Intact in situ Preparation of Drosophila melanogaster Lymph Gland for Comprehensive Analysis of Larval Hematopoiesis

Materials and Reagents

1. Fly stocks

   1. Canton-S was used as the wild type reference strain

   2. dome-Gal4,UAS2xEGFP (provided by Utpal Banerjee, University of California Los Angeles)

   3. srpHemo-Gal4-UAS-GFP (National Centre for Biological Sciences (NCBS), Fly Facility).

      
      

2. Materials

   1. Fine paint brush (number- 2)

   2. Glass cavity dish 40 x 40 mm (Atom Scientific, catalog number: SDCE4040-1)

   3. Sylgard (Sigma-Aldrich, catalog number: 761036) or equivalent

   4. Micro test plate- 96 well (Tarsons, catalog number: 941196) or equivalent

   5. 35 mm Petri dish (Tarsons, catalog number: 460035) or equivalent

      
      

3. Reagents

   Tissue dissection and fixation

   1. 10x Phosphate Buffered Saline (PBS, pH 7) stock (see Recipe)

   2. Paraformaldehyde (Fisher Scientific, catalog number: 23995)

      
      

4. Immunostaining and mounting

   1. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

   2. Normal Goat serum (GeNei, catalog number: NS1)

   3. Primary antibodies: mouse anti-P1/NimC1 and mouse anti-Hemese (provided by Istvan Ando, Biological Research Center of the Hungarian Academy of Sciences), rabbit anti-Asrij (Kulkarni et al., 2011).

   4. Secondary antibodies: Alexa fluor 568 goat anti-mouse (Invitrogen, catalog number: A11004)

      Alexa fluor 488 goat anti-rabbit (Invitrogen, catalog number: A11008)

   5. Phalloidin conjugated to Alexa fluor 633 (Invitrogen, catalog number: A22284)

   6. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride, ThermoFisher Scientific, catalog number: D1306)

   7. Glycerol (Merck, catalog number:BP229-1)

   8. Neutral red (Merck, catalog number: N4638)

      
      

5. RNA in situ hybridization

   1. In situ hybridization probe: tep4 (Rodrigues et al., 2021)

   2. 20x Saline-Sodium Citrate buffer (20x SSC, pH-7) (see recipe)

   3. Methanol (Merck, catalog number: 34860)

   4. Tween-20 (Sigma-Aldrich, catalog number: P9416)

   5. Molecular grade water-UltraPure DNase/RNase-free distilled water (Invitrogen, catalog number: 10977015)

   6. Formamide (Sigma-Aldrich, catalog number: 11814320001)

   7. tRNA (Sigma-Aldrich, catalog number: 10109517001)

   8. Heparin (Sigma-Aldrich, catalog number: H3149)

   9. Roche blocking agent (Sigma-Aldrich, catalog number: 11096176001)

   10. CHAPS (Sigma-Aldrich, catalog number: C9426)

   11. EDTA (Fisher Scientific, catalog number: S311-100)

   12. NaCl (Fisher Scientific, catalog number: S25542)

   13. MgCl₂ (Fisher Scientific, catalog number: BP214-500)

   14. Anti-DIG conjugated to alkaline phosphatase (Sigma-Aldrich, catalog number: 11093274910)

   15. NBT/BCIP (Promega, catalog number: S3771)

   16. FastRed substrate kit (Abcam, catalog number: ab64254)

Equipment

1. Stereomicroscope (Olympus SZ51, magnification range 0.8x-4x)

2. Fine forceps (Fine Science Tools, Dumont #5, catalog number: 11252-20)

3. Spring scissors 2.5 mm cutting edge (Fine Science Tools, catalog number: 15000-08)

4. Insect pins (Fine Science Tools, Minutien, 0.1 mm, stainless steel, catalog number: 26002-10)

5. Confocal microscope (Zeiss, LSM 880)

Software

1. ImageJ

2. Adobe Photoshop CS5 (Adobe Systems)

Procedure

1. Larval fillet preparation for obtaining intact lymph gland (LG):

   Whole LG preparations can be obtained for the first, second, third instar larvae and pupae using this method of dissection. Fly breeding and crosses were performed using standard protocols and larvae were reared to the appropriate stage on standard cornmeal agar medium under non saturating density. Figure 1A shows the relative size of the first, second and third instar larvae.

   
   

   
   Figure 1. Schematic representation of larval LG dissection. (A) Representative image of first, second and third instar larvae. (B-F) Step-wise schematic representation of LG dissection from wandering third instar larvae. BL: brain lobes, VNC: ventral nerve cord, LG: lymph gland and PC: pericardial cells. Number of pericardial cells may vary between LG lobes.Larvae and LG images are not depicted to scale but enlarged for clarity.

   
   

   1. Using a fine paint brush transfer the larvae to a cavity dish/ Petri plate containing water, then rinse the larvae to get rid of any food particles.

   2. Transfer the larvae to a clean cavity dish and place on ice for 20-30 min to immobilize the larvae. Immobilization (optional) helps to pin the larvae (see step 4).

   3. Place cooled larva with the dorsal side up on the Sylgard dish and view it through a stereomicroscope at magnification 4x (zoom range 0.8x-4x), focusing on the dorsal cuticle. All subsequent steps are to be performed while viewing larval tissue under the microscope.

   4. Restrain larva by inserting insect pins firmly through it near the anterior and the posterior spiracles and through the Sylgard dish (Figure 1B). Add a drop of PBS (about 200 µl) to prevent the larva from desiccating.

   5. Using fine dissection scissors make a small incision in the cuticle on the right side near the posterior end. Insert the scissors into the incision end and slit the cuticle laterally (Figure 1C, D).

   6. Lift the loose end of the cuticle with the help of fine forceps, extend it to the left side and cut along the left lateral edge of the cuticle (Figure 1E).

   7. Carefully remove the viscera. Locate the LG that is attached to the brain lobes in the anterior, flanking the dorsal vessel, followed by rows of pericardial cells at the posterior end (Figure 1F). Side illumination with dual goose-neck light source can help distinguish the refringent LG.

   8. Add a fresh drop of PBS and gently remove it to get rid of any tissue debris. Repeat if required.

   9. Replace PBS with 200 µl fixative (4% paraformaldehyde, see recipe) and incubate for 20 min at room temperature. Remove the fixative, wash three times with 1x PBS then carefully remove the pins, slowly lift the LG preparation (with the underlying ventral cuticle) from the posterior end and transfer to a 96-well plate or a 1.5 ml microcentrifuge tube for immunostaining or in situ procedures.

      
      

2. Pupal fillet preparation for obtaining intact lymph gland (LG):

   Whole LG can be prepared from 0 to 20h after pupa formation (APF). In our experience, most lobes histolyze by 15h APF (Rodrigues et al., 2021).

   1. Place pupa on the Sylgard dish with the dorsal side facing up. All subsequent steps are to be performed while viewing the pupal tissue under the stereomicroscope.

   2. Insert a fine insect pin firmly near the anterior spiracle through the Sylgard dish (Figure 2A). Add a drop of PBS (about 200 µl) to prevent the pupa from desiccating.

      
      

      
      Figure 2. Schematic representation of pupal LG dissection. (A-D) Step-wise schematic representation of pupal LG dissection. BL: brain lobes, VNC: ventral nerve cord, LG: lymph gland and PC: pericardial cells. Number of pericardial cells may vary between LG lobes. Pupa and LG images are not depicted to scale but enlarged for clarity.

      
      

   3. Using fine dissection scissors cut horizontally along the posterior spiracles (Figure 2A).

   4. Insert the scissor into the incised end and make a slit laterally along the right side of the cuticle, followed by incision along the anterior part as shown by the dotted lines (Figure 2B).

   5. Lift the loose end of the cuticle and carefully extend it to the left side and cut along the left lateral edge of the cuticle along the dotted lines (Figure 2B).

   6. Carefully remove the visceral organs without damaging the LG (Figure 2C). The cardiac tube and pericardial cells are a good landmark to locate the remaining LG lobes (Figure 2D). Add a fresh drop of PBS to remove any tissue debris, repeat if required.

   7. Follow step 9 onwards of larval LG dissection and proceed for immunostaining or in situ procedures.

      
      

3. Neutral red staining of lymph gland (LG) for easier visualization:

   1. Dissect and fix larval or pupal LG as described above. Wash thrice in 1x PBS for 8-10 min each.

   2. Add 100 µl of 0.2% neutral red solution (0.2% neutral red in 1x PBS) and incubate at room temperature for 5-7 min or till LG is visibly stained.

   3. Wash off neutral red with 1x PBS. Brain lobes and LG lobes should be easily visible. First, second, third instar and pupal LG can be easily identified by neutral red staining (Figure 3A-D).

      
      

      
      Figure 3. LG preparations stained with neutral red. (A-D) Hemi-dissected fillet preparation with intact primary and posterior lobes stained with neutral red. LG lobes are intensely stained (red) as shown for first instar (A) second instar (B) third instar (C) larvae and pupa (D). BL: brain lobes, VNC: ventral nerve cord, LG: lymph gland, Pri: primary lobes, Sec: secondary lobes, Tert: tertiary lobes and asterisks indicate pericardial cells. Scale bar: 100μm

      
      

4. Immunostaining of lymph gland (LG):

   1. All steps are performed by placing the 96-well dish on a flat bed rocker.

   2. Wash 10-12 fixed LG preparations in a 96-well dish in 200 µl 1x PBS, three times for 10-15 min each.

   3. Add 200 µl of 0.1% Triton X-100 in 1x PBS (0.1% PTX, see recipe) as a permeabilizing agent to the LG preparations incubate for 15-20 min.

   4. Discard PTX, add 100 µl of 20% normal goat serum in 1x PBS as a blocking agent and, incubate for 30 min.

   5. Remove the blocking agent and add 40 µl of primary antibody at the desired concentration diluted in 1x PBS (or blocking agent). Incubate at 4°C overnight.

   6. Remove the primary antibody, wash the LG preparations with 1x PBS, three times for 10-15 min each.

   7. Discard PBS, add 100 µl of 20% normal goat serum and incubate for 30 min.

   8. Replace with 40 µl of secondary antibody (usually 1:400) in 1x PBS and incubate for 2 h at room temperature.

   9. Remove secondary antibody, wash three times in 200 µl of 1x PBS for 10-15 min each. Proceed to the mounting step (see below, procedure section F).

      
      

5. In situ hybridization of lymph gland (LG):

   1. Wash 15-20 fixed LG preparations in a microcentrifuge tube in 500 µl 1x PBS three times for 10-15 min each.

   2. For long term storage, wash LG preparations 4 times in methanol and store in methanol at -20°C. Rinse the samples once with 50% methanol – 50% PBS and three times in 1x PBS to resume the experiment.

   3. Wash LG preparations in 500 µl of 0.1% Tween-20 in 1x PBS (PBT) for three times, 10-15 min each (See recipe for 0.1% PBT).

   4. Equilibrate LG preparations in 500 µl of equal volumes of 0.1% PBT and hybridization buffer (HB). (See recipe for hybridization buffer)

   5. Discard the PBT-HB solution and, pre-incubate in 500 µl HB for 1 h at 65°C.

   6. Remove HB and replace with 200 µl HB with 1 µl of Digoxigenin (DIG)-labelled RNA probe (Dilution of the probe needs to be adjusted empirically depending on the probe concentration and level of expression of the gene of interest). Hybridise overnight at 65°C.

   7. Remove HB carefully and wash the LG preparations in 500-800 µl of HB for 1 h at 65°C.

   8. Wash LG preparations in 500-800 µl equal volumes of 0.1% PBT and HB for 30 min at 65°C, followed by three quick washes in 0.1% PBT at room temperature.

   9. Follow up with three washes in 0.1% PBT for 10 min each at room temp.

   10. Block non-specific binding with 1% bovine serum albumin (BSA) or normal goat serum (NGS) in 0.1% PBT for 30 min.

   11. Incubate with anti-DIG antibody coupled to Alkaline Phosphatase diluted (1:1000) in the blocking solution for 2 h at room temp.

   12. Remove the anti-DIG blocking solution mixture, followed by three quick rinses in 0.1% PBT and then three 10 min washes in 0.1% PBT.

   13. Equilibrate for 10 min in 500 μl freshly prepared staining buffer (SB) (see recipe for staining buffer).

   14. Visualize with NBT/BCiP in 1 ml SB (6.5 μl NBT + 3.5 μl BCiP) at room temperature or 37°C until staining is visible.

   15. Wash 3 times in 1x PBS and mount the LG preparations as indicated below.

       
       

6. Mounting intact lymph gland (LG):

   1. Place the fixed and stained larval fillet preparation in a glass bottom 35 mm Petri dish. These can also be made by punching a hole of 1 cm diameter in a 35 mm Petri dish and gluing a coverslip to the bottom of the dish (Figure 4). Add a small drop (10-20 µl) of 70% glycerol with DAPI (1:500) or mounting medium to prevent the samples from desiccating.

   2. Using a pair of forceps hold the filleted cuticle at the anterior end. With another fine forceps carefully detach the brain lobes from rest of the cuticle. The LG is attached via the ring gland to the brain lobes and is flanking the dorsal vessel (Figure 1F). This procedure will partially dislodge it from the cuticle.

   3. Next, using a pair of forceps, hold the cuticle at the posterior end and with another forceps carefully detach and slide the posterior part of the dorsal vessel along with the pericardial cells on to the coverslip. The tissue is very fragile and the whole procedure has to be done gently while viewing through a stereomicroscope.

   4. Once the LG is loosened from the anterior and the posterior ends carefully move the LG away from the ventral cuticle. Do not directly lift or hold the LG. Grasp the brain lobes at the anterior and the pericardial cells at the posterior end when moving the LG.

   5. Multiple lymph glands can be mounted in this way in a single dish (Figure 4) and can be subjected to high resolution imaging analysis.

      
      

      
      Figure 4. LG preparations mounted in a 35 mm coverslip bottom dish. 35 mm coverslip bottom dish shows seven third instar larval LG preparations stained with neutral red. Black dotted lines indicate LG lobes. LG: lymph gland and PC: pericardial cells.

Data analysis

1. Representative results

   The Drosophila third instar larval LG is a multi-lobed organ that flanks the dorsal vessel. The first pair of lobes called as the primary lobes are followed by 2-3 pairs of posterior lobes- the secondary, tertiary and (rarely) quaternary lobes separated by pericardial cells that function as nephrocytes. For simplicity we refer to the secondary, tertiary and quaternary lobes as the posterior lobes. Rows of pericardial cells line the dorsal vessel at the posterior end.

       To demonstrate that this method of dissection maintains tissue integrity, we analyzed markers for the whole LG, cardiac tube and the pericardial cells. Phalloidin marks actin and is useful for visualizing the integrity of LG lobes and the dorsal vessel (Figure 5A). Hemese, a generic blood cell marker (Kurucz et al., 2003), is expressed in all cells of the primary and most cells of the posterior lobes (Figure 5B). Asrij (Kulkarni et al., 2011) another pan-hemocyte marker marks LG hemocytes of the primary and the posterior lobes (Figure 5C). srpHemo-Gal4 containing a regulatory region of the serpent gene is active in the embryonic hemocytes (Bruckner et al., 2004) and additionally, is strongly expressed in the pericardial cells (Figure 5D). However, very few cells in the primary lobes express srpHemo-Gal4 (Figure 5D). With the help of the above-mentioned markers we confirm this method of dissection is useful for isolating intact LG primary and posterior lobes.

   
   

   
   Figure 5. Validation of intact whole LG preparations using pan hemocyte, progenitor and differentiation markers. Whole LG preparations from wandering third larval instar include primary and posterior lobes. (A) Phalloidin (yellow) marks actin and is used for identifying LG blood cells and the cardiac tube. Pan hemocyte markers (B) Hemese (red) and (C) Asrij (green) are expressed in the LG lobes and help to identify intact lobes. (D) srpHemo-Gal4 (green) is strongly expressed in the pericardial cells. (E) dome (green) marks progenitors in the primary and the posterior lobes, P1/ Nimrod C1 (red) marks plasmatocytes. (F) in situ hybridization for tep4 (red) shows high expression in the MZ, secondary lobes and some cells of the tertiary lobes. Pri: primary lobes, Sec: secondary lobes, Tert: tertiary lobes. (A-F) Scale bar: 100μm

   
   

   For in-depth analysis of tissue integrity, we further examined known progenitor (MZ) and differentiation (CZ) markers as defined in the anterior lobes. We performed reporter, immunostaining or RNA in situ analysis to analyze marker expression. As described previously, progenitor marker domeless (Jung et al., 2005) is expressed in the MZ, secondary lobes and in some cells of the tertiary lobes (Figure 5E). Differentiation marker, P1/ Nimrod C1 (Kurucz et al., 2007) expression is restricted to the CZ of the primary lobes (Figure 5E). RNA in situ for another progenitor marker, tep4 (Rodrigues et al., 2021) reveals high expression in the MZ, the secondary lobes and in some cells of the tertiary lobes (Figure 5F). Using the fillet method of LG dissection, we obtained intact primary and posterior lobes as observed with MZ and CZ markers.

       Using this method of dissection, we have successfully separated primary and posterior lobes and performed RNA sequencing analysis (Figure 6). This has led to the identification of new markers of LG blood cells (Rodrigues et al., 2021). Maintaining tissue integrity and quality of the sample depends on critical steps in the dissection process. Though the dissections are demanding, identifying the LG and performing quick dissections (2-3 min) has been achieved routinely with 2-3 days of practice even by inexperienced summer interns. This detailed protocol aims to establish a standard method for LG dissection and comprehensive analysis that can also be adapted for isolating LG from early larval instars and pupal stages.

   
   

   
   Figure 6. Schematic representation of dissection strategy and workflow for RNA-sequencing analysis. Step-wise representation of dissection strategy and RNA-sequencing for obtaining transcriptomic profiles for the primary and the posterior lobes separately. Pri: primary lobes, Sec: secondary lobes, Tert: tertiary lobes and Post: posterior lobes.

   
   

2. Discussion

   Unlike vertebrate HSPCs, most Drosophila blood progenitors reside in a single hematopoietic organ, the lymph gland, which also harbors their differentiated progeny. The small size of the LG (~1.5–2 mm in length) makes a complete and comprehensive in situ analysis of its resident progenitor and differentiated pools possible. Yet most of our understanding of larval LG hematopoiesis is derived from in-depth analysis restricted to the anterior-most lobes, called the primary lobes. Till recently, cellular and molecular mechanisms that operate in the primary lobe were thought to be applicable to the posterior lobes. However, anterior and posterior lobes have significant differences in gene expression and mutant phenotypes (Khadilkar et al., 2014; Kulkarni et al., 2011; Rodrigues et al., 2021). Also, the posterior lobes are physically separated from the anterior lobe PSC and differentiated hemocytes that are involved in regulating progenitor fate in the anterior lobes (Figure 1F). Further, a larger pool of blood progenitors resides in the posterior lobes (Rodrigues et al., 2021). Hence incomplete analysis of the LG overlooks important developmental and functional information regarding hematopoiesis that could be relevant to vertebrate hematopoiesis. To aid dissection and analysis of the complete intact LG, we describe a detailed protocol that will also be useful in analyzing the entire progenitor population. LG primary lobes are specified at the embryonic stage whereas, the posterior lobes form during the second instar larval stage and continue to develop till the wandering larval stage. Hence, this method of dissection allows simultaneous analysis of developmental hematopoiesis in a single animal (Rodrigues et al., 2021).

       We demonstrated the utility of our method by analyzing pan-hemocyte, progenitor and differentiation markers at the RNA and protein level. Obtaining whole intact LG lobes is essential for understanding progenitor heterogeneity in physiological and immune conditions (Rodrigues et al., 2021). This protocol can be used for studying the ontogeny, development and immune aspects of LG blood cells. It can also be used for isolating distinct lobes for follow up biochemical and omics assays (Rodrigues et al., 2021; Sinha et al., 2019). Therefore, this protocol will be particularly valuable to gain new and more complete insights into the regulation of blood cell progenitor fate.

Notes

1. We use neutral red stained preparations only for the ease of visualization during training and practicing LG dissection and not for immunostaining or in situ hybridization experiments.

2. Avoid placing the coverslip on the LG preparation as this can damage the tissue. We prefer to use coverslip glass- bottom dishes for mounting. With experience, 8-10 LG preparations can be mounted in a single coverslip- bottom dish (Figure 4).

3. Fillet method of LG dissection retains the ventral larval cuticle containing sessile hemocyte clusters that can be retrieved at the sample mounting step (see procedure, section F, step 4), allowing simultaneous analysis of blood cells emerging from distinct anlage (Khadilkar et al., 2014).

4. This protocol helps to isolate 3 organ systems: dorsal vessel, pericardial cells and LG from the same animal in a single sample preparation.

5. As anterior/primary lobe progenitors are specified by the first instar and secondary and tertiary lobes at subsequent stages, this method allows analysis of developmental stages in a single animal.

Recipes

1. 10x PBS

   Dissolve sodium chloride (NaCl) –18.9 g, sodium hydrogen phosphate (Na₂HPO₄) – 2.48 g, Monosodium phosphate (NaH₂PO₄·2H₂O) – 1.17 g in autoclaved distilled water, adjust volume to 250 ml. Adjust pH to 7. Store at room temperature.

2. 4% Paraformaldehyde (PFA)

   Dissolve 4 g PFA powder in 80ml of 1x PBS and adjust the volume to 100 ml with 1x PBS solution. Place the mixture at 60 °C in a water bath to dissolve the PFA. Store at 4°C.

3. 0.1% Triton X-100 in PBS (PTX)

   Add 0.1 ml Triton X-100 in 100 ml 1x PBS. Store at room temperature.

4. 0.1% Tween-20 in PBS (PBS-T)

   Add 0.1 ml Tween-20 in 100 ml 1x PBS. Store at room temperature.

5. 20x Saline-sodium citrate buffer (SSC)

   Dissolve sodium chloride (NaCl) - 43.82 g, sodium citrate (Na₃C₆H₅O₇) - 22.05 g, adjust volume to 200 ml with molecular grade (DNase/ RNase free) water. Adjust pH to 7. Autoclave and store at -20°C.

6. Hybridization buffer

   Dissolve, Formamide- 50 ml, 20x SSC- 10 ml, yeast RNA (50 mg/ml)- 2 ml, heparin (0.05 g/ml)- 100μl, Roche blocking reagent (10%)- 20 ml (prepared according to manufacturer instruction), CHAPS (10%)- 1 ml, EDTA (0.5M)- 1 ml, Tween-20 (10%)- 1 ml, adjust the volume to 100 ml using molecular grade water (DNase/ RNase free). Store at -20°C.

7. Staining buffer

   Dissolve, 5M sodium chloride (NaCl)- 200µl, 1M magnesium chloride (MgCl₂)- 500μl, Tris-HCl pH 9.5- 1ml, Tween-20 (10%)- 1μl, adjust volume to 10ml with autoclaved distilled water.

Acknowledgments

We thank the Drosophila community for fly stocks and antibodies; National Centre for Biological Sciences, Fly Facility for stocks; JNCASR Imaging facility and our laboratory members for valuable inputs and suggestions. Schematics created with BioRender.com. This work was funded by the Indo-French Centre for the Promotion of Advanced Research (IFCPAR/CEFIPRA) grant to MSI and LW; MSI’s work was also supported by SERB grant, J C Bose award project and Jawaharlal Nehru Centre for Advanced Scientific Research.

Author contributions: DR, LW and MSI performed experiments; DR, KVR, LW and MSI wrote the manuscript; MSI and LW obtained funding and facilities for the work

Competing interests

The authors declare no conflict of interest

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