Monodelphis Research Articles

The UT family of MHC class I loci unique to non-eutherian mammals has limited polymorphism and tissue specific patterns of expression in the opossum.

BackgroundThe Major Histocompatibility Complex (MHC) class I family of genes encode for molecules that have well-conserved structures, but have evolved to perform diverse functions. The availability of the gray, short-tailed opossum, Monodelphis domestica whole genome sequence has allowed for analysis of MHC class I gene content in this marsupial. Utilization of a novel method to search for MHC related domain structures revealed a previously unknown family of MHC class I-related genes. These genes, named UT1-17, are clustered on chromosome 1 in the opossum, unlinked to the MHC region. UT genes are only found in marsupial and monotreme genomes, consistent with being ancient in mammals yet lost in eutherian mammals. This study investigates the expression and polymorphism of the UT loci in the opossum to gain insight into their possible function.ResultsOf the 17 opossum UT genes, most have restricted tissue transcription patterns, with the thymus and skin being the most common sites. Full-length structure of 11 UT transcripts revealed genes varying between five and eight exons, typical for class I family members. There were only two alternative splice variants found. The UT genes also have limited polymorphism and little evidence of positive selection. One locus, UT8, was chosen for further analysis due to its conservation amongst marsupials and generic characteristics. UT8 transcription is limited to developing αβ thymocytes, and is absent from mature αβ T cells in peripheral lymphoid tissues.ConclusionThe overall characteristics and features of UT genes including low polymorphism and restricted tissue expression make it likely that the molecules encoded by UT genes perform roles other than antigenic peptide presentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0181-9) contains supplementary material, which is available to authorized users.

Conserved and divergent expression patterns of markers of axial development in the laboratory opossum, Monodelphis domestica.

Previous comparative studies suggest that the requirement for Nodal in epiblast and hypoblast development is unique to mammalians. Expression of anterior visceral endoderm (AVE) genes in the visceral endoderm and of their orthologs in the hypoblast may be unique to mammalians and avians, and is absent in the reptilian hypoblast. Axis formation in reptiles is signaled by the formation of the posterior marginal epiblast (PME), which expresses a series of primitive streak genes. To assess the phylogenetic origin of Nodal and AVE gene expression and axis formation in amniotes, we examined marker gene expression in gray short-tailed opossum, a metatherian. Nodal was expressed in neither epiblast nor hypoblast of opossum embryos. No AVE genes were expressed in the opossum hypoblast. Attainment of polarity in the embryonic disk was signaled by Nodal, Wnt3a, Fgf8, and Bra expression in the PME at 8.5 days post-coitus. Nodal expression in epiblast or hypoblast may be unique to eutherians. AVE gene expression in visceral endoderm and hypoblast may have been independently acquired in eutherian and avian lineages. PME formation appears to be the event that signals axis formation in reptilian and metatherian embryos, and thus may be an ancestral characteristic of basal amniotes. Developmental Dynamics 245:1176-1188, 2016. © 2016 Wiley Periodicals, Inc.

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

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

Identification of the mRNA encoding interleukin-6 and its receptor, interleukin-6 receptor α, in five marsupial species

Expressed coding sequences for interleukin-6 (IL-6) and interleukin-6 receptor α (IL-6R) were examined in five marsupial species. Full length expressed coding sequences for IL-6 and IL-6R were identified and characterized in the gray short-tailed opossum (Monodelphis domestica). For IL-6, ∼225 bp fragments of the mRNA sequence were identified in the red-tailed phascogale (Phascogale calura), kultarr (Antechinomys laniger), and stripe-faced dunnart (Sminthopsis macroura), while ∼563 bp fragments of mRNA encoding IL-6R were identified in the red-tailed phascogale, kultarr, stripe-face dunnart and fat-tailed dunnart (Sminthopsis crassicaudata). Relative expression levels of IL-6 and IL-6R were examined in the heart, muscle, lung, liver, spleen and kidney of adult red-tailed phascogales, and IL-6 gene expression was found to be significantly higher in the lung and spleen than the other tissues examined, while the expression of IL-6R was significantly higher in the liver, lung and spleen. These results now serve as a reference point for examining the role and levels of IL-6 and IL-6R in the health and disease of these marsupial species. The pro-tumorigenic nature of IL-6 is of particular interest, and the identification of these IL-6 and IL-6R coding sequences provides a platform for further work to evaluate the potential role of IL-6 in marsupial cancers.

The evolution of whisker-mediated somatosensation in mammals: Sensory processing in barrelless S1 cortex of a marsupial, Monodelphis domestica.

Movable tactile sensors in the form of whiskers are present in most mammals, but sensory coding in the cortical whisker representation has been studied almost exclusively in mice and rats. Many species that possess whiskers lack the modular "barrel" organization found in the primary somatosensory cortex (S1) of mice and rats, but it is unclear how whisker-related input is represented in these species. We used single-unit extracellular recording techniques to characterize receptive fields and response properties in S1 of Monodelphis domestica (short-tailed opossum), a nocturnal, terrestrial marsupial that shared its last common ancestor with placental mammals over 160 million years ago. Short-tailed opossums lack barrels and septa in S1 but show active whisking behavior similar to that of mice and rats. Most neurons in short-tailed opossum S1 exhibited multiwhisker receptive fields, including a single best whisker (BW) and lower magnitude responses to the deflection of surrounding whiskers. Mean tuning width was similar to that reported for mice and rats. Both symmetrical and asymmetrical receptive fields were present. Neurons tuned to ventral whiskers tended to show broad tuning along the rostrocaudal axis. Thus, despite the absence of barrels, most receptive field properties were similar to those reported for mice and rats. However, unlike those species, S1 neuronal responses to BW and surround whisker deflection showed comparable latencies in short-tailed opossums. This dissimilarity suggests that some aspects of barrel cortex function may not generalize to tactile processing across mammalian species and may be related to differences in the architecture of the whisker-to-cortex pathway. J. Comp. Neurol. 524:3587-3613, 2016. © 2016 Wiley Periodicals, Inc.

Differential morphology of the superior olivary complex of Meriones unguiculatus and Monodelphis domestica revealed by calcium-binding proteins.

In mammals, the superior olivary complex (SOC) of the brainstem is composed of nuclei that integrate afferent auditory originating from both ears. Here, the expression of different calcium-binding proteins in subnuclei of the SOC was studied in distantly related mammals, the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica) to get a better understanding of the basal nuclear organization of the SOC. Combined immunofluorescence labeling of the calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k, and calretinin as well as pan-neuronal markers displayed characteristic distribution patterns highlighting details of neuronal architecture of SOC nuclei. Parvalbumin was found in almost all neurons of SOC nuclei in both species, while calbindin and calretinin were restricted to specific cell types and axonal terminal fields. In both species, calbindin displayed a ubiquitous and mostly selective distribution in neurons of the medial nucleus of trapezoid body (MNTB) including their terminal axonal fields in different SOC targets. In Meriones, calretinin and calbindin showed non-overlapping expression patterns in neuron somata and terminal fields throughout the SOC. In Monodelphis, co-expression of calbindin and calretinin was observed in the MNTB, and hence both CaBPs were also co-localized in terminal fields within the adjacent SOC nuclei. The distribution patterns of CaBPs in both species are discussed with respect to the intrinsic neuronal SOC circuits as part of the auditory brainstem system that underlie the binaural integrative processing of acoustic signals as the basis for localization and discrimination of auditory objects.

Preliminary genomic survey and sequence analysis of the complement system in non-eutherian mammals

The complement system is a major mediator of the vertebrate immune system, which functions in both innate and specific immune responses. It comprises more than 30 proteins working to remove foreign cells by way of anaphylatoxins, opsonins or the membrane attack complex. Over the last few years, whole genome sequences of non-eutherian mammals (marsupials and a monotreme), the gray short-tailed opossum (Monodelphis domestica), tammar wallaby (Macropus eugenii), Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus) and platypus (Ornithorhynchus anatinus), have become publicly available. Using these sequences, we have identified an array of complement components in non-eutherians using online search tools and algorithms. Of 57 complement and complement-related genes investigated, we identified 46 in the gray short-tailed opossum genome, 27 in the tammar wallaby genome, 44 in the Tasmanian devil genome, 47 in the koala genome and 40 in the platypus genome. The results of this study confirm the presence of key complement components in the immune repertoire of non-eutherian mammals and provide a platform for future studies on immune protection in young marsupials.

Marsupials and monotremes possess a novel family of MHC class I genes that is lost from the eutherian lineage

BackgroundMajor histocompatibility complex (MHC) class I genes are found in the genomes of all jawed vertebrates. The evolution of this gene family is closely tied to the evolution of the vertebrate genome. Family members are frequently found in four paralogous regions, which were formed in two rounds of genome duplication in the early vertebrates, but in some species class Is have been subject to additional duplication or translocation, creating additional clusters. The gene family is traditionally grouped into two subtypes: classical MHC class I genes that are usually MHC-linked, highly polymorphic, expressed in a broad range of tissues and present endogenously-derived peptides to cytotoxic T-cells; and non-classical MHC class I genes generally have lower polymorphism, may have tissue-specific expression and have evolved to perform immune-related or non-immune functions. As immune genes can evolve rapidly and are subject to different selection pressure, we hypothesised that there may be divergent, as yet unannotated or uncharacterised class I genes.ResultsApplication of a novel method of sensitive genome searching of available vertebrate genome sequences revealed a new, extensive sub-family of divergent MHC class I genes, denoted as UT, which has not previously been characterized. These class I genes are found in both American and Australian marsupials, and in monotremes, at an evolutionary chromosomal breakpoint, but are not present in non-mammalian genomes and have been lost from the eutherian lineage. We show that UT family members are expressed in the thymus of the gray short-tailed opossum and in other immune tissues of several Australian marsupials. Structural homology modelling shows that the proteins encoded by this family are predicted to have an open, though short, antigen-binding groove.ConclusionsWe have identified a novel sub-family of putatively non-classical MHC class I genes that are specific to marsupials and monotremes. This family was present in the ancestral mammal and is found in extant marsupials and monotremes, but has been lost from the eutherian lineage. The function of this family is as yet unknown, however, their predicted structure may be consistent with presentation of antigens to T-cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1745-4) contains supplementary material, which is available to authorized users.

Peramorphic males and extreme sexual dimorphism in Monodelphis dimidiata (Didelphidae)

The southern short-tailed opossum, Monodelphis dimidiata, is a species known not only for its semelparous life cycle, but also for the extreme sexual dimorphism of adults, where males are not only larger, but also have distinctive morphological characters in their skull. Using geometric morphometrics and a suite of statistical tests, I analyzed the postweaning ontogenetic development of this species in order to evaluate the age-class where sexual dimorphism becomes significant and the amount of change exhibited by both sexes. My results showed that M. dimidiata partly follows the ontogenetic pattern described for didelphids by previous authors. The character that escapes the general pattern is rostral length, which becomes shorter instead of lengthening throughout the development. This change could be related to an increment in the bite force in the anterior part of the dentition. The amount of sexual dimorphism found for this species is larger than the reported previously for other American marsupials, and I also found a higher rate of growth in males at the attaining of sexual maturity. Based on my results and the information available for other didelphids, I can suggest that M. dimidiata males undergo through a process of hypermorphosis, resulting in a peramorphic condition. It is possible that the extreme sexual dimorphism present in this species is related to reproductive success, specially taking into account their semelparous life cycle.

The Marsupial Intervertebral Disc: An Anatomical Characterization

The intervertebral disc (IVD) functions in the support and movement of the vertebrate body. It is composed of the collagenous annulus fibrosis (AF), which surrounds the inner nucleus pulposus (NP). The NP is primarily water and proteoglycans. Cartilage end plates attach the IVD to the adjacent vertebrae. The IVD undergoes numerous changes with aging (degeneration), which can result in back pain. Unfortunately, few effective options exist to treat back pain caused by disc degeneration. This has highlighted the need for methods to repair, regenerate, or prevent disc degeneration. Before these therapies are realized, basic questions on the genetic and regulatory mechanisms of IVD formation and degeneration need to be answered. The use of a wide variety of mammalian models can provide interesting insights, and much work has been done using the mouse. The goal of this study is to develop the gray short-tailed opossum (Monodelphis domestica) as a new mammalian model for study of the formation, maturation, and degeneration of the IVD. The opossum is born at an early stage of development, equivalent to an E10.5-12.5 mouse. Anterior structures are well-developed, while posterior ones (hindlimbs) are delayed at birth. We hypothesize that the IVDs are not formed in the posterior of the body, giving us a unique opportunity to study and manipulate the formation of the IVDs ex utero (in mice, IVDs are formed prenatally). Using in situ hybridization, histology, and skeletal staining, we are investigating the formation of the IVDs. We will also determine if the opossum undergoes age-related degeneration of its IVDs. Successful completion of this work will provide a baseline for future studies and also assess the viability of the opossum as a model for IVD research. Funding: NSF & IGB.

A Histological and Molecular Analysis of Tooth Regeneration in Monodelphis domestica

We aimed to gain insight into the differences between dental organs with different replacement capacities in the same animal, the gray short-tailed opossum (Monodelphis domestica), with regards to the fate of the successional dental lamina. The methods used to aid our research included three-dimensional reconstructions of serial histological sections, immunohistochemistry assays for studying expression of epithelial stem cell marker Sox2, proliferation, apoptosis, basement membrane integrity and innervation around the canine (non-replacing) and third premolar (replacing) dental organs and laminae. Our results show that the dental successional lamina developed at multiple tooth positions, with the laminae attached to non-replacing teeth starting to regress, in association with lack of Sox2 expression, reduced proliferation, increased apoptosis, disruption of the basement membrane and loss of innervation. In comparison, the successional lamina on the replacing tooth expressed Sox2, exhibited increased proliferation and reduced apoptosis and constant innervation throughout the studied stages. We believe these differences underlie the survival of the successional dental lamina and its capacity to fuel the formation of a new tooth.

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.

Cell-type phylogenetics and the origin of endometrial stromal cells.

A challenge of genome annotation is the identification of genes performing specific biological functions. Here, we propose a phylogenetic approach that utilizes RNA-seq data to infer the historical relationships among cell types and to trace the pattern of gene-expression changes on the tree. The hypothesis is that gene-expression changes coincidental with the origin of a cell type will be important for the function of the derived cell type. We apply this approach to the endometrial stromal cells (ESCs), which are critical for the initiation and maintenance of pregnancy. Our approach identified well-known regulators of ESCs, PGR and FOXO1, as well as genes not yet implicated in female fertility, including GATA2 and TFAP2C. Knockdown analysis confirmed that they are essential for ESC differentiation. We conclude that phylogenetic analysis of cell transcriptomes is a powerful tool for discovery of genes performing cell-type-specific functions.

Evolution of mammalian sensorimotor cortex: thalamic projections to parietal cortical areas in Monodelphis domestica.

The current experiments build upon previous studies designed to reveal the network of parietal cortical areas present in the common mammalian ancestor. Understanding this ancestral network is essential for highlighting the basic somatosensory circuitry present in all mammals, and how this basic plan was modified to generate species specific behaviors. Our animal model, the short-tailed opossum (Monodelphis domestica), is a South American marsupial that has been proposed to have a similar ecological niche and morphology to the earliest common mammalian ancestor. In this investigation, we injected retrograde neuroanatomical tracers into the face and body representations of primary somatosensory cortex (S1), the rostral and caudal somatosensory fields (SR and SC), as well as a multimodal region (MM). Projections from different architectonically defined thalamic nuclei were then quantified. Our results provide further evidence to support the hypothesized basic mammalian plan of thalamic projections to S1, with the lateral and medial ventral posterior thalamic nuclei (VPl and VPm) projecting to S1 body and S1 face, respectively. Additional strong projections are from the medial division of posterior nucleus (Pom). SR receives projections from several midline nuclei, including the medial dorsal, ventral medial nucleus, and Pom. SC and MM show similar patterns of connectivity, with projections from the ventral anterior and ventral lateral nuclei, VPm and VPl, and the entire posterior nucleus (medial and lateral). Notably, MM is distinguished from SC by relatively dense projections from the dorsal division of the lateral geniculate nucleus and pulvinar. We discuss the finding that S1 of the short-tailed opossum has a similar pattern of projections as other marsupials and mammals, but also some distinct projections not present in other mammals. Further we provide additional support for a primitive posterior parietal cortex which receives input from multiple modalities.

Retraction: Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin.

The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.

Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin.

The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.

Arrested development of the dorsal column following neonatal spinal cord injury in the opossum, Monodelphis domestica.

Developmental studies of spinal cord injury in which regrowth of axons occurs across the site of transection rarely distinguish between the recovery of motor-controlling pathways and that of ascending axons carrying sensory information. We describe the morphological changes that occur in the dorsal column (DC) of the grey short-tailed opossum, Monodelphis domestica, following spinal cord injury at two early developmental ages. The spinal cords of opossums that had had their mid-thoracic spinal cords completely transected at postnatal day 7 (P7) or P28 were analysed. Profiles of neurofilament immunoreactivity in transected cords showing DC development were differentially affected by the injury compared with the rest of the cord and cytoarchitecture was modified in an age- and site-dependent manner. The ability of DC neurites to grow across the site of transection was confirmed by injection of fluorescent tracer below the injury. P7 transected cords showed labelling in the DC above the site of original transection indicating that neurites of this sensory tract were able to span the injury. No growth of any neuronal processes was seen after P28 transection. Thus, DC is affected by spinal injury in a differential manner depending on the age at which the transection occurs. This age-differential response, together with other facets of remodelling that occur after neonatal spinal injury, might explain the locomotor adaptations and recovery observed in these animals.

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

BackgroundX 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0267-z) contains supplementary material, which is available to authorized users.

Development of the ethmoid in Caluromys philander (Didelphidae, Marsupialia) with a discussion on the homology of the turbinal elements in marsupials.

Homology of turbinals, or scroll bones, of the mammalian ethmoid bone is poorly known and complicated by a varied terminology. Positionally, there are two main types of ossified adult turbinals known as endoturbinals and ectoturbinals, and their cartilaginous precursors are called ethmoturbinals and frontoturbinals, respectively. Endoturbinals are considered to be serially homologous due to similarity in their developmental patterns. Consequently, endoturbinals from mammals with differing numbers of elements cannot be individually homogenized. In this study, the development of the ethmoid of Caluromys philander, the bare-tailed woolly opossum, is described based on serial sections of six pouchlings ranging in age from 20 to 84 days postnatal (PND-84), and computed tomography images of an adult skull. I found that four ethmoturbinals initially develop as seen in PND-20 and PND-30 individuals but by PND-64 an interturbinal (corresponding to endoturbinal III in adults) is present between ethmoturbinals II and III. This developmental pattern is identical to that of Monodelphis domestica, the gray short-tailed opossum, and is probably also present in the marsupials Didelphis marsupialis, and Thylacinus cynocephalus based on work of previous authors. These data suggest that endoturbinal III has a developmental pattern that differs from other endoturbinals, and the name interturbinal should be retained for the adult structure in recognition of this difference. These results may prove useful for homologizing this individual turbinal element across marsupials, the majority of which have five endoturbinals as adults. This might also explain the presumed placental ancestral condition of four endoturbinals if the marsupial interturbinal is lost.

A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity.

BackgroundThe koala, Phascolarctos cinereus, is a biologically unique and evolutionarily distinct Australian arboreal marsupial. The goal of this study was to sequence the transcriptome from several tissues of two geographically separate koalas, and to create the first comprehensive catalog of annotated transcripts for this species, enabling detailed analysis of the unique attributes of this threatened native marsupial, including infection by the koala retrovirus.ResultsRNA-Seq data was generated from a range of tissues from one male and one female koala and assembled de novo into transcripts using Velvet-Oases. Transcript abundance in each tissue was estimated. Transcripts were searched for likely protein-coding regions and a non-redundant set of 117,563 putative protein sequences was produced. In similarity searches there were 84,907 (72%) sequences that aligned to at least one sequence in the NCBI nr protein database. The best alignments were to sequences from other marsupials. After applying a reciprocal best hit requirement of koala sequences to those from tammar wallaby, Tasmanian devil and the gray short-tailed opossum, we estimate that our transcriptome dataset represents approximately 15,000 koala genes. The marsupial alignment information was used to look for potential gene duplications and we report evidence for copy number expansion of the alpha amylase gene, and of an aldehyde reductase gene.Koala retrovirus (KoRV) transcripts were detected in the transcriptomes. These were analysed in detail and the structure of the spliced envelope gene transcript was determined. There was appreciable sequence diversity within KoRV, with 233 sites in the KoRV genome showing small insertions/deletions or single nucleotide polymorphisms. Both koalas had sequences from the KoRV-A subtype, but the male koala transcriptome has, in addition, sequences more closely related to the KoRV-B subtype. This is the first report of a KoRV-B-like sequence in a wild population.ConclusionsThis transcriptomic dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-786) contains supplementary material, which is available to authorized users.