Shitikov A D, Parshina E A, Zaraisky A G, Tereshina M B
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Department of Regenerative Medicine, Pirogov Russian National Research Medical University, Moscow, Russia.
Front Cell Dev Biol. 2024 Dec 9;12:1487644. doi: 10.3389/fcell.2024.1487644. eCollection 2024.
Whole-mount hybridization (WISH) is a widely used method that supports the concept of "seeing is believing" by enabling the visualization of gene expression patterns in whole-mount multicellular samples or sections. This technique is essential in the study of epimorphic regeneration in cold-blooded vertebrates, where complex three-dimensional organs such as tails, limbs, and eyes are completely restored after loss. The tadpoles of the frog serve as a convenient model for studying regeneration, as they can regenerate their tails within a week after amputation. Modern high-throughput sequencing methods have identified various cell populations involved in the regeneration process and determined the repertoire of genes activated during this time. Specifically, a population of reparative myeloid cells expressing as a marker gene has been shown to be crucial for the initial stages of tail regeneration in tadpoles. The validation of these data and further examination using WISH offers the advantage of providing detailed information on the spatial and temporal dynamics of target gene expression levels. However, detecting mRNA by WISH can be challenging when mRNA levels are very low, transcripts are localized in hard-to-access areas, or tissue samples are prone to background staining, as is the case with regenerating tail samples. Here, we describe additional treatments for regenerating tail samples that minimize background staining and enhance the visualization of cells containing target RNA through hybridization. Using an optimized WISH protocol on tadpole tail regenerates, we obtained novel data on the expression pattern during the first day post-amputation at the regeneration-competent stage 40 and the regeneration-incompetent stage 47 (refractory period). The significant differences in the expression patterns indicate that activity is positively correlated with regeneration competence.
全胚胎原位杂交（WISH）是一种广泛应用的方法，通过在完整的多细胞样本或切片中可视化基因表达模式，支持“眼见为实”的理念。这项技术在冷血脊椎动物的再生研究中至关重要，在这类研究中，尾巴、四肢和眼睛等复杂的三维器官在缺失后能完全恢复。青蛙蝌蚪是研究再生的便捷模型，因为它们在截肢后一周内就能再生尾巴。现代高通量测序方法已鉴定出参与再生过程的各种细胞群体，并确定了在此期间激活的基因库。具体而言，已证明一群表达作为标记基因的修复性髓样细胞对蝌蚪尾巴再生的初始阶段至关重要。利用WISH对这些数据进行验证并进一步检测，具有提供目标基因表达水平时空动态详细信息的优势。然而，当mRNA水平非常低、转录本位于难以接近的区域或组织样本容易出现背景染色时，如再生尾巴样本的情况，通过WISH检测mRNA可能具有挑战性。在这里，我们描述了对再生尾巴样本的额外处理方法，这些方法可将背景染色降至最低，并通过杂交增强含有目标RNA的细胞的可视化。在蝌蚪尾巴再生样本上使用优化的WISH方案，我们获得了关于在截肢后第一天再生能力阶段40和再生无能力阶段47（不应期）的表达模式的新数据。表达模式的显著差异表明活性与再生能力呈正相关。
