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Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development
Markus J Strasser, Natalia C Mackenzie, Karin Dumstrei, La-Iad Nakkrasae, Jürg Stebler, Erez Raz
BMC Developmental Biology , 2008, DOI: 10.1186/1471-213x-8-58
Abstract: Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number.Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network.Primordial germ cells (PGCs) are progenitor cells that migrate from their site of specification to the site of the developing gonad where they differentiate into the gametes, sperm and egg. PGCs are normally specified during early development either by inductive cues (in mammals and in Urodele amphibians) [1] or by inheritance of cytoplasmic determinants (e.g. in Drosophila, C. elegans, zebrafish and Xenopus). These cytoplasmic determinants are comprised of electron dense material containing maternal RNAs and proteins, collectively termed germ plasm [2,3]. Transplantation experiments in Drosophila demonstrated that the germ plasm is sufficient for the induction of the germ cell fate [4]. Similarly, in zebrafish it has been shown that removal of germ plasm leads to loss of germ cell specification [5]. In zebrafish, the germ plasm is localized to the first and second cleavage furrows of the early embryo and this localization depends on the function of actin and microtubules [6,7]. At the 32-cell stage the germ plasm is incorporated into four cells that become the progenitors of the germ cell lineage [8]. During di
Circadian Proteins CLOCK and BMAL1 in the Chromatoid Body, a RNA Processing Granule of Male Germ Cells  [PDF]
Rita L. Peruquetti, Sara de Mateo, Paolo Sassone-Corsi
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042695
Abstract: Spermatogenesis is a complex differentiation process that involves genetic and epigenetic regulation, sophisticated hormonal control, and extensive structural changes in male germ cells. RNA nuclear and cytoplasmic bodies appear to be critical for the progress of spermatogenesis. The chromatoid body (CB) is a cytoplasmic organelle playing an important role in RNA post-transcriptional and translation regulation during the late steps of germ cell differentiation. The CB is also important for fertility determination since mutations of genes encoding its components cause infertility by spermatogenesis arrest. Targeted ablation of the Bmal1 and Clock genes, which encode central regulators of the circadian clock also result in fertility defects caused by problems other than spermatogenesis alterations. We show that the circadian proteins CLOCK and BMAL1 are localized in the CB in a stage-specific manner of germ cells. Both BMAL1 and CLOCK proteins physically interact with the ATP-dependent DEAD-box RNA helicase MVH (mouse VASA homolog), a hallmark component of the CB. BMAL1 is differentially expressed during the spermatogenic cycle of seminiferous tubules, and Bmal1 and Clock deficient mice display significant CB morphological alterations due to BMAL1 ablation or low expression. These findings suggest that both BMAL1 and CLOCK contribute to CB assembly and physiology, raising questions on the role of the circadian clock in reproduction and on the molecular function that CLOCK and BMAL1 could potentially have in the CB assembly and physiology.
Generation of Human Induced Pluripotent Stem Cells Using Epigenetic Regulators Reveals a Germ Cell-Like Identity in Partially Reprogrammed Colonies  [PDF]
Akshi Goyal, Shawn L. Chavez, Renee A. Reijo Pera
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0082838
Abstract: Previous studies have shown that induced pluripotent stem cells (iPSCs) can be derived from fibroblasts by ectopic expression of four transcription factors, OCT4, SOX2, KLF4 and c-MYC using various methods. More recent studies have focused on identifying alternative approaches and factors that can be used to increase reprogramming efficiency of fibroblasts to pluripotency. Here, we use nucleofection, morpholino technologies and novel epigenetic factors, which were chosen based on their expression profile in human embryos, fibroblasts and undifferentiated/differentiated human embryonic stem cells (hESCs) and conventionally generated iPSCs, to reprogram human fibroblasts into iPSCs. By over expressing DNMT3B, AURKB, PRMT5 and/or silencing SETD7 in human fibroblasts with and without NANOG, hTERT and/or SV40 overexpression, we observed the formation of colonies resembling iPSCs that were positive for certain pluripotency markers, but exhibited minimal proliferation. More importantly, we also demonstrate that these partially-reprogrammed colonies express high levels of early to mid germ cell-specific genes regardless of the transfection approach, which suggests conversion to a germ cell-like identity is associated with early reprogramming. These findings may provide an additional means to evaluate human germ cell differentiation in vitro, particularly in the context of pluripotent stem cell-derived germ cell development, and contribute to our understanding of the epigenetic requirements of the reprogramming process.
Electron Beam Dynamics in 4GLS  [PDF]
P. H. Williams,G. Hirst,B. D. Muratori,H. L. Owen,S. L. Smith
Physics , 2007,
Abstract: Studies of the electron beam dynamics for the 4GLS design are presented. 4GLS will provide three different electron bunch trains to a variety of user synchrotron sources. The 1 kHz XUV-FEL and 100 mA High Average Current branches share a common 540 MeV linac, whilst the 13 MHz IR-FEL must be well-synchronised to them. An overview of the injector designs, electron transport, and energy recovery is given, including ongoing studies of coherent synchrotron radiation, beam break-up and wakefields. This work is being pursued for the forthcoming Technical Design Report due in 2008.
Using Ants as a Genetic Crossover Operator in GLS to Solve STSP  [PDF]
Hassan Ismkhan
Computer Science , 2014,
Abstract: Ant Colony Algorithm (ACA) and Genetic Local Search (GLS) are two optimization algorithms that have been successfully applied to the Traveling Salesman Problem (TSP). In this paper we define new crossover operator then redefine ACAs ants as operate according to defined crossover operator then put forward our GLS that uses these ants to solve Symmetric TSP (STSP) instances.
Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference  [PDF]
Ferenc Jankovics, László Henn, ágnes Bujna, Péter Vilmos, Kerstin Spirohn, Michael Boutros, Miklós Erdélyi
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0098579
Abstract: In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general.
基于测量模型结构方程模型GLS与WLS比较  [PDF]
焦辛妮, 王长义, 汪东伟, 刘艳
中国公共卫生 , 2015, DOI: 10.11847/zgggws2015-31-01-32
Abstract: ?目的探讨结构方程模型的广义最小二乘法(GLS)和加权最小二乘法(WLS)2种参数估计方法在不同特征数据中的性能差异。方法设定包含15个显变量、3个潜变量但未包含内生显变量、内生潜变量的真模型和误设模型,运用SAS9.1软件的IML模块生成模拟数据,通过CALIS过程进行模型拟合,采用两类错误频率对GLS法和WLS法的性能进行评价。结果分布特征为正态分布、指数分布和二项分布的数据,在采用相关矩阵和协方差矩阵时,GLS和WLS的两类错误频率均随相关系数或样本含量的增加呈下降趋势;在数据特征相同的条件下,2种矩阵分析均表现为GLS两类错误频率之和小于WLS法;GLS在r=0.3且n≥750即显变量个数的50倍时,或在r=0.5且n≥300即显变量个数的20倍时3种分布的两类错误频率之和均<0.05;WLS相关矩阵分析的结果相对于协方差矩阵分析而言稳定性较差,其协方差矩阵分析表现为不论相关系数如何,只要n≥750即两类错误频率之和<0.05。结论GLS法和WLS法对参数的估计均为无偏的和渐进有效的,数据条件和矩阵的不同会影响其参数估计结果,在应用过程中应根据实际情况合理选择。
Growth Kinetics and Optical and Mechanical Properties of Glycine Lithium Sulphate (GLS) Crystals  [PDF]
S. Suresh, A. Ramanand, D. Jayaraman, S.M. Navis Priya
Journal of Minerals and Materials Characterization and Engineering (JMMCE) , 2010, DOI: 10.4236/jmmce.2010.912077
Abstract: Glycine Lithium Sulphate (GLS) is one of the potential materials for Non-linear optical property applications. Single crystals of Glycine Lithium Sulphate (GLS) with very high degree of transparency were grown from aqueous solution by slow evaporation technique. Single crystal X-ray diffraction analysis reveals that the crystal belongs to orthorhombic system with the space group Pna21. The density measurements were carried out by both theoretical and experimental methods. The optical absorption study reveals the transparency of the crystal in the entire visible region and the cut off wavelength has been found to be 350 nm. The dependence of extinction coefficient (K) and refractive index (n) on the absorption has also been reported. The mechanical properties were studied using Vickers microhardness tester. The dielectric studies were also reported for grown crystals. The photoconductivity reveals the negative nature of the photocurrent in these crystals.
GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line  [PDF]
Sophia Millonigg,Ryuji Minasaki equal contributor,Marco Nousch equal contributor,Christian R. Eckmann
PLOS Genetics , 2014, DOI: doi/10.1371/journal.pgen.1004647
Abstract: To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.
Specification of primordial germ cells in medaka (Oryzias latipes)
Amaury Herpin, Stefan Rohr, Dietmar Riedel, Nils Kluever, Erez Raz, Manfred Schartl
BMC Developmental Biology , 2007, DOI: 10.1186/1471-213x-7-3
Abstract: PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression – the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species.In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm.Taken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs.Two basic strategies for the specification of primordial germ cells (PGCs) have been described. In Drosophila, C. elegans and Xenopus the inheritance of germ plasm with associated germ cell determinants appears to be required for PGC formation [1-3]. In these
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