巴斯德研究所，INSERM，法新社国立德RECHERCHE ANR（格兰特的淋巴细胞&#39;），兴中未来投资计划和"香格里拉法甲勒驳癌症"的支持。

伦理声明:所有实验均根据巴斯德研究所伦理宪章，批准由法国农业部执行，欧盟的指导方针。一个机械手与小啮齿动物外科手术培训，由农业部法国教育部认证，完成所有手术干预。 注:见附件表1显示了5步计划程序。 1.选用胚胎<ol><li>使用36 C57BL / 6 CD45.2女性，女12例CD45.1，12 C57BL / 6 CD45.1男性和12 C57BL / 6 CD45.2男性。穿越女雌雄基因型会产生胚胎CD45.1 / 2和CD45.1作为TSP或CD45.2用作收件人胸腺叶源的来源。众议院单独所有男性。 </li><li>为了获得胚胎E18 TSP隔离，放置2女在笼子里有一只公CD45.1下午6时许，嫁接实验前21天。检查阴道塞的存在，第二天，中午之前。国家环保总局打分插入女性。 </li><li>以获得E14胚胎由TSP定植，则重复上述（1.2）的方法，用雄性CD45.2，接枝实验前17天。 </li><li>以获得的胚胎以分离E13的TSP，则重复上述（1.2）的方法，用雄性C57BL / 6 CD45.1，接枝实验前16天。 </li></ol> 2.解剖胚胎在一个水平层流罩注:在嫁接实验前两天。 <ol><li>至少5分钟期间通过颈脱位或由CO 2气体的渐进施用牺牲怀孕女性，在一个饱和密闭室。通过心肺运动最终逮捕确保死刑。湿用70％乙醇的腹部。 </li><li>做一个纵行切口在皮肤在中线腹部和提拉肌肤除了打开它。不接触消化道打开与镊子和剪刀腹膜。拉出双歧uter我们并从与剪刀阴道分离它。 </li><li>将子宫在含有40-50毫升DPBS一个90×15毫米的培养皿和切割横向每个胚的蜕膜之间。 </li><li>随着对精尖剪刀和细镊子取出子宫的肌肉膜，取出胚胎的胎盘和卵黄囊之间切割，取出amnios，这是保持胚胎。 </li><li>将胚胎在培养皿中90×15毫米含有的HBSS + 1％胎牛血清（FCS）。比E15更旧的胚胎，除去头上的一把剪刀之前，任何进一步的解剖。 </li></ol> 3.隔离胸腺<ol><li>根据双目放大镜，将胚胎，趴在仰卧位（腹面），在湿纱布床上用品。 </li><li>插入一个镊子纵向地沿胸骨的软骨，捏和打开胸电网。用弯钳，拉胸壁一边想象的Thymic裂片上气管的每一侧。 </li><li>小心地用细镊子捏下面各持有叶，将它们放置在含有1ml HBSS + 1％FCS培养皿。隔离裂片和带有两​​个1毫升注射器+＆G⅜"针除去周围结缔组织，而不破坏胶囊。 </li><li>在3毫升HBSS + 1％FCS的洗叶，以消除血细胞。 注:E15之前，胸腺裂片位于中间的气管和后来熔丝的每一侧，以构成双叶的器官。 </li></ol> 4.悬浮细胞<ol><li>梳理出的叶双目放大镜下，用安装在1毫升注射器2台26摹针，在HBSS + 1％FCS。通过尼龙网过滤细胞悬浮液。 </li></ol> 5.用染色荧光抗体和细胞分选注意:所有的抗体被预先滴定以获得种群最佳定义（titrat离子可以与抗体批次而变化）。 <ol><li>孵育胸腺细胞（E13或E18）为15-30分钟，在4℃下用100微升生物素化的抗体进行染色谱系阳性细胞的鸡尾酒（抗CD19，抗Gr1的，抗TER119，抗NK1.1 ，抗CD11c的，抗CD3，抗CD4，抗CD8，抗CD25）。 </li><li>洗去多余的抗体降速管用4ml的HBSS + 1％FCS在280 XG 7分钟，消除上清，重新悬浮在新鲜HBSS + 1％FCS沉淀。重复离心。 </li><li>在15分钟，在4℃下孵育E18生物素标记的细胞与链霉微珠和洗涤细胞的HBSS 1％FCS的两倍。重悬在2ml的HBSS + 1％FCS中细胞。最E13胸腺细胞是谱系阴性，因此不需要之前细胞分选的MACS富集。 </li><li>通过对LS柱的细胞，根据生产商的说明。当所有的细胞悬液进入列，加入2mL HBSS + 1％FCS。恢复将4ml柱的流过和离心将细胞在280×g下7分钟。 </li><li>重悬要么耗尽E18或总E13胸腺细胞在50μl的TSP抗体混合物（抗CD117的APC，抗CD135 PE，抗CD127 PECy7，抗CD24 FITC，抗CD44 APCCy7和链霉太平洋蓝的粒料），并孵育20分钟，在4℃下在黑暗中。 </li><li>用4ml的HBSS + 1％FCS和洗涤细胞重悬浮细胞于1ml HBSS + 1％FCS中用碘化丙锭。 </li><li>排序的TSP林- CD44 + CD117 + CD24 低 CD127 + CD135 +细胞在流式细胞仪（4激光器）到1.5 mL管含有200微升完全培养基+ 20％FCS。门首先根据其大小和粒度的FSC和SSC通道的细胞。消除死细胞（碘化丙啶染色），门出双峰和得分荧光指数尺度。大约100或600的TSP可以从一个E13或者一个E18胸腺，respectiv获得伊利。 </li></ol> E14殖民6.胸腺肺叶祖:悬滴法<ol><li>照射（30格雷= 30戈瑞）E14从胚胎CD45.2胸腺叶，培养前3小时。 注:E14或E15胸腺裂片已成功地被用作T细胞祖细胞的收件人。使用后妊娠天导致过早坏死因器官的大尺寸收件人胸腺裂片而胸腺裂片在较早妊娠天很差发展，可能是由于上皮细胞的发育不完全。 </li><li>离心机排序的TSP并重新悬浮细胞在培养基（OPTI-MEM，10％FCS的完全培养基，青霉素（50单位/ ml），链霉素（50微克/毫升）和2β巯基乙醇（50μM）），使得35微升包含500 TSP。 </li><li>放置细胞悬浮液的35微升滴60孔酶标板的每一个其它公。 注:35微升液滴可以熔化，如果他们被放置在相邻的孔中。 </li><li>将一个辐射波瓣每一滴的表面上。关闭寺崎板和转动板倒置以让细胞达到下降通过重力根尖极。 </li><li>击中轻轻倒置板的上侧，迫使瓣滑动到液滴的前端缘。孵育倒置酶标板在湿润的培养箱中48小时，在37℃+ 5％CO 2。定植后，无论是文化E14 thymi在FTOC 7，或移植在收件人成年小鼠8的肾囊。 </li></ol> 7.胚胎胸腺器官培养<ol><li>放置3个ml完全培养基在35毫米的培养皿。发布Isopore表面滤膜过滤漂浮在介质上。将重构裂片上用钳子（不超过8裂片上一个膜）膜的边缘。 </li><li>将含有胸腺裂片90毫米的培养皿内，连同35毫米的菜培养皿，无盖，含2水:毫升R，以确保最佳的湿度。孵育12天在37℃+ 5％CO 2。 </li></ol> 8.在移植肾包膜在无菌条件下注:肾实质被结缔组织形成胶囊包围。子囊区域是特别丰富的血液和淋巴管，从而提供一个合适的环境移植物的发展（ 例如胸腺裂片，胰岛或新生儿心脏）。移植物通常是在左肾，因为它比右肾更容易获得。 CD3 - / -雄性小鼠作为收件人从而避免移植物抗宿主反应，由于连接到Y染色体次要组织相容性抗原，因为E15之前性别决定是不容易做到。 <ol><li>消毒仪器和佩戴沿手术无菌手套。通过腹膜内注射氯胺酮10毫克/毫升+赛拉嗪1毫克的溶液麻醉小鼠/毫升在PBS中稀释（每10g体重50至100μl），用1ml注射器。通过检查缺乏叉指反射的验证麻醉。放置在每只眼睛一滴水力Optimune凝胶，以防止在麻醉过程中干燥。 注:溶液应即席制备和注射前温热至室温。麻醉持续至少20分钟。麻醉的程度应该都沿手术来控制。麻醉可以通过腹腔内注射一半剂量为每20分钟被延长。 </li><li>将鼠标在其右侧，下一个水平层流罩。消毒手术区域用70％乙醇和10％的碘化物dermic溶液。用钳子部的老鼠毛沿2厘米长度正好后腿的关节的上方。剃须手术区。 </li><li>用手术刀，使第一，皮肤组织的切口1.5厘米长度，然后用剪刀剪断肌肉组织。施加轻微压力，切口两侧，这将迫使肾脏出腹腔。 </li><li>应用生理盐水用棉芽，以保持湿润肾脏。如果您在暴露器官困难，用平头镊子拔出周围的脂肪组织到肾脏附后。用棉签放在下面的器官维持肾脏出了腹膜后腔。 </li><li>有两个细镊子，使胶囊2-3毫米的孔（避免触及实质，以防止出血）。在整个手术过程中保持暴露肾囊连续湿润。 </li><li>插入仔细裂片肾囊下，通过保持打开的胶囊的壁并朝向边缘极包膜下滑动接枝。将高达每肾脏6瓣。 </li><li>替换复古腹腔肾脏。缝合肌肉腱膜，然后用医生个人的结皮。湿用10％的碘化聚维酮溶液的伤口。把鼠标放在加热PAð在37℃。 </li><li>调查移植鼠标，直到完全恢复从麻醉中通过心脏，呼吸和运动晶须，粘膜颜色，直到总回收率看到自给自足的运动控制。 注意:我们建议注射的镇痛溶液（Buprenorphin，0.1毫克/公斤）内肌肉刚刚手术后和2天手术后，以防止手术后的疼痛。 </li><li>保持经营的动物隔离，直到完全恢复。检查隔日伤口的线圈，以验证和预防感染。从未观察到伤口感染下此过程。 注:外周血细胞从CD3每周分析- / -接枝小鼠允许我们检测来自群体源自使用CD45同种异型标记和T细胞谱系特异性抗体（ 图3）或者E13或E18祖胸腺。 </li></ol> TSP的后代通过流式细胞仪分析9 <ol><l我&gt;单独逗FTOCs获得单细胞悬液（见协议第4节）。 </li><li>染色细胞悬浮液用含有抗体识别CD45.1 PECy7，CD45.2 AmCyan，CD4 APCCy7，CD8 APC，CD3太平洋蓝，Vγ5FITC，PEVδ1的抗体混合物和孵化在第5节个人裂片。 </li><li>重悬的细胞在HBSS + 1％FCS +碘化丙啶和流式细胞仪中的流动分析（见结果在图2中）。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

两个主要的测定法可用于分析T细胞分化的体外 。最近报道是造血祖细胞与骨髓基质细胞，OP9的共培养，表达Noth1的配位体，δ-样1或4 12，这2-D测定是容易执行的，高效的和灵敏的，允许分析在单个细胞水平。然而，它既不支持T细胞发育超出DP的阶段也没有γδDETC 13的产生，这两者需要与胸腺上皮的直接相互作用。 FTOCs长期以来一直用于分析T细胞发育<sup...

T淋巴细胞，轴承αβ或γδT细胞受体（TCR），区分在一个专门的机构，胸腺。充分发展胸腺分为两个不同的区域:皮质，其中胸腺祖发展和地点的胸腺细胞重新排列高效TCR的β和α链基因是从程序性死亡（被称为阳性选择的过程）获救;和髓质，在这里选择的胸腺细胞与太强的反应，以自配体被删除（负选择）1,2。胸腺来源于第三咽囊以为稍后由间充质细胞3包围的内胚层层。它是由起始于天胚胎E12，此后，连续招募所需的正常T细胞发育4造血祖细胞定植。胸腺移民通过连续的发育阶段发展，通过严格监管的程序，发起并策划迈ntained由胸腺Notch信号传导途径的相互作用时的活化与其配体，δ-样4，表达于胸腺上皮细胞（TECs的）5。 胸腺细胞发育开始于所谓的CD4 - CD8 -双阴性（DN）的阶段。 ，DN2（CD25 + CD44 +），DN3（CD25 + CD44 - ）和DN4（CD25 - CD44 - ） - DN胸腺细胞可以根据CD25和CD44的表达到DN1（CD44 + CD25）进一步细分。 CD24（HSA）和CD117（的c-Kit）进一步细分DN1隔成5个亚群，其中DN1a和b对应于早期胸腺祖细胞（ETP）。胸腺细胞重新排列T细胞受体δ，β和α链在DN阶段，并进行预-TCR选择（DN3，DN4阶段）。他们进一步迁移到的CD4 + CD8 +双阳性（DP）室，其中的TcRα链重新排列之前正和负塞莱ction。在这个阶段，大多数胸腺细胞被消除，只有一小部分（3-5％）达到的CD4 +或CD8 +成熟T细胞室。 淋巴分化途径过程通过造血干细胞的生成多潜能祖细胞（MPP）和淋巴引发的多潜能祖细胞（LMPP）失去的红细胞和巨核细胞潜力6阶段。 LMPP由缺乏分化血细胞标记物表型定义（谱系阴性，林- ），的c-Kit（CD117）的表达，的Sca-1和Flt3的/ FLK2（CD135）和没有白细胞介素的可检测水平的（ IL）-7受体α链（IL-7rα或CD127）。 LMPPs进一步分化为普通淋巴祖（CLP）7，到这个阶段已经失去了产生骨髓细胞的能力。电保留淋巴细胞（B和T细胞），NK细胞，DC和先天淋巴样细胞（ILC）的潜力，并从LMPP相差CD1的表达27，以及缺乏高水平的Sca-1的。 虽然胸腺解决祖细胞（TSP）的性质已经被广泛讨论8，它成为近日明确表示，TSP变化表型，分化潜能和功能，整个开发9。我们的体外和体内测定进行到TSP，通过FACS分离细胞从任E13（第一波）或E18（第二波）排序表征。胎儿胸腺器官培养（FTOC）与E13的E18和祖人数相等，不同的轴承异型标志殖民，使跟随他们类似的发展环境和子代细胞揭示内在特性，这两种类型的祖细胞之间的不同照射胸腺叶。无论是通过E13 E18或定植TSP胸腺叶允许开发无选择，由于双方的祖细胞之间的竞争。 在体内移植的殖民胸腺裂片进一步表明还牛逼他E13的成熟后代和E18 TSP 在体内生物不同的特性。从第一波的TSP快速生成T细胞，但引起低的数字αβγδ和T细胞。在后者我们检测Vγ5Vδ1树突上皮T细胞（DETC）中，具有一个不变的TcR，迁移到它们施加在伤口愈合的函数和胚胎发育10中仅产生的表皮。与此相反，TSP从第二波需要更长的时间，以产生高数量的TcR + T细胞和不能生成DETC。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>90 x 15 mm and 35 x 15 mm plastic tissue culture petri dishes.</td>
			<td>TPP</td>
			<td>T93100/T9340</td>
			<td>Sampling</td>
		</tr>
		<tr>
			<td>26GA 3/8 IN 0.45x10mm syringes with needles Beckton-Dickinson Plastipak.</td>
			<td>BD Plastipak</td>
			<td>300015</td>
			<td>Cell suspension</td>
		</tr>
		<tr>
			<td>Nylon mesh bolting cloth sterilized 50/50 mm pieces.</td>
			<td>SEFAR NITEX</td>
			<td>03-100/32</td>
			<td>Filtration of cells</td>
		</tr>
		<tr>
			<td>Ethanol 70%.</td>
			<td>VWR</td>
			<td>83801.36</td>
			<td>Sterility Actions</td>
		</tr>
		<tr>
			<td>Iodide Povidone 10% (Betadine)&#160;</td>
			<td>MEDA Pharma</td>
			<td>314997.8</td>
			<td>Sterility Actions</td>
		</tr>
		<tr>
			<td>Ketamine 100mg/ml (stock solution)</td>
			<td>MERIAL</td>
			<td>&#160;-</td>
			<td>Anesthesic</td>
		</tr>
		<tr>
			<td>Xylazine 100mg/ml in PBS (stock solution)</td>
			<td>Sigma</td>
			<td>X1251-1G</td>
			<td>Anesthesic and muscle relaxant</td>
		</tr>
		<tr>
			<td>Buprenorphin 0.3mg/ml stock solution</td>
			<td>AXIENCE</td>
			<td>&#160;-</td>
			<td>Morphinic analgesic</td>
		</tr>
		<tr>
			<td>Ophtalmic gel (0.2% cyclosporin)</td>
			<td>Schering-Plough Animal Health</td>
			<td>&#160;-</td>
			<td>Eyes protection</td>
		</tr>
		<tr>
			<td>DPBS (+ CaCl2, MgCl2)</td>
			<td>GIBCO Life Technology</td>
			<td>14040-174</td>
			<td>to isolate embryos</td>
		</tr>
		<tr>
			<td>HBSS Hanks&#39; Balanced Salt Solution (+ CaCl2, MgCl2)</td>
			<td>GIBCO Life Technology</td>
			<td>24020-091</td>
			<td>to wash out the blood and dissect the embryos</td>
		</tr>
		<tr>
			<td>OPTI-MEM I GlutaMAX I</td>
			<td>GIBCO Life Technology</td>
			<td>51985-026</td>
			<td>Medium for cultures</td>
		</tr>
		<tr>
			<td>F&#339;tal Calf serum</td>
			<td>EUROBIO</td>
			<td>CVFSVF00-0U</td>
			<td>additive for cultures</td>
		</tr>
		<tr>
			<td>Penicilin and Streptomycin</td>
			<td>GIBCO Life Technology</td>
			<td>15640-055</td>
			<td>Antibiotics for cultures</td>
		</tr>
		<tr>
			<td>2 b mercapto ethanol</td>
			<td>GIBCO Life Technology</td>
			<td>31350010</td>
			<td>additive for cultures</td>
		</tr>
		<tr>
			<td>LEICA MZ6 Dissection microscope</td>
			<td>LEICA</td>
			<td>MZ6 10445111</td>
			<td>Occular W-Pl10x/23</td>
		</tr>
		<tr>
			<td>Cold lamp source</td>
			<td>SCHOTT VWR</td>
			<td>KL1500 compact</td>
			<td>Two goose neck fibers adapted</td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>Dissecting Pad</td>
		</tr>
		<tr>
			<td>Spoon, round and perforated</td>
			<td>Fine Science Tools</td>
			<td>10370-18</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Fine Iris Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Vannas spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Forceps : Dumont #5/45 Inox 11 cm</td>
			<td>F.S.T.</td>
			<td>11253-25</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Two pairs of fine straight watchmakers&#8217; forceps Dumont #5 11 cm fine tips.</td>
			<td>Fine Science Tools</td>
			<td>11295-20</td>
			<td>Dissection tools</td>
		</tr>
		<tr>
			<td>Polyamid thread with needle 6-0&#160;&#160; C-3 3/8c</td>
			<td>ETHICON</td>
			<td>F2403</td>
			<td>for sutures</td>
		</tr>
		<tr>
			<td>Needle-holder</td>
			<td>MORIA/F.S.T.</td>
			<td>12060-02</td>
			<td>for sutures</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>VWR</td>
			<td>100229-100</td>
			<td>To maintain mouse temperature during anesthesy</td>
		</tr>
		<tr>
			<td>Membrane Isopore RTTPO2500</td>
			<td>DUTSCHER</td>
			<td>44210</td>
			<td>For FTOC</td>
		</tr>
		<tr>
			<td>Terasaki 60 wells plates</td>
			<td>FISHER</td>
			<td>1x270 10318801</td>
			<td>For hanging drop technique</td>
		</tr>
		<tr>
			<td>Gauze swabs steriles 7.5cmx7.5cm</td>
			<td>Hydrex</td>
			<td>11522</td>
			<td>To apply disinfecting solution</td>
		</tr>
		<tr>
			<td>Fluorescence or biotin labelled antibodies</td>
			<td>BD Biosciences, Biolegend or e-Biocsiences&#160;</td>
			<td>Clone Number see table below</td>
			<td>Staining cells</td>
		</tr>
		<tr>
			<td>MACS Columns/Streptavidin Microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401/130-048-101</td>
			<td>Cell depletion&#160;</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Charles Rivers Laboratories (CD45.1) and Janvier Labs&#160; (CD45.2)</td>
			<td>C57BL/6 CD45.1 OR CD45.2</td>
			<td>Source of cells and thymic lobes&#160;</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>CDTA Orl&#233;ans, France</td>
			<td>CD 3 epsilon Ko CD45.2</td>
			<td>&#160;Grafting experiment</td>
		</tr>
	</tbody>
</table>

characterization of thymic settling progenitors in the mouse embryo using in vivo and in vitro assays

表征胸腺解决祖重要的是要了解T细胞发育的前期阶段，胸腺，必须设计一种T细胞置换淋巴细胞减少患者的策略。我们研究了胸腺从13鼠胚胎日和18 thymi沉降祖细胞由两个体外互补和体内技术，无论是基于"悬滴"的方法。这种方法允许辐照的殖民胸腺胎儿与叶和E13 / E18还是通过CD45异型标记，从而跟随他们的后代区分胸腺祖。定植混合种群允许分析的祖细胞生物学特性细胞自主的分歧，而与殖民人口要么尽可能去除选择性的竞争压力。殖民胸腺瓣也可以在免疫缺陷雄性受体小鼠允许成熟T细胞后代的体内分析，如叔种群动态接枝他外周免疫系统和不同的组织和器官的殖民化。胎儿胸腺器官培养透露，E13祖细胞迅速发展成为成熟的全部CD3 +细胞，引起了规范γδT细胞亚群，被称为树突状上皮性T细胞。相比之下，E18祖细胞有延迟分化和无法生成树突上皮T细胞。 / - -胸腺-接枝的CD3外周血监测小鼠进一步表明，E18胸腺沉降祖生成，随着时间的推移，更大的成熟T细胞比它们的E13对应的号码，无法在短期内胎儿胸腺可以理解的特征器官培养。

为了选择一个方法来消耗内源性的胸腺细胞，允许殖民祖最好的发展胸腺叶，我们比较了在照射后殖民胸腺叶T细胞重建的水平或5天的脱氧鸟苷（D-GUA）治疗。结果表明，尽管有在培养的第9天无差异，照射裂片含有更多的T细胞比具有的d卦处理，在这样12.天，照射是小于d卦治疗更为合适后获得T细胞发育胸腺定植（ 图1）。研究E13和E18的TSP的发育潜能，我们拓殖E14照射胸腺裂片与两种类...

Watch this Scientific Journal Video about Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays at JoVE.com

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 ...

characterization-thymic-settling-progenitors-mouse-embryo-using-vivo

Research

JoVE Journal

Developmental Biology

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

8.2K 次观看.  Institut Cochin. 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. 

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

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 ...

Live Imaging of Mouse Secondary Palate Fusion

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

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

小鼠第二腭融合的实时成像

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

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

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 ...

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

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

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

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

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

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

确定小鼠肝脏中的胆汁密度

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...