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.

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&#160;
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...

<ol>
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The authors have nothing to disclose.

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...

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, pharyngeal teeth, and branchial bones contribute to efficient foraging on different types of food.
Sticklebacks have undergone an adaptive radiation after ancestral oceanic forms colonized freshwater lakes and creeks throughout the northern hemisphere. The shift in diet from small zooplankton in the ocean to larger prey in freshwater has resulted in dramatic trophic variation in several craniofacial traits6. While many studies have focused on external craniofacial differences in sticklebacks7-13, important craniofacial changes evolve repeatedly in the internal branchial skeleton. The ability to create fertile hybrids between morphologically distinct stickleback populations provides an excellent opportunity to map the genetic basis of evolved changes to the branchial skeleton.
One trophic trait of ecological significance is the patterning of gill rakers, periodic dermal bones that line the anterior and posterior faces of the branchial bones and are used to filter prey items. Fish that typically feed on small prey items tend to have longer and more densely spaced gill rakers compared to fish that feed on larger prey14,15. Variation in gill rakers has been reported both within and between species14-19, and aspects of gill raker patterning contribute to trophic niches and fitness16. Decades of research have extensively documented gill raker number and length variation in threespine sticklebacks17-21; however, these studies typically focus on the first row of gill rakers. Recent work has shown modularity in the genetic control of gill raker number across the branchial skeleton22,23 and across a single row in gill raker spacing23 and length24 highlighting the importance of studying more than row one or a single gill raker to understand the developmental genetic basis of gill raker reduction.
A second trophic trait of both ecological and biomedical significance is the patterning of pharyngeal teeth. Teeth in fishes can be located in both the oral jaw and in the branchial skeleton, known as pharyngeal teeth. Oral teeth are used primarily for prey capture while pharyngeal teeth are used for mastication and prey manipulation25-27. Both sets form via shared developmental mechanisms and are considered developmentally homologous28. Interesting modularity occurs whereby some species, such as zebrafish, lack oral and dorsal pharyngeal teeth29 while other species have multiple toothed ceratobranchials, pharyngobranchials, and sometimes toothed basihyal and hypobranchials30. In sticklebacks, pharyngeal teeth are found ventrally on the fifth ceratobranchial and dorsally on the anterior and posterior pharyngobranchials31. Kinematics on stickleback feeding show the oral jaw is used primarily for prey capture and facilitating suction feeding9 leaving mastication to the pharyngeal jaw. In cichlids, lower pharyngeal jaw morphology varies dramatically32,33 and has been shown to be adaptive and correlated with trophic niche34. Multiple freshwater stickleback populations have evolved dramatic increases in ventral pharyngeal tooth number23,35,36. Recent work has demonstrated that the developmental genetic basis of this evolved tooth gain is largely distinct in two independently derived populations of freshwater sticklebacks36. Unlike mammalian teeth, fish regenerate their teeth continuously throughout adult life37. Both of these previously described high toothed freshwater populations have evolved an accelerated tooth replacement rate, providing a rare vertebrate system to study the genetic basis of regeneration36.
A third trophic trait that has evolved repeatedly in freshwater sticklebacks is longer epibranchial and ceratobranchial bones, the branchial arch segmental homologs of the upper and lower jaw, respectively38. Longer branchial bones confer a larger buccal cavity and likely are adaptive for allowing larger prey items to be consumed. Furthermore, in other fish, epibranchial bones are important for depression of the dorsal pharyngeal tooth plates25. Like gill rakers and pharyngeal teeth, the branchial bones are internal and thus, difficult to easily visualize or quantify.
Here we present a detailed protocol to dissect and flat-mount the branchial skeleton, allowing easy visualization and quantification of a variety of important craniofacial traits. While this protocol describes a stickleback dissection, this same method works on a variety of other fishes.

<table>
	<tbody>
		<tr>
			<td>Sodium Hydroxide (KOH)</td>
			<td>EMD</td>
			<td>PX1480-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Alderich</td>
			<td>G7893-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin (NBF)</td>
			<td>Azer Scientific</td>
			<td>NBF-4-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S</td>
			<td>EMD</td>
			<td>116-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses 22x60mm</td>
			<td>VWR</td>
			<td>16004-350</td>
			<td></td>
		</tr>
		<tr>
			<td>100x10mm Glass Petri Dish</td>
			<td>Kimble Chase</td>
			<td>23064-10010</td>
			<td>To dissect samples on</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Ellsworth Adhesives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>Can be poured into glass or plastic petri dishes to make dissecting plates</td>
		</tr>
		<tr>
			<td>Modeling Clay</td>
			<td>Sargent Art</td>
			<td>22-4000</td>
			<td>1lb cream</td>
		</tr>
		<tr>
			<td>Scintillation Vials (case of 500)</td>
			<td>Wheaton</td>
			<td>66021-668</td>
			<td>Borosilicate Glass with Screw Cap</td>
		</tr>
		<tr>
			<td>Forceps-Dumont #5 Inox (Biologie tip)</td>
			<td>FST</td>
			<td>11252-20</td>
			<td>Dumostars are an alternative</td>
		</tr>
		<tr>
			<td>Dissecting Scissors&#160;</td>
			<td>FST</td>
			<td>15003-08</td>
			<td>Alternate sizes are available depending on size of sample</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Leica</td>
			<td>S6E with KL300 LED</td>
			<td>Many other models work nicely, having a flat base helps</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes 1.7mL</td>
			<td>Denville</td>
			<td>C-2170</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardboard slide tray</td>
			<td>Fisher</td>
			<td>12-587-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and flat-mounting of the threespine stickleback branchial skeleton

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.

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...

Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

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 ...

The overall goal of this procedure is to dissect and flat-mount the Stickleback Branchial Skeleton. This method can help answer key questions in the fields of development, genetics and evolution. Such as how animals adapt to new environments and how variation in adult morphology arises during development.The main advantage of this technique is that by dissecting out and flat-mounting the branchial skeleton, on can visualize a variety of trophic traits that can't be studied in whole-mount. In preparation, review the relevant head skeletal morphology of the three spine stickleback. After preparing the fish for the dissection, begin by inserting sharp number five forceps into the side of the eye, at a 45 degree angle to puncture the membrane covering the eye.Then, peel away the eye membrane, like the lid of a yogurt container. Next, insert open forceps behind the eye, grab a hold of the optic nerve, and remove the eye. Be careful to avoid puncturing the eye, or it will leak melanin.Repeat this process with the eye on the opposite side. Then, place the blade of a small scissors under the posterior side of the opercle flap, and cut through the soft tissue dorsally above the opercle, towards the eye socket. Next cut through the frontal bone, which is dorsal to the eye socket.Then, cut through the mid line parasphenoid bone, around the center of the eye socket. Repeat the opercle cut on the opposing side. For the next steps, the pelvic spines can be used as handles.They fold out, by gently pulling them directly away from the body. Now, insert the forceps under the opercle, and slowly peel the face away from the body. Trimming away any soft tissue that is still attached in the process.Be careful around the gill rakers, and take care not to disrupt them. It's easy to accidentally grab the first ceratobranchial bone, along with the percoll when removing the facial skeleton. Look under their percoll as you peel it forward to be sure you don't have a hold of anything extra.Now, with forceps, detach the ceratohyals from both sides of the midline basihyal, while peeling away and removing the anterior cranial facial skeleton. Repeat on the opposing side. The anterior cranial facial skeleton includes the entire jaw, the entire hyoid skeleton, the underlying dorsal and endochondrial elements and the anterior part of the skull.Next, insert closed forceps just below the gut tube, and drag them anteriorally. Teasing apart the remaining muscles and ligaments attached to the branchial skeleton in the process. Then, use the tip of the closed forceps to scrape away the muscles that attach the dorsal branchial skeleton to the ventral brain case.Scrape with a posterior to anterior motion. Repeat the muscle removal process on the opposite side of the stickleback. Next, remove the branchial skeleton, by grasping the base of the gut tube and pulling anteriorally.Separate the gut tube using a perpendicular cut, posterior to the fifth ceratobranchial. A common mistake is to grasp the gut tube too anterior which leaves the ventral frontal tube way behind. Be sure to grasp deep in posterior in the thoracic cavity and you can always remove the excess gut tube later.After removing any remaining bone fragments from the brain case on the dorsal side of the branchial skeleton. Insert scissors into the branchial basket, and make a dorsal cut between the bi-lateral sets of dorsal tooth plates. Keep this cut centered to avoid causing damage to the dorsal tooth plates.Next, make two shallow lateral cuts in the rubbery gut lumen at the posterior end of the branchial skeleton. At the antieror end of the gut tube. To assist with opening the branchial skeleton.Now, transfer the skeleton into a microsentrifuge tube loaded with 50 percent glycerol, zero point two five percent potassium hydroxide for gentle clearing. Or instead, transfer it to pure glycerol if no further clearing is required. If re staining is needed, details are given in the text protocol.To prepare to mount, change the solution to pure glycerol, and shake the tube for at least five minutes. Then, remove the branchial skeleton and place it near the bottom of a 22 by 60 millimeter glass cover slip with the dorsal side up. Now, transfer a few drops of glycerol onto the skeleton.Next, roll out two small balls of modeling clay, and play one on either end of the cover slip, they function as spacers. Then, loosely place a second cover slip over the preparation, with enough pressure to flatten the anterior branchial skeleton. Now, peel open the left dorsal flap, including the dorsal tooth plates, and slide the flap between the cover slips.Then do the same with the right dorsal flap. And push the entire branchial skeleton away from the edge of the cover slip. To complete the procedure, lightly press on the top cover slip to flatten the clay balls.Just enough so they keep the branchial skeleton mounted flat. But, not so much that it is crushed. If during the process, a row of rakers is obscured, slide forceps between the coverslips and reorient the ceratobranchial or the entire preparation.When flat mounted in pure glycerol, the preparation can be stored flat at room temperature for at least a decade. This protocol results in a dissected and flat-mounted branchial skeleton. Where a variety of important trophic traits can be chronified.From a dorsal view, all rows of gill rakers, all pharyngeal tooth plates, and nearly all branchial bones can be easily visualized and chronified. Alizarin red S, also floureses on a rhodamine or similar red filter, allowing for double labeling with other markers and alternative methods for visualization. In this image, gill rakers are marked with carats.Teeth are marked with arrow heads, and bones are marked with asterisks. Once mastered, the dissection can be done in a few minutes and be performed on hundreds of animals relatively quickly. And while we've demonstrated a stickleback branchial skeleton dissection, we hope this procedure can be used as a guide for a variety of fish species.By dissecting out the branchial skeleton, we can quickly and easily visualize a variety of important trophic traits, not visible in whole mounts. Allowing for a better understanding of the development and evolution of the cranial facial skeleton.

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Research

JoVE Journal

Biology

9.7K visualizações.  University of California, Berkeley. The overall goal of this procedure is to dissect and flat-mount the Stickleback Branchial Skeleton. This method can help answer key questions in the fields of development, genetics and evolution. Such as how animals adapt to new environments and how variation in adult morphology arises during development.The main advantage of this technique is that by dissecting out and flat-mounting the branchial skeleton, on can visualize a variety of trophic traits that can't be studied in whole-mou...