A live-imaging protocol for tracking intestinal stem cell divisions in the Drosophila melanogaster pupal midgut

Summary Establishing a long-term ex vivo observation of the intestinal stem cell (ISC) is crucial to help understand the formation and homeostasis of the intestinal epithelium. Here, we present a protocol for tracking the division of Drosophila pupal ISCs during pupal midgut development. We describe steps for dissecting, mounting, and live imaging the pupal midgut. We then detail procedures for fluorescence quantification of each cell. This protocol can be applied to other fluorescently tagged proteins. For complete details on the use and execution of this protocol, please refer to Wu et al.1


SUMMARY
Establishing a long-term ex vivo observation of the intestinal stem cell (ISC) is crucial to help understand the formation and homeostasis of the intestinal epithelium.Here, we present a protocol for tracking the division of Drosophila pupal ISCs during pupal midgut development.We describe steps for dissecting, mounting, and live imaging the pupal midgut.We then detail procedures for fluorescence quantification of each cell.This protocol can be applied to other fluorescently tagged proteins.For complete details on the use and execution of this protocol, please refer to Wu et al. 1

BEFORE YOU BEGIN
The protocol below uses Drosophila pupae with the following genotype: esg-Gal4, 10xUAS-Myr::tdTomato/+; Pros::GFP/+.Red 10xUAS-Myr::tdTomato is expressed in esg + progenitor cells 2 and green Pros::GFP is expressed in pupal enteroendocrine (EE) cells. 1,3esg-Gal4,tub-Gal80 ts , 10xUAS-Myr::tdTomato/+; Pros::GFP/+, enables 10xUAS-Myr::tdTomato and other UAS driven construct expression under the control of temperature. 4To study real-time expression and localization of other proteins in the midgut, fluorescently tagged proteins can be constructed using the knock-in method. 5In the study by Wu et al., UAS-RNAi lines were crossed into this system to knock down genes of interest exclusively in esg + progenitor cells.To inactivate Gal80 ts and activate UAS-mediated gene expression, experimental samples were kept at 29 C for 2 days prior to experimentation.Note that pupal development is approximately 1.3 times faster at 29 C than at 25 C. 6 Preparation of specific stage pupae for experiments Timing: 2 days before 1.Pick up late third instar larvae (LL3, stage during which the larva wanders out of the food and climbs) with a brush, and transfer them to new vials of standard cornmeal food, with about 30 larvae in each vial and kept at 25 C.
2. Collect white pupae (0 h APF) from LL3 vials and place these pupae in a vial every half hour, and note when these pupae were collected.Keep the vials containing pupae at 25 C (Figure 1A).
Note: Staging pupae is necessary to obtain reproducible results.In this protocol, we perform experiments using pupae between 46 h and 52 h after puparium formation (APF) at 25 C (Figure 1A).
Culture flies at 25 C on standard cornmeal food, 1 and transfer to new vials every 2 days to control the number of eggs.Female LL3 are selected as follows: LL3 are transferred to PBS with a brush.Under the microscope, white transparent dots (testis, arrowhead in Figure 1B) are present on both sides of the posterior part of male larvae, but not in female larvae (Figure 1C).

REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, peptides, and recombinant proteins This step is performed in a sterile laminar flow hood at 25 C. Add 8.5 mL Schneider's Drosophila medium and 1.5 mL FBS to a 15 mL sterilized centrifuge tube, and then add 2 mg human insulin and mix well, adjusting the pH to 7.0 with 1 M NaOH.Since 500 mL of LIB is an appropriate volume for a live imaging experiment, transfer 5 mL of LIB to ten 1.5 mL centrifuge tubes containing 500 mL each.The remaining 5 mL LIB is used to prepare the Live Imaging Gel (LIG).
Note: The ten 1.5 mL centrifuge tubes containing 500 mL each can be stored at 4 C and used for one week.

Preparation of Live Imaging Gel (LIG) used for pupal midgut cultivation
Timing: 10 min Add 0.5 g gelatin to the 5 mL LIB and heat on a metal bath at 50 C for 5 min.After the gelatin has melted, transfer the LIG to ten tubes of 500 mL each and store at 4 C.
Note: This medium is critical for maintaining stem cell survival in the Drosophila pupal gut during time-lapse imaging.Aliquots of this medium should be stored at 4 C and can be used for one week.

STEP-BY-STEP METHOD DETAILS Preparation of live imaging dish
Timing: 20 min 1.Turn on the metal bath and heat to 37 C to melt the LIG by heating it on the metal bath for 10 min (Figure 2A).The temperature used to melt the LIG may increase to 40 C to prevent the LIG from solidifying too quickly.2. Using a glass cutter, cut a 22 mm 3 22 mm coverslip into two pieces from the center (Figure 2B).

Note:
The thickness of our coverslips is about 0.14 mm, and the coverslips are cut to make room for the dissected intestine.The coverslip can be cut with a glass cutter, or simply break the coverslip in half by hand (be careful not to injure your hand, and some of the coverslip may break).
3. Pipette 10 mL of melted LIG into the lumox dish 50 and quickly cover it with an 11 mm 3 22 mm coverslip (Figures 2C-2E).4. Repeat step 3 to place another coverslip, keeping the two coverslips approximately 5 mm apart (Figure 2F).
Note: Providing an ideal environment for the intestine is critical for prolonging the survival time of the dissected pupal guts, and the live imaging dish we prepared here is air-permeable and nutritious, which is beneficial for the development of the dissected guts.
Transfer the stage pupa to a dissecting microscope Timing: 5 min 5. Use the brush to gently pry several properly staged pupae from the side of the vial (Figure 3A).6. Transfer these pupae to the dissecting plate (Figure 3B). 7. Place the pupae under a dissecting microscope (Figure 3C).Note: We can get the exact developmental time of the pupae we want to study by timing from the white pupae.Since the developmental stages of the pupae are different at different times, we can also determine if the pupae are developing normally by the morphology (e.g.46 h APF Drosophila eyes are yellowish, while at 52 h APF eyes deepen in color in the yw, hs-Flp; esg-Gal4, 10XUAS-Myr::tdTomato; pros::GFP/TM6B.Tb) (Figures 3D and 3E).
Dissect the pupa to obtain the pupal midgut Timing: 5 min 8. Add 40 mL of LIB to the center of a dissecting plate and transfer one pupa into the LIB under a dissecting microscope (Figure 4A).9.Under the dissecting microscope, with the anterior of the pupa facing left if dissecting righthanded (right if dissecting left-handed), zoom in to enlarge the view of the pupa (Figure 4B).10.Gently grasp the pupa from the anterior part using one pair of blunt, flat-ended forceps (Figure 4C).11.Using another pair of fine-tipped forceps, with the forceps open, gently insert one tip of the forceps onto the top of the meconium (Figures 3D, 4D, and 4E).12. Once the tip is inserted approximately one-third to one-half of the way down the pupa, pull back to expose the midgut, being careful not to break it.13.Remove the head of the pupa by holding the head and neck of the pupa with two pairs of forceps respectively and pulling in opposite directions (arrow indicated in Figure 4F).14.Use the flat-ended forceps to gently press down on the surface of the pupa to hold it in place, and use the fine-tipped forceps to peel off the anterior shell (Figure 4G).15.Repeat step 14 to peel off the posterior shell (Figure 4H).16.Gently rinse the midgut by aspirating LIB through a 200 mL pipette to flush the midgut out of the pupal stage tissue.At this point the midgut is attached to the crop and reproductive system (Figure 4I).Mount the pupal midgut on the live imaging dish Timing: 10 min 17.Washing the 20 mL strippette tip 3 times with LIB.Then, gently aspirate the dissected pupal midgut (in LIB) under the microscope using a 20 mL pipette gun.18. Place the live imaging dish under a stereo microscope and transfer the dissected pupal midgut (together with associated tissues in LIB) to the gap between the two coverslips using the pipette gun (Figures 5A and 5B).19.Add 10 mL 37 C LIG to the transferred midgut, gently mix with LIB, aspirate and discard 10 mL of the mixed liquid.Adjust the position of the midgut so that it is not folded (Figure 5C).20.Repeat step 19 twice.

Note:
The operator should complete steps 17 to 20 within 1 min; otherwise, the LIG will solidify before the experiment is completed.
21. Allow the LIG to solidify for 2 min at 25 C. 22. Add 20 mL of 37 C LIG to the space between the two 11 3 22 mm coverslips and quickly cover the midgut with a 22 3 22 mm coverslip, gently pressing the 22 3 22 mm coverslip with forceps to bring the coverslip as close to the midgut as possible (Figures 5D and 5E).23.After 2 min, when the LIG has stabilized, seal the 22 3 22 mm coverslips with approximately 80 mL of halocarbon oil 27 using a 20 mL pipette gun to prevent water evaporation (Figure 5F).

Live imaging of the pupal midgut under a confocal microscope
Timing: 3 h, varies by experiment Protocol 24.The live imaging dish is placed without a lid, with the coverslip upside down so that the objective touches the coverslip (Figures 6A and 6B).25.Image the mounted live imaging dish on a Zeiss LSM 800 confocal microscope equipped with a Plan-Apochromat 63x/1.40Oil DIC M27 objective using the Immersol 518 F imaging medium (Figures 6A and 6B).Use definite focus to avoid focus drift, acquire time-lapse images using ZEN 2.1 with time-lapse module.26.Take an image of the pupal midgut from the parts indicated by rectangles (Figure 6C).
Note: The laser wavelengths of the green and red channels are 488 and 561, with a laser power of 2% and 0.2%, the detection wavelengths are 410-525 and 580-700, respectively, these channels are taken in frame, 8-12 mm z-stack images (2 mm intervals) of 512 3 512 pixels (0.198 mm 3 0.198 mm) are acquired every 60 s with a pixel time of 1.03 ms.

Adjust the total imaging time according to the requirements of the experiment and imaging.
Note: Imaging the midgut can last for 3 h or longer.We choose 3 h because after 3 h of imaging we can hardly detect the pISC divisions This section images the ISC division in the Drosophila pupal midgut at 25 C. Experiments can also be performed at temperatures between 18 C and 29 C, if required.
28.When live imaging is complete, place the mounted dish in 50 C hot water for 5 min to melt the LIG, then rinse the dish with water, dry, and reuse.
Note: An example of live imaging of the pupal ISC division is shown in Figure 7.A non-inverted microscope can also be used for live imaging of the pupal midgut.

EXPECTED OUTCOMES
The outcome of this protocol is a continuous video of the pupal ISCs. 1 The frame rate and the duration of imaging can be adjusted to capture events for 3 h after dissection.Asymmetric division of Pros::GFP is observed in wild type progenitors.Gene knockdown or overexpression in the progenitor cells of the midgut may alter this process.

QUANTIFICATION AND STATISTICAL ANALYSIS
Timing: 1 h, varies by experiment

LIMITATIONS
This is a live imaging protocol, so all image acquisition and analysis is dependent on genetically encoded fluorescently labeled proteins.This protocol is performed ex-vivo; therefore, the dissected intestinal tissue exhibits the ISC division for about 3 h due to the lack of in-vivo physical and physiological environment.Further optimization is needed for longer imaging.

Potential solution
Use a pair of blunt-ended forceps and a pair of fine-tipped forceps to facilitate dissection.The bluntended forceps are used to hold the pupa stable, while the fine-tipped forceps are used to dissect the midgut.The method of dissecting the pupal midgut is different for each developmental stage.The method given in this study is applicable to dissection of 46 h-52 h APF pupae (25 C).When  dissecting pupae from earlier time points, such as 42 h APF, midguts are more likely to break when prying with forceps, using more pointed forceps will cause less compression of the gut, thus increasing the rate of successful dissection.In addition, when dissecting pupae earlier than 30 h APF, usually at the moment of forceps puncture of the abdomen, intra-pupal tissues gush out of the puncture as the intra-pupal organs are under positive pressure.At this point, LIB is aspirated using a pipette gun to rinse the mixed tissues, and the pupal midgut, which is at an early stage of development, can be distinguished by the green color of the meconium and transferred to the dish using the pipette gun.
For dissection of 80 h APF or later pupae, the dissection method is the same as for adult midgut dissection, and the midgut is transferred to the dish using forceps.At all times, try to avoid prying the midgut with forceps.To gain experience in dissection, it is recommended to practice dissecting the adult midgut first, then the late pupal midgut, and finally the early pupal midgut.

Problem 2
The image is not clear (Step 27).

Potential solution
The image will not be clear if the gut is far away from the 22 3 22 mm coverslip.When fixing the midgut with LIG, adjust the position of the midgut to be as close to the 22 3 22 mm coverslip as possible.One thing to keep in mind is not to use too much LIG to cover the midgut, which can cause the sample to be too far away from the 22 3 22 mm coverslip.Be careful to adjust the midgut to the correct position without damaging it.

Problem 3
Difficulty in finding cell types at the correct development stage when imaging (Step 27).

Potential solution
Selection of cell types at the correct developmental stage is based on the developmental time, cell morphology and Pros::GFP expression.For example, to study the first asymmetric division of stem cells, the pupa should be dissected at 46 h APF, when the stem cells are dispersed, cell spacing is relatively uniform, expression of Pros is low or not detectable, and the cell morphology changes from irregular to round.To study the symmetric division of enteroendocrine mother cells (EMCs), the pupa can be dissected at 52 h APF, when newly formed stem cells and EMCs are close together, Pros is absent from stem cells, and Pros accumulates in EMCs.Because live imaging only captures cell events in 3 h, it is important to image cells that are ready to divide.Finally, to ensure reproducible results, the culture temperature of the fly pupae should be as stable as possible.

Problem 4
The pupal midgut is difficult to image for 3 h (Step 27).

Potential solution
If the midgut is dying rapidly, as epithelial cells start to loosen, nuclei stop moving and intestinal muscles stop contracting, then increasing the speed and quality of dissection is likely to be needed to improve the integrity of the midgut and reduce the amount of time the midgut is not bathing in media.Shorter exposure times and lower laser settings may help reduce damage due to phototoxicity.If the pupal midgut drifts out of focus in the z-axis, manually bring the sample into focus.In addition, we have also developed an adult FlyVAB imaging strategy for long-term intravital imaging of the adult midgut. 7

Figure 1 .
Figure 1.Collect white pupae (A) Collect LL3 and transfer to new vials.Store these LL3 vials at 25 C and collect white pupae (0 h APF) from LL3 vials every half hour.Transfer these pupae to new vials and make note of when pupae were collected (Figure 1A).(B) A testis (white transparent dot structure indicated by the arrow) on the mid-posterior part of a male LL3.(C) A female LL3.

Figure 2 .
Figure 2. Prepare live imaging dish (A) Melt the LIG by heating it on the metal bath at 37 C for 10 min.(B) Cut a 22 mm 3 22 mm coverslip into two pieces from the middle.(C) Drop 10 mL of melted LIG onto the dish.(D and E) Quickly cover the LIG with a pre-cut coverslip.(F) Repeat steps (C-E) and cover the other pre-cut coverslip, spacing the two coverslips approximately 5 mm apart.

Figure 3 .
Figure 3. Prepare pupae for dissection (A) Gently lift the staged pupae from the side of the vial.(B) Transfer these pupae to the dissecting plate.(C) Pupae prepared for dissection under a dissecting microscope.(D and E) Dorsal and lateral views of male and female pupae at 46 h APF and 52 h APF (arrow indicates meconium).Typically, males have a darker yellow tail color and are smaller individuals compared to female pupae of the same developmental stage.

Figure 5 .
Figure 5. Mount of pupal midgut (A) Transfer of the dissected midgut to the live imaging dish.(B) Zoom view of the area indicated by the arrow in (A).(C) Replace LIB with LIG.(D) Add LIG between the two 11 3 22 mm coverslips and cover the midgut with the 22 3 22 mm coverslip and press it with forceps to stabilize it.(E) Zoom view of the midgut mounted in (D), an air bubble under the slide but not on the midgut does not affect the survival and imaging of the midgut.(F) Seal these coverslips with halocarbon oil 27 (dashed lines indicate the range of oil).

Figure 6 .
Figure 6.Live imaging of pupal midgut (A) Pupal midgut mounted on a coverslip and imaged by inverted confocal microscopy.(B) Zoom view of the imaged mounted dish.(C) Imaging areas are indicated by rectangles.

Figure 7 .
Figure 7. Asymmetric distribution of Pros::GFP during the pupal ISC division Single frames from time-lapse movies of an ISC undergoing asymmetric division.Red, tdTomato; green, Pros::GFP.Arrow indicates basal localization of Pros::GFP.

Figure 8 .
Figure 8. Select ROIs for quantification analysis (A) The ROI area selected in the progenitor cell (pISC).(B) The ROI area selected in one daughter cell (ISCp).(C) The ROI area selected in the other daughter cell (EMC).M1-M3 are ROIs, B1-B1 00 and B2-B2 00 are areas for background intensity determination.