Marsupials have monoallelic MEST expression with a conserved antisense lncRNA but MEST is not imprinted.

The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles.

Conserved H3K27me3-associated chromatin remodelling allows STRA8 but not MEIOSIN expression in mammalian germ cells.

In brief: Apart from mice, meiosis initiation factors and their transcriptional regulation mechanisms are largely unknown in mammals. This study suggests that STRA8 and MEIOSIN are both meiosis initiation factors in mammals, but their transcription is epigenetically regulated differently from each other. Abstract: In the mouse, the timing of meiosis onset differs between sexes due to the sex-specific regulation of the meiosis initiation factors, STRA8 and MEIOSIN. Before the initiation of meiotic prophase I, the Stra8 promoter loses suppressive histone-3-lysine-27 trimethylation (H3K27me3) in both sexes, suggesting that H3K27me3-associated chromatin remodelling may be responsible for activating STRA8 and its co-factor MEIOSIN. Here we examined MEIOSIN and STRA8 expression in a eutherian (the mouse), two marsupials (the grey short-tailed opossum and the tammar wallaby) and two monotremes (the platypus and the short-beaked echidna) to ask whether this pathway is conserved between all mammals. The conserved expression of both genes in all three mammalian groups and of MEIOSIN and STRA8 protein in therian mammals suggests that they are the meiosis initiation factors in all mammals. Analyses of published DNase-seq and chromatin-immunoprecipitation sequencing (ChIP-seq) data sets confirmed that H3K27me3-associated chromatin remodelling occurred at the STRA8, but not the MEIOSIN, promoter in therian mammals. Furthermore, culturing tammar ovaries with an inhibitor of H3K27me3 demethylation before meiotic prophase I affected STRA8 but not MEIOSIN transcriptional levels. Our data suggest that H3K27me3-associated chromatin remodelling is an ancestral mechanism that allows STRA8 expression in mammalian pre-meiotic germ cells.

Evolution of Embryo Implantation Was Enabled by the Origin of Decidual Stromal Cells in Eutherian Mammals.

Mammalian pregnancy evolved in the therian stem lineage, that is, before the common ancestor of marsupials and eutherian (placental) mammals. Ancestral therian pregnancy likely involved a brief phase of attachment between the fetal and maternal tissues followed by parturition-similar to the situation in most marsupials including the opossum. In all eutherians, however, embryo attachment is followed by implantation, allowing for a stable fetal-maternal interface and an extended gestation. Embryo attachment induces an attachment reaction in the uterus that is homologous to an inflammatory response. Here, we elucidate the evolutionary mechanism by which the ancestral inflammatory response was transformed into embryo implantation in the eutherian lineage. We performed a comparative uterine transcriptomic and immunohistochemical study of three eutherians, armadillo (Dasypus novemcinctus), hyrax (Procavia capensis), and rabbit (Oryctolagus cuniculus); and one marsupial, opossum (Monodelphis domestica). Our results suggest that in the eutherian lineage, the ancestral inflammatory response was domesticated by suppressing one of its modules detrimental to pregnancy, namely, neutrophil recruitment by cytokine IL17A. Further, we propose that this suppression was mediated by decidual stromal cells, a novel cell type in eutherian mammals. We tested a prediction of this model in vitro and showed that decidual stromal cells can suppress the production of IL17A from helper T cells. Together, these results provide a mechanistic understanding of early stages in the evolution of eutherian pregnancy.

Soybean Yellow Stripe-like 7 is a symbiosome membrane peptide transporter important for nitrogen fixation.

Legumes form a symbiosis with rhizobia that convert atmospheric nitrogen (N2) to ammonia and provide it to the plant in return for a carbon and nutrient supply. Nodules, developed as part of the symbiosis, harbor rhizobia that are enclosed in a plant-derived symbiosome membrane (SM) to form an organelle-like structure called the symbiosome. In mature nodules exchanges between the symbionts occur across the SM. Here we characterize Yellow Stripe-like 7 (GmYSL7), a Yellow stripe-like family member localized on the SM in soybean (Glycine max) nodules. It is expressed specifically in infected cells with expression peaking soon after nitrogenase becomes active. Unlike most YSL family members, GmYSL7 does not transport metals complexed with phytosiderophores. Rather, it transports oligopeptides of between four and 12 amino acids. Silencing GmYSL7 reduces nitrogenase activity and blocks infected cell development so that symbiosomes contain only a single bacteroid. This indicates the substrate of YSL7 is required for proper nodule development, either by promoting symbiosome development directly or by preventing inhibition of development by the plant. RNAseq of nodules where GmYSL7 was silenced suggests that the plant initiates a defense response against rhizobia with genes encoding proteins involved in amino acid export downregulated and some transcripts associated with metal homeostasis altered. These changes may result from the decrease in nitrogen fixation upon GmYSL7 silencing and suggest that the peptide(s) transported by GmYSL7 monitor the functional state of the bacteroids and regulate nodule metabolism and transport processes accordingly. Further work to identify the physiological substrate for GmYSL7 will allow clarification of this role.

Male germline development in the tammar wallaby, Macropus eugenii.

Male germ cells undergo two consecutive processes - pre-spermatogenesis and spermatogenesis - to generate mature sperm. In eutherian mammals, epigenetic information such as DNA methylation is dynamically reprogrammed during pre-spermatogenesis, before and during mitotic arrest. In mice, by the time germ cells resume mitosis, the majority of DNA methylation is reprogrammed. The tammar wallaby has a similar pattern of germ cell global DNA methylation reprogramming to that of the mouse during early pre-spermatogenesis. However, early male germline development in the tammar or in any marsupial has not been described previously, so it is unknown whether this is a general feature regulating male germline development or a more recent phenomenon in mammalian evolutionary history. To answer this, we examined germ cell nuclear morphology and mitotic arrest during male germline development in the tammar wallaby (Macropus eugenii), a marsupial that diverged from mice and humans around 160 million years ago. Tammar pro-spermatogonia proliferated after birth and entered mitotic arrest after day 30 postpartum (pp). At this time, they began moving towards the periphery of the testis cords and their nuclear size increased. Germ cells increased in number after day 100 pp which is the time that DNA methylation is known to be re-established in the tammar. This is similar to the pattern observed in the mouse, suggesting that resumption of germ cell mitosis and the timing of DNA methylation reprogramming are correlated and conserved across mammals and over long evolutionary timescales.

The mammalian decidual cell evolved from a cellular stress response.

Among animal species, cell types vary greatly in terms of number and kind. The number of cell types found within an organism differs considerably between species, and cell type diversity is a significant contributor to differences in organismal structure and function. These observations suggest that cell type origination is a significant source of evolutionary novelty. The molecular mechanisms that result in the evolution of novel cell types, however, are poorly understood. Here, we show that a novel cell type of eutherians mammals, the decidual stromal cell (DSC), evolved by rewiring an ancestral cellular stress response. We isolated the precursor cell type of DSCs, endometrial stromal fibroblasts (ESFs), from the opossum Monodelphis domestica. We show that, in opossum ESFs, the majority of decidual core regulatory genes respond to decidualizing signals but do not regulate decidual effector genes. Rather, in opossum ESFs, decidual transcription factors function in apoptotic and oxidative stress response. We propose that rewiring of cellular stress responses was an important mechanism for the evolution of the eutherian decidual cell type.

Embryo implantation evolved from an ancestral inflammatory attachment reaction.

The molecular changes that support implantation in eutherian mammals are necessary to establish pregnancy. In marsupials, pregnancy is relatively short, and although a placenta does form, it is present for only a few days before parturition. However, morphological changes in the uterus of marsupials at term mimic those that occur during implantation in humans and mice. We investigated the molecular similarity between term pregnancy in the marsupials and implantation in eutherian mammals using the gray short-tailed opossum (Monodelphis domestica) as a model. Transcriptomic analysis shows that term pregnancy in the opossum is characterized by an inflammatory response consistent with implantation in humans and mice. This immune response is temporally correlated with the loss of the eggshell, and we used immunohistochemistry to report that this reaction occurs at the materno-fetal interface. We demonstrate that key markers of implantation, including Heparin binding EGF-like growth factor and Mucin 1, exhibit expression and localization profiles consistent with the pattern observed during implantation in eutherian mammals. Finally, we show that there are transcriptome-wide similarities between the opossum attachment reaction and implantation in rabbits and humans. Our data suggest that the implantation reaction that occurs in eutherians is derived from an attachment reaction in the ancestral therian mammal which, in the opossum, leads directly to parturition. Finally, we argue that the ability to shift from an inflammatory attachment reaction to a noninflammatory period of pregnancy was a key innovation in eutherian mammals that allowed an extended period of intimate placentation.

Endometrial recognition of pregnancy occurs in the grey short-tailed opossum ( Monodelphis domestica).

In human pregnancy, recognition of an embryo within the uterus is essential to support the fetus through gestation. In most marsupials, such as the opossums, pregnancy is shorter than the oestrous cycle and the steroid hormone profile during pregnancy and oestrous cycle are indistinguishable. For these reasons, it was assumed that recognition of pregnancy, as a trait, evolved in the eutherian (placental) stem lineage and independently in wallabies and kangaroos. To investigate whether uterine recognition of pregnancy occurs in species with pregnancy shorter than the oestrous cycle, we examined reproduction in the short-tailed opossum ( Monodelphis domestica), a marsupial with a plesiomorphic mode of pregnancy. We examined the morphological and gene expression changes in the uterus of females in the non-pregnant oestrous cycle and compared these to pregnancy. We found that the presence of an embryo did not alter some aspects of uterine development but increased glandular activity. Transcriptionally, we saw big differences between the uterus of pregnant and cycling animals. These differences included an upregulation of genes involved in transport, inflammation and metabolic-activity in response to the presence of a fetus. Furthermore, transcriptional differences reflected protein level differences in transporter abundance. Our results suggest that while the uterus exhibits programmed changes after ovulation, its transcriptional landscape during pregnancy responds to the presence of a fetus and upregulates genes that may be essential for fetal support. These results are consistent with endometrial recognition of pregnancy occurring in the opossum. While the effects on maternal physiology appear to differ, recognition of pregnancy has now been observed in eutherian mammals, as well as, Australian and American marsupials.

Comparative genomics of hormonal signaling in the chorioallantoic membrane of oviparous and viviparous amniotes.

In oviparous amniotes (reptiles, birds, and mammals) the chorioallantoic membrane (CAM) lines the inside of the egg and acts as the living point of contact between the embryo and the outside world. In livebearing (viviparous) amniotes, communication during embryonic development occurs across placental tissues, which form between the uterine tissue of the mother and the CAM of the embryo. In both oviparous and viviparous taxa, the CAM is at the interface of the embryo and the external environment and can transfer signals from there to the embryo proper. To understand the evolution of placental hormone production in amniotes, we examined the expression of genes involved in hormone synthesis, metabolism, and hormone receptivity in the CAM of species across the amniote phylogeny. We collected transcriptome data for the chorioallantoic membranes of the chicken (oviparous), the lizards Lerista bougainvillii (both oviparous and viviparous populations) and Pseudemoia entrecasteauxii (viviparous), and the horse Equus caballus (viviparous). The viviparous taxa differ in their mechanisms of nutrient provisioning: L. bougainvillii is lecithotrophic (embryonic nourishment is provided via the yolk only), but P. entrecasteauxii and the horse are placentotrophic (embryos are nourished via placental transport). Of the 423 hormone-related genes that we examined, 91 genes are expressed in all studied species, suggesting that the chorioallantoic membrane ancestrally had an endocrine function. Therefore, the chorioallantoic membrane appears to be a highly hormonally active organ in all amniotes. No genes are expressed only in viviparous species, suggesting that the evolution of viviparity has not required the recruitment of any specific hormone-related genes. Our data suggest that the endocrine function of the CAM as a placental tissue evolved in part through co-option of ancestral gene expression patterns.

Seahorse Brood Pouch Transcriptome Reveals Common Genes Associated with Vertebrate Pregnancy.

Viviparity (live birth) has evolved more than 150 times in vertebrates, and represents an excellent model system for studying the evolution of complex traits. There are at least 23 independent origins of viviparity in fishes, with syngnathid fishes (seahorses and pipefish) unique in exhibiting male pregnancy. Male seahorses and pipefish have evolved specialized brooding pouches that provide protection, gas exchange, osmoregulation, and limited nutrient provisioning to developing embryos. Pouch structures differ widely across the Syngnathidae, offering an ideal opportunity to study the evolution of reproductive complexity. However, the physiological and genetic changes facilitating male pregnancy are largely unknown. We used transcriptome profiling to examine pouch gene expression at successive gestational stages in a syngnathid with the most complex brood pouch morphology, the seahorse Hippocampus abdominalis. Using a unique time-calibrated RNA-seq data set including brood pouch at key stages of embryonic development, we identified transcriptional changes associated with brood pouch remodeling, nutrient and waste transport, gas exchange, osmoregulation, and immunological protection of developing embryos at conception, development and parturition. Key seahorse transcripts share homology with genes of reproductive function in pregnant mammals, reptiles, and other live-bearing fish, suggesting a common toolkit of genes regulating pregnancy in divergent evolutionary lineages.