Department of Biology, University of Maryland, College Park, Maryland, USA.
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
J Exp Zool B Mol Dev Evol. 2024 May;342(3):164-177. doi: 10.1002/jez.b.23216. Epub 2023 Aug 8.
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.
大理石无须鱲（Kmar），一种属于鲤形目鱲科的硬骨鱼，具有一系列在其他鱼类中观察不到的独特表型和行为。这些表型中有许多是离散的，且高度可塑——在个体内部随时间变化，在某些情况下甚至是可逆的。大理石无须鱲及其可杂交的姐妹种，两性无须鱲（K. hermaphroditus），是已知唯一的自交可育脊椎动物。这种不寻常的性模式有可能为脊椎动物性发育的调控提供独特的见解，也适合遗传学研究。大理石无须鱲很容易适应实验室环境，维护需求低。然而，其体内受精和小的卵窝大小限制了其在实验中的应用。为了支持大理石无须鱲作为遗传模型，我们比较了几种替代的饲养技术，以最大限度地提高早期卵裂期胚胎的回收量。我们发现，频繁的卵收集可以提高产量，而蛋白酶处理则可以提高孵化成功率。我们完成了正向诱变筛选，并获得了几个突变系，这些突变系是该模型遗传学研究的重要工具。其中几个将作为有用的隐性可育标记，用于标记杂交。重要的是，突变型 kissylips 的产卵率是野生型的两倍。将频繁的卵收集与 kissylips 突变背景相结合，可以大大提高早期胚胎的产量。这些改进足以允许对早期发育进行实验分析，并成功地用 CRISPR/Cas9 核酸酶对一个内源性酪氨酸酶基因进行单等位基因和双等位基因的靶向敲除。这些工具将共同促进这个迷人鱼类的现代发育遗传学研究，为研究可塑性的调控提供未来的见解。
