Complementation of lethal mutations

advertisement
Fly Genetics (fall 2012)
Pat O’Farrell ofarrell@cgl.ucsf.edu - 6-4707
Lecture 2 Screens & genetics of development
Note terrific review saying much of what I want to say. 1. D. St Johnston (2002) The
art and design of genetic screens: Drosophila melanogaster. Nat. Rev. Genet. 3, 176–188.
Genetics in this lecture:
• A synergy of the biology of Drosophila and its powerful genetics broke open the field of
developmental biology.
• A game changing genetic screen for embryonic mutations.
• Combining molecular and epistasis data to get clear conclusions.
• Making genetic mosaics using mitotic recombination reveals unexpected lineage barriers, reveals
the existence of epigenetics and defines its biological roles.
• Sensitized backgrounds and a focused approach to genetic screens
Developmental
Principles of building body form:
• Cells in different positions do different things.
• A grid of localized developmental regulators creates a coordinate system that tells a cell where it
is in an embryo. These patterning regulators are conserved among metazoans.
• Cells "remember" early embryonic position via a stable epigenetic program that causes cells from
different locations to do different things. The genes governing this epigenetic program were
defined in flies and are the focus of the biochemistry of chromatin structure.
General reading:
A superb review that covers a large part of what I am trying to get across in my lectures.
Eric Wieschaus Nobel Lecture – excellent description of thought behind the extraordinary screen
for all of the genes that acted zygotically control embryonic patterning (repeat of lect 1 suggestion).
http://nobelprize.org/nobel_prizes/medicine/laureates/1995/wieschaus-lecture.html
A paper that has influenced the direct of research in most branches of biology – a real game
changer. Nüsslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in
Drosophila. Nature. 1980 Oct 30;287(5785):795-801
Screening for alleles of l(epi)
Part c of the popsicle quiz.
Assume that you have a balanced stock of your starting lethal allele (let’s assume that it is on the
second chromosome and you use the Curly marked balancer SM5 to keep the stock).
l(epi)/SM5
Your problem is to select new alleles of this gene.
1. Mutagenize +/+
and cross to
L/SM5
= */L & */SM5
Pick one male fly (since you pick one fly for step 2, you are isolating and effectively “cloning an
individual * chromosome, here indicated as *1) and cross to fly with marked chromosomes.
2. *1/SM5
X l(epi)/SM5
= *1/ l(epi) & *1/SM5 & l(epi)/SM5 & SM5/SM5
not Curly
Curly
dead
If there are nonCurly progeny the *1 chromosome is viable “across from l(epi)” and therefore does
not carry a lethal allele of l(epi). Note: without balancers you would be searching for cross that
gave 25% lethality (i.e. 75% of normal number of progeny – really hard); here you just screen for
the presence (or absence) of flies with straight wings (incredibly easy).
In the screen for general lethals (end of last lecture) you distinguished relevant and non-relevant
vials (the ones with straight winged flies) in the progeny of the third cross (an F3 screen), while
here, the allele screen allows you to distinguish interesting and irrelevant vials after the second
cross (an F2 screen).
If you do step 2 many times (using a separate vial each time) you can clone many * chromosomes
and if you discard all vials that yield straight winged flies, you will be left with a bunch of vials each
containing a particular * chromosome (in trans to the Balancer) that is lethal over l(epi).
Assuming there are no recessive lethals other than l(epi) on the chromosome II that carries l(epi)
(is a realistic genetic caution – why?), all remaining * chromosomes will carry a lethal allele of
l(epi).
How do you retrieve the new mutant and tell it apart from the original l(epi) (ie. distinguish the two
types of Curly flies in the progeny of step 2)? You need at least one additional marker in the cross
*1/SM5
X l(epi)/SM5. In the above cross I achieved this by making green SM5 and black
SM5. Notice that you can tell track the non-balancer chromosome by tracking one of the
balancers. There is an obligate switch in the partnership of the green Balancer and the
chromosome being balanced so that before the cross the SM5-GFP was with the starting l(epi)
chromosome and after the cross it is with the newly mutant chromosome. To set up a stock of the
new mutant, pick the green, Curly flies and simply let them mate (make sure your females are
virgin when you set this up!). Remember that the swap of partnerships of chromosomes in a cross,
exemplified above by the GFP balancer, is a general and a reliable feature of Mendelian genetics.
It’s nice that we have lots of balancers available.
The Times are Changing
Screens for Mutants
Eras
*
Discovery
of the
unexpected
1. Genetics era: mutants were important
as markers to study genetics - segregation,
linkage, recombination, mutation and to
generate maps
*
*
2. Gene Discovery: mutations were originally the
way genes where discovered and defined
3. Dissect biology: mutations are
used as tools to figure out how
biological processes work
4. New era: all “genes” known - use mutations,
RNAi etc in conjunction with genetic manipulations
to figure out mechanisms of processes
A Little development
Genomic information dictates body structure. But how
do genes direct production of large scale form and
structure? How is space patterned? How are organs
and limbs sized and shaped?
In 1979Not a single developmental regulator was known.
Two centuries of study of development gave us
descriptions of development but not mechanism.
Signaling had been documented but no signal identified.
A Genetic Challenge
Find the genes governing development

Remember most of the molecular hardware for metabolism and normal cell function is already
deposited in the egg.
How many genes would be required to make the patterned embryo that hatches out of the egg
case 24 hr later?
What would these genes be required for?
Would their phenotypes (and eventual molecular characterization tell us anything about
development)?
Could approach this in comprehensive fashion, and find all of the required genes?
Considerations –
• Expect the mutations of interest to be lethal, likely embryonic lethal, and not to hatch.
• Interesting ones should make a cuticle with defective pattern.
• To get mutations in all of the genes that are involved, have to assay enough mutations to assay
a hit in every gene of the genome. Practical level – >5 times the allele frequency ~ 99% chance of
getting any one gene that is required and has ~ average allele frequency.
Saturation Screening
This was an unprecedented screen in terms of size, quality and ultimately impact. In doing the
screen the investigators fully vested themselves in the notion that they were dissected a process
and instead of just getting a mutation that affected it, they wanted to identify mutations in all genes
that affected the process. This grand view was give them remarkable insights into the process of
development.
EFFICIENCY: The beginning step in any standard screen in Drosophila is to mutagenize and
“collect” mutagenized chromosomes over a balancer as we have been through.
Mutagenize +/+
and cross to
L/SM5
= */L & */SM5
You then pick single males of */SM5 and set up crosses in single vials to screen for mutants. If
you screen for new allele of an existing mutation you will only get mutants in one gene and thus
only a few mutant. For this you have to set up thousands of crosses, each in it own vial. If you
were to screen for mutation in several genes at the same time (for example screening for mutations
in any of 20 genes removed by deletion) you would get 20 times as many mutations for similar
amount of work. OR extending this logic, if you screened for all the mutation affecting
development you could get a huge return on your investment.
THOROUGHNESS: In the above mutagenesis – if you mutagenize enough flies and screen
enough crosses to isolate an allele of a gene, you will done enough so that the probability of
getting a mutant in your gene is high. For example, at an “allele frequency” (measure of
mutagenesis efficiency) of 1/1000, you are likely(>99%) to find an allele if you screen 5,000 and
you often will get several alleles. If this is true for you gene, it is equally true for all the genes in the
chromosome. So if you screen 5, 000 mutagenized chromosomes for mutations that affect to
development, you have >99% chance of getting mutants in each of the genes that is involved in the
process. In other words, you will get pretty much all of them = saturation mutagenesis.
CLEVER: They developed new tricks to make it easier to handle the massive output.
How much they did and Yield (summary for chromosome II):
They screened 5,764 candidate mutagenized chromosomes.
These included 4,217 chromosomes that included a lethal.
Because some chromosomes have more than one lethal hit, this corresponds to 7,600 lethal hits.
25% of lethal lines included an embryonic lethal – i.e. 25% of the embryos did not hatch.
272 mutants (about 3.5% of lethal hits) had visible defects in the cuticular pattern.
These fell into 61 complementation groups.
Doing the Screen – the details
Presented for Chromosome II
Eric Wieschaus and Christiane Nüsslein-Volhard streamlined procedures to very efficiently identify
genes that affected the ability of the embryos to hatch and to screen for defects in the embryonic
cuticle.
They used a chromosome II Balancer
called CyO (atypical name, but typical
Balancer), and a dominant temperature
sensitive mutation (DTS) to help them
get rid of a class of unwanted progeny
and thereby save some work. This
was their set up (simplified a bit).

In the F1, mutagenized chrsomosomes
were over either CyO or DTS. Even
though the DTS is not balancer they
did not worry about recombination at
this step. Why? They set up 10,000 of
these F1 crosses.
They eliminated all the DTS progeny in
the F2. If the mutagenized
chromosome included a lethal, the only
viable progeny would be the newly
mutant chromosome over CyO. If
straight-winged progeny were included,
the cross was eliminated.
Embryos homozygous for the Balancer
hatch and die as larvae. To find
embryonic lethals, they looked at the
eggs when they should have hatched
(24 h after egg laying). If 25% fail to
hatch, there is an embryonic lethal.
They then looked at all candidate
embryonic lethals (?) for pattern defects.
Denticles – traction hairs on surface of larvae illustrate pattern
Dark field showing belts of ventral denticles as arranged in
wild type larva (or mature embryo). These are easily
visualized.
A detail view showing the finer features of pattern
in a denticle belt
Dilks and DiNardo
These patterns of denticles allowed investigators to distinguish the different
segments, and the details of organization within segments. Consequently they were
able to identify and distinguish mutants that altered these aspects of embryonic
pattern (among others).
What they found – zygotic patterning gene phenotypes
This is panel of mutant types from the Nature paper (see reading) that helped inform our
appreciation of development of pattern.
Expression and pattern
The genes discovered in the screen are required zygotically and they control development by
controlling where and when things happen. The gene products do not possess the spatial and
temporal information, rather this is encoded in their program of expression. Thus, the gene
products stimulate something to happen and where and when they do this depends on where and
when the genes are expressed. Where and when they are expressed turns out to due to the
regulatory interactions among the genes themselves – that is the pattern is emergent property of
the system. Fortunately, as the genetics suggested the organization of this system is simple in
principle even though it is complex in its details.
Here is the story
Molecular epistasis
Principle; Genes don’t act before they are expressed. If one mutation affects the onset of
expression of another, it acts upstream of it.
Mutations of pair rule genes delete every other segment. fushi tarazu , ftz, deletes odd
numbered abdominal segments, and paired, prd, deletes from the middle of an odd
numbered segment to the middle of an even numbered segement. If you stain for the
engrailed protein whose expression is usually induced in every segment primordial, you
find that ftz mutant embryos are missing half the engrailed stripes (called the even
numbered stripes just because they are numbered differently than the abdominal
segments) and prd embryos are missing the odd numbered engrailed stripes.
Engrailed antibody staining
wild-type
ftz
Odd stripes only
Therefore, ftz and prd function upstream of engrailed.
prd
Even stripes only
Mosaic analysis
Mutations can be made homozygous in the background of normal (heterozygous) fly by the
induction of recombination between a centromere and gene of interest.
Just marking cells (e.g. with little a versus A) tells us things about
lineage and growth during development.
Example –
The wings of flies have little hairs – one hair/cell that are orderly
arranged
Several mutations disturb these hairs. For example multiple
wing hair (mwh) has multiple and disorderly hairs. Clones
homozygous for mwh are readily visible on a background of
normal hairs (Fig right: a big clone of mwh/mwh in the anterior
part of the wing). When clones are induced by early X-ray
treatment, one tends to get few large clones, and when they are
induced later they occur at higher frequency but are smaller.
Why?
Clones reveal whether cell fate is coupled to
lineage (stereotyped positions of clones), or not.
Though elongated by morphogenic movements,
the wing clones are random, except that there is
line that they never cross, a “line of clonal
restriction”. This line defined two compartments,
the anterior and the posterior. This division is of
major importance in development.
More on Mosaic analysis
The Minute technique. The are a number of different genes (~60) with a similar dominant
phenotype: they have small (minute) bristles and slow growth. Many of these encode ribosomal
proteins and it is thought that they globally reduce protein synthesis when in one copy
(haploinsufficient). They are lethal when homozygous. Mitotic recombination of a Minute
heterozygote (M/+) will give one sister cell that is wild-type and is referred to as a “Minute plus” or
M+ clone, and one sister that is M/M and dies. The surviving M+ clone out grows the surrounding
cells and contributes disproportionately to the final structures, but all the pattering is normal. Such
M+ clones can fill an entire compartment (at least the surface visible part) indicating that one cell
had the potential to produce all the structures and was able to out compete all the other cells. But
size is normal and the clone never crosses into other compartments
Engrailed expression (left
antibody staining of wing
disc & right reporter
expression in wing) marks
the posterior compartment.
engrailed Specifies the Posterior Compartment of Each Segment
Normal
clones
Blue: enclones
Red: engof
clones
Sensitized screen/Dominant modifier Screens
Normal screens are work intensive. One of the best streamlined screening methods uses
mutations to cripple a pathway so that it gives visible phenotype that gets more or less severe with
even subtle changes in the activity of the crippled pathway = sensitized background.
FYI – an extraordinary fact. Removing or inactivating one copy of almost any diploid gene in flies
reduces the level of the product and the activity of the gene in half, or very close to half.
In a good sensitized background a heterozygous mutation of a gene in the crippled pathway (or a
modifier of the activity of the pathway) will change the phenotype – make the phenotype more
severe if the gene contributes positively to the pathway, or less severe if it is a negative regulator
of the pathway.
The benefit – you don’t have to use thousands of vials and carry your screen for multiple
generations, because now you are screening for dominant phenotypes (though the mutations
usually are recessive in normal background).
Hypomorphic mutants – give a weak phenotypes of two types.
Low penetrance – only a few of the flies show the phenotype
Low expressivity – all flies show a weak phenotype
Low expressivity phenotypes are useful for sensitization and screening and it is most sensitive if
the grade of phenotype is easily scored. The eye is especially well suited to this type of screening.
This scanning EM illustrates the 800 ommatidia of the fly eye. The eye is dispensable and
phenotypes representing cell cycle, development, receptor tyrosine kinase signaling, growth, cell
patterning, nerve cell function can all be screened and scored in clones.
to no eye
Wild type
Increasingly severe phenotype
Download