Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Summary

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Abstract

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Introduction

Lipids are essential for cell survival. In addition to their traditional role as integral components of cellular membrane systems, lipids also play crucial functions in energy supply and signaling transduction throughout the life cycles of individual animals¹. Thus, lipid metabolism must conform to strict regulations to maintain physiological hemostasis in cells. It is known that dysregulation of lipid metabolism results in various diseases, such as diabetes and fatty liver. Despite the great importance of lipid metabolism in animal health, the mechanisms underlying lipid metabolism regulation remain largely unknown.

Protocol

1. Egg Laying

1. Prepare the standard cornmeal food for egg laying.
   NOTE: For the recipe and cooking procedure for standard cornmeal food used here, see the previously published details¹⁵.
2. Prepare fresh yeast paste by adding 6 mL of distilled water to 4 g of active dried yeast in a 50 mL centrifugal tube. Use a spatula to mix and make a paste.
3. Make egg-laying bottles by filling the cornmeal food into the bottles and spread approximately 1 g of yeast paste onto the surface of the cornmeal food with a spatula.
4. Place the flies of desired genotypes in an egg-laying bottle and place it i....

Results

Successful execution of this procedure should result in clear lipid droplets staining that reveals the number and size of lipid droplets in the oenocytes. Figure 1A,A',A'' shows that there are few detectable lipid droplets (green dots) in the oenocytes of normal feeding larvae during different developmental stages. Figure 1B,B',B'' shows increased lipid droplets (green dots) amount in the oenocyt.......

Discussion

Among those outlined above, there are several critical steps in this protocol, with the egg laying time period being one of these. As a lipid-mobilizing tissue, the oenocyte is highly sensitive to nutrition status^6,8. Prolonged egg-laying time periods may result in crowed larvae and increased food competition, leading to inaccurate results. The 1 h egg-laying time period used in this protocol allows the larvae to develop without nutrition competition. A much larg.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL centrifuge tube	Corning	430829	50 mL
6 cm Petri dish	Thermo Fisher	150326	6 cm
Agar			For fly food
Aluminum foil	N/A	N/A	Protect smaple from light
BODIPY 493/503	Invitrogen	D3922	Lipid droplet staining dye
Confocal microscope	Leica	Leica TSC SP5	Confocal imaging
Corn syrup			For fly food
Cornmeal			For fly food
Coverslip	Citoglas	10212424C	20 × 20 mm, 0.13-0.17 thick
Dissection pin	N/A	N/A
Dissection plate	N/A	N/A
Filter paper	N/A	N/A	Diameter: 11 cm
Fixation buffer	N/A	N/A	4% Paraformaldehyde (PFA) in 1x PBS
Forcep	Dumont	11252-30	#5
Incubator	Jiangnan	SPX-380	For fly culture
Microcentrifuge tube	Axygen	MCT-150-C	1.5 mL
Microscopy slide	Citoglas	10127105P-G
Mounting medium	VECTASHIELDAntifade Mounting Medium	H-1000	Antifade mounting medium
Nail polish	PanEra	AAPR419	Seal the coverslip
Paintbrush	N/A	N/A
PBS	N/A	N/A	1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,1.8 mM KH2PO4, pH 7.4)
Rotator	Kylin-Bell Lab Instruments	WH-986
Scissor	Smartdata Medical	SR81	Vannas spring scissor
Soy flour			For fly food
Spatula	N/A	N/A
Standard cornmeal food	N/A	N/A	Accoding to Bloomington standard cornmeal food recipe
Stereo microscope	Leica	Leica S6E	For tissue dissection
Wipe paper	N/A	N/A
Yeast			For fly food
yw	Kept as lab stock	N/A	Drosophila