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Aug 2020
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Visualization and Quantitation of Wg trafficking in the Drosophila Wing Imaginal Epithelium
果蝇翅成虫上皮中Wg运输的可视化和定量研究   

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Abstract

Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. Here, we summarize and present three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help us to understand how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion.


Graphic abstract:



Figure 1. Visualization of extracellular and intracellular Wg trafficking in Drosophila wing imaginal discs. While staining of extracellular Wg without permeabilization exclusively visualizes Wg bound to the extracellular surface (left), Wg uptake and endosomal trafficking can be visualized using an antibody uptake assay (middle). Dynamic Wg release can be visualized by performing a non-permeabilizing staining at a permissive temperature that sustains secretory Wg transport (right).

Keywords: Wingless/Wnt secretion (Wnt 分泌), Morphogen signaling (成形素信号), Drosophila wing imaginal disc (果蝇的翅成虫盘), Recycling assay (回收试验), Extracelluar wingless (胞外wingless蛋白), Imaginal disc dissection (成虫盘解剖)

Background

The developing epithelium of the Drosophila wing imaginal disc (WID) is a well-established model system to study the intracellular trafficking of Wnt proteins. Imaginal discs are epithelial sac-like structures of Drosophila larvae that give rise to adult body parts during pupal transformation. Development of the fly wing from WID depends on the correct formation of several wing axes. To this end, several morphogen gradients are established. The dorsal-ventral wing axis is defined by Wg (Drosophila Wnt-1) secretion from a narrow cell stripe of Wg-expressing cells along the dorsal-ventral boundary. Upon secretion, Wg travels across the WID epithelium over short and long distances to activate target gene transcription in a dose-dependent manner. To ensure Wg gradient formation and proper wing development, Wg secretion and spread from the polarized WID epithelial monolayer are tightly regulated.


Upon translation, Wg is lipid-modified in the endoplasmic reticulum (ER) and transported via the Golgi toward the apical plasma membrane with its cargo receptor Evi/Wls (Bänziger et al., 2006; Bartscherer et al., 2006). While Wg can be transferred to neighboring cells by cell-cell contacts (Alexandre et al., 2014), long-range Wg secretion and signal transduction depend on Wg entry into the endosomal system (Strigini and Cohen, 2000; Pfeiffer et al., 2002). To this end, Wg re-enters the cell after apical presentation through a dynamin-independent endocytic route that delivers Wg into endosomal compartments (Hemalatha et al., 2016). Basal routes for secondary Wg secretion have also been described (Yamazaki et al., 2016) and Wg colocalizes with Rab4-recycling endosomes on the apical WID cell side (Gao et al., 2017; Linnemannstöns et al., 2020; Witte et al., 2020). Apical, secondary Wg release from Rab4-recycling endosomes could therefore fuel the Wg gradient independently of apicobasal transport (Witte et al., 2020). Consequently, interference with the apical release of Wg significantly hinders Wg gradient formation (Chaudhary and Boutros, 2019; Linnemannstöns et al., 2020).


The visualization of Wg trafficking to the cell surface and in post-endocytic endosomal compartments is an important tool to understand how the long- and short-range Wg signal is generated. Here, we present three main methods that have been established to unravel which of the proposed secretory routes feed the Wg signal: (1) the analysis of steady-state extracellular Wg levels [initially established by Strigini and Cohen (2000)]; (2) the visualization of intracellular Wg transport after endocytic uptake [modified from Hemalatha et al. (2016)]; and (3) the visualization of Wg recycling (Witte et al., 2020) (Figure 1). All three methods are based on immunofluorescence staining of dissected Drosophila WID, but different approaches are used to visualize Wg in the respective WID compartment (Figure 2). Briefly, while (1) and (3) make use of non-permeabilizing staining protocols, albeit at different temperatures, to specifically visualize surface bound extracellular Wg, (2) uses the endocytic uptake of antibody-labeled Wg to visualize its endosomal transport. Using UAS/Gal4-driven gene expression in combination with an engrailed promotor to drive genetic manipulations specifically in posterior WID allows the direct quantitation of anterior and posterior phenotypes within the same WID.


Multiple intracellular trafficking routes have been proposed to contribute to Wg signal transfer. It is therefore especially interesting to see how extracellular and intracellular Wg distributions react to interference with different aspects of the Wg trafficking machinery. As genetic manipulations are comparatively easy in Drosophila, the WID provides an appropriate means to probe this system. We hope that the methods presented herein contribute to gaining further insight into the evolutionarily conserved secretory Wg/Wnt trafficking pathway in order to fully understand how Wnt signals can be transferred over short and long distances. Moreover, these protocols are not limited to Wg and can be used to investigate the trafficking of other morphogens, such as Hedgehog (Hh) or Decapentaplegic (Dpp).



Figure 2. Overview of the different staining approaches. After WID dissection, extracellular Wg staining is performed on ice without permeabilization. The Wg uptake assay is performed at 22°C and includes acid washing to remove extracellularly bound signal, as well as permeabilization. Wg recycling is monitored at 22°C in the absence of permeabilization. After staining, WID samples are again dissected and mounted for downstream imaging and quantitation.

Materials and Reagents

  1. Drosophila vials (Kisker Biotech GmbH, catalog number: 789013)

  2. Plugs (Flugs wide plastic vials, Kisker Biotech GmbH, catalog number: 789035)

  3. 1.5 ml Reaction tubes (Eppendorf 3810X, catalog number: 0030 125.150)

  4. Microscope slides (LABSOLUTE, catalog number: 7 695 002)

  5. Cover slips (Roth, catalog number: H878)

  6. Ice and ice bucket

  7. Drosophila third instar larvae

  8. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: S0615)

  9. Schneider’s insect medium (Sigma-Aldrich, catalog number: S0146)

  10. Shields and Sang M3 insect medium (Sigma-Aldrich, catalog number: S3652)

  11. 16% Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710-S)

  12. Mouse anti-Wg (Developmental Studies Hybridoma Bank, catalog number: 4D4)

  13. Goat anti-mouse Alexa Fluor 647 (1:500, Invitrogen, catalog number: A21236)

  14. Hoechst 33342 (1:500, Thermo Scientific, catalog number: 62249)

  15. Mowiol (Calbiochem, catalog number: 475904)

  16. Corn meal (Spielberger Mühle, catalog number: SP061388)

  17. Soy flour (Spielberger Mühle, catalog number: SP061388)

  18. Dry yeast (Bierhefe Pulver, Cenovis)

  19. Malt extract (Lindenmeyer, Demeter)

  20. Sugar beet syrup (Bauckhof, Demeter)

  21. Agar-Agar (Roth, catalog number: 5210.2)

  22. Propionic acid (Roth, catalog number: 6026.3)

  23. Nipagin (700 ml 99% ethanol) (Roth, catalog number: 9065.4)

  24. Nipagin (C8H8O3) (Fluka, catalog number: 54752-1KG-F)

  25. NaCl (Roth, catalog number: 3957.2)

  26. KCl (Roth, catalog number: 6781.1)

  27. Na2HPO4·2H2O (Roth, catalog number: 4984.1)

  28. KH2PO4 (Fluka, catalog number: 15645940)

  29. Triton X-100 (Roth, catalog number: 3051.4)

  30. Glycine (Roth, catalog number: 3908.3)

  31. HCl (Roth, catalog number: K025.1)

  32. Fly food (see Recipes)

  33. PBS (see Recipes)

  34. PBTx (see Recipes)

  35. Glycine- HCl buffer (see Recipes)

  36. Mowiol (see Recipes)

Equipment

  1. Drosophila incubator (RUMED, type 3201)

  2. Brush (e.g., davinci, size 2)

  3. Forceps (Roth, catalog number: K342.1)

  4. Fluorescence stereomicroscope (depending on genetic set-up) (e.g., SZX12, equipped with U-RFL-T, Olympus)

  5. Dissection microscope such as Stemi 2000 (Zeiss) equipped with light source (e.g., KL-200, Zeiss)

  6. Confocal microscope such as LSM780 confocal laser-scanning microscope (Zeiss), equipped with Plan Neofluar 63×/oil NA 1.4 and PlanNeofluar 10× NA 0.3 objectives (Zeiss)

  7. Thermoblock (Eppendorf, Thermomixer compact)

  8. Rocker (Heidolph, POLYMAX 1040)

Software

  1. Zen Black (Zeiss), Version 14.0.0.0 (or alternative software for image aquisition)

  2. Fiji/ImageJ (NIH) (Schneider et al., 2012; Rueden et al., 2017), Version 1.53c (for image quantitation)

  3. Microsoft Excel, Microsoft Office Professional Plus 2016 (or alternative software for data analysis)

Procedure

  1. Setting up Drosophila crosses to obtain third instar larvae

    1. Choose an appropriate genetic system for the gene manipulation of interest.

      Note: We recommend using the UAS/Gal4-system to drive RNAi or protein expression alongside GFP specifically in posterior WID under an engrailed promotor (enGal4, UAS-GFP). In this way, the posterior half of the WID epithelium (marked by GFP) suffers the desired genetic manipulation, while the anterior WID serves as an internal control. Using this set-up enables a direct and reliable comparison of signal strength and distribution between the anterior control and posterior treatment side of the same WID. It therefore reduces the variability associated with immunofluorescence-based detection and quantitation of phenotypes in an in vivo model system.

    2. Prepare vials for Drosophila culture (see Recipes). Let fly vials come to room temperature (RT) before use.

    3. Collect males and virgin females of the desired fly strains.

    4. Set up crosses of 20 virgin females and 10-20 males in fresh food vials.

    5. Keep crosses at 18°C, 20°C, or 25°C. As UAS/Gal4-mediated expression is temperature-dependent, with a minimal Gal4 activity at 16°C and a maximal activity at 29°C, the temperature can be adjusted to obtain the required expression level.

    6. Transfer flies to fresh food vials every 48 h to obtain synchronized larval populations.

    7. Culture vials at the desired temperature until wandering third instar larvae emerge. At 25°C, wandering third instar larvae can be collected on days 5 and 6.


  2. Dissection of Wing Imaginal Discs (Part 1)

    1. Collect third instar larvae of the required genotype in PBS (see Recipes) using a brush or forceps.

      Notes:

      1. Depending on the fly strains used, larvae of the correct genotype may have to be selected based on marker expression. If using EnGal4, UAS-GFP/CyO x UAS-RNAi/UAS-RNAi, GFP-positive larvae have to be selected using a fluorescence stereomicroscope.

      2. If desired, larvae can be sexed based on the visibility of testis in males.

    2. Transfer larvae to fresh PBS to wash off food or dirt remnants.

    3. Keep larvae in PBS on ice to anesthetize the animals and slow their movement.

    4. Transfer one larva to a drop of PBS or Schneiders medium on a clean glass slide.

    5. Use a dissection microscope and two pairs of fine forceps for WID dissection.

    6. Position the larva in your field of view with the mouth-hooks toward the right and the tail toward the left (Figure 3A).

      Note: The following protocol describes an easy method for WID dissection from the perspective of a right-handed person. If your left hand is dominant, mirroring the following description may increase your precision.

    7. Grab the larva with both pairs of forceps at 1/3 to 1/2 of its body length (Figure 3B).

    8. Firmly pull on the posterior larval end to tear the larva apart (Figure 3C). Discard the posterior half of the larva and reposition the anterior half in your field of view (Figure 3D). If large organ parts of the gut and fatbody have spilled out and occlude your view, carefully remove them.

    9. Hold on to the mouth-hooks with your right pair of forceps and insert the left pair of forceps into the body opening (Figures 3E and 3F).

    10. Carefully push with the left pair of forceps toward the mouth-hook to gently slip the larva onto the right pair of forceps, thereby turning it inside-out (Figure 3G). The anterior larval half should now be fully inverted and pulled over the right pair of forceps with the internal tissue, including the WID exposed (Figures 3H1 and 3H2).

    11. Grab the inverted mouth-hooks with the left pair of forceps and slide it off the right forceps. Carefully remove the gut, salivary gland, and fat body (Figure 3I1). Be careful not to accidentally remove the WID, especially if you remove strands of the trachea. The WID should remain attached to the larva.

      Note: Internal tissues do not have to be fully removed at this step, as a second dissection will be performed before mounting the samples. Do, however, make sure that WID are not covered by remaining tissue parts to ensure uniform staining and fixation.

    12. Try to identify the two WID, one on either side of the larva. They are translucent teardrop-shaped structures and are often hanging on the trachea, sometimes floating at a distance to the larval main body (Figures 3I2 and 3I3).



      Figure 3. Dissection of anterior larvae for WID isolation. The anterior half of third instar larvae is isolated (A-D) and inverted onto one pair of forceps (E-H). Subsequently, WID (encircled in yellow) are retained at the anterior larval half, while other organ parts are removed (I). The resulting samples are transferred to medium before further staining and fixation.


    13. Transfer the inverted anterior larval parts including WID to an Eppendorf tube containing Schneider’s or M3 medium on ice, depending on the downstream application (Figure 2).

    14. Collect at least 15-20 WID samples per condition.

      Note: Do not keep WID on ice for more than 30 min before proceeding with the next step in order to minimize tissue degradation.


  3. Wing Imaginal Disc Staining

    1. Visualization of steady state extracellular Wg

      1. Collect WID samples in 200 µl M3 medium without FBS in an Eppendorf tube.

      2. Add 100 µl mouse anti-Wg antibody (DSHB 4D4 supernatant), corresponding to a dilution of 1:3, and incubate on ice for 60 min.

      3. Remove staining solution carefully and rinse three times with 1 ml ice-cold PBS. This is best achieved by aspirating the medium with a 1,000 µl pipet doubled-tipped with a 1,000 µl and 200 µl pipet tip.

        Note: Due to their attachment to the anterior body part, WID samples will quickly settle down in the Eppendorf cup, thus allowing the removal and exchange of staining and washing solutions by pipetting.

      4. Remove PBS and fix in 500 µl 4% PFA in PBS for 5 min on ice, followed by 15 min at RT with gentle agitation/on a rocker.

      5. Remove fixing solution and wash three times for 10 min in 1 ml PBS.

      6. Remove PBS and block in 500 µl PBS + 5% FBS for 30 min.

      7. Incubate with secondary antibody diluted in 5% FBS in PBS overnight at 4°C or 2 h at RT (anti-mouse-A568 + Hoechst 1:500).

      8. Remove staining solution and wash three times for 10 min in 1 ml PBS.

    1. Visualization of Wg endocytosis and trafficking

      1. Collect WID samples in 200 µl S2 medium + 10% FCS.

      2. Add 50 µl mouse anti-Wg antibody (DSHB 4D4, dilution 1:5) and incubate at 22°C for 5-90 min with gentle agitation.

        Note: For visualization of the endocytic pool of Wg in combination with total Wg, endogenously tagged GFP-Wg on the cell surface can be labeled by an anti-GFP antibody. Counterstaining with an A568-labeled secondary antibody (under C2.10) allows subsequent discrimination between (dual-color) GFP-Wg after endocytosis and (single-color) GFP-Wg that has not been present on the cell surface during Step C1.

      3. Remove staining solution carefully and rinse twice with 1 ml PBS.

      4. Remove PBS and acid wash WID samples with 0.1 M Glycine-HCl buffer (pH 3.5, see Recipes) for 30 s at RT to remove extracellularly bound antibody.

      5. Remove Glycine-HCl buffer and rinse three times with 1 ml PBS.

      6. Remove PBS and fix in 500 µl 4% PFA in PBS for 20 min at RT with gentle agitation/on a rocker.

      7. Remove fixing solution and rinse once with 1 ml PBS.

      8. Wash twice for 15 min with 1 ml PBTx (see Recipes) to permeabilize the tissue.

        Note: Permeabilization in this step enables the secondary antibodies used in Step C2j to access the cell and bind to the intracellular pool of anti-Wg-labelled, and thus endocytosed Wg.

      9. Remove PBTx and block 30 min in 500 µl 5% FBS in PBTx.

      10. Incubate with secondary antibody diluted in 5% FBS in PBTx overnight at 4°C or 2 h at RT (anti-mouse-A568 + Hoechst 1:500).

      11. Remove staining solution and wash thrice for 10 min in 1 ml PBTx.

    1. Visualization of Wg recycling

      1. Collect WID samples in 200 µl M3 medium.

      2. Add 100 µl mouse anti-Wg antibody (dilution 1:3) and incubate at 22°C for 45-60 min with gentle agitation.

      3. Remove staining solution carefully and rinse three times with 1 ml PBS.

      4. Remove PBS and fix in 500 µl 4% PFA in PBS for 20 min at RT with gentle agitation/on a rocker.

      5. Remove fixing solution and wash three times for 10 min in 1 ml PBS.

      6. Remove PBS and block in 500 µl PBS + 5% FBS for 30 min.

      7. Incubate with secondary antibody diluted in 4% FBS in PBS overnight at 4°C or 2 h at RT (anti-mouse-A568 + Hoechst 1:500).

      8. Remove staining solution and wash thrice for 10 min in 1 ml PBS.


  4. Dissection of Wing Imaginal Discs (Part 2) and Mounting

    1. Transfer WID samples with 1-2 drops of PBS onto a glass slide using a Pasteur pipet.

    2. Use a dissection microscope and two pairs of fine forceps for WID dissection.

    3. Try to locate the WID on the sample. Ideally, two WID per sample can be spotted approximately where a larval “shoulder” would be expected (at around 1/3 of the length of the anterior sample), sometimes attached to the trachea (Figure 4A).

      Note: After fixation, the WID appear more white than translucent and are usually easier to spot.

    4. Carefully detach the WID from the remaining anterior larval body part. Do not hold on to the WID tissue directly with your forceps. Instead, hold on to the main specimen part with your left-hand forceps and find the tissue strands attaching the WID to the larva. Then shear them off in a scissor-like fashion or detach the WID without pulling on it directly.

    5. Proceed with all WID samples, then remove all remaining tissue parts except for the WID from the glass slide (Figures 4B and 4C).



      Figure 4. Dissection of WID for mounting. WID are transferred to a glass slide. All larval parts except for WID are removed without disturbing the integrity of the WID tissue.


    6. Soak up PBS from the glass slide with the tip of a thin and soft tissue.

      Note: The capillary force may move your WID toward the tissue. Try not to pick up WID with the tissue.

    7. Add 60 µl Mowiol (see Recipes) to the glass slide with a cut-off pipet tip.

    8. Carefully mount with a cover slip, placing one edge of the cover slip over the sample and lowering down the other side with forceps. Try to avoid mounting air bubbles.

      Note: Air bubbles trapped under the cover slip can be carefully pushed aside by applying gentle pressure.

    9. Let Mowiol dry at 4°C for at least 12 h before imaging. Store slides at 4°C.


  5. Imaging acquisition

    1. Use a confocal microscope equipped with 10× and 63× magnification objectives that are capable of acquiring z-stacks.

    2. Identify appropriate WID using the 10× objective. Take an overview image if wanted (not needed for quantitation).

    3. Aquire z-stacks from the apical WID surface (just below the peripodial membrane) to the basal WID surface with a step size of 0.5-1 µm using the 63× objective.

    4. Image at least 5-10 WID per condition. Keep laser intensity and detector gain constant (see Figure 5 for representative examples of all three staining protocols).

      Note: If using the enGal4 system, laser intensity and detector gain can be adjusted for optimal illumination, as downstream signal analysis will involve normalization of the anterior to the posterior WID.

    5. Save image data in a file format compatible with the use of Fiji/ImageJ [such as .czi for Zen (Zeiss)].



      Figure 5. Example Wg staining using the different approaches. Extracellular Wg staining of enGal4>UAS-GFP, UAS-Ykt6 RNAi (A), as well as Wg uptake assay (B) and Wg recycling assay (C) of enGal4>UAS-GFP, UAS-Klp98A RNAi. While Ykt6 knockdown interferes with Wg secretion and therefore with extracellular Wg levels (Gross et al., 2012), Klp98A knockdown interferes with apicobasal Wg transport and promotes apical accumulation of Wg inside endosomes as well as apical Wg recycling (Witte et al., 2020). The posterior knockdown compartment is marked by the coexpression of GFP. The anterior compartment serves as a control. Maximum intensity projection of apical to basal z-stack (A) or individual apical sections the WID epithelium (B, C). Scale bars, 20 μm.

Data analysis

  1. Open WID z-stacks in Fiji/ ImageJ. Depending on the microscope software/image format used, the Bio-format plugin may be needed to open your file.

    Note: The following protocol relies on normalization of posterior to anterior signal intensities and only applies if enGal4 was used to drive the genetic manipulation of interest.

  2. Go to Image → Transform → Rotate and rotate the z-stack until the Wg-expressing cell stripe is aligned horizontally (Figure 6A).

  3. Draw a rectangular region of interest (ROI) (Figure 6B1), then go to Edit → Selection → Specify to adjust its size to 150 × 150 pixels or another appropriate size (Figure 6B2).

  4. Save the ROI into the ROI manager by going to Edit → Selection → Add to manager (Figure 6B3).

  5. Repeat steps 3 and 4 to create a set of two identical ROIs. You can save your ROI set for later analysis using the More → Save option of the manager.

  6. Position one ROI on the anterior side and one ROI on the posterior side of the Wg-expressing cell stripe. If co-expressing UAS-GFP in posterior WID, use the green channel for orientation (Figure 6B4).

    Note: Make sure the “show all” option in the ROI manager is checked.

  7. Split image channels using the channels tool or by going to Image → Color → Split Channels (Figure 6C1).

  8. On the ROI manager, go to More → Multi Measure to measure the intensity of the Wg signal in both ROIs and each slice of your z-stack simultaneously (Figure 6C2). A popup window with a data table will open.

    Note: After measuring, you can save an image including the position of your ROIs by using the Flatten option in the ROI manager before saving the image file.

  9. Copy and paste the multi-measured data to an Excel sheet (or an alternative software for data handling). For each ROI, you will receive data regarding ROI size, as well as average, minimal, and maximal signal intensity for each slice of your stack (Figure 6C3).

    Note: If your data table has different entries, use Fiji → AnalyzeSet measurements to adjust the measurement parameters.



    Figure 6. Quantitation of Wg signal intensity after enGal4-driven genetic manipulation. The Wg-expressing cell stripe is aligned horizontally (A) and appropriate anterior and posterior regions of interest (ROI) are selected (B). Then, Wg signal intensity in each ROI is measured individually for each slice of the z-stack and normalized to the anterior wildtype control (C).


  10. Open your original data file in Fiji and select the slices you are interested in (e.g., 3 most apical slices below the peripodial membrane, 3 most basal slices, whole stack, …) and mark the corresponding slices in your Excel sheet.

  11. Calculate the average intensity of the mean signal in the anterior and posterior ROI of the relevant slices.

  12. Normalize the posterior signal intensity to the anterior signal by dividing by the anterior signal average.

    Note: Normalizing the anterior to the posterior signal intensity is possible because the anterior WID side serves as an internal wildtype control in an enGal4-driven genetic manipulation setup.

  13. Using steps 1-12, collect data from at least 5-10 WID.

  14. Plot a graph in the software of your choice (e.g., Excel, Graphpad, …).

Recipes

  1. Fly food

    1. 712 g corn meal, 95 g soy flour, 168 g dry yeast, 450 g malt extract, 400 g sugar beet syrup, 50 g Agar-Agar, 45 ml propionic acid, 150 ml Nipagin solution (prepared from 300 ml H2O + 100 g Nipagin (C8H8O3), filled up to 10 L using ddH2O).

    2. Heat water until nearly boiling, then add soy flour, dry yeast, and Agar-Agar

    3. Stir in malt extract and sugar beet syrup

    4. Stir in corn meal

    5. Reduce heat and cook for 45-60 min with closed lid, stirring occasionally

    6. Cool down fly food to 60°C, then add propionic acid and nipagin

    7. Fill Drosophila vials 20-30% with fly food and dry at RT overnight. Then plug vials and store at 4°C

  2. PBS

    137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4 ·2 H2O, 1.46 mM KH2PO4

    Dissolve in ddH2O and adjust pH to 7.4

  3. PBTx

    PBS + 0.1% Triton X-100

  4. Glycine-HCl buffer

    0.1 M Glycine in ddH2O, adjust pH to 3.5 with HCl

  5. Mowiol

    Dissolve 0.25 g/ml in PBS

Acknowledgments

Research in the laboratory of J.C.G. is supported by the Deutsche Forschungsgemeinschaft-funded Research Center [SFB1324/1 (331351713) and GR4810/2-1]; the research program of the Georg-August-Universität Göttingen of University Medical Center; and a postdoctoral fellowship to K.L. by the Dorothea Schlözer Program, University Medical Center, Georg-August-Universität Göttingen. This protocol is based on work from Witte et al. (2020) and Linnemannstöns et al. (2020). We thank Dr. Vera Terblanche (Department of Evolutionary Developmental Genetics, GAU Göttingen) for providing access to her groups camera-equipped stereomicroscope.

Competing interests

The authors declare no competing or financial interests.

Ethics

No Ethics Committee approval is required for Drosophila work.

References

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  3. Bartscherer, K., Pelte, N., Ingelfinger, D. and Boutros, M. (2006). Secretion of Wnt Ligands Requires Evi, a Conserved Transmembrane Protein. Cell 125(3): 523-533.
  4. Chaudhary, V. and Boutros, M. (2019). Exocyst-mediated apical Wg secretion activates signaling in the Drosophila wing epithelium. PLoS Genet 15(9): e1008351.
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  13. Yamazaki, Y., Palmer, L., Alexandre, C., Kakugawa, S., Beckett, K., Gaugue, I., Palmer, R. H., and Vincent, J.-P. (2016). Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs. Nat Cell Biol 18(4): 451-457.

简介

[摘要]分泌型Wnt转运可在果蝇翅性心盘(WID)的极化上皮单层中研究。在该组织中,Wg (果蝇Wnt -I)在被内化到内体途径之前先出现在其源细胞的顶表面上。远距离的Wg分泌和扩散取决于内体区室的二次分泌,但是对Wg的内吞后确切命运知之甚少。这里,我们总结和用于基于免疫荧光的可视化和孔定量本三个协议吨胞内和胞外的不同池的通货膨胀的Wg在WID:(1)稳态细胞外的Wg ; (2)内体隔室内的动态Wg贩运; (3)动态Wg释放到细胞表面。使用基因驱动程序系统专门在WID (EnGal4)的后部进行基因操纵,可提供强大的内部控制,从而可以直接比较同一WID的操纵室和操纵室的信号强度。Ť这里脱颖而出,这也规避了高度的染色通常与整体组织样本有关的变异。结合在果蝇中很容易实现的Wg途径成分的基因操作,这些方法为解剖分泌性Wg转运提供了工具集,并可以帮助我们了解Wnt蛋白如何沿着内体区室进行短时和长时性转运。范围信号分泌。



图形摘要:


图1 。可视化果蝇翼假想盘中的细胞外和细胞内Wg交易。在不透化的情况下,对细胞外Wg进行染色时,只能观察到与细胞外表面结合的Wg (左),而可以使用抗体摄取测定法(中)观察到Wg的摄取和内体运输。动态Wg释放可以通过在允许分泌Wg转运的允许温度下进行非透化染色来观察(右图)。



[背景]果蝇翅假想盘(WID)的上皮发育是研究Wnt蛋白在细胞内运输的成熟模型系统。想象中的椎间盘是果蝇幼虫的上皮囊状结构,在p转化过程中会产生成年的身体部位。从WID展开飞翼取决于几个机翼轴的正确形成。为此,建立了几个形态发生剂梯度。背腹翼轴由沿着背腹边界从表达Wg的狭窄细胞条带中的Wg (果蝇Wnt-1)分泌限定。分泌后,Wg以短距离和长距离穿过WID上皮,以剂量依赖性方式激活靶基因转录。Ť o确保Wg的梯度形成和适当的翼发展,Wg的从偏振光WID上皮米分泌和扩散onolayer紧密调节。

一旦翻译,Wg的是脂质修饰中的烯doplasmic网(ER )和运输经由高尔基体朝向顶端质膜及其货物受体的Evi / WLS (Bänziger等人,2006; Bartscherer等人,2006) 。虽然Wg可以通过细胞与细胞之间的接触转移到邻近细胞(Alexandre等,2014),但Wg的长距离分泌和信号转导取决于Wg进入内体系统(Strigini和Cohen,2000; Pfeiffer等,2000)。 2002)。为此,Wg在顶端呈现后通过不依赖动力的内吞途径重新进入细胞,该途径将Wg递送到内体区室中(Hemalatha等人,2016)。乙ASAL路由二次Wg的分泌都也被描述(山崎等人,2016)和Wg的共定位与Rab4-回收在顶WID单元侧内体(高等人,2017;Linnemannstöns等人,2020;维特等等,2020)。因此,从Rab4循环内体释放出的根尖,继发性Wg可能会独立于后肢基底膜转运而增加Wg梯度(Witte等人,2020)。因此,干扰Wg的顶端释放会显着阻碍Wg梯度的形成(Chaudhary和Boutros,2019年;Linnemannstöns等人,2020年)。

可视化的Wg转运到细胞表面和内吞后内体区室中是一种重要的工具,可以了解如何产生长程和短程Wg信号。在这里,我们介绍了已建立的三种主要方法,以揭示提议的分泌途径中哪些会喂入Wg信号:(1)稳态细胞外Wg水平的分析[最初由Strigini和Cohen (2000 )建立] ;(2)内吞摄取后细胞内Wg转运的可视化[改编自Hemalatha等。(2016)] ;(3)可视化的汞回收(Witte等,2020)(图1)。这三种方法均基于对果蝇WID的免疫荧光染色,但是使用了不同的方法来可视化各个WID隔室中的Wg (图2)。简而言之,虽然(1)和(3)尽管在不同温度下仍使用非透化染色方案来特异性地显现表面结合的细胞外Wg ,但(2)使用抗体标记的Wg的内吞摄取来观察其内体转运。组合使用UAS / GAL4-驱动的基因表达用启动子engrailed基因特异性驱动遗传操作在后WID允许直接孔定量吨相同WID内前,后表型通货膨胀。

已经提出了多种细胞内运输途径来促进Wg信号转移。因此,特别有趣的是,观察细胞外和细胞内Wg的分布如何对Wg贩运机制不同方面的干扰做出反应。由于在果蝇中遗传操作相对容易,因此WID提供了一种探测该系统的合适方法。我们希望法本文提出有助于获得荷兰国际集团进一步洞察进化上保守的分泌的Wg / Wnt信号运输途径,以充分了解的Wnt信号可以在短距离和长距离传输。此外,这些协议不限于Wg ,还可以用于调查其他形态发生子的运输,例如刺猬(Hh )或十足瘫痪(Dpp )。





图2.不同染色方法的概述。WID解剖后,在冰上进行细胞外Wg染色而不透化。的Wg的摄取试验是在22执行℃,并包括酸洗涤以除去细胞外结合的信号,以及透。在没有透化的情况下,在22°C下监控Wg的回收利用。染色后,WID样品再次解剖并安装用于downstrea米成像和孔定量吨通货膨胀。

关键字:Wnt 分泌, 成形素信号, 果蝇的翅成虫盘, 回收试验, 胞外wingless蛋白, 成虫盘解剖



材料和试剂


果蝇小瓶(Kisker Biotech GmbH,目录号:789013)
塞(Flugs宽塑料小瓶,Kisker生物技术公司,目录号:789035)
1.5 ml反应管(Eppendorf 3810X,目录号:0030 125.150)
显微镜载玻片(LABSOLUTE,目录号:7695002)
盖玻片(Roth,货号:H878)
冰和冰桶
果蝇第三研究所幼虫
胎b绵羊小号erum(FBS)(Sigma公司- Aldrich公司,目录号:S0615)
Schneider的我NSECT米edium(西格玛- Aldrich公司,目录号:S0146)
屏蔽件和桑M3我NSECT米edium(西格玛- Aldrich公司,目录号:S3652)
16%多聚甲醛(电子显微镜科学,目录号:15710-S)
小鼠抗Wg (发育研究杂交瘤库,目录号:4D4)
山羊抗小鼠Alexa Fluor 647(1:500,Invitrogen,目录号:A21236)
Hoechst 33342(1:500,Thermo Scientific,目录号:62249)
Mowiol (Calbiochem ,目录号:475904)
玉米米EAL(斯皮尔伯格Mühle的,目录号:SP061388)
大豆粉(SpielbergerMühle,目录号:SP061388)
干酵母(Bierhefe普尔菲,Cenovis )
麦芽提取物(Lindenmeyer ,Demeter)
甜菜糖浆(Bauckhof ,Demeter)
琼脂-琼脂(Roth,目录号:5210.2)
丙酸(Roth,目录号:6026.3)
尼泊金(700毫升99%乙醇)(Roth,目录号:9065.4)
尼泊金(C 8 H 8 O 3 )(Fluka ,目录号:54752-1KG-F)
NaCl (Roth,目录号:3957.2)
氯化钾(Roth,6781.1)
Na 2 HPO 4 · 2H 2 O(Roth,目录号:4984.1)
KH 2 PO 4 (Fluka ,货号:15645940)
海卫一X-100(Roth,货号:3051.4)
甘氨酸(Roth,目录号:3908.3)
HCl (Roth,目录号:K025.1)
飞食(请参阅食谱)
P BS (请参阅食谱)
PBTx (请参阅食谱)
甘氨酸-HCl缓冲液(请参见食谱)
Mowiol (请参阅食谱)


设备


果蝇我ncubator(RUMED,类型3201)
刷(例如。,达芬奇,大小2)
镊子(Roth,货号:K342.1)
荧光小号tereo米icroscope(取决于遗传的建立)(例如。,SZX12,配备有U型RFL-T,奥林巴斯)
解剖显微镜如STEMI配备有光源2000(Zeiss)对(例如,KL-200,Zeiss)对
共焦显微镜,例如LSM780共焦激光扫描显微镜(Zeiss),配备了Plan Neofluar 63 × /油NA 1.4和PlanNeofluar 10 × NA 0.3物镜(Zeiss)
Thermoblock (Eppendorf,紧凑型Thermomixer)
摇杆(Heidolph ,POLYMAX 1040)




软件


禅黑色(蔡司),版本14.0.0.0(或替代软件进行图像AQUISITION )
斐济/ ImageJ的(NIH)(Schneider等人,2012。; Rueden酒店等人。,2017),版本1.53c(用于图像孔定量吨通货膨胀)
Microsoft Excel,Microsoft Office Professional Plus 2016(或用于数据分析的替代软件)


程序


设置果蝇杂交获得第三龄幼虫
选择合适的遗传系统进行感兴趣的基因操作。
注意:我们建议使用UAS / Gal4-系统驱动RNAi或蛋白质与GFP一起在激进的启动子(enGal4,UAS-GFP)下的WID后特异性表达。这样,WID上皮的后半部分(用GFP标记)遭受了所需的遗传操作,而前WID充当了内部对照。使用此设置可以直接可靠地比较同一WID的前控制侧和后处理侧之间的信号强度和分布。它第erefore减少与immunoflu相关的变异性orescence基于检测和孔定量吨在体内模型系统表型通货膨胀。


准备用于果蝇培养的小瓶(请参阅R ecipe s )。使用前,让蝇瓶达到室温(RT )。 
收集所需果蝇品系的雄性和雌性雌性。
在新鲜食品小瓶中设置20个原始女性和10-20个男性的杂交。
保持杂交在18℃,20℃ ,或25℃。由于UAS / Gal4介导的表达是温度依赖性的,在16°C时具有最小的Gal4活性,在29°C时具有最大的活性,因此可以调节温度以获得所需的表达水平。
每48小时将苍蝇转移到新鲜的食物小瓶中,以获取同步的幼虫种群。
在所需温度下培养小瓶,直到出现三龄幼虫游荡。在25℃下,徘徊第三龄幼虫可以在天收集5和6 。


机翼幻影盘的解剖(第1部分)
用刷子或镊子将所需基因型的三龄幼虫收集在PBS中(请参阅R ecipe s )。
注意小号:


根据所用的蝇蝇菌株,可能必须根据标记物表达选择正确基因型的幼虫。如果使用EnGal4,UAS-GFP / CyO x UAS-RNAi / UAS-RNAi,则必须使用荧光立体显微镜选择GFP阳性幼虫。
如果需要,可以根据男性睾丸的可见性对幼虫进行性别鉴定。
将幼虫转移到新鲜的PBS中,以洗去食物或残留的污垢。
将幼虫放在冰上的PBS中以麻醉动物并减慢其运动。
将一个幼虫转移到干净的载玻片上的一滴PBS或Schneiders培养基中。
使用解剖显微镜和两对细镊子进行WID解剖。
将幼虫放置在您的视野中,将钩子朝右,将尾巴朝左(图3A)。
注意:以下协议从惯用右手的人的角度描述了一种简单的WID解剖方法。如果你的左手是显性的,镜像以下描述M AY提高精度。


用两对镊子抓住幼虫,使其长在其体长的1/3至1/2处(图3B)。
用力拉住幼虫的后部,将幼虫撕开(图3C)。丢弃幼虫的后半部分,并在视野中重新放置前半部分(图3D)。如果肠道和脂肪体的大器官部位溢出并遮挡了您的视线,请小心将其取下。
坚持到嘴钩用右手用镊子和插入左钳子进入人体开口(图小号3E和3F)。
小心地将左对镊子推向钩子,以将幼虫轻轻滑到右对镊子上,从而将其内外翻(图3G)。前一半幼虫现在应充分倒置并拉到右一对钳子的与内部组织,包括WID露出(图小号3H1和3H2)。
用左对镊子抓住倒钩,然后将其滑出右镊子。小心取出肠,唾液腺,和肥胖的身体(图3I1)。小心不要意外移除WID,尤其是当您移除气管丝时。WID应该保持附着在幼虫上。
注意:此步骤不必完全去除内部组织,因为在安装样品之前将进行第二次解剖。做,但是,确保WID未涵盖的其余的组织部分,以保证均匀染色和固定。


尝试识别两个WID,一个在幼虫的两侧。它们是半透明泪滴形状的结构,并且通常挂在气管,有时浮在距离幼虫主体(图小号3I2和3I3)。




图3.用于WID隔离的前幼虫的解剖。将第三龄幼虫的前半部分隔离(AD)并倒入一对钳子(EH)中。随后,将WID(黄色圆圈)保留在幼虫前半部分,同时去除其他器官部分(I)。在进一步染色和固定之前,将所得样品转移到培养基中。


将倒置的前幼虫部分(包括WID)转移到装有冰上施耐德氏或M3培养基的Eppendorf管中,具体取决于下游应用(图2)。
每个条件至少收集15-20个WID样本。
注意:在进行下一步之前,请勿将WID在冰上放置30分钟以上,以最大程度地减少组织降解。


机翼梦幻碟片染色
稳态细胞外Wg的可视化
将WID样品收集在200μl不带FBS的M3培养基中的E ppendorf管中。
加入100 µl小鼠抗Wg抗体(DSHB 4D4上清液)(对应于1:3的稀释度),并在冰上孵育60分钟。
小心地除去染色溶液,并用1 ml冰冷的PBS冲洗3次。这是最好通过抽吸用1的介质来实现,000微升吸管一倍尖与1 ,000微升和200微升吸管尖。
注意:由于WID样品附着在身体的前部,因此它们会迅速落在Eppendorf杯中,从而可以通过移液去除和交换染色和洗涤溶液。


除去PBS,并在冰上在500 µl 4%PFA的PBS中固定5分钟,然后在室温下轻轻摇动/在摇杆上固定15分钟。
除去固定液,并在1 ml PBS中洗涤3次,每次10分钟。
除去PBS,然后在500 µl PBS + 5%FBS中锁定30分钟。
与稀释于5%FBS的PBS中的二抗在4°C孵育过夜或在室温2 h(anti-mouse-A568 + Hoechst 1:500)孵育。
除去染色溶液,并在1 ml PBS中洗涤3次,每次10分钟。
Wg内吞和贩运的可视化
在200 µl S2培养基+ 10%FCS中收集WID样品。
加入50 µl小鼠抗Wg抗体(DSHB 4D4,稀释度为1:5),并在22°C温和搅拌孵育5-90分钟。
注意:对于内吞池的可视化的Wg结合总的Wg ,内源性标记GFP- Wg的在细胞表面上可以标记由一个抗GFP抗体。用(下C 2-10)的A568标记的第二抗体复染允许之间的后续歧视(双-彩色)GFP- Wg的胞吞作用后和(单-彩色)GFP- Wg的尚未存在于细胞表面上时小号TEP Ç 1。


小心地除去染色溶液,并用1 ml PBS冲洗两次。
除去PBS并用0.1M甘氨酸-HCl缓冲液酸洗WID样品(pH值3.5 ,见配方小号)30秒在RT以除去细胞外结合的抗体。
除去甘氨酸-HCl缓冲液,并用1 ml PBS冲洗3次。
除去PBS,在室温下轻轻摇动/在摇杆上,在PBS中加入500 µl 4%PFA固定20分钟。
除去定影液并用1 ml PBS冲洗一次。
清洗两次,用1毫升15分钟PBTx (见配方小号)透化组织。
注意:此步骤中的通透性可使S tep C 2j中使用的二抗进入细胞并结合到细胞内的抗Wg标记的Wg上,从而被Wg内吞。


取出PBTx并在500 µl 5%FBS的PBTx中锁定30分钟。
与在PBTx中的5%FBS中稀释的二级抗体在4°C过夜孵育或在室温2小时孵育(抗小鼠A568 + Hoechst 1:500)。
除去染色溶液,并在1 ml PBTx中清洗三次,每次10分钟。
可视化的汞回收
在200 µl M3培养基中收集WID样品。
加入100 µl小鼠抗Wg抗体(稀释度为1:3),并在22°C温和搅拌下孵育45-60分钟。
小心地除去染色溶液,并用1 ml PBS冲洗3次。
除去PBS,在室温下轻轻摇动/在摇杆上,在PBS中加入500 µl 4%PFA固定20分钟。
除去固定液,并在1 ml PBS中洗涤3次,每次10分钟。
除去PBS,然后在500 µl PBS + 5%FBS中锁定30分钟。
与稀释于PBS中4%FBS中的二抗一起在4°C孵育过夜,或在RT孵育2小时(抗小鼠A568 + Hoechst 1:500)。
除去染色溶液,并在1 ml PBS中清洗三次,每次10分钟。


翼成虫盘的夹层(部分2)和中号ounting
使用巴斯德吸管将含1-2滴PBS的WID样品转移到载玻片上。
使用解剖显微镜和两对细镊子进行WID解剖。
尝试在样本上找到WID。理想情况下,每个样本可以发现两个WID,大约在预期会出现幼虫“肩”的地方(大约是前样本长度的1/3),有时会附着在气管上(图4A)。
注意:固定后,WID看起来比半透明的更白,通常更容易发现。


小心地将WID与剩余的幼虫前体部分分开。不要用镊子直接抓住WID组织。取而代之的是,用左手镊子抓住主要标本部分,然后找到将WID附着到幼虫上的组织束。然后以类似剪刀的方式将其剪下,或在不直接拉动WID的情况下将其分离。
与所有WID样品进行,然后除去除了WID从载玻片(图所有剩余组织部分小号4B和4C)。




图4. WID的安装剖视图。WID被转移到载玻片上。除去WID以外的所有幼体部分,而不会影响WID组织的完整性。


用薄而柔软的组织的尖端从载玻片上吸收PBS。
注:毛细力米唉移动你的WID朝组织。尽量不要用纸巾捡起WID。


添加60微升的Mowiol (见ř ecipe小号)到载玻片与截止吸管尖。
小心地用盖玻片安装,将盖玻片的一个边缘放在样品上,然后用镊子将其另一侧放下。尽量避免安装气泡。
注意:轻轻施加压力,可以小心地将捕获在盖玻片下面的气泡推开。


让Mowiol在成像前于4°C干燥至少12 h。将载玻片存放在4°C下。


成像一个cquisition
使用配备了10倍和63倍放大物镜的共焦显微镜,这些物镜能够采集z叠层。
使用10 ×目标确定适当的WID 。以一个全貌图像,如果想(不需要孔定量牛逼通货膨胀)。
使用63 ×物镜,以0.5-1 µm的步长从顶WID表面(恰好在周膜下方)获取Z-stack,步长为0.5-1 µm 。
每个条件下的图像至少5-10 WID。保持激光强度和检测器增益不变(所有三种染色方案的代表性示例,请参见图5)。
注意:如果使用enGal4系统,可以调整激光强度和检测器增益以获得最佳照明,因为下游信号分析将涉及WID前后的标准化。


将图像数据保存为与使用Fiji / ImageJ兼容的文件格式(例如。[ Zen(Zeiss)的czi ] 。




图5.实施例一MPL ë Wg的染色使用不同的方法。enGal4> UAS-GFP,UAS-Ykt6 RNAi的细胞外Wg染色(A),以及enGal4> UAS-GFP,UAS-Klp98A RNAi的Wg摄取测定(B)和Wg回收测定(C)。虽然Ykt6敲低会干扰的Wg分泌和第erefore与细胞外的Wg的水平(建筑等人,2012),Klp98A敲低会干扰apicobasal Wg的运输和促进的顶端积累的Wg内内涵体以及心尖的Wg循环(维特等人, 2020)。GFP的共表达标志着后部基因敲除区室。前室用作对照。顶端至基础Z堆栈(A)或WID上皮的单个顶端部分(B,C)的最大强度投影。比例尺,20 μ米。


数据分析


在斐济/ ImageJ中打开WID z堆栈。根据所使用的显微镜软件/图像格式,生物格式插件米AY需要打开你的文件。
注意:以下方案依赖于前后信号强度的标准化,只有在使用enG al4驱动感兴趣的基因操作时,才适用。


转到图像→变换→旋转并旋转z堆栈,直到表达Wg的单元格条纹水平对齐(图6 A)。
绘制一个感兴趣的矩形区域(ROI)(图6 B1),然后转到“编辑” →“选择” →“指定”以将其大小调整为150 × 150像素或其他合适的大小(图6 B2)。
通过转到编辑→选择→添加到管理器将ROI保存到ROI管理器中(图6 B3)。
重复步骤3和4,以创建两个相同的ROI集合。您可以使用管理器的“更多” →“保存”选项来保存ROI集,以供以后分析。
位置上的前侧的一个ROI和对的后侧一个ROI的Wg -表达细胞条纹。如果在后WID中共表达UAS-GFP,则使用绿色通道进行定向(图6 B4)。
注:中号阿克确保“全部显示”在ROI管理选项被选中。


使用通道工具或转到图像→色彩→拆分通道来拆分图像通道(图6 C1)。
在ROI管理器上,转到“更多→多重测量”以同时测量ROI和z堆栈的每个切片中Wg信号的强度(图6 C2)。将打开一个带有数据表的弹出窗口。
注意:测量后,可以在保存图像文件之前使用ROI管理器中的Flatten选项保存包括ROI位置在内的图像。


将多次测量的数据复制并粘贴到Excel工作表(或用于数据处理的替代软件)中。对于每个ROI,将收到关于ROI大小的数据,以及平均,最小,和最大的信号强度为您的堆栈(图中的每个切片6 C3)。
注意:如果数据表中的条目不同,请使用斐济→分析→设置测量来调整测量参数。




图6.孔定量吨的通货膨胀的Wg enGal4驱动基因操作后的信号强度。的Wg的表达细胞条纹被水平(A)对准和适当的前部和被选择的关注区域(ROI)后部区域(B)。然后,针对z堆栈的每个切片分别测量每个ROI中的Wg信号强度,并将其归一化为前野生型对照(C)。


在斐济打开您的原始数据文件,然后选择您感兴趣的切片(例如,周膜下面的3个最顶端的切片,3个最基础的切片,整个堆栈等),然后在Excel工作表中标记相应的切片。
计算相关切片的前后ROI中平均信号的平均强度。
通过除以前信号平均值,将后信号强度归一化为前信号。
注意:由于前WID侧在enGal4驱动的遗传操作设置中充当内部野生型控件,因此可以对后信号强度进行标准化。


使用步骤1-12,至少从5-10 WID收集数据。
在您选择的软件绘制的图形(例如,Excel中,GRAPHPAD ,...)。


菜谱


飞食物
712克玉米粉,95克大豆粉,168克干酵母,450克麦芽提取物,400克甜菜糖浆,50克琼脂,45毫升丙酸,150毫升Nipagin溶液(由300毫升H 2 O + 100克Nipagin(C 8 H 8 O 3 ),用ddH 2 O填充至10L 。
水加热至沸点接近,再加入黄豆粉,干酵母,以及琼脂
搅拌麦芽提取物和甜菜糖浆
搅拌玉米粉
减少热量并在密闭的盖子下煮45-60分钟,偶尔搅拌
将飞行食品冷却至60°C,然后添加丙酸和尼泊金
将果蝇小瓶装满果蝇食品20-30%,并在室温下干燥过夜。然后塞上小瓶并在4°C下储存
PBS
137 mM NaCl ,2.7 mM KCl ,8 mM Na 2 HPO 4 · 2 H 2 O,1.46 mM KH 2 PO 4


溶于ddH 2 O,并将pH值调节至7.4


PBTx
PBS + 0.1%Triton X-100


甘氨酸盐酸盐缓冲液
ddH 2 O中的0.1 M甘氨酸,用HCl调节pH值至3.5


Mowiol
将0.25 g / ml溶于PBS


致谢


研究在JCG的实验室由德国支持研究联合会资助的研究中心[SFB1324 / 1(331351713)和GR4810 / 2-1]。的研究计划的乔治-奥古斯特安大学哥廷根的大学医疗中心; 和博士后到KL由多萝西娅Schlözer计划,大学医学中心,乔治-奥古斯特安大学哥廷根。该协议基于Witte等人的工作。(2020 )和Linnemannstöns等。(2020年)。我们感谢Vera Terblanche博士(GAUGöttingen进化发展遗传学系)为她的小组配备摄像头的体视显微镜的使用提供了方便。


利益争夺


作者声明没有任何竞争或经济利益。


伦理


无è thics Ç ommittee批准所需的果蝇的工作。


参考


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Bartscherer,K.,Pelte,N.,Ingelfinger,D.和Boutros,M.(2006)。Wnt配体的分泌需要Evi,一种保守的跨膜蛋白。细胞125 (3) :523 - 533。
Chaudhary,V.和Boutros,M.(2019年)。囊外介导的顶端Wg分泌激活果蝇翅上皮细胞的信号传导。PL ø小号遗传学15 (9) :e1008351。
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施耐德(CA),拉斯班(WS)和埃利斯里(KW)(2012)。NIH Image to ImageJ:25年的图像分析。自然方法,9(7),671–675。https://doi.org/10.1038/nmeth.2089
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Witte,L.,Linnemannstoens,K.,Schmidt,K.,Honemann-Capito,M.,Grawe,F.,Wodarz,A.和Gross,JC(2020)。驱动蛋白Klp98A介导了顶端Wg的转运。开发147(15):dev186833 。
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引用:Witte, L., Linnemannstöns, K., Honemann-Capito, M. and Gross, J. C. (2021). Visualization and Quantitation of Wg trafficking in the Drosophila Wing Imaginal Epithelium. Bio-protocol 11(11): e4040. DOI: 10.21769/BioProtoc.4040.
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