Turner Meghan A, Gravina Nicholas M, Moretti Bruno, Dima Sadia, Martini Gabriella, Reeves Greg, Garcia Hernan G
Biophysics Graduate Group, University of California at Berkeley, Berkeley, CA 94720, USA.
Department of Physics, University of California at Berkeley, Berkeley, CA 94720, USA.
bioRxiv. 2025 May 13:2025.05.12.653543. doi: 10.1101/2025.05.12.653543.
Over the last two decades, new and imaging technologies have uncovered the inherently dynamic nature of transcriptional regulation in embryonic development and, in particular, in the fruit fly . These technologies have made it possible to characterize the subnuclear and single-molecule dynamics of transcription factors. However, a lack of appropriate fluorescent protein fusions has, until now, limited these studies to only a few of the dozens of important transcription factors in the fruit fly gene regulatory network dictating early development. Here, we report the creation of four new fluorescent protein fusions to Dorsal, a member of the NF-B/Rel family that initiates dorsal-ventral patterning. We generated and characterized two bright fluorescent protein fusions for Dorsal, meGFP and mNeonGreen, and two photoconvertible fluorescent protein fusions, mEos4a and Dendra2. We show that removal of the DsRed2 cassette commonly used to mark the CRISPR integration restores endogenous Dorsal mRNA and protein levels and enables the fusion allele to rescue a null allele, meeting the gold standard for endogenous function of a tagged protein in a fruit fly. We then demonstrate that our bright fluorescent protein fusions can be used to dissect the spatiotemporal dynamics of stable Dorsal clusters that traverse the nucleoplasm and uncovered that these clusters preferentially interact with active sites of Dorsal-modulated transcription. We further demonstrate that our photoconvertible fluorescent protein fusions make it possible to detect individual molecules of Dorsal in the nuclei of developing embryos. These new fluorescent protein fusions constitute a valuable resource for the community to elucidate the role of Dorsal activator dynamics in dictating fruit fly early embryonic development.
在过去二十年中，新的成像技术揭示了胚胎发育过程中，尤其是果蝇胚胎发育过程中转录调控内在的动态本质。这些技术使得表征转录因子的亚核和单分子动力学成为可能。然而，直到现在，由于缺乏合适的荧光蛋白融合体，这些研究仅限于果蝇基因调控网络中数十个重要转录因子中的少数几个，这些转录因子决定了早期发育。在这里，我们报告了四种与背腹形态发生起始因子 Dorsal（NF-κB/Rel 家族成员）的新荧光蛋白融合体的创建。我们生成并表征了两种用于 Dorsal 的明亮荧光蛋白融合体 meGFP 和 mNeonGreen，以及两种光转换荧光蛋白融合体 mEos4a 和 Dendra2。我们表明，去除通常用于标记 CRISPR 整合的 DsRed2 盒可恢复内源性 Dorsal mRNA 和蛋白水平，并使融合等位基因能够挽救无效等位基因，这符合果蝇中标记蛋白内源性功能的黄金标准。然后，我们证明我们的明亮荧光蛋白融合体可用于剖析穿越核质的稳定 Dorsal 簇的时空动态，并发现这些簇优先与 Dorsal 调节转录的活性位点相互作用。我们进一步证明，我们的光转换荧光蛋白融合体能够检测发育中胚胎细胞核中的单个 Dorsal 分子。这些新的荧光蛋白融合体为该领域阐明 Dorsal 激活剂动力学在果蝇早期胚胎发育中的作用提供了宝贵资源。
