Drosophila, Genetics, Behavior, and Optogenetics David Deitcher - - PowerPoint PPT Presentation

Drosophila, Genetics, Behavior, and Optogenetics

David Deitcher BioNB 4910

Se Seymou

• ur 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 Paralyzedts- 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

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

Drosophila melanogaster basic genetic structure

• 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

Adapted from Painter, 1934

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 1st 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

• 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 Mutants

• 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

Traditional Transgenic

Gene of Interest Enhancer Promoter

Polymerase

Transcription

TF

TF TF

• 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

Transgenics: GAL4/ UAS Binary System

GAL4 Enhancer Promoter

Polymerase

Transcription Reporter Promoter

Polymerase

Transcription

UAS UAS UAS UAS UAS

GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4 GAL4

R R R

TF

TF TF

TATA box ATG Start Codon

• 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

Green fluorescent protein (GFP)

Larval Drosophila CNS Larval Drosophila muscles

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 UAS- ChannelRhodopsin 1 copy of GAL4 and UAS

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 and Supplies How to

• pen an

account and

• rder stocks

Really useful detailed information

• n 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 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) …...

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

***Nuts and bolts: before poking anything, check for visible contractions in response to light Pulver et al., 2011, Adv. Physiol. Edu

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

• ther wavelengths

Inagaki et al. (2014) Nat. Methods

Augustin et al., 2011- JOVE

Neural circuit for flight in Drosophila

Orr et al., 2014- J. Neuroscience

Bang-sensitive Drosophila

Lee and Wu, 2002

Currently known bang-sensitive lines

Song and Tanouye, 2008

Tanouye and Wyman (1980) j. Neurophysiol. von reyn et al. (2014) Nat. Neuro. The Giant Fiber system in adult flies

GF specific GAL4 to drive expression of Chrimson

Peripherally synapsing interneuron (PSI) tergotrochanteral motorneuron (TTM)

Jahrling et al., 2010- Front. Neurosci

Layout of the flying muscles in Drosophila

Ephys preparation to record from adult fly muscles

A genetically tractable escape circuit for educators

Titlow, Johnson, Pulver (In press) JUNE

https://joshtitlow.wordpress.com/

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