Germline Stem Cells Research Articles

TRIM-NHL protein, NHL-2, modulates cell fate choices in the C. elegans germ line

Many tissues contain multipotent stem cells that are critical for maintaining tissue function. In Caenorhabditis elegans, germline stem cells allow gamete production to continue in adulthood. In the gonad, GLP-1/Notch signaling from the distal tip cell niche to neighboring germ cells activates a complex regulatory network to maintain a stem cell population. GLP-1/Notch signaling positively regulates production of LST-1 and SYGL-1 proteins that, in turn, interact with a set of PUF/FBF proteins to positively regulate the stem cell fate. We previously described sog (suppressor of glp-1 loss of function) and teg (tumorous enhancer of glp-1 gain of function) genes that limit the stem cell fate and/or promote the meiotic fate. Here, we show that sog-10 is allelic to nhl-2. NHL-2 is a member of the conserved TRIM-NHL protein family whose members can bind RNA and ubiquitinate protein substrates. We show that NHL-2 acts, at least in part, by inhibiting the expression of PUF-3 and PUF-11 translational repressor proteins that promote the stem cell fate. Two other negative regulators of stem cell fate, CGH-1 (conserved germline helicase) and ALG-5 (Argonaute protein), may work with NHL-2 to modulate the stem cell population. In addition, NHL-2 activity promotes the male germ cell fate in XX animals.

Redundant mechanisms regulating the proliferation vs. differentiation balance in the C. elegans germline.

The proper production of gametes over an extended portion of the life of an organism is essential for a high level of fitness. The balance between germline stem cell (GSC) proliferation (self-renewal) and differentiation (production of gametes) must be tightly regulated to ensure proper gamete production and overall fitness. Therefore, organisms have evolved robust regulatory systems to control this balance. Here we discuss the redundancy in the regulatory system that controls the proliferation vs. differentiation balance in the C. elegans hermaphrodite germline, and how this redundancy may contribute to robustness. We focus on the various types of redundancy utilized to regulate this balance, as well as the approaches that have enabled these redundant mechanisms to be uncovered.

Regulation, functions and transmission of bivalent chromatin during mammalian development.

Cells differentiate and progress through development guided by a dynamic chromatin landscape that mediates gene expression programmes. During development, mammalian cells display a paradoxical chromatin state: histone modifications associated with gene activation (trimethylated histone H3 Lys4 (H3K4me3)) and with gene repression (trimethylated H3 Lys27 (H3K27me3)) co-occur at promoters of developmental genes. This bivalent chromatin modification state is thought to poise important regulatory genes for expression or repression during cell-lineage specification. In this Review, we discuss recent work that has expanded our understanding of the molecular basis of bivalent chromatin and its contributions to mammalian development. We describe the factors that establish bivalency, especially histone-lysine N-methyltransferase 2B (KMT2B) and Polycomb repressive complex 2 (PRC2), and consider evidence indicating that PRC1 shapes bivalency and may contribute to its transmission between generations. We posit that bivalency is a key feature of germline and embryonic stem cells, as well as other types of stem and progenitor cells. Finally, we discuss the relevance of bivalent chromtin to human development and cancer, and outline avenues of future research.

Non-neutral clonal selection and its potential role in mammalian germline stem cell dysfunction with advancing age.

The concept of natural selection, or "survival of the fittest", refers to an evolutionary process in nature whereby traits emerge in individuals of a population through random gene alterations that enable those individuals to better adapt to changing environmental conditions. This genetic variance allows certain members of the population to gain an advantage over others in the same population to survive and reproduce in greater numbers under new environmental pressures, with the perpetuation of those advantageous traits in future progeny. Here we present that the behavior of adult stem cells in a tissue over time can, in many respects, be viewed in the same manner as evolution, with each stem cell clone being representative of an individual within a population. As stem cells divide or are subjected to cumulative oxidative damage over the lifespan of the organism, random genetic alterations are introduced into each clone that create variance in the population. These changes may occur in parallel to, or in response to, aging-associated changes in microenvironmental cues perceived by the stem cell population. While many of these alterations will be neutral or silent in terms of affecting cell function, a small fraction of these changes will enable certain clones to respond differently to shifts in microenvironmental conditions that arise with advancing age. In some cases, the same advantageous genetic changes that support survival and expansion of certain clones over others in the population (viz. non-neutral competition) could be detrimental to the downstream function of the differentiated stem cell descendants. In the context of the germline, such a situation would be devastating to successful propagation of the species across generations. However, even within a single generation, the “evolution” of stem cell lineages in the body over time can manifest into aging-related organ dysfunction and failure, as well as lead to chronic inflammation, hyperplasia, and cancer. Increased research efforts to evaluate stem cells within a population as individual entities will improve our understanding of how organisms age and how certain diseases develop, which in turn may open new opportunities for clinical detection and management of diverse pathologies.

Novel intrinsic factor Yun maintains female germline stem cell fate through Thickveins.

SummaryGermline stem cells (GSCs) are critical for the reproduction of an organism. The self-renewal and differentiation of GSCs must be tightly controlled to avoid uncontrolled stem cell proliferation or premature stem cell differentiation. However, how the self-renewal and differentiation of GSCs are properly controlled is not fully understood. Here, we find that the novel intrinsic factor Yun is required for female GSC maintenance in Drosophila. GSCs undergo precocious differentiation due to de-repression of differentiation factor Bam by defective BMP/Dpp signaling in the absence of yun. Mechanistically, Yun associates with and stabilizes Thickveins (Tkv), the type I receptor of Dpp/BMP signaling. Finally, ectopic expression of a constitutively active Tkv (TkvQD) completely suppresses GSC loss caused by yun depletion. Collectively, these data demonstrate that Yun functions through Tkv to maintain GSC fate. Our results provide new insight into the regulatory mechanisms of how stem cell maintenance is properly controlled.

Identification of Lsd1-interacting non-coding RNAs as regulators of fly oogenesis.

Lysine-specific demethylase 1 (Lsd1) plays a key role in balancing cell proliferation and differentiation. Lsd1 has been recently reported to associate with specific long noncoding RNAs (lncRNAs) to account for oncogenic gene expression in cancer cells. However, how lncRNA-Lsd1 interplay affects cell-specific differentiation remains elusive invivo. Here, through Lsd1 specific RNA immunopecipitation sequencing (RIP-seq) experiments, we identify three long hairpin RNAs as Lsd1-interacting non-coding RNAs (LINRs) from fly ovaries. Knocking out LINR-1 and LINR-2 affects fly egg production, while each of the LINR deletion mutant females produce eggs with reduced hatch rate, indicating important functions of LINRs in supporting oogenesis. At the cellular level, LINR-2 regulates the differentiation of germline stem cells and follicle progenitors likely though modulating the expression and function of Lsd1 invivo. Our identification of ovarian LINRs presents a physiological example of dynamic lncRNA-Lsd1 interplay that regulates stem cell/progenitor differentiation.

Nonrandom sister chromatid segregation mediates rDNA copy number maintenance in Drosophila.

Although considered to be exact copies of each other, sister chromatids can segregate nonrandomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate nonrandomly during asymmetric division of male germline stem cells (GSCs) in Drosophila melanogaster. Here, we demonstrate that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA binding protein Indra are required for nonrandom sister chromatid segregation (NRSS). We provide the evidence that NRSS, following unequal sister chromatid exchange, is a mechanism by which GSCs recover rDNA copy number, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA.

Asymmetric Histone Inheritance: Establishment, Recognition, and Execution.

The discovery of biased histone inheritance in asymmetrically dividing Drosophila melanogaster male germline stem cells demonstrates one means to produce two distinct daughter cells with identical genetic material. This inspired further studies in different systems, which revealed that this phenomenon may be a widespread mechanism to introduce cellular diversity. While the extent of asymmetric histone inheritance could vary among systems, this phenomenon is proposed to occur in three steps: first, establishment of histone asymmetry between sister chromatids during DNA replication; second, recognition of sister chromatids carrying asymmetric histone information during mitosis; and third, execution of this asymmetry in the resulting daughter cells. By compiling the current knowledge from diverse eukaryotic systems, this review comprehensively details and compares known chromatin factors, mitotic machinery components, and cell cycle regulators that may contribute to each of these three steps. Also discussed are potential mechanisms that introduce and regulate variable histone inheritance modes and how these different modes may contribute to cell fate decisions in multicellular organisms.

Synchronizing spermatogenesis in the mouse.

The formation of spermatozoa starts with a germ-line stem cell creating a pool of progenitor cells or undifferentiated spermatogonia. There is a requirement for these progenitor cells to be stimulated by retinoic acid (RA) to enter differentiation and ultimately form spermatocytes, undergo meiosis, form spermatids, and ultimately spermatozoa. After the stimulation by RA, which occurs at sites in the seminiferous tubules, it takes ~35days to complete this complex process. As a result, the adult testis contains germ cells in all possible states of differentiation, and the isolation of individual cell types or study of functional aspects of the cycle of the seminiferous epithelium is very difficult. We describe the use of WIN 18446-an inhibitor of RA synthesis followed by injection of RA as a mechanism for the synchronization of spermatogenesis to one to three stages of the cycle of the seminiferous epithelium. The result is that only one to four germ cell types are prevalent during the first wave of spermatogenesis. In the adult only a predictable few stages of the cycle are present throughout the entire testis enriching the targeted cells or stages of the cycle.

Transcriptome analysis of mouse male germline stem cells reveals characteristics of mature spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are adult stem cells in the testis of male animals and have the ability in self-renewal and differentiation. SSCs are derived from primordial germ cells (PGCs) that are mitotically arrested in the embryo before birth. Following the birth of the animal, PGCs resume mitosis and migrate from the centre of the seminiferous tubules to the basement membrane. The descendent of PGCs (also called gonocytes) establish stable SSC colonies in about a week postnatally in order to support the life-long spermatogenesis. Whether SSCs at different developmental stages differ in their molecular and cellular characteristics is currently unclear. In the presented study, we conducted bioinformatics analyses using transcriptomics data established previously in the laboratory on OCT4 (encoded by the pluripotent gene Pou5f1) expressing SSCs from the neonatal (3 days-post-partum, 3-dpp), juvenile (7-dpp) and adult (2~3-month) mice, including screen of differentially expressed genes (DEGs), protein-protein interaction (PPI) network analysis of DEGs and clustering of sub-networks from PPI. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses were also performed on clustered sub-networks of the PPI. In addition, all genes were analyzed using GSEA (gene set enrichment analysis) based on GO, KEGG and HALLMARK gene sets. The results showed that SSCs have a large number of DEGs among OCT4-positive SSCs from neonatal, juvenile and adult mice. The distinguishable biological functions encoded by these DEGs include biosynthesis and energy metabolism, immune response, cell junction and expression of migration and cell differentiation-related genes. Significant changes in the cell membrane composition of OCT4-positive SSCs may not only cause hypersensitive immune reactions but also affect the cell-cell contact and responses to secreted cytokines in the extracellular environment. The results also suggest that OCT4-positive SSCs may shift metabolic state from oxidative phosphorylation to glycolysis and significantly reduce the transcription of genes related to ribosome formation during aging. These results provide new clues for future research on the regulatory mechanisms of male germline stem cell development, growth and aging.

A Krüppel-like factor is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians.

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell–generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell–like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.

Auxin-Inducible Degron System Reveals Temporal-Spatial Roles of HSF-1 and Its Transcriptional Program in Lifespan Assurance.

HSF-1 is a key regulator of cellular proteotoxic stress response and is required for animal lifespan. In C. elegans, HSF-1 mediated heat shock response (HSR) declines sharply on the first day of adulthood, and HSF-1 was proposed to function primarily during larval stages for lifespan assurance based on studies using RNAi. The tissue requirement for HSF-1 in lifespan, however, is not well understood. Using the auxin-inducible degron (AID) system, we manage to uncouple the roles of HSF-1 in development and longevity. In wild-type animals, we find HSF-1 is required during the whole self-reproductive period for lifespan. This period is extended in long-lived animals that have arrested germline stem cells (GSC) or reduced insulin/IGF-1 signaling (IIS). While depletion of HSF-1 from any major somatic tissues during development results in severe defects, HSF-1 primarily functions in the intestine and likely neural system of adults to support lifespan. Finally, by combining AID and genome-wide transcriptional analyses, we find HSF-1 directly activates the transcription of constitutively-expressed chaperone and co-chaperone genes among others in early adulthood, which underlies its roles in longevity assurance.

Interchromosomal interaction of homologous Stat92E alleles regulates transcriptional switch during stem-cell differentiation

Pairing of homologous chromosomes in somatic cells provides the opportunity of interchromosomal interaction between homologous gene regions. In the Drosophila male germline, the Stat92E gene is highly expressed in a germline stem cell (GSC) and gradually downregulated during the differentiation. Here we show that the pairing of Stat92E is always tight in GSCs and immediately loosened in differentiating daughter cells, gonialblasts (GBs). Disturbance of Stat92E pairing by relocation of one locus to another chromosome or by knockdown of global pairing/anti-pairing factors both result in a failure of Stat92E downregulation, suggesting that the pairing is required for the decline in transcription. Furthermore, the Stat92E enhancer, but not its transcription, is required for the change in pairing state, indicating that pairing is not a consequence of transcriptional changes. Finally, we show that the change in Stat92E pairing is dependent on asymmetric histone inheritance during the asymmetric division of GSCs. Taken together, we propose that the changes in Stat92E pairing status is an intrinsically programmed mechanism for enabling prompt cell fate switch during the differentiation of stem cells.

Genetic analysis of Caenorhabditis elegans Haspin-like genes shows that hasp-1 plays multiple roles in the germline.

ABSTRACTHaspin is a histone kinase that promotes error-free chromosome segregation by recruiting the chromosomal passenger complex (CPC) to mitotic and meiotic chromosomes. Haspin remains less well studied than other M-phase kinases, and the models explaining Haspin function have been developed primarily in mitotic cells. Here, we generate strains containing new conditional or nonsense mutations in the Caenorhabditis elegans Haspin homologs hasp-1 and hasp-2 and characterize their phenotypes. We show that hasp-1 is responsible for all predicted functions of Haspin and that loss of function of hasp-1 using classical and conditional alleles produces defects in germline stem cell proliferation and spermatogenesis, and confirms its role in oocyte meiosis. Genetic analysis suggests that hasp-1 acts downstream of the Polo-like kinase plk-2 and shows synthetic interactions between hasp-1 and two genes expected to promote recruitment of the CPC by a parallel pathway that depends on the kinase Bub1. This work adds to the growing understanding of Haspin function by characterizing a variety of roles in an intact animal.

Drosophila MESR4 Gene Ensures Germline Stem Cell Differentiation by Promoting the Transcription of bag of marbles

Ovarian germline stem cells (GSCs) of Drosophila melanogaster provide a valuable in vivo model to investigate how the adult stem cell identity is maintained and the differentiation of the daughter cells is regulated. GSCs are embedded into a specialized cellular microenvironment, the so-called stem cell niche. Besides the complex signaling interactions between the germ cells and the niche cells, the germ cell intrinsic mechanisms, such as chromatin regulation and transcriptional control, are also crucial in the decision about self-renewal and differentiation. The key differentiation regulator gene is the bag of marbles (bam), which is transcriptionally repressed in the GSCs and de-repressed in the differentiating daughter cell. Here, we show that the transcription factor MESR4 functions in the germline to promote GSC daughter differentiation. We find that the loss of MESR4 results in the accumulation of GSC daughter cells which fail to transit from the pre-cystoblast (pre-CB) to the differentiated cystoblast (CB) stage. The forced expression of bam can rescue this differentiation defect. By a series of epistasis experiments and a transcriptional analysis, we demonstrate that MESR4 positively regulates the transcription of bam. Our results suggest that lack of repression alone is not sufficient, but MESR4-mediated transcriptional activation is also required for bam expression.

Cistanche deserticola polysaccharides extracted from Cistanche deserticola Y.C. Ma promote the differentiation of mouse female germline stem cells in vitro

Ethnopharmacological relevanceTraditional Chinese herbal medicine Cistanche deserticola Y.C. Ma has been recorded and treatment for infertility and impotence since ancient times, which is widely distributed in northwest China, and is mainly composed of phenylethanol glycosides, iridoids, lignans, polysaccharides, alkaloids, etc. C. deserticola polysaccharides (CDPs) is one of its main active ingredients, studies of its effect on germline stem cells are limited so far.Aim of the study: The aim of this study was to clarify that CDPs promoted the differentiation of FGSCs in vitro, and to initially clarify its possible cell signaling pathways. Material and methodsThe cells were randomly divided into two groups. Normal FGSCs culture medium and the optimal concentration of CDPs (0.5 μg/mL) were added for culture, which was the selected treatment concentration that could promote cell differentiation on the basis of maintaining cell viability. After treatment for different time periods (12 h, 24 h, 36 h, 48 h), the cell proliferation and differentiation were evaluated by CCK-8, real-time PCR (qPCR), cell immunofluorescence and Western blot. Subsequently, RNA-Seq and data analysis were used to preliminarily analyze and verify the different genes and possible signal pathways. ResultsUnder the treatment of CDPs, cell viability was relatively better, and the expression of meiotic markers stimulated by retinoic acid gene 8 protein (Stra8) and synaptonemal complex protein 3 (Sycp3) significantly increased. In addition, their cell morphology was more similar to oocytes. Comparison of gene expression in FGSCs identified key differential expression genes (DEGs) by RNA-Seq that consisted of 549 upregulated and 465 downregulated genes. The DEGs enriched in the functional categories of germline cell development and relevant signaling pathways, which jointly regulate self-renewal and differentiation of FGSCs. The transforming growth factor β (TGF-β) signaling pathway and bone morphogenetic protein (BMP) signaling pathway might be activated to synergistically influence cell differentiation during the CDPs treatment of FGSCs. ConclusionThese findings indicated that CDPs could promote the differentiation of FGSCs in vitro and could be regulated by different DEGs and signal transduction. Preliminary mechanism studies have shown that CDPs can exert their biological activities by regulating the TGF-β and BMP signaling pathways.

Functional Divergence of the bag-of-marbles Gene in the Drosophila melanogaster Species Group.

In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and Drosophila simulans, but not in the outgroup species Drosophila ananassae. Here, we test the null hypothesis that bam’s differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, we demonstrate that bam is not necessary for oogenesis or spermatogenesis in Drosophila teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. We further report tests of neutral sequence evolution at bam in additional species of Drosophila and find a positive, but not perfect, correlation between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between bam’s critical gametogenesis role being newly derived in D. melanogaster, D. simulans, Drosophila yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.

Vasa Is a Potential Germ Cell Marker in Leopard Coral Grouper (Plectropomus leopardus).

Vasa (Ddx4, DEAD box polypeptide 4), an extremely specific marker of germ cells in vivo, is an ATP-dependent RNA helicase that plays an essential role in germ cell development and gametogenesis. However, the expression and function information about this gene in groupers remains lacking. Here, vasa homolog termed Plvasa was isolated and identified Plvasa as a putative germ cell marker in the leopard coral grouper, (Plectropomus leopardus). Results indicated that Plvasa contained 17 exons in the genomic sequence and 9 conserved motifs of the DEAD-box protein by sequence analysis. The sequence comparison, phylogenetic analyses and synteny analyses showed that Plvasa was homologous with other teleosts. Additionally, the expression of Plvasa was significantly higher in gonads than in other tissues in adult individuals (p < 0.05). Further, the distribution of Plvasa revealed that it was only expressed in the germ cells, such as spermatids, germline stem cells and oocytes at different stages, and could not be detected in the somatic cells of gonads. The current study verified that the Plvasa gene is a valuable molecular marker of germ cells in leopard coral grouper, which potentially plays an important role in investigating the genesis and development of teleost germ cells.

Lipofection of Non-integrative CRISPR/Cas9 Ribonucleoproteins in Male Germline Stem Cells: A Simple and Effective Knockout Tool for Germline Genome Engineering.

Gene editing in male germline stem (GS) cells is a potent tool to study spermatogenesis and to create transgenic mice. Various engineered nucleases already demonstrated the ability to modify the genome of GS cells. However, current systems are limited by technical complexity diminishing application options. To establish an easier method to mediate gene editing, we tested the lipofection of site-specific Cas9:gRNA ribonucleoprotein (RNP) complexes to knockout the enhanced green fluorescent protein (Egfp) in mouse EGFP-GS cells via non-homologous end joining. To monitor whether gene conversion through homology-directed repair events occurred, single-stranded oligodeoxynucleotides were co-lipofected to deliver a Bfp donor sequence. Results showed Egfp knockout in up to 22% of GS cells, which retained their undifferentiated status following transfection, while only less than 0.7% EGFP to BFP conversion was detected in gated GS cells. These data show that CRISPR/Cas9 RNP-based lipofection is a promising system to simply and effectively knock out genes in mouse GS cells. Understanding the genes involved in spermatogenesis could expand therapeutic opportunities for men suffering from infertility.

Daidzein Activates Akt Pathway to Promote the Proliferation of Female Germline Stem Cells through Upregulating Clec11a.

Female germline stem cells (FGSCs) have been successfully isolated and characterized from postnatal mammalian and human ovarian tissues. However, the effects and mechanisms of action of natural small-molecule compounds on FGSCs are largely unknown. Here, we found that daidzein promoted the viability and proliferation of FGSCs. To elucidate the mechanism underlying this, we performed RNA-Sequence in daidzein-treated FGSCs and controls. The results showed that there were 153 upregulated and 156 downregulated genes in daidzein treatment. We confirmed the expression of some genes related to cell proliferation in the sequencing results by RT-PCR, such as Type C lectin domain family 11 member a (Clec11a), Mucin1 (Muc1), Glutathione peroxidase 3 (Gpx3), and Tet methylcytosine dioxygenase 1 (Tet1). The high expression of Clec11a at the protein level after daidzein treatment was also confirmed by western blotting. Furthermore, recombinant mouse Clec11a (rmClec11a) protein was shown to promote the viability and proliferation of FGSCs. However, knockdown of Clec11a inhibited the viability and proliferation of FGSCs, which could not be rescued by the administration of daidzein. These results indicate that daidzein promoted the viability and proliferation of FGSCs through Clec11a. In addition, both daidzein and rmClec11a activated the Akt signaling pathway in FGSCs. However, Clec11a knockdown inhibited this pathway, which could not be rescued by daidzein administration. Taken together, our findings revealed that daidzein activates the Akt signaling pathway to promote cell viability and proliferation through upregulating Clec11a. This study should deepen our understanding of the developmental mechanism of FGSCs and female infertility.