Name: in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences
Uploaded: 2017-03-22T15:00:00
Description: 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

This work was supported by an ARC DECRA to PRJ (DE120101311) and by a Deutsche Forschungsgemeinschaft (DFG) research grant to LP (PO 1440/1-1). The Australian Regenerative Medicine Institute is supported by funds from the State Government of Victoria and the Australian Federal Government. We acknowledge Dr Jeremy Ng Chi Kei, who conducted experiments described here as published in Kei et al., 2016. We are grateful for provisions of transgenic fish from Prof. Higashijima and thank Profs. Turner and Rupp for the provision of the pCS2+ plasmid and Dr. Wilkinson for generating H2B-RFP and H2A-GFP constructs. We thank FishCore facility staff (Monash University) for taking care of our animals.

All procedures were carried out according to the provisions of the Australian National Health and Medical Research Council code of practice for the care and use of animals and were approved by the institutional ethics committees.
1. Preparation of Zebrafish
<ol>
	<li>Adult fish pairing
	<ol>
		<li>Set up adult fish as pairs (or two pairs) in appropriately sized breeding tanks the evening prior to use.</li>
		<li>To keep the female separated from the male, use a divider to enable the fish to ...

<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>

Understanding the extent to which neighboring cells influence timing of the expression of crucial cell fate determining factors is essential when aiming to efficiently instruct embryonic or induced multipotent stem cells to differentiate into a specific post-mitotic cell type or even patterned tissue. Furthermore, examining these molecular events in the developing cells of the living animal additionally provides relevant dynamic (temporal and spatial) information on the particular cellular contexts associated with these ...

The ability to visualize important developmental processes in an in vivo vertebrate has contributed to making the zebrafish a key model for studying normal and disease conditions (reviewed in 1,2). In particular, the neural retina is an accessible part of the central nervous system. The retina lends itself to easily perform studies of neurogenesis due to its highly organized, yet relatively simple structure, and its highly conserved neuron types across vertebrate species 3. Dynamics of cellular behaviors such as proliferation, cell cycle exit, asymmetric cell division, fate specification, differentiation, and neural circuitry formation can be followed throughout the entire process of retinogenesis, which is completed in the central retina of the zebrafish by 3 d postfertilization (dpf) 4,5,6,7.
Furthermore, the functional requirements of different genes in each of the above mentioned stages can be concomitantly assessed in the zebrafish retina, providing an advantage over other vertebrate models in which phenotypes resulting from application of gene knockout techniques can only be assessed upon post-mortem examination of fixed tissues. In particular, the use of transgenic lines in which we can visualize and monitor the expression of fluorescent proteins as reporter transgenes in the retina, allows us to obtain temporal resolution of gene expression that underlies the genesis of a particular neuronal cell type. Due to the rapid development of zebrafish, these events can be visualized during the entire developmental period, thereby providing deeper insights into the temporal importance of gene expression in relation to neuronal cell identity acquisition and cell behavior.
Finally, these approaches can be combined efficiently in the zebrafish with the generation of chimera via transplantations, resulting in insights into two key aspects of gene function. Firstly, examining cells transplanted from a donor embryo, in which a particular gene was knocked down while they develop in an unlabeled wild type environment, allows us to obtain relevant information about gene function in a cell-autonomous manner. This leads to important insights about the function of retinal fate determinant factors within the progenitor cells they are normally expressed in. This is exemplified by the examination of the developmental fate outcome of progenitors that can no longer generate functional protein from these genes 4,6,8,9. Utilizing this approach, we have shown that many fate determinant factors (e.g., Vsx1, Atoh7, Ptf1a, Barhl2) act cell-autonomously to drive specific retinal neuronal fates; the lack of gene expression primarily leads to a fate switch, such that the cells with gene knockdown remain viable by adopting an alternate cell fate 4,6,7,10. Secondly, such chimeric experiments can be used to assess how wild type progenitors behave when they develop within different genetic environments. For example, by comparing the development of WT cells that usually express a gene of interest (and reporter transgene) in a WT versus manipulated host environment (e.g., gene knockout / knockdown), the resulting effects on gene expression and cell fate can be assessed. The lack of certain neuron types in the host environment, for instance, has been shown to influence wild type progenitor behavior in a cell non-autonomous manner, to bias them towards differentiating into the underrepresented or missing neuron types 4,7,11,12. Given that retinal neurons are born in a conserved histogenic order by the sequentially timed expression of specific neuronal fate determinant genes (fate gene expression) (reviewed in 13), we used these methods to demonstrate how the timing of fate gene expression in wild type progenitors is affected when such progenitors develop in retinal host environments with induced aberrant cellular compositions. Here, we outline these approaches as evidence for how the combination of relatively standard and widely used techniques enables the examination of the timing of fate gene expression in developing retinal progenitors 8,9.
This protocol describes an experimental approach combining time-lapse imaging with the ease of performing transplantation in the ex vivo developing zebrafish embryo to follow individual mosaically labeled cells throughout the entire period of developmental retinogenesis. By performing functional gene manipulations either in the host embryo, donor embryo, both or neither, one can assess the cell autonomy of gene function. This approach can be adapted widely to similar research questions in any other system for which the individual components outlined here are suitable.

<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

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 throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

This work presents an experimental protocol to assess changes in gene expression timing when wild type retinal progenitors develop within a morphant host embryo. The experimental host embryos are Ptf1a morphants, which lack the intermediate born horizontal and amacrine interneurons of the retina 7,26. These have been compared to control host embryos, which were injected with a standard control morpholino.
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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

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

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

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

The overall goal of these procedures is to study how changes in gene expression, either within developing cells or in their surrounding environment, influence temporal processes during development. This method can help answer key questions, such as whether the timing of fate specification is controlled by genes expressed cell-autonomously within a cell, or cell non-autonomously in neighboring cells. The main advantage of this technique lies in the ability to follow individual progenitor cells in an in vivo vertebrate as the cells are developing in genetically distinct environments.Demonstrating the procedure will be Stefanie Dudczig, a research assistant from my laboratory. After preparing zebrafish embryos, as well as micro-injection needles with Morpholinos, or mRNA, according to the text protocol, immerse the needle tip in a drop of mineral oil in a dish. Measure the injection volume by pressing the foot pedal, and adjust the gas pressure and/or duration time until the injection volume is equivalent to one nanoliter, as measured with a micrometer slide, or alternatively, with the eyepiece graticule.Place a microscope slide in a Petri dish, and with a transfer pipette, deposit single-cell stage embryos along the slide edge. Next, with a fine transfer pipette, remove as much liquid as possible to prevent movements during injections. Then, using a Microloader pipette tip with half of the thin tip cut off, align the embryos into a single column.Then, lower the needle towards a single-cell stage embryo and penetrate the chorion and yolk in one smooth stroke. Once the needle tip is centered in the yolk, press the foot pedal once and microinject one nanoliter of H2AGFP or H2BRFP mRNA into the yolk of the single-stage embryo. Remove the injection needle, move the dish containing the embryos to bring the next embryo into position, and continue injecting until the entire column of embryos are injected.After injecting all of the embryos, tilt the dish at a 30 to 45 degree angle, and use a gentle stream of E3 medium in a squeeze bottle to transfer the injected embryos to a new, clean Petri dish. At three hours post-fertilization, under a fluorescent microscope at 2X to 5X magnification, use a RFP filter to check the donor embryos for labeling signal. Select well-developed embryos with a strong fluorescent signal.After preparing injection plates and Petri dishes according to the text protocol, cut the tip of a long-form, finely-pulled glass tip Pasteur pipette to a length that allows for comfortable maneuvering by using a diamond knife to scratch the needle while rotating it until the tip falls off. Ensure that the cut is straight. Then, so as not to damage dechorionated embryos, fire-polish the glass transfer pipette by rotating the cut surface in a Bunsen burner to generate smooth ends.To dechorionate embryos, place well-developed host and donor embryos in two separate 10 milliliter Erlenmeyer glass beakers, and remove as much liquid as possible. Add 5 milliliters of the previously prepared 0.5 milligram per milliliter protease solution to each beaker, and gently swirl the embryos while looking at them under a dissecting microscope. While continuing to swirl, as soon as the first embryo is out of the chorion, use E3 medium to perform three quick rinses to remove the protease solution by pouring out most of the liquid, and refilling the beaker by gently pouring in E3 along the side.Stop the enzymatic dechorionation by rapid but gentle rinsing as soon as the first few dechorionated embryos can be observed. For the transplantations, choose only dechorionated embryos that have a symmetric cell mass and no obvious deformations or damage to the yolk. To carry out blastula transplantation, mount the needle in the needle holder and connect it to the tubing of the transplantation rig.Test the seal by manually operating the syringe to suck liquid in and out of the needle. Next, with a glass pipette, transfer the donor embryos into the first column of the agarose mold. Then transfer the host embryos into columns two to six of the agarose mold.Gently rest the transplantation needle onto the blastomere cells of a donor embryo, and slowly draw up about 20 to 50 cells into the transplantation needle. Avoid drawing up the yolk. Using the micromanipulator, lift the transplantation needle from the donor embryo.Move the injection dish to the left, and insert the transplantation needle tip through the side of the cell mass, into the animal pole of the first host embryo. Deposit five to 10 cells near the surface according to a fate mapping showing that this area gives rise to the forebrain eye field. To mount the embryos for imaging, draw up a maximum of five embryos into a plastic transfer pipette and let the embryos accumulate in the tip by gravity.Touch the transfer pipette onto the surface of a previously prepared plastic tube containing 1%low-melt agarose, allowing the embryos to sink into the tube while limiting the amount of E3 transfer. Next, transfer the five embryos from the agarose plastic tube to a Petri dish with 0.5 to one milliliter of agarose, and using the same transfer pipette, remove excess agarose so that only a small drop remains. Use a micro-loading pipette with the tip broken off to align the embryos laterally, until the agarose solidifies.Then continue mounting up to five embryos at a time, in individual agarose drops. If imaging with an upright microscope, avoid placing embryos within one centimeter of the Petri dish circumference. Use additional 1%low-melt agarose to join the agarose drops to each other in the side of the dish to prevent any dislodging and lateral movement during imaging.Choose embryos that have the correct mounting angle and a strong heartbeat, and a few transplanted donor cells primarily in the anterior ventral quadrant of the developing retina where the wave of gene expression begins. Carry out imaging according to the text protocol. As shown in this figure, micro-injection of one nanoliter of mRNA into the yolk of single-cell stage embryos results in fluorescent reporter protein expression by three hours post-fertilization, at which stage the brightest donors are selected.At 24 hours post-fertilization, embryos with transplanted donor cells in the relevant retinal locations were subjected to time-lapse imaging for 16 to 24 hours. This confocal image of a single optical Z-section shows overlay of the bright field in red channels at the developing eye, with a few labeled donor cells in red within an otherwise unlabeled host environment. These micrographs are from time-lapse imaging of the developing wild-type donor cells from atoh7:gapGFP transgenic embryos transplanted into an unlabeled wild-type host.Transgene expression onset is indicated for a few cells as seen here. Once mastered, the micro-injections can be done in two hours, and embryo selection, preparation and transplantation can be completed in three hours on the same day. Following or in parallel to these procedures, we can also fix embryos at specific ages for post-mortem analysis.For example, we can quantify what proportions of donor cells are expressing a certain gene or protein, have a specific morphology, or have migrated to a certain location. After watching this video, you should have a good understanding of how to genetically manipulate zebrafish embryos and generate chimeras between genetically distinct donor and host. You should also be able to mount the embryos for in vivo time-lapse imaging to answer questions that cannot be answered in fixed tissue.

Research

JoVE Journal

Developmental Biology

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

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