Studying Neuronal Function using the Flies and Mice

Why flies?

We know a great deal about their simple nervous systems:

lineages, expression patterns, patterns of connectivitytranscriptional regulation.

We have more than 100 years of genetics--the Drosophila genome is the best annotated.

Little genetic redundancy.

Using the a misexpression system co-opted from yeast geneticists,we can “mis”express human genes in the fly at particular times/places.

Using fluorescent proteins in similar misexpression contexts, we can combine mutant backgrounds with fluorescent reporters.

This makes it possible for the fly to tell us what is wrong with it.

More Generally, flies…

• Short life cycle and simple culture conditions are easy to control.

• Behavioral observations, including olfactory, visual, tactile, and auditory cues and quantitative and qualitative modeling.

• Neurogenetics – Fly has long established genetic models for genetic manipulation.

• Neurophysiology - Drosophila has provided detailed neuronal architecture. However, their tiny neurons has presented more than a few formidable challenges to unravel function.

• Neuroinformatics – High throughput technologies has generated the most up-to-date and comprehensive databases describing the genetics of the organism and can be found at Flybase: http://flybase.bio.indiana.edu. Many other databases have supplemented this one.

Short Life cycle and simple and myriad culture conditions

• From 2 flies, can get thousands in 20 days.• Life-cycle is temperature-dependent; e.g., 21

days at 16°C to < 10 days at 25°C.• Entire protocol books have been published on

just this organism for culturing, e.g., Drosophila Protocols (Sullivan et al., 2000) or Fly Pushing (Greenspan, 2000).

Behavior• Assayable behavior starts ~24 hr after fertilization.• At this (larval) stage, as behavior transitions from the

initially instinctive behaviors to those that more complex ones refined by experience.

• Assays can be grouped according to the sensory modality they primarily use and these cover all primary senses: visual, olfactory, gustatory, tactile, gravitational, and auditory.

• Flies can also exhibit several types of learning and memory – 2 general types of learning: associative (habituation and sensitization; S -> R) and non-associative (results from only 1 environmental stimulus, usually extremely noxious or pleasant).

Neurogenetics

• Both classical and modern transgenic approaches.

• Stable transgenic lines can be generated that use random insertions close to genomic enhancers to drive subsequent transgenic constructs.

• P{GAL4} is the most commonly used of these lines.

Enhancer-trap element

TF GAL4

Local genomic enhancers

UAS

Construct of any gene of interest

When the enhancer is active, GAL4 is expressedand activates transcription of the construct downstreamof the UAS.Routinely use a UAS-lacZ or UAS-GFP reporter to characterizethe temporal and spatial activation pattern of a new P{GAL4}insert.Once characterized, a known P{GAL4} strain can then be crossedto fly strains containing any or multiple UAS constructs.

promoter/enhancer-gal 4

UAS-reporter

X

Wild type

Transgenic

UAS

In yeastGalatose is an alternative energy source--

--One that requires new enzyme synthesis.

The intracellular portion of the Galactose RIs translocated to the nucleus and acts as a TF.Gal4

UAS-reporters can include human genes for misexpressionAND fluorescent proteins, etc., for cellular detection.

Ectopic Gene expression using GAl4

UAS=UpstreamActivatingSequence

Fly Line Description Use Locale

Reporter Genes

UAS-nuclear LacZ E. Coli β-galactosidase with a nuclear localization signal

Nuclear reporter protein

Nucleus

UAS-eGFPnuclear Enhanced GFP with a nuclear localization signal

Fluorescent nuclear reporter protein

Nucleus

UAS-mCD8-GFP Fusion protein between mouse lymphocyte marker CD8 and GFP

Outstanding labeling of neuronal processes

Membrane

Fly Stocks Carrying GAL-4 Responsive Genes

Fly Line Description Use Locale

Cell Death Inducer

UAS-hid Head involution defective expression

Induces cell death

UAS-rpr Reaper expression Induces cell death

UAS-p35 P35 expression Rescues cell death induced by hid

Fly Stocks Carrying GAL-4 Responsive Genes(cont’d)

Fly Line Description Use Locale

Targeted Suppression of Neuronal ActivityUAS-TeTxLC Tetanus toxin light

chainBlocks synaptic vesicle release

Synapse

UAS-Shi1st Temp-sensitive mutant in Shibire (Dynamin)

Deplete synaptic vesicles; Conditional block of neurotransmission

Site of endocytosis; cell membrane

UAS-dORKΔ-C Constituitively open K+ -specific rectifier channel

Electrically silences neurons

Membrane

Fly Stocks Carrying GAL-4 Responsive Genes(cont’d)

UAS-dORKΔ-NC Inactivated K+-specific channel

Does not conduct K+; used as control

Membrane

Using Enhancer-trap reporters for uncovering neuronal substructures.

• Mosaic Analysis with a Repressible Cell Marker (MARCM) (Lee and Luo, 2001) – using the P{GAL} construct, examines morphological patterns over time.

--Induction of single- and two-cell clones at various time points during development allows one to determine the projection patterns of any given neuron or group of neurons of interest that are generated at different stages of CNS development.

--Has been invaluable for analyzing the antennal lobes and mushroom bodies in Drosophila.

Functional Analysis of Neurons in the CNS

1. Selectively disrupting the molecular and cellular components of neurons of interest and determine how these disruptions affect its function in a given behavior.

2. Directed expression of the gene of interest in subsets of the cells in specific mutant backgrounds. Can distinct features of the mutant phenotype be rescued by wild-type expression in particular subsets of cells?

Functional Analysis of Neurons in the CNS using the GAL4/UAS System:

• Target expression of neuronal activity [(1) on the previous slide]. Examples include:-- Synaptobrevin-dependent neurotransmitter release.-- GTPase.-- K+ Channels.

Phenocopying by RNAi• “Heritable”-RNAi utilizing the GAL4/UAS

system has been successfully used to study:-- Larval and prepupal development-- Adult behavioral rhythms.-- GABAB receptor role in Drosophila.

Selective Ablation of Drosophila Neurons

• The GAL4 line used in conjunction with the UAS-cell death genes reaper (rpr) and head involution defective (hid) to ablate your neurons of choice.

• p35 encodes a caspase inhibitor that can rescue rpr- or hid—mediated cell death.

But, what about time…?Targeted Expression Systems Requiring Co-factors

• One of the major problems with the GAL4/UAS system is that often early dominant effects of mis- or overexpressed transgenes can preclude behavioral analysis in adult animals.

• P{switch} permits temporal, as well as spatial control over a given UAS transgene.

GAL4~LBD of thehprogR~p65 TXNdomain

P-elementenhancerdetection

Permits spatial control

RU486 activates thisexpression system

Gene Switch

Neurophysiology

• Tiny neurons – a major drawback – has proven to be a major stumbling block in much progress aimed at characterizing neuronal function in flies.

• Fluorescent and luminescent dyes are technically challenging as the dyes are non-selective limits temporal and spatial resolution of, say, [Ca2+]s.

• Fluroescence Resonance Energy Transfer (FRET) has shown the most promise where one uses transgenic reporters based on modified GFP constructs that differ as a function of [Ca2+]s.

Neuroinformatics and High-throughput Technologies

• FlyBase http://www.flybase.net/• BDGP http://fruitfly.org/• EDGP http://edgp.ebi.ac.uk/

Mouse history

• Asian musculus and European domesticus mice dominate the world but have evolved separately over ~ 1 Million years

• Mixing in Abbie Lathrop’s schoolhouse created all our commonly used mice from these two distinct founder groups

Mouse History

• Modern “house mice” emerged from Asia into the fertile crescent as agriculture was born

Mouse history

Why mice?

• Mammals, much better biological model

• Easy to breed, feed, and house

• Can acclimatize to human touch

• Most important: we can experiment in many ways not possible in humans

What do theywant with me?

Mice are close to humans

Kerstin Lindblad-TohWhitehead/MIT Center for Genome Research

Mouse sequence reveals great similarity with the human genome

Extremely high conservation: 560,000 “anchors”

Mouse-Human Comparisonboth genomes 2.5-3 billion bp long> 99% of genes have homologs> 95% of genome “syntenic”

Genomes are rearranged copiesof each other

Roughly 50% of bases change in the evolutionary time from mouse to human

Mouse sequence reveals great similarity with the human genome

Extremely high conservation: 560,000 “anchors”

Anchors (hundreds of bases with >90% identity)represent areas of evolutionary selection…

…but only 30-40% of the highly conservedsegments correspond to exons of genes!!!

What we can do

• Directed matings• Inbred lines and crosses

• Knockouts• Transgenics• Mutagenesis• Nuclear transfer

• Control exposure to pathogens, drugs, diet, etc.

YIKES!!!

Methods Useful for Mouse Neurogenetics

1. Comparative Genomics

• Human: 23 chromosomes (22 autosomes + the sex chromosomes X & Y.

• Mouse: 20 chromosomes (19 autosomes + the sex chromosomes X & Y.

• Recall earlier slide.

2. Classical Mouse Mutants and Positional Cloning

• Spontaneous or radiation-induced mutations; but lethal or mild mutations historically tended to be overlooked.

• Positional Cloning – Extremely tedious procedure – high-resolution mapping, followed by sequencing of sets of overlapping genomic DNA clones is necessary unless there is some physiological hint that allows one to focus on a candidate gene.

3. Induced Random Mutations: The ENU Screening Project

• Ethylnitrosurea induces single bp exchanges.• Offspring carry multiple paternally inherited

point mutations.• Common Aim: saturate the genome with an

unbiased spectrum of mutants => Shotgun production of mutants.

4. Transgenes

• Genes of interest (in expression plasmids) are added to the genome of a recipient animal.

• Injected into the pronucleus of zygote.• Zygote are then transferred into the genital tract of

foster mothers.• Site of transgene insertion is more or less random.• To minimize the influence of the genetic environ on a

given transgene, insert it, including its normal chromosomal environ, in the form of a large genomic DNA fragment.

• YACs or BACs often used for this purpose.

• Transgenic Mouse: Generic term for an engineered mouse that has a normal DNA sequence for a gene replaced by an engineered sequence or a sequence from another organism.

Transgenic:• Introduction of foreign or altered gene: transgenic

Over-expression, mis-expression, dominant-negativeNormal allele also present - product from two alleles

(a) protein function(b) What DNA sequences are important for the regulation of the transcription of the gene(c) Gene rescue, gene therapy

Knockout:• Replace normal with mutant allele:

Gene knock-out - removal of a part of or a whole geneNo normal allele - product of manipulated allele only

Transgenic and Knockout Animals

Generating Transgenic Mice