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Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis

Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of te...

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Autores principales: Elagoz, Ali M., Styfhals, Ruth, Maccuro, Sofia, Masin, Luca, Moons, Lieve, Seuntjens, Eve
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9196907/
https://www.ncbi.nlm.nih.gov/pubmed/35711315
http://dx.doi.org/10.3389/fphys.2022.882413
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author Elagoz, Ali M.
Styfhals, Ruth
Maccuro, Sofia
Masin, Luca
Moons, Lieve
Seuntjens, Eve
author_facet Elagoz, Ali M.
Styfhals, Ruth
Maccuro, Sofia
Masin, Luca
Moons, Lieve
Seuntjens, Eve
author_sort Elagoz, Ali M.
collection PubMed
description Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA in situ hybridization chain reaction version 3.0 (HCR v3.0) in combination with immunohistochemistry (IHC), followed by fructose-glycerol clearing and light sheet fluorescence microscopy (LSFM) imaging on Octopus vulgaris embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods.
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spelling pubmed-91969072022-06-15 Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis Elagoz, Ali M. Styfhals, Ruth Maccuro, Sofia Masin, Luca Moons, Lieve Seuntjens, Eve Front Physiol Physiology Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA in situ hybridization chain reaction version 3.0 (HCR v3.0) in combination with immunohistochemistry (IHC), followed by fructose-glycerol clearing and light sheet fluorescence microscopy (LSFM) imaging on Octopus vulgaris embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods. Frontiers Media S.A. 2022-05-30 /pmc/articles/PMC9196907/ /pubmed/35711315 http://dx.doi.org/10.3389/fphys.2022.882413 Text en Copyright © 2022 Elagoz, Styfhals, Maccuro, Masin, Moons and Seuntjens. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Physiology
Elagoz, Ali M.
Styfhals, Ruth
Maccuro, Sofia
Masin, Luca
Moons, Lieve
Seuntjens, Eve
Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis
title Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis
title_full Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis
title_fullStr Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis
title_full_unstemmed Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis
title_short Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis
title_sort optimization of whole mount rna multiplexed in situ hybridization chain reaction with immunohistochemistry, clearing and imaging to visualize octopus embryonic neurogenesis
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9196907/
https://www.ncbi.nlm.nih.gov/pubmed/35711315
http://dx.doi.org/10.3389/fphys.2022.882413
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