Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation and loss
- Stanford University School of Medicine, United States
- Stanford University, United States
Figures
Figure 1 with 3 supplements
Extended imaging of the midgut in live Drosophila adults.
(A) Adult female midgut in situ, sagittal view. The white highlighted area indicates region R4a-b, also known as P1-2, (Buchon et al., 2013a; Marianes and Spradling, 2013)) of the midgut that will …
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Figure 1—source data 1
Durations, genotypes, animal ages, and animal viability for movies analyzed in this study.
- https://doi.org/10.7554/eLife.36248.006
Figure 1—figure supplement 1
Mounts for upright, inverted and light-sheet microscopes.
(A–B) Mount for upright microscopes. (A) Schematic of an animal in the mount on a microscope stage. (A′) Isometric illustrations of mount components: (1) modified petri dish, (2) metal shim with …
Figure 1—figure supplement 2
Specifications for abdomen cutouts.
The metal shim of the imaging mount includes a cutout through which the dorsal abdomen is inserted. ‘Fat’ (left) and ‘skinny’ (right) cutouts accommodate differently sized female abdomens. This …
Figure 1—figure supplement 3
Cell viability during extended imaging.
Cell viability during extended imaging was evaluated using the cell-death stain Sytox Green. (A) Positive control. To induce cell death, midguts were dissected out of the animals and cultured ex …
Figure 2
Comprehensive, fate-specific tracking and analysis of individual cells.
(A–B) ‘Fate sensor’ midguts enable the live identification of cell types. (A) Stack projection of a single time point from a 10 hr movie (Video 7). Nuclei are distinguishable for four midgut cell …
Figure 3 with 1 supplement
Real-time kinetics of enterocyte extrusion and stem cell mitosis.
(A–E) Morphometric analysis of a single-enterocyte extrusion. (A) Time-lapse sequence (top) and schematic (bottom) showing a planar view of an extrusion event. The basal region of the extruding cell …
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Figure 3—source data 1
Raw data for Figure 3F and 3H and for mitotic index calculations.
- https://doi.org/10.7554/eLife.36248.018
Figure 3—figure supplement 1
Enterocyte extrusion occurs via ratcheted constriction of a basal junctional ring.
(A–C) Cross-sectional area of the basal junctional ring over time for three enterocyte extrusions (blue, green, red). Pulses of ring constriction (colored background) alternate with pulses of ring …
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Figure 3—figure supplement 1—source data 1
Raw data for Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.36248.017
Figure 4 with 1 supplement
Real-time orientations of stem-cell divisions in three reference frames.
(A–E) Horizontal-vertical orientations are horizontally biased. (A) Schematic of horizontal (0°) and vertical (90°) orientations. See Figure 4—figure supplement 1. (B) Live orientations of 10 …
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Figure 4—source data 1
Raw data for Figure 4B, 4C, 4D, 4G, 4H, and 4K.
- https://doi.org/10.7554/eLife.36248.021
Figure 4—figure supplement 1
Measurement of horizontal-vertical spindle orientation in space.
Horizontal-vertical orientation of the mitotic spindle was measured as the angle at which the presumptive spindle axis intersected a plane tangent to the basal surface of the mitotic cell. Spindle …
Figure 5
Whole-population and single-cell analyses of real-time Notch activation.
(A–C) A threshold level of Notch activation distinguishes stem cells and enteroblasts. (A) Single-cell measurements of the Notch reporter GBE-Su(H)-GFP:nls from live movies. Cells additionally …
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Figure 5—source data 1
Raw measurements for Figure 5.
- https://doi.org/10.7554/eLife.36248.023
Figure 6 with 1 supplement
Dynamics of cell contact and Notch reporter activation in sibling cells after birth.
(A) Contacts between newborn siblings are highly variable. Eighteen pairs of sibling cells (rows A–R) were tracked from birth (t = 0.0 hr) to the end of imaging. Color shows the likelihood of …
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Figure 6—source data 1
Raw data for Figure 6.
- https://doi.org/10.7554/eLife.36248.027
Figure 6—figure supplement 1
Comparison of cell-cell contact and inter-nuclear distance for live pairs of progenitor cells.
(A) Examples of contacting and separated progenitor pairs. Contact is revealed using esg-driven LifeactGFP (green) to label the actin cytoskeleton of progenitor cells. Inter-nuclear distances …
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Figure 6—figure supplement 1—source data 1
Raw measurements for Figure 6—figure supplement 1.
- https://doi.org/10.7554/eLife.36248.026
Author response image 1
Hourly mitotic indices.
Individual points show the mitotic index for hours 1-14 of live imaging. Earlier hours are denoted by lighter colors, and later hours are denoted by darker colors. Box shows the mean of the hourly …
Videos
Video 1
Narrated, step-by-step tutorial illustrating the preparation of an animal for midgut imaging in the fly mount.
https://doi.org/10.7554/eLife.36248.007
Video 2
Movie showing the association of the trachea (cyan) with the midgut tube (red).
Smaller tracheal branches encircle the tube and move in concert with peristaltic contractions. A large tracheal branch (upper right) is continuous with smaller branches. The large branch does not …
Video 3
Volumetric movie of the midgut illustrates the wide-field, high-resolution images that are acquired.
Numerous physiological contractions of the midgut are evident. A midgut-associated tracheal branch is visible in the lower left of the video. Scale bar, 70 µm.
Video 4
After 16 hr of continuous imaging, the animal is alive and responsive.
https://doi.org/10.7554/eLife.36248.010
Video 5
Cell viability during extended imaging.
As cells die, they become marked by the cell death stain Sytox Green, which is continuously present in the imaging media. After 11 hr of imaging, an individual midgut enterocyte changes from Sytox– …
Video 6
Movie clip of midgut before (left) and after (right) stack registration.
Before registration, blurred cells from tissue movements are evident during timepoints from 20–60 min. After registration, the blurring is negligible. Cyan, all nuclei (ubi-his2ab::mRFP); yellow, …
Video 7
Ten-hour movie of a ‘fate sensor’ midgut (esgGal4, UAS-his2b::CFP, GBE-Su(H)-GFP:nls; ubi-his2av::mRFP).
See Figure 2A–B). Nuclei are distinguishable for four midgut cell types: stem cells (red pseudocolor), enteroblasts (yellow-green pseudocolor), enterocytes (gray, polyploid), and enteroendocrine …
Video 8
Twelve-hour movie of a single-enterocyte extrusion.
The epithelium is oriented with its basal surface toward the microscope objective and its apical surface further away. The basal region of the extruding enterocyte (orange pseudocolor at t=0, 127.5, …
Video 9
Orthoview of extrusion shown in Video 8.
The nucleus of the extruding enterocyte (magenta) ejects out of the epithelium (t=150–165 min) and penetrates into the lumen (t=165–265 min). It subsequently recoils and eventually comes to rest on …
Video 10
Four-hour movie of an enteroendocrine cell extrusion.
The epithelium is oriented with its basal surface toward the microscope objective and its apical surface further away. The basal region of the extruding cell (tan pseudocolor at t=0, 75 min) is …
Video 11
Mitosis of a putative stem cell.
Green, actin (esg >LifeactGFP); yellow, E-cadherin (ubi-DE-cadherin::YFP); red, nuclei (ubi-his2av::mRFP). Each time point is the partial projection of a confocal stack. Scale bar, 10 µm
Video 12
Orthoview of a mitosis with two horizontal-vertical re-orientations.
The first re-orientation occurs between metaphase (24° at 7.5 min) and anaphase (60° at 15 min). The second re-orientation occurs between anaphase (62° at 22.5 min) and telophase (2° at 30 min). …
Video 13
Orthoview of a second mitosis with two horizontal-vertical re-orientations.
The top panel shows condensed chromatin of the dividing cell (ubi-his2ab::mRFP). The red line indicates the spindle axis. The cyan line indicates the basal plane, as revealed by the basement …
Video 14
Division of a stem cell that contacts two enteroblasts.
Division orientation aligns with the axis between the two enteroblast nuclei (magenta, GBE-Su(H)-GFP:nls). At cytokinesis (t=15–22.5 min), the new daughter nuclei hurl into the enteroblast nuclei, …
Video 15
Real-time enteroblast transition.
In the incipient enteroblast (blue dotted circle), GBE-Su(H)-GFP:nls is initially undetectable (GFP:RFP=0.014 at t=0.0 hr). Over time, its GFP intensity increases, eventually reaching the …
Video 16
A low-contact sibling pair (Pair P; Figure 6A,B) does not activate GBE-Su(H)-GFP:nls.
Following their birth at t=0.0 hr, the two siblings move apart and have probably lost contact by t=1.4 hr (inter-nuclear distance >15.5 µm; c.f. Figure 6—figure supplement 1). The mother stem cell …
Video 17
A high-contact sibling pair (Pair A, Figure 6A,C) does not activate GBE-Su(H)-GFP:nls.
Following their birth at t=0.0 hr, the two siblings probably remain in contact (inter-nuclear distance <6.0 µm; c.f. Figure 6—figure supplement 1) for at least 6.0 hr. The mother stem cell is …
Video 18
A sibling pair exhibits asymmetric Notch activation (Pair L, Figure 6A,D).
Following their birth at t=0.0 hr, the two siblings are probably in contact from t=2.6–3.6 hr, in indeterminate contact from t=3.6–9.0 hr, and separated after t=9.0 hr. The mother stem cell is …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | esgGal4 | Kyoto DGGR | DGRC:112304; FLYB:FBti0033872; | FlyBase symbol: w[*]; P{w[+mW.hs]=GawB}NP0726/CyO |
| Genetic reagent (D. melanogaster) | ubi-his2av::mRFP | Bloomington Drosophila Stock Center | BDSC:23650; FLYB:FBti0077846; RRID:BDSC_23650 | FlyBase symbol: w[*]; P{w[+mC]=His2Av-mRFP1}III.1 |
| Genetic reagent (D. melanogaster) | breathlessGal4, UAS-cyt-GFP | Other | w; btl-Gal4, UAS-cytGFP shared by Mark Metzstein | |
| Genetic reagent (D. melanogaster) | UAS-LifeactGFP | Bloomington Drosophila Stock Center | BDSC:35544; FLYB:FBti0143326; RRID:BDSC_35544 | FlyBase symbol: y[1] w[*]; P{y[+t*] w[+mC]=UAS-Lifeact-GFP}VIE-260B |
| Genetic reagent (D. melanogaster) | UAS-his2b::CFP | PMID: 24850412 | w; UAS-his2b::CFP/ (Cyo); + -- shared by Yoshihiro Inoue | |
| Genetic reagent (D. melanogaster) | GBE-Su(H)-GFP:nls | PMID: 22522699 | w?; mw, GBE-Su(H)-GFPnls/(Cyo); Dr/TM6B -- from (de Navascués et al., 2012) shared by Joaquin de Navascues | |
| Genetic reagent (D. melanogaster) | act5c-spaghetti squash::GFP | PMID:12105185 | w?; act5c-sqh::GFP; Dr/TM6C -- shared by Denise Montell | |
| Genetic reagent (D. melanogaster) | ubi-E-cadherin::YFP | PMID: 24855950 | w; ubi-E-cadherin::YFP; + -- shared by Denise Montell | |
| Chemical compound, drug | Concanavalin-A-Alexa647 | Invitrogen | Invitrogen:C21421 | 25 μg/ml final concentration |
| Chemical compound, drug | Sytox Green | ThermoFisher | ThermoFisher:S7020 | 1 μM final concentration |
| Chemical compound, drug | SiR-tubulin | Cytoskeleton | Cytoskeleton:CY-SC002 | 0.5 μM final concentration |
| Chemical compound, drug | Human insulin | Sigma Aldrich | Sigma-Aldrich:I0516 | 100 μg/ml final concentration |
| Software, algorithm | Fiji | Other | RRID:SCR_002285 | StackRegfrom Arganda-Arganda-Carreras et al., 2006b; Correct 3D drift from Parslow et al. (2014); Bioformats plugin |
| Software, algorithm | Bitplane Imaris | Other | RRID:SCR_007370 | Surpass module; Surface Recognition Wizard; Measurement Points tool |
Additional files
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Source code 1
Registration macros utilizing the ImageJ plugin StackReg to perform three channel stack registration over time.
In this macro, the XY negative space around the image is increased by a user-defined amount to account for the shifting of stack slices during the registration process. The movie is then collapsed into an RGB format and StackReg is performed on each time point using a loop function. Once completed, corrected time points are concatenated, converted back to three color hyperstacks, and then the ImageJ plugin Correct 3D Drift is applied to correct for global volume movement of the tissue over time. The macro is in *.ijm format which can be opened and viewed in ImageJ.
- https://doi.org/10.7554/eLife.36248.039
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Transparent reporting form
- https://doi.org/10.7554/eLife.36248.040
