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Fly Genetics (fall 2012)
Pat O’Farrell [email protected] - 6-4707
Lecture 3 How genes function in space and time & Intro to the “Genetic tool box”
Genetics concepts new to this section:
P-element tranformation – the fly version of transformation.
The GAL4/UAS two component expression system.
Using FLP mediated site specific transformation at FRTs – clonal loss and gain of function.
Positive marking of clones.
RNAi – the fly version
Developmental
Principle: Cells whose fate is determined produce clones that are limited to specific tissues/areas.
Reciprocally, the distribution of daughter cells reveals the developmental potential of an earlier cell.
General reading:
This is very lucid description of key developmental concepts highlighted by clonal analysis in
Drosophila. Since I really screwed up the presentation of this, I recommend you take a look at this
pdf on line.
Crick FH, Lawrence PA. (1975) Compartments and polyclones in insect
development. Science 189, 340-7
Development of P element mediated transformation: Rubin GM and Spradling AC (1982) Genetic
Transformation of Drosophila with Transposable Element Vectors. Science 218, 348-353
The single hop insertional mutagenesis strategy: Cooley, L., Kelley, R., and Spradling A. (1988)
Insertional mutagenesis of the Drosophila genome with single P elements. Science 239, 1121- 8
This recent review outlines many of the diverse tools that have been developed on the basis of
regulated expression of transgenes by the GAL4 transcription factor. del Valle Rodríguez A,
Didiano D, Desplan C. (2011) Power tools for gene expression and clonal analysis in
Drosophila.Nat Methods. 9(1):47-55.
A technical paper that gives an excellent description of the process of producing positively marked
clones, which can also be used for ectopic expression of genes in specific cells and for RNAi
knockdown in specific cells.
Wu, JS and Luo L (2006) A protocol for mosaic analysis with
a repressible cell marker (MARCM) in Drosophila. Nature Protocols 1, 2583
Hybrid dysgenesis/Some strange biology
http://www.math.princeton.edu/~jgevertz/public_html_Pelement/indexpage.html
http://engels.genetics.wisc.edu/Pelements/Pt.html
For a few decades bizarre phenomena were reported when laboratory Drosophila melanogaster
were crossed to flies from the wild. If wild male flies were crossed to laboratory females, the
progeny had shriveled gonads, reduced fertility, significant recombination in males and increased
mutation.
Infection by Transposition Element
Remarkable detective work led to the realization that the wild flies
where carrying a transposition element and that laboratory flies
had been sheltered from a world wide sweep of transmission of
this transposition element. The dysgenesis was a reflection of
the infection strategies of the element.
Transposition elements: Pieces of DNA equipped with
mechanisms that lead to their movement from one DNA
sequence to another. They are considered selfish pieces of DNA
that parasitize other replicating molecules. There are three
modes of transposition, conservative (or cut and paste),
replicative and retrotransposition. The P element uses cut-andpaste.
The P element encodes an enzyme called transposase that cuts
out P elements by recognizing a 31-bp inverted terminal repeat
sequence at the ends of the element. The excision of the Pelement leaves a double strand break that needs to be repaired.
If excision occurs in G2, the break is likely to be repaired from the
sister, which still has its copy of the P element. Repair using the
homologous sequences on the sister restores the original P
element by gene conversion and leaves the excised copy to find
a new home. The transposase cuts a target sequence with a
staggered cut and end to end joining inserts the P and fill-in of
the staggers creates a short direct repeat at the site of insertion.
Regulation – the transposase includes an intron whose excision
is suppressed in somatic cells so that transposition is effectively
limited to the germline. It is the high level of germline
transposition activity that is thought to contribute to dysgenesis.
However, dysgenesis is down regulated once the P has
successfully taken up residence in a strain. This appears to be
due to repression of transposase by a product that accumulates in the infested strains. The
repressor appears to be generated as the result of an entirely different category of RNA
mechanism called PIWI interacting RNA (or piRNA) but there may also be an involvement of RNAi.
P element as a Transgenesis Tool
Transposase source: To control transposition the
transposase that catalyzes the event has been
separated from the transposing sequences. A
mutated derivative of a P element inserted at 99B
can no longer transpose acts as simple stable
transposase source. It is called delta 2-3 in
reference to its lack of an intron. Importantly, it can
be followed by its association a dominant third
chromosome marker, Kinked (Ki).
P element vectors: Plasmids that replicate in E.
coli (thin line) but with the Cis sites for
transposition — 31 bp sequences defining the two
ends of transposing sequence bracketing a marker
gene detectable in fly plus other useful stuff.
Generally, the marker is a w+ gene called
mini-white or just white (w). It is a
streamlined version of the endogenous white
gene. It behaves like a weak white gene
complementing w- mutants. The intensity of
eye color (orange to red) is a convenient
indicator of the number of P elements in a
strain. Special vectors give great flexibility.
Insertion of DNA: Injection of w+ marked P
element constructs into embryos with 2-3
results in transposition from the plasmid to the
genome. This occurs in a few cells, so the
flies arising from these injected embryos,
called G0 flies, are usually still white eyed.
However, if the marked P element inserts into
a germ cell precursor, some of the progeny
will carry the insert and have red eyes (w+).
Select w+ progeny that lack Ki — these will
lack the chromosome with the transposase
(2-3) so that the newly transposed element
will be stable. Cross w+ progeny to Balancers
to find out what chromosome it is on.
There are many applications. For example,
the upstream regulatory region of a gene with
a tissue specific, or position specific pattern of
expression can be placed up stream of a
reporter such as GFP and the regulatory
capacity of the upstream sequences assayed.
Mobilizing a P – to mutagenize/trap etc.
It is useful to hop insertional elements all over the genome. For this, use a chromosome that has
any dominant marker (Dom) and the [w+] P element you want to hop (ammunition chromosome).
Cross w-; Dom [w+] P flies to w-; 2-3 Ki flies and select males that are w-; Dom [w+] P; 2-3 Ki —
transposition occurs in these males. Cross these to any w- stock and search for progeny flies with
reddish eyes (w+) that lack Dom and lack Ki. Since no recombination in males, Dom and [ w+] P
as well as Ki and 2-3 will remain linked. Thus, progeny without Dom should lack w+ unless it has
transposed to a different chromosome. So w+ without Dom = movement of element. Furthermore,
the selected flies (lacking Ki) will lack transposase (2-3) so the new insertions are stable.
P element mutagenesis – a gene knockout collection: Insertions gives mutations that can
quickly be characterized molecularly. Elements are mobilized as above and if they insert in a gene
they will inactivate the gene partially or completely. Several labs worked together to knock out all
of the genes in the fly. They mobilized P elements or other transposition elements (e.g. minos).
New insertions were mapped to the genome sequence by inverse PCR and insertions that
interrupted coding sequences are held in a collection available to everyone. Most fly genes have
been knocked out or at least tagged with a nearby insertion element.
e.g Genes and insertions in 100 kb of genome (see FlyBase)
.
Enhancer trapping:
When a reporter gene is under the control of a minimal promoter,
it is usually inactive, but if the element inserts near endogenous
enhancers, it “traps” them, so that they direct expression of the
reporter. Many of the endogenous enhancers have refined
developmental programs of expression, and the reporter now
recapitulates these programs. This reveals the programs and
provides fantastic markers for cells, tissues etc, which can be
used in additional genetic analyses – e.g. screens for alterations
in the development of a
cell or tissue type.
Fig. An enhancer
trap marks dentridritic
projections of specific
sensory cells in the
epidermis of the larvae (right). The image to the left shows
the projections of one cell.
Two component systems e.g. The GAL4 System
The GAL4 transcription factor of
S. cerevisiae works in other
species where it will specifically
induce transcription of
promoters associated with the
appropriate enhancer UASGAL4.
When used in flies UASGAL4 is
usually referred to simply as
UAS. If a gene is under the
control of a UAS associated
minimal promoter, it can be
introduced into Drosophila on a
P element, but it usually will not
be expressed in the absence of
GAL4. If a second strain is
made that carries a P element
expressing GAL4, and the two
are crossed together, the UAS
associated promoter will be
activated.
GAL4 Drivers – the “two component system” of expression:
P elements in which GAL4 was expressed from a minimal promoter were constructed. When
introduced into flies these often were not expressed, but just as described for enhancer trapping, if
these elements are mobilized by 2-3 expression, they will move to new sites and at some of these
sites they will trap enhancers. A cross to a UAS-GFP line (reporter) will reveal cases in which
GAL4 is now expressed and will also reveal the spatial and temporal pattern of the expression.
Lines that express GAL4 in patterns are called drivers, because they can drive any UAS constructs
to be expressed in the same pattern. As we will see this has allowed targeted gene expression in
specific cells, targeted recombination, and targeted loss of gene function. Thousands of GAL4
drivers have been made, either by the described trapping procedure or by an alternative process in
which known enhancers are placed upstream of GAL4 to design a driver with known behavior. This
bipartite GAL4/UAS expression system has proved extraordinarily powerful.
Adding temporal control to the GAL4 system:
Several strategies have been created to control the timing of activation of UAS transgenes, but the
most flexible is based on GAL80. GAL80 binds to GAL4 and inhibits transcriptional activation. Its
widespread expression using the tubulin promoter blocks GAL4 dependent transcription. One of
its uses stems from the availability of ts allele of GAL80. In a three element system in which a
GAL4 dependent transcription of a UAS transgene is restrained due to the presence of a PtubGAL80ts, expression will be extinguished at low temperatures (18 -23 C) where the GAL80ts is
active and will induced at high temperatures where GAL80 repression is inactivated. This allows
control of gene expression in space (using the GAL4 driver specificity) and time (using temperature
shifts) to control expression of transgenes.
FLP/FRT System
The yeast FLP recombinase acts at FRT sites to induce recombination. This activity has been put
to great use in flies.
FLP based mitotic recombination to produce clones:
There were numerous disadvantages of using X-rays to induce mitotic recombination. For
example, there is significant cell lethality due to X-rays, the position of the induced recombination
event is random and frequency is low.
Now days, recombination is induced by FLP. Chromosomes have been produced with FRT sites
inserted at the base of each major chromosome arm (near the centromere). The figure shows an
example in which an FRT (blue box) is at the 'base" of the X chromosome and FLP mediated
recombination produces a twin spot with two marked cells – one marked with body color marker
yellow (y) and one marked with a twisted bristle marker singed (sn).
This is used to make marked clones, but it can also be used in mutant screens.
Numerous genes that are required for development of the eye are also required for earlier
processes such as embryogenesis. How can you genetically dissect the involvement of such
genes in eye development?
One solution is to make clones of the mutant later in development. If the
mutation is not “a cell lethal mutation” you should be able to get viable
cells. If the gene were absolutely required for cell division, how big would
your clones be? If you get clones you can examine the phenotype in a
tissue. To make this more directed, you could express FLP specifically in
the eye, so that clones would be produced in this location. How would
you do this? Can you explain how you might use the system you worked
out to do a screen for new mutations that are required for proper
development of the eye?
Note this is what an eye looks like if the fly carries a eyelessGAL4 (eye
specific), a UAS-FLP, heterozygous for w+ insert on II R, and has an FRT
at base of II R. In this case there is no mutation on II R that is required
for development of the eye.
A Little about the fly eye
The following is a cool site with a beautiful animation
illustrating the cellular anatomy of the fly eye, or
more precisely the anatomy of an ommatidium – one
of the 800 units that comprise the compound eye of a
fly.
http://www.sdbonline.org/fly/vdevlhom/movie.htm
Because each ommatidia is a sophisticated
multicellular structure and 800 of these units are
precisely arrayed in the normal eye, it constitutes a
remarkably sensitive reporter for accurate
developmental and cell biological performance. The
level of integrity of eye structure allows grading
scoring of defects from extremely subtle to incredibly
severe.
LEFT: Example of analysis of essential genes in
eye clones. Using eyeless FLP, clones are
produced specifically in the eye. P, Q and R show
pairs of images – the left is a light micrograph in
which the sister clones of mitotic recombination
(induced by FLP) are marked red (w+) or white (w-).
The image on the left is a scanning electron
micrograph that shows the lenses of the individual
ommatidia and the inter-ommadidial hair. In P and
P` the white tissue is wild type, in Q and Q` it is
mutant for mitochondrial enzyme (cytochrome
oxidase). Clearly the mutation messes up
development of ommatidia, but the mutant cells
largely survive. Not shown here are data suggesting
that the mitochondrial defect induces a "checkpoint"
that arrests the cell cycle by promoting p53
dependent destruction of cyclin E (key cell cycle
protein). R and R` show that when the fly has only
one copy of one of the key subunits of the
proteosome (protein degradation system
hypothesized to degrad cyclin E) the phenotype of
the cytochrome oxidase loss is partially suppressed
– check out the ommatida in white region of R
versus smooth in Q. .
Making the eye homozygous for one chromosome arm
e.g. 2L
Wt
FRT GRM-hid
FRT GMR-hid/FRT
+ EGUF/+
FRT GMR-hid CL/FRT
+EGUF
GMR = a promoter that drives expression late in eye development
hid – a gene whose expression induces an apoptotic program
Here GMR is directly driving hid expression and the construct is on 2L, and an FRT is on the base
of the same chromosome arm.
B) When hid expression is driven by GMR almost all eye cells are produced but then die
EGUF = shorthand for eyeless-GAL4, UAS-FLP
eyeless is a promoter that drives early and continuous expression in the eye precursor cells.
The EGUF constructs are not on chromosome 2
C) In this eye there is an FRT on both maternal and paternal 2Ls and recombination promoted by
EGUF produces two populations of cells – ones with and ones without GMR-hid. When
development reaches the late stages of eye development – half of the cells commit apoptosis
when they express hid. Thus the eye is reduced.
CL = a cell lethal mutation (also on 2L along with GMR-hid). Note that this is recessive lethal, and
the cells will only die when they become homozygous.
D) Early expression of FLP begins to produce homozygous cells early during development. The
cells homozygous for CL die and surviving cells compensate by increased growth and proliferation.
The population of heterozygous cells declines as the cells proliferate, because with each cycle,
FLP has a chance (~50%) of converting the cells to homozygosity. The CL/CL cells will die and be
replaced. The "w.t." cells will accumulate. Finally, late in development GMR-hid turns on and kills
the few remaining heterozygous cells, and the cells that are homozygous for the chromosome arm
lacking GMR-hid, and CL go on to produce a normal eye.
This eye is now homozygous for one arm of one chromosome. So what?
Well if the chromosome had been mutagenized and carried a mutant, it would be homozygous just
in the eye. Phenotypes could be scored, and because you don't need two flies to breed a
homozygote, the screen can be done with single flies (mass breeding, no vials and one less
generation – work it out – it makes things easier and allows screening for a new category of
mutants).
SEE - Stowers RS, et al. (2002) Axonal transport of mitochondria to synapses depends on milton,
a novel Drosophila protein. Neuron. 36(6):1063-77
Flip out cassette – Clonal expression
FLP is also useful in different strategy in which one induces clonal expression and/or loss of
expression of transgene.
FLP mediated recombination between tandem FRT elements excises the intervening DNA and
leaves single FRT element. In the design illustrated yellow, y, includes transcriptional termination
signals and is bracketed by FRTs. FLP expression induces excision of y, simultaneously allowing
expression of X. If X expression is compatible with cell survival, you will see y marked clones in
the adult that also are expressing X.
Positively Marked Clones
Genetic markers work well but cannot be followed immediately after making a clone, preventing
analysis of the early consequence of change in gene function. For this molecular markers such as
GFP are useful. However, while recessive genetic markers are only detected in the homozygous
cells of the clone, GFP is detected in the heterozygous cells. Consequently, the usual way of
setting up clones with molecular markers is to follow the loss of the marker in the clone. It is,
however, very difficult to follow small negative clones in a sea of positive signal.
The GAL80
repressor provides
a solution. A cell
that loses GAL80
can now express
GAL4 dependent
genes and thus
can be marked by
expression of a
UAS-GFP (see
diagram).
This is called the
MARCM technique
for “Mosaic
Analysis with a
Repressible Cell
Marker”
It is powerful
enough to mark
even single cells.
It is flexible
because the
marked cell will
also express any
other UAS gene
that is present.
RNAi in flies
Long dsRNA
Flies
works
Worms
works
Cell autonomy
autonomous
Transitive/*
intransitive
Amplification
Intransitive *
Spreads throughout
(except nervous system)
Transitive
Apparently not
Amplifies signal
Mammals
Doesn’t work: induces interferon
response that nonspecifically
shuts down translation (viral
defense strategy)
?
?
?
* Transitive: If you make dsRNA complementary to the right half of transcript, the biological
response will produce RNAi against both the left and right half of the transcript. This is apparently
due to RNA "replicase" (RNA dependent RNA polymerase) that can extend RNA primers to
produce new regions of dsRNA and hence program RISC complexes with RNA from all contiguous
sequences.
Intransitive: You only get RNAi to dsRNA sequences added. Importance: If you use
dsRNA representing the nonconserved 3` untranslated region of the actin 5C gene, RNAi against
the conserved part of the gene will not be produced and the treatment will not target the other 4
highly similar Actin genes.
RNAi in cell culture
Cell culture provides advantages for RNAi screening and RNAi in Drosophila cell culture is
uniquely simple.
Advantages of Drosophila S2 cells:
1. Take up long dsRNA directly from media
2. Because long dsRNA works, library construction is simple – robotic PCR from genomic
DNA, followed by symmetric PCR to dsRNAs of about 500bp to all of the genes of the
genome.
3. The whole genome can be screened in a time frame of 2 weeks to 2 months.
4. RNAi in S2 cells also provides a terrific system for experimental dissection. One, two, three
or more dsRNAs can be used to knockout combinations of genes, and film cellular
phenotypes (e.g. cytokinesis defect) can be filmed in real time using GFP tagged markers.
Echard, A., Hickson, GRX, Foley, E., and O’Farrell, PH. (2004) Terminal Cytokinesis Events Uncovered after an RNAi
Screen. Curr. Biol. 14(18):1685-93
Foldback transgenes
http://www.vdrc.at/rnai-library/
Two libraries –1. The GD Library:
2. The second library has inserts (10,714) in a common site. Similar except a new transgenic
technology that uses a phage site specific recombinase (phiC31) to insert RNAi constructs at
specific docking sites. The benefit is that the context effect on gene expression is removed.
Disadvantage – can’t be combined in large numbers because they are allelic.
In addition to VDRC, there are two other large libraries of RNAi constructs
– one in Boston and one in Kyoto. Boston also provides reagents and technical help for
cell based RNAi screens
RNAi knockdowns – in space and time
Since RNAi transgenes are under the control of UAS, they can be expressed ubiquitously or in any
tissue with the appropriate driver and they can be regulated by GAL80ts for temporal control, or by
GAL80 in the MARCM system to specifically inactivate the gene in individual marked cells.
Using gene dose, and temperature the severity of knockdown can be adjusted.
RNAi by injection
dsRNA can be injected into the early embryo and maternal RNAs can be eliminated or
accumulation of zygotic product blocked – maternally provided proteins are of course immune to
this.
Triple RNAi of the three mitotic cyclins of Drosophila by dsRNA injection into the anterior (left) pole
blocks subsequent mitoses and the domain of action expands with time to halt successive waves
of mitosis.
McCleland ML and O’Farrell, PH (2008) RNAi of Mitotic Cyclins in Drosophila Uncouples the Nuclear and Centrosome
Cycle. Curr Biol. 18(4):245-54
There are three large banks of RNAi stocks – Vienna (VDRC), Boston (DRSC) and Japan (NIG-FLY).