Transcript Studying Neuronal Function using the Flies and Mice

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 connectivity
transcriptional 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 nonassociative (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.
Construct of any gene
of interest
Enhancer-trap element
UAS
TF GAL4
Local genomic
enhancers
When the enhancer is active, GAL4 is expressed
and activates transcription of the construct downstream
of the UAS.
Routinely use a UAS-lacZ or UAS-GFP reporter to characterize
the temporal and spatial activation pattern of a new P{GAL4}
insert.
Once characterized, a known P{GAL4} strain can then be crossed
to fly strains containing any or multiple UAS constructs.
Ectopic Gene expression using GAl4
In yeast
Galatose is an alternative energy source-One that requires new enzyme synthesis.
The intracellular portion of the Galactose R
Is translocated to the nucleus and acts as a TF.
Gal4
UAS=
Upstream
Activating
Sequence
UAS
promoter/enhancer-gal 4
Wild type
Transgenic
X
UAS-reporter
UAS-reporters can include human genes for misexpress
Fly Stocks Carrying GAL-4 Responsive Genes
Fly Line
Description
Use
Locale
Reporter Genes
UAS-nuclear LacZ
E. Coli βgalactosidase with
a nuclear
localization signal
Nuclear reporter
protein
Nucleus
UAS-eGFPnuclear
Enhanced GFP with
a nuclear
localization signal
Fluorescent nuclear Nucleus
reporter protein
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
(cont’d)
Fly Line
Description
Use
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
Locale
Fly Stocks Carrying GAL-4 Responsive Genes
(cont’d)
Fly Line
Description
Use
Locale
Targeted
Suppression of
Neuronal Activity
UAS-TeTxLC
Tetanus toxin light
chain
Blocks 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
Inactivated K+specific channel
Electrically silences
neurons
Membrane
UAS-dORKΔ-NC
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.
Gene Switch
P-element
enhancer
detection
GAL4~LBD of the
hprogR~p65 TXN
domain
RU486 activates this
expression system
Permits spatial control
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 Highthroughput 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 they
want with me?
Mice are close to humans
Kerstin Lindblad-Toh
Whitehead/MIT Center for Genome Research
Mouse sequence reveals great similarity with
the human genome
Extremely high conservation: 560,000 “anchors”
Mouse-Human Comparison
both genomes 2.5-3 billion bp long
> 99% of genes have homologs
> 95% of genome “syntenic”
Genomes are rearranged copies
of 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 conserved
segments 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 and Knockout Animals
Transgenic:
• Introduction of foreign or altered gene: transgenic
Over-expression, mis-expression, dominant-negative
Normal 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 gene
No normal allele - product of manipulated allele only
Generating Transgenic Mice