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Hung-chang Shen - One of the best experts on this subject based on the ideXlab platform.
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overview of MARCM related technologies in drosophila neurobiological research
Current protocols in protein science, 2020Co-Authors: Tsai-chi Hsu, Hung-chang ShenAbstract:Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.
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Overview of MARCM‐Related Technologies in Drosophila Neurobiological Research
Current protocols in protein science, 2020Co-Authors: Tsai-chi Hsu, Hung-chang ShenAbstract:Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.
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Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM
Journal of Visualized Experiments, 2017Co-Authors: Hung-chang Shen, Tsai-chi Hsu, Pei-chi ChungAbstract: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.
Liqun Luo - One of the best experts on this subject based on the ideXlab platform.
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Single-Neuron Labeling Using the Genetic MARCM Method
CSH Protocols, 2007Co-Authors: Liqun LuoAbstract:INTRODUCTIONOur brain is composed of hundreds of billions of neurons, each of which has an elaborate shape and a complex pattern of connections. To untangle this complexity, it is often useful to visualize one neuron at a time. This protocol describes single-neuron labeling using the MARCM system (Mosaic Analysis with a Repressible Cell Marker). The system was developed in Drosophila, but could, in principle, also work in other genetic model organisms such as Caenorhabditis elegans, zebrafish, or mice.
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A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila
Nature Protocols, 2006Co-Authors: Liqun LuoAbstract:Mosaic analysis with a repressible cell marker (MARCM) is a genetic technique used in Drosophila to label single cells or multiple cells sharing a single progenitor. Labeled homozygous mutant cells can be generated in an otherwise unlabeled heterozygous animal. Mutant or wild-type labeled cells can also be made to express one or more transgenes. Major applications of MARCM include (i) lineage analysis, (ii) investigating gene function in single or small populations of cells and (iii) neuronal circuit tracing. Our laboratory uses MARCM primarily to label and genetically manipulate neurons; however, this protocol can be adapted to any cell of interest. The protocol involves generating two fly stocks with the necessary genetic elements for MARCM analysis and subsequently generating MARCM clones. Labeled clones can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells. NOTE: In the PDF version of this article initially published online, the first “FRT” and the “Mutation” labels in Figure 1b were transposed. In both the PDF and HTML versions, “mutant” was omitted from the label on the right, which should read “Labeled homozygous mutant daughter cell”. The figure has been corrected in all versions of the article.
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Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development
Trends in Neurosciences, 2001Co-Authors: Tzumin Lee, Liqun LuoAbstract:We have modified an FLP/FRT-based genetic mosaic system to label either neurons derived from a common progenitor or isolated single neurons, in the Drosophila CNS. These uniquely labeled neurons can also be made homozygous for a mutation of interest within an otherwise phenotypically wild-type brain. Using this new mosaic system, not only can normal brain development be described with unprecedented single cell resolution, but also the underlying molecular mechanisms can be investigated by identifying genes that are required for these developmental processes.
Tsai-chi Hsu - One of the best experts on this subject based on the ideXlab platform.
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overview of MARCM related technologies in drosophila neurobiological research
Current protocols in protein science, 2020Co-Authors: Tsai-chi Hsu, Hung-chang ShenAbstract:Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.
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Overview of MARCM‐Related Technologies in Drosophila Neurobiological Research
Current protocols in protein science, 2020Co-Authors: Tsai-chi Hsu, Hung-chang ShenAbstract:Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.
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Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM
Journal of Visualized Experiments, 2017Co-Authors: Hung-chang Shen, Tsai-chi Hsu, Pei-chi ChungAbstract: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.
Tzumin Lee - One of the best experts on this subject based on the ideXlab platform.
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Reverse Genetics by Loss-of-Function Mosaic Analysis in Drosophila
CSH Protocols, 2013Co-Authors: Chih-fei Kao, Tzumin LeeAbstract:Abstract Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. One major application of MARCM, as described here, is to study cell-autonomous function(s) of a gene within single cells or a group of cells in otherwise unperturbed organisms. A mutation of interest distal to one FRT site is put in trans to a tubP-GAL80-containing chromosome arm that carries the same FRT. The resulting MARCM clones, which are negative for tubP-GAL80 and thus specifically marked, will become homozygous for the mutation in otherwise heterozygous organisms. By including a UAS-transgene, one can perform rescue experiments in the mutant MARCM clones. Conversely, if the mutation is placed on the same chromosome arm as tubP-GAL80, MARCM-labeled cells will be homozygous wild-type and may lie adjacent to sister cells that are homozygous mutant. This variant, called reverse MARCM, allows one to determine non-cell-autonomous effects of a mutation.
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Genetic Mosaic Screens in Drosophila Mushroom Bodies
CSH Protocols, 2013Co-Authors: Chih-fei Kao, Tzumin LeeAbstract:Genetic mosaics in Drosophila typically involve derivation of homozygous daughter cells from heterozygous precursors through mitotic recombination. MARCM (mosaic analysis with a repressible cell marker) couples loss of heterozygosity with derepression of a marker gene, permitting unique labeling of specific homozygous daughter cells. The generation of GAL80-minus homozygous daughter cells in otherwise heterozygous tissues allows GAL4-dependent activation of upstream activation sequence (UAS)-reporter specifically in the homozygous cells of interest. To make MARCM clones, organisms must carry at least five genetic elements (flippase [FLP], flippase recognition targets [FRTs], tubP-GAL80, GAL4, and UAS-marker) in specific configurations. In neurons whose progenitors can be efficiently targeted for mitotic recombination, genetic mosaic screens can be used to systematically uncover cell-autonomous genes that are required for development or function. This technique involves the generation of numerous FRT lines carrying various independent mutations, followed by derivation and phenotypic analysis of MARCM clones using these mutant FRT lines in combination with an MARCM-enabling stock that carries all the other genetic elements required for MARCM. Mutants of interest are recovered based on the MARCM phenotypes, which are imaged live using diverse fluorescent markers. Mutant genes that underlie the phenotypes of interest can then be identified by conventional genetics including derivation and analysis of series of recombinant chromosomes. Besides chemical mutagenesis, genes on a particular FRT chromosome may be randomly disrupted by P element insertion. This protocol describes procedures specifically used for genetic mosaic screens in the mushroom bodies (MBs).
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Twin-spot MARCM to reveal the developmental origin and identity of neurons
Nature Neuroscience, 2009Co-Authors: Chun-hong Chen, Lei Shi, Yaling Huang, Tzumin LeeAbstract:The mosaic analysis with repressible cell markers (MARCM) technique allows for lineage tracing in Drosophila. Here, the authors report an improvement on this technique, twin-spot MARCM, which permits high-resolution lineage tracing of both sister clones. A comprehensive understanding of the brain requires the analysis of individual neurons. We used twin-spot mosaic analysis with repressible cell markers (twin-spot MARCM) to trace cell lineages at high resolution by independently labeling paired sister clones. We determined patterns of neurogenesis and the influences of lineage on neuron-type specification. Notably, neural progenitors were able to yield intermediate precursors that create one, two or more neurons. Furthermore, neurons acquired stereotyped projections according to their temporal position in various brain sublineages. Twin-spot MARCM also permitted birth dating of mutant clones, enabling us to detect a single temporal fate that required chinmo in a sublineage of six Drosophila central complex neurons. In sum, twin-spot MARCM can reveal the developmental origins of neurons and the mechanisms that underlie cell fate.
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Genetic mosaic with dual binary transcriptional systems in Drosophila.
Nature Neuroscience, 2006Co-Authors: Sen-lin Lai, Tzumin LeeAbstract:MARCM (mosaic analysis with a repressible cell marker) involves specific labeling of GAL80-minus and GAL4-positive homozygous cells in otherwise heterozygous tissues. Here we demonstrate how the concurrent use of two independent binary transcriptional systems may facilitate complex MARCM studies in the Drosophila nervous system. By fusing LexA with the VP16 acidic activation domain (VP16) or the GAL4 activation domain (GAD), we obtained both GAL80-insensitive and GAL80-suppressible transcriptional factors. LexA::VP16 can mediate MARCM-independent binary transgene induction in mosaic organisms. The incorporation of LexA::GAD into MARCM, which we call dual-expression-control MARCM, permits the induction of distinct transgenes in different patterns among GAL80-minus cells in mosaic tissues. Lineage analysis with dual-expression-control MARCM suggested the presence of neuroglioblasts in the developing optic lobes but did not indicate the production of glia by postembryonic mushroom body neuronal precursors. In addition, dual-expression-control MARCM with a ubiquitous LexA::GAD driver revealed many unidentified cells in the GAL4-GH146-positive projection neuron lineages.
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Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development
Trends in Neurosciences, 2001Co-Authors: Tzumin Lee, Liqun LuoAbstract:We have modified an FLP/FRT-based genetic mosaic system to label either neurons derived from a common progenitor or isolated single neurons, in the Drosophila CNS. These uniquely labeled neurons can also be made homozygous for a mutation of interest within an otherwise phenotypically wild-type brain. Using this new mosaic system, not only can normal brain development be described with unprecedented single cell resolution, but also the underlying molecular mechanisms can be investigated by identifying genes that are required for these developmental processes.
Heng Zhang - One of the best experts on this subject based on the ideXlab platform.
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α-Ada and other AP-2 subunits are critical for dendrite pruning in ddaC neurons.
2018Co-Authors: Wenhui Zong, Yan Wang, Quan Tang, Heng ZhangAbstract:(A–F) Live confocal images of control (A), α-ada3 MARCM (B), α-ada3 MARCM rescue (C), BapΔ1 MARCM (D), BapΔ1 MARCM rescue (E), and AP-2μNN20 MARCM (F) ddaC neurons at WP and 16 h APF. ddaC somas are indicated by red arrowheads. (G) Quantification of percentage of severing defect and fragmentation defect in control and mutant ddaC neurons at 16 h APF. (H) Quantification of total length of unpruned dendrites at 16 h APF. The number of samples (n) in each group is shown on the bars. Error bars represent SEM. Scale bar (A) represents 50 μm. ***p < 0.001 as assessed by one-way ANOVA test. The individual numerical values for panels G and H can be found in S1 Data. The genotypes can be found in S1 Text. α-Ada, α-Adaptin; α-ada3, α-adaptin3; AP-2μNN20, adaptor protein-2 μ subunitNN20; APF, after puparium formation; BapΔ1, β-adaptinΔ1; FRT, flippase recognition target; GFP, green fluorescent protein; MARCM, mosaic analysis with a repressible cell marker; O/E, overexpression; WP, white prepupal.
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Imac plays a crucial role in dendrite pruning of ddaC neurons.
2018Co-Authors: Wenhui Zong, Yan Wang, Quan Tang, Heng ZhangAbstract:(A–F) Live confocal images of control (A), imac RNAi #1 (B), imac170 MARCM (C), imac170 MARCM rescue (D), non-induced ImacG102E (E), and induced ImacG102E (F) ddaC neurons at WP and 16 h APF. ddaC somas are marked by red arrowheads. (G) Quantification analysis of percentage of severing defect and fragmentation defect in control and imac mutant ddaC neurons at 16 h APF. (H) Quantification of total length of unpruned dendrites at 16 h APF. The number of samples (n) in each group is shown on the bars. Error bars represent SEM. Scale bar (A) represents 50 μm. ***p < 0.001 as assessed by one-way ANOVA test. The individual numerical values for panels G and H can be found in S1 Data. The genotypes can be found in S1 Text. APF, after puparium formation; imac, immaculate connections; MARCM, mosaic analysis with a repressible cell marker; O/E, overexpression; RNAi, RNA interference; WP, white prepupal.