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 (No. 2)

   2. Glass cavity dish 40 × 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

   6. 1.5 ml microcentrifuge tube (Axygen, catalog number: MCT-150-C)

      
      

3. Reagents
   Tissue dissection and fixation

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

   2. Na₂HPO₄ (Merck, catalog number: S9763)

   3. NaH₂PO₄·2H₂O (Merck, catalog number: 71505)

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

   5. 10× Phosphate Buffered Saline (PBS, pH 7) stock (see Recipes)

   6. 4% Paraformaldehyde (PFA) (see Recipes)

      
      
   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)

   9. 0.1% Triton X-100 in PBS (PTX) (see Recipes)

      
      
   RNA in situ hybridization

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

   2. Methanol (Merck, catalog number: 34860)

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

   4. Sodium citrate (Na₃C₆H₅O₇) (HiMedia, catalog number: TC249)

   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)

   17. 0.1% Tween-20 in PBS (PBS-T) (see Recipes)

   18. 20× Saline-Sodium Citrate buffer (20× SSC, pH 7) (see Recipes)

   19. Hybridization buffer (see Recipes)

   20. Staining buffer (see Recipes)

Equipment

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

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, model: 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, and third instar larvae, as well as for pupae, using this method of dissection. Fly breeding and crosses were performed using standard protocols. 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.

   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 A4).

   3. Place cooled larva with the dorsal side up on the Sylgard dish and view it through a stereomicroscope at magnification 4× (zoom range 0.8-4×), 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 (approximately 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 and 1D).

   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 region 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.

      
      

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

      
      

   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 of fixative (4% paraformaldehyde, see Recipes) and incubate for 20 min at room temperature. Remove the fixative and wash three times with PBS. 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 20 h after pupa formation (APF). In our experience, most lobes histolyze by 15 h 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 (approximately 200 µl) to prevent the pupa from desiccating.

   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 an 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 and repeat if required.

      
      

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

      
      

   7. Follow Step A9 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 PBS for 8-10 min each.

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

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

      
      

      
      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). Yellow arrowhead indicates lymph gland. BL: brain lobes, VNC: ventral nerve cord, LG: lymph gland, Pri: primary lobes, Sec: secondary lobes, Tert: tertiary lobes. Asterisks indicate pericardial cells. Scale bar: 100 μm

      
      

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 of PBS, three times for 10-15 min each.

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

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

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

   6. Remove the primary antibody and wash the LG preparations with 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 PBS and incubate for 2 h at room temperature.

   9. Remove secondary antibody and wash in 200 µl of PBS, three times 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 of PBS, three times for 10-15 min each.

   2. For long term storage, wash LG preparations in methanol four times and store in methanol at -20°C. To resume the experiment, rinse the samples once with a mixture of methanol and PBS (1:1) and three times in PBS.

   3. Wash LG preparations in 500 µl of 0.1% Tween-20 in PBS (PBS-T), three times for 10-15 min each (see Recipes for 0.1% PBS-T).

   4. Equilibrate LG preparations in 500 µl of equal volumes of 0.1% PBS-T and hybridization buffer (HB) (see Recipes for hybridization buffer).

   5. Discard the PBS-T-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)-labeled 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). Hybridize 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% PBS-T and HB for 30 min at 65°C, followed by three quick washes in 0.1% PBS-T at room temperature.

   9. Follow up with three washes in 0.1% PBS-T 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% PBS-T for 30 min.

   11. Incubate with anti-DIG antibody coupled to Alkaline Phosphatase diluted (1:1,000) 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% PBS-T. Then, wash in 0.1% PBS-T, three times for 10 min.

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

   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 (Figure 4).

   15. Wash three times in PBS and mount the LG preparations as indicated (see below, procedure section F).

       
       

       
       Figure 4. Schematic representation of RNA in situ hybridization procedure. Stepwise representation of lymph gland RNA in situ hybridization protocol. Pri: primary lobes, Sec: secondary lobes, Tert: tertiary lobes. Mounted LG preparation shows expression of Latran as revealed by NBT/BCIP.

       
       

6. Mounting intact lymph gland (LG)

   1. Place the fixed and stained larval fillet preparation in a coverslip 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 5). Add a small drop (10-20 µl) of 70% glycerol with DAPI (1:500) or mounting medium to prevent the samples from desiccating.

      
      

      
      Figure 5. LG preparations mounted in a 35 mm coverslip bottom dish. Seven third instar larval LG preparations stained with neutral red at a 35 mm coverslip bottom dish. Black dotted lines indicate LG lobes. LG: lymph gland, and PC: pericardial cells.

      
      

   2. Using a pair of forceps, hold the filleted cuticle at the anterior end. With another fine forceps, carefully detach the brain lobes from the rest of the cuticle. The LG is attached via the ring gland to the brain lobes and flanks 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 onto the coverslip. The tissue is very fragile, and the whole procedure must 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. When moving the LG, grasp the brain lobes at the anterior end and the pericardial cells at the posterior end.

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

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 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 6A). 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 6B). Asrij (Kulkarni et al., 2011), another pan-hemocyte marker, marks LG hemocytes of the primary and the posterior lobes (Figure 6C). srpHemo-Gal4, containing a regulatory region of the serpent gene, is active in the embryonic hemocytes (Bruckner et al., 2004) and strongly expressed in the pericardial cells (Figure 6D). However, very few cells in the primary lobes express srpHemo-Gal4 (Figure 6D). With help from the above-mentioned markers, we confirmed that this method of dissection is useful for isolating intact LG primary and posterior lobes.

   
   

   
   Figure 6. 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: 100 μm.

   
   

      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 previously described, the progenitor marker domeless (Jung et al., 2005) is expressed in the MZ, secondary lobes, and in some cells of the tertiary lobes (Figure 6E). Differentiation marker P1/ Nimrod C1 (Kurucz et al., 2007) expression is restricted to the CZ of the primary lobes (Figure 6E). 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 6F). Using the fillet method of LG dissection, we obtained intact primary and posterior lobes, as observed with MZ and CZ marker staining. Intact LG lobes are also useful in the analysis of the number of niche cells, progenitors, and differentiated blood cells in each lobe. Therefore, 3D images can be reconstructed using the IMARIS software. Using the 2D Slice module, the diameter is defined for cell nuclei marked by DAPI. The Spots module is employed to estimate the number of DAPI⁺ nuclei. Differentiated cells marked by P1 or progenitors marked by dome are analyzed using the surface module. The surface module is also used for 3D rendering, with the distance between the 3D surface (P1 or dome) and spot (DAPI⁺) defined by the 2D slice module. Next, using the module “Find spots closer to surface” or “Find spots away from surface,” the number of spots (DAPI⁺) positive or negative for a particular marker can be determined.

      Using this method of dissection, we have successfully separated primary and posterior lobes and performed RNA sequencing analysis (Figure 7). This has led to the identification of new markers of LG blood cells (Rodrigues et al., 2021). Maintenance of 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 aimed 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 7. Schematic representation of dissection strategy and workflow for RNA-sequencing analysis. Stepwise 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. Until 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 (Kulkarni et al., 2011; Khadilkar et al., 2014; Rodrigues et al., 2021). Additionally, the posterior lobes are physically separated from the anterior lobe PSC, and differentiated hemocytes are involved in regulating progenitor fate in the anterior lobes (Figure 1F). Furthermore, 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, which 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 until 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 levels. 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 (Sinha et al., 2019; Rodrigues et al., 2021). 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-bottom dishes for mounting. With experience, 8-10 LG preparations can be mounted in a single coverslip-bottom dish (Figure 5).

3. The fillet method of LG dissection retains the ventral larval cuticle containing sessile hemocyte clusters that can be retrieved at the sample mounting step (see Step F4), allowing simultaneous analysis of blood cells emerging from distinct anlage (Khadilkar et al., 2014).

4. This protocol helps to isolate three 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, while secondary and tertiary lobes only develop at subsequent stages, this method allows analysis of developmental stages in a single animal.

Recipes

1. 10× PBS

   NaCl 18.9 g

   Na₂HPO₄ 2.48 g

   NaH₂PO₄·2H₂O 1.17 g

   Dissolve in autoclaved distilled water.

   Adjust volume to 250 ml.

   Adjust pH to 7.0.

   Store at room temperature.

2. 4% Paraformaldehyde (PFA)

   Dissolve 4 g PFA powder in 80 ml of 1× PBS.

   Adjust the volume to 100 ml with 1× 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 PBS.

   Store at room temperature.

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

   Add 0.1 ml Tween-20 in 100 ml PBS.

   Store at room temperature.

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

   NaCl 43.82 g

   Na₃C₆H₅O₇ 22.05 g

   Dissolve in molecular grade (DNase/RNase free) water.

   Adjust volume to 200 ml with molecular grade (DNase/RNase free) water.

   Adjust pH to 7.0.

   Autoclave and store at -20°C.

6. Hybridization buffer

   Formamide 50 ml

   20× SSC 10 ml

   Yeast RNA (50 mg/ml) 2 ml

   Heparin (0.05 g/ml) 100 μl

   Roche blocking reagent (10%) 20 ml (prepared according to the manufacturer’s instructions).

   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

   5 M NaCl 200 µl

   1 M MgCl₂ 500 μl

   Tris-HCl pH 9.5 1 ml

   Tween-20 (10%) 1 μl

   Adjust volume to 10 ml with autoclaved distilled water.

Acknowledgments

We thank the Drosophila community for fly stocks and antibodies; the National Centre for Biological Sciences, Fly Facility for stocks, the JNCASR Imaging facility, and our laboratory members for valuable input and suggestions. Schematics were 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 a 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. Original research paper: Differential activation of JAK-STAT signaling reveals functional compartmentalization in Drosophila blood progenitors. DOI: 10.7554/eLife.61409.

Competing interests

The authors declare no conflicts of interest.

References

1. Banerjee, U., Girard, J. R., Goins, L. M. and Spratford, C. M. (2019). Drosophila as a Genetic Model for Hematopoiesis. Genetics 211(2): 367-417.
2. Benmimoun, B., Polesello, C., Haenlin, M. and Waltzer, L. (2015). The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche. Proc Natl Acad Sci U S A 112(29): 9052-9057.
3. Bruckner, K., Kockel, L., Duchek, P., Luque, C. M., Rorth, P. and Perrimon, N. (2004). The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell 7(1): 73-84.
4. Crisan, M. and Dzierzak, E. (2016). The many faces of hematopoietic stem cell heterogeneity. Development 143(24): 4571-4581.
5. Crozatier, M., Ubeda, J. M., Vincent, A. and Meister, M. (2004). Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. PLoS Biol 2(8): E196.
6. Dey, N. S., Ramesh, P., Chugh, M., Mandal, S., Mandal, L. (2016). Dpp dependent Hematopoietic stem cells give rise to Hh dependent blood progenitors in larval lymph gland of Drosophila. Elife 5: e12295.
7. Ema, H., Morita, Y. and Suda, T. (2014). Heterogeneity and hierarchy of hematopoietic stem cells. Exp Hematol 42(2): 74-82 e72.
8. Ghosh, S., Singh, A., Mandal, S. and Mandal, L. (2015). Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response. Dev Cell 33(4): 478-488.
9. Granick, J. L., Simon, S. I. and Borjesson, D. L. (2012). Hematopoietic stem and progenitor cells as effectors in innate immunity. Bone Marrow Res 2012: 165107.
10. Grigorian, M., Liu, T., Banerjee, U. and Hartenstein, V. (2013). The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland. Dev Biol 384(2): 301-312.
11. Grigorian, M., Mandal, L. and Hartenstein, V. (2011). Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the Drosophila lymph gland. Dev Genes Evol 221(3): 121-131.
12. Holz, A., Bossinger, B., Strasser, T., Janning, W. and Klapper, R. (2003). The two origins of hemocytes in Drosophila. Development 130(20): 4955-4962.
13. Honti, V., Csordas, G., Markus, R., Kurucz, E., Jankovics, F. and Ando, I. (2010). Cell lineage tracing reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments in Drosophila melanogaster. Mol Immunol 47(11-12): 1997-2004.
14. Jung, S. H., Evans, C. J., Uemura, C. and Banerjee, U. (2005). The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132(11): 2521-2533.
15. Khadilkar, R. J., Rodrigues, D., Mote, R. D., Sinha, A. R., Kulkarni, V., Magadi, S. S. and Inamdar, M. S. (2014). ARF1-GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis. Proc Natl Acad Sci U S A 111(13): 4898-4903.
16. Kulkarni, V., Khadilkar, R. J., Magadi, S. S. and Inamdar, M. S. (2011). Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis. PLoS One 6(11): e27667.
17. Kurucz, E., Markus, R., Zsamboki, J., Folkl-Medzihradszky, K., Darula, Z., Vilmos, P., Udvardy, A., Krausz, I., Lukacsovich, T., Gateff, E., et al. (2007). Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes. Curr Biol 17(7): 649-654.
18. Kurucz, E., Zettervall, C. J., Sinka, R., Vilmos, P., Pivarcsi, A., Ekengren, S., Hegedus, Z., Ando, I. and Hultmark, D. (2003). Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila. Proc Natl Acad Sci U S A 100(5): 2622-2627.
19. Lanot, R., Zachary, D., Holder, F. and Meister, M. (2001). Postembryonic hematopoiesis in Drosophila. Dev Biol 230(2): 243-257.
20. Mandal, L., Banerjee, U. and Hartenstein, V. (2004). Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm. Nat Genet 36(9): 1019-1023.
21. Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V. and Banerjee, U. (2007). A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446(7133): 320-324.
22. Rodrigues, D., Renaud, Y., VijayRaghavan, K., Waltzer, L. and Inamdar, M. S. (2021). Differential activation of JAK-STAT signaling reveals functional compartmentalization in Drosophila blood progenitors. Elife 10: e61409.
23. Rugendorff, A., Younossi-Hartenstein, A. and Hartenstein, V. (1994). Embryonic origin and differentiation of the Drosophila heart. Rouxs Arch Dev Biol 203(5): 266-280.
24. Sanchez Bosch, P., Makhijani, K., Herboso, L., Gold, K. S., Baginsky, R., Woodcock, K. J., Alexander, B., Kukar, K., Corcoran, S., Jacobs, T., et al. (2019). Adult Drosophila Lack Hematopoiesis but Rely on a Blood Cell Reservoir at the Respiratory Epithelia to Relay Infection Signals to Surrounding Tissues. Dev Cell 51(6): 787-803 e785.
25. Sinha, A., Khadilkar, R. J., S, V. K., Roychowdhury Sinha, A. and Inamdar, M. S. (2013). Conserved regulation of the Jak/STAT pathway by the endosomal protein asrij maintains stem cell potency. Cell Rep 4(4): 649-658.
26. Sinha, S., Ray, A., Abhilash, L., Kumar, M., Sreenivasamurthy, S. K., Keshava Prasad, T. S. and Inamdar, M. S. (2019). Proteomics of Asrij Perturbation in Drosophila Lymph Glands for Identification of New Regulators of Hematopoiesis. Mol Cell Proteomics 18(6): 1171-1182.
27. Tepass, U., Fessler, L. I., Aziz, A. and Hartenstein, V. (1994). Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development 120(7): 1829-1837.
28. Ward, E. J. and Skeath, J. B. (2000). Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo. Development 127(22): 4959-4969.
29. Wright, D. E., Wagers, A. J., Gulati, A. P., Johnson, F. L. and Weissman, I. L. (2001). Physiological migration of hematopoietic stem and progenitor cells. Science 294(5548): 1933-1936.