Immunostaining and Dye Penetration Experiments to Define Core Pleated Septate Junction Proteins in Drosophila Embryonic Epithelia

Summary

These protocols outline how to characterize the organization and occluding function of pleated septate junctions in control and mutant Drosophila embryonic epithelia.

Abstract

The purpose of these protocols is to evaluate the organization and barrier function of pleated Septate Junctions (pSJs) in the ectodermally-derived epithelial tissues of Drosophila embryos. pSJ form an occluding barrier on the lateral membrane of ectodermal tissues including the epidermis, salivary glands, trachea, and hindgut that is functionally similar to the tight junction in vertebrate tissues. In Drosophila, 30 proteins have been identified that are required for the formation of pSJs. Core pSJ proteins are initially localized along the length of the lateral membrane but become strongly enriched at the apical lateral region of the junction, just basal to the adherens junction, by stage 16 of embryogenesis. By late stage 15 of embryogenesis, these tissues possess a fully functional occluding junction. This paper will describe immunostaining and dye permeability protocols that can be used to interrogate the organization and function of pSJs in control and mutant embryos. Successful employment of these protocols can be used to demonstrate the occluding function of pSJs, and to test whether an unknown gene encodes a core component of the junction.

Introduction

A critical function of epithelial tissues is to compartmentalize distinct regions within an organism. Creation of these distinct environments is essential for organ function and for protecting the organism from external threats. The occluding junction formed between the cells of an epithelium is responsible for establishing and maintaining these environments by preventing the paracellular flow of solutes between the apical and basal sides of that epithelium^1,2. In vertebrate organisms, the occluding junction is referred to as the tight junction, whereas it is referred to as the septate junction (SJ) in inverte....

Protocol

1. Staging of embryos

1. Cross healthy male and female flies of the appropriate genotypes (see Table of Materials for examples) and place them in embryo collection cages with fresh apple juice plates smeared with yeast paste. Let the flies acclimate to egg collection cages for a day or two before starting embryo collections.
2. Collect embryos on apple juice plates and allow them to develop to stage 16 (~13 h after egg laying (AEL) to 16 h AEL at 25 °C). Allow the adults to lay fertilized eggs for 1-2 h on apple juice plates, remove the plate from the egg collection cage, and then age them to 13-16 AEL at 25 °C. Alternatively....

Results

The protocols presented here are used to investigate the organization and function of pSJs in ectodermal tissues in stage 16 Drosophila embryos. These protocols are only applicable to pSJs and cannot be used to interrogate sSJs. A mutation in a gene that results in failure to localize other pSJ proteins to the pSJ region in stage 16 embryos and to create an occluding junction at stage 16 is either in a core pSJ gene or an accessory pSJ gene. The main distinction between the two is whether the encoded protein is subcellul.......

Discussion

Here we present a protocol to identify core and accessory pSJ proteins using two relatively simple assays that can be achieved in modestly appointed labs with fluorescence microscopy capabilities. The first assay examines the molecular organization of the pSJ in mature polarized epithelia using confocal imaging of fixed mutant embryos. The second assay tests the integrity of the pSJ's occluding function by injecting dye-labeled 10 kDa dextran molecules into stage 16 living embryos. The simplicity of these protocols a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Capillary tube for needles (Boro 1 X 0.5/Fiber )	FHC	30-30-1
Confocal microscope	Leica	Stellaris 5
Cover glass 22 x 22 2 thickness for injections	Fisher Scientific	12-540-B
Cover glass 22 x 30 1.5 thickness for imaging	Corning	2980-233
Culture tube for immunostaining (6 x 50 mm)	Fisher Scientific	14-958-A
Embryo collection cage	Genesee Scientific	59-100
Fluorescence stereomicroscope	Leica	MZ10 F
Halocarbon oil (700)	Genesee Scientific	59-131
Heptane	Fisher Scientific	H350-4
Inverted fluorescence microscope	Leica	DMi8
Mesh baskets for dechorionating embryos	Genesee Scientific	46-101
Methanol	Fisher Scientific	A412-4
Micromanipulator	Narishige	MN151
Microscope slides	Fisher Scientific	12-544-2
Needle puller	David Kopf instruments	model 720
Normal Donkey Serum	Jackson ImmunoReseaerch	017-000-121
Nylon mesh (120 um)	Genesee Scientific	57-102
Objective lens HC PL FLUOTR 10X/0.32 PH 1	Leica	11506537
Objective lens HC PL APO 10x/0.40 CS2	Leica	15506407
Objective lens HC PL APO 40x/1.30 CS2	Leica	15506358
Petri plates 60 x 15 mm	Corning	351007
Platform rocker	Globe Scientific	GTR-FS
Primary antibody (Coracle)	DSHB	C566.9
Primary antibody (Coracle)	DSHB	C615.16
Primary antibody (E cadherin)	DSHB	DCAD2
Primary antibody (Mcr)	Ward lab, CWRU	guinea pig polyclonal
Rhodamine labeled 10 kDa dextran	Molecular Probes	D1824
Secondary antibody (donkey anti-guinea pig Cy3)	Jackson ImmunoReseaerch	706-165-148
Secondary antibody (donkey anti-mouse Cy2)	Jackson ImmunoReseaerch	715-225-151
Secondary antibody (donkey anti-rat Cy5)	Jackson ImmunoReseaerch	712-175-150
Table top shaker	New Brunswick Scientific	C2 platform shaker
Tegosept (methyl 4-hydroxybenzoate)	TCI	H0216
Triton X-100	Fisher Scientific	BP151500
Vials for fixation (20 ml WHEATON liquid scintillation vials)	Fisher Scientific	03-341-73C
Whatman 3MM  Chromatography Paper	Fisher Scientific	05-716-6H
Fly stocks	Full genotype	stock number	Source
cor4	w*; P{neoFRT}43D cora4/CyO	52232	BDSC
w1118	w[1118]	5905	BDSC
Solution	Recipe
10x PBS	Mix 90 g of NaCl, 20 g of Na2HPO4, and 8.3 g of NaH2PO4.H2O and dH20 to 1L. Filter sterilize. Dilute to 1X with dH2O for washing steps in immunostaining.
10% Triton X-100 solution	Mix 1 ml of Triton X-100 to 9 ml of dH2O on a platform rocker until dissolved. Store at room temperature.
50% bleach	Mix 10 ml of commercial bleach (7.5% sodium hypochlorite) to 10 ml of water prior to dechorionating embryos.
Apple juice plates	Boil 15 g Drosophila agar in 725 ml dH2O. Add 25 g sugar and 250 ml apple juice and mix until blended. Let the solution cool to 55 C and then add 1.3 g tegosept and gently mix. Pour into 60 x 15 petri plates and let cool. Store at 4 C.
Blocking solution	Mix 9.8 ml of 1X PBS with 100 ul of 10% Triton X-100 and 100 ul of Normal Donkey Serum in a 15 conical tube.
Embryo wash solution	Make 10X embryo wash solution by mixing 70 g NaCl and 2 ml Triton X-100 to 1L of dH2O. Dilute to 1X with dH2O for working solution.
Fix	Preheat 10 ml of 1X PBS in the microwave. Add 0.4 g  of paraformaldehyde and pipet to mix. Add 10 ul of 14N NaOH to speed dissolving. Place on a 90 C heatblock until dissolved. Once no solid paraformaldehyde remains add 10 ul of 14N HCL to adjust pH to neutral. Always wear a lab coat, gloves and googles when using paraformaldehyde.
Mounting media	Mix 9 ml of glycerol and 1 ml of 1M Tris base pH 8.0. Add 0.05 g n-propyl-gallate,  mix and heat to dissolve. Aliquot 0.5 ml into eppendorf tubes.
Rhodamine dextran solution	Make fresh dye solution by dissolving Rhodamine-labeled 10 kDa Dextran to 1 mg/ml in 0.5M Sodium Phosphate, pH 7.5 and 5 mM Potassium Chloride.
Yeast paste	Mix 50 g of commercial yeast (Red Star) with a pinch of sucrose and enough water to make a thick paste. Store at 4 C.