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 he....

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. Usi....

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 dev.......

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

List of materials used in this article

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