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.

Unique reproductive strategy in the swamp wallaby.

Reproduction in mammals requires distinct cycles of ovulation, fertilization, pregnancy, and lactation often interspersed with periods of anoestrus when breeding does not occur. Macropodids, the largest extant species of marsupials, the kangaroos and wallabies, have a very different reproductive strategy to most eutherian mammals whereby young are born at a highly altricial stage of development with the majority of development occurring over a lengthy lactation period. Furthermore, the timings of ovulation and birth in some species occurs within a very short interval of each other (sometimes hours). Female swamp wallabies have an oestrous cycle shorter than their pregnancy length and were, therefore, speculated to mate and form a new embryo before birth thereby supporting two conceptuses at different stages of pregnancy. To confirm this, we used high-resolution ultrasound to monitor reproduction in swamp wallabies during pregnancy. Here, we show that females ovulate, mate, and form a new embryo prepartum while still carrying a full-term fetus in the contralateral uterus. This embryo enters embryonic diapause until the newborn leaves the pouch 9 mo later. Thus, combined with embryonic diapause, females are continuously pregnant and lactating at the same time throughout their reproductive life, a unique reproductive strategy that completely blurs the normal staged system of reproduction in mammals.

The tammar wallaby: a non-traditional animal model to study growth axis maturation.

Maturation of the growth hormone (GH)/insulin-like growth factor 1 (IGF1) axis is a critical developmental event that becomes functional over the peripartum period in precocial eutherian mammals such as sheep. In mice and marsupials that give birth to altricial young, the GH/IGF1 axis matures well after birth, suggesting that functional maturation is associated with developmental stage, not parturition. Recent foster-forward studies in one marsupial, the tammar wallaby (Macropus eugenii), have corroborated this hypothesis. 'Fostering' tammar young not only markedly accelerates their development and growth rates, but also affects the timing of maturation of the growth axis compared with normal growing young, providing a novel non-traditional animal model for nutritional manipulation. This review discusses how nutrition affects the maturation of the growth axis in marsupials compared with traditional eutherian animal models.

The Basal Radial Glia Occurs in Marsupials and Underlies the Evolution of an Expanded Neocortex in Therian Mammals.

A hallmark of mammalian brain evolution is the emergence of the neocortex, which has expanded in all mammalian infraclasses (Eutheria, Marsupialia, Monotremata). In eutherians, neocortical neurons derive from distinct neural stem and progenitor cells (NPCs). However, precise data on the presence and abundance of the NPCs, especially of basal radial glia (bRG), in the neocortex of marsupials are lacking. This study characterized and quantified the NPCs in the developing neocortex of a marsupial, the tammar wallaby (Macropus eugenii). Our data demonstrate that its neocortex is characterized by high NPC diversity. Importantly, we show that bRG exist at high relative abundance in the tammar indicating that this cell type is not specific to the eutherian neocortex and that similar mechanisms may underlie the formation of an expanded neocortex in eutherian and marsupial mammals. We also show that bRG are likely to have been present in the therian ancestor, so did not emerge independently in the eutherian and marsupial lineages. Moreover, our data support the concept that changes in multiple parameters contribute to neocortex expansion and demonstrate the importance of bRG and other NPCs for the development and expansion of the mammalian neocortex.

Genome of the Tasmanian tiger provides insights into the evolution and demography of an extinct marsupial carnivore.

The Tasmanian tiger or thylacine (Thylacinus cynocephalus) was the largest carnivorous Australian marsupial to survive into the modern era. Despite last sharing a common ancestor with the eutherian canids ~160 million years ago, their phenotypic resemblance is considered the most striking example of convergent evolution in mammals. The last known thylacine died in captivity in 1936 and many aspects of the evolutionary history of this unique marsupial apex predator remain unknown. Here we have sequenced the genome of a preserved thylacine pouch young specimen to clarify the phylogenetic position of the thylacine within the carnivorous marsupials, reconstruct its historical demography and examine the genetic basis of its convergence with canids. Retroposon insertion patterns placed the thylacine as the basal lineage in Dasyuromorphia and suggest incomplete lineage sorting in early dasyuromorphs. Demographic analysis indicated a long-term decline in genetic diversity starting well before the arrival of humans in Australia. In spite of their extraordinary phenotypic convergence, comparative genomic analyses demonstrated that amino acid homoplasies between the thylacine and canids are largely consistent with neutral evolution. Furthermore, the genes and pathways targeted by positive selection differ markedly between these species. Together, these findings support models of adaptive convergence driven primarily by cis-regulatory evolution.

Effects of nutritional manipulation on body composition in the developing marsupial, Macropus eugenii.

When 60-day-old tammar wallaby pouch young (Macropus eugenii) are fostered to mothers at 120 days of lactation, their growth, developmental rate and maturation of their GH/IGF axes are markedly accelerated. To determine the effect of fostering on energy intake, body composition and fat accretion, we first measured total body fat and lean mass in these young. Next, we mimicked the triglyceride oleic and palmitic acid composition of 120-day milk by supplementing 60 day young with these fatty acids and comparing their growth with that of growth accelerated young. There was no difference in the weight or growth axis maturation of supplemented young but there was significantly more body fat in these and in the growth-accelerated fostered young than in controls. We conclude that the accelerated growth and GH/IGF axis maturation observed previously in fostered young is most likely due to increased milk consumption and earlier access to specific nutrients.

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.

Growth axis maturation is linked to nutrition, growth and developmental rate.

Maturation of the mammalian growth axis is thought to be linked to the transition from fetal to post-natal life at birth. However, in an altricial marsupial, the tammar wallaby (Macropus eugenii), this process occurs many months after birth but at a time when the young is at a similar developmental stage to that of neonatal eutherian mammals. Here we manipulate growth rates and demonstrate in slow, normal and fast growing tammar young that nutrition and growth rate affect the time of maturation of the growth axis. Maturation of GH/IGF-I axis components occurred earlier in fast growing young, which had significantly increased hepatic GHR, IGF1 and IGFALS expression, plasma IGF-I concentrations, and significantly decreased plasma GH concentrations compared to age-matched normal young. These data support the hypothesis that the time of maturation of the growth axis depends on the growth rate and maturity of the young, which can be accelerated by changing their nutritional status.

Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials.

Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials-which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya-also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell-cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto-maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses-displaying strong expression in the uterine glands where retroviral particles can be detected-plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades.

Ultrasonography of wallaby prenatal development shows that the climb to the pouch begins in utero.

Marsupials have a functional placenta for a shorter period of time compared to that of eutherian species, and their altricial young reach the teats without any help from the mother. We have monitored the short intrauterine development of one marsupial, the tammar wallaby, with high-resolution ultrasound from reactivation of the 100-cell diapausing blastocyst to birth. The expanding blastocyst could be visualized when it had reached a diameter of 1.5 mm. From at least halfway through pregnancy, there are strong undulating movements of the endometrium that massage the expanding vesicle against the highly secretory endometrial surface. These unique movements possibly enhance exchange of uterine secretions and gases between the mother and embryo. There was a constant rate of development measured ultrasonographically from mid-gestation, regardless of when the blastocyst reactivated. Interestingly climbing movements by the fetus began in utero about 3 days before birth, mimicking those required to climb to the pouch.

Limited genetic diversity preceded extinction of the Tasmanian tiger.

The Tasmanian tiger or thylacine was the largest carnivorous marsupial when Europeans first reached Australia. Sadly, the last known thylacine died in captivity in 1936. A recent analysis of the genome of the closely related and extant Tasmanian devil demonstrated limited genetic diversity between individuals. While a similar lack of diversity has been reported for the thylacine, this analysis was based on just two individuals. Here we report the sequencing of an additional 12 museum-archived specimens collected between 102 and 159 years ago. We examined a portion of the mitochondrial DNA hyper-variable control region and determined that all sequences were on average 99.5% identical at the nucleotide level. As a measure of accuracy we also sequenced mitochondrial DNA from a mother and two offspring. As expected, these samples were found to be 100% identical, validating our methods. We also used 454 sequencing to reconstruct 2.1 kilobases of the mitochondrial genome, which shared 99.91% identity with the two complete thylacine mitochondrial genomes published previously. Our thylacine genomic data also contained three highly divergent putative nuclear mitochondrial sequences, which grouped phylogenetically with the published thylacine mitochondrial homologs but contained 100-fold more polymorphisms than the conserved fragments. Together, our data suggest that the thylacine population in Tasmania had limited genetic diversity prior to its extinction, possibly as a result of their geographic isolation from mainland Australia approximately 10,000 years ago.

Maturation of the growth axis in marsupials occurs gradually during post-natal life and over an equivalent developmental stage relative to eutherian species.

The separation of a nutrition-responsive insulin-like growth factor (IGF) system and a growth hormone (GH) responsive IGF system to control pre- and post-natal growth of developing mammals may originate from the constraints imposed by intra-uterine development. In eutherian species that deliver relatively precocial young, maturation of the GH regulatory system is coincident with the time of birth. We measured the hepatic expression of the four key growth axis genes GH-receptor, IGF-1 and -2, and IGFBBP-3, and plasma protein concentrations of IGF-1 from late fetal life through to adult stages of a marsupial, the tammar wallaby. The data clearly show that maturation of GH-regulated growth in marsupials occurs gradually over the course of post-natal life at an equivalent developmental stage to that of precocial eutherian mammals. This suggests that the timing of GH-regulated growth in marsupials is not related to parturition but instead to the relative developmental stage.

Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolution of mammalian reproduction and development.

BACKGROUND: We present the genome sequence of the tammar wallaby, Macropus eugenii, which is a member of the kangaroo family and the first representative of the iconic hopping mammals that symbolize Australia to be sequenced. The tammar has many unusual biological characteristics, including the longest period of embryonic diapause of any mammal, extremely synchronized seasonal breeding and prolonged and sophisticated lactation within a well-defined pouch. Like other marsupials, it gives birth to highly altricial young, and has a small number of very large chromosomes, making it a valuable model for genomics, reproduction and development. RESULTS: The genome has been sequenced to 2 × coverage using Sanger sequencing, enhanced with additional next generation sequencing and the integration of extensive physical and linkage maps to build the genome assembly. We also sequenced the tammar transcriptome across many tissues and developmental time points. Our analyses of these data shed light on mammalian reproduction, development and genome evolution: there is innovation in reproductive and lactational genes, rapid evolution of germ cell genes, and incomplete, locus-specific X inactivation. We also observe novel retrotransposons and a highly rearranged major histocompatibility complex, with many class I genes located outside the complex. Novel microRNAs in the tammar HOX clusters uncover new potential mammalian HOX regulatory elements. CONCLUSIONS: Analyses of these resources enhance our understanding of marsupial gene evolution, identify marsupial-specific conserved non-coding elements and critical genes across a range of biological systems, including reproduction, development and immunity, and provide new insight into marsupial and mammalian biology and genome evolution.

Placental expression of pituitary hormones is an ancestral feature of therian mammals.

BACKGROUND: The placenta is essential for supplying nutrients and gases to the developing mammalian young before birth. While all mammals have a functional placenta, only in therian mammals (marsupials and eutherians) does the placenta closely appose or invade the uterine endometrium. The eutherian placenta secretes hormones that are structurally and functionally similar to pituitary growth hormone (GH), prolactin (PRL) and luteinizing hormone (LH). Marsupial and eutherian mammals diverged from a common ancestor approximately 125 to 148 million years ago and developed distinct reproductive strategies. As in eutherians, marsupials rely on a short-lived but functional placenta for embryogenesis. RESULTS: We characterized pituitary GH, GH-R, IGF-2, PRL and LHß in a macropodid marsupial, the tammar wallaby, Macropus eugenii. These genes were expressed in the tammar placenta during the last third of gestation when most fetal growth occurs and active organogenesis is initiated. The mRNA of key growth genes GH, GH-R, IGF-2 and PRL were expressed during late pregnancy. We found significant up-regulation of GH, GH-R and IGF-2 after the start of the rapid growth phase of organogenesis which suggests that the placental growth hormones regulate the rapid phase of fetal growth. CONCLUSIONS: This is the first demonstration of the existence of pituitary hormones in the marsupial placenta. Placental expression of these pituitary hormones has clearly been conserved in marsupials as in eutherian mammals, suggesting an ancestral origin of the evolution of placental expression and a critical function of these hormones in growth and development of all therian mammals.

The concept of superfetation: a critical review on a 'myth' in mammalian reproduction.

Superfetation is understood as another conception during an already ongoing pregnancy. This implies the existence of young of different developmental stages within the female reproductive tract during certain periods of pregnancy. Nevertheless, a clear definition of the term as well as distinct criteria to identify the occurrence of superfetation in a species is missing. The variable anatomy of mammalian reproductive tracts seems to make the occurrence of superfetation more or less likely but impedes the simple evaluation of whether it is present or not. Additionally, adequate determination methods are missing or are difficult to apply at the right time. Superfetation or rather superfetation-like pregnancies are reported for numerous species including humans, livestock and rodents. The usual criteria to assume a case of superfetation include the finding of discordantly developed young within the uterus during post mortem or parturition of young after a birth interval shorter than the assumed pregnancy length. Often the occurrence of superfetation is concluded because other explanations of reproductive artifacts are missing. Even severe reproductive pathologies are often confused with superfetation. True superfetation or superfetation as a reproductive strategy may exist in some mammals. In the American mink (Neovison (Mustela) vison) and the European badger (Meles meles) superfetation occurs in combination with embryonic diapause. In the European brown hare (Lepus europaeus), superfetation has long been assumed to exist but evidence is still controversial. Superfetation definitely occurs in certain species of poeciliid and zenarchopterid fish, some of which also exhibit viviparity and maternal care. In mammals, the evolution of such a reproductive mechanism poses many interesting evolutionary, endocrine, microbial and immunological questions that require further investigation. Here we review the scant and at times ancient literature on this poorly understood topic. The different manifestations of superfetation are defined and reliable criteria to detect superfetation are outlined. Also, the differentiation of superfetation into a reproductive strategy or as a disrupted, abnormal reproductive function is discussed. Due to the different discussed functional aspects of superfetation, it is appropriate to establish a more detailed scheme to classify the true natural superfetation cases into superfertilization, superconception and superfetation proper. To date, there is no mammal species known for which superfetation proper in terms of finding discordantly developed fetuses has been conclusively demonstrated to be not only a rare occurence but an evolved reproductive strategy.

Early onset of ghrelin production in a marsupial.

Ghrelin regulates appetite in mammals and can stimulate growth hormone (GH) release from the pituitary. In rats and humans, ghrelin cells appear in the stomach during late fetal life. Nevertheless, the role of ghrelin in early mammalian development is not well understood. Marsupials deliver highly altricial young that weigh less than 1g so they must feed and digest milk at a comparatively immature stage of development. Since they complete their growth and differentiation while in the pouch, they are accessible models in which to determine the time course of ghrelin production during development. We examined the distribution of gastric ghrelin cells, plasma ghrelin concentrations and pituitary expression of the ghrelin receptor (ghsr-1alpha) and GH in the tammar wallaby, Macropus eugenii. There were ghrelin immunopositive cells in the developing mesenchyme of the stomach from day 10 post partum (pp) to day 150pp. Subsequently ghrelin protein in the fore-stomach declined and was absent by day 250pp but remained in the gastric cells of the hind-stomach. Ghrelin was detected in the developing pancreas from day 10pp but was absent by day 150pp and in the adult. Pituitary ghsr-1alpha expression and plasma concentrations of ghrelin increased significantly up to day 70-120pp while GH expression was also elevated, declining with GH to reach adult levels by day 180pp. These results demonstrate an early onset of gastric ghrelin expression in the tammar in concert with a functional stomach at a relatively earlier stage than that of developmentally more mature eutherian young.

Exon 3 of the growth hormone receptor (GH-R) is specific to eutherian mammals.

Growth hormone receptor (GH-R) plays a critical role in the control of growth and metabolism in all vertebrates. GH-R consists of 9 coding exons (2-10) in all eutherian mammals, while the chicken only has 8 coding exons, and does not have an orthologous region to exon 3 in eutherians. To further understand the evolutionary origins of exon 3 of the GH-R in eutherians we cloned the full-length GH-R sequence in a marsupial, the tammar wallaby to determine whether exon 3 was present or absent in marsupial liver cDNA. There was no evidence for the presence of an exon 3 containing mRNA in sequence of tammar pouch young and adult livers. We next examined the genomes of the platypus (a monotreme mammal) and the grey short-tailed opossum (another marsupial). Like the tammar, the GH-R gene of neither species contained an exon 3. GH receptor can obviously function in the absence of this exon, raising speculation about the function of this domain, if any, in eutherians. A comparison of exon 3 protein sequences within 16 species of eutherian mammals showed that there was approximately 75% homology in the domain but only 3 of the 21 amino acids were identical (Leu12, Gln13 and Pro17). Interestingly, we detected greater evolutionary divergence in exon 3 sequences from species that have variants of GH or prolactin (PRL) in their placentas. These data show that exon 3 was inserted into the GH-R after the divergence of the marsupial and eutherian lineages at least 130 million years ago.

Perturbed growth and development in marsupial young after reciprocal cross-fostering between species.

Cross-fostering of marsupial young between species can potentially facilitate propagation of endangered or rare marsupial species by artificially increasing the number of progeny produced. The present study compares the growth and development of normal and cross-fostered tammar and parma wallabies. Tammars cross-fostered into the pouches of parmas grew at a similar rate to naturally reared tammar young and had developmental milestones at a similar age. However, parma young cross-fostered between the day of birth and 15 days post-partum into tammars that were carrying young of equivalent developmental stages did not grow normally and were lost from the pouch. Parma young cross-fostered at 30 days survived, but had significantly reduced growth rates and their developmental milestones were delayed compared with normally reared parma young. Thus, growth can be affected by cross-fostering, even between species like tammars and parmas that are of similar size and have similar lactation lengths. The results of the present study suggest that maternal milk regulates the timing of development of each species and a mis-match in the time that each young receives critical milk components can have a marked effect on their growth and development.