A single-cell transcriptome atlas of marsupial embryogenesis and X inactivation.

Single-cell RNA sequencing of embryos can resolve the transcriptional landscape of development at unprecedented resolution. To date, single-cell RNA-sequencing studies of mammalian embryos have focused exclusively on eutherian species. Analysis of mammalian outgroups has the potential to identify deeply conserved lineage specification and pluripotency factors, and can extend our understanding of X dosage compensation. Metatherian (marsupial) mammals diverged from eutherians around 160 million years ago. They exhibit distinctive developmental features, including late implantation1 and imprinted X chromosome inactivation2, which is associated with expression of the XIST-like noncoding RNA RSX3. Here we perform a single-cell RNA-sequencing analysis of embryogenesis and X chromosome inactivation in a marsupial, the grey short-tailed opossum (Monodelphis domestica). We resolve the developmental trajectory and transcriptional signatures of the epiblast, primitive endoderm and trophectoderm, and identify deeply conserved lineage-specific markers that pre-date the eutherian-marsupial divergence. RSX coating and inactivation of the X chromosome occurs early and rapidly. This observation supports the hypothesis that-in organisms with early X chromosome inactivation-imprinted X chromosome inactivation prevents biallelic X silencing. We identify XSR, an RSX antisense transcript expressed from the active X chromosome, as a candidate for the regulator of imprinted X chromosome inactivation. Our datasets provide insights into the evolution of mammalian embryogenesis and X dosage compensation.

Proteomic analysis of opossum Monodelphis domestica spinal cord reveals the changes of proteins related to neurodegenerative diseases during developmental period when neuroregeneration stops being possible.

One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is still unclear why the ability to regenerate CNS is lost during evolution and development and why it becomes very limited in adult mammals. A convenient model to study cellular and molecular basis of this loss is neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate postnatal spinal cord after injury in the first two weeks of their life, after which this ability abbruptly stops. Using comparative proteomic approach we identified the proteins that are differentially distributed in opossum spinal tissue that can and cannot regenerate after injury, among which stand out the proteins related to neurodegenerative diseases (NDD), such as Huntington, Parkinson and Alzheimer's disease, previously detected by comparative transcriptomics on the analog tissue. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot (WB), and the changes in the expression of related genes were analysed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy. To our knowledge, this is the first report on proteins differentially present in developing, non-injured mammalian spinal cord tissue with different regenerative capacities. The results of this study indicate that the proteins known to have an important role in the pathophysiology of neurodegeneration in aged CNS, could also have an important phyisological role during CNS postnatal development and in neuroregeneration process.

Monodelphis domestica Induced Pluripotent Stem Cells Reveal Metatherian Pluripotency Architecture.

Marsupials have been a powerful comparative model to understand mammalian biology. However, because of the unique characteristics of their embryology, marsupial pluripotency architecture remains to be fully understood, and nobody has succeeded in developing embryonic stem cells (ESCs) from any marsupial species. We have developed an integration-free iPSC reprogramming method and established validated iPSCs from two inbred strains of a marsupial, Monodelphis domestica. The monoiPSCs showed a significant (6181 DE-genes) and highly uniform (r2 [95% CI] = 0.973 ± 0.007) resetting of the cellular transcriptome and were similar to eutherian ESCs and iPSCs in their overall transcriptomic profiles. However, monoiPSCs showed unique regulatory architecture of the core pluripotency transcription factors and were more like marsupial epiblasts. Our results suggest that POU5F1 and the splice-variant-specific expression of POU5F3 synergistically regulate the opossum pluripotency gene network. It is plausible that POU5F1, POU5F3 splice variant XM_016427856.1, and SOX2 form a self-regulatory network. NANOG expression, however, was specific to monoiPSCs and epiblasts. Furthermore, POU5F1 was highly expressed in trophectoderm cells, whereas all other pluripotency transcription factors were significantly downregulated, suggesting that the regulatory architecture of core pluripotency genes of marsupials may be distinct from that of eutherians.

Sex-biased microRNA expression in mammals and birds reveals underlying regulatory mechanisms and a role in dosage compensation.

Sexual dimorphism depends on sex-biased gene expression, but the contributions of microRNAs (miRNAs) have not been globally assessed. We therefore produced an extensive small RNA sequencing data set to analyze male and female miRNA expression profiles in mouse, opossum, and chicken. Our analyses uncovered numerous cases of somatic sex-biased miRNA expression, with the largest proportion found in the mouse heart and liver. Sex-biased expression is explained by miRNA-specific regulation, including sex-biased chromatin accessibility at promoters, rather than piggybacking of intronic miRNAs on sex-biased protein-coding genes. In mouse, but not opossum and chicken, sex bias is coordinated across tissues such that autosomal testis-biased miRNAs tend to be somatically male-biased, whereas autosomal ovary-biased miRNAs are female-biased, possibly due to broad hormonal control. In chicken, which has a Z/W sex chromosome system, expression output of genes on the Z Chromosome is expected to be male-biased, since there is no global dosage compensation mechanism that restores expression in ZW females after almost all genes on the W Chromosome decayed. Nevertheless, we found that the dominant liver miRNA, miR-122-5p, is Z-linked but expressed in an unbiased manner, due to the unusual retention of a W-linked copy. Another Z-linked miRNA, the male-biased miR-2954-3p, shows conserved preference for dosage-sensitive genes on the Z Chromosome, based on computational and experimental data from chicken and zebra finch, and acts to equalize male-to-female expression ratios of its targets. Unexpectedly, our findings thus establish miRNA regulation as a novel gene-specific dosage compensation mechanism.

Gene expression profiling of postnatal lung development in the marsupial gray short-tailed opossum (Monodelphis domestica) highlights conserved developmental pathways and specific characteristics during lung organogenesis.

BACKGROUND: After a short gestation, marsupials give birth to immature neonates with lungs that are not fully developed and in early life the neonate partially relies on gas exchange through the skin. Therefore, significant lung development occurs after birth in marsupials in contrast to eutherian mammals such as humans and mice where lung development occurs predominantly in the embryo. To explore the mechanisms of marsupial lung development in comparison to eutherians, morphological and gene expression analysis were conducted in the gray short-tailed opossum (Monodelphis domestica). RESULTS: Postnatal lung development of Monodelphis involves three key stages of development: (i) transition from late canalicular to early saccular stages, (ii) saccular and (iii) alveolar stages, similar to developmental stages overlapping the embryonic and perinatal period in eutherians. Differentially expressed genes were identified and correlated with developmental stages. Functional categories included growth factors, extracellular matrix protein (ECMs), transcriptional factors and signalling pathways related to branching morphogenesis, alveologenesis and vascularisation. Comparison with published data on mice highlighted the conserved importance of extracellular matrix remodelling and signalling pathways such as Wnt, Notch, IGF, TGFß, retinoic acid and angiopoietin. The comparison also revealed changes in the mammalian gene expression program associated with the initiation of alveologenesis and birth, pointing to subtle differences between the non-functional embryonic lung of the eutherian mouse and the partially functional developing lung of the marsupial Monodelphis neonates. The data also highlighted a subset of contractile proteins specifically expressed in Monodelphis during and after alveologenesis. CONCLUSION: The results provide insights into marsupial lung development and support the potential of the marsupial model of postnatal development towards better understanding of the evolution of the mammalian bronchioalveolar lung.

Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation.

In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.

Chromosome-wide profiling of X-chromosome inactivation and epigenetic states in fetal brain and placenta of the opossum, Monodelphis domestica.

Evidence from a few genes in diverse species suggests that X-chromosome inactivation (XCI) in marsupials is characterized by exclusive, but leaky inactivation of the paternally derived X chromosome. To study the phenomenon of marsupial XCI more comprehensively, we profiled parent-of-origin allele-specific expression, DNA methylation, and histone modifications in fetal brain and extra-embryonic membranes in the gray, short-tailed opossum (Monodelphis domestica). The majority of X-linked genes (152 of 176 genes with trackable SNP variants) exhibited paternally imprinted expression, with nearly 100% of transcripts derived from the maternal allele; whereas 24 loci (14%) escaped inactivation, showing varying levels of biallelic expression. In addition to recently reported evidence of marsupial XCI regulation by the noncoding Rsx transcript, strong depletion of H3K27me3 at escaper gene loci in the present study suggests that histone state modifications also correlate strongly with opossum XCI. In contrast to mouse, the opossum did not show an association between X-linked gene expression and promoter DNA methylation, with one notable exception. Unlike all other X-linked genes examined, Rsx was differentially methylated on the maternal and paternal X chromosomes, and expression was exclusively from the inactive (paternal) X chromosome. Our study provides the first comprehensive catalog of parent-of-origin expression status for X-linked genes in a marsupial and sheds light on the regulation and evolution of imprinted XCI in mammals.

Functional conservation of the lncRNA NEAT1 in the ancestrally diverged marsupial lineage: Evidence for NEAT1 expression and associated paraspeckle assembly during late gestation in the opossum Monodelphis domestica.

Long non-coding RNAs (lncRNAs) are widely expressed and play various roles in cell homeostasis. However, because of their low conservation at the sequence level, recapitulating lncRNA evolutionary history is often challenging. While performing an ultrastructural analysis of viral particles present in uterine glands of gestating opossum females, we serendipitously noticed the presence of numerous structures similar to paraspeckles, nuclear bodies which in human and mouse cells are assembled around an architectural NEAT1/MENϵ/ß lncRNA. Here, using an opossum kidney (OK) cell line, we confirmed by immuno-electron microscopy the presence of paraspeckles in marsupials. We then identified the orthologous opossum NEAT1 gene which, although poorly conserved at the sequence level, displays NEAT1 characteristic features such as short and long isoforms expressed from a unique promoter and for the latter an RNase P cleavage site at its 3'-end. Combining tissue-specific qRT-PCR, in situ hybridization at the optical and electron microscopic levels, we show that (i) NEAT1 is paraspeckle-associated in opossum (ii) NEAT1 expression is strongly induced in late gestation in uterine/placental extracts (iii) NEAT1 induction occurs in the uterine gland nuclei in which paraspeckles were detected. Finally, treatment of OK cells with proteasome inhibitors induces paraspeckle assembly, as previously observed in human cells. Altogether, these results demonstrate that paraspeckles are tissue-specific, stress-responding nuclear bodies in marsupials, illustrating their structural and functional continuity over 200 My of evolution throughout the mammalian lineage. In contrast, the rapid evolution of the NEAT1 transcripts highlights the relaxed constraint that, despite functional conservation, is exerted on this lncRNA.

The opossum MHC genomic region revisited.

The gray short-tailed opossum Monodelphis domestica is one of the few marsupial species for which a high quality whole genome sequence is available and the major histocompatibility complex (MHC) region has been annotated. Previous analyses revealed only a single locus within the opossum MHC region, designated Modo-UA1, with the features expected for encoding a functionally classical class I α-chain. Nine other class I genes found within the MHC are highly divergent and have features usually associated with non-classical roles. The original annotation, however, was based on an early version of the opossum genome assembly. More recent analyses of allelic variation in individual opossums revealed too many Modo-UA1 sequences per individual to be accounted for by a single MHC class I locus found in the genome assembly. A reanalysis of a later generation assembly, MonDom5, revealed the presence of two additional loci, now designated Modo-UA3 and UA4, in a region that was expanded and more complete than in the earlier assembly. Modo-UA1, UA3, and UA4 are all transcribed, although Modo-UA4 transcripts are rarer. Modo-UA4 is also relatively non-polymorphic. Evidence presented support the accuracy of the later assembly and the existence of three related class I genes in the opossum, making opossums more typical of mammals and most tetrapods by having multiple apparent classical MHC class I loci.

Rate of evolutionary change in cranial morphology of the marsupial genus Monodelphis is constrained by the availability of additive genetic variation.

We tested the hypothesis that the rate of marsupial cranial evolution is dependent on the distribution of genetic variation in multivariate space. To do so, we carried out a genetic analysis of cranial morphological variation in laboratory strains of Monodelphis domestica and used estimates of genetic covariation to analyse the morphological diversification of the Monodelphis brevicaudata species group. We found that within-species genetic variation is concentrated in only a few axes of the morphospace and that this strong genetic covariation influenced the rate of morphological diversification of the brevicaudata group, with between-species divergence occurring fastest when occurring along the genetic line of least resistance. Accounting for the geometric distribution of genetic variation also increased our ability to detect the selective regimen underlying species diversification, with several instances of selection only being detected when genetic covariances were taken into account. Therefore, this work directly links patterns of genetic covariation among traits to macroevolutionary patterns of morphological divergence. Our findings also suggest that the limited distribution of Monodelphis species in morphospace is the result of a complex interplay between the limited dimensionality of available genetic variation and strong stabilizing selection along two major axes of genetic variation.

Paternal X inactivation does not correlate with X chromosome evolutionary strata in marsupials.

BACKGROUND: X chromosome inactivation is the transcriptional silencing of one X chromosome in the somatic cells of female mammals. In eutherian mammals (e.g. humans) one of the two X chromosomes is randomly chosen for silencing, with about 15% (usually in younger evolutionary strata of the X chromosome) of genes escaping this silencing. In contrast, in the distantly related marsupial mammals the paternally derived X is silenced, although not as completely as the eutherian X. A chromosome wide examination of X inactivation, using RNA-seq, was recently undertaken in grey short-tailed opossum (Monodelphis domestica) brain and extraembryonic tissues. However, no such study has been conduced in Australian marsupials, which diverged from their American cousins ~80 million years ago, leaving a large gap in our understanding of marsupial X inactivation. RESULTS: We used RNA-seq data from blood or liver of a family (mother, father and daughter) of tammar wallabies (Macropus eugenii), which in conjunction with available genome sequence from the mother and father, permitted genotyping of 42 expressed heterozygous SNPs on the daughter's X. These 42 SNPs represented 34 X loci, of which 68% (23 of the 34) were confirmed as inactivated on the paternally derived X in the daughter's liver; the remaining 11 X loci escaped inactivation. Seven of the wallaby loci sampled were part of the old X evolutionary stratum, of which three escaped inactivation. Three loci were classified as part of the newer X stratum, of which two escaped inactivation. A meta-analysis of previously published opossum X inactivation data revealed that 5 of 52 genes in the old X stratum escaped inactivation. CONCLUSIONS: We demonstrate that chromosome wide inactivation of the paternal X is common to an Australian marsupial representative, but that there is more escape from inactivation than reported for opossum (32% v 14%). We also provide evidence that, unlike the human X chromosome, the location of loci within the oldest evolutionary stratum on the marsupial X does not correlate with their probability of escape from inactivation.

Genome-wide histone state profiling of fibroblasts from the opossum, Monodelphis domestica, identifies the first marsupial-specific imprinted gene.

BACKGROUND: Imprinted genes have been extensively documented in eutherian mammals and found to exhibit significant interspecific variation in the suites of genes that are imprinted and in their regulation between tissues and developmental stages. Much less is known about imprinted loci in metatherian (marsupial) mammals, wherein studies have been limited to a small number of genes previously known to be imprinted in eutherians. We describe the first ab initio search for imprinted marsupial genes, in fibroblasts from the opossum, Monodelphis domestica, based on a genome-wide ChIP-seq strategy to identify promoters that are simultaneously marked by mutually exclusive, transcriptionally opposing histone modifications. RESULTS: We identified a novel imprinted gene (Meis1) and two additional monoallelically expressed genes, one of which (Cstb) showed allele-specific, but non-imprinted expression. Imprinted vs. allele-specific expression could not be resolved for the third monoallelically expressed gene (Rpl17). Transcriptionally opposing histone modifications H3K4me3, H3K9Ac, and H3K9me3 were found at the promoters of all three genes, but differential DNA methylation was not detected at CpG islands at any of these promoters. CONCLUSIONS: In generating the first genome-wide histone modification profiles for a marsupial, we identified the first gene that is imprinted in a marsupial but not in eutherian mammals. This outcome demonstrates the practicality of an ab initio discovery strategy and implicates histone modification, but not differential DNA methylation, as a conserved mechanism for marking imprinted genes in all therian mammals. Our findings suggest that marsupials use multiple epigenetic mechanisms for imprinting and support the concept that lineage-specific selective forces can produce sets of imprinted genes that differ between metatherian and eutherian lines.

Molecular phylogeny of short-tailed opossums (Didelphidae: Monodelphis): taxonomic implications and tests of evolutionary hypotheses.

Short-tailed opossums (genus Monodelphis) represent one of the most speciose clades of New World marsupials, with 26 currently recognized species that collectively range from eastern Panama to northern Argentina. Here we present the first phylogenetic analyses of the genus based on dense taxonomic sampling and multiple genes. From most sampled species we obtained >4800bp of DNA sequence from one mitochondrial gene (CYTB), two autosomal exons (IRBP exon 1, BRCA1 exon 11), one autosomal intron (SLC38 intron 7), and one X-linked intron (OGT intron 14). Maximum-parsimony, maximum-likelihood and Bayesian analyses of these data strongly support the monophyly of Monodelphis and recover six major clades within the genus. Additionally, our analyses support previous suggestions that several nominal taxa are synonyms of other species (M. "sorex" of M. dimidiata, M. "theresa" of M. scalops, M. "rubida" and M. "umbristriata" of M. americana, and M. "maraxina" of M. glirina). By contrast, four unnamed lineages recovered by our analyses may represent new species. Reconstructions of ancestral states of two discrete characters-dorsal pelage color pattern and habitat-suggest that the most recent common ancestor of Monodelphis was uniformly colored (with unpatterned dorsal pelage) and inhabited moist forest. Whereas some dorsal pelage patterns appear to have evolved homoplastically in Monodelphis, dorsal stripes may have had a unique historical origin in this genus.

SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex.

(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums.

Identification of the RSX interactome in a marsupial shows functional coherence with the Xist interactome during X inactivation.

The marsupial specific RSX lncRNA is the functional analogue of the eutherian specific XIST, which coordinates X chromosome inactivation. We characterized the RSX interactome in a marsupial representative (the opossum Monodelphis domestica), identifying 135 proteins, of which 54 had orthologues in the XIST interactome. Both interactomes were enriched for biological pathways related to RNA processing, regulation of translation, and epigenetic transcriptional silencing. This represents a remarkable example showcasing the functional coherence of independently evolved lncRNAs in distantly related mammalian lineages.

Splicing is dynamically regulated during limb development.

Modifications to highly conserved developmental gene regulatory networks are thought to underlie morphological diversification in evolution and contribute to human congenital malformations. Relationships between gene expression and morphology have been extensively investigated in the limb, where most of the evidence for alterations to gene regulation in development consists of pre-transcriptional mechanisms that affect expression levels, such as epigenetic alterations to regulatory sequences and changes to cis-regulatory elements. Here we report evidence that alternative splicing (AS), a post-transcriptional process that modifies and diversifies mRNA transcripts, is dynamic during limb development in two mammalian species. We evaluated AS patterns in mouse (Mus musculus) and opossum (Monodelphis domestica) across the three key limb developmental stages: the ridge, bud, and paddle. Our data show that splicing patterns are dynamic over developmental time and suggest differences between the two mammalian taxa. Additionally, multiple key limb development genes, including Fgf8, are differentially spliced across the three stages in both species, with expression levels of the conserved splice variants, Fgf8a and Fgf8b, changing across developmental time. Our data demonstrates that AS is a critical mediator of mRNA diversity in limb development and provides an additional mechanism for evolutionary tweaking of gene dosage.

Convergent and divergent evolution of genomic imprinting in the marsupial Monodelphis domestica.

BACKGROUND: Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin specific monoallelic gene expression. It is postulated to have evolved in placental mammals to modulate intrauterine resource allocation to the offspring. In this study, we determined the imprint status of metatherian orthologues of eutherian imprinted genes. RESULTS: L3MBTL and HTR2A were shown to be imprinted in Monodelphis domestica (the gray short-tailed opossum). MEST expressed a monoallelic and a biallelic transcript, as in eutherians. In contrast, IMPACT, COPG2, and PLAGL1 were not imprinted in the opossum. Differentially methylated regions (DMRs) involved in regulating imprinting in eutherians were not found at any of the new imprinted loci in the opossum. Interestingly, a novel DMR was identified in intron 11 of the imprinted IGF2R gene, but this was not conserved in eutherians. The promoter regions of the imprinted genes in the opossum were enriched for the activating histone modification H3 Lysine 4 dimethylation. CONCLUSIONS: The phenomenon of genomic imprinting is conserved in Therians, but the marked difference in the number and location of imprinted genes and DMRs between metatherians and eutherians indicates that imprinting is not fully conserved between the two Therian infra-classes. The identification of a novel DMR at a non-conserved location as well as the first demonstration of histone modifications at imprinted loci in the opossum suggest that genomic imprinting may have evolved in a common ancestor of these two Therian infra-classes with subsequent divergence of regulatory mechanisms in the two lineages.

Evolution of vertebrate interferon inducible transmembrane proteins.

BACKGROUND: Interferon inducible transmembrane proteins (IFITMs) have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. RESULTS: Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. CONCLUSIONS: Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.

Histone H3 trimethylation at lysine 9 marks the inactive metaphase X chromosome in the marsupial Monodelphis domestica.

In somatic cells of female marsupial and eutherian mammals, X chromosome inactivation (XCI) occurs. XCI results in the transcriptional silencing of one of the two X chromosomes and is accompanied by specific covalent histone modifications attributable to the inactive chromatin state. Because data about repressed chromatin of the inactive X chromosome (Xi) in marsupials are sparse, we examined in more detail the distribution of active and inactive chromatin markers on metaphase X chromosomes of an American marsupial, Monodelphis domestica. Consistent with data reported previously both for eutherian and marsupial mammals, we found that the Xi of M. domestica lacks active histone markers-H3K4 dimethylation and H3K9 acetylation. We did not observe on metaphase spreads enrichment of the Xi with H3K27 trimethylation which is involved in XCI in eutherians and was detected on the Xi in the interphase nuclei of mature female M. domestica in an earlier study. Moreover, we found that the Xi of M. domestica was specifically marked with H3K9 trimethylation, which is known to be a component of the Xi chromatin in eutherians and is involved in both marsupials and eutherians in meiotic sex chromosome inactivation which has been proposed as an ancestral mechanism of XCI.

Heterochrony in somitogenesis rate in a model marsupial, Monodelphis domestica.

Marsupial newborns are highly altricial and also show a wide array of shifts in the rate or timing of developmental events so that certain neonatal structures are quite mature. One particularly notable feature is the steep gradient in development along the anterior-posterior axis such that anterior structures are generally well developed relative to posterior ones. Here, we study somitogenesis in the marsupial, Monodelphis domestica, and document two heterochronies that may be important in generating the unusual body plan of the newborn marsupial. First, we demonstrate a 4-fold change in somitogenesis rate along the anterior-posterior axis, which appears to be due to somitogenesis slowing posteriorly. Second, we show that somitogenesis, particularly in the cervical region, initiates earlier in Monodelphis relative to other developmental events in the embryo. The early initiation of somitogenesis may contribute to the early development of the cervical region and forelimbs. Other elements of somitogenesis appear to be conserved. When compared to mouse, we see similar expression of genes involved in the clock and wavefront, and genes of the Wnt, Notch, and fibroblast growth factor (FGF) pathways also cycle in Monodelphis. Further, we could not discern differences in somite maturation rate along the anterior-posterior axis in Monodelphis, and thus rate of maturation of the somites does not appear to contribute to the steep anterior-posterior gradient.