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.

The embryo of the silky shrew opossum, Caenolestes fuliginosus (Tomes, 1863): First description of the embryo of Paucituberculata.

The development of caenolestid marsupials (order Paucituberculata) is virtually unknown. We provide here the first description of Caenolestes fuliginosus embryos collected in the Colombian Central Andes. Our sample of four embryos comes from a single female caught during a fieldtrip at Río Blanco (Manizales, Caldas), in 2014. The sample was processed for macroscopic description using a Standard Event System and for histological descriptions (sectioning and staining). The grade of development of the lumbar flexure and coelomic closure differed between embryos, two of them being more advanced than the others (similar to McCrady's stages 30 and 29, respectively). The pericardial and peritoneal cavities were present, the hepatic anlage was organized in hepatic cords, the heart was in its final position, and the mesonephros was functional. Compared to other Neotropical marsupials, an early appearance of the frontonasal-maxillary fusion and the cervical growth (thickness) was observed; however, absorption of the pharyngeal arches into the body and lung development was delayed. Besides these differences, embryos were similar to equivalent stages in Didelphis virginiana and Monodelphis domestica. Previous proposals of litter size of four for C. fuliginosus are supported.

Exogenous retinoic acid induces digit reduction in opossums (Monodelphis domestica) by disrupting cell death and proliferation, and apical ectodermal ridge and zone of polarizing activity function.

BACKGROUND: Retinoic acid (RA) is a vitamin A derivative. Exposure to exogenous RA generates congenital limb malformations (CLMs) in species from frogs to humans. These CLMs include but are not limited to oligodactyly and long-bone hypoplasia. The processes by which exogenous RA induces CLMs in mammals have been best studied in mouse, but as of yet remain unresolved. METHODS: We investigated the impact of exogenous RA on the cellular and molecular development of the limbs of a nonrodent model mammal, the opossum Monodelphis domestica. Opossums exposed to exogenous retinoic acid display CLMs including oligodactly, and results are consistent with opossum development being more susceptible to RA-induced disruptions than mouse development. RESULTS: Exposure of developing opossums to exogenous RA leads to an increase in cell death in the limb mesenchyme that is most pronounced in the zone of polarizing activity, and a reduction in cell proliferation throughout the limb mesenchyme. Exogenous RA also disrupts the expression of Shh in the zone of polarizing activity, and Fgf8 in the apical ectodermal ridge, and other genes with roles in the regulation of limb development and cell death. CONCLUSION: Results are consistent with RA inducing CLMs in opossum limbs by disrupting the functions of the apical ectodermal ridge and zone of polarizing activity, and driving an increase in cell death and reduction of cell proliferation in the mesenchyme of the developing limb.

Comparative gene expression analyses reveal heterochrony for Sox9 expression in the cranial neural crest during marsupial development.

Compared to placental mammals, marsupials have short gestation period, and their neonates are relatively immature. Despite these features, marsupial neonates must travel from the birth canal to the teat, suckle and digest milk to complete development. Thus, certain organs and tissues of marsupial neonates, such as forelimbs to crawl and jaw elements to suckle, must develop early. Previous reports showed that cranial neural crest (CNC) cells, as the source of ectomesenchyme of jaw elements, are generated significantly early in gray short-tailed opossum (Monodelphis domestica) compared to other amniote models, such as mouse. In this study, we examined the expression of genes known to be important for neural crest formation, such as BMP2/BMP4 (neural crest inducer), Pax7 (neural border specifier), Snail1 and Sox9/Sox10 (neural crest specifier) in Monodelphis domestica, and compared the expression patterns with those in mouse, chicken, and gecko embryos. Among those genes, the expression of Sox9 was turned on early and broadly in the premigratory CNC cells, and persisted in the ectomesenchyme of the cranial anlagen in opossum embryos. In contrast, Sox9 expression diminished in the CNC cells of other animals at the early phase of migration. Comparison of the onset of Pax7 and Sox9 expression revealed that Sox9 expression in the prospective CNC was earlier and broader than Pax7 expression in opossum, suggesting that the sequence of border specification and neural crest specification is altered. This study provides the first clue for understanding the molecular basis for the heterochronic development of the CNC cells and jaw elements in marsupials.

Constraints on Mammalian forelimb development: insights from developmental disparity.

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark-based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo-wide constraint.

The evolution of class V POU domain transcription factors in vertebrates and their characterisation in a marsupial.

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.

An endoderm-specific transcriptional enhancer from the mouse Gata4 gene requires GATA and homeodomain protein-binding sites for function in vivo.

Several transcription factors function in the specification and differentiation of the endoderm, including the zinc finger transcription factor GATA4. Despite its essential role in endoderm development, the transcriptional control of the Gata4 gene in the developing endoderm and its derivatives remains incompletely understood. Here, we identify a distal enhancer from the Gata4 gene, which directs expression exclusively to the visceral and definitive endoderm of transgenic mouse embryos. The activity of this enhancer is initially broad within the definitive endoderm but later restricts to developing endoderm-derived tissues, including pancreas, glandular stomach, and duodenum. The activity of this enhancer in vivo is dependent on evolutionarily-conserved HOX- and GATA-binding sites, which are bound by PDX-1 and GATA4, respectively. These studies establish Gata4 as a direct transcriptional target of homeodomain and GATA transcription factors in the endoderm and support a model in which GATA4 functions in the transcriptional network for pancreas formation.

Germ cell restricted expression of chick Nanog.

Nanog is a pluripotency-associated factor expressed in embryonic stem cells and in the epiblast and primordial germ cells of the mouse embryo. We have identified the chick orthologue of Nanog and found that its expression is limited to primordial germ cells in the early embryo and is not found throughout the epiblast. Genomic analysis has shown that Nanog is an amniote-specific gene and is absent from anamniotes and invertebrates. Furthermore, other pluripotency associated genes that are located in close proximity to Nanog in human and mouse are absent from the chick genome. Such observations lead to a scenario of sequential addition of novel genes to a genomic region associated to pluripotency. These results have profound implications for the study of the evolution of pluripotent lineages in the embryo and of vertebrate stem cells.

Transposon-free regions in mammalian genomes.

Despite the presence of over 3 million transposons separated on average by approximately 500 bp, the human and mouse genomes each contain almost 1000 transposon-free regions (TFRs) over 10 kb in length. The majority of human TFRs correlate with orthologous TFRs in the mouse, despite the fact that most transposons are lineage specific. Many human TFRs also overlap with orthologous TFRs in the marsupial opossum, indicating that these regions have remained refractory to transposon insertion for long evolutionary periods. Over 90% of the bases covered by TFRs are noncoding, much of which is not highly conserved. Most TFRs are not associated with unusual nucleotide composition, but are significantly associated with genes encoding developmental regulators, suggesting that they represent extended regions of regulatory information that are largely unable to tolerate insertions, a conclusion difficult to reconcile with current conceptions of gene regulation.

Ontogenesis of the scapula in marsupial mammals, with special emphasis on perinatal stages of didelphids and remarks on the origin of the therian scapula.

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.

The role of androgens in development of the scrotum of the grey short-tailed Brazilian opossum ( Monodelphis domestica).

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

Early differentiation and migration of cranial neural crest in the opossum, Monodelphis domestica.

Marsupial mammals are born at a highly altricial state. Nonetheless, the neonate must be capable of considerable functional independence. Comparative studies have shown that in marsupials the morphogenesis of many structures critical to independent function are advanced relative to overall development. Many skeletal and muscular elements in the facial region show particular heterochrony. Because neural crest cells are crucial to forming and patterning much of the face, this study investigates whether the timing of cranial neural crest differentiation is also advanced. Histology and scanning electron microscopy of Monodelphis domestica embryos show that many aspects of cranial neural crest differentiation and migration are conserved in marsupials. For example, as in other vertebrates, cranial neural crest differentiates at the neural ectoderm/epidermal boundary and migrates as three major streams. However, when compared with other vertebrates, a number of timing differences exist. The onset of cranial neural crest migration is early relative to both neural tube development and somite formation in Monodelphis. First arch neural crest cell migration is particularly advanced and begins before any somites appear or regional differentiation exists in the neural tube. Our study provides the first published description of cranial neural crest differentiation and migration in marsupials and offers insight into how shifts in early developmental processes can lead to morphological change.

Removal of the shell coat affects maintenance of epithelia in blastocysts of the Brushtail Possum in vitro.

The aim of the present study was to investigate the role of the marsupial shell coat in embryonic development because it may be a potential target for immunocontraceptive control of the brushtail possum. Conceptuses from 52 female possums were collected between 1995 and 1997 in New Zealand and Australia. Development was examined in representative stages from cleavage to the early trilaminar blastocyst. The effect of coat removal by microdissection was examined by comparing development in vivo (n = 29), with development in vitro, both with (n = 10) and without (n = 13) shell coat removal. Conceptuses were monitored and photographed in culture, fixed and examined by transmission electron microscopy. The ultrastructure of uni-, bi- and trilaminar blastocyst stages in vivo and in vitro and of the early stages of hypoblast and embryonic endoderm formation are described for the first time in the possum. Shell coat removal had little impact on most cleavage stages, as the intact mucoid coat appeared to provide structural support to the embryo. Common features of unilaminar coat-free specimens after culture were rounding up and detachment of cells from the blastocyst epithelium and the loss of cell surface projections. The most remarkable features of the shell-free trilaminar blastocysts in comparison with the in vivo and in vitro controls were the hydration of many cells, and the large-scale disruption and modification of numerous epithelia, particularly of the younger, or newly forming populations. The shell appears to be functionally important after blastocyst formation, particularly after breakdown of the mucoid coat. After shell removal, conceptus response in vitro suggested that the shell played a role in maintaining structural integrity. The shell was found to be necessary for normal embryonic development.

Early development of the neural plate, neural crest and facial region of marsupials.

Marsupial mammals have a distinctive reproductive strategy. The young are born after an exceptionally short period of organogenesis and are consequently extremely altricial. Yet because they must be functionally independent in an essentially embryonic condition, the marsupial neonate exhibits a unique suite of adaptations. In particular, certain bones of the facial region, most cranial musculature and a few additional structures are accelerated in their development. In contrast, central nervous system structures, especially the forebrain, are markedly premature at birth, resembling an embryonic d 11 or 12 mouse. This review examines the developmental processes that are modified to produce these evolutionary changes. The focus is on the early development of the neural plate, neural crest and facial region in the marsupial, Monodelphis domestica, compared with patterns reported for rodents. Neural crest begins differentiation and migration at the neural plate stage, which results in large accumulations of neural crest in the facial region at an early stage of development. The early accumulation of neural crest provides the material for the accelerated development of oral and facial structures. The first arch region is massive in the early embryo, and the development of the olfactory placode and frontonasal region is advanced relative to the forebrain region. The development of the forebrain is delayed in marsupials relative to the hindbrain or facial region. These observations illustrate how development may be modified to produce evolutionary changes that distinguish taxa. Further, they suggest that development is not necessarily highly conserved, but instead may be quite plastic.

Sex difference in target seeking behavior of developing cremaster muscles and the resulting first visible sign of somatic sexual differentiation in marsupial mammals.

Cremaster muscles are present in both male and female developing and adult marsupial mammals. They are complex structures and composed of several distinct bundles of striated muscle fibers provided with: (1) a distinct and extensive innervation; (2) a distinct blood vascular supply; (3) a distinct tendineous origin on the anterosuperior iliac spine; and (4) distinct target structures. The muscles thus seem to be separate anatomical entities and not a part of one or more of the layers of the ventral abdominal wall musculature. Cremaster muscles in males are elongated, are larger than in females, and for the most part are a component of the funiculus spermaticus. They insert on the distal part of the tunica vaginalis. The distal parts of the muscles in females are flattened ("fan shaped") and insert over a broad area on the dorsal borders of the mammary glands. Muscles in males have no relation whatsoever to the male mammary glandular rudiments. Muscles in females are attached at the base of the uterine round ligament. The remarkable sex difference in target structures of marsupial cremaster muscles becomes noticeable during perinatal life when outgrowing muscles take a different path in males and females. The initial appearance of this sexually dimorphic trait precedes the sexual differentiation of the genital ducts and external genitalia. In fetal males, the cremaster muscles grow in the direction of the site where scrotal bulges initially appear in the subcutaneous layers and later on the inguinal skin surface. They also take the gubernacular core of the ventral abdominal wall and the attached peritoneal epithelium with them during this outgrowth process. Consequently, this results in the development of a slitlike evagination of the abdominal lumen as the primary step to development of the processus vaginalis, while the testis and adjacent mesonephros and its duct are still attached to the posterior abdominal wall. In fetal females, the outgrowing cremaster muscles pass along the gubernacular core and, subsequently, this structure develops further as the tip (attached to the tubo-uterine junction) of the intra-abdominally protruding and further developing uterine round ligament. The female cremaster muscles grow further into caudal direction to shape a dorsal border of the developing mammary glands. The early onset of this sexually dimorphic outgrowth of cremaster muscles indicates that the "classical hormones" of sexual differentiation (anti-Müllerian hormone [AMH] and steroidal androgens) are not involved in this process. It could thus depend on primary genetic control with male development associated with the male-limited activity of genes on the Y-chromosomes and female development as the default process. Alternatively, the process in males could be under the control of an as yet unidentified third fetal testicular hormone involved in sexual differentiation processes which must then show an unexpectely early (i.e., perinatal) onset of its secretion.

An ultrastructural study of the role of an extracellular matrix during normal cleavage in a marsupial, the brushtail possum.

In marsupials, the mechanisms of lineage allocation into pluriblast and trophoblast are related to conceptus polarity and polarized discharge of extracellular matrix (ECM). The brushtail possum, Trichosurus vulpecula, a major pest species in New Zealand, is being intensively studied to develop an immunocontraceptive control method. Of 23 specimens examined, 11 were examined by electron microscopy to study the presence and role of the ECM in lineage allocation in the possum. A number of polarized features in the zygote identified the future embryonic and abembryonic poles. Pronuclei, in a broad band of mitochondrion-rich cortical cytoplasm, lay in the embryonic hemisphere, and numerous electron-lucent vesicles characterized the abembryonic cytoplasm. These vesicles seemed to contribute to the ECM. During cleavage, cells lay near the zona in the embryonic hemisphere, and ECM accumulated chiefly in the abembryonic hemisphere. Cell-zona adhesion facilitated by microvillous and club processes occurred at the early 4-cell stage, and cell-cell adhesion commenced at the late 4-cell stage. The first two cleavages were meridional, equal, and accompanied by elimination into the cleavage cavity of much of the electron-lucent vesicular material in the form of several membrane-bound yolk masses. The third cleavage was unequal, with both meridional and latitudinal planes. The first differences between trophoblast and pluriblast lineages appeared at the 8-cell stage. Later cleavage planes were latitudinal or oblique. Conceptus polarity, polarized discharge of ECM, and localized cell-zona adhesion were related to the first lineage allocation in the possum.