De novo transcriptome assembly and genome annotation of the fat-tailed dunnart (Sminthopsis crassicaudata).

Marsupials exhibit distinctive modes of reproduction and early development that set them apart from their eutherian counterparts and render them invaluable for comparative studies. However, marsupial genomic resources still lag far behind those of eutherian mammals. We present a series of novel genomic resources for the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-like marsupial that, due to its ease of husbandry and ex-utero development, is emerging as a laboratory model. We constructed a highly representative multi-tissue de novo transcriptome assembly of dunnart RNA-seq reads spanning 12 tissues. The transcriptome includes 2,093,982 assembled transcripts and has a mammalian transcriptome BUSCO completeness score of 93.3%, the highest amongst currently published marsupial transcriptomes. This global transcriptome, along with ab initio predictions, supported annotation of the existing dunnart genome, revealing 21,622 protein-coding genes. Altogether, these resources will enable wider use of the dunnart as a model marsupial and deepen our understanding of mammalian genome evolution.

Embryology of the fat-tailed dunnart (Sminthopsis crassicaudata): A marsupial model for comparative mammalian developmental and evolutionary biology.

BACKGROUND: Marsupials are a diverse and unique group of mammals, but remain underutilized in developmental biology studies, hindering our understanding of mammalian diversity. This study focuses on establishing the fat-tailed dunnart (Sminthopsis crassicaudata) as an emerging laboratory model, providing reproductive monitoring methods and a detailed atlas of its embryonic development. RESULTS: We monitored the reproductive cycles of female dunnarts and established methods to confirm pregnancy and generate timed embryos. With this, we characterized dunnart embryo development from cleavage to birth, and provided detailed descriptions of its organogenesis and heterochronic growth patterns. Drawing stage-matched comparisons with other species, we highlight the dunnarts accelerated craniofacial and limb development, characteristic of marsupials. CONCLUSIONS: The fat-tailed dunnart is an exceptional marsupial model for developmental studies, where our detailed practices for reproductive monitoring and embryo collection enhance its accessibility in other laboratories. The accelerated developmental patterns observed in the Dunnart provide a valuable system for investigating molecular mechanisms underlying heterochrony. This study not only contributes to our understanding of marsupial development but also equips the scientific community with new resources for addressing biodiversity challenges and developing effective conservation strategies in marsupials.

Therian origin of INSL3/RXFP2-driven testicular descent in mammals.

Introduction: During early development in most male mammals the testes move from a position near the kidneys through the abdomen to eventually reside in the scrotum. The transabdominal phase of this migration is driven by insulin-like peptide 3 (INSL3) which stimulates growth of the gubernaculum, a key ligament connecting the testes with the abdominal wall. While all marsupials, except the marsupial mole (Notoryctes typhlops), have a scrotum and fully descended testes, it is unclear if INSL3 drives this process in marsupials especially given that marsupials have a different mechanism of scrotum determination and position relative to the phallus compared to eutherian mammals. Methods: To understand if INSL3 plays a role in marsupial testicular descent we have sequenced and curated the INSL3 gene and its receptor (RXFP2) in a range of marsupials representing every order. Furthermore, we looked at single cell RNA-seq and qPCR analysis of INSL3 in the fat-tailed dunnart testis (Sminthopsis crassicaudata) to understand the location and timing of expression during development. Results: These data show a strong phylogenetic similarity between marsupial and eutherian orthologues, but not with monotreme INSL3s which were more similar to the ancestral RLN3 gene. We have also shown the genomic location of INSL3, and surrounding genes is conserved in a range of marsupials and eutherians. Single cell RNA-seq and qPCR data show that INSL3 mRNA is expressed specifically in Leydig cells and expressed at higher levels during the testicular descent phase in developing marsupials. Discussion: Together, these data argue strongly for a therian origin of INSL3 mediated testicular descent in mammals and suggests that a coordinated movement of the testes to the abdominal wall may have preceded externalization in marsupials and therian mammals.

CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.

Base editors are a type of double-stranded break (DSB)-free gene editing technology that has opened up new possibilities for precise manipulation of mitochondrial DNA (mtDNA). This includes cytosine and adenosine base editors and more recently guanosine base editors. Because of having low off-target and indel rates, there is a growing interest in developing and evolving this research field. Here, we provide a detailed update on DNA base editors. While base editing has widely been used for nuclear genome engineering, the growing interest in applying this technology to mitochondrial DNA has been faced with several challenges. While Cas9 protein has been shown to enter mitochondria, use of smaller Cas proteins, such as Cas12a, has higher import efficiency. However, sgRNA transfer into mitochondria is the most challenging step. sgRNA structure and ratio of Cas protein to sgRNA are both important factors for efficient sgRNA entry into mitochondria. In conclusion, while there are still several challenges to be addressed, ongoing research in this field holds the potential for new treatments and therapies for mitochondrial disorders.

Population genomics of a predatory mammal reveals patterns of decline and impacts of exposure to toxic toads.

Mammal declines across northern Australia are one of the major biodiversity loss events occurring globally. There has been no regional assessment of the implications of these species declines for genomic diversity. To address this, we conducted a species-wide assessment of genomic diversity in the northern quoll (Dasyurus hallucatus), an Endangered marsupial carnivore. We used next generation sequencing methods to genotype 10,191 single nucleotide polymorphisms (SNPs) in 352 individuals from across a 3220-km length of the continent, investigating patterns of population genomic structure and diversity, and identifying loci showing signals of putative selection. We found strong heterogeneity in the distribution of genomic diversity across the continent, characterized by (i) biogeographical barriers driving hierarchical population structure through long-term isolation, and (ii) severe reductions in diversity resulting from population declines, exacerbated by the spread of introduced toxic cane toads (Rhinella marina). These results warn of a large ongoing loss of genomic diversity and associated adaptive capacity as mammals decline across northern Australia. Encouragingly, populations of the northern quoll established on toad-free islands by translocations appear to have maintained most of the initial genomic diversity after 16 years. By mapping patterns of genomic diversity within and among populations, and investigating these patterns in the context of population declines, we can provide conservation managers with data critical to informed decision-making. This includes the identification of populations that are candidates for genetic management, the importance of remnant island and insurance/translocated populations for the conservation of genetic diversity, and the characterization of putative evolutionarily significant units.

Evolutionary origin of vertebrate OCT4/POU5 functions in supporting pluripotency.

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse embryonic stem cells, we find that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. The specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, cell phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.

A Chromosome-Scale Hybrid Genome Assembly of the Extinct Tasmanian Tiger (Thylacinus cynocephalus).

The extinct Tasmanian tiger or thylacine (Thylacinus cynocephalus) was a large marsupial carnivore native to Australia. Once ranging across parts of the mainland, the species remained only on the island of Tasmania by the time of European colonization. It was driven to extinction in the early 20th century and is an emblem of native species loss in Australia. The thylacine was a striking example of convergent evolution with placental canids, with which it shared a similar skull morphology. Consequently, it has been the subject of extensive study. While the original thylacine assemblies published in 2018 enabled the first exploration of the species' genome biology, further progress is hindered by the lack of high-quality genomic resources. Here, we present a new chromosome-scale hybrid genome assembly for the thylacine, which compares favorably with many recent de novo marsupial genomes. In addition, we provide homology-based gene annotations, characterize the repeat content of the thylacine genome, and show that consistent with demographic decline, the species possessed a low rate of heterozygosity even compared to extant, threatened marsupials.

Annotation of immune genes in the extinct thylacine (Thylacinus cynocephalus).

Advances in genome sequencing technology have enabled genomes of extinct species to be sequenced. However, given the fragmented nature of these genome assemblies, it is not clear whether it is possible to comprehensively annotate highly variable and repetitive genes such as those involved in immunity. As such, immune genes have only been investigated in a handful of extinct genomes, mainly in human lineages. In 2018 the genome of the thylacine (Thylacinus cynocephalus), a carnivorous marsupial from Tasmania that went extinct in 1936, was sequenced. Here we attempt to characterise the immune repertoire of the thylacine and determine similarity to its closest relative with a genome available, the Tasmanian devil (Sarcophilus harrisii), as well as other marsupials. Members from all major immune gene families were identified. However, variable regions could not be characterised, and complex families such as the major histocompatibility complex (MHC) were highly fragmented and located across multiple small scaffolds. As such, at a gene level we were unable to reconstruct full-length coding sequences for the majority of thylacine immune genes. Despite this, we identified genes encoding functionally important receptors and immune effector molecules, which suggests the functional capacity of the thylacine immune system was similar to other mammals. However, the high number of partial immune gene sequences identified limits our ability to reconstruct an accurate picture of the thylacine immune repertoire.

Of eyes and embryos: subfunctionalization of the CRX homeobox gene in mammalian evolution.

ETCHbox genes are fast-evolving homeobox genes present only in eutherian (placental) mammals which originated by duplication and divergence from a conserved homeobox gene, Cone-rod homeobox (CRX). While expression and function of CRX are restricted to the retina in eutherian mammals, ETCHbox gene expression is specific to preimplantation embryos. This dramatic difference could reflect the acquisition of new functions by duplicated genes or subfunctionalization of pleiotropic roles between CRX and ETCHbox genes. To resolve between these hypotheses, we compared expression, sequence and inferred function between CRX of metatherian (marsupial) mammals and ETCHbox genes of eutherians. We find the metatherian CRX homeobox gene is expressed in early embryos and in eyes, unlike eutherian CRX, and distinct amino acid substitutions were fixed in the metatherian and eutherian evolutionary lineages consistent with altered transcription factor specificity. We find that metatherian CRX is capable of regulating embryonically expressed genes in cultured cells in a comparable way to eutherian ETCHbox. The data are consistent with CRX having a dual role in eyes and embryos of metatherians, providing an early embryonic function comparable to that of eutherian ETCHbox genes; we propose that subfunctionalization of pleiotropic functions occurred after gene duplication along the placental lineage, followed by functional elaboration.

Transcriptomic Analysis of MAP3K1 and MAP3K4 in the Developing Marsupial Gonad.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.

Conceptus Coats of Marsupials and Monotremes.

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

Pre-gastrula Development of Non-eutherian Mammals.

Marsupials and monotremes differ from eutherian mammals in many features of their reproduction and development. Some features appear to be representative of transitional stages in evolution from therapsid reptiles to humans and mice, particularly with respect to the extraembryonic tissues that have undergone remarkable modifications to accommodate reduced egg size and quantity of yolk/deutoplasm, and increasing emphasis on viviparity and placentation. Trophoblast and hypoblast contribute the epithelial layers in most of the extraembryonic membranes and are the first two lineages to differentiate from the embryonic lineage. How they are specified varies greatly among mammals, perhaps largely due to heterochrony in the stage at which they must function. Differences probably also exist in the stage at which lineages are specified relative to the stage at which they fully commit to differentiation. The dogma of sequential commitment to trophoblast and hypoblast with progressive loss of potency may not be a fundamental feature of early mammalian development, but merely a recently acquired developmental pattern in eutherians, or at least mice.

Expression of STRA8 is conserved in therian mammals but expression of CYP26B1 differs between marsupials and mice.

The first sign of mammalian germ cell sexual differentiation is the initiation of meiosis in females and of mitotic arrest in males. In the mouse, retinoic acid induces ovarian Stra8 expression and entry of germ cells into meiosis. In developing mouse testes, cytochrome P450 family 26, subfamily b, polypeptide 1 (CYP26B1) produced by the Sertoli cells degrades retinoic acid, preventing Stimulated by Retinoic Acid Gene 8 (Stra8), expression and inhibiting meiosis. However, in developing humans, there is no evidence that CYP26B1 acts a meiosis-inhibiting factor. We therefore examined aspects of the retinoic acid/STRA8/CYP26B1 pathway during gonadal development in the tammar wallaby, a marsupial, to understand whether retinoic acid stimulation of STRA8 and CYP26B1 degradation of retinoic acid was conserved between widely divergent mammals. In tammar ovaries, as in human ovaries and unlike the pattern in mice, CYP26B1 expression was not downregulated before the onset of meiosis. Exposure of pre-meiotic tammar ovaries to exogenous retinoic acid in vitro upregulated STRA8 expression compared to controls. We conclude that retinoic acid and STRA8 are conserved factors that control the initiation of meiosis amongst mammals but the role of CYP26B1 as a meiosis-inhibiting factor may be specific to rodents. The identity of the marsupial meiosis-inhibiting factor remains unknown.

Embryo arrest and reactivation: potential candidates controlling embryonic diapause in the tammar wallaby and mink†.

Embryonic diapause is a period of developmental arrest which requires coordination of a molecular cross-talk between the endometrium and blastocyst to ensure a successful reactivation, but the exact mechanisms are undefined. The objectives of this study were to screen the tammar blastocyst for potential diapause control factors and to investigate the potential for members of the epidermal growth factor (EGF) family to coordinate reactivation. A select number of factors were also examined in the mink to determine whether their expression patterns were conserved across diapause species. The full-length sequences of the tammar genes of interest were first cloned to establish their level of sequence conservation with other mammals. The uterine expression of EGF family members EGF and heparin-binding EGF (HBEGF) and their receptors (EGFR and erb-b2 receptor tyrosine kinase 4 (ERBB4)) was determined by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Both HBEGF and EGF were significantly upregulated at reactivation compared to diapause. In the blastocyst, the expression of the potential diapause factors Forkhead box class O family members (FOXO1, FOXO3, and FOXO4), tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the EGF family were examined by RT-PCR and immunofluorescence. Nuclear (and hence active) FOXO expression was confirmed for the first time in a mammalian diapause blastocyst in both the tammar and the mink-CDKN1A was also expressed, but TP53 is not involved and EGFR was not detected in the blastocyst. These results indicate that the EGF family, FOXOs, and CDKN1A are promising candidates for the molecular control of embryonic diapause in mammals.

The mammalian blastocyst.

The blastocyst is a mammalian invention that carries the embryo from cleavage to gastrulation. For such a simple structure, it exhibits remarkable diversity in its mode of formation, morphology, longevity, and intimacy with the uterine endometrium. This review explores this diversity in the light of the evolution of viviparity, comparing the three main groups of mammals: monotremes, marsupials, and eutherians. The principal drivers in blastocyst evolution were loss of yolk coupled with evolution of the placenta. An important outcome of blastocyst development is differentiation of two extraembryonic lineages (trophoblast and hypoblast) that contribute to the placenta. While in many species trophoblast segregation is often coupled with blastocyst formation, in marsupials and at least some Afrotherians, these events do not coincide. Thus, many questions regarding the conservation of molecular mechanisms controlling these events are of great interest but currently unresolved. For further resources related to this article, please visit the WIREs website.

Cell fate in animal and human blastocysts and the determination of viability.

Understanding the mechanisms underlying the first cell differentiation events in human preimplantation development is fundamental for defining the optimal conditions for IVF techniques and selecting the most viable embryos for further development. However, our comprehension of the very early events in development is still very limited. Moreover, our knowledge on early lineage specification comes primarily from studying the mouse model. It is important to recognize that although mammalian embryos share similar morphological landmarks, the timing and molecular control of developmental events may vary substantially between species. Mammalian blastocysts comprise three cell types that arise through two sequential rounds of binary cell fate decisions. During the first decision, cells located on the outside of the developing embryo form a precursor lineage for the embryonic part of the placenta: the trophectoderm and cells positioned inside the embryo become the inner cell mass (ICM). Subsequently, ICM cells differentiate into embryonic lineages that give rise to a variety of tissues in the developing foetus: either the epiblast or extraembryonic primitive endoderm. Successful formation of all three lineages is a prerequisite for implantation and development to term. A comprehensive understanding of the lineage specification processes in mammals is therefore necessary to shed light on the causes of early miscarriages and early pregnancy pathologies in humans.

Different Species Choose Their Own Paths to Pluripotency.

Pluripotency is well defined functionally but ambiguously defined at the molecular level. In this issue of Developmental Cell, Boroviak and colleagues (2015) use a multi-species approach to differentiate between fundamental features of pluripotency in mammals and those that exhibit evolutionary plasticity.

Characterisation of major histocompatibility complex class I genes at the fetal-maternal interface of marsupials.

Major histocompatibility complex class I molecules (MHC-I) are expressed at the cell surface and are responsible for the presentation of self and non-self antigen repertoires to the immune system. Eutherian mammals express both classical and non-classical MHC-I molecules in the placenta, the latter of which are thought to modulate the maternal immune response during pregnancy. Marsupials last shared a common ancestor with eutherian mammals such as humans and mice over 160 million years ago. Since, like eutherians, they have an intra-uterine development dependent on a placenta, albeit a short-lived and less invasive one, they provide an opportunity to investigate the evolution of MHC-I expression at the fetal-maternal interface. We have characterised MHC-I mRNA expression in reproductive tissues of the tammar wallaby (Macropus eugenii) from the time of placental attachment to day 25 of the 26.5 day pregnancy. Putative classical MHC-I genes were expressed in the choriovitelline placenta, fetus, and gravid endometrium throughout the whole of this period. The MHC-I classical sequences were phylogenetically most similar to the Maeu-UC (50/100 clones) and Maeu-UA genes (7/100 clones). Expression of three non-classical MHC-I genes (Maeu-UD, Maeu-UK and Maeu-UM) were also present in placental samples. The results suggest that expression of classical and non-classical MHC-I genes in extant marsupial and eutherian mammals may have been necessary for the evolution of the ancestral therian placenta and survival of the mammalian fetus at the maternal-fetal interface.

The POU-er of gene nomenclature.

The pluripotency factor POU5F1 (OCT4) is well known as a key regulator of stem cell fate. Homologues of POU5F1 exist throughout vertebrates, but the evolutionary and functional relationships between the various family members have been unclear. The level to which function has been conserved within this family provides insight into the evolution of early embryonic potency. Here, we seek to clarify the relationship between POU5F1 homologues in the vertebrate lineage, both phylogenetically and functionally. We resolve the confusion over the identity of the zebrafish gene, which was originally named pou2, then changed to pou5f1 and again, more recently, to pou5f3. We argue that the use of correct nomenclature is crucial when discussing the degree to which the networks regulating early embryonic differentiation are conserved.

Paf receptor expression in the marsupial embryo and endometrium during embryonic diapause.

The control of reactivation from embryonic diapause in the tammar wallaby (Macropus eugenii) involves sequential activation of the corpus luteum, secretion of progesterone that stimulates endometrial secretion and subsequent changes in the uterine environment that activate the embryo. However, the precise signals between the endometrium and the blastocyst are currently unknown. In eutherians, both the phospholipid Paf and its receptor, platelet-activating factor receptor (PTAFR), are present in the embryo and the endometrium. In the tammar, endometrial Paf release in vitro increases around the time of the early progesterone pulse that occurs around the time of reactivation, but whether Paf can reactivate the blastocyst is unknown. We cloned and characterised the expression of PTAFR in the tammar embryo and endometrium at entry into embryonic diapause, during its maintenance and after reactivation. Tammar PTAFR sequence and protein were highly conserved with mammalian orthologues. In the endometrium, PTAFR was expressed at a constant level in the glandular epithelium across all stages and in the luminal epithelium during both diapause and reactivation. Thus, the presence of the receptor appears not to be a limiting factor for Paf actions in the endometrium. However, the low levels of PTAFR in the embryo during diapause, together with its up-regulation and subsequent internalisation at reactivation, supports earlier results suggesting that endometrial Paf could be involved in reactivation of the tammar blastocyst from embryonic diapause.