Kryptolebiasmarmoratus(Kmar),一种硬骨鱼,为类鲤鱼,有一套在其他鱼类中没有观察到的独特表型和行为。这些表型中的许多都是离散的,并且随着时间的推移在个体中是高度可塑性变化的,在某些情况下是可逆的。Kmar及其干扰性姐妹物种,K.雌雄同体,是唯一已知的自育脊椎动物.这种不寻常的性模式有可能为脊椎动物性发育的调节提供独特的见解,也适合遗传学。Kmar很容易适应实验室,几乎不需要维护。然而,它的内部施肥和小离合器尺寸限制了它的实验使用。为了支持Kmar作为一个遗传模型,我们比较了替代饲养技术,以最大程度地恢复早期卵裂期胚胎。我们发现频繁收集鸡蛋可以提高产量,蛋白酶处理促进了最大的孵化成功。我们完成了正向诱变筛选,并回收了几个突变系,这些突变系在该模型中用作遗传学的重要工具。几个将用作标记杂交的有用可行的隐性标记。重要的是,突变的kissylips以两倍于野生型的速度产下胚胎。将频繁的卵子收集与kissylips突变体背景相结合,可以大大提高早期胚胎产量。这些改进足以允许早期发育的实验分析以及用CRISPR/Cas9核酸酶成功地进行内源性酪氨酸酶基因的单等位基因和双等位基因靶向敲除。总的来说,这些工具将促进这种迷人鱼类的现代发育遗传学,导致未来对可塑性调节的见解。
Kryptolebias marmoratus (Kmar), a teleost fish of the order
Cyprinodontiformes, has a suite of unique phenotypes and behaviors not observed in other fishes. Many of these phenotypes are discrete and highly plastic-varying over time within an individual, and in some cases reversible. Kmar and its interfertile sister species, K. hermaphroditus, are the only known self-fertile vertebrates. This unusual sexual mode has the potential to provide unique insights into the regulation of vertebrate sexual development, and also lends itself to genetics. Kmar is easily adapted to the lab and requires little maintenance. However, its internal fertilization and small clutch size limits its experimental use. To support Kmar as a genetic model, we compared alternative husbandry techniques to maximize recovery of early cleavage-stage embryos. We find that frequent egg collection enhances yield, and that protease treatment promotes the greatest hatching success. We completed a forward mutagenesis screen and recovered several mutant lines that serve as important tools for genetics in this model. Several will serve as useful viable recessive markers for marking crosses. Importantly, the mutant kissylips lays embryos at twice the rate of wild-type. Combining frequent egg collection with the kissylips mutant background allows for a substantial enhancement of early embryo yield. These improvements were sufficient to allow experimental analysis of early development and the successful mono- and bi-allelic targeted knockout of an endogenous tyrosinase gene with CRISPR/Cas9 nucleases. Collectively, these tools will facilitate modern developmental genetics in this fascinating fish, leading to future insights into the regulation of plasticity.