica timed by videotaping mating animals, proceeded at similar rates in vivo and in vitro. As in other marsupials, cleavage in this opossum is characterized by a polarized conceptus. This polarity is expressed in the distribution of organelles in the zygote and the localization of secretion of the extracellular matrix material into the cleavage cavity and of the initial cell-zona attachment. Because cell-cell adhesion follows cell-zona adhesion, a unilaminar blastocyst forms without the development of an intervening morula stage.