MeLiS, CNRS UMR 5284 - INSERM U1314, Université Claude Bernard Lyon 1, 8 avenue Rockefeller, 69008, Lyon, France.
BMC Biol. 2024 Jun 3;22(1):131. doi: 10.1186/s12915-024-01922-0.
Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity.
We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons.
We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.
精细地表征基因表达模式对于理解胚胎发育的许多方面至关重要。鸡胚胎是发育生物学中一种成熟且有价值的动物模型。从第三天到第六天（E3 到 E6）的胚胎期对于许多器官的发育至关重要。杂交链反应 RNA 荧光原位杂交（HCR RNA-FISH）能够在包括各种动物模型胚胎在内的厚样本中进行多重 RNA 检测。然而，其应用受到组织不透明性的限制。
我们优化了 HCR RNA-FISH 方案，以有效地标记从 E3.5 到 E5.5 的鸡胚整体胚胎中的 RNA，并将其适应乙基肉桂酸（ECi）组织透明化。我们表明，ECi 透明化后进行光片成像 HCR RNA-FISH 允许在胚胎组织内进行 RNA 表达分析，具有良好的灵敏度和空间分辨率。最后，在 HCR RNA-FISH 之后可以进行全胚胎免疫荧光染色，例如可以检测轴突与其环境之间的复杂空间关系，或监测 GFP 电转染神经元。
我们通过优化 HCR RNA-FISH 并将其与组织透明化和 3D 成像相结合，将 HCR RNA-FISH 的应用扩展到了较老的鸡胚。免疫染色的整合使得将基因表达与经典的细胞标志物结合、将表达与形态分化相关联以及在获得或丧失功能的情况下描绘基因表达成为可能。总之，这种联合程序进一步扩展了 HCR RNA-FISH 技术在鸡胚胎学中的潜力。
