A high throughput CRISPR perturbation screen identifies epigenetic regulators impacting primordial germ cell development.

Certain environmental factors can impact fertility and reproductive parameters such as the number and quality of sperm and eggs. One possible mechanism is the perturbation of epigenetic landscapes in the germline. To explore this possibility, we conducted a CRISPRi screen of epigenetic-related genes to identify those that specifically perturb the differentiation of embryonic stem cells (ESCs) into primordial germ cell-like cells (PGCLCs), exploiting a highly scalable cytokine-free platform. Of the 701 genes screened, inhibition of 53 decreased the efficiency of PGCLC formation. NCOR2, a transcriptional repressor that acts via recruitment of Class I and Class IIa histone deacetylases (HDACs) to gene targets, was particularly potent in suppressing PGCLC differentiation. Consistent with evidence that histone deacetylation is crucial for germline differentiation, we found that the HDAC inhibitors (HDACi) valproic acid (VPA; an anti-convulsant) and sodium butyrate (SB; a widely-used dietary supplement) also suppressed ESC>PGCLC differentiation. Furthermore, exposure of developing mouse embryos to SB or VPA caused hypospermatogenesis. Transcriptome analyses of HDACi-treated, differentiating ESC>PGCLC cultures revealed suppression of germline-associated pathways and enhancement of somatic pathways. This work demonstrates the feasibility of conducting large-scale functional screens of genes, chemicals, or other agents that may impact germline development.

Scalable and Efficient Generation of Mouse Primordial Germ Cell-like Cells.

Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as high-throughput genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpressing 4 transcriptional factors - Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. The overexpression of Nanog enhances the PGC regulatory network and suppresses differentiation of somatic lineages, enabling a significant improvement in the efficiency of PGCLC production. Transcriptomic analysis reveals that differentiated PGCLCs exhibit similarities to in vivo PGCs and are more advanced compared to cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture and could differentiate into spermatogonia-like cells in vitro. Importantly, the ability to produce PGCLCs at scale, without using costly cytokines, enables biochemical and functional genomic screens to dissect mechanisms of germ cell development and infertility.

Gene regulation during meiosis.

Meiosis is essential for gamete production in all sexually reproducing organisms. It entails two successive cell divisions without DNA replication, producing haploid cells from diploid ones. This process involves complex morphological and molecular differentiation that varies across species and between sexes. Specialized genomic events like meiotic recombination and chromosome segregation are tightly regulated, including preparation for post-meiotic development. Research in model organisms, notably yeast, has shed light on the genetic and molecular aspects of meiosis and its regulation. Although mammalian meiosis research faces challenges, particularly in replicating gametogenesis in vitro, advances in genetic and genomic technologies are providing mechanistic insights. Here we review the genetics and molecular biology of meiotic gene expression control, focusing on mammals.

Integrative genome-scale analyses reveal post-transcriptional signatures of early human small intestinal development in a directed differentiation organoid model.

BACKGROUND: MicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. RESULTS: We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. CONCLUSION: The present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.

Practical strategies for robust and inexpensive imaging of aqueous-cleared tissues.

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting complicated and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardised 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes cause minimal spherical aberration with objective numerical apertures less than 0.65. Furthermore, we describe methods for aligning and assessing the light sheets, discriminating fluorescence from autofluorescence, identifying chromatic artefacts due to differential scattering and removing streak artefacts such that they do not confound downstream 3D object segmentation analyses, with mouse embryo, liver and heart imaging as demonstrated examples.

In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes.

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.

Female infertility from oocyte maturation arrest: assembling the genetic puzzle.

Assisted reproduction procedures often encounter an issue called oocyte maturation arrest (OMA), which is manifested as failed IVF/ICSI attempts using oocytes from some infertile women. In this issue of EMBO Molecular Medicine, Wang et al identify infertile women bearing novel DNA sequence variants in a gene called PABPC1L, which is essential for translation of maternal mRNAs. By conducting a series of in vitro and in vivo experiments, they demonstrated certain variants as being causal for OMA, confirming a conserved requirement for PABPC1L in human oocyte maturation. This study offers a promising therapeutic target for treating OMA patients.

Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome.

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most common chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA21 that directly contribute to cognitive deficits remain incompletely understood. Here, we found that the HSA21-encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued deficits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes.

Genome Maintenance in Mammalian Stem Cells.

Various stem cells in the body are tasked with maintaining tissue homeostasis throughout the life of an organism and thus must be resilient to intrinsic and extrinsic challenges such as infection and injury. Crucial to these challenges is genome maintenance because a high mutational load and persistent DNA lesions impact the production of essential gene products at proper levels and compromise optimal stem cell renewal and differentiation. Genome maintenance requires a robust and well-regulated DNA damage response suited to maintaining specific niches and tissues. In this review, we explore the similarities and differences between diverse stem cell types derived from (or preceding) all germ layers, including extraembryonic tissues. These cells utilize different strategies, including implementation of robust repair mechanisms, modulation of cell cycle checkpoints best suited to eliminating compromised cells, minimization of cell divisions, and differentiation in response to excessive damage.

Bone mass and adaptation to mechanical loading are sexually dimorphic in adult osteoblast-specific ERα knockout mice.

Estrogen receptor-alpha (ERα) regulates bone mass and is implicated in bone tissue's response to mechanical loading. The effects of ERα deletion in mice depend on sex, anatomical location, and the cellular stage at which ERα is removed. Few studies have investigated the effect of age on the role of ERα in skeletal maintenance and functional adaptation. We previously demonstrated that bone mass and adaptation to loading were altered in growing 10-week-old female and male mice lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO). Here our goal was to determine the effects of ERα and mechanical loading in skeletally-mature adult mice. We subjected 26-week-old skeletally-mature adult pOC-ERαKO and littermate control (LC) mice of both sexes to two weeks of in vivo cyclic tibial loading. ERα deletion in male mice did not alter bone mass or the response to loading. Adult female pOC-ERαKO mice had reduced cancellous and cortical bone mass and increased adaptation to high-magnitude mechanical loading compared to LC mice. Thus, ERα deletion from mature osteoblasts reduced the bone mass and increased the mechanoadaptation of adult female but not male mice. Additionally, compared to our previous work in young mice, adult female mice had greatly reduced mechanoadaptation and adult male mice retained most of their mechanoadaptation with age.

Genome maintenance during embryogenesis.

Genome maintenance during embryogenesis is critical, because defects during this period can be perpetuated and thus have a long-term impact on individual's health and longevity. Nevertheless, genome instability is normal during certain aspects of embryonic development, indicating that there is a balance between the exigencies of timely cell proliferation and mutation prevention. In particular, early embryos possess unique cellular and molecular features that underscore the challenge of having an appropriate balance. Here, we discuss genome instability during embryonic development, the mechanisms used in various cell compartments to manage genomic stress and address outstanding questions regarding the balance between genome maintenance mechanisms in key cell types that are important for adulthood and progeny.

Human MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging.

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

Strategies to Identify Genetic Variants Causing Infertility.

Genetic causes are thought to underlie about half of infertility cases, but understanding the genetic bases has been a major challenge. Modern genomics tools allow more sophisticated exploration of genetic causes of infertility through population, family-based, and individual studies. Nevertheless, potential therapies based on genetic diagnostics will be limited until there is certainty regarding the causality of genetic variants identified in an individual. Genome modulation and editing technologies have revolutionized our ability to functionally test such variants, and also provide a potential means for clinical correction of infertility variants. This review addresses strategies being used to identify causative variants of infertility.

Conditional surrender in one generation: determining the reproductive roles of mouse embryo lethal genes by embryo complementation.

The laboratory mouse is the most widely used animal model for studying the genetics and biology of mammalian development and reproduction. Embryonic stem cell (ESC) gene targeting technology, and the sophisticated genomic manipulations it allowed, was unique to this organism for a long period of time; this was a major factor in the mouse's rise to pre-eminence as a model system over the past three decades or so. The recent advent of CRISPR/Cas9 technology has democratized the application of genome editing to essentially all organisms. Nevertheless, the scientific infrastructure behind the mouse still makes it the organism of choice for studying molecular mechanisms of mammalian development, and for modeling human development and disease.

Sexually dimorphic DNA damage responses and mutation avoidance in the mouse germline.

Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared with somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon derepression. We also used whole-genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells (Fancm-/- ) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.

A novel function for CDK2 activity at meiotic crossover sites.

Genetic diversity in offspring is induced by meiotic recombination, which is initiated between homologs at >200 sites originating from meiotic double-strand breaks (DSBs). Of this initial pool, only 1-2 DSBs per homolog pair will be designated to form meiotic crossovers (COs), where reciprocal genetic exchange occurs between parental chromosomes. Cyclin-dependent kinase 2 (CDK2) is known to localize to so-called "late recombination nodules" (LRNs) marking incipient CO sites. However, the role of CDK2 kinase activity in the process of CO formation remains uncertain. Here, we describe the phenotype of 2 Cdk2 point mutants with elevated or decreased activity, respectively. Elevated CDK2 activity was associated with increased numbers of LRN-associated proteins, including CDK2 itself and the MutL homolog 1 (MLH1) component of the MutLγ complex, but did not lead to increased numbers of COs. In contrast, reduced CDK2 activity leads to the complete absence of CO formation during meiotic prophase I. Our data suggest an important role for CDK2 in regulating MLH1 focus numbers and that the activity of this kinase is a key regulatory factor in the formation of meiotic COs.

Variants in RABL2A causing male infertility and ciliopathy.

Approximately 7% of men worldwide suffer from infertility, with sperm abnormalities being the most common defect. Though genetic causes are thought to underlie a substantial fraction of idiopathic cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia and Bardet-Biedl syndrome, also suffer from infertility because cilia and sperm flagella share several characteristics. Here, we identified two deleterious alleles of RABL2A, a gene essential for normal function of cilia and flagella. Our in silico predictions and in vitro assays suggest that both alleles destabilize the protein. We constructed and analyzed mice homozygous for these two single-nucleotide polymorphisms, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), and found that they exhibit ciliopathy-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects and hydrocephalus. Our study provides a paradigm for triaging candidate infertility variants in the population for in vivo functional validation, using computational, in vitro and in vivo approaches.

Most Commonly Mutated Genes in High-Grade Serous Ovarian Carcinoma Are Nonessential for Ovarian Surface Epithelial Stem Cell Transformation.

High-grade serous ovarian carcinoma (HGSOC) is the fifth leading cause of cancer-related deaths of women in the United States. Disease-associated mutations have been identified by the Cancer Genome Atlas Research Network. However, aside from mutations in TP53 or the RB1 pathway that are common in HGSOC, the contributions of mutation combinations are unclear. Here, we report CRISPR mutagenesis of 20 putative HGSOC driver genes to identify combinatorial disruptions of genes that transform either ovarian surface epithelium stem cells (OSE-SCs) or non-stem cells (OSE-NSs). Our results support the OSE-SC theory of HGSOC initiation and suggest that most commonly mutated genes in HGSOC have no effect on OSE-SC transformation initiation. Our results indicate that disruption of TP53 and PTEN, combined with RB1 disruption, constitutes a core set of mutations driving efficient transformation in vitro. The combined data may contribute to more accurate modeling of HGSOC development.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 (WT1) gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene SRY is present in many cases, the etiology is unknown in most SRY-negative patients. We performed exome sequencing on 78 individuals with 46,XX TDSD/OTDSD of unknown genetic etiology and identified seven (8.97%) with heterozygous variants affecting the fourth zinc finger (ZF4) of Wilms' tumor 1 (WT1) (p.Ser478Thrfs*17, p.Pro481Leufs*15, p.Lys491Glu, p.Arg495Gln [x3], p.Arg495Gly). The variants were de novo in six families (P = 4.4 × 10-6), and the incidence of WT1 variants in 46,XX DSD is enriched compared to control populations (P < 1.8 × 10-4). The introduction of ZF4 mutants into a human granulosa cell line resulted in up-regulation of endogenous Sertoli cell transcripts and Wt1Arg495Gly/Arg495Gly XX mice display masculinization of the fetal gonads. The phenotype could be explained by the ability of the mutated proteins to physically interact with and sequester a key pro-ovary factor ß-CATENIN, which may lead to up-regulation of testis-specific pathway. Our data show that unlike previous association of WT1 and 46,XY DSD, ZF4 variants of WT1 are a relatively common cause of 46,XX TDSD/OTDSD. This expands the spectrum of phenotypes associated with WT1 variants and shows that the WT1 protein affecting ZF4 can function as a protestis factor in an XX chromosomal context.