资助由国立卫生研究院 (NIH: K01-DK087814-01A1 到 D.L.S.), 加利福尼亚州立大学生物技术教育 & 研究计划 (CSUPERB: 脊椎动物造血龛的分子控制到 D.L.S.)来自加州州立大学的研究生研究办公室 (A.C.B.)。

加州州立大学动物保育和使用委员会 (IACUC) 咨询委员会批准了下文所述的所有方法。
1. 漂白 48 hpf 斑马鱼胚胎
<ol><li>与 15 cm (w) x 15 cm (l) x 7 cm (h) 塑料容器创造3洗站: 1) 与1毫升的5% 漂白在1000年毫升胚培养基 (E3; 见表 1), 2) 具无菌 E3 和 3) 具无菌 E3。确保每个容器有500毫升的溶液淹没胚胎。 注意: 对于每个测试的条件, 至少需要10胚胎。这种洗涤程序可容纳多达200胚胎。</li>
	<li>用转移吸管将胚胎放进茶叶过滤器中, 在洗涤站 #1 浸泡蛋5分钟, 取出茶叶滤网 (内胚), 放入洗涤站 #2 5 分钟;重复洗涤站 #3 为最后5分钟。</li>
	<li>最后一次洗涤后, 将其从茶叶过滤器中的胚胎翻转过来, 将其倒置到干净的 10 cm 培养皿中, 然后用无菌 E3 和转移吸管轻轻冲洗茶叶滤网的胚。</li>
</ol>2. Dechorionate 胚胎
<ol><li>用转移吸管, 去除和丢弃尽可能多的 E3 从培养皿和添加500μ l dechorionation 蛋白酶 (10 毫克/毫升) 的胚胎。室温孵育5分钟, 轻轻拍打培养皿的一侧, 完全去除 chorions。</li>
	<li>用一个血清吸管, 加入20毫升的无菌 E3 来稀释蛋白酶。允许胚胎沉淀, 用转移吸管移除 E3。重复这个洗涤步骤3次, 以消除所有的 dechorionation 蛋白酶的痕迹。</li>
</ol>3. 准备胚胎标本
<ol><li>使用 P1000 吸管, 将10胚胎放入单一的无菌1.5 毫升离心管。</li>
	<li>用吸管除去 E3 并丢弃。 注意事项: 移液管作为胚胎, 可以很容易地在下列洗涤步骤中移位和丢弃。</li>
</ol>4. 准备分离的胚胎
<ol><li>将样品转移到层流罩, 并通过加入1毫升无菌 E3 清洗胚胎。允许胚胎沉淀到管子底部, 用 P1000 吸管除去上清液。重复共3洗。</li>
	<li>最后一次洗涤后, 删除 E3 和丢弃。添加1毫升的10毫米糖 (E3), 以去除粘液涂层 (和任何孢子, 酵母, 或可能被困在它的细菌) 周围的胚胎斑马鱼。水平放置离心管, 室温下在层流罩内孵育25分钟。</li>
</ol>5. 胚胎分离
<ol><li>洗涤样品3次与1毫升无菌 DPBS (与 Ca2 +和 Mg2 +)。最后一次洗涤后添加500μ l DPBS (与 Ca2 +和 Mg2 +), 并添加5μ l 5 毫克/毫升 (26 U/毫升) 离解蛋白酶。 注意: 此酶需要 Ca2 +和 Mg2 +, 以确保 DPBS 含有这些离子。</li>
	<li>在 180 rpm 的水平轨道振动筛上孵育37° c 的样品, 在层流罩和研制胚中进行60分钟的取样, P1000 直到样品完全分离。 注意: 定期检查胚胎以确定它们何时分离。胚液应有一定数量的组织存在;它不应该是完全同源的。它是可能的过度消化, 将毁坏 HSPCs (参见补充图 1)。</li>
</ol>6. 游离胚胎的制备
<ol><li>将10分离的胚胎移到5毫升聚苯乙烯圆底管的顶部, 有35μ m 的细胞过滤器盖。</li>
	<li>使用吸管, 用无菌 PBS 冲洗培养管 (没有 Ca2 +或 Mg2 +), 并将解决方案传递给5毫升聚苯乙烯管, 其中含有6.1 步的过滤细胞。重复直到4毫升的液体是存在于5毫升聚苯乙烯管。 注意: 在这个阶段, 只要不包含 Ca2 +或 Mg2 + , 就可以使用 PBS、DPBS 或其他等等等渗缓冲解决方案。
</li>
	<li>离心管在4° c 和 300 x g 为 5 min 粒度均匀细胞样品。用吸管, 从圆底管上取出并丢弃上清液。注意不要扰乱在试管底部的细胞颗粒。重100μ l PBS 中的细胞。</li>
</ol>7. 纤维素的制备
<ol><li>生成1x 完整纤维素 (表 1) 库存解决方案, 并在3毫升注射器和 16 G 针的无菌圆底14毫升管中添加2.5 毫升。对每个测试的条件使用一个管。</li>
	<li>
添加细胞因子, 小分子, 或其他代理, 以调查每个样本。
	<ol>
<li>为髓质分化, 添加1% 鲤血清和0.3 毫克/毫升重组斑马鱼粒细胞集落刺激因子 (Gcsf)34到纤维素培养基。请参见博et al.37以充分说明如何生成鲤鱼血清和重组细胞因子。</li>
		<li>为红分化, 加入1% 鲤血清和0.1 毫克/毫升重组斑马鱼促红细胞生成素 (Epo)38纤维素培养基。</li>
		<li>要检查多向祖, 添加 Epo 和 Gcsf。 注意: 细胞因子和添加剂不应总体积的10% 以上, 因为培养基不会粘性足以辨别个体的菌落。</li>
	</ol>
</li>
</ol>8. 将游离胚胎加入纤维素
<ol><li>使用吸管, 添加100μ l 分离的10胚胎到表面的准备纤维素与重组细胞因子和鲤鱼血清。盖子和涡旋轻轻地完全质样品。每管现在应该包含10胚胎的游离组织。</li>
	<li>使用3毫升注射器和16克针, 分1毫升的样本分为2独立的 35 mm 培养皿。确保样品完全分散在培养皿中。对每个示例重复此操作。</li>
	<li>将 35 mm 培养皿中的细胞纤维素成15厘米的培养皿。每15厘米的菜, 添加一个35毫米的 Petri 板与5毫升的无菌水 humidify 样品。孵育在32° c 和 5% CO2为 7-10 天。</li>
</ol>9. HSPC 源集落形成单元 (CFUs) 的可视化
<ol><li>在孵化7天后, 将样品放置在倒置显微镜下, 在 40-100X 处进行可视化和计数。在这一点上, 个别的殖民地可以与吸管分离, 并受到染色和/或基因分析。</li>
</ol>

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L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 133, 11-53 (2016).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat Immunol. 4, 1238-1246 (2003).</li><li>Kobayashi, I., 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=Characterization+and+localization+of+side+population+(SP)+cells+in+zebrafish+kidney+hematopoietic+tissue.">Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue.</a> Blood. 111, 1131-1137 (2008).</li><li>Henninger, J., 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=Clonal+fate+mapping+quantifies+the+number+of+haematopoietic+stem+cells+that+arise+during+development.">Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development.</a> Nat Cell Biol. 19, 17-27 (2017).</li><li>Tamplin, O. J., 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+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160, 241-252 (2015).</li></ol>

作者没有什么可透露的。

斑马鱼模型系统已成为研究原始和最终脊椎动物造血的有效、有效和廉价的模型。快速、廉价和目前很少的技术难题的产生可以用来测试小分子, 分析突变的胚胎, 以及阐明对 HSPC 生物学重要的分子通路。体外HSPCs 从成人斑马鱼中电镀是一种研究诱变、细胞因子和造血缺损的有效方法。这项协议的基础上, 这些研究, 但利用胚胎斑马鱼。将这些化验结果应用于胚胎斑马鱼, 可以更快地收集数据、使用更少的物理空间、使用更少的药物 (用于药物筛选), 并允许检测 mRNA 过度表达和/或吗 (MO) 的特定基因通路的击倒。重要的是, 它也允许研究的造血阶段, 只发生在早期脊椎动物的发展。
由于这种方法的灵活性, 它可以很容易地被修改, 以满足多种不同的需求。对该协议的一个修改是对发展时序的调制;改变胚胎使用的年龄允许 HSPC 世代的定量在时间。这也可以允许研究 HSPCs 的不同子集, 如 erythromyeloid 祖6, 它出现在 36 hpf 之前, 与善意干细胞3,4,5,8, 出现之后.目前还没有人能够在发育中的胚胎中分离和鉴定更多限制性的斑马鱼 HSPC 亚群, 但是这些实验是允许的。重要的是, 在改变动物的发育阶段, 添加到纤维素中的细胞数量也是最优化的;太少的动物不会产生殖民地, 而太多的人会创造出无数的 HSPC 殖民地。添加不同的细胞因子是另一种修改, 可以适应特定的实验。添加 Epo 将允许定量的红殖民地, 而 Gcsf 将允许计数髓细胞殖民地。添加这两种细胞因子将允许多血统的 CFU 世代。重要的是, 这些化验结果允许测试的假定细胞因子及其对 HSPC 增殖和分化的影响。这些化验也可以修改, 以执行大规模的药物测试;胚胎在发育过程中可以在不同的时间点接触到不同浓度的化合物, 以确定这些化学物质对血液生产是否有正面或负面的影响。最后的方法, 这些实验可以修改的是, 胚胎可以注射在一个细胞的阶段与特定的 mRNA 或 MOs, 这将过度或击倒特定的基因, 分别。在发展为 24-48 小时后, 这些胚胎可以受到这些化验, 以确定是否增益或失去功能的特定基因发挥了重要作用, HSPC 的形成, 增殖和分化。同样重要的是要注意, 利用不同的斑马鱼转基因菌株将有助于速度和方便的收集和解释数据。例如, 如果一个人对红开发感兴趣, 很容易在gata1:D 失调胚胎40中进行这些实验, 其中有红特异的转基因 Gata1 转录因子推动 DsRed荧光.这样, 在分析红菌落时, 可以利用荧光显微镜。同样, 这些研究可以在有多个转基因的动物身上进行, 同时寻找多个细胞谱系。这项技术将有助于识别多向 HSPCs;虽然红和髓样的殖民地很容易区分的形态学, 它是很难确定的殖民地, 包含 bipotential 祖, 没有可视化的存在多血统特定的转基因表达在同一殖民地.
虽然这些化验有很多好处, 但在每次进行这些实验时, 必须设置足够的控制;技术上的细微变化可能会改变结果和对发现的解释。例如, 如果您的技术不精确, 则在不同的时间执行实验时, 可以获得不同数量的 HSPCs;要处理这种可能性, 应该始终包括一个适当的控制。如果进行实验来测试 mo 对发育的影响, 那么控制胚胎 (无论是 uninjected 还是注入控制钼) 都应该与实验样品一起处理。如果实验是为了测试某种特定药物的效果而设计的, 那么应将未经处理的样品作为对照。最后, 如果测试一个特定的细胞因子对发育的影响, 那么同样的离合器的动物也应该同时进行处理和检查, 而不需要将细胞因子添加到文化中。
虽然这种体外化验是简单的执行和结果容易解释, 一些潜在的问题可能会出现在过程中。如果在7天的文化中没有发现殖民地, 可能会发生一些问题。首先, 检查, 以确保细胞因子是纯的, 并在适当的浓度;根据细胞因子的产生, 可能需要改变浓度并优化其活性。通常出现的另一个问题是过度消化胚胎, 这会破坏 HSPCs;注意消化步骤, 并改变时间, 如果没有殖民地被反复看到。最后, 纤维素和鲤鱼血清的混合可能是一个问题, 所以要确保所有成分的添加和混合正确。除了技术问题, 重要的是要认识到殖民地可能需要更长的发展在某些情况下;通常小的殖民地在另外的3天文化以后是更加明显的。过去的10天不建议, 因为纤维素开始干燥不久之后。另一个常见的问题是, 有时殖民地往往收集周围的 35 mm 培养皿边缘;彻底扫描盘子, 尤其是边缘, 是必不可少的。此外, 重要的是要记住, 这些殖民地是发展在一个厚, 粘性的材料;当聚焦显微镜时, 将焦点上下调整以观察稍有不同的平面--这些菌落不会坐在塑料盘子的底部。
这些体外化验有很多优点, 但也有一些局限性。最大的问题是, 这些化验不能确切证明, 一个祖是一个 HSC-只能通过长期重建的辐照动物证明。此外, 不是所有的细胞因子已经定义和调查的斑马鱼, 这限制了 HSPCs 的类型, 可以调查。例如, 没有发现斑马鱼淋巴支持的细胞因子, 以防止在这些化验中的 B 和 T 细胞生产。此外, 在培养多能 HSPCs 时, 如果添加特定的细胞因子 (或数量) 能使某些造血通路的分化下降, 还不清楚。另一个潜在的警告是, 这种化验不能有效地辨别是否 HSPCs 实际上存在于一个样本中, 但由于某种原因无法区分 (或其他不可行)。与大多数体外检测一样, 应始终进行进一步的实验以验证结果。除了这些问题, 许多信息可以从这些克隆化验中得到。
这些化验是重要的, 因为它们可以快速和有效地筛选造血缺损。研究人员可以用 MOs 和板块消化的动物来摧毁某些基因通路, 以观察基因是否在造血中起作用。此外, 突变鱼 (以前产生或新产生的突变屏幕) 可以快速和容易地评估。这些化验还允许有效的药物筛选化验活, 整个生物体, 以确定 HSPC 生物学的调制, 同时也能够观察, 如果药物有负面影响的发展或生存。这些化验还允许定量的 HSPCs 在发展中的斑马鱼在不同的 timepoints。虽然多项研究试图定量 HSC 数字在成人7,27,30,41和胚胎斑马鱼3,4,42,43, 没有研究定量斑马鱼的不同数量的血统限制 HSPCs。此外, 这些化验可以用来检查和调节分子通路的重要的血液细胞的形成和分化, 这一过程仍然是谜。

造血是使生物体存活所需的大量成熟血细胞的过程。这是一个关键的发展过程, 涉及分化的造血干细胞 (干细胞) 到各种发育限制细胞类型, 包括成熟的血液。这些干细胞必须自我更新, 使系统永远不会枯竭, 他们必须坚持从早期胚胎发育直至死亡。在脊椎动物中, 需要不断分化和增殖造血干和祖细胞 (HSPCs), 以充分补充大多数的血液细胞后有丝分裂和每天回收。干细胞先分化成限制性祖细胞的亚群, 生成成熟的血细胞;常见的淋巴祖细胞 (CLPs)1, 最终产生 T、B 和 NK 淋巴细胞, 以及常见的髓细胞祖 (中医)2 , 产生粒、红细胞、巨噬和巨。这些祖细胞致力于产生特定的细胞谱系, 并进一步分化为更发育的限制性前体细胞, 如髓红祖 (欧洲议员), 产生红细胞和血小板, 或粒细胞巨噬细胞祖 (gmp), 可产生嗜、嗜酸性粒细胞、嗜中性和巨噬细胞2。识别和隔离这些祖细胞允许识别重要的分子通路参与造血分化和许多造血疾病, 如白血病出现时, 这些前体细胞未能正确区分.
在过去的几十年中, 斑马鱼 (斑马鱼) 模型系统已经成为胚胎和成人造血研究的关键研究工具。斑马鱼是适于遗传分析, 是 phylogenetically 最低的脊椎动物模型物种, 有类似的血管和造血系统的人。斑马鱼胚胎发育前子宫,并在48小时后受精 (hpf) 生成 HSPCs3,4,5,67, 8。斑马鱼也高度肥沃, 雌性在一个单一的离合器中放置超过100鸡蛋, 允许大样本大小和实验复制。斑马鱼的胚胎是光学透明的, 允许对造血系统进行显微可视化。斑马鱼的一些荧光转基因线标记干细胞如runx1: EGFP 鱼类9、 cd41: EGFP 鱼10和kdrl:mCherry;cmyb: GFP3双阳性动物, 允许现场直播, 实时可视化 HSC 的出现和扩展在体内3,4,7,8, 9。斑马鱼的快速生成时间和发育前子宫导致其在诱变研究中的应用11,12,13,14,15和药物筛选16,17,18,19,20 , 用于对人类血液疾病有治疗前景的化合物。总的来说, 造血系统的保护, 转基因线的存在和容易的发展, 和快速的再生时间使斑马鱼成为一个廉价, 快速, 灵活, 理想的造血研究模式。
在哺乳动物的造血系统中已经开发出许多分离和检测干细胞的方法。调查人员可以利用细胞表面感受器的组合来标记干细胞21,22,23,24, 以及利用干细胞的能力来流出染料25,26。在它们被标记后, 荧光活化细胞分选 () 允许他们的物理分离。证明细胞是 HSC 需要照射宿主动物来破坏内源性 HSPCs, 移植假定的干细胞, 并观察所有成熟的血细胞类型的长期的多谱系重组。这些检测方法在小鼠中很好的工作, 因为有许多细胞表面抗体对造血细胞和近交系小鼠菌株, 促进免疫匹配的移植。然而, 很少有斑马鱼造血细胞表面抗体产生27, 阻碍了干细胞的识别和分离。最常见的方法来标记和孤立斑马鱼干细胞是与转基因动物, 藉以细胞特异启动子序列驾驶荧光蛋白的表达。研究在背主动脉腹壁上进行了可视化和枚举, 利用这一技术的显微镜干细胞3,4,8,9。其他实验室产生了斑马鱼的克隆菌株28,29 , 并在 MHC 匹配的动物中进行了成功的移植30。然而, 这些技术是成本高昂的许多实验室, 在技术上是困难的, 是费时的。为了解决这些问题, 实验室已经生成了几个体外化验, 以测试 HSPCs 的存在、增殖率和差异化能力31、32、33, 34、35、36。这些化验显示 HSPCs 的增殖和分化在体外31,32,33,34,35,36, 抢救造血缺损36, 以及一种有效的发现和检测细胞因子的方法33,34,35。它们也被用来鉴定负责 HSPC 生物学的基因31,32。在这项研究中, 我们进一步采取这些化验, 允许定量的 HSPCs 在发展中的斑马鱼胚胎。这些化验也可以用来定量的数量 HSPCs 突变动物和动物治疗的造血破坏性药物。从本质上讲, 这些化验是快速的, 目前很少的技术挑战, 是廉价的方法, 定量 HSPC 数字, 检查其增殖, 并调查块的分化。

<table><tbody><tr><td>10% Bovine Serum Albumin in Iscove&#039;s MDM</td><td>StemCell Technologies&amp;nbsp;</td><td>9300</td><td></td></tr><tr><td>DPBS (10x) with Calcium&lt;sup&gt;2+&lt;/sup&gt; and Magnesium&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080-055</td><td></td></tr><tr><td>HyClone PBS (1x)</td><td>GE Healthcare Life Sciences</td><td>sh30256.01</td><td></td></tr><tr><td>&amp;nbsp;DMEM 500 mL</td><td>Corning CellGrow</td><td>10-017-CV</td><td></td></tr><tr><td>&amp;nbsp;Ham&#039;s F12 500 mL</td><td>Corning CellGrow</td><td>10-080-CV</td><td></td></tr><tr><td>FBS 500 mL</td><td>Gemini Bio-Products</td><td>100-108</td><td></td></tr><tr><td>HEPES 100 mL (1 M)</td><td>Gibco life technologies</td><td>15-630-080</td><td></td></tr><tr><td>Penicillin/streptomycin (5000 U/mL&lt;br/&gt; and 5000 mg/mL) with L-Glutamine (200 mM)</td><td>Corning Mediatech</td><td>30-009-CI</td><td></td></tr><tr><td>Gentamycin Sulfate 10 mL (50 mg/mL)</td><td>Corning Mediatech</td><td>30-005-CR</td><td></td></tr><tr><td>1.5 mL MCF tube</td><td>FisherBrand</td><td>05-408-129</td><td></td></tr><tr><td>3 mL 23gx1 injection needle with Luer lock</td><td>BD Safety Glide</td><td>305905</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube with cell strainer cap</td><td>Corning Falcon</td><td>352235</td><td></td></tr><tr><td>Methocel MC</td><td>Sigma-Aldrich</td><td>64630</td><td></td></tr><tr><td>14 mL Polystyrene round bottom tube</td><td>Corning Falcon</td><td>352057</td><td></td></tr><tr><td>10 mm polystyrene easygrip Petri dish</td><td>Corning Falcon</td><td>351008</td><td></td></tr><tr><td>Librease TM</td><td>Roche Sigma-Aldrich</td><td>5401119001</td><td>dissociation protease</td></tr><tr><td>Pronase</td><td>Roche Sigma-Aldrich</td><td>11459643001</td><td>dechorionation protease</td></tr><tr><td>15 cm Petri dish</td><td>Corning Falcon</td><td>351058</td><td></td></tr><tr><td>AB</td><td>Zebrafish International Resource Center (ZIRC)</td><td>ZL1</td><td>zebrafish strain used</td></tr><tr><td>Dithiolthreitol (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr></tbody></table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

造血是一种必需的细胞过程, 造血干细胞和祖细胞 (HSPCs) 分化成含有成熟血液的不同细胞谱系的众多。隔离和识别这些 HSPCs 很困难, 因为它们被定义为事后; 它们只能在分化成特定的细胞谱系后定义。在过去的几十年里, 斑马鱼 (斑马鱼) 已经成为研究造血的模范有机体。斑马鱼胚胎发育为宫内, 48 小时后受精 (hpf) 已经产生了明确的 HSPCs. 通过移植和随后的研究, 对 HSPC 分化和增殖能力进行了评估。重组的造血系统, 除了可视化专业的转基因线与共焦显微镜。然而, 这些化验是成本高昂, 技术上的困难, 和耗时的许多实验室。开发一个体外模型来评估 HSPCs 将是成本效益高, 速度更快, 并目前较少的困难相比, 先前所描述的方法, 允许实验室迅速评估突变和药物屏幕, 影响 HSPC 生物学。这本新的体外测定 HSPCs 的方法是通过电镀全斑马鱼胚胎和添加外源因素, 促进只有 HSPC 分化和增殖。胚胎被分离成单个细胞, 并镀有 HSPC 支持的集落刺激因子, 导致它们产生由单个祖细胞产生的菌落形成单元 (CFUs)。这些分析应允许更仔细地检查的分子通路, 负责 HSPC 增殖, 分化和调控, 这将使研究人员了解的基础脊椎动物造血和其失调在疾病期间。

为了评估 HSPC 的数量在胚胎斑马鱼, 48 hpf 胚胎被消化, 镀在纤维素与外源性造血支持生长因子, 并孵育7天 (图 1A)。7天后, 对菌落形成单元 (CFUs) 进行了枚举 (图 1B) 和映像 (图 1C)。通过控制不同的细胞因子增加, 你可以控制的类型的殖民地产生: Epo 是必要的红分化和 Gcsf 是必要的髓细胞分化。鲤鱼血清类似于胎儿牛血清在哺乳动物组织培养中的应用;它是一般生长因子的来源。在图 1中, 添加了 Epo、Gcsf 和鲤鱼血清, 允许红、髓细胞和混合 erythromyeloid 菌落的计数。这些殖民地可以看出, 由于其形状和颜色;红殖民地是紧的, 小, 紧凑, 并且有红色由于 hemoglobinization。髓细胞的菌落更大, 其边缘有更多的有运动性的干细胞, 使它们出现皱褶。
在 7-10 天的孵化后, 成功地用 HSPC 支持因子处理的胚胎斑马鱼的电镀会产生 CFUs。重要的是, CFUs 的数量与被镀的胚胎的数量直接相关 (图 1B)。每毫升电镀4或8胚胎 (这与实验开始时的10或20胚胎相关), 分别产生大约1200或 2400 CFUs。我们的协议建议使用10胚胎, 因为在进行更大的实验时, 计算 2400 CFUs 是困难和费时的。由于消化整个胚胎的方法学, 杂屑存在于纤维素, 但可以很容易地从实际的殖民地, 在显微镜下更高的放大率。其他细胞碎片也可能存在, 但唯一的 CFUs 目前将 HSPC 派生, 由于增加了 HSPC 支持的细胞因子。如果 CFUs 很小, 而且不易被人看到, 这些文化可以被培养多达10天, 以鼓励增加增长。然而, 增加孵化不会产生更多的 CFUs;如果 CFUs 不存在, 这表明细胞因子的活性丧失, 超过消化的胚胎 (和随后的破坏 HSPCs; 参见补充图 1), 或不正确的样品电镀。每次执行此实验时都必须运行适当的控件, 以确保技术上的细微变化不会在 HSPC 枚举中产生错误的差异。例如, 如果进行了一项实验来分析 morphant 胚胎中 HSPCs 的数量, 则应与正常的兄弟姐妹一起准备一个样本。如果进行一项实验来比较药物对 HSPCs 的影响, 则应准备和分析与未处理的兄弟姐妹一起进行的样品。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56836/56836fig1.jpg"> 图 1:从 48 hpf 斑马鱼胚胎中枚举 HSPC 衍生 CFUs。(A)纤维素的胚胎电镀概述, HSPC 支持的细胞因子生成造血 CFUs。48 hpf 斑马鱼胚胎被分离和镀1% 完全纤维素与促红细胞生成素 (Epo), 粒细胞集落刺激因子 (Gcsf), 鲤血清, 允许 erythromyeloid 分化。(B)从每毫升4或8胚浓度的样本中 CFUs 菌落形成单位的计数 (分别启动10或20胚胎的试验)。枚举是在100X 的倒置显微镜下进行的。条形图表示 CFUs 的平均数目, 误差线表示标准偏差。(C)纤维素胚电镀的菌落形态学代表。菌落的形态学表明了组成菌落的细胞类型;图像是代表性的红和髓细胞殖民地。CFUs 拍摄于400X。缩放条形图 = 50 μ m。 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56836/56836fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/56836/56836supfig1.jpg"> 补充图 1: 用离解蛋白酶消化胚胎。(A)消化前10个胚胎的图像 (左; 未消化), 经过充分消化 (中间; 消化), 过量消化后 (右; 过度消化)。(B)在(A)中的已消化和经过消化的管道的图像 (7.3X) 中放大。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56836/56836supfig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

在这里, 我们提出了一个简单的方法, 定量造血干细胞和祖细胞 (HSPCs) 在胚胎斑马鱼。HSPCs 从分离的斑马鱼被镀在纤维素与支持因素, 分化成成熟的血液。这使得检测血液缺陷和允许药物筛选很容易进行。

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

development-an-vitro-assay-to-quantitate-hematopoietic-stem

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

7.9K 次观看.  California State University, Chico. 在这里, 我们提出了一个简单的方法, 定量造血干细胞和祖细胞 (HSPCs) 在胚胎斑马鱼。HSPCs 从分离的斑马鱼被镀在纤维素与支持因素, 分化成成熟的血液。这使得检测血液缺陷和允许药物筛选很容易进行。

视频: Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

科研

发育生物学

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

生物学

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

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 ...

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 ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

使用流动细胞测量来检测和量化正在发育的斑马鱼中改变的血液形成

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

在侧群的分离的Myc诱导的T细胞急性淋巴细胞白血病斑马鱼

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

免疫与感染

<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 ...