Imaging Dpp Release from a Drosophila Wing Disc

Summary

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

Abstract

The transforming Growth Factor-beta (TGF-β) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-β superfamily includes TGF-β, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-β superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Introduction

Bone Morphogenetic Proteins (BMPs) are essential for early embryogenesis and patterning of adult structures. BMPs are produced and secreted to affect transcription of target genes needed for growth and cell differentiation in responding cells. Decapentaplegic (Dpp) is a Drosophila homolog of BMP4 that is important for development of embryonic and adult structures like the wing^1,2,3,4. Several groups have focused on the role of Dpp in patterning adult fly wings because 1) the wings are comprised of two transparent epithelia sheets wi....

Protocol

1. Collecting Eggs to Generate Larvae for Dissection

1. Cross 30-40 virgin female Dpp-GAL4/TM6 Tb Hu flies to 10-15 male Sp/CyO-GFP; UAS-Dpp-GFP/TM6 Tb Hu.
   NOTE: Any two genotypes containing Dpp-GAL4 and UAS-Dpp-GFP may be used as long as the balancers have larval markers allowing for selection of the appropriate progeny during the larval stage.
2. To collect eggs, flip the crossed flies into a fresh vial of food and allow them to lay for 3–4 h before removing them from the vial. To encourage egg laying, a small amount of yeast paste made of granulated yeast mixed with a small amount of water can be added to the top of the....

Results

Figure 2 shows representative live-imaging results of this protocol. When the protocol is successful, Dpp-GFP can be seen as a stripe down the center of the wing disc with nuclei visible as non-fluorescent circles within the Dpp-GFP region (Figure 2). Dpp-GFP release is visible as fluorescent puncta that appear and disappear. We have observed Dpp-GFP fluorescence appearing and disappearing in the cell bodies and far from the cell bodies. Dpp signaling is depende.......

Discussion

BMPs such as Dpp make their significant impact when they bind a complex of membrane bound receptors to cause a cascade of intracellular signaling in neighboring or apparently distant cells. Dr. Thomas Kornberg’s lab has shown that cells that produce the Dpp signal contact cells that receive the signal using actin based thin filapodia-like structures called cytonemes^15,24,25. These data suggest that developmental cell-cell .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Baker's yeast	Red Star
CaCl2 dyhydrate	Fisher Scientific	C79-500
Coverslips	VWR	484-457
Double-sided tape	Scotch
Drosophila Agar Type II	Apex	66-104
Drosophila melanogaster: Dpp-GAL4/TM6 Tb Hu			This stock will soon be made available at Bloomington Drosophila Stock Center
Drosophila melanogaster: Sp/CyO-GFP; UAS-Dpp-GFP/TM6 Tb Hu			This stock will soon be made available at Bloomington Drosophila Stock Center
Dumont Tweezers #5	World Precision Instruments	500233	Forceps for dissecting
HEPES	Sigma Aldrich	H3375
KCl	Fisher Scientific	AC193780010
Light Corn Syrup	Karo
Malt Extract	Breiss
MgCl2	Fisher Scientific	AC223210010
Microscope slides	Sigma Aldrich	S8400
NaCl	Fisher Scientific	S271-500
NaHCO3	RPI	S22060-1000.0
Nail polish	Electron Micsroscopy Sciences	72180
Propionic Acid	VWR	U330-09
Soy Flour	ADM Specialty Ingredients	062-100
Sucrose	Fisher Scientific	S5-3
Sucrose	Fisher	S512
Tegosept	Genesee Scientific	20-259
Trehalose dyhydrate	Chem-Impex International, Inc.	00766
Yellow Corn Meal	Quaker
Zeiss LSM 780 confocal microscope	Zeiss		Microscope for live imaging
Zeiss SteREO Discovery.V8 microscope	Zeiss		Microscope for dissections