Abstract
To fully understand how animals develop, it is often necessary to remove the function of a particular gene in a specific cell type or subset of cells. In Drosophila melanogaster, mosaic animals have been widely utilized to study cell fate, growth and patterning, and restriction of cell fate. This chapter describes using FLP recombinase to generate mosaic Drosophila, discussing the chromosomes and cross scheme, how to induce the clones, how to properly identify the appropriate progeny, and how to prepare and analyze the tissues, clones, and phenotypes. It then presents three examples, applying this technique to study Hedgehog signaling. The first example describes moderate-sized costal clones in imaginal discs, using green fluorescent protein (GFP) as a marker and dppLacZ and Engrailed expression as phenotypic reporters. The second describes filling the adult eye with roadkill mutant clones, using white as a marker and scoring morphology. The third describes clonal misexpression of a truncated form of Smoothened, using GFP and yellow as markers.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Postlethwait, J. H. (1976) Clonal analysis of Drosophila cuticle patterns. In The Genetics and Biology of Drosophila (Ashburner, M. and Wright, T. R. F., eds), Vol. 2c, Academic Press, New York, pp. 359–441.
Ashburner, M. (1989) Drosophila: A Laboratory Handbook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Golic, K. G. and Lindquist, S. (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59, 499–509.
Xu, T. and Rubin, G. M. (1993) Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237.
Brand, A. H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.
Garcia-Bellido, A. and Dapena, J. (1974) Induction, detection and characterization of cell differentiation mutants in Drosophila. Mol. Gen. Genet. 128, 117–130.
Stowers, R. S. and Schwarz, T. L. (1999) A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype. Genetics 152, 1631–1639.
Moreno, E., Basler, K., and Morata, G. (2002) Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416, 755–759.
Motzny, C. K. and Holmgren, R. (1995) The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways. Mech. Dev. 52, 137–150.
Capdevila, J. and Guerrero, I. (1994) Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings. EMBO J. 13, 4459–4468.
Crozatier, M. and Vincent, A. (1999) Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to notch signaling. Development 126, 1495–1504.
Diez del Corral, R., Aroca, P., Gmez-Skarmeta, J.L., Cavodeassi, F., and Modolell, J. (1999) The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila. Genes Dev. 13, 1754–1761.
Bentrop, J., Schwab, K., Pak, W. L., and Paulsen, R. (1997) Site-directed mutagenesis of highly conserved amino acids in the first cytoplasmic loop of Drosophila Rh1 opsin blocks rhodopsin synthesis in the nascent state. EMBO J. 16, 1600–1609.
Ito, K., Awano, W., Suzuki, K., Hiromi, Y., and Yamamoto, D. (1997) The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells. Development 124, 761–771.
Lee, T. and Luo, L. (2001) Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci. 24, 251–254.
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.
Methot, N. and Basler, K. (2000) Suppressor of fused opposes hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus. Development 127, 4001–4010.
Hooper, J. E. (2003) Smoothened translates Hedgehog levels into distinct responses. Development 130, 3951–3963.
Struhl, G. and Basler, K. (1993) Organizing activity of wingless protein in Drosophila. Cell 72, 527–540.
Chou, T. B. and Perrimon, N. (1992) Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131, 643–653.
Methot, N. and Basler, K. (1999) Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96, 819–831.
Chen, Y. and Struhl, G. (1996) In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex. Cell 87, 553–563.
Sisson, J. C., Ho, K. S., Suyama, K., and Scott, M. P. (1997) Costal2, a novel kinesin-related protein in the Hedgehog signaling pathway. Cell 90, 235–245.
Li, W., Ohlmeyer, J. T., Lane, M. E., and Kalderon, D. (1995) Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80, 553–562.
Lefers, M. A., Wang, Q. T., and Holmgren, R. A. (2001) Genetic dissection of the Drosophila Cubitus interruptus signaling complex. Dev. Biol. 236, 411–420.
Jiang, J. and Struhl, G. (1995) Protein kinase A and Hedgehog signaling in Drosophila limb development. Cell 80, 563–572.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Bankers, C.M., Hooper, J.E. (2007). Clonal Analysis of Hedgehog Signaling in Drosophila Somatic Tissues. In: Horabin, J.I. (eds) Hedgehog Signaling Protocols. Methods Inmolecular Biology™, vol 397. Humana Press. https://doi.org/10.1007/978-1-59745-516-9_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-516-9_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-692-4
Online ISBN: 978-1-59745-516-9
eBook Packages: Springer Protocols