Establishing Germ Cell Fate in Xenopus

确定爪蟾生殖细胞的命运

• 批准号: 7988443
• 负责人: MARY LOU KING
• 金额: $ 13.27万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988443
• 关键词: Address; Adult; Affinity Chromatography; Animal Model; Biochemical; Biological Assay; Biological Preservation; Caenorhabditis elegans; Cardiovascular Diseases; Cells; Chromatin Structure; Cloning; Complex; Coupled; Couples; Data; Degenerative Disorder; Development; Developmental Biology; Diabetes Mellitus; Drosophila genus; Elongation Factor; Embryo; Endoderm; Endoderm Cell; Event; Family member; Figs - dietary; Gene Expression Regulation; Gene Transfer; Genes; Genetic; Genetic Transcription; Genome; Germ Cells; Germ Layers; Goals; Grant; Hour; Knowledge; Messenger RNA; Methods; Microinjections; Modeling; Modification; Molecular; Monitor; Mothers; Nuclear; Oocytes; Parkinson Disease; Pathway interactions; Play; Positive Transcriptional Elongation Factor B; Proteins; Public Health; RNA; Rana; Regenerative Medicine; Regulatory Pathway; Repression; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Somatic Cell; Specific qualifier value; Staging; Stem cells; Structure of primordial sex cell; System; Testing; Therapeutic; Time; Transcript; Transcription Elongation; Translational Repression; Translations; Work; Xenopus; base; blastomere structure; cDNA Library; cell type; gene discovery; gene repression; in vivo; insight; mutant; pluripotency; prevent; research study; stem cell biology; transcription factor; xenopus development

DESCRIPTION (provided by applicant): Understanding cellular mechanisms that preserve totipotency or pluripotency in cells are central goals of both stem cell and developmental biology. Primordial germ cells (PGCs), precursors to the gametes, are the ultimate stem cell as they retain full developmental potential while other embryonic cells become progressively restricted in their fates. Our long term goal is to establish the key regulatory pathways that operate during the specification and differentiation of PGCs in the vertebrate model organism Xenopus. Genetic studies in Drosophila and C. elegans and molecular embryological studies in Xenopus have identified gene products in PGCs that appear to protect them from inappropriate somatic differentiation. How the identified gene products are "networked" and importantly, the real mechanism through which these preserve totipotency, remain largely unknown. The present application will address this gap in our knowledge. Xenopus is the system of choice for these studies because it offers a unique combination of total accessibility at both the biochemical and embryological level. Based on our previous data, we hypothesize that during the time that germ layers are established in the embryo, totipotency is preserved in PGCs by a combination of targeted translational repression of specific maternal mRNAs and global repression of mRNA transcription controlled by Xcat2. To test our central hypothesis we will complete the following specific aims: 1) Determine how maternal VegT function is restricted in PGCs by assessing VegT stability, translation, and nuclear localization in isolated PGCs using a combination of Real Time RT-PCR, injected tagged VegT transcripts and immunolocalization. 2) Determine the role Xcat2 plays as a translational repressor in preserving PGC totipotency by identifying the RNAs that co-purify with Xcat2 using a pull-down, RT-PCR, and cloning approach. Mis-expression of these RNAs in PGCs will be tested for their effects on PGC fate. 3) Determine what mechanism is responsible for the transient repression of transcription in primordial germ cells by selecting for Xcat2 interacting factors that repress transcription in vivo functional assays. Relevance of this research to public health: Stem cells are of high therapeutic value because of their ability to develop into a wide variety of cell types that could be used in the treatment of degenerative diseases including diabetes, Parkinson's and cardiovascular disease. Our studies on PGCs, prime exemplars of totipotency, will explore how different mechanisms cooperate to preserve genetically naive or ground states in cells. Our findings will be relevant to how adult somatic stem cells maintain a pluripotent condition, important issues in stem cell biology.

描述（由申请人提供）：了解保持细胞全能性或多能性的细胞机制是干细胞和发育生物学的中心目标。原始生殖细胞（PGC）是配子的前体，是最终的干细胞，因为它们保留了全部发育潜力，而其他胚胎细胞的命运逐渐受到限制。我们的长期目标是建立在脊椎动物模式生物爪蟾中 PGC 规范和分化过程中发挥作用的关键调控途径。对果蝇和线虫的遗传学研究以及对非洲爪蟾的分子胚胎学研究已经确定了 PGC 中的基因产物，这些基因产物似乎可以保护它们免受不适当的体细胞分化。所鉴定的基因产物如何“联网”，重要的是，这些基因产物保持全能性的真正机制仍然很大程度上未知。本申请将解决我们知识上的这一空白。非洲爪蟾是这些研究的首选系统，因为它在生化和胚胎学水平上提供了完全可及性的独特组合。根据我们之前的数据，我们假设在胚胎中胚层建立期间，通过结合特定母体 mRNA 的靶向翻译抑制和 Xcat2 控制的 mRNA 转录的全局抑制，PGC 中保留了全能性。为了检验我们的中心假设，我们将完成以下具体目标：1) 通过结合实时 RT-PCR、注射标记 VegT 转录本和免疫定位来评估分离的 PGC 中的 VegT 稳定性、翻译和核定位，确定 PGC 中母体 VegT 功能如何受到限制。 2) 通过使用下拉、RT-PCR 和克隆方法鉴定与 Xcat2 共纯化的 RNA，确定 Xcat2 作为翻译抑制子在保留 PGC 全能性中所发挥的作用。将测试这些 RNA 在 PGC 中的错误表达对 PGC 命运的影响。 3) 通过选择抑制体内转录的 Xcat2 相互作用因子，确定何种机制导致原始生殖细胞中转录的瞬时抑制。这项研究与公共健康的相关性：干细胞具有很高的治疗价值，因为它们能够发育成多种细胞类型，可用于治疗退行性疾病，包括糖尿病、帕金森氏症和心血管疾病。我们对 PGC（全能性的主要范例）的研究将探索不同的机制如何合作以保存细胞中的遗传初始状态或基态。我们的研究结果将与成体干细胞如何维持多能状态相关，这是干细胞生物学中的重要问题。

项目成果

RNA transport to the vegetal cortex of Xenopus oocytes.

• DOI: 10.1006/dbio.1996.0249
• 发表时间: 1996-10
• 期刊: Developmental biology
• 影响因子: 2.7
• 作者: Yi Zhou;M. L. King

Xenopus autosomal recessive hypercholesterolemia protein couples lipoprotein receptors with the AP-2 complex in oocytes and embryos and is required for vitellogenesis.

非洲爪蟾常染色体隐性高胆固醇血症蛋白将脂蛋白受体与卵母细胞和胚胎中的 AP-2 复合物偶联，是卵黄发生所必需的。

• DOI: 10.1074/jbc.m308870200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Zhou,Yi;Zhang,Jian;King,MaryLou
• 通讯作者: King,MaryLou

Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning.

• DOI: 10.1242/dev.122.12.4119
• 发表时间: 1996-12
• 期刊: Development
• 影响因子: 4.6
• 作者: Jian Zhang;M. L. King

Hermes RNA-binding protein targets RNAs-encoding proteins involved in meiotic maturation, early cleavage, and germline development.

Hermes RNA 结合蛋白靶向参与减数分裂成熟、早期分裂和种系发育的 RNA 编码蛋白。

• DOI: 10.1111/j.1432-0436.2006.00155.x
• 发表时间: 2007
• 期刊: Differentiation; research in biological diversity
• 作者: Song,Hye-Won;Cauffman,Karen;Chan,AgnesP;Zhou,Yi;King,MaryLou;Etkin,LaurenceD;Kloc,Malgorzata
• 通讯作者: Kloc,Malgorzata

Patterns of localization and cytoskeletal association of two vegetally localized RNAs, Vg1 and Xcat-2.

两种植物定位 RNA Vg1 和 Xcat-2 的定位模式和细胞骨架关联。

• DOI: 10.1242/dev.121.1.201
• 发表时间: 1995
• 期刊: Development (Cambridge, England)
• 作者: Forristall,C;Pondel,M;Chen,L;King,ML
• 通讯作者: King,ML