Mouse Germline Research Articles

Author Correction: Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

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

Mammalian germ cells are determined after PGC colonization of the nascent gonad

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer.

A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling.

The response to environmental stresses by eukaryotic organisms includes activation of protective biological mechanisms, orchestrated in part by transcriptional regulators. The tri-member Hypoxia Inducible Factor (HIF) family of DNA-binding transcription factors include HIF-2, which is activated under conditions of oxygen or glucose deprivation. Although oxygen-dependent protein degradation is a key mechanism by which HIF-1 and HIF-2 activity is regulated, HIF-2 is also influenced substantially by the coupled action of acetylation and deacetylation. The acetylation/deacetylation process that HIF-2 undergoes employs a specific acetyltransferase and deacetylase. Likewise, the supply of the acetyl donor, acetyl CoA, used for HIF-2 acetylation originates from a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2). Although Acss2 is predominantly cytosolic, a subset of the Acss2 cellular pool is enriched in the nucleus following oxygen or glucose deprivation. Prevention of nuclear localization by a directed mutation in a putative nuclear localization signal in Acss2 abrogates HIF-2 acetylation and blunts HIF-2 dependent signaling as well as flank tumor growth for knockdown/rescue cancer cells expressing ectopic Acss2. In this study, we report generation of a novel mouse strain using CRISPR/Cas9 mutagenesis that express this mutant Acss2 allele in the mouse germline. The homozygous mutant mice have impaired induction of the canonical HIF-2 target gene erythropoietin and blunted recovery from acute anemia. Surprisingly, Acss2 protein levels are dramatically reduced in these mutant mice. Functional studies investigating the basis for this phenotype reveal multiple protein instability domains in the Acss2 carboxy terminus. The findings described herein may be of relevance in the regulation of native Acss2 protein as well as for humans carrying missense mutations in these domains.

Mutation Induction in Humans and Mice: Where Are We Now?

The analysis of mutation induction in human families exposed to mutagens provides the only source of reliable estimates of factors contributing to the genetic risk of human exposure to mutagens. In this paper, I briefly summarize the results of recent studies on the pattern of mutation induction in the human and mouse germline. The results of recent studies on the genome-wide effects of exposure to mutagens on mutation induction in the mammalian germline are presented and discussed. Lastly, this review also addresses the issue of transgenerational effects of parental exposure to mutagens on mutation rates in their non-exposed offspring, which are known as transgenerational instability. The possible contribution of transgenerational instability to the genetic risk of human exposure to mutagens is discussed.

The Complex Association of FcγRIIb With Autoimmune Susceptibility.

FcγRIIb is the only inhibitory Fc receptor and controls many aspects of immune and inflammatory responses. The observation 19 years ago that FcγRIIb−/− mice generated by gene targeting in 129 derived ES cells developed severe lupus like disease when backcrossed more than 7 generations into C57BL/6 background initiated extensive research on the functional understanding of this strong autoimmune phenotype. The genomic region in the distal part of Chr1 both in human and mice in which the FcγR gene cluster is located shows a high level of complexity in relation to the susceptibility to SLE. Specific haplotypes of closely linked genes including the FcγRIIb and Slamf genes are associated with increased susceptibility to SLE both in mice and human. Using forward and reverse genetic approaches including in human GWAS and in mice congenic strains, KO mice (germline and cell type specific, on different genetic background), knockin mice, overexpressing transgenic mice combined with immunological models such as adoptive transfer of B cells from Ig transgenic mice the involved genes and the causal mutations and their associated functional alterations were analyzed. In this review the results of this 19 years extensive research are discussed with a focus on (genetically modified) mouse models.

Germline Selection by Meiosis Defends the Transmission of Defective Mitochondria DNA

Germline selection of mtDNA is of fundamental importance in maternal inheritance of mitochondrial DNA (mtDNA), as the selection eliminates sever mtDNA mutations. However, evidence for germline selection by meiosis comes from incomplete sequencing of mtDNA in human first polar body (PB1). Here, we found various variants, including pathogenic mutation sites, present on whole genome of mtDNA in human PB1 compared with its oocyte. Meanwhile, we found mitochondria in PB1 were defective, suggesting mtDNA mutations lead to defective mitochondria in PB1. The defective mitochondria transfer in mouse germline, including cumulus-oocyte-complexes at germinal vesicle and oocytes at meiosis II stage, showed that active purifying selection of the defective mitochondria into PB1 and PB2 occurred during the first and second meiosis. Thus, twice meiosis is the last defense system for purifying selection of mtDNA mutations during oogenesis. PB1 and PB2 would be final destination of deleterious mtDNA mutations in germline selection.

Locating and Characterizing a Transgene Integration Site by Nanopore Sequencing.

The introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnn (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

The GRB2-associated binding protein 3 is required for IL-2 and IL-15 induced NK cell expansion and limits trophoblast invasion during pregnancy

Abstract The Grb2-associated binding protein 3 (Gab3) is a scaffolding protein that belongs to the Grb2-assocated binding family of proteins along with Gab1 and Gab2. Gab3 is expressed exclusively in immune cells including NK cells, CD8+T cells, mast cells and macrophages, however the function of Gab3 has remained elusive. Based on two independent mouse models; a hypomorph germline mouse model carrying a missense mutation in the PH domain of Gab3 and complete Gab3-KO mice, we uncovered a key role for Gab3 in peripheral NK cell function. Specifically, loss of Gab3 results in impaired IL-2/IL-15-induced NK cell priming and expansion, due to a selective impairment in MAPK- but not STAT5-signaling. In vivo, we show that Gab3 is required for recognition and elimination of “missing-self” and tumor targets. Gab3-deficient mice exhibit impaired uterine NK cell expansion that is associated with abnormal spiral artery remodeling and increased trophoblast invasion in the decidua basalis. This coincides with stillbirth, retained placenta, maternal hemorrhage and undelivered fetoplacental units at term. Thus, our studies identify Gab3 as a novel component required for IL-2/IL-15-mediated NK cell priming and expansion that is essential for anti-tumor responses and for controlling fetal trophoblast invasion during pregnancy.

Genome-wide identification and characterization of long noncoding and circular RNAs in germline stem cells

Germline stem cells are germ cells at an early developmental stage, so their development is key to ensuring human reproduction. There is increasing evidence that long noncoding RNA (lncRNA) and circular RNA (circRNA) play important roles in the development of germ cells. This data descriptor provides unique lncRNA and circRNA transcriptomic information for mouse germline stem cells. Using the Illumina HiSeqx 2000 system, a total of 511,836,732 raw reads were generated. High-quality transcripts, lncRNAs, and circRNAs were identificated and quantified using the reads, and more precise annotations of lncRNAs (especially 9357 novel lncRNAs) and circRNAs were performed in the germline stem cells. We then analyzed the transcript structures, genetic variants, and the interaction between circRNA and microRNA to provide the basis for subsequent functional experiments. This comprehensive dataset will help advance data sharing and deepen our understanding of mouse germline stem cells, providing a theoretical foundation for research on germ cell development and human reproduction, among others.

Spontaneous de novo germline mutations in humans and mice: rates, spectra, causes and consequences.

Germline mutations are the origin of genetic variation and are widely considered to be the driving force of genome evolution. The rates and spectra of de novo mutations (DNMs) directly affect evolutionary speed and direction and thereby establish species-specific genomic futures in the long term. This has resulted in a keen interest in understanding the origin of germline mutations in mammals. Accumulating evidence from next-generation sequencing and family-based analysis indicates that the frequency of human DNMs varies according to sex, age and local genomic context. Thus, it is likely that there are multiple causes and drivers of mutagenesis, including spontaneous DNA lesions, DNA repair status and DNA polymerase errors. In this review, recent studies of human and mouse germline DNMs are discussed, and the rates and spectra of spontaneous germline DNMs in the mouse mutator lines Pold1exo/exo and TOY-KO (Mth1-/-/Ogg1-/-/Mutyh-/- triple knockout) are summarized in the context of endogenous causes and mechanisms.

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

A gene drive biases the transmission of one of two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems were recently developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity1–4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (e.g., to model multigenic human diseases). However, such a system has not yet been demonstrated in mammals. Here, we utilize an “active genetic” element that encodes a guide RNA embedded in the mouse Tyrosinase gene to evaluate whether targeted gene conversion can occur when CRISPR/Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and male germline, these breaks are not resolved by homology directed repair. However, Cas9 expression limited to the female germline forms double strand breaks that are resolved by homology directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate feasibility of CRISPR/Cas9-mediated systems that bias inheritance in mice, which have potential to transform the use of rodent models in basic and biomedical research.

Genetic basis for primordial germ cells specification in mouse and human: Conserved and divergent roles of PRDM and SOX transcription factors.

Primordial germ cells (PGCs) are embryonic precursors of sperm and egg that pass on genetic and epigenetic information from one generation to the next. In mammals, they are induced from a subset of cells in peri-implantation epiblast by BMP signaling from the surrounding tissues. PGCs then initiate a unique developmental program that involves comprehensive epigenetic resetting and repression of somatic genes. This is orchestrated by a set of signaling molecules and transcription factors that promote germ cell identity. Here we review significant findings on mammalian PGC biology, in particular, the genetic basis for PGC specification in mice and human, which has revealed an evolutionary divergence between the two species. We discuss the importance and potential basis for these differences and focus on several examples to illustrate the conserved and divergent roles of critical transcription factors in mouse and human germline.

A conserved enhancer regulates Il9 expression in multiple lineages

Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.

Partial loss of psychiatric risk gene Mir137 in mice causes repetitive behavior and impairs sociability and learning via increased Pde10a.

Genetic analyses have linked MicroRNA-137 (MIR137) to neuropsychiatric disorders, including schizophrenia and autism spectrum disorder (ASD). MiR-137 plays important roles in neurogenesis and neuronal maturation, but the impact of miR-137 loss of function in vivo remains unclear. Here we show the complete loss of miR-137 in the mouse germline (gKO) or nervous system (cKO) leads to postnatal lethality, while heterozygous gKO and cKO mice remain viable. Partial loss of miR-137 in heterozygous cKO mice results in dysregulated synaptic plasticity, repetitive behavior, and impaired learning and social behavior. Transcriptomic and proteomic analyses revealed that the miR-137 mRNA target, Phosphodiesterase 10a (Pde10a), is elevated in heterozygous KO mice. Treatment with the PDE10A inhibitor papaverine or knockdown of Pde10a ameliorates the deficits observed in the heterozygous cKO mice. Collectively, our results suggest that MIR137 plays essential roles in postnatal neurodevelopment, and that dysregulation of miR-137 potentially contributes to neuropsychiatric disorders in humans.

Multisite de novo mutations in human offspring after paternal exposure to ionizing radiation

A genome-wide evaluation of the effects of ionizing radiation on mutation induction in the mouse germline has identified multisite de novo mutations (MSDNs) as marker for previous exposure. Here we present the results of a small pilot study of whole genome sequencing in offspring of soldiers who served in radar units on weapon systems that were emitting high-frequency radiation. We found cases of exceptionally high MSDN rates as well as an increased mean in our cohort: While a MSDN mutation is detected in average in 1 out of 5 offspring of unexposed controls, we observed 12 MSDNs in altogether 18 offspring, including a family with 6 MSDNs in 3 offspring. Moreover, we found two translocations, also resulting from neighboring mutations. Our findings indicate that MSDNs might be suited in principle for the assessment of DNA damage from ionizing radiation also in humans. However, as exact person-related dose values in risk groups are usually not available, the interpretation of MSDNs in single families would benefit from larger molecular epidemiologic studies on this new biomarker.

OTX2 restricts entry to the mouse germline.

SummaryThe successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast1. Induction requires BMP4 signalling to prospective PGCs2 and the intrinsic action of PGC transcription factors (TFs)3–6. However, the molecular mechanisms connecting BMP4 to induction of the PGC TFs responsible for segregating PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Down-regulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro dramatically increases PGCLC differentiation efficiency and prolongs the period of PGC competence. In the absence of Otx2 activity, PGCLC differentiation becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC TF Blimp1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that OTX2 functions repressively upstream of PGC TFs, acting as a roadblock to limit entry of epiblast cells to the germline to a small window in space and time, thereby ensuring correct numerical segregation of germline cells from the soma.

Effects of hydroxyurea on CNV induction in the mouse germline.

Copy number variants (CNVs) are important in genome variation and genetic disease, with new mutations arising frequently in the germline and somatic cells. Replication stress caused by aphidicolin and hydroxyurea (HU) is a potent inducer of de novo CNVs in cultured mammalian cells. HU is used extensively for long-term management of sickle cell disease. Here, we examined the effects of HU treatment on germline CNVs in vivo in male mice to explore whether replication stress can act as a CNV mutagen in germline mitotic divisions as in cultured cells and whether this would support a concern for increased CNV mutations in offspring of men treated with HU. Several trials of HU administration were performed by oral gavage and subcutaneous pump, with CNVs characterized in C57BL/6 x C3H/HeJ hybrid mouse offspring by microarray and mate-pair sequencing. HU had a short half-life of ~14 min and a narrow dose window over which studies could be performed while maintaining fertility. Tissue histopathology and reticulocyte micronucleus assays verified that doses had a substantial tissue and genetic toxicity. CNVs were readily detected in offspring that originated in both paternal and maternal mouse strains, as de novo and inherited events. However, HU did not increase CNV formation above baseline levels. These results reveal a high rate of CNV mutagenesis in the mouse germline but do not support the hypothesis that HU would increase CNV formation during mammalian spermatogenesis, perhaps due to highly toxic effects on sperm development or experimental variables related to HU pharmacology in mice. Environ. Mol. Mutagen. 59:698-714, 2018. © 2018 Wiley Periodicals, Inc.

Germline DNA replication timing shapes mammalian genome composition.

Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.

Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

PO-333 Somatic engineering of mammary gland epithelial cells using CRISPR/Cas9 for rapid testing of breast cancer susceptibility genes in mouse models

IntroductionBreast cancer is one of the most commonly diagnosed malignancies in women. In Western countries it is the second leading cause of cancer death among women. Five to ten percent of all breast cancers have a hereditary component, and approximately twenty five percent of all hereditary breast and ovarian cases are attributable to mutations in the breast cancer 1 or breast cancer 2 (BRCA1 or BRCA2) genes. In vivo studies represent a highly comprehensive and relevant setting for studying the complexity of this human disease. Genetically engineered germline mouse models of breast cancer paved the way for improved basic and translational research, but their generation is slow and expensive.Material and methodsTo overcome these limitations, our lab previously developed CRISPR/Cas9-mediated somatic genome editing approaches to test the role of candidate factors in the adult mammary tissue. In particular, a conditional Cas9-expressing mouse model was used for intraductal injection of sgRNAs encoding lentiviruses (LV-sgRNA), which allowed for somatic gene disruption of tumour suppressor genes in mammary epithelial cells and for evaluating the effect on tumour formation and disease progression (Annunziato et al. 2016).Results and discussionsWe will extend this approach to analyse candidate susceptibility genes in triple negative breast cancer. Using a K14Cre;p53F/F;Cas9 mouse model, this LV-sgRNA intraductal platform provides the opportunity to test in vivo several new (candidate) susceptibility genes and study the effects of tumour suppressor domain deletions.ConclusionThrough these studies, we hope to shed light on several genetic alterations and how they affect tumour suppression and drug response.