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>
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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. 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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>

The authors declare that they have no competing financial interests.

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.

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9.8K ビュー.  Academia Sinica. 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 individua...