Maintenance Of Primordial Germ Cells Research Articles

The mitochondrial protein Sod2 is important for the migration, maintenance, and fitness of germ cells.

To maintain a range of cellular functions and to ensure cell survival, cells must control their levels of reactive oxygen species (ROS). The main source of these molecules is the mitochondrial respiration machinery, and the first line of defense against these toxic substances is the mitochondrial enzyme superoxide dismutase 2 (Sod2). Thus, investigating early expression patterns and functions of this protein is critical for understanding how an organism develops ways to protect itself against ROS and enhance tissue fitness. Here, we report on expression pattern and function of zebrafish Sod2, focusing on the role of the protein in migration and maintenance of primordial germ cells during early embryonic development. We provide evidence that Sod2 is involved in purifying selection of vertebrate germ cells, which can contribute to the fitness of the organism in the following generations.

LIN28 is essential for the maintenance of chicken primordial germ cells

Understanding the mechanism of stem cell maintenance underlies the establishment of long-term and mass culture methods for stem cells that are fundamental for clinical and agricultural applications. In this study, we use chicken primordial germ cell (PGC) as a model to elucidate the molecular mechanisms underlying stem cell maintenance. The PGC is a useful experimental model because it is readily gene-manipulatable and easy to test gene function in vivo using transplantation. Previous studies to establish a long-term culture system have shown that secreted factors such as FGF2 are required to maintain the self-renewal capability of PGC. On the other hand, we know little about intracellular regulators responsible for PGC maintenance. Among representative stem cell factors, we focus on RNA-binding factors LIN28A and LIN28B as possible central regulators for the gene regulatory network essential to PGC maintenance. By taking advantage of the CRISPR/Cas9-mediated gene editing and a clonal culture technique, we find that both LIN28A and LIN28B regulate the proliferation of PGC in vitro. We further showed that colonization efficiency of grafted PGC at the genital ridges, rudiments for the gonads, of chicken embryos were significantly decreased by knockout (KO) of LIN28A or LIN28B. Of note, overexpression of human LIN28 in LIN28-KO PGC was sufficient to restore the low colonization rates, suggesting that LIN28 plays a key role in PGC colonization at the gonads. Transcriptomic analyses of LIN28-KO PGC reveal that several genes related to mesenchymal traits are upregulated, including EGR1, a transcription factor that promotes the differentiation of mesodermal tissues. Finally, we show that the forced expression of human EGR1 deteriorates replication activity and colonization efficiency of PGCs. Taken together, this work demonstrates that LIN28 maintains self-renewal of PGC by suppressing the expression of differentiation genes including EGR1.

The Puf-A Protein Is Required for Primordial Germ Cell Development.

Puf-A, a nucleolar Puf domain protein, is required for ribosome biogenesis. A study of Puf-A in zebrafish has shown that Puf-A is highly expressed in primordial germ cells (PGCs) and participates in PGC development. However, it remains unclear how Puf-A governs PGC development in mammals. Here, we generated transgenic mice carrying inducible Puf-A shRNA and obtained double heterozygous mice with Puf-A shRNA and Oct4-EGFP to examine the behavior of PGCs. It was found that the knockdown of Puf-A led to the loss of a considerable number of PGCs and a slowdown of the movement of the remaining PGCs. Puf-A and NPM1 colocalized in clusters in the nuclei of the PGCs. The silencing of Puf-A resulted in the translocation of NPM1 from nucleolus to nucleoplasm and the hyperactivation of p53 in the PGCs. The PGCs in Puf-A knockdown embryos showed a significant increase in subpopulations of PGCs at G1 arrest and apoptosis. Moreover, the expression of essential genes associated with PGC maintenance was decreased in the Puf-A knockdown PGCs. Our study showed that Puf-A governed PGC development by regulating the growth, survival, and maintenance of PGCs. We also observed the alterations of NPM1 and p53 upon Puf-A knockdown to be consistent with the previous study in cancer cells, which might explain the molecular mechanism for the role of Puf-A in PGC development.

Zygotic nanos3 Mutant Medaka (Oryzias latipes) Displays Gradual Loss of Germ Cells and Precocious Spermatogenesis During Gonadal Development.

Nanos is one of the components of germ plasm and is evolutionarily conserved from flies to mammals. In medaka (Oryzias latipes), maternally provided nanos3 is essential for maintenance of primordial germ cells (PGCs). Here, we generated nanos3 loss-of-function mutants by using transcription activator-like effector nuclease (TALEN) and examined the function of zygotic nanos3 in medaka. Zygotic nanos3 homozygous (-/-) mutants derived from nanos3 heterozygous mothers formed germ cells. However, after hatching, the number of germ cells decreased considerably, resulting in infertility of both nanos3-/- females and males. Surprisingly, both nanos3-/- XX and XY mutants underwent precocious spermatogenesis during early gonadal development, as seen in loss-of-function mutants of foxl3, the germline sex-determination gene, in medaka. Therefore, in addition to the maintenance of germ cells, these results suggest that zygotic nanos3 affects the proper regulation of germline sex in XX medaka.

Heading towards a dead end: The role of DND1 in germ line differentiation of human iPSCs.

The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) that plays important roles in survival and fate maintenance of primordial germ cells (PGCs) and in the development of the male germline in zebrafish and mice. Dead end was shown to be expressed in human pluripotent stem cells (PSCs), PGCs and spermatogonia, but little is known about its specific role concerning pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to determine if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines carrying biallelic loss of function mutations and analysed their differentiation potential towards PGCLCs and their gene expression on RNA and protein levels via RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation, or trilineage differentiation potential, but yielded reduced numbers of PGCLCs as compared with their parental iPSCs. RNAseq analysis of mutated PGCLCs revealed that the overall gene expression remains like non-mutated PGCLCs. However, reduced expression of genes associated with PGC differentiation and maintenance (e.g., NANOS3, PRDM1) was observed. Together, we show that DND1 iPSCs maintain their pluripotency but exhibit a reduced differentiation to PGCLCs. This versatile model will allow further analysis of the specific mechanisms by which DND1 influences PGC differentiation and maintenance.

Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg-/- embryos.

Fanconi anemia (FA) is a rare human genetic disorder characterized by bone marrow failure, predisposition to cancer and developmental defects including hypogonadism. Reproductive defects leading to germ cell aplasia are the most consistent phenotypes seen in FA mouse models. We examined the role of the nuclear FA core complex gene Fancg in the development of primordial germ cells (PGCs), the embryonic precursors of adult gametes, during fetal development. PGC maintenance was severely impaired in Fancg−/− embryos. We observed a defect in the number of PGCs starting at E9.5 and a strong attrition at E11.5 and E13.5. Remarkably, we observed a mosaic pattern reflecting a portion of testicular cords devoid of PGCs in E13.5 fetal gonads. Our in vitro and in vivo data highlight a potential role of Fancg in the proliferation and in the intrinsic cell motility abilities of PGCs. The random migratory process is abnormally activated in Fancg−/− PGCs, altering the migration of cells. Increased cell death and PGC attrition observed in E11.5 Fancg−/− embryos are features consistent with delayed migration of PGCs along the migratory pathway to the genital ridges. Moreover, we show that an inhibitor of RAC1 mitigates the abnormal migratory pattern observed in Fancg−/− PGCs.

Inactivation of REV7 enhances chemosensitivity and overcomes acquired chemoresistance in testicular germ cell tumors.

REV7 is a multitasking protein involved in replication past DNA lesions, cell cycle regulation, and gene expression. REV7 is highly expressed in the adult testis and plays an essential role in primordial germ cell maintenance in mice. In this study, we analyzed whether REV7 can be a molecular target for the treatment of testicular germ cell tumors (TGCTs), in which acquired chemoresistance is a major cause of treatment failure. Strong expression of REV7 was detected in human TGCT tissues by immunohistochemistry. REV7 depletion in the TGCT cell lines suppressed cell proliferation and increased sensitivity to cisplatin and doxorubicin. cDNA microarray analysis revealed that REV7 depletion downregulated genes in the DNA repair gene set and upregulated genes in the apoptosis gene set. REV7 depletion-provoked chemosensitivity was associated with DNA double-strand break accumulation and apoptosis activation. In addition, inactivation of REV7 in cisplatin-resistant TGCT cells recovered chemosensitivity at almost equal levels as parental cells in vitro and in vivo. Our results indicate that inactivation of REV7 enhances chemosensitivity and overcomes chemoresistance in TGCT cells, suggesting REV7 as a potential therapeutic target in chemoresistant TGCTs.

The Importance of Small Non-Coding RNAs in Human Reproduction: A Review Article.

BackgroundMicroRNAs (miRNA) play a key role in the regulation of gene expression through the translational suppression and control of post-transcriptional modifications.AimPrevious studies demonstrated that miRNAs conduct the pathways involved in human reproduction including maintenance of primordial germ cells (PGCs), spermatogenesis, oocyte maturation, folliculogenesis and corpus luteum function. The association of miRNA expression with infertility, polycystic ovary syndrome (PCOS), premature ovarian failure (POF), and repeated implantation failure (RIF) was previously revealed. Furthermore, there are evidences of the importance of miRNAs in embryonic development and implantation. Piwi-interacting RNAs (piRNAs) and miRNAs play an important role in the post-transcriptional regulatory processes of germ cells. Indeed, the investigation of small RNAs including miRNAs and piRNAs increase our understanding of the mechanisms involved in fertility. In this review, the current knowledge of microRNAs in embryogenesis and fertility is discussed.ConclusionFurther research is necessary to provide new insights into the application of small RNAs in the diagnosis and therapeutic approaches to infertility.

Estrogen Receptor β2 Oversees Germ Cell Maintenance and Gonadal Sex Differentiation in Medaka, Oryzias latipes

SummaryIn vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

Transcription of nanos-1 in Zebrafish Embryos is not Affected by Bisphenol A: Evaluated Using Quantitative Real-Time PCR

The presence of primordial germ cells (PGCs) is crucial for proper gonad formation in zebrafish (Danio rerio). The many aspects of PGC migration that allow these cells to reach the proper location at the gonadal ridge include receptors, ligands, germ plasm components, and internal maintenance of PGCs. Any one of these factors could be affected by endocrine-disrupting chemicals (EDCs), which have been shown to alter the directed migration of these cells during early embryonic development. Based on recent research wherein the EDC bisphenol A (BPA) inhibited normal PGC migration, we have used the same dose of BPA to determine the impact of BPA on a gene central to proper germ cell migration. Zebrafish embryos were exposed to BPA, and the levels of the target gene nanos-1 were analyzed using quantitative real-time PCR (q-PCR). The target gene nanos-1 is a critically important germplasm component that allows for survival and proper migration of PGCs. The q-PCR results showed that BPA did not affect the transcription level of nanos-1 in zebrafish embryos. KEYWORDS: Zebrafish; Zebrafish Embryos; nanos-1; Primordial Germ Cells; PGC Migration; Gonad Development; Endocrine-Disrupting Chemicals; Bisphenol A; Sex Determination

Expression pattern of nanos, piwil, dnd, vasa and pum genes during ontogenic development in Nile tilapia Oreochromis niloticus

Primordial germ cells (PGCs) are specified by maternally provided determinants in fish. PGCs migrate then into prospective gonadal sites during early development and give rise to germ cell lineage. PGC disrupted animals do not sexually mature which has a range of commercial as well as environmental benefits. To find potential target genes for sterilisation of Nile tilapia, relative mRNA abundance patterns and tissue distribution of four nanos, two piwil, dnd1, vasa and three pum genes were investigated during ontogenic development from unfertilised eggs to newly hatched larvae and in adult tissues, respectively. The ontogenic pattern of RNA abundance revealed that all the investigated gene transcripts are maternally deposited to varying degrees, except for nanos2 which is not expressed in eggs. The ontogenic patterns of relative RNA abundance could be grouped into three categories. The first one, including nanos3, piwil1, piwil2, dnd1 and vasa, showed abundant transcript levels during early developmental stages which are then degraded during the period of maternal to zygotic transition between blastula and gastrula stages with a reduction in expression of four to five orders of magnitude by hatching stage. Another, including pum2 and pum3, showed similar patterns to the first group, but the transcript levels are reduced by only two orders of magnitude. The third group, including nanos1a, nanos1b and pum1, was characterised by a zygotic increase. nanos2 had no detectable transcripts until hatching stage. The tissue screening of nanos1a, nanos1b, pum1, pum2 and pum3 showed that they are expressed in various tissues, implying their potential pleiotropic effects in these tissues apart from gonads. In contrast, nanos3, piwil1, piwil2, dnd1 and vasa appeared to be exclusively expressed in gonads (both ovary and testis), and nanos2 showed testis-specific expression. Based on these results nanos3, piwil1, piwil2, dnd1 and vasa were prioritised among the 11 selected genes as potential target genes for sterilisation in Nile tilapia as they have no significant zygotic expression during embryogenesis, they are expressed exclusively in gonads and maternally deposited. These features suggest a potential role of these genes in the specification and maintenance of PGCs during the ontogenic development of Nile tilapia.

Noncanonical translation via deadenylated 3' UTRs maintains primordial germ cells.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A)-tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine’s effect on PGCs arises from translation repression through primordazine-response elements in the 3′UTRs. Systematic dissection of primordazine’s mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A)-tails. In addition to poly(A)-tail dependent translation (PAT), early embryos perform poly(A)-tail independent non-canonical translation (PAINT) via deadenylated 3′UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent but not in proliferating mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.

Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

NANOG plays a pivotal role in pluripotency acquisition and lineage specification in higher vertebrates, and its expression is restricted to primordial germ cells (PGCs) during early embryonic development. Mammalian NANOG self-associates via conserved tryptophan-repeat motifs in the C-terminal domain (CTD) to maintain pluripotency. Avian NANOG, however, lacks the conserved motifs, and the molecular mechanism underlying the biologic function is not clearly understood. Here, using spectroscopic and biochemical methods as well as cell-based assays, we report that chicken NANOG (cNANOG) oligomerizes through its CTD via a novel folding-upon-binding mechanism. The CTD of cNANOG is disordered as a monomer and associates into an α-helical multimer driven by intermolecular hydrophobic interactions. Mutation of key aromatic residues in the CTD abrogates the self-association, leading to a loss of the proliferation of chicken PGCs and blastoderm cells. Our results demonstrate that the CTD of cNANOG belongs to a novel IDP that switches into a helical oligomer via self-association, enabling the maintenance of PGCs and blastoderm cells.-Choi, H. J., Kim, I., Lee, H. J., Park, Y. H., Suh, J.-Y., Han, J. Y. Chicken NANOG self-associates via a novel folding-upon-binding mechanism.

Overexpression of DYRK1A, a Down Syndrome Candidate gene, Impairs Primordial Germ Cells Maintenance and Migration in zebrafish

DYRK1A, located on chromosome 21, is a major candidate gene of Down syndrome (DS, trisomy21), and its overexpression is associated with abnormal phenotype of Down syndrome patients. The defects of gonads and germ cells in Down Syndrome suggest that overexpression of DYRK1A has potential effect on primordial germ cells (PGCs) development. Human and zebrafish DYRK1A protein sequence possess 75.6% similarity and same function domains, suggesting the evolutional conservation. Here, we used zebrafish model to detect the definite role of excessive expression of DYRK1A in PGCs development during embryogenesis. We injected DYRK1A mRNA into embryos and detected the PGCs marker gene vasa and nanos1. Results showed depletion in numbers and disordering migration of PGCs in human or zebrafish DYRK1A overexpressed zebrafish embryos. Quantitative proteome analysis indicated that embryonic proteins were significantly altered in DYRK1A overexpressed embryos. Of note, ca15b and piwil1, two identified critical factors for PGCs development, showed ectopic expression induced by overexpressed DYRK1A. In brief, we demonstrate that overexpression of DYRK1A, a candidate gene of Down’s syndrome, impairs PGCs development during early embryogenesis by altering key factors in embryos. Importantly, our work may provide a conceivable mechanism for the gonads and germ cells defects of Down syndrome patients.

Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3′UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability.

Improvement of Chicken Primordial Germ Cell Maintenance In Vitro by Blockade of the Aryl Hydrocarbon Receptor Endogenous Activity.

Primordial germ cells (PGCs) are the undifferentiated progenitors of gametes. Germline competent PGCs can be developed as a cell-based system for genetic modification in chickens, which provides a valuable tool for transgenic technology with both research and industrial applications. This implies manipulation of PGCs, which, in recent years, encouraged a lot of research focused on the study of PGCs and the way of improving their culture. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that besides mediating toxic responses to environmental contaminants plays pivotal physiological roles in various biological processes. Since a novel compound that acts as an antagonist of this receptor has been reported to promote expansion of hematopoietic stem cells, we conducted the present study with the aim of determining whether addition of an established AHR antagonist to the standard culture medium used nowadays for in vitro chicken PGCs culture improves ex vivo expansion. We have found that addition of α-naphthoflavone in culture medium promotes the amplification of undifferentiated cells and that this effect is exerted by the blockade of AHR action. Our results constitute the first report of the successful use of a readily available AHR antagonist to improve avian PGCs expansion, and they further extend the knowledge of the effects of AHR modulation in undifferentiated cells.

Interaction of NANOS2 and NANOS3 with different components of the CNOT complex may contribute to the functional differences in mouse male germ cells.

ABSTRACTNANOS2 and NANOS3 belong to the Nanos family of proteins that contain a conserved zinc finger domain, which consists of two consecutive CCHC-type zinc finger motifs, and contribute to germ cell development in mice. Previous studies indicate that there are redundant and distinct functions of these two proteins. NANOS2 rescues NANOS3 functions in the maintenance of primordial germ cells, whereas NANOS3 fails to replace NANOS2 functions in the male germ cell pathway. However, the lack of a conditional allele of Nanos3 has hampered delineation of each contribution of NANOS2 and NANOS3 to the male germ cell pathway. In addition, the molecular mechanism underlying the distinct functions of these proteins remains unexplored. Here, we report an unexpected observation of a transgenic mouse line expressing a NANOS2 variant harboring mutations in the zinc finger domain. Transcription of Nanos2 and Nanos3 was strongly compromised in the presence of this transgene, which resulted in the mimicking of the Nanos2/Nanos3 double-null condition in the male gonad. In these transgenic mice, P-bodies involved in RNA metabolism had disappeared and germ cell differentiation was more severely affected than that in Nanos2-null mice, indicating that NANOS3 partially substitutes for NANOS2 functions. In addition, similar to NANOS2, we found that NANOS3 associated with the CCR4-NOT deadenylation complex but via a direct interaction with CNOT8, unlike CNOT1 in the case of NANOS2. This alternate interaction might account for the molecular basis of the functional redundancy and differences in NANOS2 and NANOS3 functions.

Abstract 5347: DNA repair protein Rev7 is required for primordial germ cell maintenance in the mouse

Abstract REV7 is involved in DNA repair, cell cycle regulation, gene expression and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function, and that downregulation of REV7 expression in human cancer cells results in enhancement of sensitivity to DNA damaging agents. It has been reported that human REV7 is highly expressed in the adult testis by northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of Rev7-deficient (Rev7-/-) mice to clarify the significance of Rev7 in mouse development. In wild-type mice (Rev7+/+), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7-/- mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7-/- embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7-/- embryo, while cell proliferation was unaffected. Gene expression analysis of E8.5 embryos showed upregulation of Oct4 and Nanog and downregulation of c-Kit and Rev3 in Rev7-/- embryos compared with those in Rev7+/- embryos. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse. Note: This abstract was not presented at the meeting. Citation Format: Yoshiki Murakumo, Naoki Watanabe, Shinji Mii, Masato Asai, Naoya Asai, Kaoru Niimi, Takuya Kato, Atsushi Enomoto, Masahide Takahashi. DNA repair protein Rev7 is required for primordial germ cell maintenance in the mouse. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2014-5347

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.