By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.
Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene’s message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ‘s provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ‘s and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ‘s and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ‘s hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.
I. RNA Whole Mount In situ Hybridization in Marine Elasmobranchs
1. Embryo Fixation and Preparation
2. Synthesis of RNA Probe
3. Embryo Pre-treatment and Hybridization
4. Post Hybridization Washes and Antibody Hybridization
5. Post Antibody Hybridization Washes
6. Detection of Probe
7. Representative Results for I. RNA Whole Mount in situ hybridization in marine elasmobranchs
RNA whole mount in situ‘s depicting expression of Sonic hedgehog (Shh) and Hoxa13 in skate embryos are shown in Figure 1. Expression of Shh in higher vertebrates is found in the notochord and gut endoderm and this expression pattern is conserved in the skate (Figure 1a) 8,9 . Marine elasmobranchs have a unique method of osmoregulation that uses the rectal gland to secrete salts. Hoxa13 expression is high in the developing rectal gland (Figure 1b) 10. Hoxa13 gene product role in patterning the rectal gland remains unknown.
II. Paraffin Embedding and Sectioning Elasmobranch Tissue
1. Harvest and Preparation of Tissue
2. Paraffin Embedding and Sectioning
III. Alcian Blue / Nuclear Fast Red Stain of Elasmobranch Tissue
1. Alcian Blue Stain for Mucins
Examples of alcian blue staining in different regions of the L. erinacea digestive tract are shown in Figure 2. Acid mucin containing globlet cells are clearly visible by the alcian blue stain throughout the digestive tract. The distribution of acid mucins differs in the different regions of the digestive tract, thus reflecting differences in function. Acidic mucins are sparsely produced in the spiral intestine and cloaca, while a high concentration of acid mucins are detected in the distal intestine (compare Figure 2a and 2c with 2b).
PBT | 1 x PBS |
0.1% Tween-20 | |
Filter-sterilize in Nalgene-type tissue culture filter units. | |
20 x SSC, pH 4.5 | 3 M NaCl |
0.3 M NaCitrate | |
Adjust pH with citric acid. | |
Hybridization solution | 50% formamide |
1.3 x SSC, pH 4.5 | |
5 mM EDTA, pH 8 | |
50 mg/ml tRNA | |
0.2% Tween-20 | |
0.5% Chaps | |
100 mg/ml heparin | |
Aliquot and store at -20 °C for up to one year. | |
Proteinase K | 10 mg/ml stock solution |
20 μl aliquots into 0.5 μl Eppendorf-like tubes and store at -20 °C. | |
Solution #1 | 50% formamide |
1.3 x SSC, pH 4.5 | |
0.2% Tween-20 | |
Store at -20 °C | |
Solution #2 | 50% formamide |
1 x SSC, pH 4.5 | |
0.2% Tween-20 | |
Store at -20 °C | |
Sheep serum | Inactivate by heating to 55 °C for 1 hr, and store in aliquots at -20 °C. |
10 x TBS | 80 g NaCl |
2 g KCl | |
250 ml, 1 M Tris-HCl, pH 7.5 | |
Add water to 1 L | |
1 x TBST | 1 x TBS |
0.1% Tween-20 | |
NTMT | 100 mM NaCl |
100 mM Tris-HCl, pH 9.5 | |
50 mM MgCl2 | |
0.1% Tween-20 | |
Make approximately 100 ml fresh before using and filter. | |
200 x NBT stock | 50 mg/ml NBT in 70% dimethyl formamide |
Store in -20 °C in 1 ml aliquots. | |
200 x BCIP stock | 25 mg/ml BCIP in water |
Store in -20 °C in 1 ml aliquots. |
Table 1. RNA whole mount in situ solutions.
Linearized plasmid | 8 μl |
10 x transcription buffer | 4 μl |
DIG-UTP nucleotide mix | 2 μl |
RNAse inhibitor | 0.5 μl |
RNA polymerase (SP6, T3 or T7) | 1 μl |
RNAse-free sterile H20 | 4.5 μl |
Total volume | 20 μl |
Table 2. Transcription reaction to generate RNA probe.
Figure 1. Shh and Hoxa13 expression in Leucoraja erinacea embryos are visualized by RNA whole mount in situ hybridization. (a) Shh expression is detected in a stage 29 skate embryo. (a’) Higher magnification of (a) reveals Shh expression in the notochord (arrowheads) and gut endoderm (arrows). (b) Hoxa13 expression is localized to the rectal gland (arrow) of a stage 29 skate embryo. (b’) Dissected digestive tract from the embryo in (b) demonstrates the specificity of Hoxa13 transcript expression to the rectal gland. e, endoderm; n, notochord; rg, rectal gland. Click here to view larger figure.
Figure 2. Distribution of acidic mucin-producing goblet cells in the skate digestive tract. (a, c) Low numbers of acidic mucin-producing goblet cells are detected by the alcian blue stain in the spiral intestine and cloaca, respectively (arrows). (b) The distal intestine contains a higher density of acidic mucin goblet cells (arrows). In all panels, nuclei are clearly visible by the nuclear fast red stain. Click here to view larger figure.
The protocols presented are classic methods for monitoring gene expression and identifying differentiated cell types, and have been adapted for use in marine elasmobranchs. Further modifications of these protocols may be needed to adapt to different elasmobranch species.
The most common concern regarding RNA whole mount in situ‘s is the risk of RNase contamination and thereby the degradation of the RNA probe and endogenous messages. Two aspects need to be considered: the synthesis of the riboprobe and its hybridization to embryonic messages, and general techniques for handling RNA. In general, contamination by ribonucleases can be avoided by using plasticware from unopened containers, and previously unused glassware. If new glassware is unavailable, treat all glassware with RNase-AWAY (VWR#17810-491). Washes are done by gently pouring off solutions through a perforated spoon and adding fresh solution to the vial. Alternatively, very small embryos or organs/tissues can be placed in a Netwell that fits into a 6-well tissue culture dish. The tissue can be moved from one solution to the next by simply lifting the Netwell from one well to the next. This helps prevent the loss of any tissue by pouring, and also preserves the architecture of the tissue. Additional suggestions for handling RNA and preventing RNase contamination can be found in Molecular Cloning; A Laboratory Manual, and on several pharmaceutical websites, including Roche (http://www.roche-applied-science.com/labfaqs/p2_1.htm) 11.
Generation of the RNA probe and pre-treatment and hybridization of embryos are the most vulnerable steps to contamination by ribonucleases. Antisense RNA probes that complement native mRNA transcript are synthesized by in vitro reverse transcription in the presence of digoxigenin-UTP. Briefly, cDNA plasmids are linearized with a unique restriction enzyme whose site is located at the 5′ end of the gene. This allows for the RNA polymerase to fall off at the end of the gene. Choose a RNA polymerase by identifying the RNA polymerase promoter available in the plasmid, just 3′ to the stop codon. While T3 and T7 RNA polymerases are often used for pBluescript, SP6 is the choice for genes subcloned into the directional pGEM plasmids. In addition to RNase inhibitor, DEPC-treated water is used in the transcription reaction and purification of the probe. Hybridization solution and steps using PBT in the pre-treatment of embryos should all be made with DEPC water. To DEPC treat water, add 1 ml of DEPC to 1 L of water in a large flask and mix well. Leave overnight in a hood and autoclave the next day. Solutions such as 10 x PBS and SSC may also be made with DEPC-treated water. After hybridization with the riboprobe, the use of RNase-free solutions and similar precautions are no longer necessary.
Additional variables worth considering are the concentration and length of proteinase K treatment, which can affect the penetration of the probe. A careful titration of the proteinase K stock is recommended to optimize treatment time for different embryonic stages. The protocol described here has been routinely and successfully used with embryos at stages 20 – 31 according to Ballard 7. For embryos that are very young or for tissue that is particularly “sticky”, the detergent Tween-20 can be replaced with Triton-X. In addition, gene-specific modifications to the protocol may be needed depending on the abundance of transcript. To reduce background, 10% dimethylformamide has been routinely added to the development solution (NTMT) by several groups 12.
In contrast to the sensitive nature of RNA in situ‘s, the joy of histology is that it is quick and pretty foolproof! Any variability in staining is likely due to inadequate fixation. To ensure that tissue is fully fixed, it is recommended that very large tissues (such as the digestive tract of an adult animal) be dissected into smaller pieces and fresh fixative solution replaced every 10 hr over a 48 hr period. It may be necessary to optimize conditions for fixation, both under and over-fixed tissues can result in poor sectioning or uneven staining. In addition, different histochemical stains work optimally with different fixatives. Thus, it is worth modifying the fixative according to the histochemical stain used 13. 4% paraformaldehyde is recommended when fixing soft tissue organs or young embryos. For older embryos or whole animals, 10% formalin is preferred. Once tissue is paraffin embedded and sectioned, it may be used for multiple purposes, including RNA in situ‘s. This allows for resolution of gene expression on the cellular level. Alternatively, sections may be stained with different histochemical stains to identify differentiated cell types.
The alcian blue and nuclear fast red staining has been successfully performed in the skate (Leucoraja erinacea), dogfish (Squalas acanthias), hagfish (Myxine glutinosa) and lamprey (Petromyzon marinus), (unpublished results) 10,14 . In addition to the alcian blue pH 2.5 stain described here, modifying the pH of the alcian blue solution can distinguish different mucosaccharides (i.e. sialo- versus sulfomucins) 13. Different mucosaccharide constituents can distinguish goblet cells by function and localization within the digestive tract 15,16 . Alcian blue stain in the digestive tract has been used to diagnose conditions such as Barrett’s esophagus and to enhance staining of pancreatic beta-cell granules 17,18 .
In summary, histochemistry and RNA whole mount in situ‘s when used together can lead to a better understanding of the link between where a gene is expressed during development and the resulting differentiated cell type. Expression of posterior Hox genes has been linked to specification of acid mucin goblet cells in the development digestive tract 10,19 . Here I provide an example of the expression of Shh and a Hox gene along with the presence of acid mucin-producing goblet cells in the L. erinacea digestive tract. These techniques can be widely applied to different species of elasmobranchs, different developmental patterning genes and different histological stains. The continued application and adaptation of techniques to marine elasmobranchs is important to keep pace with the broad use of these animals for biomedical research models in physiology, endocrinology, toxicology and genomics.
The authors have nothing to disclose.
I wish to thank the many undergraduate students who have worked in my laboratory and contributed to the evolution of these protocols. NAT has received support from the Skidmore-Union Network, a project established with a NSF ADVANCE PAID grant.
Name of the reagent | Company | Catalogue number | Comments |
10 x transcription buffer | Roche | 11-465-384-001 | |
DIG-RNA labeling mix | Roche | 11-277-073-910 | |
RNAse inhibitor | Roche | 03-335-399-001 | |
RNA polymerase – SP6 | Roche | 10-810-274-001 | |
DNAseI, RNAse-free | Roche | 10-776-785-001 | |
Yeast RNA | Invitrogen | 15401-029 | |
CHAPS | EMD-Millipore | 220201 | |
heparin | Sigma-Aldrich | H4784 | |
DEPC (diethyl pyrocarbonate) | Research Organics | 2106D | |
Moria Perforated Spoon | Fine Science Tools | 10370-17 | |
Netwell inserts | Electron Microscopy Sciences | 64713-00 | Netwells for use in 6-well tissue culture dishes |
6-well tissue culture plate | Corning | 3516 | |
Glass scintillation vials with screw-cap lids | Weaton Science Products | 986540 | |
formamide | Fisher | BP227500 | |
Proteinase K | Invitrogen | 59895 (AM2542) | |
NBT | 11585029001 | ||
BCIP | Roche | 11585002001 | |
Hydrogen peroxide, 30% | EMD | HX0635-1 | |
Sheep serum | VWR | 101301-478 | |
glutaraldehyde | Sigma-Aldrich | G5882 | |
tRNA | Roche | 10-109-541-001 | |
Anti-DIG Fab Fragments | Roche | 1137-6623 | |
Table 3. Reagents and equipment for RNA whole mount in situ‘s. | |||
1% Alcian Blue 8GS, pH 2.5 | Electron Microscopy Sciences | 26323-01 | |
Nuclear Fast Red | Electron Microscopy Sciences | 26078-05 | |
DPX Mountant | Electron Microscopy Sciences | 13510 | |
Paraffin (Paraplast X-tra) | McCormick Scientific | 39503002 | |
10% Formalin, NBF | VWR | 95042-908 | |
Glass scintillation vials with screw-cap lids | Weaton Science Products | 986540 | |
Stainless steal base molds | Tissue-Tek | 4161-4165 | Multiple sizes available. |
Cassettes | Tissue-Tek | 4170 | |
Slide warmer | Fisher-Scientific | 12-594Q | |
Tissue Embedder | Leica Microsystems | EG1160 | |
Microtome, rotary | Leica Microsystems | RM2235 | |
Tissue-Tek Slide Staining Set | Electron Microscopy Sciences | 62540-01 | |
Tissue-Tek 24-Slide Holder | Electron Microscopy Sciences | 62543-06 | |
Superfrost*Plus slides | Fisherbrand | 12-550-17 | |
Table 4. Reagents and equipment for Alcian Blue stain. |