MARCM

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Mosaic analysis with a repressible cell marker, or MARCM, is a genetics technique for creating individually labeled homozygous cells in an otherwise heterozygous Drosophila melanogaster . [1] It has been a crucial tool in studying the development of the Drosophila nervous system. This technique relies on recombination during mitosis mediated by FLP-FRT recombination. As one copy of a gene, provided by the balancer chromosome, is often enough to rescue a mutant phenotype, MARCM clones can be used to study a mutant phenotype in an otherwise wildtype animal.

Contents

Crosses

A visual depiction of MARCM Mosaic analysis with a repressible cell marker(MARCM).gif
A visual depiction of MARCM

In order to label small populations of cells from a common progenitor, MARCM uses the GAL4-UAS system. A marker gene such as GFP is placed under control of a UAS promoter. GAL4 is ubiquitously expressed in these flies, thus driving marker expression. In addition, GAL80 is driven by a strong promoter such as tubP. Gal80 is an inhibitor of GAL4, and will suppress GFP expression under normal conditions. This tubP-GAL80 element is placed distal to an FRT site. A second FRT site is placed in trans to the GAL80 site, usually with a gene or mutation of interest distal to it. Finally, FLP recombinase is driven by an inducible promoter such as heat shock.

When FLP transcription is induced, it will recombine the chromosomes at the two FRT sites in cells undergoing mitosis. These cells will divide into two homozygous daughter cells—one carrying both GAL80 elements, and one carrying none. The daughter cell lacking GAL80 will be labeled due to expression of the marker via the GAL4-UAS system. All subsequent daughter cells from this progenitor will also express the marker.

Labs will often have MARCM-ready lines which have the inducible FLP, GAL80 distal to a FRT site, GAL4, and UAS-Marker. These can be readily crossed with flies that have a mutation of interest distal to a FRT site. [2]

A Drosophila crossing scheme to produce progeny for MARCM studies Typical MARCM cross.jpg
A Drosophila crossing scheme to produce progeny for MARCM studies

Uses

By taking advantage of MARCM, one can easily trace all the cells that have been generated from a single progenitor. This is a useful tool in tracking development and specific cell lineages in various environmental conditions. Applications for MARCM include studying neuronal circuits, [3] clonal analysis, [4] genetic screens, [5] spermatogenesis, [6] growth cone development, [7] neurogenesis, [8] and tumor metastasis. [9]

Many advances in the understanding of Drosophila development have been achieved through MARCM. The development, lineages, and characterizations of secondary axon tracts, [10] anatomical maps of cholinergic neurons in the visual systems, [11] lineages giving rise to a thoracic hemineuromere scaffold and the developmental framework for CNS architecture, [12] the role of Delta in developmental programming in the ventral nerve cord, [13] the wake-promoting octopaminergic cells in the medial protocerebrum, [14] genes involved in neuronal morphogenesis of the mushroom bodies, [15] and the regulation of commissural axon guidance [16] have all been identified through MARCM techniques.

Variations

There are many variations of MARCM. Twin-spot MARCM allows for the labeling of sister clones with two separate markers, thus allowing for a higher resolution of lineage tracing. [17] In reverse MARCM, the mutation of interest is placed on the same chromosome as GAL80, so that the wild-type homozygous clones will be labeled. [18] Flip-Out MARCM highlights individual cells inside of mutant clones (ref "Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons," Neuron, 2002). The Q system allows for GAL4 independent MARCM by using the QF/QS system. [19] Lethal MARCM allows for the generation of large marked homozygous populations by including a lethal mutation near the GAL80 site. [20] Dual-expression control MARCM uses the LexA-VP16 transcriptional system in concordance with GAL4-UAS. [21] MARCM is also often used as a genetic screen. [15]

See also

References

  1. ↑ Wu, JS; Luo L (2007-01-11). "A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila". Nature Protocols. 1 (6): 2583–9. doi:10.1038/nprot.2006.320. PMID   17406512. S2CID   205463607.
  2. ↑ Marin, E.C.; Jefferis GS; Komiyama T; Zhu H; Luo L (2002-04-19). "Representation of the glomerular olfactory map in the Drosophila brain". Cell. 109 (2): 243–55. doi: 10.1016/S0092-8674(02)00700-6 . PMID   12007410.
  3. ↑ Lai, SL; Awasaki T; Ito K; Lee T. (2008-09-01). "Clonal analysis of Drosophila antennal lobe neurons: diverse neuronal architectures in the lateral neuroblast lineage". Development. 135 (17): 2883–2893. doi: 10.1242/dev.024380 . PMID   18653555.
  4. ↑ Baer, MM; Bilstein A; Leptin M. (2007-07-01). "A clonal genetic screen for mutants causing defects in larval tracheal morphogenesis in Drosophila". Genetics. 176 (4): 2279–91. doi:10.1534/genetics.107.074088. PMC   1950631 . PMID   17603107.
  5. ↑ Kiger, AA; White-Cooper H; Fuller MT. (2000-10-12). "Somatic support cells restrict germline stem cell self-renewal and promote differentiation". Nature. 407 (6805): 750–4. Bibcode:2000Natur.407..750K. doi:10.1038/35037606. PMID   11048722. S2CID   4349276.
  6. ↑ Ng, J; Nardine T; Harms M; Tzu J; Goldstein A; Sun Y; Dietzl G; Dickson BJ; Luo L. (2002-03-28). "Rac GTPases control axon growth, guidance and branching". Nature. 416 (6879): 422–47. Bibcode:2002Natur.416..442N. doi:10.1038/416442a. PMID   11919635. S2CID   4418881.
  7. ↑ Ben Rokia-Mille, S.; Tinette S; Engler G; Arthaud L; Tares S; Robichon A. (2008-06-11). "Continued neurogenesis in adult Drosophila as a mechanism for recruiting environmental cue-dependent variants". PLOS ONE. 3 (6): e2395. Bibcode:2008PLoSO...3.2395B. doi: 10.1371/journal.pone.0002395 . PMC   2405948 . PMID   18545694. Open Access logo PLoS transparent.svg
  8. ↑ Pagliarini, RA; Xu, T. (2003-10-09). "A genetic screen in Drosophila for metastatic behavior". Science. 302 (5648): 1227–31. Bibcode:2003Sci...302.1227P. doi: 10.1126/science.1088474 . PMID   14551319. S2CID   41410051.
  9. ↑ Lovick, JK; Ngo KT; Omoto JJ; Wong DC; Nguyen JD; Hartenstein V. (2013-12-15). "Postembryonic lineages of the Drosophila brain: I. Development of the lineage-associated fiber tracts". Developmental Biology. 384 (2): 228–57. doi:10.1016/j.ydbio.2013.07.008. PMC   3886848 . PMID   23880429.
  10. ↑ Varija Raghu, S; Reiff DF; Borst A. (2011-01-01). "Neurons with cholinergic phenotype in the visual system of Drosophila". The Journal of Comparative Neurology. 519 (1): 162–76. doi:10.1002/cne.22512. PMID   21120933. S2CID   8518278.
  11. ↑ Truman, JW; Schuppe H; Shepherd D; Williams DW. (2004-10-15). "Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila". Development. 131 (20): 5167–84. doi: 10.1242/dev.01371 . PMID   15459108.
  12. ↑ Cornbrooks, C; Bland C; Williams DW; Truman JW; Rand MD. (2006-12-22). "Delta expression in post-mitotic neurons identifies distinct subsets of adult-specific lineages in Drosophila". Developmental Neurobiology. 67 (1): 23–38. doi: 10.1002/dneu.20308 . PMID   17443769. S2CID   2444513.
  13. ↑ Crocker, A; Shahidullah M; Levitan IB; Sehgal A. (2010-03-11). "Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior". Neuron. 65 (5): 670–81. doi:10.1016/j.neuron.2010.01.032. PMC   2862355 . PMID   20223202.
  14. 1 2 Reuter, JE; Nardine TM; Penton A; Billuart P; Scott EK; Usui T; Uemura T; Luo L. (2003-03-15). "A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis". Development. 130 (6): 1203–13. doi: 10.1242/dev.00319 . PMID   12571111.
  15. ↑ McGovern, VL; Seeger, MA. (2003-10-20). "Mosaic analysis reveals a cell-autonomous, neuronal requirement for Commissureless in the Drosophila CNS". Development Genes and Evolution. 213 (10): 500–4. doi:10.1007/s00427-003-0349-1. PMID   12928898. S2CID   6282587.
  16. ↑ Yu, HH; Chen CH; Shi L; Huang Y; Lee T. (2009-06-14). "Twin-spot MARCM to reveal the developmental origin and identity of neurons". Nature Neuroscience. 12 (7): 947–53. doi:10.1038/nn.2345. PMC   2701974 . PMID   19525942.
  17. ↑ Lee, T; Winter C; Marticke SS; Lee A; Luo L (2000). "Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis". Neuron. 25 (2): 307–16. doi: 10.1016/S0896-6273(00)80896-X . PMID   10719887.
  18. ↑ Potter, CJ; Tasic B; Russler EV; Liang L; Luo L. (2010-04-30). "The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis". Cell. 141 (3): 536–48. doi:10.1016/j.cell.2010.02.025. PMC   2883883 . PMID   20434990.
  19. ↑ Zhu, H; Luo L. (2004-04-08). "Diverse functions of N-cadherin in dendritic and axonal terminal arborization of olfactory projection neurons". Neuron. 42 (1): 63–75. doi: 10.1016/S0896-6273(04)00142-4 . PMID   15066265.
  20. ↑ Lai, SL; Lee T. (2006-05-09). "Genetic mosaic with dual binary transcriptional systems in Drosophila". Nature Neuroscience. 9 (5): 703–9. doi:10.1038/nn1681. PMID   16582903. S2CID   10780729.
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