We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1.高通量吗啉设计策略<ol><li>鉴定感兴趣的基因（多个）（ 例如 ，候选基因的方法，顺式调控分析，RNA测序和/或蛋白质组差动屏幕）。 </li><li>使用基因组，转录组和可用的基因表达数据上经常更新的网站（ 如 SpBase http://www.echinobase.org 20和S紫石基因搜寻的http:///urchin.nidcr.nih.gov/blast/index html的），以确定的时空表达谱与所讨论的发育机制重叠。如果没有表达数据是可用的，然后生成的qPCR引物和/ 或原位探针的反义来评估基因表达模式。 </li><li>确定的空间和时间表达模式之后，获取从基因组网站的DNA序列。 <ol><li>为了生成翻译阻断吗啉代寡核苷酸序列获得ØF中的5&#39;未翻译的区域（5&#39;非编码区）直接上游上SpBase或S.紫石基因组搜索网站提供表达序列标签（EST）数据库起始密码子。如果此信息是用于感兴趣的基因不可用，则执行5&#39;RACE。 </li><li>要设计一个剪接阻塞吗啉，搜索包括外显子和使用SpBase或S.紫石基因组搜索工具脚手架BLASTN工具内含子的基因组序列。 </li></ol></li><li>使用该寡核苷酸设计网站http://oligodesign.gene-tools.com以设计所需的25个碱基对吗啉代序列。 <ol><li>对于平移阻挡吗啉，使用mRNA序列从5&#39;到3&#39;为平移靶序列的来源。包括5&#39;非编码区序列（约70个核苷酸的转录起始位点的上游）加上起始密码子标记的Wi编码区（起始位点的下游）的25个碱基第括号（ATG）。 </li><li>对于拼接阻塞吗啉代，选择内含子 - 外显子或外显子 - 内含子边界序列，并包括50个碱基（外显子序列的25基地和内含子序列的25个碱基）的边界区域周围。扫描具有吗啉代序列的基因组中，以验证该序列是唯一的。 </li></ol></li></ol> 2，吗啉寡聚核苷酸显微注射<ol><li>通过加入100微升不含核酸酶的水进入300纳摩尔啉小瓶制备吗啉代寡核苷酸的3 1mM储备溶液。 注:请不要使用DEPC处理过的水的再悬浮，因为焦碳酸二乙酯会损坏吗啉。 </li><li>用于第一重构降速含有30秒全速股票寡核苷酸溶液的管形瓶（14000 - 16000 XG），简要地涡旋，热在65℃下进行5至10分钟，短暂涡旋，并保持啉库存在室温温度下至少1小时。吗啉原液<html> s的-20℃贮存至+4℃。 </li><li>制备含有吗啉代寡核苷酸以所需浓度注射溶液。该溶液通常含有20％的甘油或125毫米氯化钾作为载体和15％FITC-葡聚糖（异硫氰酸荧光素葡聚糖的10,000MW 2.5毫克/μl原液）。 FITC-葡聚糖和其他荧光葡聚糖偶联物通常用于通过荧光显微镜，以确定注射的胚胎。在-20℃储存注射液。 </li><li>热在热块或水浴至少2在65℃的吗啉溶液 - 5分钟。 </li><li>旋转简要30秒全速啉溶液（14000 - 16000 XG）全速1分钟，离心机，混匀（14000 - 16000 XG）至少10分钟。 </li><li>负载啉溶液注射针。有关详细显微注射的协议，请参阅Stepicheva，宋，2014年21和欢呼声和Ettensohn，2004年"&gt; 22。 </li></ol> 3.固定和原位协议在紫石S.胚胎24小时受精后（HPF） 注:此协议从阿里纳斯-梅纳等进行修改。 2000年23塞西等。 ，2014年24。 <ol><li> 固定术 <ol><li>加入数滴固定液（见下文），含胚胎的井，用移液器轻轻混匀，并允许他们定居。除去固定液，然后用固定剂的两个附加180微升洗涤液混合胚胎。 注意:对胚胎轻柔吹打向上和向下几次洗涤液中调匀是很重要的。 如果不这样做可以降低信噪比。 </li><li>离开胚胎在第二固定剂洗涤50分钟到1小时，在室温（RT）下在4％的电子显微级低聚甲醛组成的10mM MOPS pH为7.0的修正，0.1％吐温-20，以及人工seawateR（ASW）。让每次最好的结果该解决方案新鲜。此外，为了便于和实用的胚胎被固定在96孔板中。 <ol><li>为20毫升固定剂使用5毫升16％的多聚甲醛，15毫升ASW，200微升的1M MOPS pH为7.0和20微升吐温20。 </li></ol></li><li>在RT洗5次具有MOPS 180微升洗涤缓冲液由0.1M的MOPS pH为7.0，0.5M NaCl和0.1％Tween-20的至少5分钟或直至胚胎下降到井的底部。再次，要彻底轻轻吹打上下数次胚胎到洗涤缓冲混合是很重要的。 MOPS洗涤缓冲液可用于2天，如果保存在4 C。 <ol><li>为40毫升的MOPS洗涤缓冲液使用4毫升的1M MOPS pH 7.0的，4毫升5M的氯化钠，32毫升的dh 2 O和40微升吐温20。 </li><li>固定的胚胎可以保存在4℃下2天。 注意:如果存储固定胚胎长，再加入0.2％的叠氮化钠在MOPS洗涤缓冲以防止细菌滋生。</li></ol></li></ol></li><li> 预杂交 <ol><li>吸出MOPS洗涤缓冲，并添加2 180微升:MOPS的1比洗涤缓冲到杂交缓冲液中，胚胎混合到通过温和移液几次该溶液中，并在室温下孵育至少20分钟。杂交缓冲液由70％的甲酰胺，0.1M MOPS pH为7.0，0.5M的NaCl，1mg / mL的BSA和0.1％吐温-20。 <ol><li>对于40毫升杂交缓冲液使用4毫升的1M MOPS pH为7.0，4毫升5 M氯化钠，4毫升的dh 2 O，0.04克BSA和40微升吐温20。涡旋拌匀，再次添加28毫升甲酰胺和旋涡。 </li></ol></li><li>取出2:1的比例的MOPS洗和杂交缓冲液，并添加1 180微升:2的比例MOPS的洗涤缓冲至杂交缓冲液，胚胎混合到该溶液中，并在室温下孵育至少20分钟。 </li><li>之前探针杂交胚胎轻轻拌匀成100 - 150微升杂交缓冲液。在50℃下孵育胚胎至少1H。 注:隔夜孵化的胚胎是可以接受的。孵育前，密封件用粘合剂片材的孔中，以防止蒸发。 </li></ol></li><li> 杂交 <ol><li>轻轻0.3纳克/微升探针和500微克/毫升酵母tRNA到杂交缓冲液中，然后涡旋创建探针溶液 - 在一个单独的试管中加入0.1。酵母tRNA加到探针溶液以降低非特异性反义探针的结合。预热该溶液至50℃，并吸出杂交缓冲液。 </li><li>混合预杂交胚入100微升探针溶液中，用粘合剂片材密封，并在50℃下杂交为取决于探头2至7天（foxq2探针可以温育2天）。 注意:孵育时间将因关闭探头。在低水平表达的一些基因需要长达7天的潜伏期。 96孔板可以被放置在一个恒温恒湿箱作为投保蒸发如果adhes香港专业教育学院表无法正确密封。 </li><li>在50℃下用新鲜的MOPS的180微升洗5次洗涤缓冲液，共3小时。然后，洗3次（15分钟）与180 MOPS的微升在RT洗涤缓冲液。请记住混合胚胎入洗轻轻吹打几次，每次缓冲区。 </li></ol></li><li> 抗体孵育 <ol><li>吸出MOPS洗涤缓冲液和在180微升封闭缓冲液由10％正常羊血清和5毫克的混合胚胎/ mL BSA的在MOPS洗涤缓冲液和在室温下孵育至少45分钟或4℃过夜。 </li><li>除去封闭缓冲液，然后胚胎混入阻断含有1缓冲液:碱性磷酸酶缀合的抗洋地黄毒苷抗体的1500稀释在封闭缓冲液稀释。孵育在室温过夜，在密封板以避免蒸发。注意:不要离开不是一蹴而就更长的抗体。 </li><li>洗胚胎6次（5分钟或直到胚胎掉落）在MOPS在室温洗涤缓冲液。胚可以在4℃下过夜贮存。 </li></ol></li><li> 原位发展 <ol><li>在RT洗胚胎3次（10分钟）用由的0.1M Tris pH值9.5，100mM NaCl的50 mM的氯化镁 ，1mM的左旋咪唑新鲜制备的pH 9.5缓冲液和0.1％吐温-20。 <ol><li> 20毫升的pH 9.5缓冲用2毫升的1M Tris pH值9.5，400微升5M氯化钠，1毫升的1摩尔氯化镁 ，20微升吐温20，16.6毫升卫生署2 O和0.0048克左旋咪唑。 </li></ol></li><li>孵育在37℃的胚胎在避光染色缓冲液。染色缓冲液由10％的二甲基甲酰胺，4.5微升/毫升4-硝基蓝四唑氯化物（NBT），和3.5微升/毫升的新鲜制备的pH 9.5缓冲5-溴-4-氯-3-吲哚磷酸（BCIP） 。 <ol><li>为1毫升染色缓冲液使用100微升二甲基甲酰胺，4.5微升的NBT和3.5微升BCIP。 </li></ol></li><li>通过在MOPS洗涤3至5次停止碱性磷酸酶反应洗涤缓冲液。反应无线个所述foxq2探针通常需要30分钟至1小时。有些探头可在Rt或4℃需要过夜培养。 </li><li>混合胚胎成70％的MOPS洗涤和30％甘油的溶液。甘油提供必要的显微镜的折射率。胚胎可以存储在该溶液中几个星期。密封塑料石蜡板，以防止水分蒸发。 </li></ol></li><li> 玻片制备和图像捕获 <ol><li>通过设置双面胶带的三个小条与条之间的小间隙上的滑动三角形制备的幻灯片。 </li><li>转移在70％MOPS洗涤和30％的甘油溶液中的胚胎的三角形的中心，并用盖玻片覆盖。 </li><li>周围盖玻片边缘运用指甲油一层密封盖玻片。 </li><li>捕获使用复合光显微镜20倍的目标和一个附加的摄像机图像。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wnt-FoxQ2-nodal+pathway+links+primary+and+secondary+axis+specification+in+sea+urchin+embryos.">A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos.</a> Dev Cell. 14, 97-107 (2008).</li><li>Molina, M. D., de Croze, N., Haillot, E., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal:+master+and+commander+of+the+dorsal-ventral+and+left-right+axes+in+the+sea+urchin+embryo.">Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.</a> Curr Opin Genet Dev. 23, 445-453 (2013).</li><li>Range, R. C., Glenn, T. D., Miranda, E., McClay, 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=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, R., 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+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. L., McClay, 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=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, 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=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, 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=Hedgehog+signaling+requires+motile+cilia+in+the+sea+urchin.">Hedgehog signaling requires motile cilia in the sea urchin.</a> Mol Biol Evol. 31, 18-22 (2014).</li><li>Technau, U., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology.">Evolutionary crossroads in developmental biology.</a> Cnidaria. Development. 138, 1447-1458 (2011).</li><li>Yaguchi, J., Takeda, N., Inaba, K., Yaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Wnt-Nodal+Signals+Regulate+the+Patterning+of+Anterior+Neuroectoderm.">Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.</a> PLoS Genet. 12, e1006001 (2016).</li><li>Duboc, V., Rottinger, E., Besnardeau, L., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+signaling+organizes+the+oral-aboral+axis+of+the+sea+urchin+embryo.">Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.</a> Dev Cell. 6, 397-410 (2004).</li><li>Bradham, C. 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=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+sea+urchin+embryos+by+using+a+CRISPR/Cas9+system.">Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.</a> Dev Biol. 409, 420-428 (2016).</li></ol>

The authors have nothing to disclose.

这里介绍的方法是..许多实验室正在早期海胆发育期间使用类似测定解剖示出使用胚胎与基因组和形态比脊椎动物复杂较少了解信号转导途径和GRNS理事基本发育机制的功率的一例信令涉及其他细胞命运规范事件的途径（ 如缺口，刺猬，TGF-β和FGF信号）27，28，29，30，31。</s...

基因调控网络（GRNS）和信号转导途径建立胚胎发育过程中的基因被用于构建成年动物体计划的空间和时间表达。细胞至细胞的信号转导途径是这些调控网络的基本组成部分，提供了由该小区进行通信的装置。这些细胞相互作用建立和胚胎1,2中提炼的和之间的各种地区管理和分化的基因的表达。分泌细胞外调制器（配体，拮抗剂），受体和共受体之间的相互作用控制的信号转导途径的活性。细胞内分子的各种各样转导导致改变的基因表达，分裂，和/或形状的小区的这些输入。虽然许多在主要途径在细胞外和细胞内水平所使用的关键分子是公知的，它是由于在很大程度上各个信号通路的复杂性的不完整的知识。此外，不同的信号传导途径常常彼此正或负的细胞外，细胞内相互作用或者，和转录水平3，4，5，6。重要的是，信号转导通路的核心组件是高度保守的所有后生动物物种，并且，显着地，大部分的主要信号传导途径经常在许多物种中特定比较从密切相关的生物门生物体时执行类似的发展功能7，8，9， 10，11。 在开发过程中的信令的研究是在任何生物一个艰巨的任务，并有是在大多数后口模型（脊椎动物，无脊椎动物脊索动物，半索动物和棘皮）学习信号通路几个显著挑战:1）在脊椎动物中有大量的可能的配体和受体/共同调制的相互作用，细胞内的转导分子，以及由于基因组12，13，14的复杂性不同的信号通路之间潜在的相互作用; 2）配合物的形态和形态发生的运动在脊椎动物常常使其更难以解释在与信号转导途径之间的功能相互作用; 3）在多数非棘皮动物脊椎动物后口模型物种分析由孕的短窗口具有一些被囊动物物种15,16的异常的限制。 该海胆胚胎具有几个上述限制并提供了许多独特的品质在体内进行信号转导途径的详细分析。这些包括如下:1）在海胆基因组的相对简单显著减少了可能的配体，受体/共受体和细胞内转导分子的数量的相互作用17; 2）控制胚层和主要胚胎轴的说明书和构图的GRNS被很好地建立在海胆的胚胎，在细胞/地区的调节上下文的帮助理解接收的信号18，19; 3）许多信号转导通路可以早期卵裂和原肠阶段之间，当胚胎是由单层上皮细胞的形态比较容易分析进行研究; 4）涉及的分子d。在信令海胆很容易操纵的途径; 5）很多海胆都妊娠一年（ 如紫色球海胆和Lytechinus 10 斑叶至11个月）。 在这里，我们提出了一个方法，系统地，高效地刻画指定和海胆胚胎模式的领土，说明这几个无脊椎动物模型系统在复杂的分子机制研究提供了优势的信号通路的组成部分。

<table><tbody><tr><td>Translational-blocking morpholino and/or splice-blocking morpholino</td><td>Gene Tools LLC</td><td>Customized</td><td>More information at www.gene-tools.com</td></tr><tr><td>Glycerol</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>FITC (dextran fluorescein isothiocyanate)</td><td>Invitrogen, Life Technologies</td><td>D1821</td><td>Make 25 mg/mL stock solution</td></tr><tr><td>Paraformaldehyde 16% solution EM Grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>MOPS</td><td>Sigma Aldrich</td><td>M1254-250G</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>23336-0010</td><td></td></tr><tr><td>Formamide</td><td>Sigma Aldrich</td><td>47671-1L-F</td><td></td></tr><tr><td>Yeast tRNA</td><td>Invitrogen</td><td>15401-029</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Sigma Aldrich</td><td>G9023-10mL</td><td></td></tr><tr><td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td><td>Roche</td><td>11 093 274 910</td><td></td></tr><tr><td>Tetramisole hydrochloride (levamisole)</td><td>Sigma Aldrich</td><td>L9756-5G</td><td></td></tr><tr><td>Tris Base UltraPure</td><td>Research Products Internationall Corp</td><td>56-40-6</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-10</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>7786-30-3</td><td></td></tr><tr><td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td><td>Roche</td><td>11 383 221 001</td><td></td></tr><tr><td>4 Nitro blue tetrazolium chloride (NBT)</td><td>Roche</td><td>11 383 213 001</td><td></td></tr><tr><td>Dimethyl Formamide</td><td>Sigma Aldrich</td><td>D4551-500mL</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9541-5KG</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma Aldrich</td><td>M7506-2KG</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma Aldrich</td><td>C1016-500G</td><td></td></tr></tbody></table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

在海胆胚胎我们已经表明，3种不同的Wnt信号分支（的Wnt /β-catenin的，Wnt基因/ JNK，和Wnt / PKC）4，25相互作用形成在管辖前后（AP）的图案形成一个Wnt信号网络。其中的一个信号事件的最重要的结果是，初始广泛表达前神经外胚层（ANE）GRN变得由原肠胚形成的开始（24高倍视野在S紫石 ）限制到周围的前极小领土。这些结果?...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

这个视频文章详细描述了可用于系统地和有效地描述在许多脊椎动物胚胎复杂的信号通路和调控网络的组件的体内方法直截了当。

简单的力量:海胆胚胎作为<em&gt;体内</em&gt;对于研究复杂的细胞与细胞间信号传导网络交互开发模式

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

the-power-simplicity-sea-urchin-embryos-as-vivo-developmental-models

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Cell Biology

Developmental Biology

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Unit 1

Principles of Cell Signaling

SCIENTISTS IN ACTION

7.8K 次观看.  Mississippi State University. 这个视频文章详细描述了可用于系统地和有效地描述在许多脊椎动物胚胎复杂的信号通路和调控网络的组件的体内方法直截了当。 

视频: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

科研

发育生物学

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

生物学

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 ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

作为一个型号的遗传生物鳉胚胎显微注射

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

发育（青蛙）胚胎中的细胞谱系引导质谱蛋白质组学

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

化学

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

从斑马鱼胚胎中产生幼稚的胚泡外植体

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

评估外胚层上皮细胞在颅面发育的信号属性

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

简单的力量:海胆胚胎作为

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