School of BioSciences, The University of Melbourne, Parkville Campus, Melbourne, Victoria 3010, Australia.
G3 (Bethesda). 2024 Nov 6;14(11). doi: 10.1093/g3journal/jkae204.
Ecdysteroids are major hormones in insects and control molting, growth, reproduction, physiology, and behavior. The biosynthesis of ecdysteroids such as 20-hydroxyecdysone (20E) from dietary sterols is well characterized, but ecdysteroid catabolism is poorly understood. Ecdysteroid kinases (EcKs) mediate the reversible phosphorylation of ecdysteroids, which has been implicated in ecdysteroid recycling during embryogenesis and reproduction in various insects. However, to date, only 2 EcK-encoding genes have been identified, in the silkworm Bombyx mori and the mosquito Anopheles gambiae. Previously, we identified 2 ecdysteroid kinase-like (EcKL) genes-Wallflower (Wall) and Pinkman (pkm)-in the model fruit fly Drosophila melanogaster that are orthologs of the ecdysteroid 22-kinase gene BmEc22K. Here, using gene knockdown, knockout, and misexpression, we explore Wall and pkm's possible functions and genetically test the hypothesis that they encode EcKs. Wall and pkm null mutants are viable and fertile, suggesting that they are not essential for development or reproduction, whereas phenotypes arising from RNAi and somatic CRISPR appear to derive from off-target effects or other artifacts. However, misexpression of Wall results in dramatic phenotypes, including developmental arrest, and defects in trachea, cuticle, and pigmentation. Wall misexpression fails to phenocopy irreversible ecdysteroid catabolism through misexpression of Cyp18a1, suggesting that Wall does not directly inactivate 20E. Additionally, Wall misexpression phenotypes are not attenuated in Cyp18a1 mutants, strongly suggesting that Wall is not an ecdysteroid 26-kinase. We hypothesize that the substrate of Wall in this misexpression experiment and possibly generally is an unknown, atypical ecdysteroid that plays essential roles in Drosophila development, and may highlight aspects of insect endocrinology that are as-yet uncharacterized. We also provide preliminary evidence that CG5644 encodes an ecdysteroid 22-kinase conserved across Diptera.
蜕皮甾酮是昆虫的主要激素，控制蜕皮、生长、繁殖、生理和行为。蜕皮甾酮（如 20-羟蜕皮甾酮（20E））从膳食固醇的生物合成已经得到很好的描述，但蜕皮甾酮的分解代谢知之甚少。蜕皮甾酮激酶（EcKs）介导蜕皮甾酮的可逆磷酸化，这与各种昆虫胚胎发生和繁殖过程中蜕皮甾酮的再循环有关。然而，迄今为止，仅在家蚕 Bombyx mori 和疟蚊 Anopheles gambiae 中鉴定出 2 个 EcK 编码基因。以前，我们在模式果蝇 Drosophila melanogaster 中鉴定出 2 个蜕皮甾酮激酶样（EcKL）基因-Wallflower（Wall）和 Pinkman（pkm），它们是蜕皮甾酮 22-激酶基因 BmEc22K 的同源物。在这里，我们使用基因敲低、敲除和异位表达，探索了 Wall 和 pkm 的可能功能，并从遗传上验证了它们编码 EcKs 的假设。Wall 和 pkm 缺失突变体是可行和可育的，这表明它们不是发育或繁殖所必需的，而 RNAi 和体细胞 CRISPR 引起的表型似乎源自脱靶效应或其他假象。然而，Wall 的异位表达导致了显著的表型，包括发育停滞以及气管、表皮和色素沉着缺陷。Wall 的异位表达不能通过 Cyp18a1 的异位表达来模拟不可逆的蜕皮甾酮分解代谢，表明 Wall 不能直接使 20E 失活。此外，在 Cyp18a1 突变体中，Wall 异位表达的表型没有减弱，这强烈表明 Wall 不是一种蜕皮甾酮 26-激酶。我们假设，在这个异位表达实验中 Wall 的底物，可能是一种未知的、非典型的蜕皮甾酮，它在果蝇发育中起着至关重要的作用，并且可能突出了昆虫内分泌学中尚未被描述的方面。我们还提供了初步证据，表明 CG5644 编码一种保守的双翅目 22-激酶。
