Mechanisms of Sex Determination in Zebrafish

问题：性发育差异(或性发育障碍)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。DSDS影响疾病易感性、心理 发展，人的感觉。关于遗传因素相互作用的机制的知识 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 具有SD系统的重要医学模型，该模型对未知的遗传变异敏感，且质量较差 了解环境影响。我们的研究表明，斑马鱼实验室品系具有微弱的， 多态的，或非遗传的SD机制，但来自印度的天然斑马鱼有一个单一的，强大的性别- 4号染色体上的关联区(Sar4)与染色体上的ZZ雄性和ZW雌性。ZZ鱼是 通常情况下，雄性和大多数ZW鱼都是雌性，但有一小部分是有生育能力的，这很可能是由于遗传原因 修饰物或环境因素。稀有的WW鱼可以是雄鱼也可以是雌鱼。我们和其他人有 在许多候选SD基因中发生了突变，但没有一个代表sr4SD因子。我们还展示了 在实验室品系中，减数分裂卵母细胞是维持雌性发育所必需的，推测是通过发送一个 向性腺胞体发出信号。我们的总体目标是确定sr4性别的分子遗传学基础。 决定因素，并发现其分子、细胞和器官水平的机制，以指导男性或女性 发展。在一种假设下，Sar4-W有一个显性的、可变渗透性的女性决定基因。 另一种假设是使用Sar4-Z剂量，两次剂量导致男性，一次剂量或不 通常给予雌性的剂量。无论是哪种情况，我们都想知道它是如何定义性别的。在初步结果中， 我们：1)收集了4株独立衍生的具有天然sr4-sd机制的菌株；2)制作了基因组。 几种天然菌株的组装；3)开发了检测sR4-Z和sR4-W的聚合酶链式反应引物；4)制作了 ZZ和ZW FISH的细菌人工染色体(BAC)，并分离出多个Sar4 BAC； 对ZZ和ZW基因型进行单细胞RNA-SEQ，鉴定出ZZ和ZW特有的细胞团。 比较sR4-Z和sR4-W发现了一种大的重组-抑制反转，许多INDELs，和 其他多态。该研究计划的总体愿景是操纵基因组和 环境揭开斑马鱼SD的神秘面纱。工作包括：1)确定sR4中的SD因素(S) 进行必要性实验，检查CRISPR/Cas9诱导的长缺失和 使用BAC转基因对SD进行靶向敲除和充分性实验以识别改变的克隆 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 在ZZ和ZW的不同发育阶段，突变的基因类型和环境控制 鱼。其重要意义将是更好地理解基因相互作用和环境对 斑马鱼的性别决定将告知人类DSD。

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.