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.

所有的鱼的工作是经美国加州大学伯克利分校的机构动物护理和使用委员会（协议号为R330）。使用在0.025％三卡因-S浸渍用0.1％碳酸氢钠39缓冲进行安乐死。所有步骤在室温下进行。 1.准备注意:锥形管或闪烁瓶，可以封紧袋口和水平放置执行步骤1.1-1.5。鱼不需要不断摇动，而是尝试将溶液轻轻倒转或摇动管子或小瓶的齿条鱼的所有侧面暴露于染色液并允许染色均匀渗入组织尽可能经常混合。不要在一个平台摇床大批量的鱼，因为液体的重压将打破振动筛。 <ol><li>固定存储在乙醇与10％的中性缓冲的福尔马林（NBF）中过夜或新鲜安乐死鱼或鱼。另外，使用4％多聚甲醛醛在1×PBS溶液，而不是10％NBF。 注意:如果中提取DNA，夹之前在乙醇固定并存储尾或胸鳍的一小部分。 </li><li>在化学罩正确修复处理，用自来水代替（即约pH 7.0）处理2小时。避免使用去离子水，因为它常常可以是酸性的，并且可以脱钙骨。 </li><li>用0.008％茜素红S中的1％的KOH在水中24小时除去水和污点鱼。对于鱼小于20mm标准长度，用0.004％茜素红S.（请茜素红S的100倍（0.8％）原液，然后可以摊薄）。 </li><li>在自来水几个小时删除染色（投入适当的废物容器罩）和地方鱼。根据需要，直到水冲洗大多明显变化的水。 </li><li>除去水和地方鱼放入50％甘油，0.25％KOH轻度结算和随后的解剖。 注意:此染色协议从先前描述的方法40,41改性。 </li></ol><p类="jove_title"&gt; 2。解剖注:参见图1相关负责人骨骼形态的审查。 <img alt="图1" src="/files/ftp_upload/54056/54056fig1.jpg" /> 图1:棘鱼骨头部形态茜素红染色罗丹明B滤色器设置在荧光成像三刺头。有用的形态标记:OP =鳃盖，Subop = subopercle，BSR的鳃=射线，术前=前鳃，Infraorb 1-3 = 1-3眶下（也称为circumorbitals或suborbitals），登特=齿骨，PREMAX =颌骨，最大值=颌骨，纳斯=鼻，纬度。 ethm =横向筛窦，PSPH =副蝶骨，弗朗=额骨。对于刺头部骨骼的更详细说明，请参见安高（1974）31。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">请点击此处查看大图</a>这个数字。 <ol><li>铺设鱼平面（ 图2A），并在〜45°角插入尖锐＃5钟表镊子进入眼的侧刺穿覆盖在眼睛的膜。 </li><li>剥离膜从眼睛，类似于剥离酸奶盖（ 图2B）的距离。 </li><li>将眼睛后方开镊子，抢人眼球后面的视神经举行，并去除眼（ 图2C）。不要刺破眼球，因为它会泄漏黑色素。如果刺破，黑色素可以在后面的步骤中被冲走。 </li><li>重复的另一边。 </li><li>从后开始，将一个小解剖剪刀刀片的鳃盖瓣下，拖剪刀片背侧以上鳃盖，然后通过对眼窝（ 图2D）切割软组织。切背到鳃盖骨。 </li><li>切额骨（背侧眼窝）（ 图2E）。 </li><li>切周围T中的中线骨蝶骨他中心眼窝（ 图2F）的。 </li><li>重复鳃盖切在相对侧。 </li><li>将镊子鳃盖下，慢慢地剥离面远离身体，修剪任何软组织仍然附着（ 图2G - H）。注意不要破坏鳃耙的第一行。 <ol><li>用血管钳，分离从中线basihyal两侧ceratohyals而剥去和移除前颅面骨（整个下巴，包括齿骨，前颌骨和上颌骨;整个舌骨骨架包括外部皮肤鳃盖，前鳃，subopercle和鳃射线和底层背侧和腹侧软骨内的元素;并且所述颅骨的前部，包括经鼻，横向筛窦和眶下骨骼，参见图1和2I）。 </li><li>骨盆棘可以从身体折叠出来，可以作为手柄，镊子抢举行of出现存在。刺锁定到位。要解锁，轻轻一拉用钳子从棘鱼体内直接走，然后轻轻弯曲向后压脊柱平贴鱼。 </li></ol></li><li>插入钳闭后，腹为鳃骨架（略低于肠道管）并拖动向前镊子，戏弄除了剩余的肌肉和韧带附着于鳃骨架（ 图2J - K）。 </li><li>采用封闭钳提示，刮掉肌肉附着背鳃骨架腹侧脑壳在后到前的方向（ 图2L）。 </li><li>在相对侧重复2.9和2.10。 </li><li>把握肠道管的基极和拉向前以除去鳃骨架和肠管（图2M - N）。 </li><li>通过使垂直切割后至第五ceratobranchial的端部（ 图20中分离肠道管</strong&gt;）。 </li><li>从在鳃骨架的背侧脑壳除去任何剩余的骨碎片后，插入剪刀到鳃篮，使双边套背侧齿板之间的背切（切断前至后）（ 图3A - D）。确保切割中心，以避免损坏背齿板。 </li><li>使橡胶肠腔在腮骨（肠管的前端）的后端两个浅横向切割，以协助打开鳃骨架（ 图3E）。 </li><li>放置鱼和所有组织片进入罐子和鳃骨架置于用50％甘油，0.25％的KOH微量离心管中，如果不要求进一步结算继续轻柔结算，或100％的甘油。标签罐和管与一个唯一的标识符，以便它们可以被追踪。结算所需的量在很大程度上是鱼，大成鱼的大小的函数（超过40米在标准长度米）通常需要额外的空地上。 </li></ol><img alt="图2" src="/files/ftp_upload/54056/54056fig2.jpg" /> 图2:棘鱼鳃骨解剖茜素红染色的三刺鱼准备清扫。映入眼帘的是由粗放结算褪色。蓝色箭头指示运动方向。 （ 一 ）刺头侧面观，前是正确的。 （B）覆盖膜眼的去除。 （C）摘除眼球。 （ 四 ）背切鳃盖上面。 （E）额骨晋级。 （F）切蝶骨。 （G - I）的面部骨骼的去除。 （J）去除腹鳃骨骼软组织连接。 （K - L）背鳃骨架连接的去除。 （&lt;STRONG&gt;米- N）鳃骨架的去除。 （O）从鳃骨架分离肠道管。参见步骤2.1到2.16的更多细节。比例尺= 5毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.鳃骨架重新染色（如有必要） <ol><li>以染色鳃骨架较暗或清除组织以上，除去50％甘油，0.25％的KOH溶液中，用1％的KOH两次（一五分钟的洗涤，随后由第二24小时洗涤，同时在平台摇动器水平地摇动）洗涤。 </li><li>用0.008％茜素红S中的1％的KOH除去1％KOH和再染色24小时。 </li><li>去除污点和用1％的KOH代替24小时。 </li><li>除去的KOH溶液中，用50％甘油，0.25％的KOH代替。 </li></ol> 4.安装鳃骨架<ol><li>删除鳃骨架从50％甘油，0.25％的KOH或100％的甘油和地点附近与背侧22毫米×60毫米的玻璃盖玻片的底朝上（ 图3F）。加上鳃骨架的前50％甘油，0.25％的KOH或100％的甘油几滴。如果从50％甘油，0.25％KOH中以100％的甘油，改变溶液转变在微量离心管和&gt; 5分钟安装到平衡组织之前摇动。 </li><li>铺开模型粘土和地方的两个小球上的盖玻片的任一端充当隔离物。 </li><li>松散放置在有足够的压力趋于平缓前鳃骨架（ 图3G）上面的第二盖玻片。 </li><li>皮尔打开左侧背瓣背包括齿板，压平，和盖玻片（ 图3H）之间滑动。 </li><li>右背瓣重复技术和盖玻片（ 图3I）的边缘推动整个鳃骨架了。 <ol><li>改变本身，同时按住背部皮瓣钳开并小心地将盖玻片上，压扁在一个平滑的运动鳃骨架。 </li><li>另外，安装鳃骨架上的一个盖玻片颠倒，张开每个背侧出横向重力所以不允许鳃骨架收备份。然后用第二区22毫米×60毫米的玻璃盖玻片覆盖并反转准备。 注:不同的安装技术往往工作的每一个人更好或更坏。每次尝试，看看有什么感觉最舒服的。 </li></ol></li><li>轻按顶部盖玻片压平足以让腮骨泥球装持平，但要注意不要挤压标本。 <ol><li>在安装过程中，ceratobranchials可以旋转和掩盖耙一排。通过滑动盖玻片并重新定向ceratobranchials或整个鳃骨架之间镊子解决这个问题。 </li></ol></li><li>储存在滑托盘PREPS持平于ROOM温度。安装在100％的甘油，用制剂可以存储桥接盖玻片之间，用于至少十年。清洁钳和异丙醇或乙醇和求职技巧剪刀。 </li></ol><img alt="图3" src="/files/ftp_upload/54056/54056fig3.jpg" /> 图3:平板安装腮骨操作和鳃骨架安装所示。蓝色箭头指示运动方向。 （A）鳃骨背侧。 （B - D）旋转和切口背齿板之间。在软组织（E）横向切割，以进一步打开肠道管的基地。 （F）鳃骨架放置在盖玻片准备安装底部。 （G）第二盖玻片放置在鳃骨架的前半部分（上述背侧齿片）。 （H - I）打开背齿板襟翼和两个盖玻片之间的滑动鳃骨架的平坦安装。参见步骤4.1至4.6的更多细节。比例尺= 5毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+development+of+complex+biomechanical+systems:+300+million+years+of+fish+jaws.">Evolution and development of complex biomechanical systems: 300 million years of fish jaws.</a> Zebrafish. 2 (4), 243-257 (2005).</li><li>Lauder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+design+and+evolution+of+the+pharyngeal+jaw+apparatus+in+euteleostean+fishes.">Functional design and evolution of the pharyngeal jaw apparatus in euteleostean fishes.</a> Zool. J. Linn. Soc. 77, 1-38 (1983).</li><li>Fraser, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ancient+gene+network+is+co-opted+for+teeth+on+old+and+new+jaws.">An ancient gene network is co-opted for teeth on old and new jaws.</a> PLoS Biol. 7 (2), e1000031 (2009).</li><li>Stock, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dentition+in+comparative+context.">Zebrafish dentition in comparative context.</a> J. Exp. Zool. B. Mol. Dev. Evol. 308, 523-549 (2007).</li><li>Liem, K., Greenwood, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+approach+to+the+phylogeny+of+the+pharyngognath+teleosts.">A functional approach to the phylogeny of the pharyngognath teleosts.</a> Am. Zool. 21 (1), 83-101 (1981).</li><li>Anker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+kinetics+of+the+head+of+the+stickleback,+Gasterosteus+aculeatus.">Morphology and kinetics of the head of the stickleback, Gasterosteus aculeatus.</a> Trans. Zool. Soc. London. 32 (5), 311-416 (1974).</li><li>Meyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometrics+and+allometry+in+the+trophically+polymorphic+cichlid+fish,+Cichlusomu+citrinelfum:+Alternative+adaptations+and+ontogenetic+changes+in+shape.">Morphometrics and allometry in the trophically polymorphic cichlid fish, Cichlusomu citrinelfum: Alternative adaptations and ontogenetic changes in shape.</a> J. Zool., Lond. 221, 237-260 (1990).</li><li>Huysseune, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+plasticity+in+the+lower+pharyngeal+jaw+dentition+of+Astatoreochromis+alluaudi+(Teleostei:+Cichlidae).">Phenotypic plasticity in the lower pharyngeal jaw dentition of Astatoreochromis alluaudi (Teleostei: Cichlidae).</a> Arch. Oral Biol. 40 (11), 1005-1014 (1995).</li><li>Muschick, M., Indermaur, A., Salzburger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+Evolution+within+an+adaptive+radiation+of+cichlid+fishes.">Convergent Evolution within an adaptive radiation of cichlid fishes.</a> Curr. Biol. 22 (24), 2362-2368 (2012).</li><li>Cleves, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolved+tooth+gain+in+sticklebacks+is+associated+with+a+cis-regulatory+allele+of+Bmp6.">Evolved tooth gain in sticklebacks is associated with a cis-regulatory allele of Bmp6.</a> Proc. Natl. Acad. Sci. 111 (38), 13912-13917 (2014).</li><li>Ellis, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+and+genetic+mechanisms+underlie+convergently+evolved+tooth+gain+in+sticklebacks.">Distinct developmental and genetic mechanisms underlie convergently evolved tooth gain in sticklebacks.</a> Development. (142), 2442-2451 (2015).</li><li>Tucker, A. S., Fraser, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+developmental+diversity+of+tooth+regeneration.">Evolution and developmental diversity of tooth regeneration.</a> Semin. Cell Dev. Biol. 25-26, 71-80 (2014).</li><li>Erickson, P. A., Glazer, A. M., Cleves, P. A., Smith, A. S., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+developmentally+temporal+quantitative+trait+loci+underlie+convergent+evolution+of+increased+branchial+bone+length+in+sticklebacks.">Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks.</a> Proc. R. Soc. B. 281, (2014).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA Guidelines for the Euthanasia of Animals. , (2013).</li><li>Bell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+phenetics+and+genetics.">Evolutionary phenetics and genetics.</a> Evol. Genet. Fishes. , 431-528 (1984).</li><li>Taylor, W. R., Van Dyke, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+procedures+for+staining+and+clearing+small+fishes+and+other+vertebrates+for+bone+and+cartilage+study.">Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study.</a> Cybium. 9 (2), 107-119 (1985).</li><li>Erickson, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+190+base+pair,+TGF-&#946;+responsive+tooth+and+fin+enhancer+is+required+for+stickleback+Bmp6+expression.">A 190 base pair, TGF-&#946; responsive tooth and fin enhancer is required for stickleback Bmp6 expression.</a> Dev. Biol. 401 (2), 310-323 (2015).</li><li>Miller, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis-Regulatory+changes+in+Kit+ligand+expression+and+parallel+evolution+of+pigmentation+in+sticklebacks+and+humans.">cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans.</a> Cell. 131 (6), 1179-1189 (2007).</li><li>Aigler, S. R., Jandzik, D., Hatta, K., Uesugi, K., Stock, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+constraint+underlie+irreversibility+of+tooth+loss+in+cypriniform+fishes.">Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.</a> Proc. Natl. Acad. Sci. U. S. A. 111 (21), 7707-7712 (2014).</li><li>Pasco-Viel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+trends+of+the+pharyngeal+dentition+in+Cypriniformes+(Actinopterygii+Ostariophysi).">Evolutionary trends of the pharyngeal dentition in Cypriniformes (Actinopterygii Ostariophysi).</a> PLoS One. 5 (6), e11293 (2010).</li></ol>

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 branchial skeletal elements in a simple two-dimensional plane for ease of imaging and quantification of multiple traits. 
One limitation of this protocol is that although teeth, gill rakers, and most endochondral bones within this flat-mounted two-dimensional prep can be easily imaged and quantified, the second through fourth epibranchials, that have much more three-dimensional shapes, are not easily visualized and would require additional dissection to accurately measure. A second limitation of this protocol is the method is minimally destructive to the fish head. However, only two bones are damaged with a single cut each, the parasphenoid and the frontal bone. All other craniofacial skeletal elements can be returned to and measured at later points in time.
This protocol, although developed for sticklebacks, can be used in many other fish species, although sometimes modifications are necessary. For example, some fish species like zebrafish have a non-ossified dorsal chewing pad44 requiring the dorsal cut to be lateral, around this plate. The posterior end of the fifth ceratobranchial is curved is some species (e.g., zebrafish and other Cypriniformes) and must be trimmed or much larger clay spacers used to allow flat mounting. Particularly large specimens can be mounted between two pieces of glass or acrylic in the same manner. Species with a particularly thick epithelial layer covering tooth plates can be restained and cleared after dissection or more aggressively cleared with 2% KOH. Fish in this situation may also be presoaked in 1 or 2% KOH prior to Alizarin staining to increase dye penetrance. 
An alternative more technologically advanced method for imaging the branchial skeleton is Micro Computed Tomography (&#181;CT)23,45. However, &#181;CT requires an expensive scanner, is much more time consuming, and requires considerable training. Instead, this dissection protocol is inexpensive, quick, and amenable to high throughput studies required for quantitative studies such as quantitative trait loci (QTL) mapping genomic regions controlling evolved changes in craniofacial patterning. Additionally, these dissections can be performed at any larval or adult time point22,35,36,38 to study skeletal development. Although fish head anatomy is complex, this simple protocol allows easy visualization of the internal branchial skeleton, a critically important component of trophic morphology.

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。

<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.

<html>该协议导致了解剖和平板安装鳃骨架（ 图4），其中的各种重要营养特质是可以量化的。从背视图，鳃耙，所有咽齿板，几乎所有的鳃骨中的所有行可以很容易地可视化和量化22 - 24,35,36,38,42。茜素红S也发出荧光的若丹明或类似红色滤波器允许与其他标记物（ 例如 ，转基因GFP的42）和可视化的另一种方法双标记。荧光迅速消失在光，所以在黑暗店用制剂，如果荧光成像或表型计划。从腹面，鳃可以可视化及色素沉着量化43。的Preps可以存储在100％甘油中多年。 4.JPG"/&gt; 图4:代表性的刺鳃骨架染色和清除鳃骨架的两个例子被示出。 （A）明场图像显示骨骼标记为红色。 （B）一罗丹明B过滤器设置下的荧光图像。耙，牙齿和骨骼的实施例​​是分别标有插入记号，箭头和星号。标尺= 2毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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.

解剖和三刺鳃骨架的平面安装

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

dissection-flat-mounting-threespine-stickleback-branchial

Nanyang Technological University

Research

JoVE Journal

Biology

9.7K 次观看.  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. 

视频: Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

在出生后的小鼠听泡解剖:中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

灵长类动物脸演变过程中的反向解剖与 dicect 显示其他隐藏数据

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...