Transcription factor-based transdifferentiation of human embryonic to trophoblast stem cells.

During the first week of development, human embryos form a blastocyst composed of an inner cell mass and trophectoderm (TE) cells, the latter of which are progenitors of placental trophoblast. Here, we investigated the expression of transcripts in the human TE from early to late blastocyst stages. We identified enrichment of the transcription factors GATA2, GATA3, TFAP2C and KLF5 and characterised their protein expression dynamics across TE development. By inducible overexpression and mRNA transfection, we determined that these factors, together with MYC, are sufficient to establish induced trophoblast stem cells (iTSCs) from primed human embryonic stem cells. These iTSCs self-renew and recapitulate morphological characteristics, gene expression profiles, and directed differentiation potential, similar to existing human TSCs. Systematic omission of each, or combinations of factors, revealed the crucial importance of GATA2 and GATA3 for iTSC transdifferentiation. Altogether, these findings provide insights into the transcription factor network that may be operational in the human TE and broaden the methods for establishing cellular models of early human placental progenitor cells, which may be useful in the future to model placental-associated diseases.

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.

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.

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.

Rsx is a metatherian RNA with Xist-like properties in X-chromosome inactivation.

In female (XX) mammals, one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY). X-chromosome inactivation in eutherian mammals is mediated by the non-coding RNA Xist. Xist is not found in metatherians (marsupials), and how X-chromosome inactivation is initiated in these mammals has been the subject of speculation for decades. Using the marsupial Monodelphis domestica, here we identify Rsx (RNA-on-the-silent X), an RNA that has properties consistent with a role in X-chromosome inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, in which both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-chromosome inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-chromosome inactivation in mammals and pose questions about the mechanisms by which X-chromosome inactivation is achieved in eutherians.

Developmental origins of mammalian spermatogonial stem cells: New perspectives on epigenetic regulation and sex chromosome function.

Male and female germ cells undergo genome-wide reprogramming during their development, and execute sex-specific programs to complete meiosis and successfully generate healthy gametes. While sexually dimorphic germ cell development is fundamental, similarities and differences exist in the basic processes governing normal gametogenesis. At the simplest level, male gamete generation in mammals is centred on the activity of spermatogonial stem cells (SSCs), and an equivalent cell state is not present in females. Maintaining this unique SSC epigenetic state, while keeping to germ cell-intrinsic developmental programs, poses challenges for the correct completion of spermatogenesis. In this review, we highlight the origins of spermatogonia, comparing and contrasting them with female germline development to emphasize specific developmental processes that are required for their function as germline stem cells. We identify gaps in our current knowledge about human SSCs and further discuss the impact of the unique regulation of the sex chromosomes during spermatogenesis, and the roles of X-linked genes in SSCs.

Operative laparoscopy as the mainstay method in management of hemodynamically unstable patients with ectopic pregnancy.

STUDY OBJECTIVE: To determine the safety and sustainability of operative laparoscopy in hemodynamically unstable women with ectopic pregnancy according to the effect of operator experience on success rates, whether the volume of hemoperitoneum affects the operative method used, and requirements for admission to the intensive care unit (ICU) and administration of blood transfusion. DESIGN: Prospective cohort study (Canadian Task Force classification II-A). SETTING: University hospital. PATIENTS: Between January 2003 and February 2010, 505 women with ectopic pregnancy (55 tubal, 4 ovarian, 7 cornual, and 1 in the cesarean scar) were seen, including 124 women with hemoperitoneum greater than 500 mL, of whom 67 were hemodynamically unstable. INTERVENTIONS: Operative laparoscopy. MEASUREMENTS AND MAIN RESULTS: The greater the volume of hemoperitoneum, the greater the likelihood of hemodynamic instability. The odds of hemodynamic instability were greater in nontubal ectopic pregnancies. The overall operative laparoscopy rate in hemodynamically unstable patients was 85%, compared with 95% in hemodynamically stable women. The volume of hemoperitoneum did not affect the operative method used. Experienced operators had a 100% success rate at operative laparoscopy in hemodynamically unstable women, compared with a 72% success rate with confident operators. A small number of women required admission to the ICU. Although the laparoscopy group required more blood transfusions, they had a shorter length of hospital stay compared with the laparotomy group. CONCLUSION: Operative laparoscopy is safe and sustainable in most women with hemodynamic instability. Women who undergo operative laparoscopy do no worse than those who undergo laparotomy, and even those who require ICU admission still benefit from the advantages of operative laparoscopy.

Isolation of spermatogenic cells from the cynomolgus macaque testis with flow cytometry.

Here, we describe a detailed protocol for the isolation of purified populations of viable spermatogenic cells derived from the non-human primate model organism Macaca fascicularis (cynomolgus). Using fluorescence-activated cell sorting (FACS), we describe methods to isolate spermatogonia and primary spermatocytes ranging across the sub-stages of meiosis prophase I. These cell populations can be used with a variety of downstream assays, including single-cell approaches such as RNA sequencing, chromatin immunoprecipitation, quantitative RT-PCR, and immunocytochemistry. For complete details on the use and execution of this protocol, please refer to Lau et al. (2020).

Single-Cell RNA Sequencing of the Cynomolgus Macaque Testis Reveals Conserved Transcriptional Profiles during Mammalian Spermatogenesis.

Spermatogenesis is highly orchestrated and involves the differentiation of diploid spermatogonia into haploid sperm. The process is driven by spermatogonial stem cells (SSCs). SSCs undergo mitotic self-renewal, whereas sub-populations undergo differentiation and later gain competence to initiate meiosis. Here, we describe a high-resolution single-cell RNA-seq atlas of cells derived from Cynomolgus macaque testis. We identify gene signatures that define spermatogonial populations and explore self-renewal versus differentiation dynamics. We detail transcriptional changes occurring over the entire process of spermatogenesis and highlight the concerted activity of DNA damage response (DDR) pathway genes, which have dual roles in maintaining genomic integrity and effecting meiotic sex chromosome inactivation (MSCI). We show remarkable similarities and differences in gene expression during spermatogenesis with two other eutherian mammals, i.e., mouse and humans. Sex chromosome expression in the male germline in all three species demonstrates conserved features of MSCI but divergent multicopy and ampliconic gene content.

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.

SETDB1 Links the Meiotic DNA Damage Response to Sex Chromosome Silencing in Mice.

Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing.

Non-Canonical and Sexually Dimorphic X Dosage Compensation States in the Mouse and Human Germline.

Somatic X dosage compensation requires two mechanisms: X inactivation balances X gene output between males (XY) and females (XX), while X upregulation, hypothesized by Ohno and documented in vivo, balances X gene with autosomal gene output. Whether X dosage compensation occurs in germ cells is unclear. We show that mouse and human germ cells exhibit non-canonical X dosage states that differ from the soma and between the sexes. Prior to genome-wide reprogramming, X upregulation is present, consistent with Ohno's hypothesis. Subsequently, however, it is erased. In females, erasure follows loss of X inactivation, causing X dosage excess. Conversely, in males, erasure leads to permanent X dosage decompensation. Sex chromosomally abnormal models exhibit a "sex-reversed" X dosage state: XX males, like XX females, develop X dosage excess, while XO females, like XY males, develop X dosage decompensation. Thus, germline X dosage compensation states are determined by X chromosome number, not phenotypic sex. These unexpected differences in X dosage compensation states between germline and soma offer unique perspectives on sex chromosome infertility.

Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome.

How the replication machinery is loaded at origins of DNA replication is poorly understood. Here, we implicate in this process the Xenopus laevis homolog (xRTS) of the RECQL4 helicase mutated in Rothmund-Thomson syndrome. xRTS, which bears homology to the yeast replication factors Sld2/DRC1, is essential for DNA replication in egg extracts. xRTS can be replaced in extracts by its human homolog, while RECQL4 depletion from mammalian cells induces proliferation failure, suggesting an evolutionarily conserved function. xRTS accumulates on chromatin during replication initiation, after prereplication-complex (pre-RC) proteins, Cut5, Sld5, or Cdc45 but before replicative polymerases. xRTS depletion suppresses the loading of RPA, the ssDNA binding protein that marks unwound origins before polymerase recruitment. However, xRTS is unaffected by xRPA depletion. Thus, xRTS functions after pre-RC formation to promote loading of replication factors at origins, a previously unrecognized activity necessary for initiation. This role connects defective replication initiation to a chromosome-fragility disorder.

Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein.

How dividing mammalian cells overcome blocks to DNA replication by DNA damage, depleted nucleotide pools, or template-bound proteins is unclear. Here, we show that the response to blocked replication requires BRCA2, a suppressor of human breast cancer. By using two-dimensional gel electrophoresis, we demonstrate that Y-shaped DNA junctions at stalled replication forks disappear during genome-wide replication arrest in BRCA2-deficient cells, accompanied by double-strand DNA breakage. But activation of the replication checkpoint kinase Chk2 is unaffected, defining an unexpected function for BRCA2 in stabilizing DNA structures at stalled forks. We propose that in BRCA2 deficiency and related chromosomal instability diseases, the breakdown of replication forks, which arrest or pause during normal cell growth, triggers spontaneous DNA breakage, leading to mutability and cancer predisposition.