小RNAs在生殖细胞发育过程中的作用

很多动物可以产生具调节作用的小RNAs,根据产生方式和作用机制可以将它们分为三类:微小RNAs(miRNAs)、与Piwi相互作用的RNAs(piRNAs)和内源小干扰RNAs(endo-siRNAs),这些小RNAs可以在生物生殖细胞发育过程中发挥重要作用。其中miRNAs的主要作用是调节蛋白质基因的表达;piRNAs主要的作用是沉默转座因子,但piRNAs主要存在于生殖细胞中;endo-siRNAs则可能具有上述两种主要作用。该文论述了这三种小RNAs在生物生殖细胞发育过程中的作用,同时也讨论了它们在治疗生物不育及其在生物节育方面的应用前景。     

Germline-specific Antigens Identified by Monoclonal Antibodies in the Nematode Caenorhabditis elegans1

Of 27 monoclonal antibodies identified to react, by indirect immunofluorescent antibody staining, with specific cells and tissues of the nematode Caenorhabditis elegans, we report here three monoclonal antibodies pertaining to the gonadal tissues. One antibody defines an antigen that is distributed over the entire embryo at earlier development and later becomes unique to the gonad, including mature oocytes. The antigens recognized by the other two are distributed asymmetrically in the posterior region of the fertilized egg's cytoplasm destined to become the germline precursor cell. Each antigen is successively segregated only to the germline precursor cells of the developing embryo and, postembryonically, is uniquely localized around the germline cell nuclei of the larvae and adults.     

The C. elegans TPR Containing Protein,TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis

Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.     

Somatic PI3K activity regulates transition to the spermatocyte stages in <Emphasis Type="Italic">Drosophila</Emphasis> testis

Spermatogenesis, involving multiple transit amplification divisions and meiosis, occurs within an enclosure formed by two somatic cells. As the cohort of germline cells divide and grow, the surface areas of the somatic cells expand maintaining a tight encapsulation throughout the developmental period. Correlation between the somatic cell growth and germline development is unclear. Here, we report standardization of a quantitative assay developed for estimating the somatic roles of target molecules on germline division and differentiation in Drosophila testis. Using the assay, we studied the somatic roles of phosphatidylinositol-3-kinase (PI3K). It revealed that the expression of PI3K^DN is likely to facilitate the early germline development at all stages, and an increase in the somatic PI3K activity during the early stages delays the transition to spermatocyte stage. Together, these results suggest that somatic cell growth plays an important role in regulating the rate of germline development.     

A Transport Model for Estimating the Time Course of ERK Activation in the C.?elegans Germline

The Caenorhabditis elegans germline is a well-studied model system for investigating the control of cell fate by signaling pathways. Cell signals at the distal tip of the germline promote cell proliferation; just before the loop, signals couple cell maturation to organism-level nutrient status; at the proximal end of the germline, signals coordinate oocyte maturation and fertilization in the presence of sperm. The latter two events require dual phosphorylation and activation of ERK, the effector molecule of the Ras/MAPK cascade. In C. elegans, ERK is known as MPK-1. At this point, none of today’s methods for real-time monitoring of dually phosphorylated MPK-1 are working in the germline. Consequently, quantitative understanding of the MPK-1-dependent processes during germline development is limited. Here, we make a step toward advancing this understanding using a model-based framework that reconstructs the time course of MPK-1 activation from a snapshot of a fixed germline. Our approach builds on a number of recent studies for estimating temporal dynamics from fixed organisms, but takes advantage of the anatomy of the germline to simplify the analysis. Our model predicts that the MPK-1 signal turns on ∼30 h into germ cell progression and peaks ∼7 h later.     

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.     

Proteomic analysis reveals the differential histone programs between male germline cells and vegetative cells in Lilium davidii

In flowering plants, male germline fate is determined after asymmetric division of the haploid microspore. Daughter cells have distinct fates: the generative cell (GC) undergoes further mitosis to generate sperm cells (SCs), and the vegetative cell (VC) terminally differentiates. However, our understanding of the mechanisms underlying germline development remains limited. Histone variants and modifications define chromatin states, and contribute to establishing and maintaining cell identities by affecting gene expression. Here, we constructed a lily protein database, then extracted and detailed histone entries into a comprehensive lily histone database. We isolated large amounts of nuclei from VCs, GCs and SCs from lily, and profiled histone variants of all five histone families in all three cell types using proteomics approaches. We revealed 92 identities representing 32 histone variants: six for H1, 11 for H2A, eight for H2B, five for H3 and two for H4. Nine variants, including five H1, two H2B, one H3 and one H4 variant, specifically accumulated in GCs and SCs. We also detected H3 modification patterns in the three cell types. GCs and SCs had almost identical histone profiles and similar H3 modification patterns, which were significantly different from those of VCs. Our study also revealed the presence of multiple isoforms, and differential expression patterns between isoforms of a variant. The results suggest that differential histone programs between the germline and companion VCs may be established following the asymmetric division, and are important for identity establishment and differentiation of the male germline as well as the VC.     

Scratching the niche that controls Caenorhabditis elegans germline stem cells

The Caenorhabditis elegans gonad provides a well-defined model for a stem cell niche and its control of self-renewal and differentiation. The distal tip cell (DTC) forms a mesenchymal niche that controls germline stem cells (GSCs), both to generate the germline tissue during development and to maintain it during adulthood. The DTC uses GLP-1/Notch signaling to regulate GSCs; germ cells respond to Notch signaling with a network of RNA regulators to control the decision between self-renewal and entry into the meiotic cell cycle.     

PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts.     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.     

Inhibition of germline proliferation during C. elegans dauer development requires PTEN, LKB1 and AMPK signalling

In C. elegans, reduced insulin-like signalling induces developmental quiescence, reproductive delay and lifespan extension. We show here that the C. elegans orthologues of LKB1 and AMPK cooperate during conditions of reduced insulin-like signalling to establish cell cycle quiescence in the germline stem cell population, in addition to prolonging lifespan. The inactivation of either protein causes aberrant germline proliferation during diapause-like ;dauer' development, whereas the loss of AMPK uncouples developmental arrest from lifespan extension. Reduced TGF-beta activity also triggers developmental quiescence independent of the insulin-like pathway. Our data suggest that these two signalling pathways converge on the C. elegans PTEN orthologue to coordinate germline proliferation with somatic development during dauer formation, via the regulation of AMPK and its upstream activator LKB1, rather than through the canonical insulin-like signalling cascade. In humans, germline mutations in TGF-beta family members, PTEN or LKB1 result in related tumour-predisposing syndromes. Our findings establish a developmental relationship that may underscore their shared, characteristic aetiology.     

Small RNAs in the animal gonad: Guarding genomes and guiding development

Germ cells must safeguard, apportion, package, and deliver their genomes with exquisite precision to ensure proper reproduction and embryonic development. Classical genetic approaches have identified many genes controlling animal germ cell development, but only recently have some of these genes been linked to the RNA interference (RNAi) pathway, a gene silencing mechanism centered on small regulatory RNAs. Germ cells contain microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs); these are bound by members of the Piwi/Argonaute protein family. piwi genes were known to specify germ cell development, but we now understand that mutations disrupting germline development can also affect small RNA accumulation. Small RNA studies in germ cells have revealed a surprising diversity of regulatory mechanisms and a unifying function for germline genes in controlling the spread of transposable elements. Future challenges will be to understand the production of germline small RNAs and to identify the full breadth of gene regulation by these RNAs. Progress in this area will likely impact biomedical goals of manipulating stem cells and preventing diseases caused by the transposition of mobile DNA elements.