Whitney Laboratory for Marine Bioscience, University of Florida, St. Augustine, FL, USA.
Department of Neuroscience and McKnight Brain Institute, University of Florida, Gainesville, FL, USA.
Methods Mol Biol. 2024;2757:215-237. doi: 10.1007/978-1-0716-3642-8_9.
In situ hybridization is a powerful and precise tool for revealing cell- and tissue-specific gene expression and a critical approach to validating single-cell RNA-seq (scRNA-seq). However, applying it to highly fragile animals such as ctenophores is challenging. Here, we present an in situ hybridization protocol for adult Pleurobrachia bachei (Cydippida)-a notable reference species representing the earliest-branching metazoan lineage, Ctenophora, sister to the rest of Metazoa. We provided expression patterns for several markers of cell phenotypes, as illustrated examples. The list includes predicted small secretory molecules/neuropeptides, WntX, genes encoding RNA-binding proteins (Musashi, Elav, Dicer, Argonaut), Neuroglobin, and selected transcription factors such as BarX. Both cell- and organ-specific expression of these genes further support the convergent evolution of many ctenophore innovations, which are remarkably distinct from tissue and organ specification in other basal metazoan lineages.
原位杂交是一种强大而精确的工具，可用于揭示细胞和组织特异性基因表达，是验证单细胞 RNA 测序 (scRNA-seq) 的关键方法。然而，将其应用于栉水母等非常脆弱的动物是具有挑战性的。在这里，我们提出了一种适用于成年 Pleurobrachia bachei（栉水母目）的原位杂交方案，该动物是一种具有代表性的早期分支后生动物谱系栉水母门的模式生物，是后生动物的姐妹群。我们提供了几个细胞表型标记物的表达模式，作为示例说明。这些标记物包括预测的小分泌分子/神经肽、WntX、编码 RNA 结合蛋白（Musashi、Elav、Dicer、Argonaut）、神经球蛋白和一些转录因子，如 BarX。这些基因的细胞和器官特异性表达进一步支持了许多栉水母创新的趋同进化，这与其他基础后生动物谱系的组织和器官特化明显不同。
