Name: a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis
Uploaded: 2016-03-15T15:00:00
Description: Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

This work was supported by funding to DJJ through DFG project #JA2108/2-1.

NOTE: The following steps outline our method for conducting an in situ experiment on embryonic and larval stages of L. stagnalis. Where a step involves the use of a hazardous chemical this is indicated by the word &#39;CAUTION&#39; and all appropriate safety procedures should be adopted. Links to representative MSDS sheets for hazardous chemicals are provided in Supplementary File 1. Recipes for all reagents are provided in Supplementary File 2.
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<ol>
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The method described here allows for the efficient visualization of RNA transcripts with presumably varying expression levels within all developmental stages of Lymnaea stagnalis. To remove embryos and larvae from their capsules we trialed a variety of chemical, osmotic shock and physical treatments reported for other encapsulated-developing model organisms. However, in our hands the method we describe here is the only high-throughput technique that removes the tough capsular membrane without damaging the embryo...

Molluscs are a group of animals that hold the interest of a broad diversity of scientific disciplines. Despite their morphological diversity1, species richness (second only to the Arthropods in terms of species number2) and relevance to a wide range of commercial3, medical4 and scientific issues5-8, there are relatively few molluscan species that can claim to be both well-equipped scientific models and easy to maintain in a laboratory environment. One mollusc that is much used by disciplines such as neurobiology9, ecotoxicology10 and more recently evolutionary biology11,12, is Lymnaea stagnalis, primarily because of its widespread distribution and extreme ease of maintenance. Despite its popularity as a &#39;model&#39; organism and its long history of use by developmental biologists13-19, the range and power of molecular tools available to the L. stagnalis scientific community lies far behind that of more traditional animal models (Drosophila, mouse, sea urchin, nematodes).
Our desire to develop Lymnaea as a molecular model stems from an interest in the molecular mechanisms that guide shell formation. This motivated us to refine a set of techniques that would allow for the efficient, consistent and sensitive visualization of gene expression during Lymnaea&#39;s development. Whole mount in situ hybridization (WMISH) is widely employed for a variety of model organisms and has been in use for more than 40 years 20. In its different guises, ISH can be employed to spatially localize specific loci on chromosomes, rRNA, mRNA and micro-RNAs.
One of the challenges we needed to address prior to refining a WMISH method for L. stagnalis was the issue of gently and efficiently extracting embryos and larvae of varying stages from the egg capsules in which they are deposited. This extraction, or &#39;decapsulation&#39;, needs to be achieved efficiently in order to collect adequate material for a given in situ experiment, while at the same time maintaining morphological and cellular integrity. While other model organisms also undergo encapsulated development, in our hands none of the methods reported for those species could be successfully employed in L. stagnalis.
The overall goals of this method are therefore: to extract L. stagnalis embryos and larvae from their capsules in a high-throughput fashion, to apply pre-hybridization treatments that optimize the WMISH signal, to prepare embryos and larvae with satisfactory WMISHsignals for imaging.

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			<td height="21" style="height:21px;width:380px;">Featherweight forceps</td>
			<td style="width:173px;">Ehlert &#38; Partner</td>
			<td style="width:104px;">#4181119</td>
			<td style="width:1547px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Silicon tubing</td>
			<td>Glasger&#228;tebau OCHS GmbH</td>
			<td>760070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillaries</td>
			<td>Hilgenberg</td>
			<td>1403547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 well tissue culture dishes</td>
			<td>Carl Roth</td>
			<td>CE55.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37% Formaldehyde</td>
			<td>Carl Roth</td>
			<td>P733.1</td>
			<td>CAUTION - May cause cancer. Toxic by inhalation, in contact with skin and if swallowed. Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediamine tetraacetic acid</td>
			<td>Carl Roth</td>
			<td>CN06.3</td>
			<td>CAUTION - CAUSES EYE IRRITATION. MAY CAUSE RESPIRATORY TRACT AND SKIN IRRITATION. Avoid breathing dust. Avoid contact with eyes, skin and clothing. Use only with adequate ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td>Carl Roth</td>
			<td>2189.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Carl Roth</td>
			<td>9127.1</td>
			<td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Carl Roth</td>
			<td>3957.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll type 400</td>
			<td>Carl Roth</td>
			<td>CN90.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyvinylpyrrolidone K30 (MW 40)</td>
			<td>Carl Roth</td>
			<td>4607.1</td>
			<td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease freeBovine Serum Albumin</td>
			<td>Carl Roth</td>
			<td>8895.1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Salmon sperm</td>
			<td>Carl Roth</td>
			<td>5434.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Carl Roth</td>
			<td>7692.1</td>
			<td>CAUTION - ADVERSE EFFECTS INCLUDE HEMORRHAGE, LOCAL IRRITATION. POSSIBLE ALLERGIC REACTION IF INHALED, INGESTED/CONTACTED. EYES/SKIN/RESPIRATORY TRACT IRRITANT. POSSIBLE HYPERSENSITIZATION. DURING PREGNANCY HAS BEEN REPORTED TO INCREASE RISK OF STILLBIRTH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase-K</td>
			<td>Carl Roth</td>
			<td>7528.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Carl Roth</td>
			<td>3790.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionised formamide</td>
			<td>Carl Roth</td>
			<td>P040.1</td>
			<td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard formamide</td>
			<td>Carl Roth</td>
			<td>6749.3</td>
			<td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethanolamine</td>
			<td>Carl Roth</td>
			<td>6300.1</td>
			<td>CAUTION - Avoid breathing vapor or mist. Avoid contact with eyes. Avoid prolonged or repeated contact with skin. Wash thoroughly after handling.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic anhydride</td>
			<td>Carl Roth</td>
			<td>4483.1</td>
			<td>CAUTION - CAUSES SEVERE SKIN AND EYE BURNS. REACTS VIOLENTLY WITH WATER. HARMFUL IF SWALLOWED. VAPOR IRRITATING TO THE EYES AND RESPIRATORY TRACT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maleic acid</td>
			<td>Carl Roth</td>
			<td>K304.2</td>
			<td>CAUTION - Very hazardous in case of eye contact (irritant), of ingestion, . Hazardous in case of skin contact (irritant), of inhalation (lung irritant). Slightly hazardous in case of skin contact (permeator). Corrosive to eyes and skin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma</td>
			<td>B6630-250ML</td>
			<td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. Harmful if swallowed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma</td>
			<td>10,800-6</td>
			<td>CAUTION - Harmful if swallowed. Harmful if inhaled. Causes serious eye irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Carl Roth</td>
			<td>3783.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blocking powder</td>
			<td>Roche</td>
			<td>11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti DIG Fab fragments AP conjugated</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Carl Roth</td>
			<td>9090.3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Nitro blue tetrazolium chloride in dimethylformamide&#160;</td>
			<td>Carl Roth</td>
			<td>4421.3</td>
			<td>CAUTION - May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-bromo-4-chloro-3-indolyl-phosphate</td>
			<td>Carl Roth</td>
			<td>A155.3</td>
			<td>CAUTION - Potentially harmful if ingested. Do not get on skin, in eyes, or on clothing. Potential skin and eye irritant.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-acetyl cysteine</td>
			<td>Carl Roth</td>
			<td>4126.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol</td>
			<td>Carl Roth</td>
			<td>6908.1</td>
			<td>CAUTION - May cause eye and skin irritation. May cause respiratory and digestive tract irritation. The toxicological properties of this material have not been fully investigated.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tergitol</td>
			<td>Sigma</td>
			<td>NP40S</td>
			<td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Sodium dodecyl sulphate</td>
			<td>Carl Roth</td>
			<td>CN30.3</td>
			<td>CAUTION - Harmful if swallowed. Toxic in contact with skin. Causes skin irritation. Causes serious eye damage. May cause respiratory irritation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Carl Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">di-Sodium hydrogen phosphate dihydrate (Na2HPO4.2H2O)</td>
			<td>Carl Roth</td>
			<td>4984.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Carl Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Tri sodium citrate dihydrate (C6H5Na3O7.2H2O)</td>
			<td>Carl Roth</td>
			<td>3580.1</td>
			<td>CAUTION - May cause eye, skin, and respiratory tract irritation. The toxicological properties of this material have not been fully investigated.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Mineral oil&#160;</td>
			<td>Carl Roth</td>
			<td style="width:104px;">HP50.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">InSituPro-Vsi&#160;</td>
			<td style="width:173px;">Intavis</td>
			<td style="width:104px;"></td>
			<td>www.intavis.de/products/automated-ish-and-ihc</td>
		</tr>
	</tbody>
</table>

a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

The representative WMISH staining patterns shown in Figure 3 were generated using the technique described above, and reflect a variety of spatial expression patterns for genes involved in a range of molecular processes ranging from shell formation (Novel gene 1, 2, 3 and 4), to cell-cell signaling (Dpp) to transcription regulation (Brachyury) across a range of developmental stages. While we have not quantified the expression levels of t...

Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

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.

Research

JoVE Journal

Biology

Double Whole Mount in situ Hybridization of Early Chick Embryos

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Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...