LHB是由威康信托基金会动态小区博士课程资助; KAR是由MRC项目基金资助MR / L002566 / 1（授予EJR和CLH）和CLH被授予ARUK资金19479.我们也想感谢成像建议欧胜生物成像设备。

该协议中的所有步骤，按照布里斯托大学的动物保健和福利的指导方针和那些英国内政部的。 1.肌肉骨骼解剖的可视化注意:在适当的年龄骨骼肌球蛋白为了显现骨架元件的形状，以量化肌肉并确定肌肉附件的准确位置，免疫染色（第1.1节）鱼（其揭示肌肉）和II型胶原（可视化软骨）。可替代地，可视化使用的转基因荧光报道线肌肉骨骼解剖结构如胶原A1记者COL2A1:mCherry 13,14形象化软骨和慢肌球蛋白重链记者smyhc:GFP 15形象化肌肉附件（第1.2节）的位置。 标志着软骨和肌肉线条的替代可以工作同样出色。 <ol><li> 荧光Immunosta进不去<ol><li> 1小时固定在磷酸盐缓冲的盐水（PBS）在过量的4％多聚甲醛（PFA）幼虫。在PBS中洗涤用0.1％吐温20（PBT）和分别在PBT 50％甲醇（MeOH）和100％的MeOH脱水5分钟。 注意:PFA是有毒的，并应根据材料的安全片进行处理。 注:幼虫可以存储在100％MeOH中，直到需要。 </li><li>水化在50％MeOH中的幼虫在PBT中5分钟。洗在PBT中5分钟。 </li><li>透在0.25％胰蛋白酶幼虫在PBT中在冰上5-6分钟。洗4X PBT为每5分钟。 </li><li>块在PBT 5％血清2-3小时。 </li><li>孵化幼虫在5％血清中的PBT 2兔抗型胶原蛋白和小鼠抗肌球蛋白抗体的推荐稀释度在室温1小时或过夜，在4℃。 注:建议稀释范围一般是抗体的数据表上。选择时引发针对不同种类彼此以及也迪菲抗体租到组织。 </li><li>洗幼虫6X在PBT 15分钟。 </li><li>块在PBT 5％血清1-2小时。 </li><li>孵育在黑暗次级抗体。在5％血清和PBT适当稀释的特异性抗体用荧光标记的抗小鼠（550）和抗 - 兔（488）的第二抗体。 </li><li>洗涤6X的每个上的10X共聚焦显微镜尽快10分钟PBT和图像。 </li></ol></li><li> 成像肌肉骨骼几何 <ol><li>腹侧安装在在微温0.3-0.5％低熔点（LMP）琼脂糖在Danieau的溶液16盖玻片幼虫。 注:转基因鱼将需要安装前和成像过程中0.02％MS222（三卡因甲磺酸盐，pH值7）服用镇静剂。 </li><li>取使用10X物镜和大约2.5X数字变焦的感兴趣区域的共焦图像栈。制备使用488纳米和561 n中的绿色和红色信道的图像米分别激光器。图像在含有1.3微米和3行的平均值z平面之间的间隔的512×512像素的分辨率。所得堆栈将包括大约100ž切片。 </li><li>将数据导出为TIFF系列。从5dpf斑马鱼幼虫肌肉和软骨元件最大突起示于图1。 </li></ol></li></ol> 2.生成三维曲面<ol style="margin-left: 40px;"><li>选择在3，4和5单丝旦为每个时间点的代表性数据集（多个样品的可视化后选择）。 </li><li>打开三维TIFF栈和选择分析软件的所有通道。右键单击软骨通道，选择图像滤波器和平滑:高斯（ 图2B）。 </li><li>在项目视图中右键点击过滤图像，然后选择"图像分割&#39;，然后&#39;编辑新的标签"。为每个材料， 即，软骨和j的新标签oint。选择使用魔术棒工具图像的软骨区（ 图2C，白色信号，紫色轮廓）。使用画笔工具，从轮廓去除噪声。 注意:如果使用魔术棒工具，单击"所有切片"。 </li><li>用画笔工具选中接合区域和分配给联合组件（ 图2C，蓝色轮廓） </li><li>一次流畅的多片在顶部菜单流畅标签选择分割。右键点击图片，选择生成表面产生一个三维表面呈现组件（ 图2D）的。 </li><li>点击表面和数据保存为一个文件hmascii进口到网格软件。 </li></ol> 3.计算肌肉力量的有限元模型用<ol style="margin-left: 40px;"><li>算从smyhc的共焦图像的肌纤维的数量:GFP转基因斑马鱼（ 图1A，慈姑，1C）并测量直径纤维ETER来计算它们的横截面面积（πR2）。 </li><li>从文献确定每块肌肉单位面积适当的力。每单位面积产生的幼虫斑马鱼骨骼肌（40 NN个/μm2）的最大的肌肉力量，使用17。 </li><li>通过用每单位面积的力的纤维的数量和它们的面积乘以计算每个解剖肌肉群的力。 见表1。 </li></ol> 4.生成网格<ol style="margin-left: 40px;"><li>导入在第2（上图）到一个软件包能够产生有限元网格生成3D模型。 </li><li>通过使用2D菜单下的收缩包装工具生成软骨和关节面的A2D网格。选择一个合适的单元尺寸。 注意:1.5-2.5之间用一个元素的大小。如果有必要，生成一系列不同尺寸的2D表面的网格来进行三维网格优化（第4节。4）。 </li><li>开展下发现网格质量检查&#39;2D&gt;工具&gt;检查元素"面板来检查网格中的重复元素，插入和穿透。在模型树通过应用程序选项卡修复二面角。 </li><li>生成不同的使用3D&gt; Tetramesh子面板单元尺寸的2D表面网格3D网格。 注:比较不同筛目大小的结果，并选择与后进一步模拟了收敛和不损害功能定义的最低目尺寸的有限元模型。 图3中的例子含有150万为下颚软骨四面体单元并具有2.0二维元件尺寸。 </li><li>变换网格使颌模型是规模按使用几何&gt;子面板的距离焦堆栈。 注:确保软骨和关节部件由出口的合并模型或通过使用关系连接在模型中。 </li></ol> 5.有限元国防部埃尔建设<ol><li>使用商业有限元（FE）软件，建立一个有限元模型。使用3D肌肉和第1节为指导生成软骨标记共聚焦栈，分配对应于肌肉附着点的节点。创建表示每个肌肉的起源和插入两个节点（ 图3）之间的载体。 </li><li>创建类型为"历史"的载荷集申请一个&#39;C负载"每块肌肉。指定牛顿（在步骤3.3计算的）的幅度，并分配相关联的向量。 图3示出了用于内收下颌（AM），量角器hyoideus（PH）和intermandibularis（IM）的连接点。 注:对于这些颚肌肉，最大收缩力，使得只有50％的各负载的是在每个站点施加的原点和插入之间分配。 </li><li>由文献确定的分配适当的弹性各向同性的材料属性。杨氏模量为软骨并且在该模型中的域间分别为1.1兆帕和0.25兆帕，泊松比为0.25，这两种18,19。 </li><li>创建类型&#39;边界&#39;的负载收藏家对模型应用的初始约束。去标签分析&gt;约束与create子面板，挑上你希望只模型的节点。选择自由度（DOF）的模型，其自然的运动范围的最佳逼近该限制的运动。 注意: 图3中的模型中运动的所有轴被约束（DOF:1，2，3表示的x，y和z，分别）在ceratohyal在该模型，并在中点锚定它在空间在其中palatoquadrate连接到斑马鱼头骨的其余部分（ 图3， 表1）的点y和z轴。模型必须在所有三个自由度中的至少一个节点的约束。 </li><li>创建"加载一步"，您要SIMUL每种类型的运动吃了（ 即开启，关闭），分析菜单下，选择所有相关的载荷和约束（第5.4节制造）（在5.2节制造）来模拟这一运动。当它出现时，从下拉菜单中选择"静态"。 </li><li>出口模式包括在一个适当的文件格式的网格，载荷，约束和材料特性，在这种情况下，".INP"的格式。 </li><li>负荷模型到有限元分析软件。创建和使用该工作模块的模型执行作业。 </li><li>应力，应变，位移等 ，在结果选项卡和可视化菜单中分析输出（ 图4和图5）。 </li></ol> 6.颚变形/位移的距离的确认<ol><li>选择3-6 的Tg（Col2a1aBAC:mcherry）转基因斑马鱼。 </li><li>轻轻麻醉用0.02％MS222幼虫，直到他们不再响应触摸但他们的心依然跳动。 </li><li>安装幼虫横向上不温不火的1％琼脂糖LMP盖玻片（而麻醉）（在Danieau的解决方案组成）。 </li><li>来自各地的头部取出琼脂糖和钳下巴。 </li><li>冲洗新鲜Danieau的解决方案（没有MS222）在幼虫的头部使用巴斯德吸管，直到正常的嘴部运动恢复删除麻醉。 </li><li>使用影片采集软件，以嘴部运动的亮场的高速视频。取1分钟左右时长的电影最高帧速，或者足以记录钳口的多个周期。 </li><li>选择帧，显示颚开到最大位移。测量麦克尔软骨的前尖端和在微米上颚（筛板的尖端）之间的距离。 </li><li>计算从多个仔稚鱼的平均位移。 </li><li>从模型中提取位移数据。使用6.8计算的平均位移来验证模型位移行为（ <strong&gt;的图4）。如果模型从预期运行敏感性分析通过顺序地改变材料性能和肌肉负荷偏离。 </li></ol>

<ol>
	<li>Rayfield, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finite+Element+Analysis+and+Understanding+the+Biomechanics+and+Evolution+of+Living+and+Fossil+Organisms.">Finite Element Analysis and Understanding the Biomechanics and Evolution of Living and Fossil Organisms.</a> Annu. Rev. Earth Planet. Sci. 35, 541-576 (2007).</li><li>Rao, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Finite Element Method in Engineering: Fifth Edition. , (2010).</li><li>Taylor, M., Prendergast, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+decades+of+finite+element+analysis+of+orthopaedic+devices:+Where+are+we+now+and+what+are+the+opportunities.">Four decades of finite element analysis of orthopaedic devices: Where are we now and what are the opportunities.</a> J Biomech. 48, 767-778 (2015).</li><li>Button, D. J., Rayfield, E. J., Barrett, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+biomechanics+underpins+high+sauropod+diversity+in+resource-poor+environments.">Cranial biomechanics underpins high sauropod diversity in resource-poor environments.</a> Proc Royal Soc London B: Biol Sci. 281. 281, (2014).</li><li>Mammoto, T., Mammoto, A., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+and+developmental+control.">Mechanobiology and developmental control.</a> Annu Rev Cell Dev Biol. 29, 27-61 (2013).</li><li>Shwartz, Y., Farkas, Z., Stern, T., Aszodi, A., Zelzer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+contraction+controls+skeletal+morphogenesis+through+regulation+of+chondrocyte+convergent+extension.">Muscle contraction controls skeletal morphogenesis through regulation of chondrocyte convergent extension.</a> Dev biol. 370, 154-163 (2012).</li><li>Rolfe, R. 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=Identification+of+mechanosensitive+genes+during+skeletal+development:+alteration+of+genes+associated+with+cytoskeletal+rearrangement+and+cell+signalling+pathways.">Identification of mechanosensitive genes during skeletal development: alteration of genes associated with cytoskeletal rearrangement and cell signalling pathways.</a> BMC genomics. 15, 48 (2014).</li><li>Roddy, K. A., Kelly, G. M., van Es, M. H., Murphy, P., Prendergast, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterns+of+mechanical+stimulation+co-localise+with+growth+and+cell+proliferation+during+morphogenesis+in+the+avian+embryonic+knee+joint.">Dynamic patterns of mechanical stimulation co-localise with growth and cell proliferation during morphogenesis in the avian embryonic knee joint.</a> J Biomech. 44, 143-149 (2011).</li><li>Haudenschild, D. R., Chen, J., Steklov, N., Lotz, M. K., D'Lima, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+chondrocyte+actin+cytoskeleton+in+living+three-dimensional+culture:+response+to+anabolic+and+catabolic+stimuli.">Characterization of the chondrocyte actin cytoskeleton in living three-dimensional culture: response to anabolic and catabolic stimuli.</a> Mol cell biomech. 6, 135-144 (2009).</li><li>Nowlan, N. C., Murphy, P., Prendergast, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+pattern+of+mechanical+stimulation+promotes+ossification+in+avian+embryonic+long+bones.">A dynamic pattern of mechanical stimulation promotes ossification in avian embryonic long bones.</a> J Biomech. 41, 249-258 (2008).</li><li>Giorgi, M., Carriero, A., Shefelbine, S. J., Nowlan, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiological+simulations+of+prenatal+joint+morphogenesis.">Mechanobiological simulations of prenatal joint morphogenesis.</a> J Biomech. 47, 989-995 (2014).</li><li>Heegaard, J. H., Beaupre, G. S., Carter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+modulated+cartilage+growth+may+regulate+joint+surface+morphogenesis.">Mechanically modulated cartilage growth may regulate joint surface morphogenesis.</a> J Ortho Res. 17, 509-517 (1999).</li><li>Hammond, C. L., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+populations+of+endochondral+osteoblasts+with+differential+sensitivity+to+Hedgehog+signalling.">Two populations of endochondral osteoblasts with differential sensitivity to Hedgehog signalling.</a> Development. 136, 3991-4000 (2009).</li><li>Mitchell, R. 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=New+tools+for+studying+osteoarthritis+genetics+in+zebrafish.">New tools for studying osteoarthritis genetics in zebrafish.</a> Osteoarthritis and cartilage / OARS, Osteoarth Res Soc. 21, 269-278 (2013).</li><li>Elworthy, S., Hargrave, M., Knight, R., Mebus, K., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+multiple+slow+myosin+heavy+chain+genes+reveals+a+diversity+of+zebrafish+slow+twitch+muscle+fibres+with+differing+requirements+for+Hedgehog+and+Prdm1+activity.">Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity.</a> Development. 135, 2115-2126 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danieau's+solution+(30&#215;).">Danieau's solution (30&#215;).</a> Cold Spring Harb Prot. , (2011).</li><li>Iorga, B., 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=Micromechanical+function+of+myofibrils+isolated+from+skeletal+and+cardiac+muscles+of+the+zebrafish.">Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish.</a> J Gen physiol. 137, 255-270 (2011).</li><li>Tanck, E., Blankevoort, L., Haaijman, A., Burger, E. H., Huiskes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+muscular+activity+on+local+mineralization+patterns+in+metatarsals+of+the+embryonic+mouse.">Influence of muscular activity on local mineralization patterns in metatarsals of the embryonic mouse.</a> J Ortho Res. 18, 613-619 (2000).</li><li>Tanck, 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=The+mechanical+consequences+of+mineralization+in+embryonic+bone.">The mechanical consequences of mineralization in embryonic bone.</a> Bone. 35, 186-190 (2004).</li><li>Brunt, L. H., Norton, J. L., Bright, J. A., Rayfield, E. J., Hammond, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finite+element+modelling+predicts+changes+in+joint+shape+and+cell+behaviour+due+to+loss+of+muscle+strain+in+jaw+development.">Finite element modelling predicts changes in joint shape and cell behaviour due to loss of muscle strain in jaw development.</a> J Biomech. 48, 3112-3122 (2015).</li><li>Roddy, K. A., Prendergast, P. J., Murphy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+influences+on+morphogenesis+of+the+knee+joint+revealed+through+morphological,+molecular+and+computational+analysis+of+immobilised+embryos.">Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos.</a> PloS one. 6, e17526 (2011).</li><li>Cuff, A. R., Bright, J. A., Rayfield, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+experiments+on+finite+element+models+of+an+ostrich+(Struthio+camelus)+cranium.">Validation experiments on finite element models of an ostrich (Struthio camelus) cranium.</a> Peer J. 3, 1294 (2015).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Musculoskeletal+patterning+in+the+pharyngeal+segments+of+the+zebrafish+embryo.">Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo.</a> Development. 124, 2945-2960 (1997).</li><li>Dowthwaite, G. P., 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+mechanism+underlying+the+movement+requirement+for+synovial+joint+cavitation.">A mechanism underlying the movement requirement for synovial joint cavitation.</a> Matrix biol. 22, 311-322 (2003).</li><li>Nia, H. T., Han, L., Li, Y., Ortiz, C., Grodzinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poroelasticity+of+cartilage+at+the+nanoscale.">Poroelasticity of cartilage at the nanoscale.</a> Biophys J. 101, 2304-2313 (2011).</li></ol>

作者什么都没有透露。 在图3-5的某些数据已经从J.Biomech，48（12），布伦特等重印，有限元建模预测在关节形状和细胞行为由于在颌发展，3112-22肌肉拉伤的损耗变化。 2015年，爱思唯尔15的许可。

有限元模型已被用于关联是在与那些接受骨形成10株骨骼元件的面积，以及软骨内骨化及关节形态8,12,21过程中受到压力区域地图。其他的研究也已经能够应用的理论增长模型联合开发过程中11,12复制更改。在这里，我们显示了建立有限元模型的相对简单的系统，斑马鱼下颌20的协议。不像收集原始图片的有限元模型，例如CT扫描22的转基因系共焦成像或免疫染色斑马鱼的替代方法允许待研究多种组织。它可以，因此，提供有关软骨肌肉附着点的直接信息。在脊椎动物斑马鱼模型是特别适合遗传和药理操纵。 FE模型斑马鱼的一代颅面软骨现在开辟了合资形态的生物力学和遗传学之间的相互作用进一步研究的可能性。 有许多的创造一种有限元模型的过程中的关键步骤;第一种是产生系统的精确三维表示。这需要足够高的分辨率，以明确界定边界成像。注意，即使使用高分辨率成像，以良好的表面有可能有理顺一些地区。另一个关键步骤是定义负载和正确的约束的正确位置。不充分约束模型将无法求解和负载的不正确的位置会造成异常运动。 原始数据的一些处理（ 图2）是必要的，因为从原始数据产生的表面就难以啮合（ 图2B）。我们过滤使用高斯滤波器（数据图2C </s仲&gt;）和我们进行了曲线的一些手动平滑以产生一组清洁轮廓可以被转换成三维表面的。太多的平滑可以产生已经失去了它的许多功能的"回炉"的表面。选择正确的元件的大小是一个反复的过程与选择过小的元件尺寸产生过大的网孔这是计算密集的。然而，选择过大的元件尺寸将产生无法概括结构的正确的形状的网格。正确的网状有这样捕获的下巴正确的形状，并聚集在一个正确的解决方案的最小单元尺寸，使用颚排量核实。它也可能是必要的修改材料特性或负载的计算，以更好地模拟了正确的位移为不同年龄和物种将具有显着不同的性质。 重要的是要记住，总有一个假设的模型和局限性是非常重要的ssumptions作出运行的有限元模型。当只建模一个或少量数目的样本，关键是要保证，因为有可能是个体之间的微小变化有代表性的样品被选择。因为只有某些夹爪元件和肌肉都包括在内，该模型是斑马鱼颅面肌肉骨骼系统的简化版本。因此，约束不得不被定位以考虑其中模型化颚元件将与颅骨的其余部分连接和模型在中心被人为约束来解决它在"空间"。这种人工约束没有从作为ceratohyal本身没有分析模型得出的解释影响。更多的颅面结构的包容性，尤其是其他夹板打开的肌肉，如sternohyals及其连接的软骨23，可以添加到模型，但是限制包括更大的模型在有限元软件运行的能力。 S ="jove_content"&gt;另一个限制是，我们还没有模拟韧带插入，虽然这可以通过弹簧8的插入来实现。在这种情况下提出的另一个假设是，该模型会表现得线性。在模型菌株的大小是可比的那些在公开的模型和施加到体外细胞10,24，用菌株是低于3500以上由约束和肌肉附着点分开-5000μɛ。因此，在该模型的相关区域的菌株的线性模型上可接受的范围内被认为。软骨不完全表现为一个线性材料和先前已被建模为一个多孔弹性材料，这使流体行为的分析模型25。扩频的肌肉附着点之间的本地节点的集群将分发的峰值力和更准确地表示肌肉插入某些肌肉。 ENT"&gt; FE的使用允许的应变和应力作用于结构的评估。作为一个技术它在许多生命科学学科，包括骨科，古生物学以及最近的发育生物学常用的。在这里，我们将介绍如何建立的FE的斑马鱼下颚。将来这些模型可以扩展至来看整个颚，包括口感。类似的技术可用于在鱼脊柱生物力学，这在迄今大多研究了运动装置建模。

有限元（FE）建模是一个工程技术，可以计算计算并映射作用于结构1株的大小和位置。该模型包括三维结构中，通过"有限元"的网格表示的，并且分析的最终结果是由许多因素，包括在网格中，机械的大小和位置的元素的结构和数量支配载荷和材料特性。材料特性描述给定类型的负载下材料的行为的某些方面;杨氏模量（E）描述的材料的弹性而泊松比描述了当一个样品拉伸到其长度的材料的宽度的比例减少。有限元建模可以用来通过考虑关于结构的独特的输入数据来计算各种各样的变量，包括位移，应力，压力和应变作用于模型&#39;氏形状，位置和负荷的大小和特定材料性质。 有限元建模被广泛应用于工程2和日益骨科3和古生物应用4。在开发生物机械力是已知的，作为在许多细胞的刺激以激活细胞反应5-8和预测都内显影器官系统的相对位置和机械刺激的幅度它是有用的，但是，目前的有限元建模已很少使用斑马鱼的发展。 既软骨和骨已被证明是机械敏感材料。例如， 在体外压缩已经发现激活软骨细胞的途径，而张力已被证明是必要的骨形成9。有限元分析（FEA）已经被利用来模拟应变作用于生物标本，其中包括作用于骨骼的骨元素在FO细则第十五10。其它发展的应用包括其使用以预测关节的形状是已暴露于理论生物机械力11,12后，并显示出小鸡膝关节形态8中本株的模式。 该协议旨在分享从共聚焦图像生成的三维曲面，网格和有限元模型，以了解发展组织的力学的经验。我们还表明验证的有限元模型虽然捕捉体内真正的关节位移信息的方法。而我们使用斑马鱼颚作为范例相同的技术可以用于可通过共聚焦或多光子成像而获得的肌肉骨骼系统的结构的三维信息的任何小生物系统上使用。

<table border="0" cellpadding="0" cellspacing="0" width="937">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coll2</td>
			<td style="width:237px;">Abcam</td>
			<td style="width:119px;">ab34712</td>
			<td style="width:365px;">Type II collagen antibody - stains all cartilage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">A4.1025 / MF20</td>
			<td style="width:237px;">Developmental studies hybridoma bank</td>
			<td style="width:119px;">A4.1025</td>
			<td>Skeletal mysoin antibody - marks all skeletal muscle&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Low melt agarose</td>
			<td style="width:237px;">Sigma&#160;</td>
			<td>A9414-5G</td>
			<td>For mounting zebrafish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS222 (Ethyl 3-aminobenzoate methanesulfonate )</td>
			<td style="width:237px;">Sigma</td>
			<td>E10521-10G</td>
			<td>To make anaesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:237px;">Fisher</td>
			<td>T/3760/48</td>
			<td>sample permeablilisation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 488Mouse IgG</td>
			<td style="width:237px;">Thermofisher</td>
			<td align="right" style="width:119px;">35502</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 550 Rabbit IgG</td>
			<td style="width:237px;">Thermofisher&#160;</td>
			<td align="right" style="width:119px;">84541</td>
			<td>Secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SP8/SP5 or SPE confocal</td>
			<td style="width:237px;">Leica&#160;</td>
			<td style="width:119px;"></td>
			<td>For imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;LAS Leica capture software&#160;</td>
			<td style="width:237px;">Leica&#160;</td>
			<td style="width:119px;"></td>
			<td>Imaging software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aviso (version 7.0.0)</td>
			<td style="width:237px;">FEI Visualization Science Group</td>
			<td style="width:119px;"></td>
			<td>3D image analysis software (Section 2)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hypermesh part of the Hyperworks package (version 10)</td>
			<td style="width:237px;">Altair Engineering</td>
			<td style="width:119px;"></td>
			<td>FE model generating software (Section 4-5)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Abaqus (version 6.14)</td>
			<td style="width:237px;">SIMULIA</td>
			<td style="width:119px;"></td>
			<td>FE analysis software (Section 5.7-5.8)</td>
		</tr>
	</tbody>
</table>

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.

免疫染色肌肉（ 图1A）和软骨（ 图1B）或转基因记者成像（ 图1C）使颚的三维结构来进行可视化，与相关联的肌肉一起。通过以高分辨率成像是可能建立捕获两个颚的三维形状（ 图2）和所述位置和负载的放置（ 图3）的模型。利用通过高速视频捕捉看到体内的位移（ 图4），我们证实在该模型的运动范围是一个现实的范围之内。 的有限元模型一次运行可以用来显示一系列数据，例如压力（ 图5A），最小和最大主应变（ 图5B - K）的。这些结果是三暗淡<html> ensional因此该模型可以被放大才能看到细节的精细图案（ 图 5E，5I）旋转，以获得相关的视图（ 图5F，5G，5J，5K）和数字切片（ 图5E&#39;，5E&#39;，5I"，5I &#39;&#39;）显示如何应力，应变或压力变化整个模型的图案。另外，也可以从模型中提取的量化数据（未示出）。通过验证该模型，并使用最准确的材料特性，负载和网眼形状的有限元模型可用于探索开发该窗期间由细胞所经历的机械环境的最佳估计。该模型的结果可直接相比，在细胞行为和基因表达20变化。 <html>重1"SRC ="/文件/ ftp_upload / 54811 / 54811fig1.jpg"/&gt; 图 1: 斑马鱼下颌中的5旦的肌肉骨骼元件的代表性图像 5dpf幼虫的下颚的代表性共聚焦栈所有前壁顶部（A）的免疫染色A4.1025其中污渍所有骨骼肌球蛋白（B）中示出免疫染色II型胶原从活幼虫表达的转基因记者COL2A1标志着所有软骨（C）堆栈:mCherry标记软骨（红色）和smyhc:GFP慢肌（绿色）。 IA:intermandibularis前，PH:量角器hyoideus，AM:收下颌，HI:hyoideus逊色，HI:hyoideus优越，CH:sternohyoideus，MC:麦克尔软骨，PQ:Palatoquadrate，CH:ceratohyal <a href="http://ecsource.jove.com/files/ftp_upload/54811/54811fig1large.jpg" target="_blank">请点击此处查看大图这一数字。</a> "FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/54811/54811fig2.jpg" /> 图2: 来自共焦数据3D表面生成图像表示从共焦数据转换成用于与接合区域的较高放大倍数的斑马鱼下颚3D表面的过渡。 （ 一 ）原焦的数据; （B）的高斯滤波器应用后数据集; （C）过滤的轮廓; （D）三维曲面。 <a href="http://ecsource.jove.com/files/ftp_upload/54811/54811fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54811/54811fig3.jpg" /> 图3: 代表网格显示约束和力矢量的5 DPF幼虫为（A）口封闭和代表的网格。（B）张口。白点表示，其中该模型被约束的地方，并且其中尺寸（ 例如，x和y或x，y和z）表示。白线表示的肌肉位置，用白色箭头表示肌肉力量的载体。红色显示软骨和黄色的域间。这个数字已经从先前公布的布伦特等 15辅料修改。 <a href="http://ecsource.jove.com/files/ftp_upload/54811/54811fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54811/54811fig4.jpg" /> 图 4: 敏感性试验 FE-模型中对于不同的软骨和域间的杨氏模量5dpf斑马鱼模拟颚位移。鄂式位移（开到微米闭合）被标记上颌骨;使用颜色键记录。每个模型（A - L）的具有软骨的不同组合（C = 1.1，3.1，或6.1兆帕）或域间（ⅰ0.25 = 0.5，0.75，或1兆帕）的属性。水平黑色箭头突出颚位移在麦克尔软骨的尖端的值（由垂直黑色箭头表示）。M和N从旦幼虫表现最小的5个视频静止，即，钳口闭合（M）和最大值 ， 也即下颌全开（N）与两个叠加的（O） -白线O代表的位移（43微米）。在0.25（A）最佳匹配域间活鱼看到的位移1.1这种情况下，相对软骨属性（O）。这个数字的AL小组此前已公布的布伦特等 15。 <a href="http://ecsource.jove.com/files/ftp_upload/54811/54811fig4large.jpg" target="_blank">请点击此处查看大VERS</a>这个数字的离子。 <img alt="图5" src="/files/ftp_upload/54811/54811fig5.jpg" /> 图5:从有限元模型代表的数据在5 DPF应用于所有的幼虫肌肉FE-模型仿真（A - C）。 （A）米塞斯（EMaxmin）（B）最小主应力（E最小P，μɛ）（C）最大主应变（E最大P.，μɛ）。下颚打开时的​​最大和最小主应变的有限元模型模拟。 （D - K）:最大主应变（ē最大P.，μɛ）在（D）腹侧颚图，（E）腹联合视图（E）显示了近-远段的位置通过麦克尔软骨关节和域间在（E&#39;）和（E&#39;&#39;），分别为。 （F）:横向下颚视图。 （G）:外侧关节视图。 （H - K）:最小主应力（的E民P，μɛ）（H）中腹下颌图，（ 我 ）联合腹面观。 （Ⅰ）表示近端-远端段的位置通过麦克尔软骨关节和（I&#39;）和（I&#39;）为（J）的域间中:横向颚图。 （K）:外侧关节视图。这个数字，已经出版的布伦特等 15。 <a href="http://ecsource.jove.com/files/ftp_upload/54811/54811fig5large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td>肌纤维的数量</td><td>肌纤维面积（微米2） </td><td>肌肉群面积（微米2） </td><td>力（N） </td></tr><tr><td> 5 DPF intermandibularis前</td><td>五</td><td> 23.8 </td><td> 119 </td><td> 4.76e-6 </td></tr><tr><td> 5 DPF量角器hyoideus </td><td> 6 </td><td> 23.8 </td><td> 142.8 </td><td> 5.71e-6 </td></tr><tr><td> 5 DPF下颌内收</td><td> 9 </td><td> 23.8 </td><td> 214.2 </td><td> 8.57e-6 </td></tr></tbody></table> 表格1: 肌肉定量分析。计算的Intermandibularis前，下颌内收和量角器Hyoideus平均肌力在5 DPF使用40 NN /μm2以下（从每17参考采取单位面积值）。 （Lorga 等 ，2011）（N = 3）。

Watch this Scientific Journal Video about Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics at JoVE.com

建立有限元模型来研究斑马鱼颌生物力学 |发育生物学|视频

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

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 ...

building-finite-element-models-to-investigate-zebrafish-jaw

Research

Education

JoVE Journal

JoVE Core

Biology

Developmental Biology

Human Biology

Musculoskeletal System

SCIENTISTS IN ACTION

9.1K 次观看.  University of Bristol. 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. 

视频: Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

科研

发育生物学

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

斑马鱼高血糖的急性和慢性模型:评估高血糖对神经发生的影响和放射性标记的分子的生物分布的方法

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...