Fang Junnan, Brockett Jovan S, Tian Weiyi, Hua Yuqing, Gavis Elizabeth R, Lerit Dorothy A
Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia.
Current affiliation: Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, Ohio.
Curr Protoc. 2025 May;5(5):e70145. doi: 10.1002/cpz1.70145.
Embryogenesis necessitates the precise orchestration of cellular events to establish tissue patterning, developmental robustness, and viability. Syncytial embryogenesis, as in Drosophila melanogaster, poses added challenges as the synchronous and rapid nuclear divisions prior to cellularization occur within a shared cytoplasm. While the first several rounds of nuclear divisions occur within the center of the embryo, the nuclei progressively migrate peripherally, giving rise to the syncytial blastoderm. This spatial choreography hinges upon the dynamic interplay of actin and microtubules. Actin and microtubules coordinate nuclear division and position while preventing deleterious nuclear collisions. Additionally, the cytoskeleton also facilitates the segregation of organelles and molecular cargoes, including cell fate determinants required for cellular differentiation. As development progresses, actin and microtubules drive cellularization events for both germline and somatic cell lineages. Cytoskeletal disruption causes developmental arrest and embryonic lethality, underscoring its importance for embryogenesis. Given the significance of the cytoskeleton to these events, its visualization remains a cornerstone of cell and developmental biology research. Indeed, studies of the Drosophila embryo cytoskeleton have yielded valuable insights into cell biological mechanisms and developmental pathways conserved in various systems. Nevertheless, achieving optimal preservation of filamentous cytoskeletal structures poses technical challenges. Here, we present an embryo fixation method tailored to enhance the visualization of actin and microtubules via standard light microscopy approaches. This protocol complements immunofluorescence and molecular labeling techniques, including the direct labeling of fluorescently tagged proteins or mRNAs. By enabling detailed analysis of the cytoskeleton, this method expands opportunities to investigate the molecular mechanisms underlying embryo development and related processes. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Preparation of embryos for immunofluorescence of actin or microtubules Basic Protocol 2: Coupling immunofluorescence of the cytoskeleton with visualization of mRNAs via smFISH.
胚胎发生需要精确协调细胞事件，以建立组织模式、发育稳健性和生存能力。像在黑腹果蝇中那样的合胞体胚胎发生带来了额外的挑战，因为在细胞化之前的同步且快速的核分裂发生在共享的细胞质中。虽然最初几轮核分裂发生在胚胎中心，但细胞核逐渐向周边迁移，形成合胞体胚盘。这种空间编排取决于肌动蛋白和微管的动态相互作用。肌动蛋白和微管协调核分裂和位置，同时防止有害的核碰撞。此外，细胞骨架还促进细胞器和分子货物的分离，包括细胞分化所需的细胞命运决定因素。随着发育的进行，肌动蛋白和微管驱动生殖系和体细胞谱系的细胞化事件。细胞骨架破坏会导致发育停滞和胚胎致死，突出了其对胚胎发生的重要性。鉴于细胞骨架对这些事件的重要性，其可视化仍然是细胞和发育生物学研究的基石。事实上，对果蝇胚胎细胞骨架的研究已经对各种系统中保守的细胞生物学机制和发育途径产生了有价值的见解。然而，实现丝状细胞骨架结构的最佳保存带来了技术挑战。在这里，我们提出一种胚胎固定方法，旨在通过标准光学显微镜方法增强肌动蛋白和微管的可视化。该方案补充了免疫荧光和分子标记技术，包括对荧光标记蛋白或mRNA的直接标记。通过能够对细胞骨架进行详细分析，该方法扩大了研究胚胎发育及相关过程潜在分子机制的机会。© 2025威利期刊有限责任公司。基本方案1：用于肌动蛋白或微管免疫荧光的胚胎制备 基本方案2：通过单分子荧光原位杂交将细胞骨架免疫荧光与mRNA可视化相结合。
