Drosophila has long been a successful model organism in multiple biomedical fields. Spatial gene expression patterns are critical for the understanding of complex pathways and interactions, whereas temporal gene expression changes are vital for studying highly dynamic physiological activities. Systematic studies in Drosophila are still impeded by the lack of spatiotemporal transcriptomic information. Here, utilizing spatial enhanced resolution omics-sequencing (Stereo-seq), we dissected the spatiotemporal transcriptomic changes of developing Drosophila with high resolution and sensitivity. We demonstrated that Stereo-seq data can be used for the 3D reconstruction of the spatial transcriptomes of Drosophila embryos and larvae. With these 3D models, we identified functional subregions in embryonic and larval midguts, uncovered spatial cell state dynamics of larval testis, and revealed known and potential regulons of transcription factors within their topographic background. Our data provide the Drosophila research community with useful resources of organism-wide spatiotemporally resolved transcriptomic information across developmental stages.

果蝇长期以来一直是多个生物医学领域的成功模式生物。空间基因表达模式对于理解复杂的途径和相互作用至关重要，而时间基因表达变化对于研究高度动态的生理活动至关重要。由于缺乏时空转录组信息，果蝇的系统研究仍然受到阻碍。在这里，利用空间增强分辨率组学测序（Stereo-seq），我们以高分辨率和灵敏度剖析了发育中的果蝇的时空转录组变化。我们证明了 Stereo-seq 数据可用于 3D 重建果蝇的空间转录组胚胎和幼虫。通过这些 3D 模型，我们确定了胚胎和幼虫中肠中的功能亚区，揭示了幼虫睾丸的空间细胞状态动态，并揭示了其地形背景中已知和潜在的转录因子调节子。我们的数据为果蝇研究界提供了跨发育阶段的有机体时空解析转录组信息的有用资源。