Name: imaging and analysis of tissue orientation and growth dynamics in the developing drosophila epithelia during pupal stages
Uploaded: 2020-06-02T13:00:00
Description: Watch this Scientific Journal Video about Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages at JoVE.com

我们要感谢马丁-布兰科实验室的成员进行了有益的讨论。我们还感谢尼克·塔彭（英国伦敦克里克研究所）、布卢明顿股票中心（美国印第安纳大学）和FlyBase（关于果蝇基因注释）。费德里卡·曼吉奥内得到了JAE-CSIC博士前研究金的支持。马丁-布兰科实验室的资金来自"科学科学和科学投资方案"（BFU2014-57019-P和BFU2017-82876-P）和拉蒙阿雷塞斯基金会。

注：该协议分为五个步骤：（1） 暂存小狗，（2） 准备小狗成像，（3） 生长的腹部上皮的活成像，（4） 生成遗传马赛克，（5） 数据处理和分析（包括描述如何从细胞结轮廓分析细胞方向动力学和细胞克隆的生长动力学的部分）。
1. 成像前分期使用果蝇小狗
<ol><li>产卵（AEL）后，在25°C（±12小时）的塑料小瓶中培养标准基的相应基因型，5天（±12小时）。 注：?...

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远程顺序是大多数功能性生理单元的基本特征。在形态生成过程中，通过集成以高时空精度实现的复杂指令来实现顺序。多级和多级约束被集成到定型的组织安排中。
极性和定向性对于开发过程中的有序空间排列至关重要。极性意味着在开发过程中对称性断裂。实现不对称是必要的，以确定胚胎前背（A/P）和多索温托（D/V）轴和成人组织26。除了这种早期作...

为了发挥自身的作用，上皮组织完全依赖于其细胞成分的空间组织。在大多数上皮上，细胞不仅相互挤合，以形成精确的鹅卵石层，而且相对于身体轴的方向。
精确组织组织的功能重要性在感觉上皮中显而易见，如脊椎动物内耳和视网膜。在第一种情况下，头发和支撑细胞以特定的轴向方向对齐，以有效感知机械输入，如声音和运动11、22。同样，光受体细胞空间组织对于通过视网膜3实现最佳光学特性至关重要。因此，细胞位置和方向的空间控制对于适当的生理功能具有特别重要的意义。
果蝇是一种全息昆虫，通过蜕变，对幼虫体结构进行完全转化，导致其成年组织。果蝇pupa是一个优秀的模型，用于各种动态事件的非侵入性活成像，包括发育细胞迁移4、细胞分裂和生长动力学5、肌肉收缩6、细胞死亡7、伤口修复8和细胞定向9。在成人果蝇中，外部上皮表现出高度的秩序。这很容易观察到的三角（即，细胞突起源自单上皮细胞）和感觉刷毛遍布苍蝇的身体表面10。事实上，三角线排列在平行行引导气流11。成人上皮的形态和单个细胞的有序排列在胚胎生成期间开始，并在胚胎阶段达到高潮。虽然在胚胎细胞分裂，杂交和形状的变化都减少组织顺序12，13，这是在发育的后期阶段，特别是在pupal阶段，当苍蝇接近成熟12,9。
不动的果蝇小狗提供了一个理想的系统来评估细胞的形状和方向变化。皮巴腹部表皮具有特殊优势。当成年头、胸腔、生殖器和附属物的前体生长并从幼虫阶段得到图案时，被集成到幼虫表皮中的组状细胞开始生长和区分，仅在幼崽14。此功能允许跟踪所有参与建立组织秩序的时空事件。
在胚胎发育期间，在每个假定腹段的相反位置指定了组织爆炸。成人的背腹部表皮来自地主位置的组震巢穴，存在于前和后舱15，16。15,当组织爆炸扩大，取代幼虫上皮细胞（LECs），反向巢在背侧中线引信形成一个汇管板17，18，19，20。17,18,19,20
本研究描述了1）一种对果蝇小狗进行解剖、安装和长期实时成像的方法，以及2）以高时空分辨率研究细胞定向和生长动力学的分析方法。此处提供了详细的协议，涵盖从初始 pupae 制备（即暂存和成像）到定向和方向特征的提取和定量所需的所有步骤。我们还描述了如何从细胞克隆的分析中推断局部组织特性。所述的所有步骤都是微创的，允许长期实时分析。此处描述的方法可以很容易地适应并应用于其他发育阶段、组织或模型生物体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analysis Software</td>
			<td>-</td>
			<td>ImageJ</td>
			<td>Analyzing data</td>
		</tr>
		<tr>
			<td>Drosophila</td>
			<td>Atpa::GFP</td>
			<td>-</td>
			<td>Strains employed for data collection</td>
		</tr>
		<tr>
			<td>Drosophila</td>
			<td>hsflp1.22;FRT40A/FRT40A Ubi.RFP.nls</td>
			<td>-</td>
			<td>Strains employed for data collection</td>
		</tr>
		<tr>
			<td>Dumont 5 Forceps</td>
			<td>FST</td>
			<td>11251-20</td>
			<td>1.5 mm diameter for dissection</td>
		</tr>
		<tr>
			<td>Glass Bottom Plates</td>
			<td>Mat Tek</td>
			<td>P35G-0.170-14-C</td>
			<td>Mounting pupae for data collection</td>
		</tr>
		<tr>
			<td>Halocarbon Oil 27</td>
			<td>Sigma-Aldrich</td>
			<td>9002-83-9</td>
			<td>mounting pupae</td>
		</tr>
		<tr>
			<td>Inverted Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>Data collection</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>DFC365FX</td>
			<td>Visualization of the pupae during dissection</td>
		</tr>
	</tbody>
</table>

imaging and analysis of tissue orientation and growth dynamics in the developing drosophila epithelia during pupal stages

在多细胞生物体中，成熟的组织和器官在其组成细胞的空间排列中表现出高度的秩序。感官上皮给出了一个显著的例子，其中相同或不同身份的细胞通过细胞粘附性（显示高度组织的平面模式）聚集在一起。单元格以相同的方向相互对齐，并在较大距离内显示等效极性。成熟的上皮体组织是在形态形成过程中建立的。为了理解成熟上皮的平面排列是如何实现的，在体内发育过程中以高时空保真度跟踪细胞方向和生长动力学至关重要。强大的分析工具对于识别和描述本地到全球的过渡也至关重要。果蝇小狗是一个理想的系统，用于评估定向细胞形状变化的基础上皮形态发生。上皮发育的皮毛构成不动体的外表面，允许对完整动物进行长期成像。此处描述的协议旨在成像和分析在皮球腹部表皮生长时全球和局部水平的细胞行为。所述方法可轻松适应其他发育阶段、组织、亚细胞结构或模型生物体的细胞行为成像。

上述协议包括为长期活体成像制备果蝇小狗，以及分析腹部表皮细胞取向和生长动力学的程序。通过应用这种方法，可以生成发育中的普帕的高分辨率电影，时间长达48小时，而不会显著光漂白或光毒性。图4显示了描绘不同时间点和从不同角度方向的pupae的腹部表皮（例如，组织爆炸和LECs）的快照。对这些电影的后续分析能够识别和量化在扩?...

Watch this Scientific Journal Video about Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages at JoVE.com

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

该协议旨在成像和分析果蝇在果蝇发生变质时，果蝇腹部上皮细胞定向和组织生长动力学。本文描述的方法可用于研究果蝇或其他模型生物体的不同发育阶段、组织和亚细胞结构。

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

在多细胞生物体内，组织在空间方向上进一步显示高度。在形态形成过程中，细胞在远距离上对齐并显示一系列优先级。要了解在发育过程中如何达到表皮的平面排列，在体内以高空间时间保真度跟踪细胞的方向和生长动力学至关重要。所述协议旨在成像和分析细胞行为在全球和地方水平在果蝇幼皮表皮。这种方法可以提供对不同研究理论、细胞生物学、形态生成和平面极性见解，并可应用于其他组织、结构和模型的分析。经过一些练习后，所有步骤都可以轻松执行。它们要求精确。此协议的可视化有助于在更短的时间内实现最佳结果。幼崽解剖是复杂的。首先，使用湿润的画笔将分阶段的白色预包装转移到新鲜的塑料小瓶中，并在25摄氏度下尽可能长时间地保留它们。然后，在钳子的帮助下，从小瓶壁上取下每个幼崽。将每个小狗的腹侧粘在用双面胶带覆盖的玻璃滑梯上。用钳子的尖端轻轻敲击头尖和钳子的背表面，以确保钳子盒附着在胶带上。在立体显微镜下开始解剖，用钳子轻轻从钳子中去除蛋白石。通过手术开口将钳子的一角插入到钳子外壳和幼崽表面之间的浅体中。在一个或多个摆动中横向将箱子从头撕到尾部，避免捏住小狗。当您继续向后部结束时，将破裂的小狗箱折叠回侧侧。小心地将钳子插入动物的下面，轻轻拉起，从打开的幼崽盒中取下幼崽。小狗粘在钳子的尖端。轻轻地握住腹侧，将幼崽转移到一小滴气体渗透卤化油顶部的玻璃底盘上。在盘子边缘卷一块湿纸巾，盖上盘子以保持湿度。根据要评估的域和工艺，将油滴放在玻璃底盘上的油滴上。对于长期活成像的早期扩展的后巢，后向横向定向的幼崽。要形象地显示它们的后期扩张和组织重塑，请对幼崽进行定向。将装有安装过的幼崽的玻璃底盘转移到显微镜阶段，并使用透光聚焦在腹部区域的表面。要采用线粒体重组来诱导腹部上皮的遗传马赛克，准备携带热冲击诱导性翻转酶的处女，特定基因组位置的FRT位点，以及一个可识别的细胞标记物，对FRT站点的致病。准备在等效基因组位置携带 FRT 位点的突变雄性。在25摄氏度的塑料小瓶中，以三到一的比例交叉几个雌雄，为期四到五天。为了在组细胞中产生体细胞，在十字架后代的第三个星幼虫阶段进行热休克处理，将含有动物的塑料小瓶淹没在37摄氏度的水浴中，在37摄氏度的热水浴中45分钟到1小时。在图像 J 软件中，使用最大强度投影功能保护 2D 中共和显微镜获取的 Z 堆栈切片。巢具有特征形状，在左侧的平面坐标系中定向，在顶部的背向中定位它们。为了通过此分析获得最佳结果，必须以高分辨率获取输入图像。若要在本地单元格边上获取质量方向值，请单击插件菜单并选择方向 J 分布选项。将高斯窗口西格玛设置为一个像素，将立方样条设置为渐变，将最小一致性设置为 20%，将最小能量设置为 1%。使用插件的颜色调查选项。将色调设置为方向，将饱和度设置为一致性，将亮度设置为输入图像。此颜色将相对于设置的平面坐标系对单元格边缘方向进行编码。接下来，在插件菜单下，使用方向 J 测量选项量化细胞方向和方向单元格到单元格对齐。在组织细胞所占据的范围内，产生20微米左右、面积均匀的相邻非重叠区域。按量值计算与 ROIS 的主导局部方向和一致性。在这项研究中，所使用的方法能够产生高分辨率的影片的发展，长达48小时，没有显著的照片漂白或照片毒性。此处显示了以 RFP NLS 及其双点为标志的野生类型克隆示例。克隆沿分段边界拉长。双克隆是并行排列的或串联排列的。野生型克隆在幼虫形成后26小时47小时形态图显示两个阶段的复杂边界形态。在百分层形成后 26 小时 47 小时，几何参数的框和胡须图显示，在此时间窗口中，平均面积和周长显著增加。克隆人的方向在扩展和改造期间保持。在幼虫形成后26小时47小时的形状参数表明，野生克隆的粗糙度、圆度和圆形几乎没有变化。一定要不损坏幼崽，否则它会在几个小时内死亡，影响活成像。本协议不要求使用沙图斯试剂或仪器。解剖时避免捏自己。通过应用这些方法，可以使用不同的想象技术长期生成高分辨率电影。光子或旋转磁盘的焦点。氯分析可用于探索非自主反应，因为随机细胞有助于识别交叉说话或细胞通信机制。这些方法可以很容易地适应研究各种形态遗传现象，包括表皮、肌肉和在变质期间神经发育的协调。

Research

JoVE Journal

Developmental Biology

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Live Imaging of Mouse Secondary Palate Fusion

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

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

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 ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Dissection and Staining of Drosophila Pupal Ovaries

Dissection and Staining of Drosophila Pupal Ovaries

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

果蝇分子和免疫组化分析制备周围嗅组织的研制

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...