作者感谢Frederic Geissmann教授和Christian Schulz教授进行了深入的讨论;汉娜·加纳博士对手稿的批判性阅读，Xavier Montagutelli博士和Jean Jaubert博士以及巴斯德研究所动物设施的工作人员，以支持畜牧业;以及他们的技术援助Pascal Dardenne和一名安进学者Vytaute Boreikaite。 EGP实验室的研究由巴斯德研究机构，CNRS，Cercle FSER（FRM和来自巴斯德研究所和REVIVE联盟的起始包）资助，LI由REVIVE联盟的博士研究生资助。

根据巴斯德研究所（CETEA）批准的机构动物护理和使用委员会进行动物手术。 1. 在 CSF1R MeriCreMer 罗莎 LSL-YFP胚胎子宫脉冲标记<ol><li> 准备4-羟基他莫昔芬（OH-TAM，50 mg / mL）的储备溶液。 <ol><li>在通风柜下，打开25毫克小瓶的OH-TAM，加入250μL乙醇（100％）。 </li><li>将OH-TAM溶液转移到具有截头的2mL微量离心管中，并以最大速度涡旋10分钟。 </li><li>在超声波浴中超声30分钟。 </li><li>在通风柜内加入250μL的PEG-35蓖麻油，得到50mg / mL的储备溶液。 注意:PEG-35蓖麻油是具有两亲性质的溶剂，其结合疏水性分子并将其溶解在含水溶剂中。 </li><li>漩涡约5分钟在超声波浴中以最大速度超声处理30分钟。 </li><li>每个微量离心管（每次注射1次）分装90μL（4.5mg）。 </li><li>储存于4℃下放置1周或在-20℃下长期先前描述的13。 </li></ol></li><li> 准备孕酮（10mg / mL） <ol><li>向25mg孕酮中加入250μL的100％乙醇以在通风柜下制备10mg /100μL悬浮液。轻轻旋转</li><li>加入2250μL高压消毒的向日葵油，在罩下制成10mg / ml孕酮溶液。 </li><li>以最大速度涡旋约5分钟，等分并储存在4°C。请注意，存储时解决方案是清晰的。 </li></ol></li><li> 他莫昔芬 注意:胚胎发育估计考虑阴道栓塞形成的日子为胚胎日（E）0.5。 E8.5单次注射诱导重组进入孕妇Csf1r MeriCreMer女性 12 。补充OH-TAM与37.5μg/ g孕酮，以降低他莫昔芬给药后的流产率。下午1点注射（早晨观察阴道塞）。 <ol><li>在注射的早晨，将OH-TAM溶液的等分试样超声处理10分钟（或直到完全重悬）。 </li><li>在通风柜内加入360μLNaCl 0.9％，得到10mg / mL OH-TAM溶液，并彻底涡旋。超声处理直到溶液澄清并完全重悬（至少30分钟）。 </li><li>预热孕酮至室温。 </li><li>在微量离心管中混合450μL的OH-TAM和225μL的孕酮。涡流。 </li><li>超声处理至少10分钟，并用25G针装入1 mL注射器。 </li><li>在动物设施中，称重怀孕的雌性（ Csf1r MeriCreMer女性处于近交的FVB遗传背景和典型的体重en 25和33g）。 </li><li>通过将计算出的体积缓慢注入鼠标来进行腹膜内注射。取出针后，轻轻按压穿刺伤口，按摩腹部分布OH-TAM。 注意:OH-TAM注射剂量为75 mg / kg，孕酮剂量为37.5 mg / kg。 表1提供注射到怀孕女性的体积。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>小鼠体重（g） </td><td>从混合物注射的总体积（μL） </td></tr><tr><td> 25 </td><td> 281 </td></tr><tr><td> 26 </td><td> 292 </td></tr><tr><td> 27 </td><td> 303 </td></tr><tr><td> 28 </td><td> 315 </td></tr><tr><td> 29 </td><td> 326 </td></tr><tr><td>三十</td><td> 338 </td></tr><tr><td> 31 </td><td> 348 </td></tr><tr><td> 32 </td><td> 360 </td> &lt;/ TR&gt; <tr><td> 33 </td><td> 371 </td></tr><tr><td> 34 </td><td> 382 </td></tr><tr><td> 35 </td><td> 394 </td></tr></tbody></table> 表1:4-OH-他莫昔芬（OH-TAM）的注射体积。单次注射的单次注射量为75μg/ g（体重）的补充有37.5μg/ g孕酮的OH-TAM。 2.卵黄囊（YS）和胎肝（FL）的解剖注意:严格的无菌技术在操作胚胎时是不必要的，除非它们将被用于长期培养。然而，工作区域必须清洁，并在吸收纸上覆盖箔。 <ol><li>制备冰冷的磷酸盐缓冲盐水（PBS）和消化混合物（含有1mg / mL胶原酶D，100U / mL脱氧核糖核酸酶I（DNase I）和3％胎牛血清的PBS）。 </li><li>牺牲怀孕的女性由cer在所需的妊娠日（ 例如胚胎阶段E10.5）的动脉脱位。 </li><li>将皮肤紧贴在生殖器上，并用剪刀在中线做一个小切口。将皮肤拉向头部，完全暴露身体，无毛皮。切开腹肌露出内脏，推动肠道露出两个子宫角。 </li><li>用中等大小的镊子抓住附着在卵巢上的脂肪垫，轻轻拉出子宫。在子宫颈的颈部切开并从腹腔抬起角。去除脂肪垫以完全释放子宫角，并从每侧的卵巢切割角。 </li><li>将角状物置于10毫米培养皿中的冰冷PBS中。在一个末端（颈部末端）快速抓住子宫肌层，并在肌肉层和蜕膜组织之间滑动精细剪刀，以释放与周围蜕膜组织的胚胎。 注意:肌肉倾向于快速收缩提取，所以这一步必须尽可能快。 </li><li>使用一对细镊子切断Reichert膜和胎盘。 </li><li> 轻轻取出卵黄囊，并将其置于含有0.5mL消化混合物的24孔组织培养板中。 注意:在这一步，可以收集胚胎血液。 <ol><li>在切断脐带和卵黄血管后立即将胚胎转移到含有10mM冰冷的乙二胺四乙酸（EDTA）的12孔组织培养板中。使用锋利的精细剪刀斩断胚胎，尽量限制组织扩张。在冰上孵育10-15分钟，收集含有血液的EDTA。 </li></ol></li><li>取出胚胎周围的羊膜</li><li>对于阶段&lt;E11.5，计算体细胞对数以更好地分期胚胎和更好的时间分辨率（每个体细胞对在〜1小时30分钟内发展）。 </li><li>切胚胎我们的头镊子或精剪刀。将头部转移到具有0.5mL消化混合物的24孔板中。 注意:进一步解剖晚期阶段的神经外胚层和脑，用于流式细胞术分析;小心清除周围血管丛。 </li><li>切割后肢上方的胚胎并移除前肢。 </li><li> 要隔离肝脏，用一对精细的镊子打开胸部。夹住心脏前方，轻轻拉动，同时使用第二镊子将器官从身体释放出来。 <ol><li>仔细分离胎儿肝脏与心脏和肠道。将胎肝转移到具有0.5mL消化混合物的24孔板中。仔细监测解剖显微镜下的转移。 </li></ol></li><li>通过聚合酶链反应测定（PCR）收集尾区（或任何其他胚胎部分）进行基因分型。 注意:可以从E10.5胚胎收获其他组织和器官，以使用流式细胞术进行类似的分析。血，头滑雪n，主动脉 - 性腺 - 中肾区（AGM），心脏和神经外胚层可以在E10.5收集。在晚期阶段，也可以收集肺，肾，脾和胰腺。以前描述了不同发展阶段AGM解剖的详细描述14 。 </li></ol> 3.流式细胞术的胚胎组织加工<ol><li>在37℃下将器官（置于消化混合物中）孵育30分钟。 </li><li>将组织和酶溶液转移到置于装有6ml FACS缓冲液（0.5％牛血清白蛋白（BSA））和2mM EDTA的1×PBS中的6孔组织培养板中的100μm筛管上。通过用2mL注射器的黑色橡胶活塞轻轻地糖化来机械解离以获得单细胞悬浮液。 注意:从现在开始，所有步骤应在4°C进行。 </li><li>用巴斯德移液管收集细胞悬浮液，并转移到15 mL管中。 </li><li> SPI在4℃下，320℃，n进行7分钟。通过抽吸弃去上清液。 </li><li> 将沉淀物重悬于60μLFc阻断缓冲液（CD16 / CD32阻断抗体，在FACS缓冲液中稀释1/50）。 <ol><li>将50μL单孔细胞悬浮液每孔转移到圆底96多孔板中。在冰上孵育至少15分钟。 注意:处理少量样品时，也可以在5 mL聚苯乙烯FACS管中进行染色。 </li><li>将剩余的10μL转移到5 mL聚苯乙烯FACS管中，以获得每个组织的样品池。该池将用作对照（ 即每个荧光染料的未染色样品和荧光减去一个（FMO）对照）。将每个荧光灯减去一个（FMO）对照（每个荧光染料一个）50μL的池转移到96多孔板。 注意:每个FMO对照包含来自抗体面板的所有荧光染料偶联抗体，除了该抗体正在测量。 FMO用于识别门边界，并控制多色面板中的光谱重叠。为了正确地解决不同组织之间的背景和自发荧光的变化，重要的是从每个组织的样品池中制备这些对照。 YFP表达的适当对照是来自Cre阴性胚胎的样品，通过PCR基因分型证实。 </li></ol></li></ol>表面抗原染色<ol><li> 在FACS缓冲液中制备抗体混合物（表2）。每个样品准备50μL抗体混合物。 <ol><li>使用以下荧光染料偶联的抗体:抗CD45.2（克隆104）;抗CD11b（克隆M1 / 70）;抗F4 / 80（克隆BM8）;抗AA4.1（克隆AA4.1）;抗体试剂盒（克隆2B8）;和抗Ter119（克隆Ter119）。 </li><li>在FACS缓冲液中为FMO对照制备六种抗体混合物。每个对照样品准备50μL抗体混合物。 没有TE:抗体混合物中的抗体稀释浓度比材料表中指定的最终浓度高两倍。对于具有6个荧光染料偶联抗体的小组，制备6个FMO对照。 表2提供了一个管的抗体混合物和FMO对照的组成。 </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td><td colspan="7">抗体积（μL）（终体积50μL） </td></tr><tr><td>抗体</td><td>克隆</td><td>抗体混合</td><td> FMO CD45.2 </td><td> FMO CD11b </td><td> FMO F4 / 80 </td><td> FMO AA4.1 </td><td> FMO套件</td><td> FMO Ter119 </td></tr><tr><td>抗CD45.2 </td><td> 104 </td><td> 1 </td><td> 0 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>抗CD11b </td><td> M1 / 70 </td><td> 0.5 </td><td> 0.5 </td><td> 0 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td></tr><tr><td>抗F4 / 80 </td><td> BM8 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 0 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td>抗AA4.1 </td><td> AA4.1 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 0 </td><td> 1 </td><td> 1 </td></tr><tr><td>防套件</td><td> 2B8 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0 </td><td> 0.5 </td></tr><tr><td>抗TER119 </td><td> TER119 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0.5 </td><td> 0 </td></tr><tr><td colspan="2"> FACS缓冲区</td><td> 45.5 </td><td> 46.5 </td><td> 46 </td><td> 46.5 </td><td> 46.5 </td><td> 46 </td><td> 46 </td></tr></tbody></table>表2:用于染色和荧光减1（FMO）对照的抗体体积。最终体积为50μL所需抗体积（μL）。 <ol start="2"><li>向96孔多孔板中的样品中加入50μL抗体混合物（染色时的最终体积为100μL）。轻轻移液两次，并在冰上孵育30分钟。 </li><li>在4℃下以320×g旋转96孔多孔板7分钟，弃去上清液。重悬于200μLFACS缓冲液中。 </li><li>用150μLFACS缓冲液重复洗涤步骤（步骤4.3）两次。 </li><li>将样品和对照（FMO和未染色的池样品）通过70μm过滤器过滤到5 ml聚苯乙烯管中。存放在冰上直到获得。 </li></ol> 5.通过流式细胞术鉴定EMP和YS衍生的巨噬细胞注意:该协议使用4激光流式细胞计数器等式进行了优化使用405nm紫激光，488nm蓝色激光，562nm黄色激光和638nm红色激光。 <ol><li>按照制造商的说明准备每个偶联抗体的补珠。使用未染色的样品和补偿珠来优化激光强度。 </li><li>为了识别门边界，对每个抗体使用FMO（荧光减去一个）对照。 </li><li>为了排除死细胞，在获取前1分钟向管（含有200μL染色细胞悬液）中加入1μLDAPI（1mg / ml）。使用强DAPI信号和前向散射参数（FSC）来排除死细胞和碎屑。 注意:使用流式细胞仪的软件在采集过程中绘制门。补偿矩阵和分析可以在使用其他市售软件进行采样后进行。 </li><li>使用前向和侧向散射（FSC vs SSC）来执行活体细胞和双倍体歧视从总事件。 </li><li>在对数刻度轴上创建荧光点图，绘制子门，首先排除红细胞（Ter119 + ），然后识别祖细胞（Kit + ）和造血细胞（CD45 + Kit neg ）（参见图1 ）。 </li><li>创建荧光直方图以量化YS（ 图2 ），胎肝（ 图3 ）和脑（ 图4 ）中不同确定群体之间YFP的标记效率。 </li></ol>

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作者没有什么可以披露的。

造血前体细胞的不同波浪在短时间内部分重叠，这使得每次发育造血波对免疫细胞的贡献的分析技术上非常具有挑战性。 他莫昔芬诱导型Cre系统提供了以时间可诱导的方式标记特定细胞并在胚胎或成年人中进行谱系分析的机会，而不需要离体或体外培养或移植。在他莫昔芬诱导的Cre菌株中，在细菌Cre重组酶和人雌激素受体（CRE-ER）的配体结合结构域的突变形式?...

造血祖细胞在发育过程中有几个相继但重叠的波浪，其骨髓后代保持成年。首先，单能的"原始"祖细胞出现在E7.5-E8.25之间鼠标卵黄囊1,2和产生胚胎的巨噬细胞而没有任何单核细胞的中间。来自原始祖细胞的巨噬细胞是否持续存在于成年大脑中，因为小神经胶质细胞仍然是主动调查的一个主题。第二，红血球前体（EMPs）出现在E8.5的卵黄囊中，进入血液并定殖胚胎。从环境管理计划卵黄囊hemogenic内皮在Runx1的依赖性的内皮到造血过渡3,4出现。虽然EMP可以分化成卵黄囊中的巨噬细胞，但它们也从胚胎期（E）9 5定殖于胎肝，并且不同分化成红细胞，巨核细胞，巨噬细胞，单核细胞粒细胞和肥大细胞6 。来自EMP的巨噬细胞在发育和成体组织中表现出增殖能力。是否EMP源性巨噬细胞分化绕过的单核细胞级仍有争议非常知之甚少其分化途径7,8。最后，造血干细胞（HSCs）在E10.5出现在主动脉 - 性腺 - 中肾区域内的胚胎中，并迁移到胎儿肝脏。长期再增殖能力的HSC仅检测到E11后（在42体节对阶段）9。在那里，他们扩大和区分E12.5直到明确的造血开始转移到骨髓，这在产后生活期间成为血细胞生产的主要部位10 。 sp妊娠和时间重叠的出现以及共同的免疫表型标志物迄今阻碍了我们区分这些胚胎造血祖细胞波的特异贡献的能力。虽然EMP和HSC都以Runx1依赖性方式产生，并表达转录因子Myb和生长因子受体Csf1r（集落刺激因子1受体，也称为巨噬细胞集落刺激因子受体）等，EMP可以区分为HSC由于缺乏淋巴细胞的潜力， 体外和体内都缺乏长期的重建潜力，缺乏表型谱系Sca- 11的表达 。需要遗传命运映射模型来表征巨噬细胞个体发育，因为它们允许以细胞特异性和时间特异性方式靶向胚胎祖细胞。我们在这里介绍我们实验室使用的命运映射协议来区分两个谱系在大多数成年组织中发现的巨噬细胞:HSC衍生的浸润巨噬细胞和不依赖HSC的宿主巨噬细胞。 组织定居巨噬细胞已被追溯到表达细胞因子受体12 CSF1R的Myb独立前体细胞和存在于使用三个互补命运-映射策略6在E8.5-E10.5胚胎。为了研究没有标记胎儿HSC的卵黄囊造血，我们使用转基因株式Cs r r r re re re er er of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of of Csf1r启动子。因此，Cre重组酶在有限的时间窗口内将在Csf1r表达细胞中活跃。当与含有lox-STOP-lox盒（Rosa26 LSL-eYFP ）下游的荧光蛋白的报道菌株一起使用时，它将导致永久性在诱导时存在的细胞的遗传标记，也是其后代。给予E8.5活性形式的他莫昔芬，4-羟基他莫昔芬（OH-TAM），标记EMP和巨噬细胞，而不标记卵黄囊单能"原始"祖细胞或胎儿HSC。因此，我们已经描述了胚胎发育过程中EMPs及其后代的免疫表型，并评估了卵黄囊衍生的巨噬细胞对成年巨噬细胞池的贡献。需要进一步的工作来表征原始祖细胞来源的巨噬细胞是否也使用这种方法进行标记，以及它们是否可以促成成年巨噬细胞池。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 Straight Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>MC19/B Pascheff-Wolff Spring Scissors&#160;</td>
			<td>Fine Science Tools</td>
			<td>15371-92</td>
			<td></td>
		</tr>
		<tr>
			<td>8.5 cm straight scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
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		<tr>
			<td>Anti-Mouse Kit-PE (clone 2B8)</td>
			<td>BD Pharmingen</td>
			<td>553355</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45.2 APC-Cy7 (clone 104)</td>
			<td>Sony</td>
			<td>1149120</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse AA4.1-APC (CD93, clone AA4.1)</td>
			<td>eBioscience</td>
			<td>17-5892</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119)</td>
			<td>Biolegend</td>
			<td>560512</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse F4/80-BV421 (clone BM8)</td>
			<td>BD Pharmingen</td>
			<td>123137</td>
			<td>1/100 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD11b PE-Cy7 (clone M1/70)</td>
			<td>BD Pharmingen</td>
			<td>552850</td>
			<td>1/200 dilution in FACs Buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Fischer Scientific</td>
			<td>12559069</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fischer Scientific</td>
			<td>11570506</td>
			<td></td>
		</tr>
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			<td>Collagenase D&#160;</td>
			<td>Roche</td>
			<td>11088882001</td>
			<td></td>
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			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527-20KU</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353224</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Fischer Scientific</td>
			<td>11874235</td>
			<td></td>
		</tr>
		<tr>
			<td>96 wells U-shape bottom tissue culture plate</td>
			<td>Dutscher Dominique</td>
			<td>353227</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Syringe Plastipak 2 mL&#160;</td>
			<td>Dutscher Dominique</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 100 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon grid 70 &#181;m cell strainers</td>
			<td>Dutscher Dominique</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>Dutscher Dominique</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup GP Millipore filtration kit, 0.2 &#956;m</td>
			<td>Dutscher Dominique</td>
			<td>51246</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>(Z)-4-HYDROXYTAMOXIFEN 25mg</td>
			<td>Sigma</td>
			<td>H7904-25MG</td>
			<td>Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P3972</td>
			<td>Always use appropriate personal protective equipment</td>
		</tr>
		<tr>
			<td>PEG-35 castor oil (Kolliphor/Cremophor EL)</td>
			<td>Sigma</td>
			<td>C5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Sunflower oil</td>
			<td>Sigma</td>
			<td>S5007-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Sigma</td>
			<td>24103-1L-R-D</td>
			<td>Always use appropriate personal protective equipment and use under the fumehood</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E9884-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI, 1 ML (1 MG/ML IN WATER)</td>
			<td>Fischer Scientific</td>
			<td>10116287</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX S B2-R3-V4-Y4 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>B75408</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX Daily QC Fluorospheres</td>
			<td>Beckman Coulter&#160;</td>
			<td>B53230</td>
			<td></td>
		</tr>
		<tr>
			<td>VersaComp Antibody Capture Bead kit (2x5 mL)</td>
			<td>Beckman Coulter&#160;</td>
			<td>B22804</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J</td>
			<td>The Jackson laboratory</td>
			<td>19098</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td>
			<td>The Jackson laboratory</td>
			<td>6148</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of erythromyeloid progenitors and their progeny in the mouse embryo by flow cytometry

巨噬细胞是免疫系统固有的专业吞噬细胞。在稳态状态下，无性巨噬细胞在成体组织中发现，其中它们作为感染和组织损伤的前线哨点。虽然其他免疫细胞从位于骨髓中的造血干细胞和祖细胞（HSPC）不断更新，但已经显示出巨噬细胞谱系，被称为驻留巨噬细胞，而不是从骨髓HSPC输入的组织中自我维持。这个谱系的例子是脑中的小胶质细胞，肝脏中的枯否细胞和表皮中的朗格汉斯细胞等。肠和结肠固有层是唯一没有HSPC独立的巨噬细胞的成体组织。最近的研究已经确定，存在的巨噬细胞来自不同于胎儿造血干细胞（HSC）的祖细胞的胚外卵黄囊造血。在卵黄囊定型造血中，红细胞生成素祖细胞（EMP）会引起红细胞和骨髓细胞，特别是驻极体巨噬细胞。 EMP仅在发育E8.5和E10.5天之间的蛋黄囊内产生，并且早在循环连接时迁移到胎肝，其中它们扩展和分化直到至少E16.5。他们的后代包括红细胞，巨噬细胞，嗜中性粒细胞和肥大细胞，但只有EMP来源的巨噬细胞持续到组织中成年。 EMP出现的瞬态性和与HSC生成的时间重叠使得这些祖细胞的分析困难。我们建立了基于巨噬细胞因子受体Csf1r启动子表达的他莫昔芬诱导的命运映射方案，通过流式细胞术在体内表征EMP和EMP来源的细胞。

通过将OH-TAM的E8.5施用于与携带Rosa26-LSL-eYFP报告物的雄性配对的Csf1r-Mer-iCre-Mer雌性中实现遗传命运测绘。在存在OH-TAM的情况下，切除盒可导致表达Csf1r的细胞中YFP的永久表达。我们从E10.5的胚胎收集了两个造血组织:卵黄囊和胎肝以及非造血组织（神经外胚层）。通过酶学和机械解离获得单细胞悬浮液，并用荧光偶联抗体染色后通过流式细胞术分析样品。排除死细胞和?...

Watch this Scientific Journal Video about Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry at JoVE.com

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

当浸润的巨噬细胞从循环前体连续招募到成体组织时，驻留的巨噬细胞在发育过程中种植其组织，在那里它们被维持而无需进一步从祖细胞输入。最近确定了居民巨噬细胞的祖细胞。在这里，我们提出了驻地巨噬细胞祖细胞的遗传命运映射方法。

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

identification-erythromyeloid-progenitors-their-progeny-mouse-embryo

Research

JoVE Journal

Developmental Biology

12.9K 次观看.  Institut Pasteur. 当浸润的巨噬细胞从循环前体连续招募到成体组织时，驻留的巨噬细胞在发育过程中种植其组织，在那里它们被维持而无需进一步从祖细胞输入。最近确定了居民巨噬细胞的祖细胞。在这里，我们提出了驻地巨噬细胞祖细胞的遗传命运映射方法。 

视频: Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

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 ...

科研

发育生物学

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

步特定老鼠的精子细胞分选流式细胞仪

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

通过机械组织分离和流式细胞术对哺乳动物雄性生殖细胞多特异性纯化的标准化方法

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

应用造血干细胞移植评估骨髓增生异常综合征的起源

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...