Advanced Techniques for Cell Lineage Labelling in Drosophila

Abstract

The ability to mark and genetically manipulate clonally related cells in live organisms is invaluable for investigating the mechanisms of tissue development, homeostasis and repair. A wide variety of techniques have been developed in Drosophila melanogaster for this purpose. These cell lineage labelling techniques range from simple methods for randomly marking cells to complex schemes for differentially labelling and genetically altering specific cells or more than one clone at a time. For example, coupled MARCM makes it possible to simultaneously label both halves of a cell lineage with positive markers; FINGR uses a combinatorial approach, using Gal4 and Gal80, to provide finer spatial control over clone induction; Flybow and Drosophila Brainbow increase the resolution and efficiency of clonal analysis through multicolour labelling; and Gā€trace differentially marks cells that currently express a driver from cells that expressed the driver in the past. These labelling techniques each have their own advantages and disadvantages. But together they create a powerful arsenal of tools for the study of many diverse topics in tissue biology.

Key Concepts:

  • Cell lineage labelling is a technique, which allows populations of clonally related cells to be traced in vivo.

  • Modern cell lineage labelling techniques in Drosophila allow cells to be labelled and genetically modified without invasive procedures.

  • Cell lineage labelling has become the gold standard for the identification of stem cells in Drosophila.

  • Recently developed cell lineage labelling techniques offer more flexibility, precision and control.

Keywords: lineage analysis; clonal analysis; mosaic analysis; Drosophila; FLP/FRT; Gal4; UAS

Figure 1.

Negative labelling systems: FLP/FRT‐based mitotic recombination system for generating negatively labelled clones in a positively labelled background. Colours signify the genotype of cells, as indicated by the cartoon cell above each genotype. The chromosome arms that undergo mitotic recombination are illustrated for a parent cell and its daughters. In the daughter cells, grey indicates an unrecombined chromosome. Unrecombined cells (light green), homozygous GFP‐positive cells (dark green), homozygous negatively labelled cells (white). Note that, in practice, there is often no visible difference in fluorescence from one versus two copies of GFP (light green and dark green).

Figure 2.

Positive labelling systems: FLP/FRT‐based mitotic recombination systems for generating positively labelled clones in a negatively labelled background. The chromosome arms that undergo mitotic recombination are illustrated for a parent cell and its daughters. In the daughter cells, grey indicates an unrecombined chromosome. In the PMML method the Actin5c promoter is divided into N‐(Act(N)) and C‐(Act(C))terminal halves. Unrecombined cells and daughter cells that do not express a label (white), daughter cells expressing the corresponding label for each method (green).

Figure 3.

Dual labelling systems: FLP/FRT‐based mitotic recombination systems for generating clones in which the two halves of a lineage are labelled with different markers. The chromosome arms that undergo mitotic recombination are illustrated for a parent cell and its daughters. For dual‐marked mitotic clones, unrecombined cells with cytoplasmic GFP (GFPc) and nuclear LacZ (lacZn) are red with a green border, daughter cells with only GFPc are white with a green border, and daughter cells with only lacZn are red with a black border. For other methods, unlabelled, unrecombined cells are white, daughter cells expressing a GFP label are green, daughter cells expressing an RFP label are red, and daughter cells expressing both GFP and RFP labels are yellow. In Twin‐spot MARCM, mi stands for miRNA. In the Twin Spot Generator method, both types of segregation that lead to labelling and are depicted. (N) and (C) refer to the corresponding terminal half of GFP or RFP.

Figure 4.

Rainbow labelling systems: Mitotic recombination systems for generating clones with multicoloured markers. Components of the systems are depicted on the left. Upside‐down lettering indicates that a coding sequence or binding site is in the opposite orientation on the chromosome. Recombination between binding sites with the same orientation results in an excision, and recombination between binding sites with opposite orientations results in an inversion. All possible inversions or excisions are indicated on the cartoons, and the marker that would be expressed as a result of each is listed on the right. In Flybow, Cerulean is only expressed if inversion 2 is followed by excision 3. Flybow uses a variant of FLP/FRT, indicated by grey lettering.

Figure 5.

Targeted labelling systems: Mitotic recombination systems that use driver expression patterns to more specifically target labelling. (a, b) The genetic components of the system are shown on the right and labelling patterns, based on the genotype and expression domain of the driver(s), are shown on the left. Colours signify the label expressed by a cell: GFP (green), RFP (red) or both (yellow). No symbol indicates an unrecombined parent cell; the cloverleaf and diamond symbols identify the cells in each of the two halves of the lineage of the parent cell that underwent recombination. The cloverleaf and diamond patterns are shown above their corresponding genotypes in the diagram on the right. The grey ‘X’ or blue ‘Y’ areas indicate the driver expression domains. Abbreviations: LexA fused with the Gal4 activation domain (LxA:GAD), LexA‐binding sites (LxABS). (c, d) The overall clonal patterns in a tissue, rather than cellular details, are shown for FLP‐out Gal80 and FINGR systems. Clones are outlined, grey area denotes the Gal4 expression domain, and areas with GFP‐expression are green. (e) For the GTRACE method, the change in the labelling pattern is shown for two time points is shown for a tissue in which a population of Gal4‐expressing cells divide to produce some Gal4 nonexpressing cells. Although the lineage relationship between specific cells cannot be determined with this method, the population of green cells must have derived from the population of yellow cells, and all cells derived from the population of Gal4‐expressing cells must be either green or yellow (all Gal4‐expressing cells and their progeny are indicated with a+). Colours are the same as in (a) and (b). (f) Flybow 2.0 is a variant on Flybow that only undergoes rainbow labelling in the region defined by the intersection of a Gal4 and FLP expression pattern. Components are similar to Flybow 1.1 except for the addition of a flip‐out cassette (red arrows). Colour coding is the same as for Flybow1.1. Black and grey lettering distinguish canonical from variant FLP/FRTs, respectively.

close

References

Ando R, Mizuno H and Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306: 1370–1373.

Arnone M and Dmochowski I (2004) Using reporter genes to study cis‐regulatory elements. Methods in Cell Biology 74: 621–652.

Bateman J and Lee A (2006) Site‐specific transformation of Drosophila via ΠC31 integrase‐mediated cassette exchange. Genetics 173: 769–777.

Beumer K and Pimpinelli S (1998) Induced chromosomal exchange directs the segregation of recombinant chromatids in mitosis of Drosophila. Genetics 150: 173–188.

Bohm RA, Welch WP, Goodnight LK et al. (2010) A genetic mosaic approach for neural circuit mapping in Drosophila. Proceedings of the National Academy of Sciences of the USA 107: 16378–16383.

Bossing T and Technau G (1994) The fate of the CNS midline progenitors in Drosophila as revealed by a new method for single cell labelling. Development 120: 1895–1906.

Brand AH and Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.

Branda C and Dymecki S (2004) Talking about a revolution: the impact of site‐specific recombinases on genetic analyses in mice. Developmental Cell 6: 7–28.

Brent R and Ptashne M (1985) A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43: 729–736.

Bunch TA, Grinblat Y and Goldstein LS (1988) Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Research 16: 1043–1061.

Call G, Olson J, Chen J et al. (2007) Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes. Genetics 177: 689–697.

Evans CJ, Olson JM, Ngo KT et al. (2009) G‐TRACE: rapid Gal4‐based cell lineage analysis in Drosophila. Nature Methods 6: 603–605.

Fischer J, Giniger E, Maniatis T et al. (1988) GAL4 activates transcription in Drosophila. Nature 332: 853–856.

Fox D and Spradling A (2009) The Drosophila hindgut lacks constitutively active adult stem cells but proliferates in response to tissue damage. Cell Stem Cell 5: 290–297.

Gohl DM, Silies MA, Gao XJ et al. (2011) A versatile in vivo system for directed dissection of gene expression patterns. Nature Methods 8: 231–237.

Golic KG and Lindquist S (1989) The FLP recombinase of yeast catalyses site‐specific recombination in the Drosophila genome. Cell 59: 499–509.

Gordon MD and Scott K (2009) Motor control in a Drosophila taste circuit. Neuron 61: 373–384.

Griffin R, Sustar A, Bonvin M et al. (2009) The twin spot generator for differential Drosophila lineage analysis. Nature Methods 6: 600–602.

Groth A, Fish M and Nusse R (2004) Construction of transgenic Drosophila by using the site‐specific integrase from phage φC31. Genetics 166: 1775–1782.

Groth A and Olivares E (2000) A phage integrase directs efficient site‐specific integration in human cells. Proceedings of the National Academy of Sciences of the USA 97: 5995–6000.

Grueber WB, Ye B, Yang C et al. (2007) Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology. Development 134: 55–64.

Hadjieconomou D, Rotkopf S, Alexandre C et al. (2011) Flybow: genetic multicolor cell labelling for neural circuit analysis in Drosophila melanogaster. Nature Methods 8: 260–266.

Hampel S, Chung P, McKellar CE et al. (2011) Drosophila Brainbow: a recombinase‐based fluorescence labelling technique to subdivide neural expression patterns. Nature Methods 8: 253–259.

Hannah A (1953) Nonautonomy of yellow in gynandromorphs of Drosophila melanogaster. Journal of Experimental Zoology 123: 523–559.

Harrison DA and Perrimon N (1993) Simple and efficient generation of marked clones in Drosophila. Current Biology 3: 424–433.

Heidmann D and Lehner C (2001) Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen‐dependent activity regulation. Development Genes and Evolution 211: 458–465.

Illmensee K (1972) Developmental potencies of nuclei from cleavage, preblastoderm, and syncytial blastoderm transplanted into unfertilized eggs of Drosophila melanogaster. Development Genes and Evolution 170: 267–298.

Janning W (1978) Gynandromorph fate maps in Drosophila. Results and Problems in Cell Differentiation 9: 1–28.

Kirilly D, Spana EP, Perrimon N et al. (2005) BMP signaling is required for controlling somatic stem cell self‐renewal in the Drosophila ovary. Developmental Cell 9: 651–662.

Lai SL and Lee T (2006) Genetic mosaic with dual binary transcriptional systems in Drosophila. Nature Neuroscience 9: 703–709.

Lee T and Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22: 451–461.

Livet J, Weissman TA, Kang H et al. (2007) Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450: 56–62.

Luan H, Peabody NC, Vinson CR et al. (2006) Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron 52: 425–436.

Lukyanov KA, Chudakov DM, Lukyanov S et al. (2005) Innovation: photoactivatable fluorescent proteins. Nature Reviews Molecular Cell Biology 6: 885–891.

Ma J and Ptashne M (1987) The carboxy‐terminal 30 amino acids of GAL4 are recognized by GAL80. Cell 50: 137–142.

Mavrakis M, Rikhy R and Lippincott‐Schwartz J (2009) Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo. Developmental Cell 16: 93–104.

McGarry T and Lindquist S (1985) The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader. Cell 42: 903–911.

McGuire SE, Le PT, Osborn AJ et al. (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302: 1765–1768.

Nakano M, Odaka K, Ishimura M, Kondo S et al. (2001) Efficient gene activation in cultured mammalian cells mediated by FLP recombinase‐expressing recombinant adenovirus. Nucleic Acids Research 29: E40.

Nystul TG and Spradling AC (2007) An epithelial niche in the Drosophila ovary undergoes long‐range stem cell replacement. Cell Stem Cell 1: 277–285.

Pignoni F and Zipursky SL (1997) Induction of Drosophila eye development by decapentaplegic. Development 124: 271–278.

Potter CJ, Tasic B, Russler EV et al. (2010) The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis. Cell 141: 536–548.

Roman G, Endo K, Zong L et al. (2001) P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster. Proceedings of the National Academy of Sciences of the USA 98: 12602–12607.

Rørth P (1998) Gal4 in the Drosophila female germline. Mechanisms of Development 78: 113–118.

Shaner NC, Steinbach PA and Tsien RY (2005) A guide to choosing fluorescent proteins. Nature Methods 2: 905–909.

Siegal M and Hartl D (1996) Transgene coplacement and high efficiency site‐specific recombination with the Cre/loxP system in Drosophila. Genetics 144: 715–726.

Skora AD and Spradling AC (2010) Epigenetic stability increases extensively during Drosophila follicle stem cell differentiation. Proceedings of the National Academy of Sciences of the USA 107: 7389–7394.

Stern C (1936) Somatic crossing over and segregation in Drosophila melanogaster. Genetics 21: 625–730.

Sulston JE, Schierenberg E, White JG et al. (1983) The embryonic cell lineage of the nematode Caenorhabditis elegans. Developmental Biology 100: 64–119.

Szüts D and Bienz M (2000) LexA chimeras reveal the function of Drosophila Fos as a context‐dependent transcriptional activator. Proceedings of the National Academy of Sciences of the USA 97: 5351–5356.

Technau GM and Campos‐Ortega JA (1987) Cell autonomy of expression of neurogenic genes of Drosophila melanogaster. Proceedings of the National Academy of Sciences of the USA 84: 4500–4504.

Venken K, Schulze K, Haelterman N and Pan H (2011) MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes. Nature Methods 8: 737–743.

Vincent JP and O'Farrell PH (1992) The state of engrailed expression is not clonally transmitted during early Drosophila development. Cell 68: 923–931.

Xu T and Rubin GM (1993) Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117: 1223–1237.

Yu HH, Chen C, Shi L et al. (2009) Twin‐spot MARCM to reveal the developmental origin and identity of neurons. Nature Neuroscience 12: 947–953.

Further Reading

Duffy J (2002) GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis 34: 1–15.

Fox D, Morris L, Nystul T et al. (2008) Lineage Analysis of Stem Cells. Stem Book [Internet]. Cambridge, MA: Harvard Stem Cell Institute, 2008–2009 Jan 31.

Lee T (2009) New genetic tools for cell lineage analysis in Drosophila. Nature Methods 6: 566–568.

Lippincott‐Schwartz J and Patterson G (2003) Development and use of fluorescent protein markers in living cells. Science 300: 87–91.

Luo L (2007) Fly MARCM and mouse MADM: genetic methods of labeling and manipulating single neurons. Rain Research Reviews 55(2): 220–227.

Pfeiffer B, Ngo T, Hibbard K et al. (2010) Refinement of tools for targeted gene expression in Drosophila. Genetics 186: 735–755.

Piston D, Patterson G, Lippincott‐Schwartz J, Claxton N and Davidson M Introduction to Fluorescent Proteins. MicroscopyU [Internet]. www.microscopyu.com/articles/livecellimaging/fpintro.html.

Theodosiou N and Xu T (1998) Use of FLP/FRT system to study Drosophila development. METHODS: A Companion to Methods in Enzymology 14: 355–365.

Zugates C and Lee T (2004) Genetic mosaic analysis in the nervous system. Current Opinion in Neurobiology 14: 647–653.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Hafezi, Yassi, and Nystul, Todd G(Mar 2012) Advanced Techniques for Cell Lineage Labelling in Drosophila. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022539]