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1Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College of Medicine
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We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
Schultz, R. D., Gumienny, T. L. Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI. J. Vis. Exp. (59), e3362, doi:10.3791/3362 (2012).
The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.
1. Preparation of DiI stain
2. Preparation of nematodes
3. Mounting and observing specimens
4. Representative Results
DiI stains the cuticle of wild-type and mutant C. elegans. The cuticular surface contains annuli separated by circumferential furrows and, in some stages, longitudinal ridges called alae. Each developmental stage has cuticular structures with distinct compositions2. Ridges or furrows of both alae and annuli fluorescently stain, depending on surface composition, throughout larval and adult stages and remain visible up to a day after recovery using this method. Background fluorescent speckles are sometimes observed (Figures 2F, G), but not routinely (Figure 1, Figure 2A-E, H, I). All images were taken with spinning disk confocal or, when noted, widefield (wf) compound microscopy.
Figure 1. DiI stains cuticle and environmentally exposed neurons. L2 stage animal. 630x magnification Mosaic image parts were captured using iVision-Mac software (BioVision Technologies, Exton, PA). Images were joined using Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). Scale bar = 10 μm. DiI also fluorescently stains amphid and phasmid sensory neurons in the head and tail respectively (arrows mark some).
Figure 2. DiI fluorescently stains C. elegans cuticle at all stages of post-embryonic development. 630x magnification. Scale bar = 10 μm. Staining of wild-type animals in A) L1; B) L2; C) dauer; D) L3; E) L4; and F) adult stages. The alae and annular ridges are fluorescently stained in L1 and dauer animals (A, C). DiI stains annular ridges in L2 animals (B). Annular furrows stain in L3 and L4 animals (D, E). The furrows of the alae and annuli are stained in adult animals (F-H). Alae are composed of two, five, or three ridges (in L1, dauer, or adult animals, respectively) that run the length of the animal (arrows)16. Annuli create circumferential ridges around the animal (arrowheads, F and J). The cuticle of adult mutant animals display moderate cuticular organization defects (G-H). G) Ridges of alae are discontinuous (wf). H) Supernumerary alae ridges are fused and branched or bifurcated (wf). Collagen gene mutants exhibit alae and annular organization defects (I-J). I) In transgenic animals overexpressing pRF4 (rol-6(su1006)), ridges of alae lie at an angle to the length of the animal. J) Annuli in transgenic animals overexpressing pRF4 (rol-6(su1006)) display an irregular pattern.
Figure 3. External morphological structures are illuminated by DiI staining. 630x magnification. Scale bars = 10 μm. DiI also highlights other exterior features, including A) adult hermaphrodite vulva, B) adult male tail rays and fan, and C) hermaphrodite tail spike (forked in this mutant background).
Figure 4. Cuticle takes longer to stain with DiI than environmentally exposed neurons. 630x magnification. Scale bar = 10 μm. After two hours of staining, amphid (not shown) and phasmid sensory neurons (arrows) are sufficiently stained. In contrast, the cuticle of younger animals is only partially stained in patches (arrowhead).
|Wash||Stain solution (+ DiI)||Incubation time||Cuticle stained|
|M9 + 0.5% Triton X-100||M9 + 0.5% Triton X-100||2 hrs||no|
|M9 + 0.5% Triton X-100||M9 + 0.5% Triton X-100||3 hrs||no|
|M9 + 0.5% Triton X-100||M9||2 hrs||partial|
|M9 + 0.5% Triton X-100||M9||3 hrs||yes|
|M9 + 0.5% Triton X-100||H20||2 hrs||partial|
|M9 + 0.5% Triton X-100||H20||3 hrs||yes|
Table 1. Cuticular staining under different conditions. Various incubation solutions and times were tested to optimize cuticular staining in animals. H20, sterile distilled water. Partial staining indicates patchy staining of larval cuticle (Figure 4), though adult cuticle stains consistently.
The DiI staining method presented here allows for a relatively quick and convenient way to visualize the cuticle in C. elegans. By repurposing and optimizing a method commonly used to image environmentally exposed sensory neurons15,17, DiI can be used to fluorescently stain both alae and annular structures (Figures 1 and 2), as well as the vulva, male tail, and hermaphrodite tail spike (Figure 3). We have found that the incubation solution and time influence the ability of DiI to consistently stain the cuticle (Table 1, Figure 4). The method for DiI staining environmentally exposed neurons uses a two-hour incubation time in M9 with Triton X-10015. An initial wash of M9 with the surfactant Triton X-100 helps remove contamination from the cuticular surface and prevents the animals from clumping. However, incubating the animals three hours in a staining solution in the same buffer prevents the dye from staining the cuticle (Table 1). This may be caused by lipids on the nematode surface being stripped off by the longer treatment in the detergent solution. Incubation of animals in water with DiI stains the cuticle, indicating that the M9 salt solution is not required for DiI staining of cuticle. Although a water-based protocol is recommended for neuronal DiI staining15, we find that animals treated this way often appear unhealthy. Washing animals briefly in 0.5% Triton X-100 in M9, followed by a three-hour incubation in staining solution made with M9, provides consistent staining of cuticular structures and maintains the well-being of the animals.
Animals can be rescued after observation and maintained, permitting direct downstream analyses. This method is a convenient tool that can be used in many studies, including, but not limited to, cuticular secretion and organization, epidermal cell development, heterochronic gene pathways, and nematode evolution.
We have nothing to disclose.
We wish to thank S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, and H-C. Hsiao for helpful discussions. This work was funded by start-up funds from the TAMHSC Department of Molecular and Cellular Medicine. The compound scope and spinning disk were purchased with funds provided by the department and the TAMHSC Office of the Dean. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources. pRF4 (rol-6(su1006)) was a gift of A. Fire.
|DiI (1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate)||Biotium, Inc.||60010||Stock dilution:
20 mg/mL in DMF
working dilution: 30 mg/mL
|Triton X-100 (Octylphenoxypolyethoxyethanol)||VWR international||EM-9410|
|M9 (22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1mM MgSO4)|
|NGM (Nematode growth medium)||IPM Scientific, Inc.||11006-501||Can be prepared following NGM agar protocol18|
|Agar-agar||EMD Millipore||1.01614||4% in water|
|Levamisole (Levamisole hydrochloride)||Sigma-Aldrich||31742||100 μM - 1 mM levamisole as required|
|Microscope slides||VWR international||16005-106|
|Microscope cover glasses||VWR international||16004-302|
|Compound scope||Carl Zeiss, Inc.||A1m||Use objectives to match the needs of the experiment|
|TRITC or other compatible filter||Chroma Technology Corp.||49005||ET - DSRed (TRITC/Cy3) sputtered filter set|
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