Single transcript imaging to assay gene expression in wholemount Drosophila melanogaster tissues

Abstract

Single molecule Fluorescence in situ Hybridization (smFISH) for mRNA provides a powerful quantitative handle on expression from endogenous gene loci. While the method has been widely applied in cells in culture, applications to primary tissue samples remain fewer, and often use involved cryosectioning. Even apart from quantitative access to absolute transcript counts in specific tissue volumes, many other advantages of smFISH can be envisaged in tissue samples. Primary among these are the ability to report on subtle differences in expression among different cell types within a tissue, and the ability to correlate the expression from different target genes. Here, we present a modified method of smFISH applicable on various primary wholemount tissues from the fruit fly Drosophila melanogaster, and show the efficacy of the method in a variety of larval and adult tissue, and embryos. We also combine smFISH in tissue with immunofluorescence to demonstrate the possibility of capturing transcriptional and translational aspects of gene expression in the same tissue. Given the widespread use of Drosophila melanogaster as a model system in Developmental Biology and Genetics, such methods are likely to be of wide interest and could yield rich information about gene expression in tissues from this organism.

Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye

“…The wash buffer was aspirated and the tissues were incubated with a hybridization mix overnight at 37°C. The probe sequences for spitz and argos is given in the previous paper [ 40 ]. The mix was removed and tissues were washed twice with wash buffer at 37°C.…”

“…A cartoon depicting the brief protocol and singly labeled 20-mer oligonucleotide probes binding to mRNA target is shown in S1A Fig . StarSearch software ( https://www.seas.upenn.edu/~rajlab/StarSearch/launch.html ) was used to count transcripts in 3D as done before [ 40 ]. The absolute counts and ratios were calculated in regions of interest on the eye field and then averaged.…”

Buffered EGFR signaling regulated by spitz to argos expression ratio is critical for patterning the Drosophila eye

“…The wash buffer was aspirated and the tissues were incubated with a hybridization mix overnight at 37°C. The probe sequences for spitz and argos is given in the previous paper (Pasnuri et al, 2018). The mix was removed and tissues were washed twice with wash buffer at 37℃.…”

“…1A. StarSearch software (https://www.seas.upenn.edu/~rajlab/StarSearch/launch.html) was used to count transcripts in 3D as done before (Pasnuri et al, 2018). The absolute counts and ratios were calculated in regions of interest on the eye field and then averaged.…”

“…These problems can be circumvented by detecting endogenous mRNA in situ, which additionally could also indicate the identity of cells that express these genes. In recent years Single-molecule Fluorescence In Situ Hybridization (smFISH) has emerged as a powerful method to sensitively and quantitatively report the gene expression in wholemount tissues, along with cell-to-cell variability (Pasnuri et al, 2018;Trcek et al, 2017;Yang et al, 2017). We have previously shown that such methods can be used to detect even low levels of gene expression in the wholemount Drosophila tissues (Pasnuri et al, 2018).…”