The overall goal of this protocol is to efficiently visualize the spatial expression patterns of individual genes in embryonic and larval whole mounts of the freshwater gastropod Lymnaea stagnalis. This method can help answer key questions in the fields of Evo-Divo and developmental biology such as where and when genes are expressed, how they interact with each other and what their punitive functions may be. The main advantage of this technique is that the removal of embryonic and larvae material from their egg capsules is quick, easy and effective.

This material is then categorized according to a developmental road map that we provide in the supplementary material and then process through stage-specific pre-hybridization steps. To begin this procedure, connect a 20-milliliter disposal syringe to a silicone tubing. Next, tape a standard microscope slide to a large inverted Petri dish.

Then, tape the silicone tubing immediately adjacent to the microscope slide. After that, place a pulled glass needle on the microscope slide. And gently insert it into the silicone tubing until the tip of the needle protrudes approximately 20%of the inner diameter of the tubing.

Once the needle is in position, tape it down to the Petri dish. Allow the free end of the silicone tubing to rest in another Petri dish that will collect the decapsulated material. To collect the egg strains from the walls of an aquarium, use a piece of flat, flexible plastic as a spatula to scrape the egg strain off the substrate.

And use a plastic tea strainer to fish the floating egg strain out of the water. After that, stage and sort the material under the microscope according to this guide. Place the egg strain onto a paper towel and make a longitudinal incision along the egg mass using the featherweight forceps.

Then, roll the egg capsules out of the egg strain and remove as much of the jelly material as possible from each capsule by pushing it around the paper towel. Next, using the featherweight forceps, transfer the egg capsules into a Petri dish containing 5 milliliters of tap water. Continue to collect de-jellied egg capsules of the desired developmental stages into this dish.

Collect enough capsules from all developmental stages for the planned WMISH experiment before proceeding to the next step. Now, relax larvae older than five days post first cleavage in a 2%weight-to-volume solution of magnesium chloride hexahydrate for 30 minutes prior to fixation. After 30 minutes, assess the degree of relaxation by submerging several larvae while still in their egg capsules and the fixative solution and monitoring their response under a microscope.

Incompletely relaxed larvae will retract into their shells. While fully relaxed larvae will not respond. Once these larvae have been relaxed, transfer the egg capsules using a wide bore plastic pipette into a sealable tube that provides 10 times the volume of egg capsules.

Next, aspirate as much liquid as possible from the tube and replace it with the fixative solution that is 10 times the volume of the settled egg capsules. Gently rotate the egg capsules in fixative at room temperature for the appropriate duration for each developmental stage. Then, discontinue the rotation and allow the capsules to sink.

Aspirate the fixative solution into an appropriate waste container. Wash the egg capsules by replacing the fixative solution with PBTw. And rotating them at room temperature for five minutes.

Aspirate the PBTw and wash two more times. After that, remove the embryos and larvae from their capsules using the apparatus. Draw the capsules into a 20-milliliter syringe.

Then attach the syringe to the tubing. Dispel the capsules through the tubing and glass needle into the collection dish. Afterward, collect the decapsulated embryos and larvae into a 1.5-milliliter tube using a micro pipette.

Subsequently, either dehydrate and store the fixed material or perform the Proteinase K, TEA and post-fixation steps. Once these steps are completed, briefly pre-hybridize the material and add the riboprobes. The importance of an appropriate Pro K treatment to the final results of an in situ experiement is paramount for Lymnaea stagnalis.

This is reflected in a wide range of Pro K concentrations required by distinct developmental stages ranging from 0 to 500 micrograms to mil. During hybridization, remove a single tube from the heat block. Rotate it rapidly between the thumb and forefinger to suspend the larvae without disturbing the oil phase, and replace it in the heat block.

Repeat this procedure once every six to 12 hours. The gene expression patterns shown here were generated using larvae from a range of developmental stages. And all samples were mounted and imaged in Murray's Clear.

Approximate ages are indicated in the top right of each image. The orientation of the view is indicated in the lower right, and gene orthology is indicated in the lower left. These results illustrate the expression patterns of genes involved in a variety of molecular processes ranging from shell formation to transcription to cell-to-cell signaling.

In all cases, a unique and clear cellular staining pattern is visible, and the results are highly consistent among individual larvae within an experiment, and highly reproducible between experiments. Once mastered, the total hands-on time of this technique requires less than two days. Most of the time is spent on collecting and preparing embryonic and larvae material for hybridization and collecting the images of the results.

While attempting this procedure, it's important to remember that every riboprobe will likely require subtle adjustments to important parameters such as the hybridization temperature, probe concentration and the duration of hybridization. After it's development, this technique allowed us to fairly characterize a suite of genes involved in shell formation that have been identified by previous transcriptomic and proteomic screens.