Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Summary

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Abstract

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be visualized simultaneously by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Introduction

In multicellular organisms, a group of cells is endowed with a specialized function in their biogenic activity that is essential for the whole body. To fulfill their missions, each tissue or organ expresses a series of genes related to their functions and communicates with other tissues to orchestrate their activities in the context of development. To characterize such specialized cellular functions and inter-organ interactions, we need to specify a group of cells along with other types of cells being kept intact in the multicellular architecture.

One example of such specialized organs is a steroidogenic organ, where many biosynthetic enzym....

Protocol

NOTE: The overall scheme of protocols is shown in Figure 1.

1. The Dissection of the Larval Ring Gland (RG)

NOTE: In D. melanogaster, which belongs to cyclorrhaphous Diptera, the PG is within a composite endocrine organ called the ring gland (RG, Figure 2D). Since it is unfeasible that the PG is surgically separated from other types of cells (discussed later), a practical target is to isolate an intact, undamaged RG by dissection.

1. Preparation of larvae at the appropriate developmental stages
   NOTE....

Results

We used the above protocols to visualize steroidogenic organs and their interactive organs in D. melanogaster larvae and adult females. The overall scheme of protocols is shown in Figure 1.

The RG, including the PG (Figure 2D), is smaller and more transparent than the brain and is located at the anterior-dorsal side of the brain (Figure 2A-C and 3A-E). To label the PG cells, several groups have generated various types of antibodies against ecdysteroidogenic e.......

Discussion

We studied ecdysteroid biosynthesis and its regulatory mechanism in D. melanogaster, and devised a protocol for dissection and immunostaining. The timing of ecdysteroid biosynthesis is affected by environmental cues through neuronal inputs³³, so it is essential to maintain the innervation of the ecdysteroidogenic organs along with the brain, VNC, and other tissues during dissection.

As described above, the D. melanogaster PG forms a complex endocr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
egg collection
tissue culture dish (55 mm)	AS ONE	1-8549-02	for grape-juice agar plates
collection cup	HIKARI KAGAKU
yeast paste	Oriental dry yeast, Tokyo
100% grape juice	Welch Food Inc.
rearing larvae
small vials (12ml, 40×23.5 mm, PS)	SARSTEDT	58.487
disposable loop	AS ONE	6-488-01
standard fly food			the recepi us on the website of Blooington stock center.
dissection
dissecting microscope	Carl Zeiss	Stemi 2000-C
dissecting microscope	Leica	S8 AP0
tissue culture dish (35 x 10 mm, non-treated)	IWAKI	1000-035
Sylgard	TORAY		coarting silicon inside dishes
Terumo needle (27G, 0.40 x 19 mm)	TERUMO	NN-2719S	A "knife" to cut the tissue
forceps, Inox, #5	Dumont, Switzerland
insect pin (0.18 mm in diameter)	Shiga Brand		for fillet dissection
micro scissors	NATSUME SEISAKUSHO CO LTD.	MB-50-10
fixation
ultrapure water	Merck Millipore
phosphate buffered saline (PBS)
Formaldehyde	Nacalai tesque	16222-65
Paraformaldehyde	Nacalai tesque	02890-45
Triton-X100	Nacalai tesque	35501-15
microtubes (1.5 ml)	INA OPTIKA	CF-0150
Incubation
As one swist mixer TM-300 (rocker)	As one	TM-300	rocker
Bovine Serum Albumin	SIGMA	9048-46-8
primary antibody
anti-Sro (guinea pig), 1:1000
anti-GFP (rabbit), 1:1000	Molecular Probes	A6455	Shimada-Niwa ans Niwa, 2014
anti-GFP (mouse mAb, GF200), 1:100	Nakarai tesque	04363-66
anti-5HT (rabbit), 1:500	SIGMA	S5545
anti-Hts 1B1 (mouse)	Developmental Studies Hybridoma Bank (DSHB)	1B1
anti-DE-cadherin (rat), 1:20	DSHB	DCAD2
anti-nc82 (mouse), 1:50	DSHB	nc82
secondary antibody
Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	Life Technologies	A-11008
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate	Life Technologies	A-11001
Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 546 conjugate	Life Technologies	A-11081
Goat anti-Guinea Pig IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate	Life Technologies	A-21435
Alexa Fluor 546 dye-conjugated phalloidin	Life Technologies	A-22283
Mounting reagents
Micro slide glass	Matsunami Glass Ind.,Ltd.	SS7213
Square microscope cover glass	Matsunami Glass Ind.,Ltd.	C218181
FluorSave reagent (Mounting reagent)	Calbiochem	345789
Transfer pipette 1 ml (Disposable dropper)	WATSON	5660-222-1S
imaging
LSM700 laser scanning microscope system	Carl Zeiss		inverted Axio Observer. Z1 SP left
image processing
LSM700 ZEN	Carl Zeiss		It is a special user interface based on the 64 bit Microsoft Windows7 operating system
ImageJ
Fly stocks
w; GMR45C06-GAL4			from Bloomington Drosophila Stock Center. (#46260)
UAS–GFP; UAS–mCD8::GFP			gifts from K. Ito, The University of Tokyo.
w[1118]
w; phantom-GAL4#22/UAS-turboRFP
w; UAS-mCD8::GFP; TRH-GAL4			see in Ref29, Alekseyenko, O. V, Lee, C. & Kravitz, E. A.(2010)
w; UAS-mCD8::GFP			from Bloomington Drosophila Stock Center. (#32188)
yw;; nSyb-GAL4			from Bloomington Drosophila Stock Center. (#51941)