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Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains

Circadian clocks are ∼24-h timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of c...

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Autores principales: Yuan, Ye, Padilla, Marc-Antonio, Clark, Dunham, Yadlapalli, Swathi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9682093/
https://www.ncbi.nlm.nih.gov/pubmed/36439243
http://dx.doi.org/10.3389/fphys.2022.1051544
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author Yuan, Ye
Padilla, Marc-Antonio
Clark, Dunham
Yadlapalli, Swathi
author_facet Yuan, Ye
Padilla, Marc-Antonio
Clark, Dunham
Yadlapalli, Swathi
author_sort Yuan, Ye
collection PubMed
description Circadian clocks are ∼24-h timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of core clock mRNAs and its role in generating ∼24-h circadian rhythms. Here we describe a streamlined single molecule Fluorescence In Situ Hybridization (smFISH) protocol and a fully automated analysis pipeline to precisely quantify the number and subcellular location of mRNAs of Clock, a core circadian transcription factor, in individual clock neurons in whole mount Drosophila adult brains. Specifically, we used ∼48 fluorescent oligonucleotide probes that can bind to an individual Clock mRNA molecule, which can then be detected as a diffraction-limited spot. Further, we developed a machine learning-based approach for 3-D cell segmentation, based on a pretrained encoder-decoder convolutional neural network, to automatically identify the cytoplasm and nuclei of clock neurons. We combined our segmentation model with a spot counting algorithm to detect Clock mRNA spots in individual clock neurons. Our results demonstrate that the number of Clock mRNA molecules cycle in large ventral lateral clock neurons (lLNvs) with peak levels at ZT4 (4 h after lights are turned on) with ∼80 molecules/neuron and trough levels at ZT16 with ∼30 molecules/neuron. Our streamlined smFISH protocol and deep learning-based analysis pipeline can be employed to quantify the number and subcellular location of any mRNA in individual clock neurons in Drosophila brains. Further, this method can open mechanistic and functional studies into how spatiotemporal localization of clock mRNAs affect circadian rhythms.
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spelling pubmed-96820932022-11-24 Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains Yuan, Ye Padilla, Marc-Antonio Clark, Dunham Yadlapalli, Swathi Front Physiol Physiology Circadian clocks are ∼24-h timekeepers that control rhythms in almost all aspects of our behavior and physiology. While it is well known that subcellular localization of core clock proteins plays a critical role in circadian regulation, very little is known about the spatiotemporal organization of core clock mRNAs and its role in generating ∼24-h circadian rhythms. Here we describe a streamlined single molecule Fluorescence In Situ Hybridization (smFISH) protocol and a fully automated analysis pipeline to precisely quantify the number and subcellular location of mRNAs of Clock, a core circadian transcription factor, in individual clock neurons in whole mount Drosophila adult brains. Specifically, we used ∼48 fluorescent oligonucleotide probes that can bind to an individual Clock mRNA molecule, which can then be detected as a diffraction-limited spot. Further, we developed a machine learning-based approach for 3-D cell segmentation, based on a pretrained encoder-decoder convolutional neural network, to automatically identify the cytoplasm and nuclei of clock neurons. We combined our segmentation model with a spot counting algorithm to detect Clock mRNA spots in individual clock neurons. Our results demonstrate that the number of Clock mRNA molecules cycle in large ventral lateral clock neurons (lLNvs) with peak levels at ZT4 (4 h after lights are turned on) with ∼80 molecules/neuron and trough levels at ZT16 with ∼30 molecules/neuron. Our streamlined smFISH protocol and deep learning-based analysis pipeline can be employed to quantify the number and subcellular location of any mRNA in individual clock neurons in Drosophila brains. Further, this method can open mechanistic and functional studies into how spatiotemporal localization of clock mRNAs affect circadian rhythms. Frontiers Media S.A. 2022-11-09 /pmc/articles/PMC9682093/ /pubmed/36439243 http://dx.doi.org/10.3389/fphys.2022.1051544 Text en Copyright © 2022 Yuan, Padilla, Clark and Yadlapalli. 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
Yuan, Ye
Padilla, Marc-Antonio
Clark, Dunham
Yadlapalli, Swathi
Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains
title Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains
title_full Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains
title_fullStr Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains
title_full_unstemmed Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains
title_short Streamlined single-molecule RNA-FISH of core clock mRNAs in clock neurons in whole mount Drosophila brains
title_sort streamlined single-molecule rna-fish of core clock mrnas in clock neurons in whole mount drosophila brains
topic Physiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9682093/
https://www.ncbi.nlm.nih.gov/pubmed/36439243
http://dx.doi.org/10.3389/fphys.2022.1051544
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