我们感谢肖恩·卡罗尔和托马斯. 沃纳为您提供飞行股票、设备的直保险丝、在拍摄中的帮助、清阿加塔的指导和伊丽莎白中岛的英语编辑。这项工作得到了 KAKENHI 17K19427 和武田科学基金会的支持。

1. 飞行股票
<ol><li>对下列所有协议使用果蝇 guttifera 。</li>
	<li>使用塑料瓶 (直径25毫米 x 高度96毫米) 和纤维素插头 (直径 23 mm x 高度 26 mm) 进行库存维护。使用标准的玉米面/糖/酵母/琼脂食品, 并遵循一个出版物描述了这个物种的三其他替代食谱2。 注: D. guttifera (股票编号 15130-1971.10) 由加州大学圣地亚哥分校的果蝇物种储备中心提供。尽管D. guttifera属于immigranstripunctata辐射, 它与23中的 "黑腹" 有很密切的关系, 但它有许多与d。.因此, 该协议可以适用于许多果蝇物种, 尽管有些物种需要特定的食物和/或技术提示来保持它们的2。</li>
</ol>2. 蛹的观察和蛹阶段的定义
注意: 观察的蛹取自在25° c 的12:12 小时光/暗循环中保持的苍蝇。班布里奇和 Bownes17描述了从原始位置蛹到一块湿润的组织纸 (97% 存活946个移动的蛹) 的低风险. D. guttifera蛹可以通过本质上相同的方法进行准备。
<ol><li>在一个塑料瓶 (直径25毫米 x 高 96 mm) 的新鲜食品 (标准玉米面/糖/酵母/琼脂食品) 中放置健康的成人, 让它们产卵. 等待7天, 以获得第三晚龄。</li>
	<li>将1毫升标准玉米面/糖/酵母/琼脂食品放入每1.5 毫升微。3针孔与2毫米间距在管内的盖子, 通过穿透一个图钉, 让呼吸。</li>
	<li>离心管内与食物 7 s 在一个迷你离心 (860 x g)。</li>
	<li>倒置和自来水瓶, 以消除所有成人的瓶子。</li>
	<li>将 5-10 毫升的 ddH2O (或反渗透水) 倒入小瓶。</li>
	<li>将幼虫用水倒入塑料培养皿 (直径90毫米 x 高15毫米)。确定第三晚龄由他们的大身体大小 (3-4 毫米在长度)。</li>
	<li>将第三龄幼虫用镊子轻轻移入管内, 用食物 (10 只幼虫/微)。在25° c 的夜间孵育。</li>
	<li>将新形成的蛹移到一张已被 ddH2O 的纸巾上, 并放置在塑料培养皿中 (直径 35 mm x 高度 10 mm)。</li>
	<li>将培养皿放在潮湿的室内 (含10毫升的 ddH2O 在底部), 并等待蛹发育到所需的阶段。</li>
	<li>将蛹移到一个塑料培养皿 (直径60毫米 x 高 15 mm) 的纸巾上。 注意: 阶段的定义主要可以根据四腹果蝇17的各个阶段进行。通常,果蝇的蛹期可以归类为 P1-P15 (ii), 尽管根据所使用的物种需要对阶段定义进行一些修改。如果可能, 删除蛹可以精确而细致地观察。请参见下面的详细信息 (步骤 3)。</li>
	<li>用立体显微镜观察蛹。拍摄的照片使用数码相机连接到立体显微镜。</li>
</ol>3. 去除蛹
注意:果蝇的蛹被称为蛹的结构所覆盖。昆虫的 Muscomorpha (苍蝇) 不脱落它的幼虫角质层在蛹;相反, 它硬化后的角质层 apolysis, 并使用它作为一个保护的蛹, 蛹24。寄生在蛹内的蛹有一个真正的蛹角质层, 这是非常柔软和脆弱的。在 apolysis 发生附近 P4 (ii) 之前, 上皮细胞和蛹被连接在一起, 因此去除蛹没有损伤是非常困难的。P5 后, 除去蛹是费力的, 但对形态学观察和蛹阶段的定义有用。这一过程的执行如下。
<ol><li>在一块纸巾上贴一块双面胶带。</li>
	<li>将蛹放在双面磁带的腹侧上 (图 1A)。</li>
	<li>找到蛹前侧与内蛹之间的空间。用镊子抓住并除去这个缝隙周围的蛹, 并露出蛹头部的前侧。</li>
	<li>将镊子的尖端与前后轴线平行移动, 将其插入。提起镊子的尖端, 使其局部折断蛹。重复此操作, 直到破损到达蛹的后部。确保蛹和蛹腿之间也形成间隙, 并打破蛹的腹侧, 并尽量减少内部蛹的损伤 (图 1B)。</li>
	<li>在打破蛹尽可能多, 取出蛹使用一个精美的画笔 (#5/0) (图 1C)。</li>
	<li>将蛹放在一张纸巾上, 用 ddH 的2O 放置在一个塑料培养皿中 (直径 60 mm x 高度 15 mm)。尽快拍照, 因为裸露的蛹易受伤害, 容易变干。 注意: 没有蛹的蛹不适合测量蛹期 (步骤 4), 因为压力 (如脱水) 和物理损伤可能会干扰正常的发育。</li>
</ol>4. 蛹阶段的测量
<ol><li>如步骤2所述, 准备蛹以测量蛹阶段的持续时间。收集蛹 1-2, 2-3, 3-4 天后, 蛹形成 (20 蛹每)。给蛹识别的个体识别号 (1-60)。在以下步骤中继续收集新形成的蛹, 以获得20多的幼蛹。给新形成的蛹提供个人识别号 (61-80)。将蛹放在一块 ddH 的2O 湿纸巾上 (3.9 厘米2) 的12孔细胞培养板 (1 蛹/井, 12 蛹/板)。用 ddH2O 填充板的内部空间, 以保持湿度, 将盖子盖上, 并将板材置于25° c、恒定光 (24:0 小时/暗) 条件下。 注意: 没有蛹的蛹不适合测量蛹期的长短。请不要删除蛹。</li>
	<li>每30分钟观察一次所有蛹的体色、刚毛、氏管和黄体的形态特征, 并记录观察阶段 (P1-P15 (ii), 根据在一个计数表中的参考17,19)。</li>
	<li>通过三 (或更多) 人的旋转移动, 继续记录超过四天 (96 小时)。</li>
	<li>统计每个特定阶段的记录数, 并计算平均值 (观察/蛹的平均计数)。然后乘以 0.5 (h), 导致估计的阶段长度 (h)。</li>
</ol>5. 测量机翼上黑斑的强度
注意: 蛹或成人翼上的黑点的强度可以通过测量光学密度 (OD) 来量化。使用已知的 ODs (阶梯密度滤镜) 的玻璃过滤器用于校准25, 这样就可以从机翼的数字图像中计算出特定区域的外径。测量现场外径和外径, 并从前者中减去后者以获得光斑强度 (ΔOD)。本文介绍了ΔOD 的解剖、测量和计算方法。此过程可以在步骤2之后完成, 独立于步骤3和步骤4。一旦你执行了步骤2并且了解了所有的蛹阶段, 你可以直接开始或重复步骤5。
<ol><li>图像准备<ol>
<li>如步骤2所述, 准备一个新的蛹的焦点阶段。用镊子取出蛹的前部。用镊子取出蛹, 并将其放入磷酸盐缓冲盐水 (PBS,表 2) 在一个塑料培养皿 (直径35毫米 x 高度 10 mm)。</li>
		<li>剪下机翼的基底关节 (基底关节是机翼的狭窄下部)。当机翼折叠时, 将其放入塑料培养皿中 (直径 35 mm x 高 10 mm), 填充 ddH2O 以渗透压延伸它 (机翼自行展开)。</li>
		<li>每10分钟从一个小瓶里收集新 eclosed 的成年人。麻醉与 co2的苍蝇使用 co2麻醉垫, 确认麻醉不动, 并削减了机翼的基础关节。</li>
		<li>将 PBS 的10µL 放在玻璃滑梯上, 将机翼放在那里, 盖上盖子滑块 (18 mm x 18 mm)。</li>
		<li>打开立体声显微镜的光。将光源设置为最高水平。设置物镜11.5X。将膜片设置为最开放状态。打开摄像机将相机设置为 (ISO: 100, 模式魁北克省 3136 x 2352 像素, 快门速度: 1/二十年代)。把焦点放在样品上, 移动显微镜的焦点旋钮。</li>
		<li>按下遥控装置的快门按钮以拍摄图像。每翼拍摄3张照片, 每个机翼必须以 campaniform 触角、纵静脉点或后 crossvein 为中心, 将机翼的远端部分定位在左侧, 机翼的前部位于上部。</li>
	</ol></li>
	<li>校准<ol>
<li>拍摄的9部分的阶梯密度滤镜使用相同的相机设置获得的机翼图像。</li>
		<li>启动 ImageJ 软件 (https://imagej.nih.gov/ij/)22。</li>
		<li>单击文件 |打开 |并选择阶梯密度滤波器的图像之一。</li>
		<li>单击图像 |类型 |8位 |将图像转换为8位图像。</li>
		<li>单击编辑 |选择 |指定 |并检查椭圆形和居中列。在宽度列中写入 100 (像素), 在高度列中 100 (像素), 1568 在X 坐标列和1176在Y 坐标列中。单击确定。</li>
		<li>单击分析 |度量值 |。对选定区域的 "平均灰色值" 进行了测量。</li>
		<li>重复5.2.3。5.2.6。8剩余的图像。</li>
		<li>单击分析 |在函数列中校准并选择Rodbard25 , 并在中间的右侧列中写入以下数字 (0.04、0.336、0.632、0.928、1.224、1.52、1.816、2.112、2.408; 这些数字取决于阶梯式密度过滤器)。</li>
		<li>检查全局校准列, 然后单击确定。 注意: 通过执行此过程, "平均灰度值" 将使用 Rodbard 函数转换为 "光学密度 (OD)"。在此步骤之后, 可以为 ImageJ 软件中的特定选定区域计算光学密度。</li>
	</ol></li>
	<li>选择测量面积 注意: 斑点通常与地标相关, 如 campaniform 器、纵静脉提示和 crossveins。机翼上的这些和其他地标可以用来选择测量区域。这里, 一个例子D. guttifera(图2) 进行了描述。<ol><li>定义点 A, campaniform 触角点。<ol>
<li>打开 campaniform 触角斑点位于图像中心的图像。单击图像 |类型 |8位 |将图像转换为8位图像。</li>
			<li>在区域选择工具中单击矩形, 然后绘制一个矩形。设置矩形的左上角, 使其附着在第三条纵静脉的后线上, 更远地从 campaniform 触角点。设置矩形的右侧, 使其位于 campaniform 触角点的右侧。</li>
			<li>单击编辑 |选择 |添加到管理器 |。检查全部显示列。</li>
			<li>在行选择工具中单击角度工具。在第三纵静脉的后线上画第一条线。设置在步骤1中绘制的矩形顶点上线条的左端点。在矩形的上侧绘制第二行。按 "m" 键来测量这一步中绘制的两条线之间的角度。在计算机屏幕上单击图像的窗口。</li>
			<li>单击编辑 |选择 |添加到管理器 |。</li>
			<li>在区域选择工具中单击矩形, 并绘制一个大约1/9 图像窗口大小的矩形。单击编辑 |选择 |旋转 |。写入步骤5.3.1.4 中测量的角度的负度数。在角度列中, 单击确定以旋转此步骤中绘制的矩形。</li>
			<li>移动在步骤5.3.1.6 中绘制的矩形。使用箭头键。将第二纵脉后线的端点设置在矩形的左侧, 并将矩形的下侧连接到第三条纵静脉的后线。确定在该过程中, 从第二纵静脉的终点到第三纵静脉的后线的垂直线。将垂直线的脚定义为点 A (图 2A)。</li>
			<li>在将光标放在点 a 上时, 记录点 a 的 x 坐标和 y 坐标, 在区域选择工具下面指示。</li>
		</ol>
</li>
		<li>点 B 的定义, 纵静脉尖点<ol>
<li>打开一个纵向静脉尖点位于图像中心的图像。单击图像 |类型 |8位 |将图像转换为8位图像。</li>
			<li>重复步骤5.3.1 中描述的相同过程。(定义点 a, campaniform 触角点) 在图像中找到点 a。</li>
			<li>单击编辑 |选择 |添加到管理器 |。</li>
			<li>在区域选择工具中单击矩形, 然后绘制一个矩形。设置矩形的左上角顶点, 位于第三条纵静脉的后线的终点。</li>
			<li>单击编辑 |选择 |添加到管理器 |。</li>
			<li>在行选择工具中单击角度工具。绘制第一线连接点 A 和第三纵脉后线的终点。将此行定义为直线 a. 在步骤5.3.2.4 中绘制的矩形的上侧绘制第二行。按 "m" 键测量两条线之间的角度。</li>
			<li>单击编辑 |选择 |添加到管理器 |。</li>
			<li>在区域选择工具中单击矩形, 并绘制一个大约1/9 图像窗口大小的矩形。单击编辑 |选择 |旋转 |。写入步骤5.3.2.6 中测量的角度的负度数。在角度列中, 单击确定以旋转此步骤中绘制的矩形。使用箭头键移动矩形。设置矩形的上侧, 使其连接到直线 A 和第四纵脉前线的终点, 使其位于矩形的左侧。</li>
			<li>单击编辑 |选择 |添加到管理器 |。</li>
			<li>在区域选择工具中单击矩形, 并绘制一个大约1/9 图像窗口大小的矩形。单击编辑 |选择 |旋转 |。在角度列中, 写入在步骤6中测量的角度的减号度数, 然后单击确定以旋转此步骤中绘制的矩形。使用箭头键移动矩形。设置矩形的左下角, 使其位于在步骤5.3.2.8 中绘制的矩形的左上顶点, 从而获得从第四纵脉的前线终点到直线的垂直线. 定义垂直线的交点和第三纵静脉的后线作为点 B (图 2B)。</li>
			<li>在将光标放在 b 点上时, 记录点 b 的 x 坐标和 y 坐标, 在区域选择工具下面指示。</li>
		</ol>
</li>
		<li>点 C、后 crossvein 斑的定义<ol>
<li>打开一个图像, 其中后 crossvein 点位于图像的中心。单击图像 |类型 |8位 |将图像转换为8位图像。</li>
			<li>在后 crossvein 和第四纵静脉的交叉区 (图 2C) 中, 将点 C 定义为第四纵脉前线的后最点。</li>
			<li>将光标放在点 c 上时, 在区域选择工具下面, 记录点 c 的 x 坐标和 y 坐标。</li>
		</ol>
</li>
		<li>点 D 的定义, 控制区域<ol>
<li>打开 campaniform 触角斑点位于图像中心的图像。单击图像 |类型 |8位 |将图像转换为8位图像。</li>
			<li>在行选择工具中单击直, 并绘制一条连接第二纵静脉前线终点和第四纵静脉后线终点的直线。将点 D 定义为这条线的交点和第三纵静脉的后线 (图 2D)。</li>
			<li>将光标放在点 d 上时, 在区域选择工具下面, 记录 d 点的 x 坐标和 y 坐标。</li>
		</ol>
</li>
	</ol></li>
	<li>测量<ol>
<li>打开一个机翼斑点的图像 (为测量点 a, 打开图像与点 a 在中心)。单击图像 |类型 |8位 |将图像转换为8位图像。</li>
		<li>在区域选择工具中单击矩形, 并绘制图像窗口大约1/9 大小的矩形。</li>
		<li>单击编辑 |选择 |指定 |。检查椭圆形和居中列。在宽度和高度列中写入 100 (像素), 在x 坐标列中写入点 a (或点 b 或点 c) 和 d 的 x 坐标, 并在y 坐标列中写入点 a (或点 b 或点 c) 和 d 的 y 坐标。单击确定。</li>
		<li>单击分析 |度量值 |。如果5.2 中描述的校准已经完成, 则 ODs 在平均值列中指示。</li>
		<li>用点 A、B 和 C 的 ODs 减去点 D 的外径计算ΔODs。 注意: D 点在机翼的透明部分, 不包括色素沉着, 因此适用于背景控制。</li>
	</ol></li>
</ol>

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作者没有利益冲突。

我们在这里描述蛹阶段的定义的协议, 去除蛹为详细的观察, 测量蛹阶段的期间和测量黑斑点的强度在翼在D. guttifera。这些协议可以适用于许多果蝇和相关的蝇类, 特别是具有翼色素沉着的物种。
深入观察和描述更详细的发展事件将使阶段进一步细分。在许多情况下, 需要解剖或切片的蛹的发展事件不适合阶段定义, 因为一个人必须杀死一个蛹的分期, 并进一步使?...

果蝇的某些形态特征在种类中是多种多样的1,2,3,4,5。我们可以通过比较这些形态的生成机制来探讨形态多样性的产生问题。这种形态的例子有幼体毛、成人性梳子、外生殖器、腹部色素沉着和翅膀色素沉着6、7、8、9、10,11,12,13,14,15. 为了研究成人之间的形态学差异, 观察和分析蛹阶段是很重要的, 因为成虫性状的命运决定于晚期幼体阶段, 随后的形态发生在蛹时期。
在果蝇的发育生物学研究中, "小时 APF" (蛹形成后的小时) 是表示蛹阶段16的常用方法。该系统采用蛹形成后的绝对时间, 便于常规实验。但是, 蛹的发育速度可能不同, 可能会受到轻微的遗传、后生或微差异的影响, 因此在蛹形成后同样的绝对时间不保证蛹在同一发展阶段。在许多情况下, 由形态学特征定义的阶段比比较多个个体更可取。特别是, 物种之间的比较需要精确的分期和相应的 (同源) 阶段之间的比较。
班布里奇和 Bownes17根据果蝇蛹的形态学特征, 认可了20蛹阶段 (P1 至 P15 (ii))。这种分期是最广泛使用的系统形态发育分期18。在先前的研究中, 我们进行了蛹分期的果蝇 guttifera , 以建立一个基础的机翼色素沉着研究19。D. guttifera的翅膀上有一个黑色的圆点图案, 它是机翼色素沉着形成的模型物种之一20。虽然我们提到了班布里奇和 Bownes 的研究17中描述的形态学标准, 但我们直接用串行观测19来测量阶段的持续时间, 而不是使用班布里奇和 Bownes 的阶段工期估计从观察的频率。在这里, 我们描述了在卜et al19中使用的果蝇的蛹阶段的蛹分期和测量的方法。
研究翼色素沉着的发育机制, 需要了解蛹或成年期色素沉着的发生。卜et al。19通过对机翼图像进行图像分析, 在蛹和成人阶段对色素沉着进行量化的光学密度 (ODs)。果蝇翅膀的色素沉着被认为是由黑黑色素堆积引起的21。为量化 ODs, 使用了灰度图像和 ImageJ 软件 (https://imagej.nih.gov/ij/)22 。为了识别和量化斑点特定的色素沉着 (ΔOD), 我们从一个斑点内的 od 中减去外径。为了使这种方法重现性和客观, OD 测量的地方应该确定使用翼静脉作为地标。在这篇文章中, 我们详细描述了这种定量的方法, 机翼色素在果蝇 guttifera。

<table><tbody><tr><td>&lt;em&gt;Drosophila guttifera&lt;/em&gt;</td><td>The Drosophila Species Stock Center at the U.C. San Diego</td><td>15130-1971.10</td><td>&lt;em&gt;Drosophila guttifera&lt;/em&gt;, a fruit fly species used in this article</td></tr><tr><td>Plastic vial</td><td>Hightech</td><td>MKC-30</td><td>Plastic vial, for fly stock maintenance</td></tr><tr><td>Buzz plugs vial and bottle closures for glass vials</td><td>Fisher Scientific</td><td>AS-271</td><td>Cellulose plug, for fly stock maintenance</td></tr><tr><td>White soft sugar</td><td>Mitsui Sugar</td><td>J-500g</td><td>White soft sugar, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Corn flour</td><td>Nippon Flour Mills</td><td>F</td><td>Corn flour, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Corn grits - C</td><td>Nippon Flour Mills</td><td>GC</td><td>Corn grits - C, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Agar powder</td><td>Matsuki Kanten Sangyo</td><td>No.602</td><td>Agar powder, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Dry beer yeast</td><td>Asahi Food &amp;amp; Healthcare</td><td>Y2A</td><td>Dry beer yeast, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Butyl p-hydroxybenzoate</td><td>Nacalai Tesque</td><td>06327-02</td><td>Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Ethanol</td><td>Wako</td><td>057-00456</td><td>Ethanol, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Flat bottom microtube</td><td>Ina Optica</td><td>CF-0150</td><td>1.5 mL microtube, for collecting pupae</td></tr><tr><td>CAPSULEFUGE</td><td>Tomy</td><td>PMC-060</td><td>Mini microcentrifuge, for collecting pupae</td></tr><tr><td>Sterilized Schale NB</td><td>Sansei Medical</td><td>01-013</td><td>Plastic Petri dish (diameter 90 mm x height 15 mm)</td></tr><tr><td>Serum tube rack</td><td>Iwaki</td><td>9796-050</td><td>Used as a moist chamber, for observation of pupa</td></tr><tr><td>Corning Falcon Easy-Grip tissue culture dish</td><td>Corning</td><td>353001</td><td>Plastic Petri dish (diameter 35 mm x height 10 mm)</td></tr><tr><td>Falcon standard tissue culture dish</td><td>Corning</td><td>353002</td><td>Plastic Petri dish (diameter 60 mm x height 15 mm)</td></tr><tr><td>Push-pin</td><td>Kokuyo</td><td>51233709</td><td>Push-pin, for making pinholes on the microtube lid</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td>Stereomicroscope, for morphological observation</td></tr><tr><td>Digital camera</td><td>Olympus</td><td>DSE-330-A</td><td>Digital camera, for imaging</td></tr><tr><td>NICETACK double sided tape</td><td>Nichiban</td><td>NW-15SF</td><td>Double sided tape, for removing puparium</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Forceps, for removing puparium</td></tr><tr><td>Van Gogh VISUAL Paint brush</td><td>Talens Japan</td><td>GWVR-#5/0</td><td>Paint brush, for removing puparium</td></tr><tr><td>Greiner CELLSTAR 12 well cell culture plate</td><td>Merck</td><td>665-180</td><td>12-well cell culture plate, for measuring durations of pupal periods</td></tr><tr><td>NaCl</td><td>Wako</td><td>191-01665</td><td>NaCl, for PBS</td></tr><tr><td>KCl</td><td>Nacalai Tesque</td><td>285-14</td><td>KCl, for PBS</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>196-02835</td><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;12H&lt;sub&gt;2&lt;/sub&gt;O, for PBS</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Nacalai Tesque</td><td>28721-55</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, for PBS</td></tr><tr><td>Stepped Neutral Density (ND) Filter 0.04 - 3.0</td><td>Edmund Optics</td><td>64-384</td><td>Stepped density filter, for calibration of pigmentation measurement</td></tr><tr><td>ImageJ software</td><td>NIH</td><td>1.8.0-101</td><td>ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)</td></tr><tr><td>FINE FROST glass slide</td><td>Matsunami Glass Ind</td><td>FF-001</td><td>Glass slide, for measurement of intensity of black spots on a wing</td></tr><tr><td>Square microscope cover glass 18 x 18</td><td>Matsunami Glass Ind</td><td>C018181</td><td>Cover slip, for measurement of intensity of black spots on a wing</td></tr></tbody></table>

methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera

不同种类的果蝇(水果蝇) 提供了研究进化机制和遗传变化的机会。特别是, 成人阶段是一个丰富的形态特征来源的种间比较, 包括翼色素沉着比较。为了研究物种间的发育差异, 需要进行细致的观察和适当的分期, 以便进行精确的比较。在这里, 我们描述的蛹周期的分期和量化的翅膀色素在一个波尔卡斑点果蝇,果蝇 guttifera的协议。首先, 我们描述了详细的形态学观察方法和定义的蛹阶段基于形态。该方法包括一种去除蛹外壳的蛹的技术, 以便对蛹形态进行细致观察。其次, 我们描述了测量定义的蛹阶段的持续时间的方法。最后, 我们用数字图像和 ImageJ 软件描述了基于图像分析的机翼色素沉着定量方法。通过这些方法, 我们可以为比较蛹阶段成人性状的发育过程奠定坚实的基础。

将D. guttifera的蛹期划分为17阶段 (P1-P15 (ii); 三个具有代表性的阶段 (P1、P5-6、P10) 的图像显示图 3, 所有17阶段都在图 4中说明。虽然班布里奇和 Bownes17在d. 黑腹中识别了20阶段, 但这些阶段中的某些步骤无法应用于d. guttifera。两个发育事件的顺序, 黄色身体的出现 (在中肠26内?...

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Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

介绍了果蝇 guttifera的分期蛹周期和机翼色素沉着的测量方法。色素沉着的分期和量化为研究成人性状的发育机制提供了坚实的基础, 并使性状发育的种间比较得以实现。

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

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Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

11.5K 次观看.  Kyoto University. 介绍了果蝇 guttifera的分期蛹周期和机翼色素沉着的测量方法。色素沉着的分期和量化为研究成人性状的发育机制提供了坚实的基础, 并使性状发育的种间比较得以实现。

视频: Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

科研

发育生物学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

神经科学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting 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 ...

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

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

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

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

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 ...

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4.
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bx&alpha;, where x and y are the size of the two traits, such as organ and body size8,9. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + &alpha;log(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of &alpha;, called the 'allometric coefficient'9,10. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and &alpha;) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12).
Not all traits scale linearly with each other or with body size (e.g., 13,14) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila, our methodology should be applicable to nearly any fully metamorphic insect.

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.

实验操作的车身尺寸估计​​形态缩放关系果蝇</em

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

生物学

<meta charset="utf8"></head><body>无蛹的 果蝇 Pupal 腿部形态发生的扩展实时成像

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

<meta charset="utf8"></head><body>作者聚焦：揭示 果蝇 发育过程中的动态上皮结构

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

果 蝇 蛹切除后蛹腿部发育的长期实时成像

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

免疫与感染

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

摘要

探索更多视频

此视频中的章节

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

可视化本体感受器

果蝇蛹卵巢的解剖与染色

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

实验操作的车身尺寸估计形态缩放关系果蝇

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹中炎症细胞动力学的长期体内追踪

定量比较顺调节元件（CRE）的活动，在转基因果蝇

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

摘要

探索更多视频

此视频中的章节

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

可视化本体感受器

果蝇蛹卵巢的解剖与染色

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

实验操作的车身尺寸估计​​形态缩放关系果蝇

果 蝇 蛹切除后蛹腿部发育的长期实时成像

果 蝇 蛹切除后蛹腿部发育的长期实时成像

果 蝇 蛹切除后蛹腿部发育的长期实时成像

果蝇蛹中炎症细胞动力学的长期体内追踪

定量比较顺调节元件（CRE）的活动，在转基因果蝇

实验操作的车身尺寸估计形态缩放关系果蝇

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像

果蝇蛹切除后蛹腿部发育的长期实时成像