Name: cell lineage analyses and gene function studies using twin-spot marcm
Uploaded: 2017-03-02T14:00:00
Description: Watch this Scientific Journal Video about Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM at JoVE.com

This work was supported by the Ministry of Science and Technology (MOST 104-2311-B-001-034) and the Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan.

1. Build tsMARCM-ready Flies Using the Required Transgenes11,13
<ol>
	<li>Generate the original version of tsMARCM-ready flies from transgenes that are carried by individual flies11 (see Table 1). Conduct standard fly genetic crossing schemes, which have been described previously, by putting multiple transgenes in the same fly stocks13.
	<ol>
		<li>In one parental line, assemble the transgenes of ...

<ol>
	<li>Ito, M., Masuda, N., Shinomiya, K., Endo, K., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+neural+projections+reveals+clonal+composition+of+the+Drosophila+brain.">Systematic analysis of neural projections reveals clonal composition of the Drosophila brain.</a> Curr Biol. 23 (8), 644-655 (2013).</li><li>Yu, H. 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=Clonal+development+and+organization+of+the+adult+Drosophila+central+brain.">Clonal development and organization of the adult Drosophila central brain.</a> Curr Biol. 23 (8), 633-643 (2013).</li><li>Goodman, C. S., Doe, C. Q., Bate, M., Arias, A. 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 Development of Drosophila melanogaster. , (1993).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22 (3), 451-461 (1999).</li><li>Lee, T., Lee, A., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+mushroom+bodies:+sequential+generation+of+three+distinct+types+of+neurons+from+a+neuroblast.">Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.</a> Development. 126 (18), 4065-4076 (1999).</li><li>Lee, T., Marticke, S., Sung, C., Robinow, S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+requirement+of+the+USP/EcR-B+ecdysone+receptor+for+mushroom+body+neuronal+remodeling+in+Drosophila.">Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila.</a> Neuron. 28 (3), 807-818 (2000).</li><li>Chiang, A. S., 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=Three-dimensional+reconstruction+of+brain-wide+wiring+networks+in+Drosophila+at+single-cell+resolution.">Three-dimensional reconstruction of brain-wide wiring networks in Drosophila at single-cell resolution.</a> Curr Biol. 21 (1), 1-11 (2011).</li><li>Jefferis, G. S., Marin, E. C., Stocker, R. F., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+neuron+prespecification+in+the+olfactory+map+of+Drosophila.">Target neuron prespecification in the olfactory map of Drosophila.</a> Nature. 414 (6860), 204-208 (2001).</li><li>Hong, W., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+wiring+specificity+in+the+fly+olfactory+system.">Genetic control of wiring specificity in the fly olfactory system.</a> Genetics. 196 (1), 17-29 (2014).</li><li>Zhu, S., 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=Gradients+of+the+Drosophila+Chinmo+BTB-zinc+finger+protein+govern+neuronal+temporal+identity.">Gradients of the Drosophila Chinmo BTB-zinc finger protein govern neuronal temporal identity.</a> Cell. 127 (2), 409-422 (2006).</li><li>Yu, H. H., Chen, C. H., Shi, L., Huang, Y., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twin-spot+MARCM+to+reveal+the+developmental+origin+and+identity+of+neurons.">Twin-spot MARCM to reveal the developmental origin and identity of neurons.</a> Nat Neurosci. 12 (7), 947-953 (2009).</li><li>Yu, H. 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=A+complete+developmental+sequence+of+a+Drosophila+neuronal+lineage+as+revealed+by+twin-spot+MARCM.">A complete developmental sequence of a Drosophila neuronal lineage as revealed by twin-spot MARCM.</a> PLoS Biol. 8 (8), (2010).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly pushing : the theory and practice of Drosophila genetics. , (1997).</li><li>Awasaki, 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=Making+Drosophila+lineage-restricted+drivers+via+patterned+recombination+in+neuroblasts.">Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts.</a> Nat Neurosci. 17 (4), 631-637 (2014).</li><li>Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+mosaics+for+lineage+analysis+in+flies.">Generating mosaics for lineage analysis in flies.</a> Wiley Interdiscip Rev Dev Biol. 3 (1), 69-81 (2014).</li><li>Wu, J. S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat Protoc. 1 (4), 2110-2115 (2006).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Lin, S., Kao, C. F., Yu, H. H., Huang, Y., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+Drosophila+lateral+antennal+lobe+neurons+reveals+notch-dependent+binary+temporal+fate+decisions.">Lineage analysis of Drosophila lateral antennal lobe neurons reveals notch-dependent binary temporal fate decisions.</a> PLoS Biol. 10 (11), e1001425 (2012).</li><li>Zhan, X. L., 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=Analysis+of+Dscam+diversity+in+regulating+axon+guidance+in+Drosophila+mushroom+bodies.">Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies.</a> Neuron. 43 (5), 673-686 (2004).</li><li>Griffin, 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=The+twin+spot+generator+for+differential+Drosophila+lineage+analysis.">The twin spot generator for differential Drosophila lineage analysis.</a> Nat Methods. 6 (8), 600-602 (2009).</li><li>Potter, C. J., Tasic, B., Russler, E. V., Liang, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Q+system:+a+repressible+binary+system+for+transgene+expression,+lineage+tracing,+and+mosaic+analysis.">The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.</a> Cell. 141 (3), 536-548 (2010).</li><li>Lai, S. L., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mosaic+with+dual+binary+transcriptional+systems+in+Drosophila.">Genetic mosaic with dual binary transcriptional systems in Drosophila.</a> Nat Neurosci. 9 (5), 703-709 (2006).</li><li>Kao, C. F., Yu, H. H., He, Y., Kao, J. C., Lee, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+deployment+of+factors+regulating+temporal+fate+in+a+diverse+neuronal+lineage+of+the+Drosophila+central+brain.">Hierarchical deployment of factors regulating temporal fate in a diverse neuronal lineage of the Drosophila central brain.</a> Neuron. 73 (4), 677-684 (2012).</li></ol>

Critical Steps within the Protocol
Steps 1.1.1, 1.2.1, 2.3, 2.5, and 3.2 are critical for obtaining good tsMARCM results. Tissue-specific-GAL4 drivers that do not express GAL4 in neural progenitors are preferred for steps 1.1.1 and 1.2.1. Avoid over-crowding the animals grown in the fly-food vials in step 2.3. Because the induction latency, the expression level of FLP upon heat-shock, and the average dividing time of neural progenitors (i.e., NBs and GMCs) are not known, it is b...

The brain, comprised of a vast number and diverse types of neurons, endows animals with the ability to perceive, process, and respond to challenges from the external world. Neurons of the adult Drosophila central brain are derived from a limited number of neural stem cells, called neuroblasts (NBs), during development1,2. Most of the NBs participating in brain neurogenesis in Drosophila undergo asymmetric division to generate self-renewing NBs and ganglion mother cells (GMCs), and the GMCs then go through another round of division to produce two daughter cells that differentiate into neurons3 (Figure 1A). Because of the intricacy of neuronal morphology and the challenges associated with identifying specific neurons, a positive mosaic labeling technology, mosaic analysis with a repressible cell marker (MARCM), was invented to enable the visualization of a single neuron or a small subset of neurons out of the population of surrounding, unlabeled neurons4.
MARCM utilizes the flippase (FLP)/FLP recognition target (FRT) system to mediate site-specific recombination between homologous chromosomes within a dividing precursor cell carrying a heterozygous allele of a repressor gene, whose expression normally inhibits the expression of a reporter gene4. After the mitotic division, the recombinant chromosomes are segregated into twin cells, such that one cell contains homozygous alleles of the repressor gene and the other cell has no repressor gene-the expression of the reporter in that cell (and its descendants) is no longer blocked4. Three clonal patterns are usually found in a MARCM experiment when FLP is stochastically induced in NBs or GMCs: single-cell and two-cell-GMC clones, which depict neuronal morphology at single-cell resolution, and multi-cellular-NB clones, which reveal entire morphological patterns of neurons derived from a common NB (Figure 1B). The MARCM technique has been widely applied in Drosophila neurobiological studies, including in neuronal type identification for reconstructing brain-wide wiring networks, neural lineage analyses for disclosing the developmental history of neurons, phenotypic characterization of gene functions involved in cell fate specification, and neuronal morphogenesis and differentiation studies5,6,7,8,9,10. Because conventional MARCM only labels one of the two daughter cells (and lineages) after the induced mitotic recombination event, potentially useful information from the unmarked side is lost. This limitation precludes the application of the basic MARCM system to high-resolution analyses of many neural lineages that switch cell fates in fast tempo or to precision analyses of gene functions in identical neurons of different animals11,12.
Twin-spot MARCM (tsMARCM) is an advanced system that labels neurons derived from a common progenitor with two distinct colors, which enables the recovery of useful information from both sides of the twin cells, thus overcoming the limitation of the original MARCM system11 (Figure 2A-2C). In the tsMARCM system, two RNA interference (RNAi)-based suppressors are situated at trans-sites of homologous chromosomes within a precursor cell, and the expression of those suppressors independently inhibit the expression of their respective reporters (Figure 2B). Following site-specific mitotic recombination mediated through the FLP/FRT system, the two RNAi-based suppressors become segregated into twin cells to permit the expression of distinct reporters (Figure 2B). Two clonal patterns, single-cell associated with single-cell clones and two-cell-GMC associated with multi-cellular-NB clones, are typically seen in a tsMARCM experiment (Figure 2C). Information derived from one side of the twin cells can be utilized as the reference for the other side, enabling high-resolution neural lineage analyses, such as birth-dating the labeled neurons, and phenotypic analyses of identical neurons in different animals for the precise investigation of neural gene function11,12. Here, we present a step-by-step protocol describing how to conduct a tsMARCM experiment, which can be used by other laboratories to broaden their studies of neural development (as well as the development of other tissues, if applicable) in Drosophila.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carbon dioxide (CO2)</td>
			<td>Local vendor</td>
			<td>n.a.</td>
			<td>Anesthetize flies</td>
		</tr>
		<tr>
			<td>CO2 pad</td>
			<td>Local vendor</td>
			<td>n.a.</td>
			<td>Sort, cross and transfer flies</td>
		</tr>
		<tr>
			<td>10x PBS pH 7.4</td>
			<td>Uniregion Bio-Tech (or other vendors)</td>
			<td>PBS001</td>
			<td>Dissect, rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>Fix fly brains</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Normal goat</td>
			<td rowspan="2">Jackson ImmunoResearch&#160;</td>
			<td rowspan="2">005-000-121</td>
			<td rowspan="2">Immunostain fly brains</td>
		</tr>
		<tr>
			<td>serum</td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD8 antibody</td>
			<td>Invitrogen</td>
			<td>MCD0800</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Rabbit anti-DsRed antibody</td>
			<td>Clonetech</td>
			<td>632496</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Mouse anti-Brp antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>nc82</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG antibody conjugated with Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11006</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG antibody conjugated with Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A11035</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG antibody conjugated with Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A21236</td>
			<td>Immunostain fly brains</td>
		</tr>
		<tr>
			<td>SlowFade gold antifade reagent</td>
			<td>Molecular Probes</td>
			<td>S36936</td>
			<td>Mount fly brains and protect quenching of fluorescence&#160;</td>
		</tr>
		<tr>
			<td>PYREX 9 depression glass spot plate</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td>Dissect, rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Sylgard 184 silicone elastomer kit</td>
			<td>World Precision Instruments</td>
			<td>SYLG184</td>
			<td>Make black Sylgard dishes to protect forceps during brain dissection</td>
		</tr>
		<tr>
			<td>Activated charcoal</td>
			<td>Sigma-Aldrich</td>
			<td>242276-250G</td>
			<td>Make black Sylgard dishes</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>Dissect and mount fly brains</td>
		</tr>
		<tr>
			<td>Micro slide</td>
			<td>Corning</td>
			<td>2948-75x25</td>
			<td>Mount fly brains</td>
		</tr>
		<tr>
			<td>Micro cover glass No.1.5</td>
			<td>VWR International</td>
			<td>48366-205</td>
			<td>Mount fly brains</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Local vendor</td>
			<td>not available</td>
			<td>Seal micro cover glass on micro slides</td>
		</tr>
		<tr>
			<td rowspan="2">Incubator&#160;</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">LTI603</td>
			<td rowspan="2">culture flies at 25&#176;C</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td rowspan="2">Water bath</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">WB212-B2</td>
			<td rowspan="2">Induce heat-shock in flies at 37&#176;C</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td rowspan="2">Orbital shaker</td>
			<td>Kansin Instruments</td>
			<td rowspan="2">OS701</td>
			<td rowspan="2">Wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>(or other vendors)</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Leica</td>
			<td>EZ4</td>
			<td>Sort, cross and transfer flies; Dissect, rinse, wash and immunostain fly brains</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss (or other vendors)</td>
			<td>LSM 700 (or other models)</td>
			<td>Image tsMARCM clones</td>
		</tr>
		<tr>
			<td>image-processing software 1 
			(e.g., Zeiss LSM image browser)&#160;</td>
			<td>Zeiss</td>
			<td>not available</td>
			<td>Project stacks of confocal images</td>
		</tr>
		<tr>
			<td>image-processing software 2 
			(e.g., Image-J)&#160;</td>
			<td>not available</td>
			<td>not available</td>
			<td>Count cell number of tsMARCM clones</td>
		</tr>
	</tbody>
</table>

cell lineage analyses and gene function studies using twin-spot marcm

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

The tsMARCM system has been used to facilitate neural lineage analyses and gene function studies by retrieving important information on neurons derived from common NBs. The system has been used to identify most (if not all) neuronal types, determine the cell number of each neuronal type, and ascertain the birth-order of these neurons11,12,18 (see the Discussion Section for detail...

Watch this Scientific Journal Video about Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM at JoVE.com

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

The overall goal of this twin-spot MARCM protocol is to conduct high resolution cell lineage analyses and better gene function studies. Brain tissue is comprised of a vast number of diverse neurons, it's challenging to study the mechanism underlying how neurons assemble together during development. The main advantage of this technique is to label neurons derived from common neuron progenitors in two distinct colors.Which permit the retrieval of the birth of the information of individual neurons. Therefore this technique can help researchers to explore the cellular and molecular mechanism underlying neural development. Demonstrating the procedure today will be Pei-Chi Chung.A technician from Professor Yu's laboratory. Begin by combining twin-spot MARCM ready males and females in a fly food vial with fresh yeast in the vial. Maintain the crossed twin-spot MARCM ready flies at 25 degrees Celsius for two days to enhance the chance of mating before collecting fertilized eggs.To avoid overcrowding when the larvae hatch, tap down the crossed twin-spot MARCM ready flies, and transfer into freshly yeasted vials. Culture the twin-spot MARCM offspring, until they reach the desired stage of development. Here, flies at the larval stage will be heat shocked to induce flippase expression.Transfer the fly vials to a 37 degrees Celsius water bath for 10, 20, 30, 40, and 50 minutes to determine the optimal heat shock for flippase expression. To generate the twin-spot MARCM clones of interest. The timing of the heat shock determines the stage of clearing generation, while the heat shock period defines how many clearings will be generated.Culture the heat shocked twin-spot MARCM animals at 25 degrees Celsius, until the desired developmental stage. Then proceed to staining and mounting. Add PBS to the forceps protection dishes.And then, transfer the twin-spot MARCM animals to the dishes. While looking through a dissection microscope, use forceps to dissect the brains from the twin-spot MARCM animals. First, remove the head from the body of the fly.Then gently remove the cuticle without damaging the brain. Next, carefully remove tracheae which appear as white colored fibers. Transfer the brains to a glass spot plate, Containing 4%formaldehyde and PBS.And fix at room temperature for 20 minutes. Begin the staining procedure by rinsing the fly brains three times with 1%PBT. And then washing with 1%PBT three times.With shaking for 30 minutes each time. Incubate the fly brains with a mixture of primary antibodies overnight at four degrees Celsius or for four hours at room temperature. Rinse and wash the fly brains three times each with 1%PBT, with shaking as before.Then incubate the fly brains with a mixture of secondary antibodies for four hours at room temperature or overnight at four degrees Celsius. After rinsing and washing the brains as before, mount the brains on a microslide with an anti-quenching reagent. And then place a cover glass to cover and protect the fly brains.Seal the edges of the micro cover glass and microslide using clear nail polish. Finally, capture fluorescent images of twin-spot MARCM clones from the samples using the confocal microscopy system of choice. This figure shows results from the twin-spot MARCM clone, a VL2p antero dorsal olfactory projection neurons derived by the induction of flippase.To mediate mytotic recombination in the adPN neuro blast at the embryonic stage. This first diagram shows the neurogenesis pattern. In this confocal microscope image of the brain, The VL2p adPNs are shown in green and are associated with multi cellular neuro blast adPNs in magenta.This image shows the cells in the lateral horn. The neuronal morphology of the antennal lobe is seen here. This image shows the data for DL1 adPNs.As seen here, the DL1 adPNs appear later in development than VL2p adPNs. Induction of twin-spot MARCM clones of DC3 adPNs demonstrate that this neuronal type appears later in development. This image shows that twin-spot MARCM event occurs in a GAL4 negative neuron within the adPN lineage.Once mastered, this technique can help researchers to analyze neural lineage and provide different markers to distinguish neurons from neuroblast clone. Furthermore twin-spot MARCM can produce homozygous mutant clones to explore molecular mechanism. In a heterozygous genetic background.

cell-lineage-analyses-and-gene-function-studies-using-twin-spot-marcm

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Tracing Gene Expression Through Detection of &amp;#946;-galactosidase Activity in Whole Mouse Embryos

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...