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

Our research investigates the developmental nonoccluding functions of septate junction proteins, focusing on identifying and characterizing new components involved in the structure and biological roles. This protocol is meant to characterize the organization and occluding function of pleated septate junctions in the ectodermal epithelia of Drosophila embryos. To begin, cross healthy male and female Drosophila flies of the appropriate genotypes, and place them in embryo collection cages with fresh apple juice plates smeared with yeast paste.

Allow the adults to lay fertilized eggs on the apple juice plates. Remove the plate from the egg collection cage and age the embryos for 13 to 16 hours after egg-laying at 25 degrees Celsius to reach stage 16. Then fix the stage 16 embryos in an appropriate fixative solution and perform immunostaining with the desired antibodies.

For best results, image the embryos on a confocal microscope with a 40x oil immersion objective. After focusing on the embryo of interest, set the laser intensity and gain to achieve a strong fluorescent signal without oversaturating any pixels. Identify stage 16 embryos that do not express balancer-encoded green fluorescent protein, using midgut morphology.

Focus the scan on the salivary gland or hindgut, and position the Z-scan to include the largest possible diameter of the lumen to visualize the full lateral membrane of the epithelial cell. Set a suitable line average and image a polarized tissue in at least 20 control and 20 Cora 4 mutant stage 16 embryos. Record data only from embryos in which the adherence junction marker is tightly localized to the apical-lateral region of the membrane.

In wild-type stage 16 embryos, assess enrichment of pleated septate junction proteins along the apical region of the lateral membrane near the adherens junction. Record the persistent distribution of the pleated septate junction marker all along the lateral membrane in the mutant. Make a fresh dye solution by dissolving rhodamine-labeled 10 kilodalton dextran to 1 milligram per milliliter in 0.5 molar sodium phosphate at pH 7.5, with 5 millimolar potassium chloride.

Using a needle puller, pull a 1-millimeter outer diameter by 0.5-millimeter inner diameter capillary tube with fiber, into a glass needle. Next, load the glass needle into the needle holder on a micromanipulator and arrange the micromanipulator next to an inverted microscope. Orient the needle as close to parallel to the microscope slide as possible, and lower it to the same Z-plane as the side of the slide.

Break the tip of the needle by striking it against the side of the microscope slide. Raise the needle above the level of the slide and remove it for loading. Next, fill the capillary tube with dye using the smallest available pipette tip.

Reload the filled needle into the needle holder. Test the flow of the 10 kilodalton dextran by injecting a small amount into a drop of halocarbon oil on the surface of a microscope slide. Using a 22 x 22 millimeter number 2 thickness cover slip, create an indented line on an apple juice plate.

Line up approximately 25 embryos each of control and Cora 4 mutant at stage 16 or 17 on the apple juice plate. Ensure that their posterior ends align along the line, and that the ventral surfaces face upward. Then apply a piece of double-sided tape to a 22 x 22 millimeter number 2 cover slip.

Touch the tape to the embryo line and lightly rub the cover slip to transfer the embryos from the plate onto the double-sided tape. Next, add a drop of halocarbon oil to a microscope slide and attach the cover slip to the slide by allowing capillary action to create a bond. Orient the cover slip closer to the side of the slide opposite any frosted portion.

Desiccate the embryos for three to five minutes in a container of desiccant, and cover the embryos completely with halocarbon oil. Mount the slide with embryos onto the inverted microscope with the embryos facing up and away from the objective. Align the micromanipulator so that the needle faces the posterior end of the embryos at an angle as close to parallel to the embryos as possible, and adjust the Z-axis of the micromanipulator so that the needle tip aligns with the midpoint of the embryo along the dorsal-ventral axis.

Next, inject the embryos by quickly moving the stage into the needle so that the needle tip enters the hemocoel. Inject approximately 0.2 to 0.5 nanoliters of dye using a 10-25-or 50-milliliter syringe. Quickly withdraw the needle and move the stage to the next embryo for dye injection.

Then add additional halocarbon oil over the embryos. Cover the embryos with a 22 x 22 millimeter number 2 cover slip to create a uniform oil layer between the cover slips and prevent crushing. Perform imaging on a fluorescent compound microscope or a confocal microscope after dextran dye injection.

On the confocal microscope, acquire a Z-stack through approximately one quarter of the embryo depth along the dorsal-ventral axis to assess the lumen of the trachea. Examine the lumen of the trachea, a salivary gland, or hindgut for accumulation of labeled dextran. Record the genotype and developmental stage of each embryo and whether labeled dextran accumulates in any tubular organ.

Note embryos in which the perivitelline space is strongly stained with dye, as these may represent inadvertent injection into that space. In wild-type embryos, macroglobulin complement-related, or Mcr, was uniformly localized along the lateral membrane at stage 13, became enriched in the apical region of the lateral membrane at stage 14, and was strongly localized to the apical-lateral region, just basal to the adherens junction by stage 16. Cortical antibody staining defined the mature pleated septate junctions in stage 16 wild-type embryos similarly to Mcr.

In Cora 4 mutant embryos, Mcr failed to achieve strong apical localization by stage 16 and remained distributed along the lateral membrane. E-cadherin localization was similar in wild-type and Cora 4 mutant stage 16 embryos. In wild-type stage 17 embryos injected with rhodamine-labeled 10 kilodalton dextran, the dye surrounded the trachea but did not penetrate the lumen.

In Cora 4 mutant stage 17 embryos, the dye accumulated in the lumen of the trachea within 10 minutes of injection. This protocol allows researchers to confidently determine whether a gene encodes the core or the accessory component of the pleated septate junctions in the Drosophila embryonic tissues. Embryo staging is absolutely crucial while performing this technique, as this requires stage 16 embryos.

The septate junctions matures over time until it is fully established in stage 16. We believe this protocol can be effectively implemented in open-ended genetic screening using RNA interference lines to identify new pleated septate junction genes.