Name: Video: Confocal Imaging and Quantitative Analysis of smFISH in Wing Discs and Adult Muscles
Uploaded: 2023-09-08T14:40:00
Duration: 4 min 16 s
Description: 393 Views. Confocal Imaging and Quantitative Analysis of smFISH in Wing Discs and Adult Muscles

confocal imaging and quantitative analysis of smfish in wing discs and adult muscles

Detection and Quantification of Single mRNA Molecules in Drosophila

Adult skeletal muscles are made of differentiated multinucleated myofibers whose contractile properties generate movements. Muscle growth, maintenance, and regeneration rely upon muscle progenitors and muscle stem cells (MuSCs) that are specified during embryonic development1. The myogenic program is finely controlled by a set of core myogenic transcription factors (TFs) (e.g., Pax3/7, MYOD, Mef2, and ZEB)2,3,4. Deciphering the molecular mechanisms regulating muscle biology is important for elucidating fundamental myology-related questions and for possible therapeutic use in treating muscle-degenerative diseases.
Drosophila has a long history as a genetic model to study myogenesis5. It has recently emerged as a new model to investigate muscle regeneration6,7,8. The muscle structure and core myogenic programs are highly conserved between flies and mammals. For example, the TFs Mef2 and Zfh1/ZEB have a conserved function in regulating muscle development and regeneration3,9,10,11. Adult Drosophila muscles, such as the Indirect Flight Muscles (IFMs), are formed from a specific population of MuSCs, known as Adult Muscle Progenitors (AMPs)12. These AMPs are specified during embryogenesis and associate with epithelial structures such as wing and leg discs. Throughout embryonic and larval stages, AMPs remain undifferentiated until metamorphosis when they engage in differentiation and fusion to form the IFMs13,14. The TF Spalt major (spalt, salm) is expressed during flight muscle development and is necessary to determine their structural identity15. Mef2 is another important myogenic TF that is essential for adult muscle formation16,17. It is expressed in the AMPs and maintained in the adult IFMs10,18. While the majority of AMPs differentiate into functional muscles, a subset escapes differentiation and forms the adult MuSCs11. Similar to vertebrates, the TF Zfh1/ZEB is required to prevent premature differentiation of the adult MuSCs and to maintain their stemness9,10.
Gene expression dynamics have been proven to regulate various muscle biological processes19,20. The advent of high-throughput single-cell and single-nuclei RNA sequencing techniques has enabled a comprehensive exploration of these transcriptional dynamics21,22. One notable limitation of these approaches is their inability to provide the spatial distribution of mRNA molecules in the multinucleated muscle fibers. These features can be investigated by single-molecule fluorescence in situ hybridization (smFISH) that reveals two types of mRNA bodies: 1. Individual mRNA molecules spread out in the cytoplasm and representing the mature RNA and 2. A maximum of two bright nuclear foci correspond to the nascent transcript and reveal the transcriptionally active alleles23. Therefore, smFISH is a method of choice for individual mRNA molecule quantification, investigating their spatial distribution and providing snapshots of the gene transcriptional dynamics.
The smFISH method relies on a set of short fluorescent oligonucleotide probes specifically designed to be complementary to the target transcript. Upon annealing, it generates high-intensity point sources permitting the detection of mRNA at a single-molecule level using confocal microscopy24. This method is increasingly applied for a wide variety of cell types, including mammalian muscle tissues19,20. However, in Drosophila like other animal models, most of the knowledge about adult muscle gene expression is derived from bulk molecular assays, which lack quantitative information on the spatial location of mRNA molecules. Here we optimized a method for performing smFISH on muscle precursor cells and adult Drosophila muscles23,25. This protocol includes a fully automated analysis pipeline for nuclei segmentation and mRNA counting and localization.

Author Spotlight: Investigating mRNA Spatial Distribution in Drosophila Muscle Tissue 

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

Watch this Scientific Journal Video about Confocal Imaging and Quantitative Analysis of smFISH in Wing Discs and Adult Muscles at JoVE.com

Confocal Imaging and Quantitative Analysis of smFISH in Wing Discs and Adult Muscles  

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Research

JoVE Journal

Developmental Biology

393 Views. Confocal Imaging and Quantitative Analysis of smFISH in Wing Discs and Adult Muscles

Video: Confocal Imaging and Quantitative Analysis of smFISH in Wing Discs and Adult Muscles  

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing single-molecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells.

Drosophila is a well-established model for studying key molecules that regulate myogenesis. However, current methods are insufficient to determine mRNA transcriptional dynamics and spatial distribution within syncytia. To address this limitation, we optimized an RNA fluorescence in situ hybridization method allowing the detection and quantification of mRNAs at a single-molecule scale.

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study ...