Name: genotyping of sea anemone during early development
Uploaded: 2019-05-13T14:30:00
Description: Watch this Scientific Journal Video about Genotyping of Sea Anemone during Early Development at JoVE.com

我们感谢匿名评论者对手稿的早期版本的评论,该版本改进了手稿。这项工作得到了阿肯色大学的资金支持。

1. 诱导产卵、体外受精和脱胶
<ol><li>在16°C的黑暗中保持海水中每千分之12的盐度(ppt),每天喂食Artemia。 </li>
	<li>在产卵诱导的前一天,将动物置于温控和光控制的培养箱中。对孵化器进行编程,使动物在25°C时暴露在8小时光下。可选:在将一小块牡蛎(<1mm 3)喂给单个动物之前,再将它们放入孵化器,以提高产卵。</li>
	<li>在16°C下将动物留在孵化器中1小时。</li>
	<li>将动物从?...

<ol>
	<li>Medina, M., Collins, A. G., Silberman, J. D., Sogin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+hypotheses+of+basal+animal+phylogeny+using+complete+sequences+of+large+and+small+subunit+rRNA.">Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (17), 9707-9712 (2001).</li><li>Erwin, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cambrian+Conundrum:+Early+Divergence+and+Later+Ecological+Success+in+the+Early+History+of+Animals.">The Cambrian Conundrum: Early Divergence and Later Ecological Success in the Early History of Animals.</a> Science. 334 (6059), 1091-1097 (2011).</li><li>Putnam, N. 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Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+knockouts+reveal+an+endogenous+role+for+ancient+neuropeptides+in+regulating+developmental+timing+in+a+sea+anemone.">CRISPR knockouts reveal an endogenous role for ancient neuropeptides in regulating developmental timing in a sea anemone.</a> eLife. 7, e39742 (2018).</li><li>He, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+axial+Hox+code+controls+tissue+segmentation+and+body+patterning+in+Nematostella+vectensis.">An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis.</a> Science. 361 (6409), 1377 (2018).</li><li>Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN+and+CRISPR/Cas9-mediated+genome+editing+in+the+early-branching+metazoan+Nematostella+vectensis.">TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis.</a> Nature Communications. 5, 5486 (2014).</li><li>Servetnick, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9-mediated+excision+of+Nematostella+brachyury+disrupts+endoderm+development,+pharynx+formation+and+oral-aboral+patterning.">Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral-aboral patterning.</a> Development. 144 (16), 2951-2960 (2017).</li><li>Kraus, Y., Aman, A., Technau, U., Genikhovich, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-bilaterian+origin+of+the+blastoporal+axial+organizer.">Pre-bilaterian origin of the blastoporal axial organizer.</a> Nature Communications. 7, 11694 (2016).</li><li>Fritzenwanker, J. H., Genikhovich, G., Kraus, Y., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+and+axis+specification+in+the+sea+anemone+Nematostella+vectensis.">Early development and axis specification in the sea anemone Nematostella vectensis.</a> Developmental Biology. 310 (2), 264-279 (2007).</li><li>Lee, P. N., Kumburegama, S., Marlow, H. Q., Martindale, M. Q., Wikramanayake, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+developmental+potential+along+the+animal-vegetal+axis+in+the+anthozoan+cnidarian,+Nematostella+vectensis,+is+mediated+by+Dishevelled.">Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled.</a> Developmental Biology. 310 (1), 169-186 (2007).</li><li>Shinzato, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+Acropora+digitifera+genome+to+understand+coral+responses+to+environmental+change.">Using the Acropora digitifera genome to understand coral responses to environmental change.</a> Nature (London). 476 (7360), 320-323 (2011).</li><li>Gold, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+the+jellyfish+Aurelia+and+the+evolution+of+animal+complexity.">The genome of the jellyfish Aurelia and the evolution of animal complexity.</a> Nature Ecology &#38; Evolution. 3 (1), 96-104 (2019).</li><li>Clevesa, P. A., Strader, M. E., Bay, L. K., Pringle, J. R., Matz, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+genome+editing+in+a+reef-building+coral.">CRISPR/Cas9-mediated genome editing in a reef-building coral.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (20), 5235-5240 (2018).</li><li>Momose, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+doses+of+CRISPR/Cas9+ribonucleoprotein+efficiently+induce+gene+knockout+with+low+mosaicism+in+the+hydrozoan+Clytia+hemisphaerica+through+microhomology-mediated+deletion.">High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion.</a> Scientific Reports. 8, 11734 (2018).</li><li>Artigas, G. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gonad-expressed+opsin+mediates+light-induced+spawning+in+the+jellyfish+Clytia.">A gonad-expressed opsin mediates light-induced spawning in the jellyfish Clytia.</a> eLife. 7, e29555 (2018).</li></ol>

这里描述了一种基于PCR的协议,用于基因型单个海合明胚胎,而不牺牲动物。产卵和脱硫后,受精卵可以发育成胃卵。每个胃肠道胚胎的腹腔区域被手术切除,分离的腹腔组织用于后续基因组DNA提取,而剩余的手术后胚胎愈合并继续发育。然后,gDNA提取物用于PCR测定,以确定每个胚胎的基因型。该方法利用海葵胚胎的口腔半部分调节和发育10,11和大多数胚胎的能力(>90%)<sup class="xref...

仙人掌代表一组不同的动物,包括水母、珊瑚和海葵。它们是二叶虫,由细胞外基质(中细胞体)分离的内皮和内皮组成。Cnidaria是一个姐妹团体,以种像比拉提利亚,传统的动物模型,如果蝇和穆斯属于1。此外,被认为发生在2月2日寒武纪前,Cnidaria-Bilateria发散。因此,对仙人掌和双栖教徒的比较研究对于深入了解他们最近共同祖先的生物学至关重要。最近,比较基因组学显示,仙人掌和双子食者共享许多发育工具包基因,如notch和bHLH,这意味着他们的共同祖先已经拥有这些基因3。然而,这些发育工具包基因在Cnidaria和Bilateria的最后共同祖先中的作用相对而言,还不太了解。为了解决这个问题,研究这些深保守的基因在仙人掌中如何发挥作用是至关重要的。
新兴的仙人掌遗传模型之一是蚂蚁内马托斯特拉的维森斯。其基因组已测序3,各种基因工具,包括形态介导基因敲除,梅甘细胞介导的转基因,CRISPR-Cas9介导基因敲和敲,现在可用于这种动物。此外,内马托斯特拉的发展也相对来说也比较了解。在胚胎生成过程中,胃化发生于阴道4,胚胎发育成自由游泳的平面幼虫。之后,平面图转化为带嘴和圆角触角的环状息肉。息肉然后增长,并达到性成熟。
CRISPR-Cas9介导的靶向诱变现因现在经常用于研究内马托斯特拉维克塞西5、6、7、8、9的基因功能。为了在Nematostella产生敲除突变体,一种含有原点特异性单导RNA和内分酶Cas9蛋白的鸡尾酒首先被注射到未受精或受精的卵子中,以产生通常显示的F0创始人动物马赛克主义。F0动物随后被提升到性成熟,并相互交叉,以产生F1种群,其中一个子集可能是敲除突变体6。或者,性成熟的F0动物可以与野生型动物交叉,以产生F1杂物动物,而F1杂物携带一个敲击等位基因在兴趣点,然后可以交叉彼此产生F2后代,四分之一其中预计将被敲除突变体5。这两种方法都需要一种方法来识别来自基因异质种群的敲除突变体。息触角可用于提取基因组DNA进行基因分型6,7。然而,在研究相关基因的发育功能和突变胚胎未达到息肉阶段的情况下(即,由于与突变相关的幼虫致死性),需要在早期发现敲除突变体。这里描述的是基于PCR的方案基因型个体动物在气态阶段不牺牲动物,这使得识别从基因异质的胚胎种群敲除突变体。整个基因分型过程的持续时间取决于要筛选的胚胎数量,但它至少需要4-5小时。

<table>
	<tbody>
		<tr>
			<td>Drosophila Peltier Refrigerated Incubator</td>
			<td>Shellab</td>
			<td>SRI6PF</td>
			<td>Used for spawning induction</td>
		</tr>
		<tr>
			<td>Instant ocean sea salt</td>
			<td>Instant ocean</td>
			<td>138510</td>
			<td></td>
		</tr>
		<tr>
			<td>Brine shrimp cysts</td>
			<td>Aquatic Eco-Systems, Inc.</td>
			<td>BS90</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine Hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Orbital Shaker, Model 3500</td>
			<td>VWR</td>
			<td>89032-092</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-HCl, 1M, pH8.0</td>
			<td>QUALITY BIOLOGICAL</td>
			<td>351-007-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>VWR</td>
			<td>BDH9258</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M pH8</td>
			<td>VWR</td>
			<td>BDH7830-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet-P40 Substitute</td>
			<td>US Biological</td>
			<td>N3500</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K solution (20 mg/mL), RNA grade</td>
			<td>ThermoFisher</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>VWR</td>
			<td>710</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting needle holder</td>
			<td>Roboz</td>
			<td>RS-6060</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten dissecting needle</td>
			<td>Roboz</td>
			<td>RS-6063</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Eppendorf Mastercycler Thermal Cyclers</td>
			<td>Eppendorf</td>
			<td>E6336000024</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>New England BioLabs</td>
			<td>N0447L</td>
			<td></td>
		</tr>
		<tr>
			<td>GLWamide universal forward primer</td>
			<td></td>
			<td></td>
			<td>5&#8217;- CATGCGGAGACCAAGCGCAAGGC-3&#8217;</td>
		</tr>
		<tr>
			<td>Reverse primer specific to glw-a</td>
			<td></td>
			<td></td>
			<td>5&#8217;-CCAGATGCCTGGTGATAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Reverse primer specific to glw-c </td>
			<td></td>
			<td></td>
			<td>5&#8217;- CGGCCGGCGCATATATAG-3&#8217;</td>
		</tr>
	</tbody>
</table>

genotyping of sea anemone during early development

这里描述的是基于PCR的协议基因型的胃肠阶段胚胎的蚂蚁阴极内人内马托斯特拉vesisis,而不牺牲动物的生命。在体外受精和脱胶后,酶在室温下发育24小时,达到早至中气期。然后,将胃粒胚胎放在含有海水的培养皿中的甘蔗胶床上。在解剖显微镜下,钨针用于从每个胚胎中手术分离一个腹腔组织片段。手术后胚胎可以愈合并继续发育。基因组DNA从分离的组织片段中提取,并用作位点特异性PCR的模板。基因型可以根据PCR产品的大小或存在/不存在的等位基因特异性PCR产品来确定。手术后胚胎然后根据基因型进行分类。整个基因分型过程的持续时间取决于要筛选的胚胎数量,但它至少需要4~5小时。该方法可用于识别来自基因异质胚胎种群的敲除突变体,并在发育过程中分析表型。

涅马托斯特拉基因组有一个单一的位点,编码神经肽GLW酰胺的前体蛋白。三个敲除突变的均位位基因在这个位子(glw-a,glw-b,和glw-c)之前已经报告5。四个携带野生型等位基因 ( + ) 和敲除等位基因的雄性在GLW 酰胺位位 ( 基因型 : + / glw- c) 与携带野生型等位基因和不同敲除的杂音雌叉同?...

Watch this Scientific Journal Video about Genotyping of Sea Anemone during Early Development at JoVE.com

Genotyping of Sea Anemone during Early Development

早期开发期间海葵基因分型

该协议的目标是在胃化过程中基因型海葵奈马托斯特拉静脉曲张,而不牺牲胚胎。

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

该协议使研究人员能够在早期胚胎形成过程中识别突变海葵，以便分析胚胎后发育表型。这项技术的主要优点是，它可以用来基因型个别海葵早期在基因上不牺牲动物的生命。演示这个程序的将是米格尔·席尔瓦，一个研究生从我的实验室。在产卵诱导前一天，将海葵线虫菌体放在温度和光控制的孵化器中，对孵化器进行编程，使动物在25摄氏度下暴露在8小时的光线下。第二天，将动物从孵化器中取出，在室温下放在长椅上，并亮着灯，以便产卵，这一点将在一个半小时到两个小时内发生。使用转移移液器谁的尖端被削减扩大开口，将卵子包从女性容器放入一个含有精子的男性容器，并留在男性容器中至少15分钟，以允许受精。受精后，将鸡蛋包装去掉，将它们放在含有3%半胱氨酸的海水中，放在一个玻璃培养皿上，轻轻搅拌12分钟。然后用塑料移液器分解团块，继续搅拌两到三分钟，直到完全脱液。将受精卵放在同一盘中16摄氏度或室温下。第二天，准备手稿中描述的DNA提取缓冲液，通过涡流混合，通过向每个管中加入20微升，将其加入PCR管中。在海水中溶解1%的加糖，倒入培养皿中盖底，并在长凳上冷却，制作凝胶床。用新鲜的海水盖住凝胶床。使用移液器将 24 小时受精后胚胎移植到含有 agarose 凝胶床的培养皿中。将钨针插入需要支架中，将其尖端浸入酒精中并放入火焰中以烧掉酒精，进行消毒。在解剖显微镜下，使用钨针去除一块大约纵的胚胎大小的表面红玫瑰，在红糖上制造凹陷。然后，使用钨针，将胚胎放入凹陷中，其侧朝下，以限制胚胎进行显微手术的运动。为了进行成功的手术，限制胚胎的运动至关重要。用钨针，手术切除一块外阴组织沿口腔外阴轴，位于对面的口腔喷口。使用P20移液器，将分离的外皮组织转移到含有20微升DNA提取缓冲液的PCR管中。将手术后胚胎转移到含有至少500微升新鲜海水的24井板井中。将含有手术后胚胎的盘子放在16摄氏度的培养箱中，直到基因分型完成。要从单个胚胎中提取基因组DNA，首先使用微型离心机短暂地旋转含有DNA提取缓冲液和分离胚胎组织的PCR管。然后在55摄氏度下孵育管子3小时，每30分钟旋转30秒，以确保细胞团的破裂，增强细胞解。在95摄氏度下解活蛋白酶K4分钟后，利用提取的基因组DNA作为模板建立PCR反应，以放大感兴趣的位置。设计手稿中描述的所需底像。设置 20 微升 PCR 反应。完成PCR后，运行红糖凝胶电泳以确定PCR产品的大小和存在或不存在，确保根据PCR产品的预期尺寸调整红糖凝胶电泳的状态。使用有关大小和存在或不存在或PCR产品PCR结果为每个手术后胚胎分配基因型，然后根据胚胎的基因型对胚胎进行排序。线虫基因组有一个位点，编码神经肽GLWamide的前体蛋白。之前曾报道过该地位有三个敲除突变等位基因。PCR结果从随机采样的胚胎之间的一个异质异质女性携带一个淘汰等位基因和异质雄性携带C敲除等位基因之间的后代显示不同的基因类型。胚胎一和二显示一个PCR波段对应预期大小的淘汰等位基因。胚胎三和六显示两个PCR波段与淘汰等位基因A和C.Embryos四，七和八显示一个PCR波段对应C淘汰等位基因的预期大小。胚胎 5 显示没有带， 表明缺乏底像结合。为了排除基因组DNA提取失败的可能性，使用反向底转运行另一个PCR，该底转可以绑定到野生类型序列。如果检测到预期大小的PCR产品，基因组DNA提取是成功的。而没有产品表明提取失败。虽然该协议是为海葵胚胎设计的，但肯定有可能将这种方法应用于其他人类，如珊瑚和水母，在那里可以获取基因组信息和胚胎。按照这个程序，识别的突变体用于评估发育表型。例如，组织学技术，如免疫污染可用于评估形态缺陷，而原位杂交，实时定量RT-PCR和RNA-seq可用于评估基因表达的缺陷。此外，活成像可用于评估胚胎后发育过程中的行为缺陷，如幼虫或早期息肉。

Research

JoVE Journal

Developmental Biology

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

协议获得合子和体细胞胚胎研究早期胚胎发育的调控中的示范豆科<em&gt;苜蓿</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

科研

发育生物学

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Studying Wnt Signaling During Patterning of Conducting Airways

研究Wnt信号传导过程中的航空公司图案

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

从斯塔利特海葵的反口Physa完全诱导再生息肉<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

简单的力量：海胆胚胎作为<em&gt;体内</em&gt;对于研究复杂的细胞与细胞间信号传导网络交互开发模式

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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 ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...