Name: generation of parabiotic zebrafish embryos by surgical fusion of developing blastulae
Uploaded: 2016-06-11T15:00:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae at JoVE.com

We thank Julie R. Perlin for helpful comments on the manuscript. D.I.S. is supported by grants from the American Society of Hematology, the Cooley's Anemia Foundation, and the NIH (K01DK085217 and R03DK100672). E.J.H. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. B.L. is a Howard Hughes Medical Institute Medical Research Fellow. B.W.B. is supported by an Irvington Fellowship from the Cancer Research Institute and a Young Investigator Award from the Conquer Cancer Foundation of ASCO. L.I.Z. is supported by grants from the NIH (R01CA103846, P01HL032262, and R01HL04880), Taub Foundation for MDS Research, Harvard Stem Cell Institute, and is an investigator of the Howard Hughes Medical Institute.

该协议被批准波士顿儿童医院的动物护理和使用委员会。这个协议是从以 ​​前出版的方法15进行修改。 1.试剂的制备（提前几天或几周） <ol><li>准备1.5％琼脂糖涂层的菜肴。 添加1.5克琼脂糖至100ml斑马鱼的E3介质中的Erlenmeyer烧瓶中。热火，琼脂糖溶解，再倒入约5mL成直径100毫米×20毫米深的培养皿中。 注意:这些都是用于胚胎dechorionation（步骤3.1），并为联体手术（步骤3.3）。在4℃保存琼脂糖涂层菜的几个星期;带他们出去了冰箱和联体手术当天早晨温至室温。 </li><li>制备4％的甲基纤维素。 将4-在100ml的E3的甲基纤维素粉末在锥形瓶中搅拌棒。将在4℃的搅拌盘烧瓶中，设置为最低速度，让它搭配为2天。 <ol><li>间歇用刮刀分手甲基纤维素的白色团块。甲基纤维素的结晶不充分溶解在该浓度。当该溶液变得清晰并出现均匀，没有白色团块残留，等分试样在-20℃下长期贮存溶液进入1.5毫升离心管并冷冻。 注意:甲基纤维素的较低浓度都可以使用;然而，这种较高的百分比，更粘稠甲基纤维素已取得了最好的结果。 </li></ol></li><li>准备高钙林格（HCR）解决方案。 让400毫升HCR溶液，混合8毫升的5M NaCl（116毫最终），400微升3M氯化钾（2.9毫最终），800微升5M的CaCl 2（10mM的终浓度），以及1M HEPES的2毫升（5毫米最终出）与390毫升E3媒体。 注:在4℃下保存HCR解决了几个星期;温至室温连体手术当天的早上。 </li><li>准备5 - 6传输修改巴斯德移液器环囊胚对。简要地加热该吸管在本生灯的结束。然后，使用大钳，弯曲吸管至约45℃的端，而它仍然是热的。 <ol><li>为了确保有可能损坏胚胎，简要地握住吸管尖在火焰周边3秒没有锋利的边缘。这将平滑/抛光移液器的结束。此改性的玻璃巴斯德移液管一起使用，在下面的步骤3.5 10毫升吸管泵（ 图1）。 </li></ol></li><li>准备玻璃针工具囊胚对手术缝合。作为斑马鱼胚胎显微注射完成3针玻璃 - 拉2。使用实验室膜各玻璃针固定到木杆或塑料处理取笑针（ 图1A）的末尾。使用镊子，断绝了针的末端处直径约20 - 30微米。使用一个阶段微米到引导该步骤（图1B）。 注:网元的大小edle前端是重要的。如果前端是太大，难以精确地控制，并且可以引起过度的破坏。如果尖端太小则难以充分地缠绕在胚胎。 </li></ol> 2.胚胎斑马鱼和收集繁殖对设置（-1天0天） <ol><li>建立成年斑马鱼的繁殖对。设置鱼夜联体手术前要进行的。使用分隔分离男性和女性。为了增加期望的胚胎，将获得，设置若干对每一行的可能性。我们发现，一般为20 - 在对50％的产卵。 </li><li>第二天早上，拉分频器，允许交配。收集用细网滤茶任何胚胎，然后将它们转移到含有E3胚胎介质18培养皿。 </li><li>根据需要微注射胚胎（ 例如，用25 PG质粒DNA，200 PG的mRNA，或用1 - 6纳克啉）收集的1小时之内。让他们生长，直到256细胞阶段。如果DNA表达构建体放置在热休克启动子的控制下（ 例如，热休克蛋白70， 图4B），由热震惊parabionts在37℃下控制的基因表达的定时在后一阶段（ 例如 ，在36高倍视野）。 注:对于荧光转基因的替代，荧光葡聚糖可以加入到DNA，RNA或吗啉代喷射混合（ 例如，葡聚糖，级联蓝在2毫克/毫升）。葡聚糖染料不会正常发展干扰和将允许适当的荧光联体融合后下被识别的注入胚。可替代地，着色菌株的联体（ 例如，AB）与非着色的应变（ 例如，卡斯帕）可以用来在稍后阶段，以确定所注入的胚胎。 </li><li>孵育胚胎在28.5°C.Use一个立体镜定期监测其发展，因为他们256细胞附近开发elopmental阶段（约2.5 HPF）。 注意:如果需要，转移胚胎到不同的温度，以确保联体伙伴（ 例如，吗啉代注射的胚胎往往发展比其未注射的同行慢移的未注射的胚胎至RT之间阶段匹配而啉注射的胚胎放置。在28.5℃，将导致其阶段几个小时的发展）调整后。 </li></ol> 3.联体斑马鱼胚胎的产生通过发展囊胚的手术融合<ol><li>作为胚胎接近256细胞阶段（约2.5 HPF），从每一个遗传背景（ 即，遗传突变，转基因系和/或基因操作）转移胚胎成单独的琼脂糖包被的培养皿（ 例如，一个盘用于吗啉代注射胚胎和对照胚胎第二盘）。另外，胚胎转移到清洁，无划痕的玻璃烧杯或玻璃培养皿。 注意:避免使用无涂层的塑料菜肴胚胎会坚持到塑料和被他们的绒毛膜一旦损坏。 </li><li>倒出E3媒体直到刚好足够的液体仍然覆盖胚胎（约7 - 10ml）中。加入200微升50毫克/从灰色链霉菌 （蛋白酶）的细胞外液中分离蛋白酶毫升混合物中。摇动轻轻胚胎每隔几分钟。 <ol><li>当胚胎的50％已经走出自己的绒毛膜，这通常需要5 - 新鲜的E3胚胎中期的 - （20毫升约15）10分钟，丰盛的体积冲洗一下三次。 <ol><li>轻轻拉起来到改性玻璃巴斯德吸管，然后轻轻地消除他们Dechorionate留在他们的绒毛膜任何胚胎。一旦胚胎已dechorionated和清洗，更换HCR解决方案的E3媒体。作为替代蛋白酶，用细镊子手动删除绒毛膜。目标是在60 - 100 dechor可用于每个实验条件ionated胚胎。 注意:这是几倍大于最终将使用更多的，但许多胚胎有可能处理或手术联体期间被损坏。一旦胚dechorionated，保持它们浸没，并且不使它们接触水的表面上，作为表面张力将导致他们被破坏。请记住，使用高剂量的蛋白酶使胚胎走出自己的绒毛膜更快，更同步的时尚。当使用蛋白酶的较低剂量，将胚需要更长的时间从绒毛膜出现并在一个不太同步方式，其可导致胚胎的一部分通过长时间暴露于蛋白酶被损坏这样做。 </li></ol></li></ol></li><li>解冻在最大速度台式离心机的甲基纤维素（在上面的步骤1.2。制造），离心10分钟以沉淀不溶的结晶，这将损坏胚胎或破裂的蛋黄秒。 <ol><li>离心后，用明确的上限比例（体积约75％）。转让1 - 甲基纤维素的1.5厘米直径的滴成被覆琼脂糖1.5％培养皿。小心避免从管的底部起草任何丸状晶体。 </li><li>使用一个标记，以预先在盘的底侧每个液滴的位置。将12 - 每100毫米直径的培养皿甲基纤维素 - （3等分的价值2）15滴。准备根据需要每个实验尽可能多的板块。 注:使用每个液滴为单囊胚对的融合。该标记将指定每个液滴一旦介质被添加-在该点液滴变得几乎不可见的位置。 </li></ol></li><li>准备每个步骤3.3以上制备琼脂糖涂层菜抗生素的补充解决方案，HCR 1个40毫升一份。以各40毫升等分试样的HCR中的50单位/毫升，氨苄青霉素，50单位/毫升，和卡那霉素加青霉素 - 链霉素，在0.5微克/毫升。 <ol><li>当准备进行囊胚融合，认真填写每个含甲基纤维素滴用40毫升抗生素补充HCR解决方案的菜肴。如果HCR溶液中加入太快，它可以破坏液滴。 </li></ol></li><li>附加改性的玻璃巴斯德吸管（将在上面步骤1.4制），以10毫升吸管泵。在一个立体镜收集来自各个背景（ 例如，一个吗啉注射胚胎和一个野生型胚胎）一dechorionated胚胎。 <ol><li>分配的两个胚胎之前，使用巴斯德吸管，使在甲基纤维素降的中间小凹陷，然后轻轻既胚胎存入抑郁症。 注意:在传送过程中，转动巴斯德吸管略微向上，使胚胎沉入巴斯德移液管的弯曲，以便避免与在移液管的顶部或底部的液体的表面的制造接触（这可能会导致到兵卫- [R破裂）。 </li></ol></li><li>沉积胚胎成甲基纤维素后，立即用木杆或塑料处理取笑针上的端部（ 图1A）的凝胶加载吸管尖小心重新定位甲基纤维素中的胚胎，使得它们的动物极点是直接抚摸对方。 <ol><li>利用通过甲基纤维素接近胚胎斯文扫地的动作对其重新定位，而不直接接触他们。让胚胎坐5分钟，使甲基纤维素会沉淀在他们身边。在此期间，装载下一对成另一种降。 注意事项:避免让与胚胎直接接触，特别注意不要触摸卵黄囊，这将容易破裂的任何部分。 </li></ol></li><li>使用玻璃针的工具，小心翼翼地绕每个胚胎在其接触点。用温和的缝制动作中，从第一胚胎细胞可以再次被拉入第二胚胎，然后。在这个运动结束，举行的地方针2 - 3秒，然后绘制它拿走非常缓慢。 注:有一个周期约2 - 3秒后的胚受伤其中两种组织似乎更粘合剂，更可能连接到彼此。因此，我们认为，保持到位针伤人后简要地有助于保持每个胚的伤员组织中的时间的初期接触伤人之后。 </li><li>允许这些胚胎坐10 - 在室温下15分钟。在此期间，不断地缝合在甲基纤维素的其他滴新对。 10后- 15分钟，评估所述第一对是否保持附着（ 图2A）。 注意:如果胚胎更长附着，重复缝合过程只要胚胎大约30％的外包阶段比年轻。这通常允许最多三次尝试胚胎缝合在一起。如果胚胎似乎一直保持附件但Connection并不是实质性的，在以加强它的连接的边缘执行附加缝合。避免摇动或在连体手术和潜伏期移动菜肴。 注意:尽管他们的早期发育阶段和感知的脆弱性，胚胎细胞块可以容忍操纵的大量（ 即，伤人和缝合），仍然正常发育。蛋黄，但是，可以只用轻轻一碰破裂，杀死胚胎。当拼接两个囊胚的玻璃针，不要使每个胚胎的细胞质量和蛋黄之间的界面接触。 </li><li>在同样的菜和媒体（不改变媒体）孵育胚胎O / N为28.5℃。到了第二天早上，胚胎将有大部分溶解甲基纤维素液滴搬出去了。删除未成功融合的胚胎。倒出培养基，并用新鲜25毫升E3替换。 注意:如果使用工作着色应变，加入500μl0.15％（50倍）苯基硫脲（PTU）至终浓度0.003％到方框色素沉着。胚胎需要在PTU仍然持续保持色素沉着的抑制。 </li></ol> 4.显微镜装置，图像采集，处理及分析<ol><li>制备0.8％的低熔点（LMP）琼脂糖:添加琼脂糖至100毫升的E30.8克LMP和热，直到完全溶解。趁热等分试样1毫升LMP琼脂糖成在37℃加热块1.5毫升离心管。在LMP琼脂糖将保持在37℃熔融。添加40微升4毫克/毫升（25倍），pH 7.0的三卡因以每次1毫升等分试样的LMP琼脂糖在37℃。注意:如果使用色素应变工作，加入20微升0.15％（50倍）PTU至每1ml等分。 <ol><li>存储剩余的LMP琼脂糖在密封50ml试管数月，在4℃。 </li></ol></li><li>麻醉联体胚胎加入1毫升4毫克/毫升（25倍），pH 7.0的三卡因被成像到25毫的E3的胚胎已经英寸</li><li>轻轻地摇晃培养皿，使胚胎会在中间池。然后，用宽嘴塑料移液管吸取了在少液尽可能胚胎。转动吸管直立并使得它们沉淀在吸移管的最底部轻轻弹起胚胎。 <ol><li>与胚胎在移液管的底部，由移液管尖轻轻触摸到琼脂糖的表面将它们转移到LMP琼脂糖1毫升等分试样。避免转移多余的液体，因为这会稀释琼脂糖。 </li></ol></li><li>从吸管排出多余液体后，用吸管琼脂糖内轻轻混合胚胎，然后用吸管给所有的琼脂糖和胚胎转移到一个玻璃底的井（1.5号盖玻片），6-孔板中。 注意:确保胚胎转移到成像板后丢弃移液器。残余琼脂糖将设置吸移管内部，renderin克它无效的进一步使用。相反，使用新的移液管各一次。 </li><li>下一个立体镜，可以使用固定在木材处理取笑针的端部的凝胶加载枪头胚胎向下朝向护罩玻璃定位（以确保它们是显微镜物镜的工作距离内），并在所需的取向成像。 注:不断重新定位，直到琼脂糖完全建立。照顾根据该一对胚胎装入联体胚胎是将被成像。鉴于联体胚胎的随机取向，对于一些对可能难以对图像两者胚胎。在这种情况下，回收利用镊子和宽嘴塑料移液管从琼脂糖胚胎，然后从不同的角度重新装入成像。 </li><li>一旦琼脂糖已设置，覆盖2 - 3毫升E3的补充有三卡因（和PTU如有必要）。 </li><li>图像用倒置的宽视场落射荧光，拉斯胚胎ER-扫描共聚焦，或旋转盘共聚焦显微镜。选择最适合于期望的成像目标。换一个全胚胎区​​，使用4倍的目标。选择较高的倍率，比如20倍的目标，将图像特定组织16，19。 注意:如果使用正置显微镜，安装在盖玻片LMP琼脂糖（类似于上文）。倒置玻璃盖玻片上具有真空润滑脂填充有相同的E3-三卡因-PTU 16,19的环的玻璃显微镜载片。 </li><li>过程中获得自由使用图像分析软件包，如NIH图像的J / FIJI 16，19的图像。 注:计数细胞的数量和量化动态，如细 ​​胞迁移和细胞分裂使用分割和跟踪算法16，19。 </li></ol>

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Physiol. 145 (2), 336-352 (1959).</li><li>Goldman, D. C., Bailey, A. S., Pfaffle, D. L., Al Masri, A., Christian, J. L., Fleming, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP4+regulates+the+hematopoietic+stem+cell+niche.">BMP4 regulates the hematopoietic stem cell niche.</a> Blood. 114 (20), 4393-4401 (2009).</li><li>Dieterlen-Li&#232;vre, F., Martin, C., Beaupain, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quail-chick+chimaeras+and+parabionts:+several+new+models+to+investigate+early+developmental+events+in+the+haemopoietic+system.">Quail-chick chimaeras and parabionts: several new models to investigate early developmental events in the haemopoietic system.</a> Folia Biol (Praha). 25 (5), 293-295 (1979).</li><li>Demy, D. L., Ranta, Z., Giorgi, J. 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Methods. 10 (3), 256-258 (2013).</li><li>Anderson, H., 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=Hematopoietic+stem+cells+develop+the+absence+of+endothelial+cadherin+5+expression.">Hematopoietic stem cells develop the absence of endothelial cadherin 5 expression.</a> Blood. 126 (26), 2811-2820 (2015).</li><li>Murayama, E., Sarris, M., Redd, M., Le Guyader, D., Vivier, C., Horsley, W., Trede, N., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NACA+deficiency+reveals+the+crucial+role+of+somite-derived+stromal+cells+in+haematopoietic+niche+formation.">NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation.</a> Nat Commun. 28 (6), 8375 (2015).</li><li>Shah, D. 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The authors declare that they have no conflict of interest.

联体融合已调查在成年鼠模型和鸡胚10-14候选基因的细胞功能的有力工具。最近，囊胚融合方法已用于产生连体斑马鱼胚胎15所述。在本协议中，基于视频的教程用于演示和更好地描述的方法为，以研究在造血发育感兴趣的候选基因的时空，细胞 - 本征的，和细胞外的角色创建不同遗传背景的连体斑马鱼的胚胎超越。 本文中所描述的方法，最近被用于跟踪的HSC...

野生型和转基因动物的遗传马赛克（嵌合体）的创建是调查细胞内在与候选基因1-6的细胞外功能的完善和经典的策略。在斑马鱼囊胚移植已被广泛使用，以产生用于细胞自治7-9的研究嵌合胚胎。取决于感兴趣的组织，但是，它可以是具有挑战性的供体细胞可预测定位到期望的组织（ 例如，血液）1-3，7-9。鼠标遗传学家早就采用连体手术的方法来产生连体生物与共享循环10-14。由于联体动物都有一个共同的血液，即来自一种动物具有不同遗传背景的其他动物的细胞的细胞之间的相互作用，可以研究10-16。近日，Karima Kissa小组优雅证明CRE的能力连体吃斑马鱼胚胎，然后用这个系统来研究造血干祖细胞（HSPC）的迁移15。此外，斑马鱼联体最近用于研究内皮钙5的造血干细胞（HSC）的出现和迁移，16的作用和斑马鱼发育17中，研究中的HSPC利基基质细胞的作用。 不像小鼠，斑马鱼的胚胎是透明的，允许联体发育过程中的细胞的直接可视化，使得系统唯一地强大。斑马鱼中的联体的效用，然而，由一个陡峭的学习曲线，并在胚胎细腻手术连体限制可以没有详细的说明和视觉示范技术上的挑战。该协议的目标是提供一套明确的，一步一步的指示陪如何产生连体斑马鱼胚胎为研究基于视频的教程颞，细胞 - 本征的，或者一个候选基因（s）的显影囊胚的手术融合的细胞外功能。重要的改进和提高parabiont生存和新的实验性应用程序的其他建议包括在内。

<table><tbody><tr><td>Methyl cellulose</td><td>Sigma</td><td>M0387</td><td></td></tr><tr><td>Individual components for E3/HCR:</td><td></td><td>For 1L: 14,61g NaCl, 0,63g KCl, 2,43g CaCl&lt;sub&gt;2&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O, 1,99g MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td></td></tr><tr><td>NaCl (Sodium chloride)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>KCl (Potassium chloride)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (Calcium chloride dihydrate)</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (Magnessium sulfate heptahydrate)</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Hepes (1M ) buffer solution</td><td>ThermoFisher</td><td>15630-080</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>&lt;strong&gt;Antibiotics:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pen/Strep</td><td>gibco by Life technologies</td><td>15140-120</td><td></td></tr><tr><td>Ampicilin sodium salt</td><td>Sigma Life Science</td><td>A0166</td><td></td></tr><tr><td>Kanamycin sulfate from Streptomyces kanamyceticus</td><td>Sigma Life Science</td><td>K1377</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>50 mg/ml Pronase from Streptomyces griseus</td><td>Roche</td><td>11459643001</td><td></td></tr><tr><td>capillary glass used for needles (Capillary Glass &amp;amp; Filaments)</td><td>Sutter Instrument</td><td>ITEM#: BF 100-50-10</td><td></td></tr><tr><td>Teasing needles with wooden handles</td><td>Fisher Scientific</td><td>S07894</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>Fisherbrand</td><td>13-678-20A</td><td></td></tr><tr><td>10 ml pipette pump (green) (Pipette Pump Pipettor)</td><td>Novatech International</td><td>F37898-0000</td><td></td></tr><tr><td>100 mm diameter/ 20 mm deep plastic petri dishes (PETRI DISH, 100/20 MM, PS, CLEAR, WITH VENTS,&lt;br/&gt; HEAVY DESIGN, 15 PCS./BAG )</td><td>Greiner Bio-one</td><td>664102</td><td></td></tr><tr><td>Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable</td><td>ThermoFisher</td><td>D-1976</td><td></td></tr><tr><td>PTU. working stock is 0.003% (50x is 0.15%). for 500 ml, 0.75 g N-Phenylthiourea</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>Tricaine (powder) (Tricaine Methanesulfonato, Tricaine-S)</td><td>Western Chemical Inc</td><td>MS 222</td><td></td></tr><tr><td>LMP agarose (Ultrapure LMP agarose)</td><td>Invitrogen</td><td>16520100</td><td></td></tr><tr><td>plastic transfer pipette (just the wide ended one I think)</td><td>Fisherbrand</td><td>137115AM</td><td></td></tr><tr><td>Glass-bottom 6-well plates used for imaging</td><td>MatTek</td><td>P06G1.5-20-F</td><td></td></tr><tr><td>plastic western gel loading tip fixed on the end of a wood-handled dissecting needle (GELoader tips)</td><td>Eppendorf</td><td>22351656</td><td></td></tr><tr><td>glass cover slips, slides and vacuum grease if mounting for an upright microscope:</td><td></td><td></td><td></td></tr><tr><td>Vaccum grease ( Dow Corning&amp;reg; high-vacuum silicone grease&lt;br/&gt; colorless, weight 5.3 oz (tube) )</td><td>Dow Corning</td><td>Z273554</td><td></td></tr><tr><td>Glass cover slips</td><td>Corning Life Sciences</td><td>2960-244</td><td></td></tr></tbody></table>

generation of parabiotic zebrafish embryos by surgical fusion of developing blastulae

不同的遗传背景的两种动物的手术间生态创建了一个独特的场景来研究细胞内在与感兴趣的候选基因，细胞的迁徙行为，并在不同的遗传设置分泌信号，细胞外源性的角色。由于联体动物有着共同的循环，从一个动物的血液或血源性因素将与其合作伙伴，反之亦然交换。因此，从一个遗传背景的细胞和分子因素可在第二遗传背景的情况下进行研究。成年小鼠的联体已被广泛用于研究衰老，癌症，糖尿病，肥胖，和大脑的发育。最近，斑马鱼胚胎的联体已被用于研究造血发育生物学。与此相反的小鼠，斑马鱼胚胎的透明特性允许细胞的直接可视化的背景下连体，使其成为调查为f的独特而强大的方法undamental细胞和分子机制。这种技术的效用，但是，通过用于产生连体斑马鱼胚胎陡峭的学习曲线限定。这个协议提供了关于如何手术融合不同的遗传背景两个斑马鱼胚胎的囊胚调查感兴趣的候选基因的作用的工序一步方法。此外，联体斑马鱼的胚胎是耐受热冲击，使得基因表达可能的时间控制。该方法不需要复杂的设置和对胚胎发育过程中研究体内细胞迁移，命运说明书中，并分化广阔的应用领域。

与先前发表的研究15一致，斑马鱼胚胎的成功联体融合取决于两个胚胎的分期和取向和甲基纤维素的浓度。只需几个简单的，廉价的工具，产生了手术融合发展囊胚，增长到连体胚胎共享的循环。这些工具包括一个修改巴斯德吸管，10毫升吸管泵，以及其中使用单独的或与一种凝胶装载尖端或玻璃微注射针通过实验室膜固定到端（ 图1）木材处理取笑针...

Watch this Scientific Journal Video about Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae at JoVE.com

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

generation-parabiotic-zebrafish-embryos-surgical-fusion-developing

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.4K 次观看.  Boston Children’s Hospital. This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

视频: Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

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

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

生物学

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

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. ...

<meta charset="utf8"></head><body>用于再生和发育研究的稳健斑马鱼细胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>作者聚焦:绘制发育和再生过程中斑马鱼神经系统中的细胞连接图

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

胚胎和幼体斑马鱼的高分辨率细胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

生产单倍体斑马鱼的胚胎通过<em&gt;体外</em&gt;施肥

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...

联体斑马鱼胚胎一代发展囊胚的手术融合

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微珠植入斑马鱼胚胎

移植嵌合斑马鱼胚胎生成

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

一种简单有效的斑马鱼胚胎移植装置

从斑马鱼胚胎中产生幼稚的胚泡外植体

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

生产单倍体斑马鱼的胚胎通过