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Feb 2021

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This protocol has an early version published in Bio-101. DOI: 10.21769/BioProtoc.4052

Intact in situ Preparation of the Drosophila melanogaster Lymph Gland for a Comprehensive Analysis of Larval Hematopoiesis
黑腹果蝇淋巴腺的完整原位制备用于综合幼虫造血分析   

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Abstract

Blood cells have a limited lifespan and are replenished by a small number of hematopoietic stem and progenitor cells (HSPCs). Adult vertebrate hematopoiesis occurs in the bone marrow, liver, and spleen, rendering a comprehensive analysis of the entire HSPC pool nearly impossible. The Drosophila blood system is well studied and has developmental, molecular, and functional parallels with that of vertebrates. Unlike vertebrates, post-embryonic hematopoiesis in Drosophila is essentially restricted to the larval lymph gland (LG), a multi-lobed organ that flanks the dorsal vessel. Because the anterior-most or primary lobes of the LG are easy to dissect out, their cellular and molecular characteristics have been studied in considerable detail. The 2-3 pairs of posterior lobes are more delicate and fragile and have largely been ignored. However, posterior lobes harbor a significant blood progenitor pool, and several hematopoietic mutants show differences in phenotype between the anterior and posterior lobes. Hence, a comprehensive analysis of the LG is important for a thorough understanding of Drosophila hematopoiesis. Most studies focus on isolating the primary lobes by methods that generally dislodge and damage other lobes. To obtain preparations of the whole LG, including intact posterior lobes, here we provide a detailed protocol for larval fillet dissection. This allows accessing and analyzing complete LG lobes, along with dorsal vessel and pericardial cells. We demonstrate that tissue architecture and integrity is maintained and provide methods for quantitative analysis. This protocol can be used to quickly and effectively isolate complete LGs from first instar larval to pupal stages and can be implemented with ease.

Keywords: Drosophila hematopoiesis (果蝇的造血功能), Complete larval lymph gland (完整的幼虫淋巴腺), Blood progenitor (血祖细胞), Dissection (解剖), Posterior lobes (神经垂体), Secondary lobes (副裂片), Tertiary lobes (三级叶)

Background

Vertebrate hematopoietic stem and progenitor cells (HSPCs) give rise to various kinds of mature blood cell types. HSPCs can be identified by surface marker expression, staining properties of vital dyes, proliferative ability, and in vivo differentiation potential (Granick et al., 2012; Ema et al., 2014; Crisan and Dzierzak, 2016). Murine and zebrafish in vivo models have proved extremely useful in understanding various aspects of vertebrate HSPC biology. Adult mouse HSPCs primarily reside in the bone marrow, although recent studies show that HSPCs can circulate in the peripheral blood (Wright et al., 2001; Granick et al., 2012). Bone marrow is primarily located in the flat bones of the pelvis, vertebrae, ribs, and cranium, and in the long bones of the tibia, femur, and humerus. However, for post-embryonic analysis, HSPCs are obtained mainly from the long bones of the tibia and femur that represent a subset of the entire progenitor population. Distribution of HSPCs across various anatomical sites makes it difficult to study the entire progenitor pool, especially in post-embryonic stages and in larger animals such as mice and humans. The Drosophila hematopoietic system has proved helpful in addressing various aspects of hematopoiesis, owing to conserved signaling mechanisms and transcriptional factors that regulate hematopoiesis (Banerjee et al., 2019).


Drosophila hematopoiesis occurs in two successive waves. First, blood cell progenitors emerge from the procephalic/head mesoderm in the early embryo and give rise to larval circulating and sessile hemocytes, which persist until adulthood (Tepass et al., 1994; Holz et al., 2003; Honti et al., 2010; Ghosh et al., 2015; Sanchez Bosch et al., 2019). The second wave of hematopoiesis takes place in a specialized larval hematopoietic organ called the lymph gland (LG), located dorsally, flanking the anterior cardiac tube/dorsal vessel (Rugendorff et al., 1994; Lanot et al., 2001; Mandal et al., 2004; Grigorian et al., 2013). Blood cell progenitors that form the LG are derived from the embryonic dorsal mesoderm. Clonal analysis suggests the presence of hemangioblast precursor cells that can give rise to LG blood cells and cells of the dorsal vessel (Mandal et al., 2004). By stage 11, Odd-skipped (Odd) is expressed in the thoracic and the abdominal segments, T1-A6 (Ward and Skeath, 2000); the thoracic clusters form the LG, and the abdominal clusters give rise to the pericardial cells (Mandal et al., 2004). At stages 11-12, expression of the homeotic gene Antennapedia is restricted to segment T3 (Mandal et al., 2007). By stages 13-16, Odd+ cells in the thoracic segment (T1-T3) coalesce to form the LG, whereas Antennapedia is expressed in 5-6 cells at the posterior boundary of the LG primordium (Mandal et al., 2007). Two Collier expressing clusters appear in the thoracic segments T2 and T3, which coalesce following germ-band retraction (Crozatier et al., 2004). Collier expression is maintained at high levels at the posterior tip of the developing LG in 3-5 cells, whereas the remaining LG cells express Collier at low levels (Crozatier et al., 2004). In the late embryo, the LG consists of a single pair of lobes, the primary/anterior lobes, each lobe containing approximately 20 cells that express Serpent and Odd (Jung et al., 2005). At the first instar larval stage, primary lobe cells proximal to the dorsal vessel, termed pre-progenitors, express Serpent, Notch, Dorothy, and STAT92e, and lack expression of domeless (dome) (Jung et al., 2005; Dey et al., 2016; Banerjee et al., 2019). By the second instar, primary lobes have increased in size, consisting of approximately 200 cells in each lobe. Additionally, 2-3 pairs of smaller lobes are formed posterior to the primary lobes and are referred to as the secondary, tertiary, and quaternary lobes (Jung et al., 2005; Banerjee et al., 2019; Rodrigues et al., 2021).


Based on morphology and molecular marker analysis, third instar larval LG primary lobes are compartmentalized into three zones. The posterior signaling center (PSC) acts as the signaling niche. The medullary zone (MZ) towards the cardiac tube consists of multipotent progenitors. A peripheral cortical zone (CZ) mainly harbors phagocytic plasmatocytes and a few crystal cells. Intermediate zone (IZ) progenitors reside in the region between the MZ and the CZ, are identified by the expression of progenitor and early differentiation markers, and lack the expression of late markers (Jung et al., 2005; Banerjee et al., 2019). The multiple posterior lobes harbor progenitors that resist differentiation upon immune challenge (Rodrigues et al., 2021). Under steady state conditions, blood cells produced in the LG are released in circulation only at the pupal stage, contributing to the pupal and adult blood cell populations (Holz et al., 2003; Grigorian et al., 2011; Ghosh et al., 2015; Sanchez Bosch et al., 2019).


While primary lobes are well characterized, the identity of the posterior lobes was ill-characterized until recently (Rodrigues et al., 2021). Based on the expression of a limited set of markers and mutant analysis, a few studies proposed that the secondary lobes are essentially composed of blood cell progenitors that differentiate at the larval/pupal transition (Jung et al., 2005; Grigorian et al., 2011; Kulkarni et al., 2011; Benmimoun et al., 2015). Studies on secondary/posterior lobes used preparation of LG samples detached from their brain/ring gland anterior attachment site with thin tungsten needles and placed on glass slides (Lanot et al., 2001). This method of sample preparation causes damage to the delicate organ and might be the reason for the partial analysis of LG lobes. To obtain the entire intact LG, we use the larval fillet method of dissection described in this protocol, which helps maintain primary and posterior lobes intact. This protocol has been invaluable for a comprehensive analysis in our previous studies (Kulkarni et al., 2011; Sinha et al., 2013; Khadilkar et al., 2014; Sinha et al., 2019; Rodrigues et al., 2021). For instance, we could show that depletion of asrij, arf1, or garz and overexpression of arf1GAP1 leads to severe phenotypes of hyperproliferation and premature differentiation in the posterior lobes, as compared to the primary lobes (Kulkarni et al., 2011; Khadilkar et al., 2014). We also employed this method of dissection for whole LG proteomic analysis, which provided a resource to identify novel regulators of hematopoiesis (Sinha et al., 2019). Furthermore, differential RNA sequencing analysis for the primary and the posterior lobes helped to identify novel progenitor markers and regulators of hematopoiesis, and unveiled the molecular heterogeneity, as well as functional compartmentalization of the LG progenitor pool present in the different lobes (Rodrigues et al., 2021). Thus far, our studies suggest that analysis of the whole LG is crucial for exploring the complete application of Drosophila LG hematopoiesis. Here, we describe detailed protocols for whole LG sample preparation that can be used for GFP expression screens, immunostaining, RNA in situ, and high-throughput analyses.

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

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简介

[摘要]血细胞寿命有限,由少量造血细胞补充干细胞和祖细胞 (HSPC)。成年脊椎动物的造血发生在骨髓、肝脏、和脾脏,几乎不可能对整个 HSPC 池进行全面分析。这果蝇血液系统得到了很好的研究,并且在发育、分子和功能上与脊椎动物。与脊椎动物不同,果蝇的胚胎后造血本质上是仅限于幼虫淋巴腺 (LG),这是一种位于背血管两侧的多叶器官。因为LG 的最前叶或初级叶很容易解剖出来,它们的细胞和分子特性进行了相当详细的研究。2-3对后叶较多脆弱而脆弱,在很大程度上被忽视了。然而,后叶含有大量血液祖细胞池,并且几个造血突变体显示出前细胞之间的表型差异和后叶。因此,对 LG 的全面分析对于彻底的了解果蝇的造血功能。大多数研究侧重于通过以下方式隔离初级叶通常会移动和损坏其他叶的方法。获得整个LG的准备,包括完整的后叶,这里我们提供了幼虫鱼片解剖的详细协议。这允许访问和分析完整的 LG 叶,以及背血管和心包细胞。我们证明组织结构和完整性得到维持,并提供定量方法分析。该协议可用于快速有效地从一龄幼虫中分离出完整的 LG到蛹阶段,可以轻松实施。

关键词:果蝇造血,完整幼虫淋巴腺,血液祖细胞,解剖,后叶、次级叶、第三叶

[背景]脊椎动物造血干细胞和祖细胞 (HSPC) 产生各种成熟的血细胞类型。HSPCs 可以通过表面标记表达、染色特性来识别活性染料、增殖能力和体内分化潜能的分析(Granick等人,2012 年;Ema等人,2014; Crisan 和 Dzierzak,2016 年)。小鼠和斑马鱼的体内模型已被证明非常有用了解脊椎动物 HSPC 生物学的各个方面。成年小鼠 HSPCs 主要存在于骨髓,尽管最近的研究表明 HSPC 可以在外周血中循环(Wright等。, 2001; 格拉尼克等人。, 2012)。骨髓主要位于骨盆的扁平骨中,椎骨、肋骨和颅骨,以及胫骨、股骨和肱骨的长骨中。然而,对于后胚胎分析,HSPCs 主要来自胫骨和股骨的长骨,代表整个祖先种群的一个子集。HSPCs 在不同解剖部位的分布站点使得研究整个祖细胞池变得困难,特别是在胚胎后阶段和较大的动物,例如老鼠和人类。在果蝇造血系统已被证明有帮助的解决造血的各个方面,由于保守的信号机制和调节造血功能的转录因子(Banerjee等,2019)。果蝇造血发生在两个连续波中。首先,血细胞祖细胞从早期胚胎中的前头/头部中胚层并产生幼虫循环和无柄血细胞,持续到成年(Tepass等人,1994 年;Holz等人,2003 年;Honti等人,2010 年;Ghosh等。, 2015; 桑切斯博世等。, 2019)。第二波造血发生在称为淋巴腺 (LG) 的特殊幼虫造血器官,位于背侧,位于前部两侧心管/背脉管(Rugendorff等人,1994; Lanot等人,2001;曼德尔等人,2004; Grigorian等阿尔。, 2013)。形成 LG 的血细胞祖细胞来自胚胎背侧中胚层。克隆分析表明存在可产生 LG 血的成血管细胞前体细胞背血管的细胞和细胞(Mandal等,2004)。通过第11阶段,奇数跳过(Odd)表示在胸段和腹段,T1-A6(Ward 和 Skeath,2000);胸椎簇形成LG 和腹部簇产生心包细胞(Mandal等,2004)。在阶段 11-如图 12 所示,同源基因 Antennapedia 的表达仅限于片段 T3(Mandal等,2007 )。经过在第 13-16 阶段,胸段 (T1-T3) 中的奇数+细胞合并形成 LG,而Antennapedia 在 LG 原基的后边界以 5-6 个细胞表达(Mandal等人,2007)。两个 Collier 表达簇出现在胸段 T2 和 T3 中,它们合并在胚带回缩之后(Crozatier等人,2004 年)。Collier 表达保持在高水平3-5 个细胞中发育中的 LG 的后尖端,而其余的 LG 细胞在低水平表达 Collier水平(Crozatier等人,2004 年)。在胚胎晚期,LG 由一对裂片组成,初级/前叶,每个叶包含大约 20 个表达 Serpent 和 Odd 的细胞(荣格等。, 2005)。在第一龄幼虫阶段,靠近背血管的初级叶细胞称为前叶细胞。祖细胞,表达 Serpent、Notch、Dorothy 和 STAT92e,缺乏 domeless(圆顶)的表达(Jung等人,2005 年;Dey等人,2016 年;Banerjee等人,2019 年)。到第二龄,初级叶有大小增加,每个叶由大约 200 个细胞组成。此外,还有 2-3 对较小的叶在初级叶之后形成,被称为二级、三级和第四纪裂片(Jung等人,2005 年;Banerjee等人,2019 年;Rodrigues等人,2021 年)。基于形态学和分子标记分析,三龄幼虫 LG 初级叶是分为三个区域。后信号中心 (PSC) 充当信号利基。朝向心管的髓质区 (MZ) 由多能祖细胞组成。一个外设皮质区(CZ)主要含有吞噬性浆细胞和少量晶体细胞。中间地带(IZ) 祖细胞位于 MZ 和 CZ 之间的区域,通过表达祖细胞和早期分化标记,缺乏晚期标记的表达(Jung et al. , 2005;班纳吉等人。, 2019)。多个后叶包含抵抗分化的祖细胞免疫攻击(Rodrigues等人,2021 年)。在稳态条件下,血细胞在LG 仅在蛹阶段在循环中释放,有助于形成蛹和成体血细胞种群(Holz等人,2003 年;Grigorian等人,2011 年;Ghosh等人,2015 年;Sanchez Bosch等人,2019 年)。虽然初级叶的特征很好,但后叶的特征却很差直到最近(Rodrigues等人,2021 年)。基于一组有限的标记和突变体的表达分析,一些研究提出次级叶基本上由血细胞组成在幼虫/蛹转变时分化的祖细胞(Jung等人,2005 年;Grigorian等人,2011 年;库尔卡尼等人。, 2011; 本米蒙等人。, 2015)。二级/后叶使用制剂的研究用细钨针从大脑/环腺前附着部位分离的 LG 样本并放置在载玻片上(Lanot等,2001)。这种样品制备方法会损坏脆弱的器官,可能是对 LG 裂片进行部分分析的原因。获得完整的LG,我们使用本协议中描述的幼虫圆角解剖方法,这有助于保持初级和后叶完好无损。该协议对于我们的综合分析非常宝贵之前的研究(Kulkarni等,2011;Sinha等,2013;Khadilkar等,2014;Sinha等,2019;罗德里格斯等人。, 2021)。例如,我们可以证明asrij、arf1或garz和arf1GAP1 的过度表达导致过度增殖和早产的严重表型与初级叶相比,后叶的分化(Kulkarni et al. , 2011; Khadilkar等。, 2014)。我们还采用这种解剖方法进行全 LG 蛋白质组学分析,提供了一种资源来识别造血的新调节剂(Sinha等,2019)。此外,初级和后叶的差异 RNA 测序分析有助于鉴定新的造血的祖标志物和调节剂,并揭示了分子异质性,以及作为存在于不同叶中的 LG 祖细胞池的功能划分(罗德里格斯等。, 2021)。到目前为止,我们的研究表明,对整个 LG 的分析对于探索果蝇LG造血的完整应用。在这里,我们描述了整个的详细协议LG 样品制备,可用于 GFP 表达筛选、免疫染色、原位RNA和高通量分析。

关键字:果蝇的造血功能, 完整的幼虫淋巴腺, 血祖细胞, 解剖, 神经垂体, 副裂片, 三级叶

材料和试剂

A. 飞股票

1. Canton-S作为野生型参考菌株

2. dome-Gal4,UAS2xEGFP(加州大学洛杉矶分校Utpal Banerjee提供)

3. srpHemo-Gal4-UAS-GFP(国家生物科学中心(NCBS),飞行设施)

B. 材料

1.细漆刷(2号)

2.玻璃腔盘40×40 mmAtom Scientific,目录号:SDCE4040-1

3. SylgardSigma-Aldrich,目录号:761036)或同等产品

4. 微量测试板,96 孔(Tarsons,目录号:941196)或等效物

5. 35 mm 培养皿(Tarsons,目录号:460035)或同等产品

6. 1.5 ml 微量离心管(Axygen,目录号:MCT-150-C

C. 试剂组织解剖和固定

1. NaClFisher Scientific,目录号:S25542

2. Na 2 HPO 4(默克,目录号:S9763

3. NaH 2 PO 4 ·2H 2 O(默克,目录号:71505

4.多聚甲醛(Fisher Scientific,目录号:23995

5. 10×磷酸盐缓冲盐水(PBSpH 7)储备(见配方)

6. 4% 多聚甲醛 (PFA)(见配方)

免疫染色和安装

1. Triton X-100Sigma-Aldrich,目录号:T8787

2.正常山羊血清(GeNei,目录号:NS1

3.一抗:小鼠抗P1/NimC1和小鼠抗Hemese(由Istvan Ando提供,匈牙利科学院生物研究中心)、兔抗AsrijKulkarni等。, 2011)

4.二抗:Alexa fluor 568山羊抗小鼠(Invitrogen,目录号:A11004Alexa fluor 488 山羊抗兔(Invitrogen,目录号:A11008

5. Alexa fluor 633 结合的鬼笔环肽(Invitrogen,目录号:A22284

6. DAPI4'6-二脒基-2-苯基吲哚,二盐酸盐,ThermoFisher Scientific,目录编号:D1306)

7.甘油(默克,目录号:BP229-1

8.中性红(默克,目录号:N4638

9. PBS (PTX) 中的 0.1% Triton X-100(见配方)

RNA原位杂交

1.原位杂交探针:tep4 (Rodrigues et al. , 2021)

2.甲醇(默克,目录号:34860

3. Tween-20Sigma-Aldrich,目录号:P9416

4.柠檬酸钠(Na 3 C 6 H 5 O 7)(HiMedia,目录号:TC249

5. 分子级水-UltraPure DNase/RNase-free 蒸馏水(Invitrogen,目录号:10977015)

6.甲酰胺(Sigma-Aldrich,目录号:11814320001

7. tRNASigma-Aldrich,目录号:10109517001

8.肝素(Sigma-Aldrich,目录号:H3149

9.罗氏封闭剂(Sigma-Aldrich,目录号:11096176001

10. CHAPSSigma-Aldrich,目录号:C9426

11. EDTAFisher Scientific,目录号:S311-100

12. NaClFisher Scientific,目录号:S25542

13. MgCl 2Fisher Scientific,目录号:BP214-500

14.与碱性磷酸酶缀合的抗DIGSigma-Aldrich,目录号:11093274910

15. NBT/BCIPPromega,目录号:S3771

16. FastRed 底物套件(Abcam,目录号:ab64254

17. PBS (PBS-T) 中的 0.1% Tween-20(见配方)

18. 20×生理盐水-柠檬酸钠缓冲液(20×SSCpH 7)(见配方)

19. 杂交缓冲液(见配方)

20. 染色缓冲液(见配方)

设备

1、体视显微镜(Olympus SZ51,放大倍数0.8-4×

2. 细镊子(Fine Science ToolsDumont #5,目录号:11252-20

3.弹簧剪刀2.5毫米刀刃(Fine Science Tools,目录号:15000-08

4.昆虫针(Fine Science ToolsMinutien0.1 mm,不锈钢,目录号:26002-10

5. 共聚焦显微镜(蔡司,型号:LSM 880)软件1.ImageJ2. Adobe Photoshop CS5Adobe 系统)

程序

A. 获得完整淋巴腺 (LG) 的幼虫鱼片制备可以获得一、二、三龄幼虫的完整LG制剂,以及蛹,使用这种解剖方法。苍蝇育种和杂交使用标准进行协议。幼虫在标准玉米面琼脂培养基上培养到适当的阶段,在非饱和密度。图 1A 显示了第一、第二和第三龄幼虫的相对大小。1. 用细刷子将幼虫转移到装有水的腔盘/培养皿中,然后冲洗幼虫以去除任何食物颗粒。

2. 将幼虫转移到干净的腔盘中,置于冰上 20-30 分钟以固定幼虫。固定(可选)有助于固定幼虫(见步骤 A4)。

3. 将冷却的幼虫背面朝上放在 Sylgard 培养皿上,并通过放大倍数为 4 倍的立体显微镜(变焦范围 0.8-4 倍),聚焦于背部角质层。全部随后的步骤将在显微镜下观察幼虫组织时进行。

4. 通过在靠近前部和后部的地方将昆虫针牢固地插入幼虫来抑制幼虫气孔,并通过 Sylgard 培养皿(图 1B)。加入一滴 PBS(约 200 µl)以防止幼虫干燥。

5. 使用精细解剖剪刀,在靠近右侧的角质层上做一个小切口。后端。将剪刀插入切口端并横向切开角质层(图 1C 1D)。

6. 用细镊子将角质层松散的一端提起,向左侧延伸,切开沿着角质层的左侧边缘(图 1E)。7. 小心地取出内脏。找到连接到前脑叶的 LG背血管两侧的区域,其次是后端的心包细胞行(图 1F)。双鹅颈光源的侧面照明可以帮助区分折射LG。图 1. 幼虫 LG 解剖示意图。(一)代表形象一、二、三龄幼虫。(BF) LG 解剖的逐步示意图来自游荡的三龄幼虫。BL:脑叶,VNC:腹神经索,LG:淋巴腺,和PC:心包细胞。LG 叶之间的心包细胞数量可能不同。幼虫和 LG 图像未按比例绘制,但为清晰起见放大。

8. 加入一滴新鲜的 PBS 并轻轻地将其除去以除去任何组织碎片。如果需要,请重复。

9. 200 µl 固定剂(4% 多聚甲醛,参见配方)替换 PBS 并孵育 20分钟在室温下。取出固定液,用 PBS 洗涤 3 次。小心移除针,从后部缓慢提起 LG 制剂(带有下方的腹侧角质层)最后,转移到 96 孔板或 1.5 ml 微量离心管中进行免疫染色或在现场手续。

B. 获得完整淋巴腺 (LG) 的蛹鱼片制备整个 LG 可以在蛹形成 (APF) 0 20 小时内制备。根据我们的经验,大多数叶通过 15 小时 APF 进行组织分解(Rodrigues等,2021)。1. 将蛹背面朝上放在 Sylgard 培养皿上。所有后续步骤都必须在立体显微镜下观察蛹组织时进行。

2. 通过 Sylgard 培养皿在前气门附近牢固插入细昆虫针(图 2A)。添加一滴 PBS(约 200 µl)以防止蛹变干。

3. 使用精细解剖剪刀,沿后气门水平切割(图 2A)。

4. 将剪刀插入切口端,沿角质层右侧横向切开一条,然后是沿前部的切口,如虚线所示(图 2B)。

5. 提起角质层松散的一端,小心地向左侧延伸,沿左侧剪开沿虚线的角质层的侧边缘 ( 2B)

6. 在不损坏 LG 的情况下小心取出内脏器官(图 2C)。心导管和心包细胞是定位剩余 LG 叶的良好标志(图 2D)。添加一个滴一滴新鲜的 PBS 以去除任何组织碎片,并在需要时重复。图 2. LG 解剖示意图。(AD) 逐步示意图蛹 LG 解剖的代表。BL:脑叶,VNC:腹神经索,LG:淋巴腺体和 PC:心包细胞。LG 叶之间的心包细胞数量可能不同。蛹和 LG 图像未按比例绘制,但为清晰起见放大。

7. 按照步骤 A9 开始幼虫 LG 解剖并进行免疫染色或原位染色程序。

C. 淋巴腺 (LG) 的中性红色染色,以便于观察

1. 如上所述解剖并固定幼虫或蛹 LG。在 PBS 中洗涤三次,每次 8-10 分钟。

2. 加入 100 µl 0.2% 中性红溶液(PBS 0.2% 中性红),室温孵育5-7 分钟或直到 LG 明显染色。

3. PBS 洗去中性红。脑叶和 LG 叶应该很容易看到。第一秒,和三龄和蛹 LG 可以很容易地通过中性红染色 ( 3A-3D) 识别。

3. 用中性红染色的 LG 制剂。(AD) 半解剖鱼片制备完整的初级和后叶被中性红染色。LG 裂片被严重染色(红色)如一龄 (A)、二龄 (B)、三龄 (C)、幼虫和蛹 (D) 所示。黄色箭头表示淋巴腺。BL:脑叶,VNC:腹神经索,LG:淋巴腺,Pri:初级叶,Sec:次级叶,Tert:三级叶。星号表示心包细胞。比例尺:100 µm

D. 淋巴腺 (LG) 免疫染色

1. 所有步骤都是通过将 96 孔培养皿放在平板摇杆上来执行的。

2. 200 µl PBS 96 孔培养皿中清洗 10-12 个固定的 LG 制剂,3 次,每次 10-15 分钟每个。

3. 200 µl 0.1% Triton X-100 PBS0.1% PTX,参见配方)作为渗透剂加入到LG 制剂孵育 15-20 分钟。

4. 弃去 PTX,加入 100 µl 20% 正常山羊血清的 PBS 作为封闭剂,封闭 30 分钟。

5. 去除封闭剂,加入 40 µl 所需浓度的一抗用 PBS(或封闭剂)稀释。在 4°C 下孵育过夜。6. 去除一抗,用 PBS 清洗 LG 制剂 3 10-15分钟每个。

7. 弃去 PBS,加入 100 µl 20% 正常山羊血清,孵育 30 分钟。

8. PBS 中的 40 µl 二抗(通常为 1:400)替换并在室温下孵育 2 小时温度。

9. 去除二抗,用 200 µl PBS 洗涤,3 次,每次 10-15 分钟。继续安装步骤(见下文,程序部分 F)。

E.淋巴腺原位杂交 (LG)

1. 500 µl PBS 在微量离心管中清洗 15-20 个固定的 LG 制剂,3 次用于每次 10-15 分钟。

2. 为了长期储存,用甲醇清洗 LG 制剂四次,并储存在甲醇中 -20°C。要继续实验,请用甲醇和 PBS 的混合物冲洗样品一次(1:1) 3 次在 PBS 中。

3. 500 µl 0.1% Tween-20 PBS (PBS-T) 清洗 LG 制剂,3 次,每次 10-15 分钟每个(参见 0.1% PBS-T 的食谱)。

4. 500 µl 等体积的 0.1% PBS-T 和杂交缓冲液中平衡 LG 制剂(HB)(参见杂交缓冲液的配方)。

5. 弃去 PBS-T-HB 溶液,在 500 µl HB 65°C 预孵育 1 小时。

6. 去除 HB 并用 1 µl 地高辛 (DIG) 标记的 RNA 探针替换 200 µl HB(探针的稀释需要根据探针浓度凭经验调整和目标基因的表达水平)。在 65°C 下杂交过夜。

7. 小心取出 HB,在 65°C 下用 500-800 µl HB 清洗 LG 制剂 1 小时。

8. 500-800 µl 等体积的 0.1% PBS-T HB 中将 LG 制剂在 65°C 下洗涤 30 分钟,然后在室温下在 0.1% PBS-T 中快速洗涤 3 次。

9. 在室温下用 0.1% PBS-T 清洗 3 次,每次 10 分钟。10. 1% 牛血清白蛋白 (BSA) 或正常山羊血清 (NGS) 阻断非特异性结合在 0.1% PBS-T 30 分钟。

11. 与稀释 (1:1,000) 的碱性磷酸酶偶联的抗 DIG 抗体孵育室温封闭液 2 小时。

12. 去除抗 DIG 封闭溶液混合物,然后在 0.1% PBS-T 中快速冲洗 3 次。然后,用 0.1% PBS-T 洗涤 3 次,每次 10 分钟。

13. 500 µl 新鲜制备的染色缓冲液 (SB) 中平衡 10 分钟(参见配方)。

14. 在室温或 37°C 下用 1 ml SB (6.5 µl NBT + 3.5 µl BCIP) 中的 NBT/BCIP 进行可视化直到染色可见(图 4)。

15. PBS 中洗涤 3 次并按指示安装 LG 制剂(见下文,程序F) 节。图 4. RNA原位杂交过程的示意图。逐步淋巴腺 RNA原位杂交方案的表示。Pri:初级叶,Sec:二级裂片,Tert:三级裂片。安装的 LG 制剂显示Latran 的表达为NBT/BCIP 透露。

F. 安装完整的淋巴腺 (LG)

1. 将固定和染色的幼虫鱼片置于盖玻片底部 35 mm 培养皿中。这些也可以通过在 35 mm 培养皿上打一个直径为 1 cm 的孔并粘合盖玻片到菜的底部(图 5)。加入一小滴 (10-20 µl) 70% 甘油DAPI (1:500) 或安装介质,以防止样品干燥。

5. 安装在 35 毫米盖玻片底盘中的 LG 制剂。七龄幼虫 LG 制剂在 35 毫米盖玻片底盘上染上中性红。黑色点缀线表示 LG 瓣。LG:淋巴腺,PC:心包细胞。

2. 用镊子夹住前端的圆角角质层。再用一把细镊子,小心地将脑叶与角质层的其余部分分开。LG 通过环形压盖连接到脑叶和背侧血管 ( 1F)。此程序将部分移走它来自角质层。

3. 接下来,用一把镊子夹住后端的角质层,再用另一把镊子,小心地将背血管的后部与心包细胞分开并滑动盖玻片上。组织非常脆弱,整个过程必须轻柔地进行,同时通过立体显微镜观察。

4. LG 从前后两端松开后,小心地将 LG 移开从腹侧角质层。请勿直接提起或握住 LG。移动LG时,抓住大脑前端为心叶,后端为心包细胞。

5. 多个淋巴腺可以通过这种方式安装在一个培养皿中(图 5),并且可以进行高分辨率成像分析。数据分析A. 代表性成果在果蝇第三龄幼虫LG是多叶形器官侧翼背脉管。首先一对叶,称为初级叶,后面是 2-3 对后叶 - 二级、三级、和(很少)四级叶 - 由充当肾细胞的心包细胞分隔。为了为简单起见,我们将二级、三级和四级叶称为后叶。行心包细胞在后端排列在背血管上。为了证明这种解剖方法保持组织完整性,我们分析了标记物整个 LG、心导管和心包细胞。鬼笔环肽标记肌动蛋白,可用于可视化 LG 叶和背血管的完整性 ( 6A)。血红素,一种通用的血细胞标记(Kurucz等人,2003 年),在原代的所有细胞和大部分后部细胞中表达裂片 ( 6B)Asrij (Kulkarni et al. , 2011),另一种泛血细胞标记物,标记 LG 血细胞初级和后叶(图 6C)。srpHemo-Gal4,包含一个调控区该蛇基因,是活跃在胚胎血细胞(布鲁克纳等人,2004)和强烈在心包细胞中表达 ( 6D)。然而,初级叶中很少有细胞表达srpHemo-Gal4 ( 6D) 。在上述标记的帮助下,我们确认这解剖方法可用于分离完整的 LG 初级和后叶。

6. 使用泛血细胞、祖细胞和分化标记。从游荡的第三龄幼虫开始的整个 LG 准备包括初级和后叶。(A) 鬼笔环肽(黄色)标记肌动蛋白,用于识别 LG血细胞和心导管。泛血细胞标记物 (B)Hemese(红色)和 (C) Asrij(绿色)在 LG 裂片中表达并有助于识别完整的裂片。(D) srpHemo-Gal4(绿色)是在心包细胞中强烈表达。(E)圆顶(绿色)标记初级中的祖细胞和后叶,P1/Nimrod C1(红色)标记浆细胞。(F)原位杂交tep4(红色)在 MZ、二级叶和一些三级细胞中高表达裂片。Pri:初级叶,Sec:次级叶,Tert:三级叶。(AF) 比例尺:100 µm。为了深入分析组织完整性,我们进一步检查了已知祖细胞 (MZ) 和分化 (CZ) 标记,如前叶中所定义。进行了记者,免疫染色或 RNA原位分析来分析标记表达。如前所述,

无穹顶祖标记(Jung等人,2005)在 MZ、次级叶和在三级叶的一些细胞(图 6E)。分化标记 P1/Nimrod C1Kurucz等人,2007) 表达仅限于初级叶的 CZ ( 6E)RNA原位再祖标记tep4Rodrigues等人,2021 年)显示 MZ 中的高表达,次要裂片,以及在第三裂片的一些细胞中(图 6F)。使用LG解剖的圆角方法,我们获得了完整的初级和后叶,如用 MZ CZ 标记染色所观察到的。完整LG 叶也可用于分析生态位细胞、祖细胞和分化细胞的数量。每个叶中的血细胞。因此,可以使用 IMARIS 软件重建 3D 图像。使用 2D Slice 模块,为由 DAPI 标记的细胞核定义直径。景点模块用于估计 DAPI +原子核的数量。以 P1 或使用表面模块分析由圆顶标记的祖细胞。表面模块也是用于 3D 渲染,具有 3D 表面(P1 或圆顶)和点之间的距离(DAPI +)由二维切片模块定义。接下来,使用模块查找更接近表面的点查找点远离表面,特定标记的正负点数 (DAPI + ) 可以是决定。使用这种解剖方法,我们成功地分离了初级和后叶,进行了 RNA 测序分析(图 7)。这导致了新的标记物的鉴定LG 血细胞(Rodrigues等,2021)。保持组织完整性和样本质量取决于解剖过程中的关键步骤。虽然解剖要求很高,识别 LG 并执行快速解剖(2-3 分钟)已通过 2-3几天的练习,即使是经验不足的暑期实习生。这个详细的协议旨在建立LG 解剖和综合分析的标准方法,也可适用于从早期幼虫龄期和蛹期分离 LG

7. RNA 的解剖策略和工作流程示意图测序分析。解剖策略和 RNA 测序的逐步表示分别获得初级和后叶的转录组谱。Pri:初级裂片,Sec:二级裂片,Tert:三级裂片,Post:后裂片。B. 讨论与脊椎动物 HSPC 不同,大多数果蝇血液祖细胞位于单个造血器官中,淋巴腺,它也包含它们分化的后代。LG 的小尺寸(~1.5-2mm 长)对其常驻祖先进行了完整而全面的原位分析,并且差异化池成为可能。然而,我们对幼虫 LG 造血的大部分理解都来源于从深入分析仅限于最前叶,称为初级叶。直到最近,在初级叶中运作的细胞和分子机制被认为是适用的到后叶。然而,前叶和后叶在基因上有显着差异。表达和突变表型(Kulkarni等人,2011 年;Khadilkar等人,2014 年;Rodrigues等人,2021)。此外,后叶与前叶 PSC 物理分离,并且分化的血细胞参与调节前叶中的祖细胞命运(图 1F)。此外,更多的血祖细胞位于后叶(Rodrigues等,2021)。因此,对 LG 的不完整分析忽略了重要的发育和功能信息关于造血,这可能与脊椎动物的造血有关。帮助解剖和完整完整的 LG 分析,我们描述了一个详细的协议,这也将是有用的分析整个祖先群体。LG 初级叶是在胚胎阶段指定的,而后叶在二龄幼虫阶段形成并继续发育直到。游荡幼虫阶段。因此,这种解剖方法允许同时分析单个动物的发育造血功能(Rodrigues等,2021)。我们通过分析泛血细胞、祖细胞和RNA和蛋白质水平的分化标志物。获得完整的 LG 叶是必不可少的用于了解生理和免疫条件下的祖细胞异质性(Rodrigues etal ., 2021)。该协议可用于研究个体发育、发育和免疫方面LG 血细胞。它还可以用于分离不同的叶以进行后续的生化和组学化验(Sinha等人,2019 年;Rodrigues等人,2021 年)。因此,该协议将特别获得关于血细胞祖细胞命运调节的新的和更完整的见解是有价值的。

笔记

1.     我们使用中性红染色制剂只是为了便于训练期间的可视化和练习 LG 解剖,而不是用于免疫染色或原位杂交实验。

2.     避免将盖玻片放在 LG 制剂上,因为这会损坏组织。我们更愿意使用盖玻片底培养皿进行安装。有经验,8-10LG制剂即可安装在单个盖玻片底盘中(图 5)。

3.     LG解剖的圆角方法保留含有无蒂血细胞的腹侧幼虫角质层可以在样品安装步骤中检索到的簇(参见步骤 F4),允许同时对来自不同原基的血细胞的分析(Khadilkar等,2014)。

4.     该协议有助于分离三个器官系统:背血管、心包细胞和 LG在单个样品制备中使用相同的动物。

5.     由于前/初级叶祖细胞由一龄指定,而二级和三级裂片仅在后续阶段发育,此方法允许分析发育阶段在单个动物中。

食谱

1. 10×PBS氯化钠 18.9 Na 2 HPO 4 2.48 NaH 2 PO 4 ·2H 2 O 1.17 g溶解在高压灭菌的蒸馏水中。将体积调节至 250 毫升。将 pH 值调整为 7.0。在室温下储存。2. 4% 多聚甲醛 (PFA) 4 g PFA 粉末溶解在 80 ml 1× PBS 中。用 1× PBS 溶液将体积调节至 100 ml。将混合物置于 60°C 的水浴中以溶解 PFA。储存在 4°C3. PBS (PTX) 中的 0.1% Triton X-100 100 ml PBS 中加入 0.1 ml Triton X-100。在室温下储存。4. 0.1% Tween-20 PBS (PBS-T) 100 ml PBS 中加入 0.1 ml Tween-20。在室温下储存。5. 20× 柠檬酸钠缓冲液 (SSC)氯化钠 43.82 Na 3 C 6 H 5 O 7 22.05 克溶于分子级(无 DNase/RNase)水中。用分子级(无 DNase/RNase)水将体积调节至 200 ml。将 pH 值调整为 7.0。高压灭菌并储存在 -20°C6. 杂交缓冲液甲酰胺 50 毫升20×SSC 10 毫升酵母 RNA (50 毫克/毫升) 2 毫升肝素 (0.05 g/ml) 100 µl罗氏封闭剂 (10%) 20 ml(根据制造商的说明制备)。CHAPS (10%) 1 毫升EDTA (0.5M) 1 毫升吐温 20 (10%) 1 毫升使用分子级水(无 DNase/RNase)将体积调节至 100 ml。储存在-20°C7. 染色缓冲液5 M 氯化钠 200 微升1 M 氯化镁2 500 微升Tris-HCl pH 9.5 1 毫升吐温 20 (10%) 1 µl用高压灭菌的蒸馏水将体积调节至 10 ml

致谢

我们感谢果蝇社区提供苍蝇种群和抗体;国家生物中心科学、股票飞行设施、JNCASR 成像设施和我们的实验室成员宝贵的意见和建议。原理图是使用 BioRender.com 创建的。这项工作是由印法高级研究促进中心 (IFCPAR/ CEFIPRA )资助授予 MSI LWMSI 的工作还得到了 SERB 赠款、JC Bose 奖励项目和贾瓦哈拉尔·尼赫鲁高级科学研究中心。作者贡献:DRLW MSI 进行了实验;DRKVRLW MSI 编写了手稿; MSI LW 为这项工作获得了资金和设施。原始研究论文:JAK-STAT 信号的差异激活揭示了果蝇的功能区室化血祖。DOI 10.7554/eLife.61409。利益争夺作者宣称没有利益冲突。

参考

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Copyright Rodrigues et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rodrigues, D., VijayRaghavan, K., Waltzer, L. and Inamdar, M. S. (2021). Intact in situ Preparation of the Drosophila melanogaster Lymph Gland for a Comprehensive Analysis of Larval Hematopoiesis. Bio-protocol 11(21): e4204. DOI: 10.21769/BioProtoc.4204.
  2. 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.
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