解剖和三刺鳃骨架的平面安装
Summary
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.
Abstract
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.
Introduction
多样性令人难以置信的大量存在于脊椎动物之间的头部骨骼，尤其是鱼类之一。在许多情况下，这有利于多样性不同喂养策略1 – 4，并可能涉及到外部和内部颅面图案重大变化。鳃骨架是在鱼的喉部内部位于并包围大部分口腔的。鳃骨架是由连续5个同源片段，其中前四个支持鳃。这五个段一起充当鱼类和他们的食物5之间的接口。变异的性状，包括鳃耙，咽齿和鳃骨众多有助于有效觅食不同种类的食物。
刺鱼都发生在整个北半球殖民淡水湖泊和小溪洋祠堂后形成的适应辐射。饮食的转变从海洋到淡水更大的猎物小浮游动物造成了戏剧性的变化营养几个颅面特征6。虽然许多研究都集中在刺鱼7外部颅面分歧– 13，重要的颅面变化在内部鳃骨骼多次演变。以形态不同刺种群之间创建育杂种的能力提供了一个绝佳的机会来映射的发展变化对鳃骨架的遗传基础。
的生态意义的一个营养特点是鳃耙，定期皮肤骨骼该行的鳃骨的前部和后部的面孔和用于过滤猎物的图案。鱼类通常在小型猎物饲料往往有更长和更密集的间隔鳃耙鱼类相比上更大的猎物14,15饲料。变异的鳃耙有报道包括了Within和物种之间14-19和鳃耙图案化方面对营养壁龛和健身16。几十年的研究已经广泛记载了threespine刺鱼17鳃耙数量和长度的变化– 21;然而，这些研究通常集中鳃耙的第一行。最近的工作在整个鳃骨架22,23和跨在鳃耙单行距23和24的长度突出学习以上排一个或一个单一的鳃耙理解的重要性，显示模块在鳃耙数的遗传控制鳃耙减少发育遗传基础。
的生态和生物医学意义第二种营养特点是咽齿的图案。在鱼类齿可位于两个所述口腔颚和在鳃骨架，被称为咽齿。口服齿主要用于对p雷伊捕捉而咽齿用于咀嚼和猎物操纵25 – 27。这两套通过共享发展机制形成和发育有28同源考虑。发生有趣的模块，因此一些品种，如斑马鱼，缺乏口腔背咽齿29，而其他物种有多个齿ceratobranchials，pharyngobranchials，有时齿basihyal和hypobranchials 30。在刺鱼，咽齿在第五ceratobranchial和背部的腹侧前发现后pharyngobranchials 31。在刺喂养运动学表明口腔颚主要用于捕获猎物和促进抽吸喂养9离开咀嚼咽颚。在鲷，低级咽颌形态学变化很大32,33并已被证明是自适应和营养生态位34相关联。多PLE淡水棘鱼种群已发展腹侧咽齿号23,35,36急剧增加。最近的工作已经证明，这个演变齿增益的发育遗传基础是在淡水刺鱼36的两个独立的派生种群很大程度上是不同的。不同于哺乳动物的牙齿，鱼不断再生他们的牙齿在整个成年生活37。这两个先前描述的高齿淡水种群的进化加速牙齿替代率，提供了难得的脊椎动物系统来研究再生36的遗传基础。
已在淡水刺鱼反复演进的第三营养性状是较长epibranchial和ceratobranchial骨头，上部和下部卡爪的腮弓节段性同源物，分别为38。长鳃骨赋予较大的口腔和可能是适应性允许更大的猎物为consumed。此外，在其他鱼类，骨头epibranchial是背咽齿板25的抑郁症很重要的。像鳃耙和咽齿，鳃骨骼内部，因此，很难轻易可视化或量化。
这里我们提出了一个详细的协议剖析和平面安装鳃骨架，允许各种重要颅面性状容易的可视化和量化。虽然这个协议描述了一个刺清扫术，此方法同样适用于其他多种鱼类。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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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