Name: cell lineage analyses and gene function studies using twin-spot marcm
Uploaded: 2017-03-02T14:00:00.000+00:00
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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 FRT40A, UAS-mCD8::GFP (the first reporter, mCD8::GFP; expression of the transgene is under the control of the upstream activation sequence promoter, which produces a membrane-tethered reporter of mouse cluster of differentiation 8 protein fused with green fluorescent protein), and UAS-rCD2RNAi (the suppressor that inhibits the expression of the second reporter; the RNAi against the expression of rat cluster of differentiation 2, rcd2) on the left arm of the 2nd chromosome. Place tissue-specific-GAL4 driver on the X or 3rd chromosome, if possible.</li>
		<li>In the other parental line, assemble the transgenes of FRT40A, UAS-rCD2::RFP (the second reporter, rCD2::RFP; the other membrane-tethered reporter consisting of rCD2 protein fused with red fluorescent protein), and UAS-GFPRNAi (the suppressor that inhibits the expression of mCD8::GFP; the RNAi against the expression of gfp) on the left arm of the 2nd chromosome. Place heat-shock (hs)-FLP or tissue-specific-FLP on the X or 3rd chromosome, if possible.</li>
	</ol>
	</li>
	<li>Generate the new version of tsMARCM-ready flies from transgenes that are carried by individual flies14 (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 an FRT site and UAS-mCD8.GFP.UAS-rCD2i (a combined transgene of mCD8::GFP and the suppressor that inhibits the expression of rCD2::RFP) together in the same arm of the 2nd or 3rd chromosome (appropriate pairs of transgenes of FRT and UAS-mCD8.GFP.UAS-rCD2i are indicated in Table 1). Place tissue-specific-GAL4 driver on chromosomes other than the chromosome of the chosen FRT site, if possible.</li>
		<li>In the other parental line, assemble the transgenes of an FRT site and UAS-rCD2.RFP.UAS-GFPi (the other combined transgene of rCD2::RFP and the suppressor that inhibits the expression of mCD8::GFP) in the same arm of the 2nd or 3rd chromosome (appropriate pairs of transgenes of FRT and UAS-rCD2.RFP.UAS-GFPi are indicated in Table 1). Place hs-FLP or tissue-specific-FLP on chromosomes other than the chromosome of the chosen FRT site, if possible.</li>
	</ol>
	</li>
</ol>
2. Cross tsMARCM-ready Flies to Generate tsMARCM Clones in Their Progeny
<ol>
	<li>Cross tsMARCM-ready flies together (e.g., put 10-15 male flies from step 1.1.1 or 1.2.1 and 20-30 virgin female flies from step 1.1.2 or 1.2.2 together) in a fly-food vial with fresh-made yeast paste on the wall of the vial.</li>
	<li>Maintain the crossed tsMARCM-ready flies at 25 &#176;C for 2 days to enhance the chance of mating before collecting fertilized eggs.</li>
	<li>Frequently transfer the crossed tsMARCM-ready flies into freshly yeasted vials to avoid crowding (tap down the flies or use carbon dioxide to anesthetize the flies to facilitate the transfer; keep no more than 80-100 fertilized eggs in a 10 mL fly-food vial to avoid too many hatched larvae).</li>
	<li>Culture the tsMARCM animals (i.e., fertilized eggs; an example of the genotype of tsMARCM animals, as used in Figure 3, is hs-FLP[22],w/w; UAS-mCD8::GFP,UAS-rCD2RNAi,FRT40A,GAL4-GH146/UAS-rCD2::RFP,UAS-GFPRNAi,FRT40A; +; +) that have been laid in the serially transferred vials at 25 &#176;C until they develop to the desired stages (e.g., embryos, larvae, or pupae).</li>
	<li>Place the transferred vials that contain tsMARCM animals at the same developmental stage to a 37 &#176;C water bath for 10, 20, 30, 40, and 50 min to determine the optimal time of heat-shock that induces the expression of FLP to generate the tsMARCM clones of interest. Skip this step if tissue-specific-FLP is being used in the tsMARCM experiment (hs-FLP is preferred for neural lineage analyses; the reasons for this preference have been described previously15). 
	NOTE: Repeat step 2.5 using the optimal time of heat-shock in future tsMARCM experiments if more tsMARCM clones of interest are wanted; generally, more FLP expression is induced in hs-FLP[22] compared to hs-FLP[1] with the same heat-shock time.</li>
	<li>Culture the heat-shocked tsMARCM animals at 25 &#176;C until they have reached the appropriate developmental stage, and then proceed to step 3.</li>
</ol>
3. Prepare, Stain, and Mount Fly Brains Containing tsMARCM Clones
<ol>
	<li>Prepare forceps-protection dishes. Mix part A (30 g) and part B (3 g) of the Silicone Elastomer Kit with activated charcoal (0.25 g). Pour the mixture into the appropriate dishes.</li>
	<li>Dissect larval, pupal, or adult brains out of the tsMARCM animals in 1x phosphate-buffered saline (PBS) using forceps on a forceps-protection dish viewed through a dissection microscope. NOTE: A protocol for fly brain dissection has been described previously16.</li>
	<li>Fix the fly brains in a glass spot plate with 1x PBS containing 4% formaldehyde at room temperature for 20 min. Process the fly brains in this glass spot plate for the remainder of the steps.</li>
	<li>Rinse the fly brains three times with 1% PBT (1x PBS containing 1% Triton X-100) and wash them three times for 30 min each in 1% PBT (rinse: remove PBT and add new PBT quickly; wash: remove PBT, add new PBT, and incubate the fly brains in the new PBT using an orbital shaker).</li>
	<li>Incubate the fly brains with the mixture of primary antibodies overnight at 4 &#176;C or for 4 h at room temperature. An example of the mixture of primary antibodies is rat anti-CD8 antibody (1:100), rabbit anti-DsRed antibody (1:800), and mouse anti-Bruchpilot (Brp) antibody (1:50) in 1% PBT containing 5% normal goat serum (NGS).</li>
	<li>Rinse the fly brains three times with 1% PBT, and then wash them three times for 30 min each with 1% PBT.</li>
	<li>Incubate the fly brains with the mixture of secondary antibodies overnight at 4 &#176;C or for 4 h at room temperature. An example of the mixture of secondary antibodies is goat anti-rat IgG antibody conjugated with green-fluorescent dye (1:800), goat anti-rabbit IgG antibody conjugated with yellow-fluorescent dye (1:800), and goat anti-mouse IgG antibody conjugated with far-red-fluorescent dye (1:800) in 1% PBT containing 5% NGS.</li>
	<li>Rinse the fly brains three times with 1% PBT, and then wash them three times for 30 min each with 1% PBT. Mount them on a micro slide with an anti-quenching reagent. Put a micro cover glass on the micro slide to cover and protect the fly brains. Seal the edges of the micro cover glass and micro slide using clear nail polish. Store these mounted and sealed fly brain specimens at 4 &#176;C.</li>
</ol>
4. Take, Process, and Analyze Fluorescent Images of tsMARCM Clones
<ol>
	<li>Capture fluorescent images of tsMARCM clones from the samples created in step 3.8 using the confocal microscopy system of choice.</li>
	<li>Project stacks of tsMARCM confocal fluorescent images into two-dimensional, flattened images using the image processing software supplied with the confocal microscopy system of choice (see Figure 3B-3E, 3G-3J, 3L-3O, and 3Q-3T for examples of flattened confocal fluorescent images).</li>
	<li>Count the cell number of the multi-cellular-NB sides of the tsMARCM clones using an appropriate image processing software (e.g., the &#34;Cell Counter&#34; function in ImageJ17; see Figure 3E, 3J, 3O, and 3T for examples of the cell bodies of neurons).</li>
</ol>

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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 best to vary the heat-shock time (10, 20, 30, 40, and 50 min) on different samples of the same genotype and developmental stage in step 2.5. This will allow for the determination of the optimal heat-shock time in order to obtain the best ratio of tsMARCM clones of interest. In addition, be careful during fly brain dissection in step 3.2 in order to obtain intact brain samples; this is crucial for accurately counting neuronal cell numbers. For researchers who are interested in using tsMARCM for studying the function of genes on other chromosome arms (e.g., 2R, 3L, and 3R), tsMARCM-ready flies should be assembled as in step 1.2 by choosing appropriate pairs of transgenes of FRT, UAS-mCD8.GFP.UAS-rCD2i,and UAS-rCD2.RFP.UAS-GFPi, which are indicated in Table 1. We also recommend that the female tsMARCM-ready flies carry hs-FLP on the X chromosome (e.g., hs-FLP[22],w; UAS-rCD2::RFP,UAS-GFPRNAi,FRT40A; +; +), which facilitates repeated tsMARCM experiments.
Modifications, Troubleshooting, and Limitations of the Technique
The efficiency and perdurance of RNAi suppressors and reporters are the major determinants for whether neural lineage analyses and gene function studies can be successfully performed in tsMARCM experiments. Generally, the combination of low perdurance of RNAi suppressors and reporters derived from neural progenitors, together with the high expression of RNAi suppressors and reporters in post-mitotic neurons, permits better tsMARCM results. Because the expression of RNAi suppressors and reporters is controlled by GAL4 drivers, researchers should select strong GAL4 drivers that express GAL4 in differentiated neurons but not in neural progenitors. For example, GAL4-OK107 is a strong GAL4 driver that expresses GAL4 in mushroom body (MB) progenitors and their derivatives, MB &#947;, &#945;&#39;/&#946;&#39;, and &#945;/&#946; neurons11,19. Unfaithful labeling of MB &#947; neurons has been observed in two clonal patterns (i.e., single-cell associated with single-cell clones and two-cell-GMC associated with multi-cellular-NB clones) when GAL4-OK107 was used in tsMARCM experiments11. Intriguingly, the above issue can be solved by using MB247-GAL4, a different MB GAL4 driver that expresses GAL4 in differentiated MB neurons11.
Not all GAL4 drivers can be used in the tsMARCM system or in other mosaic labeling systems (e.g., MARCM, Q-MARCM, and twin-spot generator)4,20,21 if the driver is not expressed in the neurons of interest. Therefore, prior to using tsMARCM for neural lineage analyses, GAL4 drivers should be tested for their coverage of the neurons of interest in the dual-expression-control MARCM with a pan-cell driver (e.g., tubulin promoter-LexA::GAD)22. We note that tsMARCM clones sometimes cannot be analyzed unambiguously, especially when a GAL4 driver with a complex neural expression pattern is used (e.g., green lateral horn (LH) neurons prevent clear visualization of the axonal elaboration pattern of the VL2p adPN in Figure 3B and 3C). However, the above issue of ambiguous patterns also occurs in other mosaic labeling systems (e.g., MARCM, Q-MARCM, and twin-spot generator)4,20,21 because the limitation originates with the GAL4 driver. We also note that, to conduct rescue experiments for the authentication of gene functions, the rescue transgenes should be engineered with the target sequences of the RNAi suppressors in 5&#39; or 3&#39; untranslated regions of the transgenic rescue constructs, or an alternative twin-spot labeling system (e.g., coupled MARCM, a combination of Q-MARCM and conventional MARCM)21 should be adopted.
Significance of the Technique with Respect to Existing/Alternative Methods
Besides tsMARCM, twin-spot generator and coupled MARCM would also, at least in theory, allow for the visualization of neurons derived from common neural progenitors in two distinct colors (a comparison of these three mosaic labeling technologies has been presented previously15). The minimum number of transgenes required for assembling twin-spot generator, the original version of tsMARCM (or the new version of tsMARCM), and coupled MARCM is 4, 8 (or 6), and 9, respectively. However, tsMARCM is the only system that has been used to conduct comprehensive neural lineage analyses12,15,18,23. Site-specific recombination does occur in non-mitotic cells in the twin-spot generator system, and this leads to the co-expression of the two reporters in the same cell, making it challenging to use this system to study the development of the central nervous system15. In contrast, mosaic clones obtained in the tsMARCM and coupled MARCM systems are derived from mitotic cells. However, because the coupled MARCM system requires two independent drivers to control the expression of the different reporters in the Q-MARCM and conventional MARCM systems, this raises concerns about the labeling of neurons derived from common neural progenitors if the two independent drivers do not faithfully label the same neurons in the neural lineages of interest15.
Future Applications or Directions after Mastering the Technique
Using tsMARCM to conduct high-resolution neural lineage analyses 
By applying the approach used in Figure 3 to systematically analyze tsMARCM clones of adPNs, 40 types of adPNs, with a total of 82 neurons, were identified in a previous study12. Among these 40 types of adPNs, 35 were uni-glomerular adPNs (i.e., innervating a single glomerulus in the antennal lobe (AL)), which relay olfactory information to other regions of the Drosophila brain12 (see Figures 2-4 in Yu et al., 2010). The other five types of adPNs were poly-glomerular adPNs (i.e., innervating multiple glomeruli in the AL), which could integrate olfactory information from the AL and provide an additional level of functional complexity to the olfactory system12 (see Figures 3E-3H and 4Z in Yu et al., 2010). Embryonic-born adPNs have only one neuron per type, whereas larval-born adPNs are represented by multiple neurons per type, suggesting that there is interplay between specificity and redundancy in the olfactory system12 (see Figures 2-4 in Yu et al., 2010). Interestingly, neurons produced within certain developmental windows are destined to die, and many types of adPNs are found with a fixed number of neurons in the ALad1 neural lineage, suggesting that the number and types of adPNs for assembling the olfactory system are tightly regulated12 (see Figures 2H, 4V, and 5 in Yu et al., 2010; also see Figure 3P-3T).
Using tsMARCM to accurately study gene functions 
Besides birth-dating adPNs, tsMARCM has also been applied to determine the birth-order of six central complex neurons labeled by GAL4-OK107. These six neurons make up a small subset of neurons in the LALv1 neural lineage that contribute to the circuitry of the Drosophila central complex to control intricate motor patterns2,11 (see Figure 4a and 4b in Yu et al., 2009). Interestingly, each of these six central complex neurons possesses a distinct neuronal morphology11 (see Figure 4d-4h and 4o in Yu et al., 2009). All of them carry distinct axonal patterns in the lateral accessory lobe (LAL). The first four neurons distribute their dendrites to sub-compartments of the noduli, a sub-structure of the central complex, whereas the remaining two neurons project their dendrites to the ellipsoid body, another sub-structure of the central complex. Specifically, the 3rd-born central complex neuron projects its dendrites to the ventral noduli, with a compact axonal pattern in the LAL, whereas the 4th-born central complex neuron distributes its dendrites to the dorsal noduli, with a broad axonal pattern in the LAL11 (see Figure 4f and 4g in Yu et al., 2009).
Beyond comprehensively identifying neurons, the power of tsMARCM would be better exemplified by studying gene functions in neurons of interest, since the tsMARCM system permits better phenotypic analyses by enabling the examination of identical neurons in different animals. For example, tsMARCM detects a single temporal fate change that requires chronologically inappropriate morphogenesis (chinmo, a BTB-zinc finger nuclear protein) in the 3rd-born central complex neurons11 (see Figure 4i, 4p, and 4w in Yu et al., 2009). If the chinmo mutation had been characterized in the original MARCM system (i.e., single green-labeled neurons, without the labeling of neurons derived from the associated sides of the twin cells as the reference), the chinmo-deficient neuron shown in Figure 4i from Yu et al., 2009 would have been misidentified as the 4th-born, rather than the 3rd-born, central complex neuron. This would have resulted in the misinterpretation of chinmo function as controlling axonal guidance rather than specifying temporal fate11 (double-arrow in Figure 4i from Yu et al., 2009). However, by using the magenta multi-cellular-NB sides of tsMARCM clones as the reference (via counting the cell numbers in Figure 4m, 4n, and 4p from Yu et al., 2009), the chinmo-deficient neuron shown in Figure 4i from Yu et al., 2009) was proven to be the 3rd-born central complex neuron, which therefore led to the correct conclusion that chinmo functions in temporal fate specification for central complex neurons11. Taken together, the above results highlight the important advantages of the tsMARCM system for high-resolution phenotypic analyses, thereby accurately disclosing gene functions in neurons (or other types of cells, if applicable) for future neural developmental studies (or the developmental studies of other tissues).

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><tbody><tr><td>Carbon dioxide (CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Local vendor</td><td>n.a.</td><td>Anesthetize flies</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; 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>Jackson ImmunoResearch&amp;nbsp;</td><td>005-000-121</td><td>Immunostain fly brains</td></tr><tr><td>serum</td><td></td><td></td><td></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&amp;nbsp;</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>Incubator&amp;nbsp;</td><td>Kansin Instruments</td><td>LTI603</td><td>culture flies at 25 &amp;deg;C</td></tr><tr><td>(or other vendors)</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td>Kansin Instruments</td><td>WB212-B2</td><td>Induce heat-shock in flies at 37 &amp;deg;C</td></tr><tr><td>(or other vendors)</td><td></td><td></td><td></td></tr><tr><td>Orbital shaker</td><td>Kansin Instruments</td><td>OS701</td><td>Wash and immunostain fly brains</td></tr><tr><td>(or other vendors)</td><td></td><td></td><td></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&lt;br/&gt; (&lt;em&gt;e.g.&lt;/em&gt;, Zeiss LSM image browser)&amp;nbsp;</td><td>Zeiss</td><td>not available</td><td>Project stacks of confocal images</td></tr><tr><td>image-processing software 2&lt;br/&gt; (&lt;em&gt;e.g.&lt;/em&gt;, ImageJ)&amp;nbsp;</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 details on using tsMARCM to conduct neural lineage analyses or gene function studies). Here, we used four tsMARCM clones of anterodorsal olfactory projection neurons (adPNs in the ALad1 neural lineage of the Drosophila olfactory system) labeled with GAL4-GH146 as examples to illustrate how to conduct a tsMARCM experiment1,2. After FLP expression was induced at the embryonic stage, a single, green VL2p adPN associated with 45 magenta adPNs was detected in a tsMARCM clone (Figure 3A-3E). In contrast, when FLP was induced later, at different larval stages, single, green adPNs (DL1 adPN or DC3 adPN) or no-GH146+ adPNs associated with different numbers of magenta adPNs (31, 21, or 15) were observed in three other tsMARCM clones (Figure 3F-3T). The birth of VL2p adPNs prior to the birth of DL1-, DC3- and no-GH146+-adPNs was determined by counting the cell numbers of adPNs in their associated multi-cellular-NB sides of the tsMARCM clones (45, 31, 21, and 15 in Figure 3E, 3J, 3O, and 3T, respectively). In addition to counting the cell numbers, the complexity of the axonal and dendritic patterns in the multi-cellular-NB sides of the tsMARCM clones can be used to determine the relative birth-order of their associated two-cell-GMC sides of the tsMARCM clones. Mosaic clones induced at early developmental stages normally contain more neurons and thus have more complex axonal and dendritic patterns, whereas mosaic clones induced at late developmental stages contain fewer neurons and thus have less complex axonal and dendritic patterns (Figure 3C-3D, 3H-3I, 3M-3N, and 3R-3S).
<img alt="Figure 1" src="/files/ftp_upload/55278/55278fig1.jpg" /> 
Figure 1: Schematic illustrations of Drosophila neurogenesis and the three types of clonal patterns found in a MARCM experiment. (A) A schematic diagram illustrating neurogenesis in most neural lineages of the Drosophila central brain: neuroblasts (NBs) undergo an asymmetric division to generate self-renewing NBs and ganglion mother cells (GMCs), and GMCs divide one more time to produce two daughter neurons (Ns). (B) Three clonal patterns are observed in a MARCM experiment: single-cell clones (a), when flippase (FLP) is induced in GMCs, and two-cell-GMC clones (b) and multi-cellular-NB clones (c), when FLP is induced in NBs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55278/55278fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55278/55278fig2.jpg" /> 
Figure 2: Schematic diagrams of the tsMARCM system. (A) tsMARCM-ready flies: one parental fly contains transgenes of an FRT site and UAS-mCD8::GFP (the first reporter), UAS-rCD2RNAi (the first suppressor that inhibits the expression of the second reporter), and a tissue-specific-GAL4; the other parental fly contains an FRT site, UAS-rCD2::RFP (the second reporter), UAS-GFPRNAi (the second suppressor that inhibits the expression of the first reporter), and hs-FLP. (B) The presence of the two RNAi-based suppressors in the progeny of the crossed tsMARCM-ready flies inhibits the expression of reporters (i.e., mCD8::GFP and rCD2::RFP in the G1 and G2 phases of panel B). The expression of FLP is induced by heat-shock, and FLP then mediates the site-specific recombination of homologous chromosomes within a dividing precursor cell upon binding to FLP recognition targets (FRTs). This results in the segregation of the RNAi-based suppressors in the twin cells after mitotic division to permit the expression of distinct reporters. (C) Two clonal patterns are observed in a tsMARCM experiment: single-cell, associated with single-cell clones (a) when FLP is induced in GMCs, and two-cell-GMC, associated with multi-cellular-NB clones (b) when FLP is induced in NBs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55278/55278fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55278/55278fig3.jpg" /> 
Figure 3: Using four tsMARCM clones of adPNs as examples of how to conduct a tsMARCM experiment in a neural lineage analysis. Four tsMARCM clones of adPNs are illustrated, with diagrams of their neurogenesis patterns (A, F, K, and P); confocal images of the overall brain (B, G, L, and Q), showing the lateral horn (LH; C, H, M, and R), the antennal lobe (AL; D, I, N, and S) and the soma (E, J, O, and T); and the patterns of their neuronal morphologies. Single adPNs (VL2p, DL1, DC3, and no-GH146+ adPNs; green) associated with multi-cellular-NB adPNs (magenta) of the tsMARCM clones were derived by the induction of FLP to mediate mitotic recombination in the adPN NB at the embryonic (A-E) and larval (F-T) stages. Note that adPNs are one of the hemi-lineages in the ALad1 neural lineage, which results in tsMARCM clones of a single, green adPN associated with magenta adPNs in Figure 3 (for unknown reasons, neurons from the other hemi-lineage of the ALad1 neural lineage die). The birth-order of VL2p, DL1, DC3, and no-GH146+ adPNs was determined by counting the cell numbers of adPNs in the magenta multi-cellular-NB sides of the tsMARCM clones (45, 31, 21, and 15 within the dashed lines of panels E, J, O, and T). The neurite patterns are usually more complex in the magenta multi-cellular-NB sides when the tsMARCM clones are induced at early developmental stages (e.g., the dendritic pattern is more complex in panel E compared to panels I, N, and S). tsMARCM analyses are sometimes confusing when neurons of other neural lineages are also labeled by the same GAL4 driver (e.g., green LH neurons, shown by double-arrows, obstruct the visualization of the axonal elaboration pattern of the VL2p adPNs in panels B and C). Brain neuropiles (shown in blue) were stained with antibody against Bruchpilot (Brp). Heat-shock time: 12 min (panels A-E), 15 min (panels F-J), and 35 min (panels K-T). Scale bar in panels B-E, G-J, L-O, and Q-S = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55278/55278fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.9K Views.  Academia Sinica. 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.

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Cancer Research

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 ...

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Genetics

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Biochemistry

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Neuroscience

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...

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

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Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)