这项工作是由ARC DECRA到PRJ（DE120101311）和德意志研究联合会（DFG）的研究经费以LP（PO 1440 / 1-1）的支持。澳大利亚再生医学研究所是由维多利亚州政府和澳大利亚联邦政府的资金支持。我们承认杰里米·伍枝记医生，谁进行公布在箕等人在这里描述的实验。 ，2016年我们是从Higashijima教授转基因鱼的规定，感激和感谢PROFS。特纳和鲁普为提供PCS2 +质粒和威尔金森博士生成H2B-RFP和H2A-GFP构造。我们感谢FishCore设施的工作人员（莫纳什大学）采取了动物的照顾。

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8, 353-367 (2007).</li><li>Scholpp, S., Poggi, L., Zigman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+on+the+stage+-+spotlight+on+nervous+system+development+in+zebrafish:+EMBO+practical+course,+KIT,+Sept.+2013.">Brain on the stage - spotlight on nervous system development in zebrafish: EMBO practical course, KIT, Sept. 2013.</a> Neural Dev. 8, 23 (2013).</li><li>Cayouette, M., Poggi, L., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+in+the+vertebrate+retina.">Lineage in the vertebrate retina.</a> Trends Neurosci. 29, 563-570 (2006).</li><li>Poggi, L., Vitorino, M., Masai, I., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influences+on+neural+lineage+and+mode+of+division+in+the+zebrafish+retina+in+vivo.">Influences on neural lineage and mode of division in the zebrafish retina in vivo.</a> J Cell Biol. 171, 991-999 (2005).</li><li>Poggi, L., Zolessi, F. R., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+retinal+differentiation.">Time-lapse analysis of retinal differentiation.</a> Curr Opin Cell Biol. 17, 676-681 (2005).</li><li>Jusuf, P. R., 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=Biasing+amacrine+subtypes+in+the+Atoh7+lineage+through+expression+of+Barhl2.">Biasing amacrine subtypes in the Atoh7 lineage through expression of Barhl2.</a> J Neurosci. 32, 13929-13944 (2012).</li><li>Jusuf, P. R., 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=Origin+and+determination+of+inhibitory+cell+lineages+in+the+vertebrate+retina.">Origin and determination of inhibitory cell lineages in the vertebrate retina.</a> J Neurosci. 31, 2549-2562 (2011).</li><li>Jusuf, P., Harris, W. A., Poggi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+retinal+progenitor+lineages+in+developing+zebrafish+embryos.">Imaging retinal progenitor lineages in developing zebrafish embryos.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Jusuf, P., Harris, W. A., Poggi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+transgenic+zebrafish+embryos+for+imaging+the+developing+retina.">Preparation of transgenic zebrafish embryos for imaging the developing retina.</a> Cold Spring Harb Protoc. 2013, (2013).</li><li>Vitorino, M., 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=Vsx2+in+the+zebrafish+retina:+restricted+lineages+through+derepression.">Vsx2 in the zebrafish retina: restricted lineages through derepression.</a> Neural Dev. 4, 14 (2009).</li><li>Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+regulation+of+neuronal+production+in+the+larval+frog+retina.">Cell-specific regulation of neuronal production in the larval frog retina.</a> J Neurosci. 7, 3317-3324 (1987).</li><li>Reh, T. A., Tully, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tyrosine+hydroxylase-containing+amacrine+cell+number+in+larval+frog+retina.">Regulation of tyrosine hydroxylase-containing amacrine cell number in larval frog retina.</a> Dev Biol. 114, 463-469 (1986).</li><li>Livesey, F. J., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+neural+cell-fate+determination:+lessons+from+the+retina.">Vertebrate neural cell-fate determination: lessons from the retina.</a> Nature reviews. Neuroscience. 2, 109-118 (2001).</li><li>Zolessi, F. R., Poggi, L., Wilkinson, C. J., Chien, C. B., Harris, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+and+orientation+of+retinal+ganglion+cells+in+vivo.">Polarization and orientation of retinal ganglion cells in vivo.</a> Neural Dev. 1, 2 (2006).</li><li>Kimura, Y., Okamura, Y., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alx,+a+zebrafish+homolog+of+Chx10,+marks+ipsilateral+descending+excitatory+interneurons+that+participate+in+the+regulation+of+spinal+locomotor+circuits.">alx, a zebrafish homolog of Chx10, marks ipsilateral descending excitatory interneurons that participate in the regulation of spinal locomotor circuits.</a> J Neurosci. 26, 5684-5697 (2006).</li><li>Kimura, Y., Satou, C., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V2a+and+V2b+neurons+are+generated+by+the+final+divisions+of+pair-producing+progenitors+in+the+zebrafish+spinal+cord.">V2a and V2b neurons are generated by the final divisions of pair-producing progenitors in the zebrafish spinal cord.</a> Development. 135, 3001-3005 (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2007).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Kei, J. N., Dudczig, S., Currie, P. D., Jusuf, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+from+each+retinal+neuron+population+drives+expression+of+subsequent+fate+determinant+genes+without+influencing+the+cell+cycle+exit+timing.">Feedback from each retinal neuron population drives expression of subsequent fate determinant genes without influencing the cell cycle exit timing.</a> J Comp Neurol. 524, 2553-2566 (2016).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. , (2009).</li><li>Zou, J., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+GFP-expressing+blastomeres+for+live+imaging+of+retinal+and+brain+development+in+chimeric+zebrafish+embryos.">Transplantation of GFP-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos.</a> J Vis Exp. , (2010).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J Vis Exp. , (2009).</li><li>Schier, A. F., Talbot, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetics+of+axis+formation+in+zebrafish.">Molecular genetics of axis formation in zebrafish.</a> Annu Rev Genet. 39, 561-613 (2005).</li><li>De Luca, E., 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=ZebraBeat:+a+flexible+platform+for+the+analysis+of+the+cardiac+rate+in+zebrafish+embryos.">ZebraBeat: a flexible platform for the analysis of the cardiac rate in zebrafish embryos.</a> Sci Rep. 4, 4898 (2014).</li><li>Lin, J. W., 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=Differential+requirement+for+ptf1a+in+endocrine+and+exocrine+lineages+of+developing+zebrafish+pancreas.">Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas.</a> Dev Biol. 274, 491-503 (2004).</li><li>Hollyfield, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+retina+in+the+killifish,+Fundulus+heteroclitus.">Histogenesis of the retina in the killifish, Fundulus heteroclitus.</a> J Comp Neurol. 144 (3), 373-380 (1972).</li><li>La Vail, M. M., Rapaport, D. H., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytogenesis+in+the+monkey+retina.">Cytogenesis in the monkey retina.</a> J Comp Neurol. 309 (1), 86-114 (1991).</li><li>Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., LaVail, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+and+topography+of+cell+genesis+in+the+rat+retina.">Timing and topography of cell genesis in the rat retina.</a> J Comp Neurol. 474 (2), 304-324 (2004).</li><li>Sharma, S. C., Ungar, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histogenesis+of+the+goldfish+retina.">Histogenesis of the goldfish retina.</a> J Comp Neurol. 191 (3), 373-382 (1980).</li><li>Stiemke, M. M., Hollyfield, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+birthdays+in+Xenopus+laevis+retina.">Cell birthdays in Xenopus laevis retina.</a> Differentiation. 58 (3), 189-193 (1995).</li></ol>

The authors have nothing to disclose.

理解的程度，邻近细胞影响的关键的细胞命运决定因子的表达的定时旨在有效地指示胚胎或诱导的多能干细胞分化成特定的有丝分裂后的细胞类型或甚至图案化的组织时是必不可少的。此外，检查在活的动物的显影细胞中的这些分子事件还提供相关的动态上与体内这些事件相关的特定蜂窝上下文（时间和空间）的信息。这种研究不能在所有脊椎动物模型可以轻松实现。 <p class="jove_content"...

<html>可视化在体内脊椎动物重要的发育过程的能力已经到使斑马鱼的关键模型为研究正常和疾病条件作出了贡献（在1综述，2）。具体地，神经视网膜是中枢神经系统的访问的一部分。视网膜适合于容易地进行神经发生的研究，由于其高度的组织，但结构较简单，并在脊椎动物物种3其高度保守的神经类型。细胞行为，如增殖，细胞周期出口，非对称的细胞分裂，命运规范，分化和神经电路形成的动力学可以遵循整个retinogenesis的整个过程中，这是在斑马鱼的中心视网膜由3天受精后完成（ dpf）的4，5，REF"&gt; 6，7。 另外，在每个上述的阶段的不同基因的功能要求，可伴随地评估在斑马鱼视网膜，用其他脊椎动物模型中，表型从基因敲除技术的应用导致提供的优点只能在验尸检查评估固定的组织。特别是，使用的转基因品系中，我们可以可视化和监控荧光蛋白如在视网膜报告基因的表达，使我们能够获得underlies一个特定的神经元细胞类型的起源的基因表达的时间分辨率。由于斑马鱼的迅速发展，这些事件可以在整个发育期间被可视化，从而提供更深入了解的基因表达相对于神经元细胞身份采集和细胞行为的时间的重要性。 最后，这些方法可有效地在通过移植的产生嵌合体的斑马鱼相结合，导致分析上市基因功能的两个关键方面。首先，检查从捐赠者的胚胎，其中一个特定基因被撞倒，而他们在未标记的野生型环境中开发移植的细胞，使我们能够获得有关基因功能的细胞自主的方式相关的信息。这导致了关于它们在此。正常表达是由祖细胞，可以不再从这些基因4,6产生功能性蛋白质的发育命运结果的检查例举的祖细胞内视网膜命运决定因素的功能是重要的见解， 8,9。利用这种方法，我们已经表明，很多命运的决定性因素（ 例如，VSX1，Atoh7，Ptf1a，Barhl2）细胞的作用-autonomously推动特定视网膜神经命运;缺乏的基因表达的主要导致命运开关，使得与基因拦截的细胞保持通过采用一个备用细胞命运4，6，7，10是可行的。其次，这种嵌合的实验可用于评估时不同遗传环境中开发类型的祖细胞如何野生表现。例如，通过比较野生型细胞的发展通常表达在一WT兴趣（和报告基因）与操纵宿主环境（ 例如，基因敲除/击倒）的基因，对基因表达和细胞命运所得效果可以评估。缺乏在主机环境中的某些神经类型的，例如，已被证明在细胞非自主的方式来影响野生型祖行为，以偏压它们朝向分化成的代表性不足Ò④短缺的神经类型4，7，11，12。鉴于视网膜神经元出生在通过特定的神经元的命运决定簇的基因的顺序定时表达式（命运的基因表达）（以13中综述）保守histogenic顺序，我们使用这些方法，以说明如何命运的基因表达在野生型祖细胞的定时当这种祖细胞在诱发异常细胞组合物视网膜主机环境发展会受到影响。这里，我们概述这些方法作为证据的相对标准和广泛使用的技术的组合如何能够命运的基因表达的时机在显影视网膜祖8,9的检查。 这个协议描述了一种实验方法组合延时成像用的放心的每形成在离体显影斑马鱼胚胎移植到跟随个别mosaically标记细胞整个发育retinogenesis的整个期间。通过执行功能基因操作或在宿主胚胎，供体胚胎，两者或两者都不是，人们可以评估基因功能的细胞自主性。这种方法可广泛适用于在任何其它系统的量这里概述的各个部件都是合适的类似研究问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td>Agarose coated dishes can be prepared a few days prior, sealed with parafilm to prevent drying and stored at 4C</td>
		</tr>
		<tr>
			<td>Agarose low melt</td>
			<td>Sigma</td>
			<td>A9414-100G</td>
			<td>Can be prepared in larger volume, microwaved to liquify and then kept molte in 40C waterbath. 1 ml aliquots can be prepared (one for each group of embryos mounted).</td>
		</tr>
		<tr>
			<td>borosillicate glass capillary tube w/o filament</td>
			<td>SDR Scientfic</td>
			<td>30-0035</td>
			<td>alternatives with similar diameter can be used&#160;</td>
		</tr>
		<tr>
			<td>borosillicate glass capillary tube with filament</td>
			<td>SDR Scientfic</td>
			<td>30-0038</td>
			<td>alternatives with similar diameter can be used&#160;</td>
		</tr>
		<tr>
			<td>glass petri dishes (60 mm diameter)</td>
			<td>Science Supply</td>
			<td>1070506</td>
			<td>any glass alternative of any size</td>
		</tr>
		<tr>
			<td>Injection tube (2m)</td>
			<td>Eppendorf</td>
			<td>524616004</td>
			<td>This connects the needle holder to the syringe during transplantation</td>
		</tr>
		<tr>
			<td>Microinjection mold (plastic mold with wedge-shaped protrusions)</td>
			<td>Adaptive Science Tools</td>
			<td>PT-1</td>
			<td>alternatives with similar shape can be use</td>
		</tr>
		<tr>
			<td>microneedle holder</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td>any alternative that fits the outside diameter of the injection needles can be used</td>
		</tr>
		<tr>
			<td>microinjector</td>
			<td>Narishige or Eppendorf Femtojet</td>
			<td></td>
			<td>Pneumatic injector using gas pressure</td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Coherent Scientific</td>
			<td>M330IR</td>
			<td>similar alternatives can be used</td>
		</tr>
		<tr>
			<td>microloader tip</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5904-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>needle puller</td>
			<td>Sutter Instruments</td>
			<td>Model P-2000</td>
			<td>Settings used for this puller are: H 430, Fil 4, Vel 50, Del 225, Pul 75. Any needle puller can be used if it results in appropriate tip dimension</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Interpath</td>
			<td>PM996</td>
			<td>any paraffin film</td>
		</tr>
		<tr>
			<td>Pasteur pipette plastic</td>
			<td>Samco Scientific</td>
			<td>202</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette glass</td>
			<td>Hirschmann Laborgeraete</td>
			<td>9260101</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>P5942-25MG</td>
			<td>&#160;Protease Type XIV</td>
		</tr>
		<tr>
			<td>N-phenyl thiourea</td>
			<td>Sigma</td>
			<td>P7629-25G</td>
			<td>PTU is toxic and can be prepared as a stock solution to avoid frequent exposure to the powder, consult MSDS before purchase / use.</td>
		</tr>
		<tr>
			<td>Qiaquick gel extraction</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>any alternatives to purify DNA can be used</td>
		</tr>
		<tr>
			<td>Rneasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>any alternatives to purify RNA can be used</td>
		</tr>
		<tr>
			<td>SP6 mMessage kit</td>
			<td>Qiagen</td>
			<td>AM1340</td>
			<td>any alternatives to transcribe DNA using SP6</td>
		</tr>
	</tbody>
</table>

in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences

遗传和技术优势作出了斑马鱼脊椎动物一个关键的模式生物，其中基因操作的后果可在体内在整个快速发育期间进行跟踪。多个进程可以研究包括细胞增殖，基因表达，细胞迁移和形态发生。重要的是，嵌合体通过移植的产生可以容易地执行，允许主机环境的影响下镶嵌标签和单个细胞的跟踪。例如，通过结合宿主胚的个体移植供体细胞中有功能的基因操作（ 例如，通过显微注射吗啉）和实时成像，外在的影响，细胞非自治信号（由经遗传修饰的环境提供）进行评估。在这里，我们证明这种方法是如何使用荧光的转基因表达的开始比较作为细胞命运determin的定时的代理在通货膨胀不同的遗传主机环境。 在本文中，我们提供的协议为显微注射斑马鱼胚胎标记供体细胞，并引起在宿主胚胎的基因敲除，用于产生嵌合胚胎移植技术的说明，以及用于制备和体内的时间推移共焦运行协议多个胚胎成像。特别是，在执行多位置成像比较事件定时例如基因表达的开始时是至关重要的。这需要从多个控制和同时处理的胚胎实验数据收集。这种方法可以很容易地扩展为在任何器官或访问的实时成像选择的组织外在影响的研究，其前提是移植可根据已建立的胚胎命运地图容易定位。

这项工作提出了一个实验性协议时，野生型视网膜祖细胞morphant胚胎主机内发展，以评估基因表达变化的时序。实验宿主胚胎Ptf1a morphants，缺乏视网膜7，26的中间生的水平和无长突的interneurons。这些已相比，控制宿主胚胎，其中注射了一个标准的控制啉。 因为在整个中枢神经系统的?...

Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

在不同的遗传背景的个体WT视网膜祖细胞的现场跟踪允许细胞非自主性的信令神经过程中贡献进行评估。这里，通过胚胎移植和体内的时间推移共焦成像的基因敲除，嵌合体的产生的组合物用于此目的。

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

in-vivo-imaging-transgenic-gene-expression-individual-retinal

Research

JoVE Journal

Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

7.6K 次观看.  The University of Melbourne. 在不同的遗传背景的个体WT视网膜祖细胞的现场跟踪允许细胞非自主性的信令神经过程中贡献进行评估。这里，通过胚胎移植和体内的时间推移共焦成像的基因敲除，嵌合体的产生的组合物用于此目的。 

视频: In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

科研

发育生物学

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

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

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

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 ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...