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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Bibliographic Details
Published in:	Frontiers in physiology Vol. 13; p. 882413
Main Authors:	Elagoz, Ali M, Styfhals, Ruth, Maccuro, Sofia, Masin, Luca, Moons, Lieve, Seuntjens, Eve
Format:	Journal Article
Language:	English
Published:	Switzerland Frontiers Media S.A 30-05-2022
Subjects:	brain development cephalopod fluorescent in situ hybridization immunohistochemistry invertebrate Physiology tissue clearing brain development fluorescent in situ hybridization light sheet imaging cephalopod tissue clearing immunohistochemistry invertebrate
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Summary:	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 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 embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent mRNA visualization. As proof of concept, neuronal ( ) and glial ( ) markers were used for multiplexed HCR v3.0. Neural progenitor ( ) and precursor ( ) 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology Edited by: Natraj Krishnan, Mississippi State University, United States Reviewed by: Robyn J. Crook, San Francisco State University, United States Camino Gestal, Spanish National Research Council (CSIC), Spain
ISSN:	1664-042X 1664-042X
DOI:	10.3389/fphys.2022.882413