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.
Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a 'whole mount' 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).
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 'model' 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'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 'decapsulation', 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.
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 'CAUTION' 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.
1. Assembly of Decapsulation Apparatus
2. Sample Collection, Fixation and Decapsulation
NOTE: All steps are carried out at RT unless otherwise noted.
3. Proteinase-K, TEA and Post-fixation
NOTE: We find performing the following steps in small baskets with a mesh floor the most efficient and gentle method for quickly exchanging time critical solutions. While these can be home made, we use baskets (medium size) that are compatible with the Intavis InSituPro-Vsi liquid handling robot (www.intavis.de/products/automated-ish-and-ihc). Such baskets can be quickly and easily moved between the wells of a 12 well tissue culture dish (TCD) in order to exchange solutions, or the solution can be aspirated from the well using a pipette. Alternatively, all solution exchanges can be performed without these baskets by simply aspirating the supernatant from the larvae. In this case a gentle swirling motion will concentrate all embryos and larvae to the center of the dish allowing the supernatant to be removed from the edge of the well. The following assumes the user is employing baskets for solution exchanges.
4. Hot Washes and Immunodetection
NOTE: While we use a liquid handling robot for the following steps, these can also easily be done manually. In this case, embryos and larvae should be kept in the 1.5 ml tubes they were hybridized in. All subsequent solution exchanges are aspirated and added with a P1,000 pipette. When performed manually each of the following steps should employ 1 ml of each solution.
5. Color Development and Mounting
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 these genes we expect that they would also vary significantly, indicating that our method can be applied against a broad variety of gene products expressed in all stages of development at various levels. Only one of the genes presented here (Dpp) has been previously described in L. stagnalis 21,22. The results we present here are largely in keeping with these previous reports, but with significantly higher spatial resolution. The spatial expression pattern of Brachyury has been described in abalone 23 and limpet 24 and in both cases was also detected in mantle cells as we find for L. stagnalis (Figure 3F). We isolated Novel genes 1 – 4 (from a proteomic screen designed to identify gene products directly involved in shell formation, and so their spatial expression patterns associated with the shell gland (Figure 3A and B) or shell field (Figure 3C and D) are completely congruent with shell-forming functions. These results indicate that the high throughput technique we have developed for removing embryos and larvae from the egg capsule, and the subsequent stage-specific permeabilization treatments, generate whole mount samples that will yield high quality in situ staining patterns for a wide variety of genes for all stages of embryonic and larval development.
Figure 1. A Summarized Ontogeny of Lymnaea stagnalis and Corresponding Fixation and Proteinase-K Treatments. Representative images of embryos and larvae from the first 7 days of development illustrate a significant increase in size (row 1) and morphological complexity (rows 2 and 3). These developmental changes translate into large differences in the appropriate fixation time and Proteinase-K concentrations that generate optimal WMISH signals. For WMISH all stages should be fixed in 3.7% formaldehyde in PBS at RT with gentle agitation within their egg capsules. All stages should then be treated with the appropriate Proteinase-K concentration for 10 min. Note that we observe significant inter-batch variation in the activity of Proteinase-K from our supplier. This variation must be accounted for by performing a round of 'calibrating' WMISH experiments where the activity of the new Proteinase-K is empirically determined. The Proteinase-K concentrations stated in the figure should therefore be treated as an initial guide, however the relative concentrations between developmental stages (for example 2 day old embryos require a Proteinase-K concentration 3 times higher than day 1 embryos) are set. Please click here to view a larger version of this figure.
Figure 2. Apparatus Used to Decapsule L. stagnalis Embryos. (A) An overview of the apparatus that can efficiently remove L. stagnalis embryos and larvae from their capsules. (B) A magnified view of the yellow boxed area in (A). A sharp glass needle is placed on the microscope slide and inserted into the silicon tubing (inner diameter 1 mm, outer diameter 3 mm) such that the tip protrudes approximately 20% of the way into the cavity of the tubing. The glass needle is then also taped to the microscope slide and the Petri dish. (C) A schematic 'plan' view of the yellow boxed section in A. Egg capsules containing fixed embryos and larvae are first collected using the 20 ml syringe. The syringe is then attached to the silicon tubing and the capsules expelled through the tubing and past the needle. Egg capsule membranes are torn by the needle and the liberated embryonic and larval material can be collected from the collection dish using a micropipette. (D) A schematic 'cross section' view of the yellow boxed section in A. The microscope slide ensures that the needle enters the silicon tubing at the correct height. (E) A representative view of larvae prior to being processed by the decapsuling apparatus. (F) A representative view of larvae that have made a single pass through the apparatus. More than 90% of the material has been effectively and gently removed from their capsules. Please click here to view a larger version of this figure.
Figure 3. Representative Images of WMISH Expression Patterns Against a Variety of Genes From a Range of L. stagnalis Developmental Stages Generated by the Method Described Here. All developmental stages were processed as described in the above method and have been mounted and imaged in BB:BA (Murray's clear). Approximate ages are indicated in the top right of each panel and the orientation is indicated in the lower right. Gene orthology (when known) is indicated in the lower left of each panel. Abbreviations: shell gland (sg); shell field (sf); mantle (mt); foot (ft); Decapentaplegic (Dpp); dpfc (days post first cleavage). All scale bars are 20 µm. Please click here to view a larger version of this figure.
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 embryos and larvae. Following decapsulation, the material can either be stored, or treated with a stage specific regimen of Proteinase-K and then hybridized to a riboprobe. Additional empirical optimization efforts (typically focused on probe concentration and hybridization temperature) may be required for each probe/target. These parameters (in addition to the fixation regimen and Proteinase-K treatments) are typically the most influential parameters of any in situ experiment (assuming that the quality of the fixed material and the RNA probe are of a high standard).
The importance of an appropriate Proteinase-K treatment to the final result of an in situ experiment is paramount for L. stagnalis. This is reflected in the wide range of Proteinase-K concentrations required by distinct developmental stages (ranging from 0 µg/ml to 500 µg/ml). It is therefore important to be able to assign a given egg string to an ontogenetic window. To this end, the guideline that we provide in Figure 1 allows for the staging of developmental material of unknown ages (a print friendly version of this figure is available in Supplementary File 3 that users may find useful to have at the microscope when staging material). We note that for other species of gastropods Proteinase-K treatments for WMISH can either be kept constant for a wide range of developmental stages 8,25,26, or can be omitted entirely 27. This is in stark contrast to the situation in L. stagnalis. Furthermore, while other research groups have previously reported WMISH expression patterns for several genes in L. stagnalis larvae (see 22,28,29) the method that we describe here yields patterns of significantly higher spatial resolution. Finally, we have observed significant inter-batch variation in the activity of the Proteinase-K from our supplier. This variation must be accounted for by performing a round of 'calibrating' WMISH experiments where the activity of the new Proteinase-K is empirically determined. All subsequent experiments with aliquots of Proteinase-K from that batch can then be freely performed.
We previously described an alternative WMISH method for L. stagnalis embryos and larvae elsewhere 12. That method detailed the use of the mucolytic agent N-acetyl-L-cysteine (NAC), a reducing agent such as Dithiothreitol (DTT) and a pre-hybridization treatment with sodium dodecyl sulfate (SDS). We found those treatments enhanced the staining patterns of some genes for some developmental stages. The fixation strategy that we recently developed and describe here (fixing larvae within their capsules) simplifies and expedites the steps required to prepare material for an in situ experiment, and apparently negates the need for empirically determining additional optimal pre-hybridization treatments with NAC, DTT or SDS. Future refinements to the technique reported here could include the visualization of microRNAs (following modifications to standard WMISH protocols previously reported 30), double or triple labeling of mRNA targets 31, and fluorescent visualization of WMISH signals 32. Arguably the greatest limitation of the technique is the overall length of time it takes to go from collecting the material, to a digital image that represents a given gene expression pattern. Due to the nature of the biochemical and biophysical events that must take place during such a process this is an inherent feature of most in situ hybridization protocols.
Lymnaea occupies a position within the Metazoa that is extremely under-represented in terms of model organisms. As a representative Spiralian, Lymnaea can bring insight into the evolution of distinct morphological features such as shell formation 12 and body handedness 33-35 and is also a valuable neuroethology 36 and neurophysiology model 9,37. Powerful techniques such as the ability to efficiently visualize gene expression patterns in situ increases the functionality of Lymnaea as a model organism, and broadens the variety of questions that it can be used to address. At a time when the generation of large sequence datasets (complete transcriptomes and even genomes) is relatively routine, such methods will become more relevant to researchers wishing to interpret the flood of sequence data from such models. While Lymnaea is a relatively derived gastropod 38, and possesses what would be considered a large genome in comparison to other model organisms (1.22 Gb 39), it has many practical and interesting features that make it an attractive model system. The methods that we describe here expand the toolbox available to Lymnaea and may be of use to other species that undergo encapsulated development.
The authors have nothing to disclose.
This work was supported by funding to DJJ through DFG project #JA2108/2-1.
Featherweight forceps | Ehlert & Partner | #4181119 | |
Silicon tubing | Glasgerätebau OCHS GmbH | 760070 | |
Glass capillaries | Hilgenberg | 1403547 | |
12 well tissue culture dishes | Carl Roth | CE55.1 | |
37% Formaldehyde | Carl Roth | P733.1 | 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. |
Ethylenediamine tetraacetic acid | Carl Roth | CN06.3 | 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 |
Magnesium Chloride | Carl Roth | 2189.1 | |
Tween-20 | Carl Roth | 9127.1 | 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. |
Sodium Chloride | Carl Roth | 3957.1 | |
Ficoll type 400 | Carl Roth | CN90.1 | |
polyvinylpyrrolidone K30 (MW 40) | Carl Roth | 4607.1 | 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. |
Nuclease freeBovine Serum Albumin | Carl Roth | 8895.1 | |
Salmon sperm | Carl Roth | 5434.2 | |
Heparin | Carl Roth | 7692.1 | 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 |
Proteinase-K | Carl Roth | 7528.1 | |
Glycine | Carl Roth | 3790.2 | |
Deionised formamide | Carl Roth | P040.1 | 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. |
Standard formamide | Carl Roth | 6749.3 | 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. |
Triethanolamine | Carl Roth | 6300.1 | CAUTION – Avoid breathing vapor or mist. Avoid contact with eyes. Avoid prolonged or repeated contact with skin. Wash thoroughly after handling. |
Acetic anhydride | Carl Roth | 4483.1 | CAUTION – CAUSES SEVERE SKIN AND EYE BURNS. REACTS VIOLENTLY WITH WATER. HARMFUL IF SWALLOWED. VAPOR IRRITATING TO THE EYES AND RESPIRATORY TRACT |
Maleic acid | Carl Roth | K304.2 | 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. |
Benzyl benzoate | Sigma | B6630-250ML | 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. |
Benzyl alcohol | Sigma | 10,800-6 | CAUTION – Harmful if swallowed. Harmful if inhaled. Causes serious eye irritation. |
Glycerol | Carl Roth | 3783.1 | |
Blocking powder | Roche | 11096176001 | |
Anti DIG Fab fragments AP conjugated | Roche | 11093274910 | |
Tris-HCl | Carl Roth | 9090.3 | |
4-Nitro blue tetrazolium chloride in dimethylformamide | Carl Roth | 4421.3 | CAUTION – May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes. |
5-bromo-4-chloro-3-indolyl-phosphate | Carl Roth | A155.3 | CAUTION – Potentially harmful if ingested. Do not get on skin, in eyes, or on clothing. Potential skin and eye irritant. |
N-acetyl cysteine | Carl Roth | 4126.1 | |
Dithiothreitol | Carl Roth | 6908.1 | 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. |
Tergitol | Sigma | NP40S | 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. |
Sodium dodecyl sulphate | Carl Roth | CN30.3 | CAUTION – Harmful if swallowed. Toxic in contact with skin. Causes skin irritation. Causes serious eye damage. May cause respiratory irritation. |
Potassium Chloride | Carl Roth | 6781.1 | |
di-Sodium hydrogen phosphate dihydrate (Na2HPO4.2H2O) | Carl Roth | 4984.1 | |
Potassium dihydrogen phosphate (KH2PO4) | Carl Roth | 3904.1 | |
Tri sodium citrate dihydrate (C6H5Na3O7.2H2O) | Carl Roth | 3580.1 | CAUTION – May cause eye, skin, and respiratory tract irritation. The toxicological properties of this material have not been fully investigated. |
Mineral oil | Carl Roth | HP50.2 | |
InSituPro-Vsi | Intavis | www.intavis.de/products/automated-ish-and-ihc |