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：性发育差异（或障碍）（DSD）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 影响疾病易感性、心理 发展和人的意识。了解遗传因素相互作用的机制 然而，其他与环境有关的直接性别决定（SD）是不完整的。斑马鱼是一种 重要的医学模型，其 SD 系统对未识别的遗传变异敏感且较差 了解环境影响。我们的研究表明，斑马鱼实验室品系具有较弱的、 多态性或非遗传性 SD 机制，但来自印度的天然斑马鱼具有单一、强的性 4 号染色体 (sar4) 上与染色体 ZZ 男性和 ZW 女性的相关区域。 ZZ鱼是 始终是雄性，大多数 ZW 鱼是雌性，但少数是可育的“neomales”，可能是由于遗传 调节剂或环境因素。稀有的WW鱼可以是雄性或雌性。我们和其他人有 在许多候选 SD 基因中发生突变，但没有一个代表 sar4 SD 因子。我们还表明 减数分裂卵母细胞在实验室菌株中是维持雌性发育所必需的，大概是通过发送 向性腺体发出信号。我们的总体目标是确定 sar4 性别的分子遗传基础 决定因素，并发现其指导男性或女性的分子、细胞和器官水平机制 发展。根据一种假设，sar4-W 具有显性的、不同外显性的女性决定基因。 另一种假设涉及 sar4-Z 剂量，两次剂量导致雄性，一次剂量或无剂量 通常给予女性的剂量。无论哪种情况，我们都想知道它如何指定性别。初步结果显示， 我们：1）收集了四个具有天然sar4 SD机制的独立衍生菌株； 2) 制作基因组 几种天然菌株的组装； 3) 开发了检测sar4-Z和sar4-W的PCR引物； 4) 制作 ZZ 和 ZW 鱼的细菌人工染色体 (BAC)，并分离出许多 sar4 BAC；和 5) 对 ZZ 和 ZW 基因型进行单细胞 RNA 测序，鉴定出 ZZ 和 ZW 特异性细胞簇。 sar4-Z 与 sar4-W 的比较发现了一个大的重组抑制倒位、许多插入缺失和 其他多态性。该研究计划的总体愿景是操纵基因组并 揭开斑马鱼 SD 之谜的环境。工作包括：1) 通过以下方式识别 sar4 中的 SD 因子： 进行必要的实验来检查 CRISPR/Cas9 诱导的长缺失的影响 SD 的靶向敲除和使用 BAC 转基因的充分性实验来识别改变的克隆 标清； 2）通过单细胞基因表达研究了解斑马鱼雄性和雌性SD的途径 ZZ 和 ZW 中的各种发育阶段、突变基因型和环境操作 鱼。其意义在于更好地理解基因相互作用和环境对基因的影响。 斑马鱼性别决定将为人类 DSD 提供信息。