Paganos P et al. (2022), FISH for All: A Fast and Efficient Fluorescent ...

ECB-ART-50347

Front Physiol 2022 Jan 01;13:878062. doi: 10.3389/fphys.2022.878062.

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FISH for All: A Fast and Efficient Fluorescent In situ Hybridization (FISH) Protocol for Marine Embryos and Larvae.

Paganos P , Caccavale F , La Vecchia C , D'Aniello E , D'Aniello S , Arnone MI .

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In situ hybridization is one the most commonly used techniques for developmental and evolutionary biology and has extensively contributed to the identification of distinct cell types and cell states, as well dissecting several molecular mechanisms involved in physiological processes. Moreover, it has been used as a tool to compare distinct gene expression patterns and, therefore, genetic programs across animal species. Nowadays, the predominance of transcriptomics in science has imposed the need to establish a reliable, fast and easy whole mount in situ hybridization protocol. Here we describe a fluorescent in situ hybridization protocol that is rapid, accurate and applicable in a great variety of marine species.

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	FIGURE 1. Expression patterns of known gene markers in echinoderm representatives through FISH. FISH of S. purpuratus larva with antisense probe for Vasa (AâA''), S. franciscanus larva with antisense probe for Fgf9/16/20 (BâB''), L. variegatus larva with antisense probe for SynB (CâC''), P. lividus larva with antisense probe for Pax6 (DâD''), A. lixula with antisense probe for ManrC1A (EâE'') and P. miniata larva with antisense probe for Cdx (FâF''). Nuclei are stained with DAPI (in grey). All images are stacks of merged confocal Z sections. A, Anus; Ao, Apical organ; Cb, Ciliary band; Cp, Coelomic pouches; Cs, Cardiac sphincter; Oe, Oral ectoderm; Pmc, Primary mesenchyme cell; St, Stomach.
	FIGURE 2. Examples of double FISH. Double FISH of S. purpuratus embryos at gastrula stage using antisense RNA probes against Cdx (A) and Pdx1 (B). The overlay of the two channels is shown in panel (C). Double FISH of S. purpuratus pluteus larvae using antisense RNA probes against Fbsl (D) and Spec1 (E). The overlay of the two channels is shown in panel (F). Nuclei are labelled with DAPI (in grey). All images are stacks of merged confocal Z sections. Abo, Aboral ectoderm; Cb, Ciliary band; Hg, Hindgut.
	FIGURE 3. Expression patterns of known gene markers in mollusk, tunicate and cephalochodate representatives through FISH. FISH of M. galloprovincialis embryos (AâA'') and larvae (BâB'') with antisense probe for Actin; C. robusta embryo with antisense probe for Hnf6 (CâC''); B. lanceolatum embryo (DâD'') and larva (EâE'') not treated and treated with Proteinase K (FâF'') with antisense probe for FoxE. Nuclei are labelled with DAPI (in grey). All images are stacks of merged confocal Z sections. Csg, club-shaped gland; M, Muscle; P, Pharyngeal endoderm; Vg, Visceral ganglion; Sv, Sensory vesicle; T, Tail. Arrow indicates positive cells in the posterior neural tube.
	FIGURE 4. Overview of the FISH procedure described in this study. Schematics of the embryos and larvae used to test the protocol are represented in gray. Color code of the staining corresponds to what used in Figures 1â3.

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