Name: methods to test endocrine disruption in drosophila melanogaster
Uploaded: 2019-07-03T14:00:00
Description: Watch this Scientific Journal Video about Methods to Test Endocrine Disruption in Drosophila melanogaster at JoVE.com

作者感谢奥索利娜·佩蒂洛的技术支持。作者感谢玛丽亚罗萨里亚·阿莱塔博士(CNR)对书目的支持。作者感谢古斯塔沃·达米诺·米塔博士将其介绍给EDC世界。作者感谢莱卡微系统公司和帕斯夸尔·罗曼诺的帮助。这项研究得到了项目PON03PE_001110_1的支持。"纳米技术定向学的Sviluppo di nanotecnoe a.rigenerazione e Ricostruzione Tissutale, 在奥东托里亚/奥多里亚/奥科利西亚的植入物 e 感理"acronimo"SORRISO"承诺:PO FESR 2014-2020 CAMPANIA;项目PO FESR坎帕尼亚2007-2013年"纳纳特诺诺诺诺托控制迪莫莱科尔生物-技术纳塔诺诺诺诺诺诺"。

1. 食物准备
<ol><li>
对于库存维持和幼虫生长,使用含有3%粉状酵母、10%蔗糖、9%预煮玉米粉、0.4%琼脂的玉米粉培养基,其后称为玉米粉培养基(CM)。
	<ol>
<li>将30克酵母放入100 mL的自来水中,煮沸,煮15分钟。</li>
		<li>另外,将90克预先煮熟的玉米粉、100克糖和4克琼脂混合成900 mL的自来水。</li>
		<li>将溶液煮沸,降低热量,连续煮5分钟。</li>
		<li>5分钟后,加入热酵母溶液,再煮15分钟。</li>
		<li>关?...

果蝇D.melanogaster被广泛用作体内模型系统,以研究环境EDC的潜在影响,如DBP28,双酚A,4-NP,4-ter-OP29,MP30,EP31, 32, DEHP33,和 EE234.有几个原因导致它作为模型在这一研究领域使用。除了作为模型系统无可争议的优势外,果蝇与人类共享高度的基因保护,而果蝇EDC测定法可能...

人类活动已经释放出大量的化学物质,这对生物体和自然生态系统来说是一个严重的问题。在这些污染物中,估计约有1,000种不同的化学物质可能改变内分泌系统的正常平衡;根据这个特性,它们被归类为干扰内分泌的化学物质(EDCs)。具体来说,根据内分泌学会最近的定义,EDCs是"一种外源性化学物质,或化学品的混合物,可以干扰激素作用的任何方面"2 。在过去的三十年里,越来越多的科学证据表明,EDC会影响动植物的繁殖和发育3,4,5,6,7, 8.此外,EDC接触与一些人类疾病的日益流行有关,包括癌症、肥胖症、糖尿病、甲状腺疾病和行为障碍9、10、11。
EDC的一般机制
由于其分子特性,EDCs的行为类似于激素或激素前体3,4,5,6,7,8,9, 10,11,12.从这个意义上说,它们可以通过模仿激素活性或阻断内源性激素结合来与激素受体结合,扰乱内分泌系统。在第一种情况下,与受体结合后,它们可以像其天然激素那样激活它。在其他情况下,EDC与受体结合会阻止其天然激素的结合,因此受体被阻断,无法再被激活,即使存在其天然激素3。因此,EDC 可以影响多个过程,例如内源性激素的合成、分泌、运输、代谢或外围作用,这些过程负责维持的平衡、繁殖、发展和/或行为。有机体。受体绑定不是迄今为止EDC描述的唯一行动方式。现在很清楚,他们也可以通过在酶通路中招募联合激活剂或核心压榨器,或者通过修改表观遗传标记来解除基因表达10、11、12、13 ,14,不仅对当代人,而且对后代的健康带来影响。
果蝇激素
已广泛研究了选定的EDC的潜在影响,无论是在野生动物物种中,还是在内分泌机制相当广为人知的几个模型系统中。对于无脊椎动物,影响生长、发育和繁殖的内分泌系统在昆虫中被广泛特征,原因有若干原因,涉及它们在生物研究领域的广泛应用、其经济重要性和最后,开发能够专门干扰害虫激素系统的杀虫剂。
特别是在昆虫中,果蝇D.黑色素气仪已被证明是一个非常强大的模型系统,以评估EDCs的潜在内分泌效应。在黑色素和脊椎动物中,激素在整个生命周期中起着重要的作用。在这个有机体中,有三个主要的荷尔蒙系统,其中涉及类固醇激素20-羟基克西酮(20E)15,16,Sesquiterpenoid青少年激素(JH)17,和神经肽和肽/蛋白质激素18.第三组由最近发现但明显涉及各种生理和行为过程的肽组成,如长寿、平衡、代谢、生殖、记忆和运动控制。20E与胆固醇衍生的类固醇激素(如雌二醇)同源,而JH与视黄酸有一些相似之处;他们两者都是德罗索菲拉19,20中比较著名的激素。它们的平衡对于协调蜕皮和蜕变,以及控制几个发育后过程(如生殖、寿命和行为21)至关重要,从而为测试内分泌提供了不同的可能性在果蝇的中断。此外,异类固醇激素和JHs是所谓的第三代杀虫剂的主要目标,这种杀虫剂旨在干扰昆虫的发育和生殖内分泌介导过程。这些化学品的激动剂或拮抗作用模式是众所周知的,因此它们可以作为评估潜在EDCs对昆虫生长、繁殖和发育影响的参考标准22。例如,Methoprene已被广泛用于控制蚊子和其他水生昆虫23,24,作为JH激动剂,抑制20E诱导的基因转录和蜕变。
除了激素,果蝇中的核受体(NR)超级家族也是众所周知的;它包括18个进化保存转录因子,涉及控制激素依赖性发育途径,以及生殖和生理学25。这些激素NR属于所有六个NR超级家族亚型,包括那些涉及神经传递26,两个视黄酸NR,和那些类固醇NR,在脊椎动物,代表EDCs27的主要目标之一。
果蝇作为研究EDC的模型系统
目前,根据分子特性,世界各地的几个环境机构将干扰内分泌系统的可能性归因于不同的人造化学品。鉴于EDC是环境和生物体的全球性和普遍问题,这一领域的研究的总体目标是减轻其疾病负担,并保护生物免受其不利影响。为了加深对一种化学物质潜在的内分泌效应的理解,有必要在体内对其进行测试。为此,D. melanogaster表示一个有效的模型系统。迄今为止,果蝇已被广泛用作体内模型,以评估几种环境EDC的影响;据报道,暴露于几种EDCs,如二丁基邻苯二甲酸酯(DBP)28,双酚A(BPA),4-非基酚(4-NP),4-吨-乙基苯酚(4-t-OP)29,甲基对羟基苯甲酸酯(MP)30,乙基对羟基苯甲酸酯(EP)31, 32、二乙基(乙酰乙酰)邻苯二甲酸酯(DEHP)33和17-α-乙酸乙酯(EE2)34,影响脊椎动物模型中的新陈代谢和内分泌功能。有几个原因导致它作为这一研究领域的一个模型。除了对其内分泌系统的深入了解外,其他优势还包括其生命周期短、成本低、基因组易于操作、研究历史悠久以及多种技术可能性(参见 FlyBase 网站,http://flybase.org/)。D. melanogaster还为轻松研究跨代效应和种群对环境因素的反应提供了强大的模型,并避免了与高等动物体内研究相关的伦理问题。此外,果蝇与人类共享高度的基因保护,这可能使果蝇EDC检测有助于预测或建议这些化学物质对人类健康的潜在影响。除了扩大对人类健康影响的理解外,果蝇还有助于评估EDC暴露于环境中的风险,如生物多样性丧失和环境退化。最后,果蝇提供了在实验室使用的额外优势,在实验室中,可能影响其发育、繁殖和寿命的因素可以加以控制,以便将任何变异归因于要测试的物质。
考虑到这一点,我们优化了简单和强大的健身测定,以确定EDC对一些果蝇激素特性的影响,如生育/生育、发育时机和成人寿命。这些测定已广泛用于一些EDCs 23,24,25,26,27。特别是,我们使用以下协议来评估暴露于合成雌激素 EE234和 BPA 和双酚 AF (BPA F)(未发布数据)的影响。这些协议可以很容易地修改,以研究给定EDC在一次的影响,以及多个EDC在D.melanogaster的综合影响。

<table>
	<tbody>
		<tr>
			<td>17&#945;-Ethinylestradiol</td>
			<td>Sigma</td>
			<td>E4876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar for Drosophila medium</td>
			<td>BIOSIGMA</td>
			<td>789148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisphenol A</td>
			<td>Sigma</td>
			<td>239658-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisphenol AF</td>
			<td>Sigma</td>
			<td>90477-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td>CA&#39; BIANCA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>BIOSIGMA</td>
			<td>789008</td>
			<td>25x95 mm</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>BIOSIGMA</td>
			<td>789009</td>
			<td>29x95 mm</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>Kaltek</td>
			<td>187</td>
			<td>22X63</td>
		</tr>
		<tr>
			<td>Embryo collection cage</td>
			<td>Crafts</td>
			<td></td>
			<td>Plexiglass cylinder (12,5 x7 cm) with an open end and the other end closed by a rectangular base in which a slot allows the insertion of special trays for laying</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>FLUKA</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Etherizer</td>
			<td>Crafts</td>
			<td></td>
			<td>cylindrical glass container with a cotton plug</td>
		</tr>
		<tr>
			<td>Glass Bottle</td>
			<td></td>
			<td></td>
			<td>250mL Bottles</td>
		</tr>
		<tr>
			<td>Glass Vials</td>
			<td>Microtech</td>
			<td>ST 10024</td>
			<td>FLAT BOTTOM TUBE 100X24</td>
		</tr>
		<tr>
			<td>Hand blender Pimmy</td>
			<td>Ariete</td>
			<td></td>
			<td>food processor</td>
		</tr>
		<tr>
			<td>Instant Success yeast</td>
			<td>ESKA</td>
			<td></td>
			<td>Powdered yeast</td>
		</tr>
		<tr>
			<td>Laying tray</td>
			<td>Crafts</td>
			<td></td>
			<td>plexiglass trays (11 x 2,6 cm) in wich to pour medium for laying</td>
		</tr>
		<tr>
			<td>Methyl4-hydroxybenzoate</td>
			<td>SIGMA</td>
			<td>H5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Falcon</td>
			<td>351016</td>
			<td>60x5</td>
		</tr>
		<tr>
			<td>Red dye no. 40</td>
			<td>SIGMA</td>
			<td>16035</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope with LED lights</td>
			<td>Leica</td>
			<td></td>
			<td>S4E</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>HIMEDIA</td>
			<td>MB025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tomato sauce</td>
			<td>Cirio</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to test endocrine disruption in drosophila melanogaster

近年来,越来越多的证据表明,所有生物体和环境都暴露于激素样的化学物质,即内分泌干扰物(EDCs)。这些化学物质可能改变内分泌系统的正常平衡,导致不利影响,以及人类中荷尔蒙紊乱的数量增加或野生动物物种的生长和繁殖减少。对于某些 EDC,有记录的健康影响和限制其使用。然而,对于他们中的大多数来说,在这个意义上仍然没有科学证据。为了验证一种化学物质在全有机体中的潜在内分泌效应,我们需要在适当的模型系统中以及果蝇,果蝇,果蝇黑色素气。在这里,我们报告详细的体内协议,以研究果蝇的内分泌中断,解决EDC对苍蝇的繁殖/生育能力,发育时间和寿命的影响。在过去几年中,我们使用这些果蝇生命特性来研究暴露于17-β-苯乙醇(EE2)、双酚A(BPA)和双酚AF(BPA F)的影响。总之,这些测定涵盖了所有果蝇生命阶段,并使得评估所有激素介导过程中的内分泌中断成为可能。成熟/生育力和发育时序测定有助于分别测量EDC对苍蝇生殖性能和发育阶段的影响。最后,寿命测定涉及慢性EDC对成人的接触,并测量他们的存活率。然而,这些生命特征也可能受到几个必须仔细控制的实验性因素的影响。因此,在这项工作中,我们建议一系列程序,我们已经优化了这些检测的正确结果。这些方法允许科学家建立内分泌中断的任何EDC或混合不同的EDCs在果蝇,虽然要确定负责这种效果的内分泌机制,可能需要进一步的文章。

在本节中,以简化方案的形式报告上述协议的关键步骤。鉴于苍蝇倾向于避免令人不快的化合物,第一件事就是测定所选EDC的味道。这可以通过混合食品着色(例如,红色食品染料40号)35与食品补充与选定的EDC在各种剂量或与溶剂单独。以这些介质喂养的苍蝇在立体显微镜下接受检查,食物摄入量由腹部着色估算(图1)。图1显示了一个典型的?...

Watch this Scientific Journal Video about Methods to Test Endocrine Disruption in Drosophila melanogaster at JoVE.com

Methods to Test Endocrine Disruption in Drosophila melanogaster

内分泌干扰化学品 (EDC) 是生物体和自然环境的严重问题。果蝇黑色素是研究体内EDC效应的理想模型。在这里,我们提出的方法,以调查果蝇的内分泌中断,解决EDC对苍蝇的生育、生育、发育时间和寿命的影响。

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

嗜血杆菌黑色素酯是一个强大的模型，用于评估化学品（如内分泌干扰物）在体内的潜在毒性和内分泌作用，这些化学物质对人类健康和自然环境构成严重问题。解决EDC对苍蝇荷尔蒙寿命特征的影响，如生育能力、生育能力、发育时间和寿命。这是一种简单、廉价、快速的方法，可以调查体内内分泌中断。果蝇的生育力、发育和寿命可能受到几个因素的影响，这些因素必须仔细控制，以确保本协议的成功结果。因此，在饲养和处理苍蝇时需要给予最大的关注。为了测定所选EDC的味道，麻醉后，将15只幼蝇从苍蝇床转移到4个平行小瓶，里面装着与食品色素混合的玉米粉介质。控制小瓶仅含有溶剂，其他三个小瓶辅以不同剂量的选定EDC。将小瓶在加湿的培养箱中保持一天，自然12小时的光线。再次麻醉苍蝇后，将固定苍蝇转移到白纸上，将其置于立体显微镜下观察。比较每个治疗组的腹部着色与对照组。为了检测苍蝇的生殖性能，为每个EDC准备三小瓶苍蝇作为父母小瓶，8个雌性苍蝇和4个雄性在10毫升玉米粉介质辅以EE2。为了控制，准备6瓶苍蝇，每瓶有8只雌性苍蝇和4只雄性苍蝇，在10毫升玉米粉中用EE2溶剂补充。后在25摄氏度的孵化器里飞行。在它们发育期间，避免幼虫过度拥挤。四天后，将每个小瓶倒置在乙醚器上，取出父母，将小瓶返回孵化器五天。在第九天的下午晚些时候，将每个小瓶倒置在乙醚器上，将小瓶中取出所有新出现的苍蝇，并在18摄氏度的孵化器中过夜。10日早晨，在嗜酒二氧化碳工作站上，小心地反转培养瓶，防止麻醉苍蝇跌落到介质上，并在棉塞和小瓶壁之间插入一根用针状针头的橡胶管。打开阀门以传递二氧化碳。在几秒钟内，在显微镜下将苍蝇转移到苍蝇床上。将处女女性和年轻男性分别收集成两组在飞床上。随机细分每组苍蝇在小子组，10个雌性或20雄每瓶充满了新鲜相应的玉米粉介质。继续孵育，收集30个处女和30名男性每个EDC，90个处女女性和90名男性控制。这些苍蝇在摄氏25度时，直到它们被封住后四天。然后每两天将它们转移到含有新鲜相应介质的新小瓶中。检查雌性小瓶中没有幼虫。然后为小瓶贴上不同系列序号的标签，用于以下治疗。麻醉后，将一个EE2溶剂处理雌性转移到含有不含EE2的新鲜玉米粉番茄介质的小瓶中，并加入一个EE2溶剂处理雄性进行控制。每次治疗，EE2 处理的男性与 EE2 溶剂处理雌性或 EE2 处理性雌性与 EE2 溶剂处理型雄性配对。房子20单十字架在25摄氏度。在随后的10天内，每天将每对交配转移到没有EDC的新鲜玉米粉番茄中小瓶中。每天目视检查每个小瓶的卵子，并在生育电子表格上报告其数量。在每个小瓶中，仔细检查新出现的苍蝇，并记录10天期间成年后代的每日数量。从最初的交配10天后，从最后一个系列小瓶中删除父母。在环闭性测定方案中，首先，每个治疗组，设置10瓶不到两天的年轻健康苍蝇，每只6只雌蝇和3只雄性在10毫升的玉米粉食品中，没有EDC。将苍蝇放在食物上24小时，并允许它们交配。然后，每个治疗组再准备10个平行小瓶，每组10毫升新鲜玉米粉，并辅以不同的EDC浓度或EE2溶剂进行控制。将交配苍蝇转移到这些新瓶。制作一个开发电子表格来记录不同的系列。让苍蝇产卵16小时。然后，将父母从小瓶中取出。在25摄氏度下孵育小瓶三到四天，或直到可见流浪幼虫。每天，通过按小瓶外在外文上按顺序书写一个数字来计算每个小瓶中新幼崽的数量，以避免数同一小瓶两次。报告三到四天新出现幼崽的数量，或直到不再有幼崽形成。从第九天开始，每天计算新兴成年人的数量，直到不再有成年人出现。报告在发展电子表格上，并根据手稿执行余下环法测定。在寿命协议中，首先设置了20个小瓶，有8个雌性，4个雄性，每个小瓶都装满了10毫升的玉米粉食物，并把它们放在25摄氏度的温度下。四天后，丢弃苍蝇，将小瓶放回孵化器。在第九天的下午晚些时候，从小瓶中取出所有新出现的苍蝇，然后将小瓶返回孵化器。16至24小时后，在轻二氧化碳麻醉下，将250只一日大小的成年苍蝇分成四组，并转移到250毫升的瓶装含有成人中度食物的瓶子中，其中3只补充不同的EDC浓度，1只单独使用EE2溶剂。将苍蝇在25摄氏度下保持两到三天，以便它们交配。然后，按性别将每组苍蝇分成两组。在每个治疗条件下，对于两性，随机细分为五个平行小瓶，密度为每瓶20人。准备一个寿命电子表格，其中从上一次转移中幸存的苍蝇数量中减去死苍蝇的数量，以便自动获得每次转移的幸存者人数。三天后，将不麻醉的苍蝇转移到含有相应食物的新小瓶中，并检查死亡情况。记录苍蝇的年龄和死苍蝇的数量。每三天重复一次转移，直到所有的苍蝇死亡。在此实验中，寿命曲线被绘制为累积生存期与每个 EDC 浓度和控制的经过天数。对于控制，在生存曲线保持相对较高的长期初始时间之后，它大约60天后呈指数级下降。EDC暴露后，被治疗苍蝇的生存曲线受到显著影响，36天后出现明显下降。建议分析的平行小瓶应该非常相似，否则很难理解要丢弃哪个小瓶。因此，我们建议小心，采用良好的实验实践，使用大量的样本量。按照此协议，还可以评估选定的内分泌干扰物的代际影响，并测试不同内分泌干扰物组合的潜在添加剂效应。

methods-to-test-endocrine-disruption-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

Methods to Test Endocrine Disruption in Drosophila melanogaster

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

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 ...

科研

发育生物学

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

饲养果蝇<em&gt;果蝇</em&gt;在无菌和悉生条件

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

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 ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

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 ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

使用微计算断层扫描的 果蝇黑色素加斯特 全动物成像

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

黑腹果蝇第三龄幼虫脑的活细胞成像

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...