Name: fluorescent in situ hybridization and 5-ethynyl-2'-deoxyuridine labeling for stem-like cells in the hydrozoan jellyfish cladonema pacificum
Uploaded: 2022-08-03T15:00:00
Description: Watch this Scientific Journal Video about Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum at JoVE.com

这项工作得到了AMED的支持，授权号为JP22gm6110025（致Y.N.）和JSPS KAKENHI资助号为22H02762（致Y.N.）。

注意：有关本协议中使用的所有材料，试剂和设备的详细信息，请参阅 材料表 。
1. 探针合成
<ol><li>核糖核酸提取<ol><li>使用切断吸头的 3.1 mL 移液管将三个在人工海水 （ASW） 中培养的活水母在 1.5 mL 管中，并尽可能多地去除 ASW。 注意：ASW是通过用氯中和剂将矿物盐混合物溶解在自来水中制备的;最终比重（S.G.）为1.018或千分之几（ppt）为~...

<ol>
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增殖细胞和干细胞是形态发生、生长和再生等各种过程中的重要细胞来源21，22。本文描述了一种通过FISH和EdU标记在水母乳中对干细胞标记物Nanos1进行共染色的方法。先前使用EdU或BrdU标记的工作表明，增殖细胞定位于触手球16，17，但它们的分子特征尚不清楚。本研究显示了增殖细胞分布和Nano...

刺胞动物被认为是一种基部分支的后生动物门，包含具有神经和肌肉的动物，使它们处于了解动物发育和生理学进化的独特位置1，2。刺胞动物分为两大类：花生动物（例如海葵和珊瑚）仅拥有扁平幼虫和无柄息肉阶段，而Medusozoa（水生动物，Staurozoa，Scyphozoa和Cubozoa的成员）通常采取自由游泳的水母或水母的形式，以及扁平幼虫和息肉。刺胞动物通常表现出很高的再生能力，其潜在的细胞机制，特别是它们拥有成体干细胞和增殖细胞，引起了人们的广泛关注3，4。最初在Hydra中鉴定，水生干细胞位于外胚层上皮细胞之间的间质空间中，通常称为间质细胞或i细胞3。
水生动物i细胞具有共同的特征，包括多能性，广泛保守的干细胞标志物（例如，纳米，Piwi，Vasa）的表达和迁移潜力3，5，6，7，8。作为功能性干细胞，i细胞广泛参与水生动物的发育、生理和环境反应，这证明了它们的高再生能力和可塑性3。虽然干细胞与i细胞相似，尚未在水生动物之外发现，即使在已建立的模式物种Nematostella中，增殖细胞仍然参与体细胞组织的维持和再生以及生殖系9。由于刺胞动物发育和再生的研究主要在息肉型动物（如Hydra，Hydractinia和Nematostella）上进行，因此水母物种中干细胞的细胞动力学和功能在很大程度上仍未得到解决。
水生动物水母 Clytia hemisphaerica是一种 世界性水母物种，在世界各地（包括地中海和北美）具有不同的栖息地，已在几项发育和进化研究中被用作实验模型动物10。 Clytia 体积小、易于处理、卵大，适用于实验室维护，以及引入遗传工具，例如最近建立的转基因和敲除方法11，为详细分析水母生物学背后的细胞和分子机制提供了机会。在 Clytia medusa 触手中，i细胞位于称为球茎的近端区域，线虫等祖细胞迁移到远端尖端，同时分化成不同的细胞类型，包括线细胞12。
在水母的口腔器官Clytia manubrium的再生过程中，存在于性腺中的Nanos1 + i细胞迁移到手稿因损伤而丢失的区域，并参与手稿的再生7。这些发现支持了Clytia中的i细胞也表现为参与形态发生和再生的功能性干细胞的观点。然而，鉴于i细胞的性质在具有代表性的息肉型动物（如Hydra和Hydractinia3）之间有所不同，因此干细胞的特征和功能可能在水母物种中多样化。此外，除了Clytia之外，其他水母的实验技术受到限制，增殖细胞和干细胞的详细动力学未知13。
水生动物水母 Cladonema pacificum 是一种新兴的模式生物，可以在没有水泵或过滤系统的情况下保存在实验室环境中。Cladonema medusa具有分支触手，这是 Cladonematidae 家族的共同特征，并且在灯泡14附近的外胚层上有一个称为ocellus的感光器官。触手分支过程发生在沿着触手的近轴侧出现的新分支部位。随着时间的流逝，触手继续伸长并分枝，较老的枝条被推向尖端15。此外， Cladonema 触手可以在截肢后的几天内再生。最近的研究表明增殖细胞和干细胞样细胞在 Cladonema16，17的触手分支和再生中的作用。然而，虽然传统的 原位 杂交（ISH）已被用于可视化 Cladonema中的基因表达，但由于其分辨率低，目前很难在细胞水平上详细观察干细胞动力学。
本文描述了一种通过FISH可视化Cladonema中的干细胞样细胞并与细胞增殖标志物EdU共染色的方法18。我们通过FISH可视化了干细胞标记5，17Nanos1的表达模式，这允许在单细胞水平上鉴定干细胞样细胞分布。此外，Nanos1表达与EdU标记的共染色使得区分活跃增殖的干细胞样细胞成为可能。这种监测干细胞样细胞和增殖细胞的方法可以应用于广泛的研究领域，包括触手分支、组织稳态和克拉多内玛的器官再生，类似的方法可以应用于其他水母物种。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol&#160;</td>
			<td>Wako</td>
			<td>137-06862</td>
			<td></td>
		</tr>
		<tr>
			<td>3.1 mL transfer pipette</td>
			<td>Thermo Scientific</td>
			<td>233-20S</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Wako</td>
			<td>029-15043</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DIG-POD</td>
			<td>Roche</td>
			<td>11207733910</td>
			<td></td>
		</tr>
		<tr>
			<td>Cladonema pacificum Nanos1 forward primer</td>
			<td></td>
			<td></td>
			<td>5&#8217;-AAGAGACACAGTCATTATCAAGC 
			GA-3&#8217;</td>
		</tr>
		<tr>
			<td>Cladonema pacificum Nanos1 reverse primer</td>
			<td></td>
			<td></td>
			<td>5&#8217;-CGACGTGTCCAATTTTACGTGCT -3&#8217;</td>
		</tr>
		<tr>
			<td>Cladonema pacificum Piwi forward primer</td>
			<td></td>
			<td></td>
			<td>5&#8217;- AAAAGAGCAGCGGCCAGAAAGA 
			AGGC -3&#8217;</td>
		</tr>
		<tr>
			<td>Cladonema pacificum Piwi reverse primer</td>
			<td></td>
			<td></td>
			<td>5&#8217;- GCGGGTCGCATACTTGTTGGTA 
			CTGGC -3&#8217;</td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye</td>
			<td>Invitrogen&#160;</td>
			<td>C10337</td>
			<td>EdU kit</td>
		</tr>
		<tr>
			<td>Coroline off</td>
			<td>GEX Co. ltd</td>
			<td>N/A</td>
			<td>chlorine neutralizer</td>
		</tr>
		<tr>
			<td>DIG Nucleic Acid Detection Kit Blocking Reagent</td>
			<td>Roche</td>
			<td>11175041910</td>
			<td>blocking buffer&#160;</td>
		</tr>
		<tr>
			<td>DIG RNA labeling mix&#160;</td>
			<td>Roche</td>
			<td>11277073910</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;</td>
			<td>Promega</td>
			<td>P117B</td>
			<td></td>
		</tr>
		<tr>
			<td>ECOS competent cell DH5&#945;</td>
			<td>NIPPON GENE</td>
			<td>316-06233</td>
			<td>competent cell</td>
		</tr>
		<tr>
			<td>Fast gene Gel/PCR Extraction kit</td>
			<td>Fast gene</td>
			<td>FG-91302</td>
			<td>gel extraction kit</td>
		</tr>
		<tr>
			<td>Fast gene plasmid mini kit</td>
			<td>Fast gene</td>
			<td>FG-90502</td>
			<td>plasmid miniprep</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Wako&#160;</td>
			<td>068-00426</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine</td>
			<td>SIGMA-ALDRICH&#160;</td>
			<td>H3393-10KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D(-)-thiogalactopyranoside (IPTG)</td>
			<td>Wako</td>
			<td>096-05143</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Invitrogen</td>
			<td>22700-025</td>
			<td>agar plate</td>
		</tr>
		<tr>
			<td>LB Broth Base</td>
			<td>Invitrogen</td>
			<td>12780-052</td>
			<td>LB medium</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Wako</td>
			<td>134-00495</td>
			<td></td>
		</tr>
		<tr>
			<td>mini Quick spin RNA columns</td>
			<td>Roche</td>
			<td>11814427001</td>
			<td>clean-up column</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako&#160;</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop OneC Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Scientific</td>
			<td>ND-ONEC-W</td>
			<td>spectrophotometer</td>
		</tr>
		<tr>
			<td>Polyoxyethlene (20) Sorbitan Monolaurate (Tween-20)</td>
			<td>Wako&#160;</td>
			<td>166-21115</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerMasher 2</td>
			<td>nippi&#160;</td>
			<td>891300</td>
			<td>homogenizer</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Nacarai Tesque&#160;</td>
			<td>29442-14</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>TaKaRa</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen&#160;</td>
			<td>74004</td>
			<td>total RNA isolation kit</td>
		</tr>
		<tr>
			<td>RQ1 RNase-Free Dnase</td>
			<td>Promega</td>
			<td>M6101</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline Sodium Citrate Buffer 20x powder (20x SSC)</td>
			<td>TaKaRa</td>
			<td>T9172</td>
			<td></td>
		</tr>
		<tr>
			<td>SEA LIFE</td>
			<td>Marin Tech</td>
			<td>N/A</td>
			<td>mixture of mineral salts</td>
		</tr>
		<tr>
			<td>T3 RNA polymerase&#160;</td>
			<td>Roche</td>
			<td>11031163001</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNA polymerase&#160;</td>
			<td>Roche</td>
			<td>10881767001</td>
			<td></td>
		</tr>
		<tr>
			<td>TAITEC HB-100</td>
			<td>TAITEC</td>
			<td>0040534-000</td>
			<td>Hybridization incuvator</td>
		</tr>
		<tr>
			<td>TaKaRa Ex Taq&#160;</td>
			<td>TaKaRa</td>
			<td>RR001A</td>
			<td>Taq DNA polymerase</td>
		</tr>
		<tr>
			<td>TaKaRa PrimeScript 2 1st strand cDNA Synthesis Kit</td>
			<td>TaKaRa</td>
			<td>6210A</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr>
			<td>Target Clone</td>
			<td>TOYOBO&#160;</td>
			<td>TAK101</td>
			<td>pTA2 Vector</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Roche</td>
			<td>10109541001</td>
			<td></td>
		</tr>
		<tr>
			<td>TSA Plus Cyanine 5</td>
			<td>AKOYA Biosciences</td>
			<td>NEL745001KT</td>
			<td>tyramide signal amplification (TSA) technique</td>
		</tr>
		<tr>
			<td>Zeiss LSM 880</td>
			<td>ZEISS</td>
			<td>N/A</td>
			<td>laser scanning confocal microscope</td>
		</tr>
	</tbody>
</table>

fluorescent in situ hybridization and 5-ethynyl-2'-deoxyuridine labeling for stem-like cells in the hydrozoan jellyfish cladonema pacificum

刺胞动物，包括海葵、珊瑚和水母，表现出不同的形态和生活方式，表现为无柄息肉和自由游泳的美杜莎。正如 在Hydra 和 Nematostella等已建立的模型中的例子，干细胞和/或增殖细胞有助于刺胞珊瑚虫的发育和再生。然而，大多数水母的潜在细胞机制，特别是在美杜莎阶段，在很大程度上尚不清楚，因此，开发一种用于识别特定细胞类型的稳健方法至关重要。本文描述了一种可视化水生动物水母 Cladonema pacificum 中干细胞样增殖细胞的方案。 Cladonema medusae拥有分枝触手，在整个成虫阶段不断生长并保持再生能力，为研究增殖和/或干细胞样细胞协调的细胞机制提供了一个独特的平台。使用干细胞标记物的全安装荧光 原位 杂交（FISH）可以检测干细胞样细胞，而使用S相标记物5-乙炔基-2'-脱氧尿苷（EdU）进行脉冲标记可以鉴定增殖细胞。结合FISH和EdU标记，我们可以检测固定动物上活跃增殖的干细胞样细胞，并且该技术可以广泛应用于其他动物，包括非模型水母物种。

Cladonema触手已被用作研究形态发生和再生的细胞过程的模型15，16，17。触手结构由上皮管组成，其中干细胞样细胞或i细胞位于近端区域，称为触手球，并且沿着近轴侧将新分支依次添加到球茎远端区域的后部（图3A）15。以前的报告表明，细胞增殖在触手球和新的分支位点使用EdU?...

Watch this Scientific Journal Video about Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum at JoVE.com

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

在这里，我们描述了一种用于可视化水母 Cladonema中干细胞样增殖细胞的协议。与干细胞标记物的全安装荧光 原位 杂交可以检测干细胞样细胞，5-乙炔基-2'-脱氧尿苷标记可以鉴定增殖细胞。一起，可以检测到活跃增殖的干细胞样细胞。

太平洋水母干细胞样细胞的荧光原位杂交和5-乙炔基-2'-脱氧尿苷标记

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and ...

Nanos1表达与EdU标记的共染色使得区分活跃增殖的干细胞样细胞成为可能。我们可以在水母Cladonema的单细胞水平上识别干细胞样细胞的分布。我们可以在同一水母样本中可视化干细胞样细胞和增殖细胞，从而可以检测增殖和非增殖的干细胞样细胞，从而了解干细胞异质性。使用包括干细胞在内的动物模型进行发育生物学和再生研究。水生动物水母Cladonema pacificum是一种新兴的模式生物，可以在没有任何专门系统的情况下保存在实验室环境中。水母蜥蜴有分枝触手。分支发生在触手的近轴侧的新部位。随着时间的流逝，触手继续伸长和分枝，较老的枝条被推向尖端。触手也可以在截肢后几天内再生。体积小，易于处理，以及成体干细胞或干细胞样细胞的存在使Cladonema成为研究干细胞在不同过程中作用的良好系统。首先，使用3.5毫升移液管将Cladonema medusae放入1.5毫升管中，并加入ASW至总体积为500微升。加入 7.5 微升 10 毫摩尔 EdU 储备溶液，并将样品在 22 摄氏度下孵育一小时。等待一个小时，并尽可能多地去除含有 EdU 的 ASW。麻醉水母后，孵育五分钟。在 ASW 中用 4% 多聚甲醛将水母固定在 4 摄氏度下过夜。对于蛋白酶处理和固定后，除去多聚甲醛并用含有0.1%吐温20的PBS洗涤样品三次，每次10分钟。取出PBST并加入杂交缓冲液。将样品在室温下在杂交缓冲液中孵育15分钟。取出杂交缓冲液并加入新鲜杂交缓冲液。在杂交培养箱中在55摄氏度下预杂交至少两个小时。取出杂交缓冲液并与含有探针的杂交缓冲液一起孵育。在杂交培养箱中在 55 摄氏度下杂交 18 至 24 小时。接下来，继续移除探头。首先去除含有探针的杂交缓冲液，然后加入洗涤缓冲液1。用洗涤缓冲液在55摄氏度下洗涤样品两次，每次15分钟。除去洗涤缓冲液一，加入洗涤缓冲液二。用洗涤缓冲液两次在55摄氏度下洗涤样品15分钟。除去两个洗涤缓冲液，然后加入2X SSC。用2X SSC在55摄氏度下洗涤样品两次，持续15分钟。取出 2X SSC，然后加入预热的 PBST。在室温下用PBST洗涤样品15分钟。对于抗DIG抗体孵育，首先去除PBST，然后加入1%封闭缓冲液。将样品在室温下孵育至少一小时，同时在摇杆上缓慢摇晃。阻塞后，移除 1%阻塞缓冲区。加入抗 DIG-POD 溶液，并将样品在 4 摄氏度下孵育过夜。要检测DIG标记的探针，请取出抗DIG-POD溶液并加入三氯化钠吐温缓冲液。用三氯化钠吐温缓冲液在室温下洗涤样品三次，持续10分钟。稀释荧光染料将偶联的酪酰胺储备溶液在扩增稀释缓冲液中制成活性Cy5酪胺溶液。尽可能多地去除三氯化钠吐温缓冲液，然后加入活性Cy5酪胺溶液。将样品在黑暗中孵育10分钟。用PBST在黑暗中洗涤样品三次10分钟。要制备 EdU 检测鸡尾酒，请混合成分。去除 PBST，然后添加 EdU 检测鸡尾酒。将样品在黑暗中孵育30分钟。用PBST在黑暗中清洗三次，每次10分钟。对于DNA染色，在PBST中稀释Hoechst以制备Hoechst溶液。删除 PBST，然后添加 Hoechst 解决方案。将样品在黑暗中孵育30分钟。用PBST在黑暗中洗涤样品三到四次，持续10分钟。接下来进行安装，使用转移移液管将水母转移到载玻片上的银行，并切断尖端。用镊子轻轻地将盖玻片放在水母上，并用透明的指甲油密封盖玻片的侧面。Nanos1表达和EdU阳性细胞的共标记揭示了触手中干细胞样细胞和增殖细胞的空间模式。EdU阳性细胞在整个触手球中分布更广，而Nanos1阳性细胞在触手球和新的分支位点局部积累更多，这表明根据发育时间和不同阶段检测到干细胞样细胞和增殖细胞的不同分布。在19.79%的细胞中观察到EdU和Nanos1的共标记，表明这些细胞是活跃增殖的干细胞群。有趣的是，在鳞茎中间和新的分支位点发现14.46%的细胞是EdU阳性Nanos1阴性，这表明存在非干细胞样增殖细胞。相比之下，观察到26.32%的细胞在球茎基部和新的分支位点呈EdU阴性Nanos1阳性，表明存在缓慢循环或静止的干细胞群，两者都不能通过EdU脉冲标记检测到。孵育时间短的EdU脉冲标记仅允许检测干细胞样细胞和非干细胞样细胞中的增殖细胞。当改变EdU的孵育时间并结合长时间的追逐实验时，可以标记慢循环干细胞或其他细胞类型，包括后代和分化细胞。

Research

JoVE Journal

Developmental Biology

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

科研

发育生物学

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

DNA的特定染色甲基绿在胚胎的荧光标记的应用

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

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 ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

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

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

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 ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

斑马鱼原位杂交染色的基因分型与定量

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...