Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Summary

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Abstract

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.

Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Introduction

The complex mechanisms regulating the transcription factors and signaling pathways that coordinate the development of the hematopoietic system and that malfunction in hematological diseases remain poorly understood. These transcription factors and signaling pathways, as well as their regulation, are highly conserved between Drosophila and mammalian hematopoiesis^1-5. Thus the Drosophila hematopoietic system represents an excellent genetic model to define the molecular mechanisms controlling hematopoiesis and underlying hematological diseases.

Similar to mammals, Drosophila generate blood cells, called hemocytes, in spatially and temporally distinct phases of hematopoiesis. Traditionally, Drosophila hematopoiesis was thought to be restricted to phases in the embryonic mesoderm and in the larval lymph gland. Recent studies provide evidence that hematopoiesis also occurs in larval sessile clusters and in the adult abdomen^6-8. All hematopoietic phases produce two types of mature hemocytes: plasmatocytes and crystal cells. Plasmatocytes are macrophage-like cells involved in phagocytosis, innate immunity, and wound healing. Crystal cells contain pro-phenoloxidases required for melanization, a reaction used in insect immune responses and wound healing. Larval hematopoiesis can generate a third mature hemocyte type, called a lamellocyte, in response to certain immune challenges such as parasitoid wasp infection^9,10. Lamellocytes are large, adherent cells that function, in conjunction with plasmatocytes and crystal cells, to encapsulate and neutralize wasp eggs laid in Drosophila larvae. In the absence of parasitization, lamellocytes are not found in wild-type larvae. Melanotic masses resemble melanized, encapsulated wasp eggs; many mutant Drosophila strains develop melanotic masses in the absence of parasitization. The presence of lamellocytes and/or melanotic masses can be indicative of hematopoietic abnormalities. In fact, the melanotic mass phenotype has been used to identify genes and pathways involved in hematopoiesis^11-14.

The larval hematopoietic system is the most extensively studied to date. It is comprised of hemocytes circulating in the hemolymph, sessile hemocyte clusters patterned under the cuticle, and hemocytes residing in the lymph gland. The lymph gland is a series of bilateral lobes attached to the dorsal vessel. Each primary lobe of the lymph gland is divided into three main zones. The outermost zone is known as the cortical zone and contains maturing hemocytes. The innermost zone is called the medullary zone and is comprised of quiescent hemocyte precursors. The third zone, the posterior signaling center, is a small group of cells at the base of the lymph gland that act as a stem cell-like niche. Early work established critical functions for Notch^15-18, Hedgehog^19,20, JAK-STAT¹⁸, and Wingless²¹ activity to regulate larval lymph gland development. More recent studies have demonstrated that BMP²², FGF-Ras²³, and Hippo^24,25 signaling also function within the larval lymph gland.

Four larval hematopoietic assays outlined here describe 1) measuring circulating hemocyte concentration, defined as number of cells per unit volume, 2) isolating and fixing circulating hemocytes for immunohistochemistry, 3) visualizing crystal cells in vivo, and 4) dissecting, fixing, and mounting lymph glands for immunohistochemistry. These assays can be used as hematopoietic readouts to assess the functions and regulations of signaling pathways in the larval hematopoietic system. While these methods have been used previously in the field, visual documentation of these assays has begun only recently^8,26-30. Several publications cited here are helpful resources describing similar methods and hematopoietic markers^26,31-33. Additionally, Trol and Viking are useful markers of the lymph gland basement membrane.

Protocol

1. Circulating Hemocyte Concentration

1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.
2. Collect larvae in dissecting dish wells filled with 1x phosphate buffered saline (PBS, Table 1).
3. For each larva, place 10 µl 1x PBS in a microcentrifuge tube on ice and 10 µl 1x PBS on a clean dissecting pad. Place the dissecting pad on an illuminated stereomicroscope base.
4. Dry an individual larva by placing it on a tissue wipe before transferring it to a PBS drop on the dissection pad.
5. Using a pair of forceps, gently tear open and carefully invert the cuticle to release the hemolymph.
   NOTE: Take care not to scrape or jab the cuticle as this could potentially release sessile hemocytes²⁹.
6. Using a pipet, collect the hemolymph from the dissecting pad. Avoid collecting larval debris such as the fat body.
7. Add the hemolymph to a microcentrifuge tube and mix by pipetting up and down.
   NOTE: Several samples can be collected at once and kept on ice for up to an hour.
8. Optional: Mix Trypan blue with the hemolymph sample in equal parts to dye and exclude dead cells. Count cells within 3 min of adding Trypan blue because it is toxic to cells.
9. Mix the sample by pipetting up and down before loading 10 µl into a hemocytometer chamber.
10. If using an automated cell counter, set appropriate parameters for cell size and circularity. For example, set a minimum cell size of 2 µm, a maximum cell size of 22 µm, and a circularity of 75 - 80% roundness to detect normal, round hemocytes³⁴. Experiment with different parameters to detect larger and spindle-shaped hemocytes, such as lamellocytes. Alternatively, use a hemocytometer to manually count hemocytes.

2. Circulating Hemocyte Immunohistochemistry

1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.
2. Place one coverslip in each well of a 6- or 12-well plate.
   NOTE: Square coverslips (22 mm) fit in 6-well plates and round coverslips (18 mm diameter) fit in 12-well plates.
3. Collect larvae in dissecting dish wells filled with 1x PBS.
4. Place 5 µl 1x PBS in the center of each coverslip.
5. Place the plate on an illuminated stereomicroscope base.
6. Dry an individual larva on a tissue wipe and then place in the PBS on the coverslip.
7. Using a pair of forceps, gently tear and carefully invert the cuticle. Before removing the larval carcass, use it to spread the hemolymph evenly.
   NOTE: Take care not to scrape or jab the cuticle as this could potentially release sessile hemocytes²⁹.
8. Repeat for all larvae, collecting the hemolymph of one larva per coverslip. Allow hemocytes to adhere to the coverslips for 5 - 8 min at room temperature RT. Do not exceed 30 min before fixation.
9. Fix hemocytes by adding 5 µl of 7.5% formaldehyde (Table 1) to each coverslip for 15 min at room temperature.
10. Wash coverslips three times with 1x PBS. Tip the plate and aspirate PBS after each wash. To prevent run-off of the permeabilization solution in the next step, use the aspirator to remove excess PBS from perimeter of the coverslips.
11. Add 100 µl permeabilization solution (Table 1) to each coverslip for 20 min at RT.
12. Tip and gently tap the plate to aspirate the permeabilization solution.
13. Dilute primary antibody with antibody solution (Table 1) according to provider's specifications. Add at least 500 µl primary antibody solution to coverslips in 12-well plates. Add at least 1.5 ml in 6-well plates. Incubate at 4 °C overnight.
14. Remove the primary antibody solution and wash coverslips with 1x PBS for 10 min on an orbital shaker, three times.
15. Dilute secondary antibody with antibody solution according to provider's specifications. Add at least 500 µl secondary antibody solution to coverslips in 12-well plates. Add at least 1.5 ml for 6-well plates. Incubate for at least 2 hr at RT.
16. Remove the secondary antibody solution and wash coverslips with 1x PBS for 10 min on an orbital shaker as in Step 2.10, three times.
17. During the wash steps, clean glass microscope slides with 70% ethanol (Table 1) using tissue wipes. For each coverslip, place 5 µl mounting buffer (Table 1) on the glass slide.
    NOTE: Two coverslips fit on one slide.
18. Aspirate the final PBS wash.
19. Use forceps to carefully remove the coverslips from the plates and place, inverted, on top of the mounting buffer.
    Note: Microscope slides can be stored at 4 °C prior to imaging. Maximum storage time depends on the stability of the antibodies used. 6- and 12-well plates can be rinsed and reused.

3. In Vivo Crystal Cell Melanization

1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2-6 hours.
2. Before beginning, set a heating source at 60 °C.
   NOTE: A thermal cycler program of 60 °C for 10 min (followed by a 25 °C hold) works best, but a water bath or other heating source is sufficient as long as the heat is distributed consistently and evenly over each larva.
3. Collect larvae in dissecting dish wells filled with 1x PBS (Table 1).
4. One at a time, dry a larva on a tissue wipe and place at the bottom of a PCR tube. Place each larva in a separate PCR tube. (See Figure 3A.)
5. For consistent results, ensure that larvae stay at the bottom of the PCR tubes by chilling larvae in the tubes at 4 °C for 10 - 15 min and/or gently tapping the tubes prior to heating.
6. Place the PCR tubes in the thermal cycler (or water bath). Heat at 60 °C for 10 min.
7. Carefully remove larvae from the PCR tubes into dissecting dish wells filled with fresh 1x PBS.
8. Dry larvae on a tissue wipe and arrange on a flat surface for imaging under a stereomicroscope.
9. Score images of larvae blindly by multiple individuals.

4. Larval Lymph Gland Immunohistochemistry

NOTE: The lymph gland is located approximately one-third length from the anterior end of a larva slightly below the brain on the dorsal side. (See arrow in Figure 3B.) The lymph gland flanks the dorsal vessel and is most easily dissected attached to the mouth hooks or to the brain. Wild-type, third instar lymph glands are very small structures; the primary lobes are approximately 100 - 200 µm in length. (See Figure 4A.)

1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.
2. Lymph gland dissection
   1. For each experimental condition, add 1 ml 1x PBS (Table 1) to one well of a 24-well plate.
   2. Add 1 drop of 0.1% PBST (Table 1) to each well using a disposable transfer pipet.
      NOTE: Detergent, such as Tween 20, is added to lower the surface tension, allowing the tissue to sink to the bottom of the well.
   3. Place the plate flat on ice.
   4. Collect larvae in dissecting dish wells filled with 1x PBS.
   5. Place a clean dissecting pad on an illuminated stereomicroscope base. Use a disposable transfer pipet to place small drops of 0.01% PBST (Table 1) on the pad. Transfer one larva to a PBST drop for dissection.
   6. Hold the larva with one pair of forceps approximately one-quarter length from the posterior end, dorsal side up.
   7. Use another pair of forceps to grab the cuticle immediately anterior to the forceps that are holding the larva. Gently pull the cuticle toward the anterior end until the mouth hooks are exposed.
      NOTE: The aim is to peel the cuticle without disturbing any internal structures.
   8. Release the cuticle and use both forceps to cut the larva in two. Remove the posterior end from the PBST drop.
      NOTE: If the cuticle does not peel all the way to the mouth hooks, cut the larva in two and remove the posterior end. Use one pair of forceps to hold the edge of the cuticle, and use the other pair of forceps to push the mouth hooks through the opening. This will invert the cuticle and expose the mouth hooks. This can be used as an alternate dissection method as well.
   9. Use one pair of forceps to pin down the cuticle (either the ventral cuticle, the dorsal cuticle flap, or both) for stability.
   10. Use another pair of forceps to grab the exposed mouth hooks and gently pull them out.
       NOTE: This will separate the cuticle from the internal structures. Ideally, the eye/antennal imaginal discs, brain, ring gland, and lymph gland flanking the dorsal vessel will remain attached to the mouth hooks.
   11. While still holding the mouth hooks, carefully remove unwanted structures such as the salivary glands, fat body, and intestine.
       NOTE: If the brain separates from the mouth hooks, the lymph gland might still be attached to and collected with the brain. In this case, hold the ventral nerve cord instead of the mouth hooks.
   12. Using the mouth hooks or ventral nerve cord as a handle, transfer the dissected complex containing the lymph gland to the well on ice. Do not exceed 30 min before fixation.
3. Fixation and immunohistochemistry
   1. Place the 24-well plate on the stereomicroscope base.
   2. Use a p200 pipette to carefully remove the PBST from the well.
      NOTE: Empty, neighboring wells are useful to temporarily deposit waste.
   3. Gently add 200 µl 3.7% formaldehyde (Table 1) down the side of the well and swirl the plate to ensure that the dissected tissues are completely submerged.
   4. Return the plate to ice for 30 min. If the lymph glands express fluorescent protein(s), keep the plate covered to prevent photobleaching.
   5. Use a p200 pipette to carefully remove the fixative.
   6. Wash by adding 200 µl 1x PBS to the well and placing the plate on an orbital shaker for 5 min at room temperature. Use a p200 pipette to carefully remove the PBS.
      NOTE: Fixed lymph glands can be left on ice in PBS until lymph glands for all experimental conditions are dissected and fixed.
   7. Repeat the wash steps two more times.
   8. Add 200 µl permeabilization solution (Table 1) to the well. Set the plate on an orbital shaker for 45 min at RT.
      NOTE: 45 min is the "gold standard" for lymph gland permeabilization but the authors have success after only 20 min.
   9. Remove the solution with a p200 pipette.
   10. Dilute primary antibody with antibody solution (Table 1) according to provider's specifications. Add 300 µl primary antibody solution to the well and ensure that the dissected tissues are completely submerged. Incubate at 4 °C overnight.
   11. Use a p200 pipette to remove the primary antibody.
   12. Wash by adding 200 µl 1x PBS to the well and placing the plate on an orbital shaker for 10 min at room temperature. Use a p200 pipette to carefully remove the PBS.
   13. Repeat the wash steps two more times.
   14. Dilute secondary antibody with antibody solution according to provider's specifications. Add 300 µl secondary antibody solution to the well and ensure that the dissected tissues are completely submerged. Incubate on an orbital shaker for at least 2 hr at RT.
   15. Use a p200 pipette to remove the secondary antibody and wash as described in Steps 4.3.12 & 4.3.13. Do not remove the PBS after the final wash.
4. Lymph gland mounting
   1. Clean glass microscope slides using 70% ethanol (Table 1) and tissue wipes.
   2. For each experimental condition, place one drop of mounting buffer (Table 1) on the glass slide.
      NOTE: Two conditions fit on one slide. Mounting buffer volume depends on the number of lymph glands to be mounted. Use 2 µl for approximately 18 - 20 lymph glands, 1 µl for 10 - 12, and 0.5 µl for 5 or less.
   3. Place the microscope slide on an illuminated stereomicroscope base.
   4. For up to two conditions at a time, transfer all of the lymph glands from the well to the mounting buffer with forceps, using the mouth hooks or ventral nerve cord as a handle.
   5. Space the dissected tissues evenly in a circular or rectangular shape, spreading the mounting buffer in the process.
   6. For each of the dissected tissues, individually, slide one tong of the forceps under the dorsal vessel and gently pull toward the periphery of the mounting buffer.
      NOTE: This will draw the lymph gland out from the rest of the dissected tissues and flatten the lymph gland on the glass.
   7. Using one tong of the forceps and a sawing motion, cut the dorsal vessel between the lymph gland and the brain.
   8. Move the rest of the dissected tissues to the opposite side of the lymph gland, at the outermost edge of the buffer.
      NOTE: The unwanted dissected tissues will eventually form a perimeter around the lymph glands and serve as a support upon which the coverslip will rest.
   9. Repeat until all of the lymph glands are separated from the dissected tissues, reserving one of the unwanted dissected tissues to place in the center.
   10. Take a coverslip between two fingers. Check that the coverslip is free of dust and fingerprints. If necessary, use a tissue wipe and 70% ethanol to clean the coverslip.
   11. Ensuring that the edge of the coverslip is parallel to the edge of the glass slide, carefully lower the coverslip over the mounting buffer.
       NOTE: Microscope slides can be stored at 4 °C prior to imaging. Maximum storage time depends on the stability of the antibodies used. 24-well plates can be rinsed and reused.

5. Imaging

1. Image fixed hemocytes and mounted lymph glands on an appropriate standard fluorescence or confocal microscope at 20X or higher magnification according to manufacturer's operating manual. Image whole larvae on a standard stereomicroscope at 2X magnification according to manufacturer's operating manual.
2. Follow software provider's instructions for deconvolution, if desired.

Solution	Composition	Storage	Comments
1x PBS	200 mg/L potassium chloride	room temperature
	200 mg/L potassium phosphate monobasic
	8,000 mg/L sodium chloride
	1,150 mg/L sodium phosphate dibasic
	dH2O
Fixative	3.7% or 7.5% formaldehyde in 1x PBS	room temperature in the dark	Formaldehyde is toxic.
Permeabilization solution/antibody diluent	0.4% Triton	4 °C	The standard formula uses 0.4% Triton but the authors use 0.1% Tween 20 with success. Use to dilute primary and secondary antibodies according to providers' recommended concentrations.
	5% bovine serum albumin, normal goat serum, or normal donkey serum
	1x PBS
70% ethanol	70% 200 proof ethanol in dH2O	room temperature
Mounting buffer	0.5% N-propyl gallate	4 °C in the dark	N-propyl gallate is harmful. DAPI is a mutagen.
	80% glycerol
	Optional: 1 μg/ml DAPI (4’,6-diamidino-2-phenylindole)
	1x PBS
0.1% PBST	0.1% Tween 20 in 1x PBS	room temperature
0.01% PBST	1:10 dilution of 0.1% PBST	room temperature

Table 1. Solutions used in this Protocol.

Results

Circulating Hemocyte Concentration

Hemocyte numbers increase throughout larval development³⁵. To illustrate that this method detects differences in hemocyte numbers and concentration, regardless of the biological cause, we measured hemocyte concentrations of delayed and non-delayed larvae. Loss of prothoracicotropic hormone (ptth) by genetic ablation of ptth-producing neurons (ptth>grim) produces a delay in larval de...

Discussion

Upon genetic or environmental alteration, the four methods described here can be used individually or in conjunction to analyze distinct processes during hematopoiesis such as signaling, survival, proliferation, and differentiation. Drosophila hematopoiesis is a dynamic process; the number of hemocytes per animal increases³⁵ and the structure and gene expression of the lymph gland changes³² during development. Prior to performing these assays, therefore, it is critical to restrict egg colle...

Materials

Name	Company	Catalog Number	Comments
PBS tablets	MP Biomedicals	2810305
dissecting dish	Corning	7220-85
microcentrifuge tube	Denville	C2170
silicone dissecting pad, made from Sylgard 184 kit	Krayden (distributed through Fisher)	NC9644388 (Fisher catalog number)	Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.
stereomicroscope	Morrell Instruments (Nikon distributor)	mna42000, mma36300	Nikon models SMZ1000 and SMZ645
tissue wipe	VWR	82003-820
forceps	Electron Microscopy Sciences	72700-DZ
p200 pipette	Eppendorf	3120000054
Countess Automated Cell Counter	Invitrogen	C10227
Countess cell counting chamber slides	Invitrogen	C10283
hemocytometer	Hausser Scientific	3200
trypan blue stain	Life Technologies	T10282
formaldehyde	Fisher	BP531-500
Triton	Fisher	BP151-500
Tween 20	Fisher	BP337-500
bovine serum albumin	Rocky Mountain Biologicals	BSA-BSH-01K
normal goat serum	Sigma	G9023-10ML
normal donkey serum	Sigma	D9663-10ML
200 proof ethanol	VWR	V1001
N-propyl gallate	MP Biomedicals	102747
glycerol	VWR	EM-4750
DAPI (4’,6-diamidino-2-phenylindole)	Fisher	62248
6-well plate	Corning	351146
12-well plate	Corning	351143
microscope cover glass, 22 mm square	Fisher	12-544-10
microscope cover glass, 18 mm circular	Fisher	12-545-100
glass microscope slides	Fisher	22-034-980
thermal cycler	Eppendorf	E950010037	Mastercycler EP Gradient S
PCR tubes	USA Scientific	1402-2700
24-well plate	Corning	351147
disposable transfer pipet	Fisher	13-711-9AM
fluorescence microscope	Zeiss		Axio Imager.Z1