Determining the potency of primordial germ cells by injection into early mouse embryos.

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution.

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.

A conserved transcriptional program for MAIT cells across mammalian evolution.

Mucosal-associated invariant T (MAIT) cells harbor evolutionarily conserved TCRs, suggesting important functions. As human and mouse MAIT functional programs appear distinct, the evolutionarily conserved MAIT functional features remain unidentified. Using species-specific tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in six species spanning 110 million years of evolution. Cross-species analyses revealed conserved transcriptional events underlying MAIT cell maturation, marked by ZBTB16 induction in all species. MAIT cells in human, sheep, cattle, and opossum acquired a shared type-1/17 transcriptional program, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating common differentiation for innate-like T cells. Distinct type-1 and type-17 MAIT subsets developed in rodents, including pet mice and genetically diverse mouse strains. However, MAIT cells further matured in mouse intestines to acquire a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity, and tissue-repair genes. Altogether, the study provides a unifying view of the transcriptional features of innate-like T cells across evolution.

Cross-species imputation and comparison of single-cell transcriptomic profiles.

Cross-species comparison and prediction of gene expression profiles are important to understand regulatory changes during evolution and to transfer knowledge learned from model organisms to humans. Single-cell RNA-seq (scRNA-seq) profiles enable us to capture gene expression profiles with respect to variations among individual cells; however, cross-species comparison of scRNA-seq profiles is challenging because of data sparsity, batch effects, and the lack of one-to-one cell matching across species. Moreover, single-cell profiles are challenging to obtain in certain biological contexts, limiting the scope of hypothesis generation. Here we developed Icebear, a neural network framework that decomposes single-cell measurements into factors representing cell identity, species, and batch factors. Icebear enables accurate prediction of single-cell gene expression profiles across species, thereby providing high-resolution cell type and disease profiles in under-characterized contexts. Icebear also facilitates direct cross-species comparison of single-cell expression profiles for conserved genes that are located on the X chromosome in eutherian mammals but on autosomes in chicken. This comparison, for the first time, revealed evolutionary and diverse adaptations of X-chromosome upregulation in mammals.

Functionalisation of conjugated macrocycles with type I and II concealed antiaromaticity via cross-coupling reactions.

Conjugated macrocycles can exhibit concealed antiaromaticity; that is, despite not being antiaromatic, under specific circumstances, they can display properties typically observed in antiaromatic molecules due to their formal macrocyclic 4n π-electron system. Paracyclophanetetraene (PCT) and its derivatives are prime examples of macrocycles exhibiting this behaviour. In redox reactions and upon photoexcitation, they have been shown to behave like antiaromatic molecules (requiring type I and II concealed antiaromaticity, respectively), with such phenomena showing potential for use in battery electrode materials and other electronic applications. However, further exploration of PCTs has been hindered by the lack of halogenated molecular building blocks that would permit their integration into larger conjugated molecules by cross-coupling reactions. Here, we present two dibrominated PCTs, obtained as a mixture of regioisomers from a three-step synthesis, and demonstrate their functionalisation via Suzuki cross-coupling reactions. Optical, electrochemical, and theoretical studies reveal that aryl substituents can subtly tune the properties and behaviour of PCT, showing that this is a viable strategy in further exploring this promising class of materials.

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.

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.

CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes.

Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.

Generating CRISPR-Cas9-Mediated Null Mutations and Screening Targeting Efficiency in Human Pluripotent Stem Cells.

CRISPR-Cas9 mutagenesis facilitates the investigation of gene function in a number of developmental and cellular contexts. Human pluripotent stem cells (hPSCs), either embryonic or induced, are a tractable cellular model to investigate molecular mechanisms involved in early human development and cell fate decisions. hPSCs also have broad potential in regenerative medicine to model, investigate, and ameliorate diseases. Here, we provide an optimized protocol for efficient CRISPR-Cas9 genome editing of hPSCs to investigate the functional role of genes by engineering null mutations. We emphasize the importance of screening single guide RNAs (sgRNAs) to identify those with high targeting efficiency for generation of clonally derived null mutant hPSC lines. We provide important considerations for targeting genes that may have a role in hPSC maintenance. We also present methods to evaluate the on-target mutation spectrum and unintended karyotypic changes. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Selecting and ligating sgRNAs into expression plasmids Basic Protocol 2: Validation of sgRNA via in vitro transcription and cleavage assay Basic Protocol 3: Nucleofection of primed human embryonic stem cells Basic Protocol 4: MiSeq analysis of indel mutations Basic Protocol 5: Single cell cloning of targeted hPSCs Basic Protocol 6: Karyotyping of targeted hPSCs.

Artificial refuges for wildlife conservation: what is the state of the science?

Artificial refuges are human-made structures that aim to create safe places for animals to breed, hibernate, or take shelter in lieu of natural refuges. Artificial refuges are used across the globe to mitigate the impacts of a variety of threats on wildlife, such as habitat loss and degradation. However, there is little understanding of the science underpinning artificial refuges, and what comprises best practice for artificial refuge design and implementation for wildlife conservation. We address this gap by undertaking a systematic review of the current state of artificial refuge research for the conservation of wildlife. We identified 224 studies of artificial refuges being implemented in the field to conserve wildlife species. The current literature on artificial refuges is dominated by studies of arboreal species, primarily birds and bats. Threatening processes addressed by artificial refuges were biological resource use (26%), invasive or problematic species (20%), and agriculture (15%), yet few studies examined artificial refuges specifically for threatened (Vulnerable, Endangered, or Critically Endangered) species (7%). Studies often reported the characteristics of artificial refuges (i.e. refuge size, construction materials; 87%) and surrounding vegetation (35%), but fewer studies measured the thermal properties of artificial refuges (18%), predator activity (17%), or food availability (3%). Almost all studies measured occupancy of the artificial refuges by target species (98%), and over half measured breeding activity (54%), whereas fewer included more detailed measures of fitness, such as breeding productivity (34%) or animal body condition (4%). Evaluating the benefits and impacts of artificial refuges requires sound experimental design, but only 39% of studies compared artificial refuges to experimental controls, and only 10% of studies used a before-after-control-impact (BACI) design. As a consequence, few studies of artificial refuges can determine their overall effect on individuals or populations. We outline a series of key steps in the design, implementation, and monitoring of artificial refuges that are required to avoid perverse outcomes and maximise the chances of achieving conservation objectives. This review highlights a clear need for increased rigour in studies of artificial refuges if they are to play an important role in wildlife conservation.

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.

Epigenetics drive the evolution of sex chromosomes in animals and plants.

We review how epigenetics affect sex chromosome evolution in animals and plants. In a few species, sex is determined epigenetically through the action of Y-encoded small RNAs. Epigenetics is also responsible for changing the sex of individuals through time, even in species that carry sex chromosomes, and could favour species adaptation through breeding system plasticity. The Y chromosome accumulates repeats that become epigenetically silenced which leads to an epigenetic conflict with the expression of Y genes and could accelerate Y degeneration. Y heterochromatin can be lost through ageing, which activates transposable elements and lowers male longevity. Y chromosome degeneration has led to the evolution of meiotic sex chromosome inactivation in eutherians (placentals) and marsupials, and dosage compensation mechanisms in animals and plants. X-inactivation convergently evolved in eutherians and marsupials via two independently evolved non-coding RNAs. In Drosophila, male X upregulation by the male specific lethal (MSL) complex can spread to neo-X chromosomes through the transposition of transposable elements that carry an MSL-binding motif. We discuss similarities and possible differences between plants and animals and suggest future directions for this dynamic field of research. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

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.

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.

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.

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.

The BCL-2 pathway preserves mammalian genome integrity by eliminating recombination-defective oocytes.

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.

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.