Drosophila, Genetics, Behavior, and Optogenetics David Deitcher BioNB 4910
Se Seymou our Be Benzer – Fa Father of Drosophila Be Behavior
Mu Mutant Behaviors Identified by by Be Benzer an and Other ers Many of these mutants encode genes crucial for nervous system function Led to subsequent cloning of genes Hyperkinetic – K+ channel beta subunit Shaker – K+ channel Paralyzed ts - voltage gated sodium channel Period – transcription factor affecting circadian rhythm
Na Natural Behaviors Behavior observed in wild type animals Screens conducted to identify mutants in the behavior (either chemical mutagens, transposon based, RNAi, or overexpression) Genes localized, cloned, and expression pattern determined
Drosophila melanogaster basic life cycle
Gender Identification Sex combs only present on male flies. External genitalia present on males. Females have a more pointed abdomen, males are rounded.
Drosophila melanogaster basic genetic structure Adapted from Painter, 1934 4 pairs of chromosomes (1 sex chromosome, 3 autosomes) • No crossing over in males • Genome sequenced ( ~15,000 genes, ~50% of fly protein sequences have human • homologs
Terminology Gene - a unit of inheritance that usually is directly responsible for one trait or character. Allele - an alternate form of a gene. Usually there are two alleles for every gene, sometimes as many a three or four. Homozygous -when the two alleles are the same. Heterozygous -when the two alleles are different, in such cases the dominant allele is expressed. Dominant- a term applied to the trait (allele) that is expressed regardless of the second allele. The name will have the 1 st letter in uppercase. Recessive - a term applied to a trait that is only expressed when the second allele is the same. Will be written in lower case. Phenotype - the physical expression of the allelic composition for the trait under study. Genotype - the allelic composition of an organism.
Mutants versus Transgenics Mutants Transgenics Missing whole genes or have changes • Foreign DNA inserted into germ line • in endogenous gene function cells Can be dominant or recessive • Heritable changes in genome • Loss of function or gain of function • Allows mixing and matching of useful • Traditionally made by chemical • proteins from different organisms mutagenesis, P-element insertions, or Allows cell specific expression of • X-rays transgenes Gene names often refer to what • Can generate cell-specific knockdown • happens when that gene is mutant with RNAi (i.e. “white” refers to the eye color mutation that causes the flies to have white rather than the wild type red eye color). Mutations affect the whole animal •
Traditional Transgenic • Identify promoter/enhancer to express your gene of interest • Construct a transposon vector with promoter driving expression of your gene • Inject embryos to obtain transformant which expresses your gene • If you want to express the gene expressed in another pattern- YOU MUST REPEAT WHOLE PROCESS TF Transcription Polymerase TF TF Promoter Enhancer Gene of Interest
Transgenics: GAL4/ UAS Binary System GAL4 GAL4 TF GAL4 Transcription Polymerase TF TF Enhancer Promoter GAL4 TATA box ATG Start Codon R R R Transcription Polymerase GAL4 GAL4 GAL4 GAL4 GAL4 Promoter Reporter UAS UAS UAS UAS UAS Promoter/enhancer sequences drive expression of GAL4, a yeast transcription factor • GAL4, on its own, has no effect on flies • Your gene interest is inserted downstream of UAS sequences, the DNA target of GAL4 • It is silent without GAL4 present • To express, cross the flies and GAL4 turns on the gene in the tissue specified by the GAL4 expression • • To express gene in different cells, cross to different GAL4 line
GAL4-UAS and genetically encoded fluorophores Larval Drosophila muscles Larval Drosophila CNS Green fluorescent protein (GFP)
How Do You Make Transgenic Flies?
GA GAL4/UAS Tools to Affect Behavior UAS- Gene Function Effect on Neurons Shibire ts Vesicle recycling Eliminates NT release after temperature shift TrpA1 Temperature activated channel APs after temperature shift EKO (Electrical Knockout) Mutant Shaker channel Reduces APs Kir2.1 Inward Rectifier Greatly reduces APs Reaper/Hid Cell death gene Eliminate selected cell developmentally Channelrhodopsin Light activated ion channel Depolarize neurons during blue light stimulation Halorhodopsin Light activated ion pump Hyperpolarizes neurons during light Chrimson Light activated ion channel Depolarize neurons during red light stimulation Many RNAi lines Knockdown of gene function Can perturb numerous functions
Example of How to Use GAL4/UAS For example: Pan-neuronal GAL4 X UAS- ChannelRhodopsin Pan-neuronal GAL4 UAS- ChannelRhodopsin Progeny: Pan-neuronal GAL4 1 copy of GAL4 and UAS UAS- ChannelRhodopsin With thousands of different GAL4 and UAS lines, the transgenic expression choices are huge!!
Where Can I Find Information? FlyBase – http://flybase.org/ A Database of Drosophila Genes & Genomes
Where do I get reagents? Bloomington Drosophila Stock Center – fly stocks and useful information http://flystocks.bio.indiana.edu $17 – 3.50 stock charges depending on # of stocks ordered. Media Recipes How to and Supplies open an account and order stocks Really useful detailed information on Balancers
Optogenetics n. genetics A science that combines optics and genetics to probe neural circuits (Wiktionary)
Phototaxis in: Chlamydomonas cell with two flagella, a large chloroplast (green), and the yellow/orange eyespot.
Optical tools for depolarizing, hyperpolarizing neurons cHARGe Halorhodopsin ChR2 (chloride pump) H134R-ChR2 NpHr C128S-ChR2 eNpHr1-3 Cheta Chef Archerhodopsin Chief (proton pump) VChR1 (multicellular algae) ReaChR ArcherhodopsinT Chrimson (proton pump) ….. …...
If you want to use Channelrhodopsin on flies you need a transparent prep Transgenicflies (freely available) Inexpensive blue light source Pulver et al., 2011, Adv. Physiol. Edu. Hornstein et al., 2009, J. Vis. Exp.
Light-evoked synaptic potentials at the larval neuromuscular junction Pulver et al., 2011, Adv. Physiol. Edu ***Nuts and bolts: before poking anything, check for visible contractions in response to light
Glutamatergic motor neurons Pat Rivlin, Ron Hoy,
Perturbing Adult Behaviors • Most interesting behaviors in Drosophila are at the adult stage • The cuticle poses a problem in using optogenetics as it absorbs blue light well • Red-shifted channelrhodopsin could provide a solution in an intact animal • Activate neurons with red light and observe behavior • Perturb natural behaviors with red light • Possibly trigger mutant behaviors with red light (i.e. seizures)
De novo sequencing of 127 algae transcriptomes What other kinds of ChR2s are out there?
One ChR2 variant that is red-shifted Why might chrimson be useful? Why do we have this new tool?
Red-orange light penetrates tissues better than other wavelengths Inagaki et al. (2014) Nat. Methods
Neural circuit for flight in Drosophila Orr et al., 2014- J. Neuroscience Augustin et al., 2011- JOVE
Bang-sensitive Drosophila Lee and Wu, 2002
Currently known bang-sensitive lines Song and Tanouye, 2008
The Giant Fiber system in adult flies GF specific GAL4 to drive expression of Chrimson Peripherally synapsing interneuron (PSI) Tanouye and Wyman (1980) j. Neurophysiol. tergotrochanteral motorneuron (TTM) von reyn et al. (2014) Nat. Neuro.
Layout of the flying muscles in Drosophila Jahrling et al., 2010- Front. Neurosci
Ephys preparation to record from adult fly muscles
A genetically tractable escape circuit for educators https://joshtitlow.wordpress.com/ Titlow, Johnson, Pulver (In press) JUNE
Light evoked escape behavior in adult flies
Light-evoked muscle action potentials at the adult fly neuromuscular junction
Moonwalker
Practical goals 1. Set up red LED controllers on rigs 2. Observe quantify behavioral responses of adult flies with chrimson expressed in different populations of neurons (control (UAS-chrimson, Moonwalker>chrimson) 3. Dissect animals with chrimson in GF neurons and bss1/+ chrimson;Vglut-GAL4 (expresses chrimson in all motoneuronsin a partially bang sensitive mutant) 4. Record light evoked activity in adult flight muscles Dissection video available here: https://www.youtube.com/watch?v=lFd3IxXnpOo). Slides thanks to Bruce Johnson, Karen Hibbard, Stephan Pulver, Ron Hoy
Recommend
More recommend
