Germline Stem Cell Establishment and the Role of Dazl in Gametogenesis

生殖干细胞的建立和 Dazl 在配子发生中的作用

• 批准号: 10752077
• 负责人: Maya Pahima
• 金额: $ 4.61万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-11-01 至 2026-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752077
• 关键词: Adopted; Adult; Alleles; Animals; Antibodies; Apoptotic; Automobile Driving; Biological; Cell Differentiation process; Cells; Cloning; Contraceptive methods; Cyst; Data; Data Set; Development; Ethylnitrosourea; Event; Female; Fertility; Fostering; Gametogenesis; Generations; Genes; Genetic; Genetic Models; Genetic Transcription; Genomics; Germ Cells; Heterozygote; Human; Infertility; Knowledge; Laboratories; Maintenance; Meiosis; Messenger RNA; Modeling; Molecular; Outcome; Ovary; Population; Process; Proteins; Publishing; RNA-Binding Proteins; Reporter Genes; Reproductive Health; Research; Research Personnel; Role; Specific qualifier value; Stem Cell Development; Structure of primordial sex cell; System; Techniques; Testing; Training; Transcript; Transgenic Organisms; Translations; Validation; Vertebrates; Woman; Zebrafish; cell type; chemotherapy; design; egg; experimental study; germline stem cells; inducible Cre; male; mutant; photoactivation; premature; preservation; prevent; progenitor; protein biomarkers; protein expression; reproductive; reproductive development; reproductive fitness; single-cell RNA sequencing; skills; stem cell fate; stem cell fate specification; stem cell population; stem cells; tool

PROJECT SUMMARY In all animals, including humans, establishment and maintenance of the germline stem cells (GSCs) is critical for life-long reproductive health and fitness. Yet, how GSCs are specified in vertebrates remains largely unknown. GSCs arise from primordial germ cells (PGCs) and serve to establish, renew, and expand the germ cell population. Differentiation of PGCs to GSCs is conserved and critical for establishment of the germline, yet a major gap in our knowledge of reproductive development and fertility remains understanding what factors influence this process. To examine early GSC development, I am using zebrafish – a genetically tractable model with high fecundity and rapid external development. Recently published data from our laboratory showed that, in zebrafish, PGCs lacking the conserved germline-specific RNA-binding protein Dazl (Deleted in Azoospermia- like) fail to differentiate into GSCs and are lost shortly thereafter. Dazl is a translational regulator which was previously implicated in meiosis; however, our finding that dazl mutant PGCs fail to differentiate raises interesting questions as to how GSCs arise from PGCs, and the specific role(s) of Dazl during this process. I hypothesize that Dazl translationally regulates its associated factors during PGC differentiation to maintain PGC germline fate and promote GSC establishment. However, Dazl has been characterized as both an activator and repressor of translation of thousands of germline targets in various contexts, making the specific mechanisms by which it promotes GSC establishment unclear. Herein, I propose to functionally define Dazl’s role in GSC establishment using a combination of genetic and molecular approaches. Specifically, my proposed research strategy will (1) trace dazl mutant germ cell fates to determine whether Dazl maintains PGC identity, (2) screen for GSC-specific markers to investigate the mechanism of GSC fate establishment, and (3) test the functions of conserved Dazl- associated mRNAs which I hypothesize are translationally regulated by Dazl during PGC differentiation. Together, these approaches will delineate Dazl’s role in PGC differentiation and GSC specification. More broadly, these studies may shed light on mechanisms to maintain, renew, and prevent premature loss of the GSC population, which has important implications for reproductive health. Completion of these aims will provide rigorous training in all aspects of genetics-based functional analysis, including generation, characterization, and validation of mutant and transgenic zebrafish lines, Cre-based lineage tracing, and genomic cloning, which will foster my development as a successful independent investigator committed to using genetic models to study reproductive development.

项目摘要 在包括人类在内的所有动物中，生殖系干细胞（GSC）的建立和维持至关重要 终身生殖健康和健身。然而，如何在脊椎动物中指定GSC在很大程度上仍然是 未知GSC起源于原始生殖细胞（PGCs），并用于建立、更新和扩增生殖细胞。 细胞群PGCs向GSC的分化是保守的，并且对于种系的建立是关键的，然而， 我们对生殖发育和生育能力的认识中的一个主要差距仍然是理解哪些因素 影响这个过程。为了研究早期GSC的发展，我使用了斑马鱼--一种遗传学上易于处理的模型 繁殖力强，外部发育快。我们实验室最近公布的数据显示， 在斑马鱼中，缺乏保守的生殖系特异性RNA结合蛋白Dazl的PGCs（在无精子症中， 类似）不能分化成GSC，并且在此后不久丢失。Dazl是一种翻译调节因子， 以前参与减数分裂;然而，我们发现dazl突变体PGCs不能分化引起了人们的兴趣， 关于GSC如何从PGCs产生的问题，以及Dazl在此过程中的具体作用。我假设 在PGC分化过程中，Dazl通过调控相关因子来维持PGC胚系 命运，促进GSC的建立。然而，Dazl已被表征为激活剂和阻遏物 在不同的背景下，成千上万的生殖系目标的翻译，使其特定的机制， 推动GSC的建立不明确。在此，我建议从功能上定义Dazl在GSC建立中的作用 使用遗传和分子方法的组合。具体而言，我提出的研究战略将（1） 追踪dazl突变体生殖细胞命运以确定Dazl是否保持PGC身份，（2）筛选GSC特异性的 标记，以探讨GSC命运建立的机制，（3）测试保守的Dazl- 相关的mRNA，我假设在PGC分化过程中由Dazl进行调节。 总之，这些方法将描绘Dazl在PGC分化和GSC特化中的作用。更 广义上说，这些研究可能有助于阐明维持、更新和防止过早丧失的机制。 GSC人口，这对生殖健康具有重要影响。这些目标的实现将为 在基于遗传学的功能分析的各个方面进行严格的培训，包括生成，表征， 突变和转基因斑马鱼品系的验证，基于Cre的谱系追踪和基因组克隆，这将 培养我作为一个成功的独立研究者的发展，致力于使用遗传模型来研究 生殖发育