Human Embryogenesis: A Comparative Perspective.

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.

Enhancer evolution across 20 mammalian species.

The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.

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

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

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TAF4b transcription networks regulating early oocyte differentiation.

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.

Gene expression across mammalian organ development.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

Extinction risk of the world's freshwater mammals.

The continued loss of freshwater habitats poses a significant threat to global biodiversity. We reviewed the extinction risk of 166 freshwater aquatic and semiaquatic mammals-a group rarely documented as a collective. We used the International Union for the Conservation of Nature Red List of Threatened Species categories as of December 2021 to determine extinction risk. Extinction risk was then compared among taxonomic groups, geographic areas, and biological traits. Thirty percent of all freshwater mammals were listed as threatened. Decreasing population trends were common (44.0%), including a greater rate of decline (3.6% in 20 years) than for mammals or freshwater species as a whole. Aquatic freshwater mammals were at a greater risk of extinction than semiaquatic freshwater mammals (95% CI -7.20 to -1.11). Twenty-nine species were data deficient or not evaluated. Large species (95% CI 0.01 to 0.03) with large dispersal distances (95% CI 0.03 to 0.15) had a higher risk of extinction than small species with small dispersal distances. The number of threatening processes associated with a species compounded their risk of extinction (95% CI 0.28 to 0.77). Hunting, land clearing for logging and agriculture, pollution, residential development, and habitat modification or destruction from dams and water management posed the greatest threats to these species. The basic life-history traits of many species were poorly known, highlighting the need for more research. Conservation of freshwater mammals requires a host of management actions centered around increased protection of riparian areas and more conscientious water management to aid the recovery of threatened species.

Riesgo de extinción de los mamíferos de agua dulce Resumen La pérdida continua de hábitats de agua dulce representa una amenaza importante para la biodiversidad mundial. Analizamos el riesgo de extinción de 166 especies de mamíferos acuáticos y semiacuáticos de agua dulce-un grupo que se documenta pocas veces como colectivo. Usamos las categorías de la Lista Roja de Especies Amenazadas de la Unión Internacional para la Conservación de la Naturaleza de diciembre 2021 para determinar el riesgo de extinción. Después comparamos este riesgo entre grupos taxonómicos, áreas geográficas y caracteres biológicos. El 30% de los mamíferos de agua dulce están categorizados como amenazados. La declinación de las tendencias poblacionales fue común (44.0%), incluyendo una mayor tasa de declinación (3.6% en 20 años) que para los mamíferos o las especies de agua dulce como conjunto. Los mamíferos acuáticos de agua dulce se encuentran en mayor riesgo de extinción que los mamíferos semiacuáticos (95% IC -7.20 a -1.11). Veintinueve especies no contaban con suficientes datos o no estaban evaluadas. Las especies grandes (95% IC 0.01 a 0.03) con distancias de dispersión amplias (95% IC 0.03 a 0.15) tuvieron un mayor riesgo de extinción que las especies pequeñas con menores distancias de dispersión. El número de procesos amenazantes asociados a alguna especie agravó su riesgo de extinción (95% CI 0.28 a 0.77). Las principales amenazas para estas especies fueron la cacería, el desmonte de tierras para tala y agricultura, la contaminación, los desarrollos residenciales y la destrucción o modificación del hábitat causados por presas o manejo hidrológico. Se sabe poco sobre los caracteres básicos de la historia de vida de muchas especies, lo que destaca la necesidad de más investigación al respecto. La conservación de mamíferos de agua dulce requiere una serie de acciones gestoras centradas en el incremento de la protección de las áreas ribereñas y una gestión hidrológica más consciente para ayudar a la recuperación de las especies amenazadas.

Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.

CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

Meiotic Silencing in Mammals.

Meiosis is essential for reproduction in sexually reproducing organisms. A key stage in meiosis is the synapsis of maternal and paternal homologous chromosomes, accompanied by exchange of genetic material to generate crossovers. A decade ago, studies found that when chromosomes fail to synapse, the many hundreds of genes housed within them are transcriptionally inactivated. This process, meiotic silencing, is conserved in all mammals studied to date, but its purpose is not yet defined. Here, I review the molecular genetics of meiotic silencing and consider the many potential functions that it could serve in the mammalian germ line. In addition, I discuss how meiotic silencing influences sex differences in meiotic infertility and the profound impact that meiotic silencing has had on the evolution of mammalian sex chromosomes.

Erratum: Genome editing reveals a role for OCT4 in human embryogenesis.

This corrects the article DOI: 10.1038/nature24033.

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Advances and challenges in genetic technologies to produce single-sex litters.

There is currently a requirement for single-sex litters for many applications, including agriculture, pest control, and reducing animal culling in line with the 3Rs principles: Reduction, Replacement, and Refinement. The advent of CRISPR/Cas9 genome editing presents a new opportunity with which to potentially generate all-female or all-male litters. We review some of the historical nongenetic strategies employed to generate single-sex litters and investigate how genetic and genome editing techniques are currently being used to produce all-male or all-female progeny. Lastly, we speculate on future technologies for generating single-sex litters and the possible associated challenges.

Disparate roost sites drive intraspecific physiological variation in a Malagasy bat.

Many species are widely distributed and individual populations can experience vastly different environmental conditions over seasonal and geographic scales. With such a broad ecological reality, datasets with limited spatial and temporal resolution may not accurately represent a species and could lead to poorly informed management decisions. Because physiological flexibility can help species tolerate environmental variation, we studied the physiological responses of two separate populations of Macronycteris commersoni, a bat widespread across Madagascar, in contrasting seasons. The populations roost under the following dissimilar conditions: either a hot, well-buffered cave or within open foliage, unprotected from the local weather. We found that flexible torpor patterns, used in response to prevailing ambient temperature and relative humidity, were central to keeping energy budgets balanced in both populations. While bats' metabolic rate during torpor and rest did not differ between roosts, adjusting torpor frequency, duration and timing helped bats maintain body condition. Interestingly, the exposed forest roost induced extensive use of torpor, which exceeded the torpor frequency of overwintering bats that stayed in the cave for months and consequently minimised daytime resting energy expenditure in the forest. Our current understanding of intraspecific physiological variation is limited and physiological traits are often considered to be fixed. The results of our study therefore highlight the need for examining species at broad environmental scales to avoid underestimating a species' full capacity for withstanding environmental variation, especially in the face of ongoing, disruptive human interference in natural habitats.

ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing.

In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.

DNA damage induces a kinetochore-based ATM/ATR-independent SAC arrest unique to the first meiotic division in mouse oocytes.

Mouse oocytes carrying DNA damage arrest in meiosis I, thereby preventing creation of embryos with deleterious mutations. The arrest is dependent on activation of the spindle assembly checkpoint, which results in anaphase-promoting complex (APC) inhibition. However, little is understood about how this checkpoint is engaged following DNA damage. Here, we find that within minutes of DNA damage checkpoint proteins are assembled at the kinetochore, not at damage sites along chromosome arms, such that the APC is fully inhibited within 30 min. Despite this robust response, there is no measurable loss in k-fibres, or tension across the bivalent. Through pharmacological inhibition we observed that the response is dependent on Mps1 kinase, aurora kinase and Haspin. Using oocyte-specific knockouts we find the response does not require the DNA damage response kinases ATM or ATR. Furthermore, checkpoint activation does not occur in response to DNA damage in fully mature eggs during meiosis II, despite the divisions being separated by just a few hours. Therefore, mouse oocytes have a unique ability to sense DNA damage rapidly by activating the checkpoint at their kinetochores.

Facultative hyperthermia during a heatwave delays injurious dehydration of an arboreal marsupial.

Heatwaves negatively impact wildlife populations and their effects are predicted to worsen with ongoing global warming. Animal mass mortality at extremely high ambient temperature (Ta) is evidence for physiological dysfunction and, to aid conservation efforts, improving our understanding of animal responses to environmental heat is crucial. To address this, I measured the water loss, body temperature and metabolism of an Australian marsupial during a simulated heatwave. The body temperature of the common ringtail possum Pseudocheirus peregrinus increased passively by ∼3°C over a Ta of 29-39°C, conveying water savings of 9.6 ml h-1 When Ta crossed a threshold of 35-36°C, possums began actively cooling by increasing evaporative water loss and thermal conductance. It is clear that facultative hyperthermia is effective up to a point, but once this point is surpassed - the frequency and duration of which are increasing with climate change - body water would rapidly deplete, placing possums in danger of injury or death from dehydration.

Mammalian X Chromosome Dosage Compensation: Perspectives From the Germ Line.

Sex chromosomes are advantageous to mammals, allowing them to adopt a genetic rather than environmental sex determination system. However, sex chromosome evolution also carries a burden, because it results in an imbalance in gene dosage between females (XX) and males (XY). This imbalance is resolved by X dosage compensation, which comprises both X chromosome inactivation and X chromosome upregulation. X dosage compensation has been well characterized in the soma, but not in the germ line. Germ cells face a special challenge, because genome wide reprogramming erases epigenetic marks responsible for maintaining the X dosage compensated state. Here we explain how evolution has influenced the gene content and germ line specialization of the mammalian sex chromosomes. We discuss new research uncovering unusual X dosage compensation states in germ cells, which we postulate influence sexual dimorphisms in germ line development and cause infertility in individuals with sex chromosome aneuploidy.

DNA damage response protein TOPBP1 regulates X chromosome silencing in the mammalian germ line.

Meiotic synapsis and recombination between homologs permits the formation of cross-overs that are essential for generating chromosomally balanced sperm and eggs. In mammals, surveillance mechanisms eliminate meiotic cells with defective synapsis, thereby minimizing transmission of aneuploidy. One such surveillance mechanism is meiotic silencing, the inactivation of genes located on asynapsed chromosomes, via ATR-dependent serine-139 phosphorylation of histone H2AFX (γH2AFX). Stimulation of ATR activity requires direct interaction with an ATR activation domain (AAD)-containing partner. However, which partner facilitates the meiotic silencing properties of ATR is unknown. Focusing on the best-characterized example of meiotic silencing, meiotic sex chromosome inactivation, we reveal this AAD-containing partner to be the DNA damage and checkpoint protein TOPBP1. Conditional TOPBP1 deletion during pachynema causes germ cell elimination associated with defective X chromosome gene silencing and sex chromosome condensation. TOPBP1 is essential for localization to the X chromosome of silencing "sensors," including BRCA1, and effectors, including ATR, γH2AFX, and canonical repressive histone marks. We present evidence that persistent DNA double-strand breaks act as silencing initiation sites. Our study identifies TOPBP1 as a critical factor in meiotic sex chromosome silencing.

Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.