我们感谢显微成像的技术支持。有些菌株是由由 NIH 研究基础设施项目 (P40 OD010440) 资助的秀丽遗传学中心提供的。这项工作得到了一所大学生物医学发现奖学金, 澳洲项目赠款 (GNT1105374), 澳洲高级研究金 (GNT1137645) 和 veski 创新研究金的支持: VIF 23 到罗杰波科克。

1. 制剂和畜牧业
注: 有关所有产品信息, 请参阅材料表。
<ol><li>OP50 大肠杆菌文化: OP50 细菌溶源性汤 (磅) (1% 胰蛋白胨, 0.5% 酵母, 0.5% NaCl, pH 7.0) 隔夜在37°c 没有抗生素. </li>
	<li>种子400µL OP50 细菌的线虫生长培养基 (NGM) 板 (1.5 克氯化钠, 8.5 克琼脂, 1.25 克蛋白胨, 1 毫升1米 CaCl2, 1 毫升5毫克/毫升胆固醇在乙醇, 1 毫升1米 MgSO4, 25 毫升1米 KPO4缓冲器), 空气干燥细菌为 48 h。</li>
	<li>在种子 NGM 板上采摘蠕虫, 在15摄氏度或20摄氏度孵化。<ol>
<li>为分析成年雌雄同体生殖, 选择喂养良好的 L4 蠕虫到细菌草坪和孵化过夜20摄氏度。</li>
		<li>在雌雄同体的精子数量, 孵化 L4 蠕虫在20°c 12 小时, 直到蠕虫达到 L4/adult 蜕皮。要使蠕虫与 L4 阶段同步, 请在漂白溶液中用大量的鸡蛋采摘成虫 (1 米氢氧化钠和漂白剂1:1 的比例) 来准备鸡蛋。让鸡蛋孵化和采摘 L4 动物的实验。</li>
	</ol>
</li>
	<li>通过将25µL 的聚 l-赖氨酸溶液放在滑梯上, 用吸管尖端将溶液涂好, 制备聚四氟乙烯显微镜幻灯片。除去多余的聚 l-赖氨酸与纸巾, 让幻灯片的空气干燥和孵化的幻灯片在65°c 15-20 分钟。</li>
</ol>2. 生殖解剖和染色6,13
<ol><li>斑点10µL 0.01% 四咪唑 (麻醉剂) 在22毫米 x 22 毫米盖玻片并且选择一天老成人蠕虫入它。</li>
	<li>解剖蠕虫在尾部, 因为它成为镇静剂使用注射器 (5 毫升) 和针 (24 "x 1"), 并确保生殖没有损坏。 注意: 解剖必须在蠕虫完全瘫痪之前进行, 这样蠕虫和蠕虫运动中的负压就能帮助生殖的弹射。对于精子的评分, 必须采取预防措施, 以防止在解剖过程中蝗受精囊针的损伤。避免接触生殖与针, 除了解剖点后, 蝗受精囊。如果生殖车厢内有断裂或生殖的任何排出物被观察到, 则生殖不应用于分析。</li>
	<li>将倒置的盖玻片放在聚 l-赖氨酸涂层的滑动上, 使解剖生殖保持在幻灯片上。</li>
	<li>将纸塔放在盖玻片的一个角上, 将盖玻片下的多余液体移开, 然后在液氮中放置1分钟的滑动. 用针与幻灯片上的 germlines 快速移除盖玻片。</li>
	<li>将幻灯片转移到-20 °c 甲醇 30-六十年代在一个房间里, 然后孵化30分钟在新配制的固定溶液 (1x 磷酸盐缓冲盐水 (PBS) 含0.08 米 HEPES pH 6.9, 1.6 毫米 MgSO4, 0.8 毫米乙二醇-双 (β氨基乙基醚)-n, n, n ', n-'-四乙酸四酸 (EGTA), 和3.7% 多聚甲醛) 室温。</li>
	<li>通过放置在包含 1x PBS 的房间, pH 值7.4 与 0.1% Tween-20 10 分钟, 洗涤幻灯片. 重复此步骤一次。</li>
	<li>将湿纸巾放在室温下的塑料盒中, 用30µL 的 germlines (30% 的山羊血清, 在1700年µL 的蒸馏 H2O 和300µL 10x PBS) 在潮湿的房间里, 在室温下放置30分钟。</li>
	<li>在30% 山羊血清中添加50µL 的 REC-8 抗体溶液, 每种样品的比值1:300。在4°c 一夜间孵化。</li>
	<li>在装有 1x PBS 的腔室中放置 0.1% Tween-20 10 分钟, 将其冲洗干净. 重复步骤一次, 通过在幻灯片的拐角处放置纸巾来去除多余的液体。</li>
	<li>通过稀释绿色荧光 (488 nm 发射波长) 共轭二级抗体 (1:1000), 罗丹明 (肌动蛋白染色, 1:4000) 和 DAPI (DNA 染色, 1:4000) 在30% 正常山羊血清中制备二级抗体溶液。</li>
	<li>在幻灯片中添加50µL 二级抗体溶液。</li>
	<li>室温下孵育1小时。</li>
	<li>在装有 1x PBS 的腔室中放置两次幻灯片, 0.1% Tween-20 10 分钟。</li>
	<li>将30µL 的固定试剂添加到幻灯片上, 并将12毫米2 x 1.5 µm 盖玻片放在顶部, 并在4°c 一夜之间干燥幻灯片。</li>
</ol>3. 共焦显微术
<ol><li>打开共焦显微镜、激光器、共振扫描仪和共焦显微镜软件。将带有染色 germlines 的幻灯片放在63X 目标上方的幻灯片架上。</li>
	<li>找到一个生殖, 聚焦在生殖的顶端, 并用共焦显微镜软件标记它。同样专注于底部的生殖和标记它建立总生殖厚度。</li>
	<li>图像的整个生殖通过定义的厚度, 每片0.5 µm. 标记完整的生殖并开始获取。扫描每个切片8次, 并平均值以提高图像质量。共振扫描仪将允许快速扫描, 以避免在成像过程中漂白。如果显微镜没有共振扫描仪, 将扫描次数减少到四, 以避免漂白。</li>
</ol>4. 核数和分布的后成像分析
注: 有关使用的软件工具和按钮的屏幕截图, 请参阅辅助图 1 。
<ol><li>将图像导入 Imaris 8.4.1 或更高版本的软件。</li>
	<li>通过确定各区域核形态, 定义生殖的有丝分裂区、过渡区、减数分裂区、卵母细胞区和蝗受精囊。</li>
	<li>使用软件的 "曲面函数" 按钮 (辅助图 1A) 选择感兴趣的区域。</li>
	<li>取消自动曲面创建向导, 并手动从三维堆栈中绘制图像的第一个和最后一个切片所感兴趣的区域。</li>
	<li>单击创建曲面。 注意: 该软件将自动开发堆栈之间的第一和最后一栈。</li>
	<li>通过单击掩码通道按钮 (辅助图 1 b-C), 屏蔽除 DAPI 之外的所有通道。</li>
	<li>使用 "现场功能" 按钮 (辅助图 1A) 定义核尺寸参数。对于有丝分裂区域, 定义一个 XY 直径为2µm, 同时留下 Z 直径未定义。对于过渡区域, 将2µm 和 Z 直径的 XY 直径定义为1.5 µm. (补充图 1D)。 注: 根据显微镜的放大倍数和规格, 对直径进行故障诊断。对于当前的协议, 使用的目标是 63X, 在成像过程中提供额外的1.70 倍放大倍数。如果目标改变, 例如 40X, 直径应该相应地改变。应使用 wildtype germlines 执行故障排除以建立正确的参数。野生型生殖臂的有丝分裂区有大约250个细胞核。应修改直径以达到此值。使用至少 15 germlines 来确定直径的最佳值。对过渡区 (150-170 核) 和减数分裂区 (600-700 核) 进行标定时, 应采用类似的方法。</li>
	<li>通过定义最小阈值 (辅助图 1E) 来限制对斑点的检测。使用 DAPI 染色的野生型 germlines, 通过增加最小三维渲染阈值来建立最小阈值, 直到第一个点出现在生殖之外。</li>
	<li>保存图像并获取由软件自动生成的表中的核数。将图像导出为 TIF 文件。</li>
</ol>5. 评分精子和染色体编号的后成像分析
<ol><li>通过选择蝗受精囊作为感兴趣的区域来执行三维渲染。要检测每个精子, 请在 X 和 Y 轴之间定义 0.75-1.0 µm 之间的光斑直径, 而 Z 直径仍未定义。使用 "斑点函数" 按钮, 如辅助图 1所示。</li>
	<li>通过滴答作响的背景减法框减去背景, 并使用软件计算的背景校正值 (辅助图 1D)。 注: Imaris 软件首先选取高强度点, 因此背景的影响是最小的, 只要感兴趣区域与背景有显著差异。第二种方法是手动定义背景校正, 在这里可以为背景提供适当的值。如果独立样本的强度需要比较, 手动背景校正将是适当的。</li>
	<li>调整三维渲染阈值以检测蝗受精囊中所有 DAPI 染色的精子(辅助图 1E)。</li>
	<li>对于染色体计数, 在开发三维模型之前, 定义 X 和 Y 轴的光斑直径 < 0.75 µm。</li>
	<li>保存图像并导出为 TIF 文件。</li>
</ol>6. 生殖骨架重建后的影像学分析
<ol><li>通过单击掩码通道按钮, 标识生殖中感兴趣的区域, 并屏蔽除罗丹明之外的所有通道。</li>
	<li>使用 "曲面函数" 按钮 (辅助图 1) 定义0.25 µm 的曲面细节。 注: 这意味着每0.25 µm 将被渲染成三维模型。表面细节可以是恒定的任何区域的生殖, 三维表面模型将创建每0.25 µm。然而, 对于卵母细胞区域, 由于该地区有明确的肌动蛋白纤维, 表面细节可以增加到 0.35-0.5 µm。</li>
	<li>通过滴答作响的背景减法框减去背景, 并使用软件计算的背景校正值 (辅助图 1D)。</li>
	<li>根据需要分析的结构调整三维渲染阈值 (辅助图 1E)。</li>
	<li>保存已开发的三维图像并导出为 TIF 文件。</li>
</ol>

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作者声明没有利益冲突。

该协议的目标是提高准确性, 减少生殖分析所需的时间。在解剖 germlines 的标准制备后, 通过计算渲染, 制备了一个三维生殖核模型。在允许观测生殖核在空间中的分布时, 三维渲染计算了生殖特定区域的核数目。我们的方法的关键方面是准确定义的大小和形状参数的原子核。这取决于染色的清晰度和图像的放大倍数。通过与已发布的手动分析数据1进行比较, 我们确认了该方法的正...

与哺乳动物的信号通路守恒使线虫成为研究多个生物过程的优秀模型1,2。在实验室中, 我们使用线虫生殖研究干细胞的发育、凋亡和基因表达。虽然生殖是一个三维结构, 但由于三维分析的耗时和劳动密集型性质, 许多研究是二维的。二维分析极有可能会在生殖中歪曲体内事件。C. 线虫成体雌雄同体有两个生殖臂, 每个手臂都有一个躯体远端尖端细胞 (DTC), 在未分化状态3,4中维持远端生殖细胞。这些生殖细胞开始分化, 因为它们离开 DTC, 逃避其影响, 并成为卵母细胞和精子, 因为它们到达生殖的近端。在此过程中, 生殖细胞核发生有丝分裂, 然后过渡到减数分裂5,6。精子生产由发育的幼虫阶段 4 (L4) 完成, 之后卵母细胞在成年期间产生。精子储存在蝗受精囊卵母细胞中, 以生成胚胎。
有多种遗传和环境因素可以影响线虫中的生殖发展, 导致细胞核数量、凋亡事件数、染色体动态、蛋白质表达和/或定位的变化7 ,8,9,10,11。对这些事件的分析需要确定基于核形态和分布的各个分化阶段。要用大量的样本量手动准确地分析这些参数, 是劳动密集型和耗时的。为了规避这些缺点, 并使分析的一致性, 我们开发了一种自动化的方法, 三维检查的线虫生殖核计数, 核分布, 蛋白质表达, 和骨架结构。将共焦显微术与三维渲染相结合, 生成了粒度和形状参数, 用于鉴定生殖细胞分化的各个阶段。此外, 这种方法可以计数的生殖细胞核和精子加评分的染色体数在每个卵母细胞。
生殖的一个关键结构是骨架, 它提供了生殖室的稳定性, 艾滋病细胞质流和保护生殖核12。利用计算渲染, 我们对生殖细胞骨架进行了三维重建, 并在生殖中发现了明显的骨架特征。在这里, 我们描述了一步一步的协议, 以说明计算分析如何结合共焦成像, 使对C. 线虫生殖的综合分析。
我们提出了一个快速的方法, 三维分析线虫生殖 (图 1)。利用三维分析, 可以研究生殖核的三维分布 (图 2和图 3), 自动计数单元格 (图 2), 重建生殖骨架 (图 3), 蛋白质的分布 (图 4), 并评分卵母细胞中蝗受精囊和染色体中的精子数量(图 5)。该方法不仅能方便准确地量化生殖, 而且能识别生理上相关的表型。

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">C. elegans strains: wild type (N2, Bristol), rnp-8(tm2435) I/hT2[bli-4(e937) let-?(q782) qIs48] (I;III), cpb-3(bt17) I, glp-1 (e2141) III&#160;</td>
			<td style="width:264px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:168px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OP50 Escherichia coli bacteria</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nematode Growth Media (NGM) plates</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyclonal rabbit anti-REC-8&#160;</td>
			<td>SDIX</td>
			<td>29470002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 conjugated antibody raised in goat</td>
			<td>Thermofisher Scientific</td>
			<td>A-21236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytoskeletal dye phalloidin&#160;</td>
			<td>Thermofisher Scientific</td>
			<td style="width:168px;">A-12380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetramisol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4</td>
			<td>Sigma Aldrich</td>
			<td style="width:168px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES buffer, pH 7.4&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:168px;">G0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X PBS pH 7.4&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P1389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37% Paraformaldehyde solution</td>
			<td>Merck Millipore</td>
			<td>1040031000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroshield fixing reagent&#160;</td>
			<td>Sigma Aldrich</td>
			<td>F6182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol&#160;</td>
			<td>Millipore&#160;</td>
			<td>1009832511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20&#176;C &#38; 25&#176;CIncubator&#160;</td>
			<td>Any brand</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope</td>
			<td style="width:264px;">Any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;&#160;</td>
			<td style="width:264px;">Any brand (Leica, Zeiss)</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Computer equipped with Imaris suit 8.4.1 or later version, full licence to use the software and Matlab software.</td>
			<td style="width:264px;">Bitplane</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Phospho buffered saline, pH 7.4</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon microscope slides&#160;</td>
			<td style="width:264px;">Tekdon&#160;</td>
			<td style="width:168px;">&#160;941-322-8288</td>
			<td></td>
		</tr>
	</tbody>
</table>

computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton

秀丽线虫(C. 线虫) 生殖用于研究几个生物学上重要的过程, 包括干细胞发育、细胞凋亡和染色体动力学。虽然生殖是一个很好的模型, 分析往往是二维由于时间和劳动力需要的三维分析。这些研究的主要读数是生殖中细胞核和蛋白质分布的数量/位置。在这里, 我们提出了一个方法来执行生殖的自动分析使用共焦显微镜和计算方法, 以确定核在每个区域的生殖的数量和位置。我们的方法还分析了生殖蛋白的分布, 使三维检测的蛋白质表达在不同的遗传背景。此外, 我们的研究显示在生殖的不同区域骨架体系结构的变化, 可以适应特定的空间发展要求。最后, 我们的方法可以自动计数的精子在每个生殖的蝗受精囊。结合起来, 我们的方法实现了对C. 线虫生殖的快速和可重现的表型分析。

图 1表示三维生殖分析所需的时间。L4 雌雄同体孵化20摄氏度被解剖, 以隔离 germlines 和染色 DAPI, 罗丹明和抗体对生殖蛋白。Germlines 是用共焦显微镜成像的。染色和共焦显微术需要大约24小时的完整生殖计算分析需要 10-15 分钟来计算细胞核的数量和位置, 确定蛋白质分布, 分析骨架结构, 并评分精子数量。完整的手动分析要求每生殖超过2小时, 并受操作...

Watch this Scientific Journal Video about Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton at JoVE.com

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

我们为秀丽线虫生殖提供三维重建的自动方法.我们的方法确定生殖中每个核的数量和位置, 并分析生殖蛋白质分布和骨架结构。

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

computational-analysis-caenorhabditis-elegans-germline-to-study

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

6.2K 次观看.  Monash University. 我们为秀丽线虫生殖提供三维重建的自动方法.我们的方法确定生殖中每个核的数量和位置, 并分析生殖蛋白质分布和骨架结构。

视频: Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

利用共聚焦分析<em&gt;非洲爪蟾</em&gt;调查的Wnt和Shh形态发生素梯度的调节剂

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

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生殖系化学毒性的利用线虫综合评估

利用共聚焦分析

方法来检查淋巴腺和血细胞中

在Active ERK的时空分析

固定化的线虫线虫分析神经元内细胞的转运

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

评估溶酶体碱化在活的秀丽线虫中的肠道

种系线虫与胸苷模拟教育的细胞周期分析

线虫中耳炎群链球菌引起氧化应激的研究