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/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/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/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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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...

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>Potassium Hydroxide (KOH)</td><td>EMD</td><td>PX1480-1</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</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>AX0485-3</td><td></td></tr><tr><td>Microscope Cover Glasses 22 mm x 60 mm</td><td>VWR</td><td>16004-350</td><td></td></tr><tr><td>100 mm x 10 mm 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>1 lb cream</td></tr><tr><td>Scintillation Vials (case of 500)</td><td>Wheaton</td><td>986586</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&amp;nbsp;</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 1.7 ml</td><td>Denville</td><td>C2170</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?...

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

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

科研

发育生物学

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

建立有限元模型，研究斑马鱼下颌生物力学

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

表征多层鱼鳞（中<em&gt; Atractosteus锅铲</em&gt;）基于纳米压痕技术，X射线CT，红外光谱和SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

生物工程

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

神经科学

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

研究斑马鱼脊椎动物分割的三维尾部外植培养

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

解剖和三刺鳃骨架的平面安装

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表征多层鱼鳞（中

连续光漂白划定单雪旺细胞在神经肌肉接头处

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

完全性脊髓损伤和脑解剖协议后续Wholemount

研究斑马鱼脊椎动物分割的三维尾部外植培养

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

平山准备观察斑马鱼胚胎标本染色，并通过分析全山