JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: April 12th, 2013

DOI:

10.3791/50165

1Department of Biological Sciences, Union College

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

  1. Skate embryos can be staged according to Ballard 7. Optimal stages for detection of gene expression depends on the tissue of interest. To track gene expression in the skate digestive tract, stages 27-30 are optimal 7.
  2. Dissect embryos into PBS, separating the embryos from the yolk sac.
  3. Fix in 30 ml of freshly made 4% paraformaldehyde (PF.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 dista.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 synthe.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
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.

  1. Forester, R., Goldstein, L. Intracellular osmoregulatory role of amino acids and urea in marine elasmobranchs. Am. J. Physiol. 230, 925-931 (1976).
  2. Shuttleworth, T., Shuttleworth, R. . Physiology of elasmobranch fishes. , 171-194 (1988).
  3. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., Somero, G. N. Living with water stress: evolution of osmolyte systems. Science. 217, 1214-1222 (1982).
  4. Ballatori, N., Villalobos, A. Defining the molecular and cellular basis of toxicity using comparative models. Toxicl. Appl. Pharmacol. 183, 207-220 (2002).
  5. Nieto, M. A., Patel, K., Wilkinson, D. G. In situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol. 51, 219-235 (1996).
  6. Jass, J. R., Walsh, M. D. Altered mucin expression in the gastrointestinal tract: a review. J Cell Mol Med. 5, 327-351 (2001).
  7. Ballard, W. W., Mellinger, J., Lechenault, H. A series of normal stages for development of Scyliorhinus canicula, the lesser spotted dogfish (Chondrichthyes; Scyliorhinidae). J. Exp. Zool. 267, 318-336 (1993).
  8. Echelard, Y., et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell. 75, 1417-1430 (1993).
  9. Riddle, R. D., Johnson, R. L., Laufer, E., Tabin, C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 75, 1401-1416 (1993).
  10. Theodosiou, N. A., Hall, D. A., Jowdry, A. L. Comparison of acid mucin goblet cell distribution and Hox13 expression patterns in the developing vertebrate digestive tract. J. Exp. Zool. B. Mol. Dev. Evol. 308, 442-453 (2007).
  11. Sambrook, J., Russell, D. W. Ch. 7. Molecular Cloning; A Laboratory Manual. 1, 7.82 (2001).
  12. Zhang, G., Miyamoto, M. M., Cohn, M. J. Lamprey type II collagen and Sox9 reveal an ancient origin of the vertebrate collagenous skeleton. Proc. Natl. Acad. Sci. U.S.A. 103, 3180-3185 (2006).
  13. Sheehan, D. C., Hrapchak, B. B. . Theory and practice of histotechnology. , (1980).
  14. Theodosiou, N. A., Simeone, A. Evidence of a rudimentary colon in the elasmobranch, Leucoraja erinacea. Dev. Genes Evol. 222, 237-243 (2012).
  15. Filipe, M. I. Mucins in the human gastrointestinal epithelium: a review. Invest. Cell Pathol. 2, 195-216 (1979).
  16. Corfield, A. P., Wagner, S. A., Clamp, J. R., Kriaris, M. S., Hoskins, L. C. Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria. Infect. Immun. 60, 3971-3978 (1992).
  17. Reid, B. J., et al. Flow-cytometric and histological progression to malignancy in Barrett's esophagus: prospective endoscopic surveillance of a cohort. Gastroenterology. 102, 1212-1219 (1992).
  18. Mowry, R. Selective staining of pancreatic beta-cell granules. Evolution and present status. Arch. Pathol. Lab Med. 107, 464-468 (1983).
  19. Roberts, D. J., Smith, D. M., Goff, D. J., Tabin, C. J. Epithelial-mesenchymal signaling during the regionalization of the chick gut. Development. 125, 2791-2801 (1998).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved