Mechanisms of Sex Determination in Zebrafish

斑马鱼性别决定机制

• 批准号: 10541144
• 负责人: JOHN H. POSTLETHWAIT
• 金额: $ 36.88万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10541144
• 关键词: Bacterial Artificial Chromosomes; Biological Assay; CRISPR/Cas technology; Cell Communication; Cells; Chromosomes; Complex; Development; Disease; Disease susceptibility; Dose; Environment; Environmental Risk Factor; Female; Fishes; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genetic Variation; Genome; Genotype; Goals; Human; India; Knock-out; Knowledge; Learning; Medical; Meiosis; Minority; Modeling; Molecular; Molecular Genetics; Mutation; Oocytes; Organ; Pathway interactions; Persons; Predisposition; Research; Sexual Development; Signal Transduction; Specific qualifier value; System; Vision; Work; Zebrafish; body system; cell type; developmental genetics; dosage; experimental study; gene interaction; insertion/deletion mutation; male; mutant; neuronal cell body; non-genetic; programs; psychologic; sex; sex determination; single-cell RNA sequencing

The Problem: Differences in (or Disorders of) Sex Development (DSD) are complex medical issues that involve multiple interacting cell types and organ systems. DSDs impact disease susceptibility, psychological development, and sense of person. Knowledge of mechanisms by which genetic factors interact with each other and with the environment to direct sex determination (SD), however, is incomplete. Zebrafish is an important medical model with a SD system that is sensitive to unidentified genetic variation and poorly understood environmental influences. Our research showed that zebrafish lab strains have weak, polymorphic, or non-genetic SD mechanisms, but natural zebrafish from India have a single, strong Sex- Associated Region on chromosome-4 (sar4) with chromosomally ZZ males and ZW females. ZZ fish are always males and most ZW fish are females, but a minority are fertile `neomales', likely due to genetic modifiers or environmental factors. Rare WW fish can be either males or females. We and others have made mutations in many candidate SD genes, but none represent the sar4 SD factor. We also showed that meiotic oocytes are necessary in lab strains to maintain female development, presumably by sending a signal to the gonadal soma. Our overall goals are to identify the molecular genetic basis of the sar4 sex determinant, and to discover its molecular, cellular, and organ level mechanisms to direct male or female development. Under one hypothesis, sar4-W has a dominant, variably penetrant, female-determining gene. An alternative hypothesis invokes sar4-Z dosage, with two doses resulting in a male and one dose or no doses usually giving females. In either case, we want to know how it specifies sex. In preliminary results, we: 1) collected four independently derived strains with the natural sar4 SD mechanism; 2) made genome assemblies for several natural strains; 3) developed PCR primers to assay sar4-Z and sar4-W; 4) made Bacterial Artificial Chromosomes (BACs) for ZZ and for ZW fish and isolated many sar4 BACs; and 5) performed single-cell RNA-seq on ZZ and ZW genotypes, which identified ZZ- and ZW-specific cell clusters. A comparison of sar4-Z to sar4-W identified a large recombination-suppressing inversion, many indels, and other polymorphisms. The overall vision of the research program is to manipulate genomes and environments to uncover the mysteries of zebrafish SD. Work includes: 1) identifying SD factor(s) in sar4 by performing necessity experiments that examine the effects of CRISPR/Cas9-induced long deletions and targeted knockouts on SD and sufficiency experiments using BAC transgenesis to identify clones that alter SD; and 2) to learn the pathways of male and female SD in zebrafish by single-cell gene expression studies in a variety of developmental stages, mutant genotypes, and environmental manipulations in ZZ and ZW fish. The significance will be a better understanding of gene interactions and environmental influences on zebrafish sex determination that will inform human DSDs.

问题：性发展（DSD）（DSD）的差异（或疾病）是复杂的医学问题 涉及多种相互作用的细胞类型和器官系统。 DSD会影响疾病易感性，心理 发展和人物感。了解遗传因素与每种遗传因素相互作用的机制知识 然而，其他并且与指导性别确定的环境（SD）是不完整的。斑马鱼是一个 具有SD系统的重要医疗模型，对身份不明的遗传变异敏感并且很差 了解环境影响。我们的研究表明，斑马鱼实验室菌株较弱， 多态性或非遗传SD机制，但来自印度的天然斑马鱼具有一个强烈的性别 - 染色体-4（SAR4）的相关区域与染色体ZZ男性和ZW女性相关区域。 ZZ鱼是 总是男性和大多数ZW鱼是女性，但少数是肥沃的“新核心”，可能是由于遗传 修饰符或环境因素。罕见的WW鱼可以是雄性或女性。我们和其他人有 在许多候选SD基因中进行突变，但没有一个代表SAR4 SD因子。我们还表明 在实验室菌株中，需要减数分裂卵母细胞维持女性发育，大概是通过发送 向性腺躯体发出信号。我们的总体目标是确定SAR4性别的分子遗传基础 决定簇，并发现其分子，细胞和器官水平机制指向雄性或女性 发展。在一个假设下，SAR4-W具有主导的，多样的渗透性，雌性决定的基因。 另一种假设调用SAR4-Z剂量，两剂导致雄性和一个剂量 剂量通常给女性。无论哪种情况，我们都想知道它是如何指定性别的。在初步结果中， 我们：1）通过天然SAR4 SD机制收集了四个独立衍生的菌株； 2）制作基因组 几种天然菌株的组装； 3）开发了PCR引物来测定SAR4-Z和SAR4-W； 4）制造 用于ZZ和ZW鱼的细菌人造染色体（BAC），并隔离了许多SAR4 BAC； 5） 在ZZ和ZW基因型上进行了单细胞RNA-Seq，该基因型鉴定了ZZ-和ZW特异性细胞簇。 SAR4-Z与SAR4-W的比较确定了大型重组抑制反转，许多indels和 其他多态性。研究计划的整体愿景是操纵基因组和 环境揭示斑马鱼SD的奥秘。工作包括：1）识别SAR4中的SD因子 执行必要实验，以检查CRISPR/CAS9引起的长删除和 使用BAC转基因识别改变的克隆的SD和足够实验的靶向敲除 SD; 2）通过单细胞基因表达研究来学习斑马鱼中男性和女性SD的途径 在各种发育阶段，突变基因型和ZZ和ZW中的环境操纵 鱼。意义将更好地理解基因相互作用和对环境的影响 斑马鱼的性决定将为人类DSD提供信息。