Drosophila, Genetics, Behavior, and Optogenetics. David Deitcher BioNB 4910

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1 Drosophila, Genetics, Behavior, and Optogenetics David Deitcher BioNB 4910

2 Seymour Benzer Father of Drosophila Behavior

3 Mutant Behaviors Identified by Benzer and Others 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

4 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

5 Drosophila melanogaster basic life cycle

6 Gender Identification Sex combs only present on male flies. External genitalia present on males. Females have a more pointed abdomen, males are rounded.

7 Drosophila melanogaster basic genetic structure Adapted from Painter, 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

8 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.

9 Mutants versus Transgenics Mutants Missing whole genes or have changes in endogenous gene function Can be dominant or recessive Loss of function or gain of function Traditionally made by chemical mutagenesis, P-element insertions, or X-rays Gene names often refer to what happens when that gene is mutant (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 Transgenics Foreign DNA inserted into germ line cells Heritable changes in genome Allows mixing and matching of useful proteins from different organisms Allows cell specific expression of transgenes Can generate cell-specific knockdown with RNAi

10 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 TF TF Enhancer Polymerase Promoter Transcription Gene of Interest

11 Transgenics: GAL4/ UAS Binary System GAL4 GAL4 GAL4 UAS TF TF TF Enhancer GAL4 GAL4 GAL4 UAS UAS UAS GAL4 UAS Polymerase Promoter TATA box Polymerase Promoter ATG Start Codon Transcription GAL4 Transcription Reporter R R R GAL4 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

12 GAL4-UAS and genetically encoded fluorophores Larval Drosophila muscles Larval Drosophila CNS Green fluorescent protein (GFP)

13 How Do You Make Transgenic Flies?

14 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 InwardRectifier 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

15 Example of How to Use GAL4/UAS For example: Pan-neuronal GAL4 Pan-neuronal GAL4 X UAS- ChannelRhodopsin UAS- ChannelRhodopsin Progeny: Pan-neuronal GAL4 UAS- ChannelRhodopsin 1 copy of GAL4 and UAS With thousands of different GAL4 and UAS lines, the transgenic expression choices are huge!!

16 Where Can I Find Information? FlyBase A Database of Drosophila Genes & Genomes

17 Where do I get reagents? Bloomington Drosophila Stock Center fly stocks and useful information $ stock charges depending on # of stocks ordered. How to open an account and order stocks Media Recipes and Supplies Really useful detailed information on Balancers

18 Optogenetics n. genetics A science that combines optics and genetics to probe neural circuits (Wiktionary)

19 Phototaxis in: Chlamydomonas cell with two flagella, a large chloroplast (green), and the yellow/orange eyespot.

20 Optical tools for depolarizing, hyperpolarizing neurons charge ChR2 H134R-ChR2 C128S-ChR2 Cheta Chef Chief VChR1 (multicellular algae) ReaChR Chrimson.. Halorhodopsin (chloride pump) NpHr enphr1-3 Archerhodopsin (proton pump) ArcherhodopsinT (proton pump)...

21 If you want to use Channelrhodopsin on flies you need a transparent prep Transgenic flies (freely available) Inexpensive blue light source Pulver et al., 2011, Adv. Physiol. Edu. Hornstein et al., 2009, J. Vis. Exp.

22 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

23 Glutamatergic motor neurons Pat Rivlin, Ron Hoy,

24 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)

25 De novo sequencing of 127 algae transcriptomes What other kinds of ChR2s are out there?

26 One ChR2 variant that is red-shifted Why might chrimson be useful? Why do we have this new tool?

27 Red-orange light penetrates tissues better than other wavelengths Inagaki et al. (2014) Nat. Methods

28 Neural circuit for flight in Drosophila Orr et al., J. Neuroscience Augustin et al., JOVE

29 Bang-sensitive Drosophila Lee and Wu, 2002

30 Currently known bang-sensitive lines Song and Tanouye, 2008

31 The Giant Fiber system in adult flies GF specific GAL4 to drive expression of Chrimson Peripherally synapsing interneuron (PSI) tergotrochanteral motorneuron (TTM) Tanouye and Wyman (1980) j. Neurophysiol. von reyn et al. (2014) Nat. Neuro.

32 Layout of the flying muscles in Drosophila Jahrling et al., Front. Neurosci

33 Ephys preparation to record from adult fly muscles

34 A genetically tractable escape circuit for educators Titlow, Johnson, Pulver (In press) JUNE

35 Light evoked escape behavior in adult flies

36 Light-evoked muscle action potentials at the adult fly neuromuscular junction

37 Moonwalker

38 1. Set up red LED controllers on rigs Practical goals 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: Slides thanks to Bruce Johnson, Karen Hibbard, Stephan Pulver, Ron Hoy