果蝇生殖腺干细胞和它们的微环境

干细胞微环境是由容纳一个或多个干细胞,并控制干细胞自我更新和子代细胞产生的组织细胞以及细胞外基质组成。干细胞必须在微环境内才能增殖,才能保持自我更新的特性。通过对果蝇生殖腺干细胞微环境的结构及其产生的信号路径（该路径可以调节干细胞自我更新）的研究,发现微环境中支持细胞和它们发出的信号路径在调节干细胞的增殖和分化中起重要的作用。     

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.     

Abscission Is Regulated by the ESCRT-III Protein Shrub in Drosophila Germline Stem Cells

Abscission is the final event of cytokinesis that leads to the physical separation of the two daughter cells. Recent technical advances have allowed a better understanding of the cellular and molecular events leading to abscission in isolated yeast or mammalian cells. However, how abscission is regulated in different cell types or in a developing organism remains poorly understood. Here, we characterized the function of the ESCRT-III protein Shrub during cytokinesis in germ cells undergoing a series of complete and incomplete divisions. We found that Shrub is required for complete abscission, and that levels of Shrub are critical for proper timing of abscission. Loss or gain of Shrub delays abscission in germline stem cells (GSCs), and leads to the formation of stem-cysts, where daughter cells share the same cytoplasm as the mother stem cell and cannot differentiate. In addition, our results indicate a negative regulation of Shrub by the Aurora B kinase during GSC abscission. Finally, we found that Lethal giant discs (lgd), known to be required for Shrub function in the endosomal pathway, also regulates the duration of abscission in GSCs.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

三种果蝇精巢生殖干细胞半衰期的研究

果蝇精巢生殖干细胞(Germline stem cells,GSCs)的数量伴随衰老呈现递减趋势。本文以两种野生型果蝇品系Oregon、W1118以及Stat基因突变体的精巢为研究材料,采用免疫荧光染色技术,标记果蝇精巢Hub组织及生殖干细胞,研究了室温(25℃)条件下三种果蝇羽化后六个时间点(第1、15、30、45、60、75 d)精巢GSC的数量。结果表明,两种野生型Oregon、W1118与stat突变体果蝇羽化后第1 d的GSC平均数分别为8.55、8.60与8.15,根据曲线图得到,两种野生型果蝇GSCs半衰期均为60 d左右,而stat突变体果蝇约为30 d,两者存在显著差异,表明stat基因突变加速了果蝇精巢GSCs的衰老。     

Selection and Maintenance of Sexual Identity in the Drosophila Germline

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene.     

A Regulatory Network of Drosophila Germline Stem Cell Self-Renewal

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Mathematical Model for Two Germline Stem Cells Competing for Niche Occupancy

In the Drosophila germline stem cell ovary niche, two stem cells compete with each other for niche occupancy to maintain stem cell quality by ensuring that differentiated stem cells are rapidly pushed out the niche and replenished by normal ones (Jin et al. in Cell Stem Cell 2:39–49, 2008). To gain a deeper understanding of this biological phenomenon, we have derived a mathematical model for explaining the physical interactions between two stem cells. The model is a system of two nonlinear first order and one second order differential equations coupled with E-cadherins expression levels. The model can explain the dynamics of the competition process of two germline stem cells and may help to reveal missing information obtained from experimental results. The model predicts several qualitative features in the competition process, which may help to design rational experiments for a better understanding of the stem cell competition process.     

Germline Stem Cells Drive Ovary Regeneration in Zebrafish

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Notch信号通路参与卵巢生殖干细胞的调控及卵巢的衰老进程

卵巢生殖干细胞(ovarian germline stem cells, OGSCs)的发现,打破了生殖医学领域传统的"固定卵泡池"理论。近年来,OGSCs新的研究成果不断涌现,但关于OGSCs体内调控机制的研究仍然较少。Notch通路广泛参与多种成体干细胞不对称分裂的过程,并与细胞衰老密切相关,但其是否参与OGSCs的体内调控机制及卵巢的衰老进程尚不清楚。本研究以原代培养技术提取OGSCs,通过荧光双标染色发现,OGSCs标志基因MVH、Oct4与Notch信号通路相关分子Notch1、Hes1在OGSCs中存在共表达;抑制Notch信号通路活性后,cck-8检测发现,OGSCs的增殖活性呈下降趋势;而以免疫组化、荧光双标、Western印迹法检测性成熟期(2月龄)、不孕和衰老(20月龄)小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达变化,发现2月龄小鼠卵巢皮层中MVH、Oct4、Notch1和Hes1的表达量较高(P<0.05),而不孕和衰老小鼠卵巢皮层中,MVH、Oct4、Notch1和Hes1的表达量均明显下降。上述结果表明,Notch信号通路在小鼠OGSCs中高表达,并可能参与调控OGSCs的增殖机制及卵巢的衰老进程。     

The Bantam microRNA Is Associated with Drosophila Fragile X Mental Retardation Protein and Regulates the Fate of Germline Stem Cells

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP). We have previously demonstrated that dFmr1, the Drosophila ortholog of the fragile X mental retardation 1 gene, plays a role in the proper maintenance of germline stem cells in Drosophila ovary; however, the molecular mechanism behind this remains elusive. In this study, we used an immunoprecipitation assay to reveal that specific microRNAs (miRNAs), particularly the bantam miRNA (bantam), are physically associated with dFmrp in ovary. We show that, like dFmr1, bantam is not only required for repressing primordial germ cell differentiation, it also functions as an extrinsic factor for germline stem cell maintenance. Furthermore, we find that bantam genetically interacts with dFmr1 to regulate the fate of germline stem cells. Collectively, our results support the notion that the FMRP-mediated translation pathway functions through specific miRNAs to control stem cell regulation.     

Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors

We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent.     

Notch信号通路与生殖干细胞研究进展

Notch信号通路是一个在进化中高度保守的信号通道,具有调控细胞增殖、分化及凋亡的作用。近年来,随着研究的不断深入,发现Notch信号通路与生殖干细胞的增殖分化及干细胞微环境的作用机理密切关联,Notch信号通路在生殖系统发育及疾病治疗中的作用机制逐渐引起人们的广泛关注。该文综合论述了Notch信号通路的生理特性及功能,重点阐述Notch信号通路在精原干细胞、卵巢生殖干细胞及生殖干细胞微环境系统中的调控机制。     

肿瘤干细胞与卵巢癌研究进展

近年来,肿瘤干细胞学说作为肿瘤发生发展的重要原因获得越来越多的认可。肿瘤干细胞是指肿瘤中存在的含量极少、具有无限增殖潜能的干细胞样肿瘤细胞,它们能自我更新、分化、迁徙,是导致肿瘤发生、发展、转移和耐药的重要原因。卵巢癌也可能是卵巢癌干细胞所致的疾病。卵巢癌干细胞的分离鉴定正处于起始阶段,针对卵巢癌干细胞的靶向治疗可能在卵巢癌治疗中具有重要作用,为临床彻底治愈卵巢癌带来希望。     

Model-Based Analysis for Qualitative Data: An Application in Drosophila Germline Stem Cell Regulation

Discovery in developmental biology is often driven by intuition that relies on the integration of multiple types of data such as fluorescent images, phenotypes, and the outcomes of biochemical assays. Mathematical modeling helps elucidate the biological mechanisms at play as the networks become increasingly large and complex. However, the available data is frequently under-utilized due to incompatibility with quantitative model tuning techniques. This is the case for stem cell regulation mechanisms explored in the Drosophila germarium through fluorescent immunohistochemistry. To enable better integration of biological data with modeling in this and similar situations, we have developed a general parameter estimation process to quantitatively optimize models with qualitative data. The process employs a modified version of the Optimal Scaling method from social and behavioral sciences, and multi-objective optimization to evaluate the trade-off between fitting different datasets (e.g. wild type vs. mutant). Using only published imaging data in the germarium, we first evaluated support for a published intracellular regulatory network by considering alternative connections of the same regulatory players. Simply screening networks against wild type data identified hundreds of feasible alternatives. Of these, five parsimonious variants were found and compared by multi-objective analysis including mutant data and dynamic constraints. With these data, the current model is supported over the alternatives, but support for a biochemically observed feedback element is weak (i.e. these data do not measure the feedback effect well). When also comparing new hypothetical models, the available data do not discriminate. To begin addressing the limitations in data, we performed a model-based experiment design and provide recommendations for experiments to refine model parameters and discriminate increasingly complex hypotheses.     

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

The laboratory rat was one of the earliest mammalian species for scientific research and used as animal disease models in physiology,toxicology,behavior,immunology,and tumor-biology for over 150 years (Jacob,1999).However,rat lags far behind mouse in generating human disease models and functional genomic studies because of the lack of authentic rat embryonic stem (ES) cells (Voigt and Serikawa,2009),whereas the first mouse ES cell line was established in 1981 (Evans and Kaufman,1981).By combining two or three kinase inhibitors which target GSK3,MEK and FGF signaling pathways in serum-free N2B27 medium,germline competent rat ES cells were first derived in 2008 (Buehr et al.,2008;Li et al.,2008).     

Lsd1 Restricts the Number of Germline Stem Cells by Regulating Multiple Targets in Escort Cells

Specialized microenvironments called niches regulate tissue homeostasis by controlling the balance between stem cell self-renewal and the differentiation of stem cell daughters. However the mechanisms that govern the formation, size and signaling of in vivo niches remain poorly understood. Loss of the highly conserved histone demethylase Lsd1 in Drosophila escort cells results in increased BMP signaling outside the cap cell niche and an expanded germline stem cell (GSC) phenotype. Here we present evidence that loss of Lsd1 also results in gradual changes in escort cell morphology and their eventual death. To better characterize the function of Lsd1 in different cell populations within the ovary, we performed Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq). This analysis shows that Lsd1 associates with a surprisingly limited number of sites in escort cells and fewer, and often, different sites in cap cells. These findings indicate that Lsd1 exhibits highly selective binding that depends greatly on specific cellular contexts. Lsd1 does not directly target the dpp locus in escort cells. Instead, Lsd1 regulates engrailed expression and disruption of engrailed and its putative downstream target hedgehog suppress the Lsd1 mutant phenotype. Interestingly, over-expression of engrailed, but not hedgehog, results in an expansion of GSC cells, marked by the expansion of BMP signaling. Knockdown of other potential direct Lsd1 target genes, not obviously linked to BMP signaling, also partially suppresses the Lsd1 mutant phenotype. These results suggest that Lsd1 restricts the number of GSC-like cells by regulating a diverse group of genes and provide further evidence that escort cell function must be carefully controlled during development and adulthood to ensure proper germline differentiation.     

雌性哺乳动物生殖干细胞：在争议中前进

100多年以来,雌性哺乳动物出生后是否存在生殖干细胞的争议尚无定论．2004年,研究人员从出生后的小鼠卵巢中发现并分离到雌性生殖干细胞(female germline stem cells,FGSCs),挑战了存在近半个世纪的理论：哺乳动物出生后不会对卵母细胞库进行更新．随后很多研究不仅指出哺乳动物出生后卵巢中新生成的卵母细胞源自FGSCs,而且发现如果将FGSCs移植回受体卵巢,它们能够产生功能性的卵母细胞并由此得到健康的后代．可是,有的研究小组重复实验或者精心设计实验,却未得到相同的结果,甚至得出相反的结果．最近,有研究者从育龄女性卵巢中分离到了在体内外都能够分化出功能性卵母细胞的FGSCs,不过这些卵母细胞的受精能力还有待证实．本文回顾了哺乳动物FGSCs的研究历程,并对这一存在已久的争论以及FGSCs研究方向和将来的运用前景展开了评述．