Refined Spatial Manipulation of Neuronal Function by Combinatorial Restriction of Transgene Expression

Selective genetic manipulation of neuronal function in vivo requires techniques for targeting gene expression to specific cells. Existing systems accomplish this using the promoters of endogenous genes to drive expression of transgenes directly in cells of interest or, in "binary" systems, to drive expression of a transcription factor or recombinase that subsequently activates the expression of other transgenes. All such techniques are constrained by the limited specificity of the available promoters. We introduce here a combinatorial system in which the DNA-binding (DBD) and transcription-activation (AD) domains of a transcription factor are independently targeted using two different promoters. The domains heterodimerize to become transcriptionally competent and thus drive transgene expression only at the intersection of the expression patterns of the two promoters. We use this system to dissect a neuronal network in Drosophila by selectively targeting expression of the cell death gene reaper to subsets of neurons within the network.

[1]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[2]  I. Kevrekidis,et al.  Optical imaging and control of genetically designated neurons in functioning circuits. , 2005, Annual review of neuroscience.

[3]  J. B. Duffy,et al.  GAL4 system in drosophila: A fly geneticist's swiss army knife , 2002, Genesis.

[4]  C. Wilson,et al.  P-element-mediated enhancer detection: a versatile method to study development in Drosophila. , 1989, Genes & development.

[5]  William Wisden,et al.  Dissecting neural circuitry by combining genetics and pharmacology , 2005, Trends in Neurosciences.

[6]  Y Wang,et al.  A regulatory system for use in gene transfer. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Ptashne,et al.  Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. , 1986, Science.

[8]  Y. Jan,et al.  Tracing neurons with a kinesin-β-galactosidase fusion protein , 1993, Roux's archives of developmental biology.

[9]  J. Truman,et al.  Increases in cyclic 3′,5′‐guanosine monophosphate (cGMP) occur at ecdysis in an evolutionarily conserved crustacean cardioactive peptide‐immunoreactive insect neuronal network , 1996, The Journal of comparative neurology.

[10]  David Landsman,et al.  B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners. , 2002, Genome research.

[11]  Benjamin H. White,et al.  A conditional tissue-specific transgene expression system using inducible GAL4 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  C. Helfrich-Förster,et al.  Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior , 2003, Development.

[13]  I. Karsch-Mizrachi,et al.  RNA interference demonstrates a role for nautilus in the myogenic conversion of Schneider cells by daughterless. , 2000, Developmental Biology.

[14]  A. Nordheim,et al.  Regulatory squelching , 1994, FEBS letters.

[15]  Chun-Yuan Ting,et al.  Drosophila N-cadherin functions in the first stage of the two-stage layer-selection process of R7 photoreceptor afferents , 2005, Development.

[16]  M. Ptashne,et al.  The carboxy-terminal 30 amino acids of GAL4 are recognized by GAL80 , 1987, Cell.

[17]  N. Perrimon,et al.  Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. , 1993, Development.

[18]  A. Plückthun,et al.  A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble. , 2000, Journal of molecular biology.

[19]  M. Gossen,et al.  Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. , 1994, Molecular and cellular biology.

[20]  R. Awatramani,et al.  Assembly of the Brainstem Cochlear Nuclear Complex Is Revealed by Intersectional and Subtractive Genetic Fate Maps , 2006, Neuron.

[21]  D. Wilkin,et al.  Neuron , 2001, Brain Research.

[22]  M. Suster,et al.  Refining GAL4‐driven transgene expression in Drosophila with a GAL80 enhancer‐trap , 2004, Genesis.

[23]  Liqun Luo,et al.  Mosaic Analysis with a Repressible Cell Marker for Studies of Gene Function in Neuronal Morphogenesis , 1999, Neuron.

[24]  H. Keshishian,et al.  Molecular genetic approaches to the targeted suppression of neuronal activity , 2001, Current Biology.

[25]  Jun Ma,et al.  Deletion analysis of GAL4 defines two transcriptional activating segments , 1987, Cell.

[26]  T. Kitamoto,et al.  Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP. , 2001, Brain research. Gene expression patterns.

[27]  Y. Jan,et al.  Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. , 1994, Genes & development.

[28]  D. Kvitsiani,et al.  Neural Circuitry that Governs Drosophila Male Courtship Behavior , 2005, Cell.

[29]  Ronald L. Davis,et al.  Spatiotemporal Rescue of Memory Dysfunction in Drosophila , 2003, Science.

[30]  R. Brent,et al.  A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor , 1985, Cell.

[31]  S. Sudo,et al.  Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Mallo.  Controlled gene activation and inactivation in the mouse. , 2006, Frontiers in bioscience : a journal and virtual library.

[33]  K. Yao,et al.  Neural Specificity of elav Expression: Defining a Drosophila Promoter for Directing Expression to the Nervous System , 1994, Journal of neurochemistry.

[34]  Philippe Soriano,et al.  Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation , 2003, Nature Genetics.

[35]  Stephen S. Gisselbrecht,et al.  New fluorescent protein reporters for use with the drosophila gal4 expression system and for vital detection of balancer chromosomes , 2002, Genesis.

[36]  J. Ewer,et al.  Genetic and hormonal regulation of the death of peptidergic neurons in the Drosophila central nervous system. , 1999, Journal of neurobiology.

[37]  M. Nitabach,et al.  Functional Dissection of a Neuronal Network Required for Cuticle Tanning and Wing Expansion in Drosophila , 2006, The Journal of Neuroscience.

[38]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[39]  I. Pastan,et al.  Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10−15 M , 2001, Protein science : a publication of the Protein Society.