1Department of Biological Sciences, DePaul University, 2Children’s Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine
LeClair, E. E., Topczewski, J. Methods for the Study of the Zebrafish Maxillary Barbel. J. Vis. Exp. (33), e1558, doi:10.3791/1558 (2009).
Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle.
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (>3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.
Zebrafish are housed and reared according to standard methods 1. Past the late larval period, the developmental stage of a zebrafish can be measured by its standard length (SL), or the straight line distance in millimeters from the anteriormost point of the upper lip to the base of the caudal fin (posteriormost hypural plate)2. At approximately one month post fertilization (10-12 mm SL), the nasal and maxillary barbels appear as transparent epithelial buds near the olfactory pits and on the posterior corners of the maxillae, respectively. As the zebrafish matures, the barbels extend into narrow, whisker-like appendages. Because the maxillary barbel is larger (2-3 mm) and easier to manipulate, our protocol applies only to this particular appendage; similar techniques, however, could be adapted to the smaller nasal barbel.
Agar Embedding of Matched Barbel Pairs
The zebrafish maxillary barbel is an underutilized tissue system for studying the growth, maintenance, and regeneration of several cell types in zebrafish. Although the barbel appendage has no human analog, the cell types it contains are highly conserved, making it possible to study skin, glands, melanocytes, circulatory vessels and nerves in an optically clear and anatomically simple cylindrical structure. Similar to the well-studied caudal fin, barbel tissue can be induced to regenerate by amputation. Using the border of the maxilla as an anatomical landmark, the amputation plane can be placed precisely, facilitating the measurement of barbel regrowth. For an experienced operator, each surgery takes only a few seconds. Recovery is rapid, and we have so far detected no short or long-term effects on fish behavior. Zebrafish with one maxillary barbel swim, eat and breed as effectively as non-surgical controls, and have comparable survival to the time of tissue collection, up to 6 months after surgery. The physiological impact of this surgery predicted to be minimal because 1) the extraoral taste buds carried on the barbel are also found on many other parts of the fish epithelium, including the lips, cheeks and head5, and 2) differentiating taste buds appear on the regenerating barbel within 72 hours (LeClair et al., unpublished data.) This makes the maxillary barbel a minimally invasive system for studying wound healing, revascularization, and reinnervation within the context of an adult vertebrate.
After surgical induction of regeneration, barbels can be collected at intervals for morphometric measurement and/or microscopic analysis of fixed tissue. Conveniently, the maxillary barbel is approximately the length (2-3 mm) and diameter (100-200 mm) of a zebrafish embryo, facilitating the application of many standard protocols, including paraffin histology, cryosectioning, whole-mount immunohistochemistry, and in situ hybridization. Taken together, these features make the maxillary barbel a highly feasible in vivo model for studying tissue repair and regeneration.
These methods were developed by E.E.L. during a research leave in the lab of J.T. Support was provided by the DePaul University Research Council and an NIH/NIDCR R01 grant to J.T. (DE016678). We gratefully acknowledge the efforts of Caroline Hunter, animal care technician in the CMRC zebrafish core facility and Paulina Pawluczuk, our undergraduate laboratory assistant.
|A stereo dissecting microscope with transmitted and incident fiber-optic illumination is required for barbel clipping and agar embedding|
|Fish system water|
|2 zebrafish crossing tanks|
|MS-222 (ethyl 3-aminobenzoate methanesulfonate; Sigma Chemical #E10521-10G)|
|sterile 120-mm Petri plate|
|wet paper towels|
|blunt metal spatula|
|Dumont #55 Bio Inox Forceps (Fine Science Tools #11255-20)|
|Mini-Vannas spring scissors (Fine Science Tools #15000-00)|
|methylene blue antifungal agent (Drs. Foster & Smith #CD-905781)|
|small aquarium fishnet|
|4% paraformaldehyde in 1x phosphate-buffered saline (PF-PBS), aliquoted into 50 mL conical tubes and stored frozen at -20Â°C until use|
|1x phosphate-buffered saline (pH 7.27-6)|
|250 mL glass flask|
|2% agarose in distilled water|
|standard small agarose gel electrophoresis rig (gel size = ~10 cm square)|
|small-toothed electrophoresis sample comb (comb size 4 x 1.5 mm)|
|warm water bath (50-60Â°C)|
|black enameled insect pins, size 000 (Fine Science Tools #26000-25)|
|1-2 pin holders (Fine Science tools #26016-12)|
|9-inch glass Pasteur pipette|
|pipette bulb or pump|
|small paper labels|
|(optional) printed calibration scale|
|wet paper towels|
|zip-closure plastic bag|
|scalpel/razor blade (to cut out agar blocks)|