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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 25mg/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 ...

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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

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科<em&gt;苜蓿</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

科研

发育生物学

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

研究Wnt信号传导过程中的航空公司图案

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...