The Wg and Dpp morphogens regulate gene expression by modulating the frequency of transcriptional bursts
- Department of Molecular Biosciences, Northwestern University, United States
- NSF-Simons Center for Quantitative Biology, Northwestern University, United States
- Department of Engineering Sciences and Applied Mathematics, Northwestern University, United States
Figures
Figure 1 with 3 supplements
smFISH analysis of sfGFP-sens mRNA levels in wing imaginal discs.
(A) Schematic of a wing disc outlining different regional domains, and the positions of boundaries between Dorsal (D) - Ventral (V) and Anterior (A) - Posterior (P) compartments of the disc. Each …
Figure 1—figure supplement 1
Development of smFISH imaging and analysis.
(A,B) Representative optical sections of wing discs probed for sfGFP mRNAs. Upper panels show 1x exposure of fluorescence from optical sections. Lower panels show same sections with 4x overexposure …
Figure 1—figure supplement 2
Determination of false-positive and false-negative rates for smFISH.
(A) Wing discs were imaged and scored for 3D fluorescent objects using the sfGFP probe set. Discs were either from animals with two copies of the sfGFP-sens transgene and two copies of the …
Figure 1—figure supplement 3
smFISH imaging of the eye imaginal disc.
(A) Schematic of the eye antennal disc complex showing the approximate location of cells that express the sens gene. Anterior is to the left. (B,C) Optical sections through a representative eye …
Figure 2 with 1 supplement
smFISH analysis of mRNA levels from Dpp-responsive genes.
(A) Schematic of wing discs highlighting the graded distribution of Dpp protein in the wing pouch, centered around the AP boundary, and the expression domain for salm, one of the targets of Dpp …
Figure 2—figure supplement 1
Detection of RNAs corresponding to the sd gene.
(A) sd mRNA number as a function of cell distance from the anterior-most border of the wing pouch. An axis orthogonal to the AP boundary is used to map cell position. Numbers refer to distance in μm …
Figure 3 with 2 supplements
Sites of nascent transcription are detected by smFISH.
(A) Sites of nascent transcription can fluoresce more brightly than single mRNA molecules due to multiple nascent transcripts localized to one gene locus. (B) Probes recognizing an omb exon generate …
Figure 3—figure supplement 1
Detection of transcription sites and their quantification.
(A) A representative frequency distribution of fluorescence intensity for 3D spots identified in one wing disc expressing sfGFP-sens. The median intensity is 28 units. (B) The same wing disc was …
Figure 3—figure supplement 2
Transcription sites and mRNA patterns in unsegmented images.
(A,B,C,D,E) Three discs were analyzed independently (green, blue, orange dots) for spots that corresponded to the mRNAs from sens (A), salm (B), omb (C), dad (D) and brk (E). Spots were binned …
Figure 4
Modeling transcription sites using bursting dynamics.
(A) Model framework showing the three rate parameters affecting transcription initiation. Two parameters affect the promoter state, while the third parameter only affects how many initiation events …
Figure 5
Transcription site detection correlates with mRNA number.
(A,B) The probability of detecting a cell with a transcription site varies with the cell's location relative to the source of morphogen. Error bars are 95% bootstrapped confidence intervals. (A) …
Figure 6 with 1 supplement
Burst frequency is regulated by Dpp and Wg.
(A,B) The average number of nascent RNAs in a transcription site does not vary with the cell's location relative to the source of morphogen. Error bars are bootstrapped 95% confidence intervals. (A) …
Figure 6—figure supplement 1
Modeling the relationship between average number of nascent RNAs in a transcription site and the probability of detecting a site for the brk, omb, salm, and sens genes.
(A) Simulations are performed where the rate parameter kini has been systematically varied so that burst size alone is variable. Resulting values for nascent RNA number and fraction of cells with a …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | white1118 | BloomingtonDrosophilaStock Center | BDSC: 3605 Flybase: FBst0003605 RRID:BDSC_3605 | |
| Gene (Drosophila melanogaster) | sensE1 | Nolo et al., 2000 | Flybase: FBal0098024 | From Hugo Bellen |
| Genetic reagent (Drosophila melanogaster) | sfGFP-sens [VK37] | Venken et al., 2006. From Hugo Bellen | Pacman construct containing sens gene with N-terminal 3xFlag-TEV-StrepII-sfGFP-FlAsH fusion tag inserted at 22A3 (VK37) | |
| Genetic reagent (Drosophila melanogaster) | dad-GFP [VK37] | BloomingtonDrosophilaStock Center | BDSC: 81273 Flybase: FBti0150281 RRID:BDSC_81273 | y w; PBac{y[+mDint2] w[+mC]=Dad GFP.FLAG} inserted at 22A3 (VK37) |
| Genetic reagent (Drosophila melanogaster) | brk-GFP [VK33] | BloomingtonDrosophilaStock Center | BDSC: 38629 Flybase: FBti0147730 RRID:BDSC_38629 | w1118; PBac{y[+mDint2] w[+mC]=brk GFP.FPTB} inserted at 65B2 (VK33) |
| Sequence-based reagent | GFP hybridization oligo probes | Biosearch Technologies | Custom probe set | Set of oligos with 3' modification mdC(TEG-Amino). Sequence of all oligos is in Supplementary file 1 |
| Sequence-based reagent | sens hybridization oligo probes | Biosearch Technologies | Custom probe set | Set of oligos with 3' modification mdC(TEG-Amino). Sequence of all oligos is in Supplementary file 1 |
| Sequence-based reagent | salm hybridization oligo probes | IDT | Custom probe set | Set of oligos. Sequence of all oligos is in Supplementary file 1 |
| Sequence-based reagent | omb hybridization oligo probes | IDT | Custom probe set | Set of oligos. Sequence of all oligos is in Supplementary file 1 |
| Sequence-based reagent | sd hybridization oligo probes | IDT | Custom probe set | Set of oligos. Sequence of all oligos is in Supplementary file 1 |
| Sequence-based reagent | omb intron hybridization oligo probes | IDT | Custom probe set | Set of oligos. Sequence of all oligos is in Supplementary file 1 |
| Sequence-based reagent | omb 5' exon hybridization oligo probes | IDT | Custom probe set | Set of oligos. Sequence of all oligos is in Supplementary file 1 |
| Chemical compound, drug | NHS-ester ATTO 633 dye | Sigma | #01464 | |
| Chemical compound, drug | NHS-ester ATTO 565 dye | Sigma | #72464 | |
| Chemical compound, drug | amino-11-ddUTP | Lumiprobe | A5040 | |
| Chemical compound, drug | Paraformaldehyde (powder) | Polysciences | 00380–1 | |
| Chemical compound, drug | Triton X-100 | Sigma Aldrich | T9284-500ML | |
| Chemical compound, drug | VectaShield | Vector Labs | H-1000 | |
| Chemical compound, drug | 4′,6-diamidino-2-phenylindole (DAPI) | Life Technologies | D1306 | |
| Chemical compound, drug | salmon sperm single stranded DNA | Invitrogen | #15632 | |
| Chemical compound, drug | vanadyl ribonucleoside | New England Biolabs | #S14025 | |
| Software, algorithm | MATLAB pipeline to process raw smFISH images with no prior preprocessing | This paper | https://github.com/elifesciences-publications/smfish_pipeline | |
| Other | Graces’ Insect Medium | Sigma | #69771 | Growth medium for organ culture |
Additional files
-
Supplementary file 1
Excel file containing the sequences of all oligonucleotide probes used for smFISH experiments in this paper.
Each worksheet lists the oligos specific for a gene, as indicated. Sequences are ordered 5' - to - 3'.
- https://cdn.elifesciences.org/articles/56076/elife-56076-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56076/elife-56076-transrepform-v2.pdf
