Makkinje Wouter P D, Simon Sabrina, Breukink Inge, Verbaarschot Patrick, Machida Ryuichiro, Schranz M Eric, van Velzen Robin
Biosystematics group, Wageningen University & Research, Droevendaalsesteeg 1, Wageningen, The Netherlands, 6708PB.
Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Sugadaira Kogen, Ueda, Nagano, 386-2204, Japan.
BMC Genomics. 2024 Dec 4;25(1):1177. doi: 10.1186/s12864-024-10934-7.
Over the course of evolution, insects have seen drastic changes in their mode of development. While insects with derived modes of development have been studied extensively, information on ancestral modes of development is lacking. To address this, we selected a member of one of the earliest lineages of extant flying insects, serving as an outgroup to the modern winged insects, the short germ, non-model mayfly Ephemera vulgata Linnaeus (Insecta: Ephemeroptera, Ephemeridae). We document the embryonic morphology throughout its development and establish a global temporal expression atlas.
DAPI staining was used to visualise developmental morphology to provide a frame of reference for the sequenced timepoints. A transcriptome was assembled from 3.2 billion Illumina RNAseq reads divided in 12 timepoints with 3 replicates per timepoint consisting of 35,091 putative genes. We identified 6,091 significantly differentially expressed genes (DEGs) and analysed them for broad expression patterns via gene ontology (GO) as well as for specific genes of interest. This revealed a U-shaped relationship between the sum of DEGs and developmental timepoints, over time, with the lowest number of DEGs at 72 hours after egg laying (hAEL). Based on a principal component analysis of sequenced timepoints, overall development could be divided into four stages, with a transcriptional turning point around katatrepsis. Expression patterns of zld and smg showed a persistent negative correlation and revealed the maternal-to-zygotic transition (MZT), occurring 24 hAEL. The onset of development of some major anatomical structures, including the head, body, respiratory system, limb, muscle, and eye, are reported. Finally, we show that the ancestral short germ sequential mode of segmentation translates to a sequential Hox gene activation and find diverging expression patterns for lab and pb. We incorporate these patterns and morphological observations to an overview of the developmental timeline.
With our comprehensive differential expression study, we demonstrate the versatility of our global temporal expression atlas. It has the capacity to contribute significantly to phylogenetic insights in early-diverging insect developmental biology and can be deployed in both molecular and genomic applications for future research.
在进化过程中，昆虫的发育模式发生了巨大变化。虽然具有衍生发育模式的昆虫已得到广泛研究，但关于祖先发育模式的信息却很缺乏。为了解决这个问题，我们选择了现存飞行昆虫最早谱系之一的成员，即短胚、非模式蜉蝣（Ephemera vulgata Linnaeus，昆虫纲：蜉蝣目，蜉蝣科），作为现代有翅昆虫的外类群。我们记录了其整个发育过程中的胚胎形态，并建立了一个全局时间表达图谱。
使用DAPI染色来可视化发育形态，为测序时间点提供参考框架。从32亿条Illumina RNA测序读数中组装了一个转录组，这些读数分为12个时间点，每个时间点有3个重复，共包含35,091个推定基因。我们鉴定出6,091个显著差异表达基因（DEG），并通过基因本体论（GO）分析它们的广泛表达模式以及感兴趣的特定基因。这揭示了随着时间推移，DEG总和与发育时间点之间呈U形关系，产卵后72小时（hAEL）时DEG数量最少。基于测序时间点的主成分分析，整体发育可分为四个阶段，在胚胎反转期左右有一个转录转折点。zld和smg的表达模式显示出持续的负相关，并揭示了母源 - 合子转变（MZT）发生在24 hAEL。报告了一些主要解剖结构，包括头部、身体、呼吸系统、肢体、肌肉和眼睛的发育起始。最后，我们表明祖先的短胚顺序分节模式转化为顺序的Hox基因激活，并发现lab和pb的不同表达模式。我们将这些模式和形态学观察结果整合到发育时间线的概述中。
通过我们全面的差异表达研究，我们证明了全局时间表达图谱的多功能性。它有能力为早期分化昆虫发育生物学的系统发育见解做出重大贡献，并可用于未来研究的分子和基因组应用中。
