Mechanisms of Sex Determination in Zebrafish

问题：性发育差异（或性发育障碍）是一个复杂的医学问题， 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 发展和人的感觉。了解遗传因素与每种因素相互作用的机制 其他的与环境直接相关的性别决定（SD）研究则是不完整的。斑马鱼是一种 具有SD系统的重要医学模型，该系统对未识别的遗传变异敏感， 了解环境的影响。我们的研究表明，斑马鱼实验室菌株有弱， 多态性，或非遗传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号染色体上的相关区域（sar 4），具有染色体ZZ男性和ZW女性。ZZ鱼 通常是雄性，大多数ZW鱼是雌性，但少数是可生育的“新雄性”，可能是由于遗传原因。 改性剂或环境因素。罕见的WW鱼可以是男性或女性。我们和其他人已经 在许多候选SD基因中进行了突变，但没有一个代表sar 4 SD因子。我们还发现， 减数分裂卵母细胞在实验室品系中是维持雌性发育所必需的，大概是通过发送一个 向性腺索马发出信号。我们的总体目标是确定sar 4性别的分子遗传基础。 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，sar 4-W有一个显性的、非显性的、决定雌性的基因。 另一种假设涉及sar 4-Z剂量，两种剂量导致雄性，一种剂量或没有 通常给女性的剂量。无论哪种情况，我们都想知道它是如何指定性别的。初步结果显示， 我们：1）收集了4株具有天然sar 4 SD机制的独立衍生菌株; 2）构建了基因组 3）开发了检测sar 4-Z和sar 4-W的PCR引物; ZZ和ZW鱼的细菌人工染色体（BAC），并分离出许多sar 4 BAC;以及5） 对ZZ和ZW基因型进行了单细胞RNA-seq，鉴定了ZZ和ZW特异性细胞簇。 sar 4-Z与sar 4-W的比较鉴定了大的重组抑制倒位，许多插入缺失， 其他多态性。该研究计划的总体愿景是操纵基因组， 环境来揭开斑马鱼SD的奥秘。工作包括：1）通过以下方法确定SAR 4中的SD因子： 进行必要性实验，检查CRISPR/Cas9诱导的长缺失的影响， SD上的靶向敲除和使用BAC转基因的充分性实验，以鉴定改变 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的各种发育阶段、突变基因型和环境操作中， 鱼.其意义将是更好地了解基因相互作用和环境对 斑马鱼的性别决定，将告知人类的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.