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Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Published: May 7th, 2016



1Department of Molecular and Cell Biology, University of California, Berkeley

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

An incredible amount of diversity exists in the head skeleton among vertebrates, especially among fishes. In many cases this diversity facilitates different feeding strategies1-4, and can involve major changes to both external and internal craniofacial patterning. The branchial skeleton is located internally in the throat of a fish and surrounds most of the buccal cavity. The branchial skeleton is comprised of 5 serially homologous segments, the anterior four of which support the gills. Together these five segments function as an interface between fish and their food5. Variation in a multitude of traits including gill rakers....

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All fish work was approved by the Institutional Animal Care and Use Committee of the University of California-Berkeley (protocol number R330). Euthanasia was performed using immersion in 0.025% Tricaine-S buffered with 0.1% sodium bicarbonate39. All steps are performed at room temperature.

1. Preparation 

Note: Perform steps 1.1-1.5 in conical tubes or scintillation vials that can seal tightly and be laid horizontally. Fish do not need to be constantly shaken, but try to mix the solution as of.......

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This protocol results in a dissected and flat mounted branchial skeleton (Figure 4) where a variety of important trophic traits can be quantified. From a dorsal view, all rows of gill rakers, all pharyngeal tooth plates, and nearly all branchial bones can be easily visualized and quantified22-24,35,36,38,42. Alizarin Red S also fluoresces on a rhodamine or similar red filter allowing double labeling with other markers (e.g., trans.......

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The branchial skeleton is a complex set of bones in the throat of a fish that manipulates, filters, and masticates food items on their way to the esophagus. Many interesting trophic traits including the patterning of gill rakers, pharyngeal teeth, and branchial bones vary across and within species. The majority of these traits are difficult to near impossible to accurately measure with the branchial skeleton in situ (e.g., gill raker length, branchial bone length). This flat-mounting protocol places all.......

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This work was funded in part by NIH R01 #DE021475 to CTM and an NSF Graduate Research Fellowship to NAE. Thanks to Miles Johnson for assistance with imaging and Priscilla Erickson for critical reading of the manuscript.


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Name Company Catalog Number Comments
Sodium Hydroxide (KOH) EMD PX1480-1
Glycerol Sigma-Alderich G7893-4L
10% Neutral Buffered Formalin (NBF) Azer Scientific NBF-4-G
Alizarin Red S EMD 116-12
Microscope Cover Glasses 22x60mm VWR 16004-350
100x10mm Glass Petri Dish Kimble Chase 23064-10010 To dissect samples on
Sylgard 184 Silicone Elastomer Kit Ellsworth Adhesives 184 SIL ELAST KIT 0.5KG Can be poured into glass or plastic petri dishes to make dissecting plates
Modeling Clay Sargent Art 22-4000 1lb cream
Scintillation Vials (case of 500) Wheaton 66021-668 Borosilicate Glass with Screw Cap
Forceps-Dumont #5 Inox (Biologie tip) FST 11252-20 Dumostars are an alternative
Dissecting Scissors  FST 15003-08 Alternate sizes are available depending on size of sample
Dissecting Microscope Leica S6E with KL300 LED Many other models work nicely, having a flat base helps
Microcentrifuge Tubes 1.7mL Denville C-2170
Cardboard slide tray Fisher 12-587-10

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