1.) Each cell in the Drosophila wing produces a hair that points towards the tip of
the wing. Researchers wondered how each cell knew which way the wing tip was.
Screens were carried out and mutants identified where the consistent hair
orientation is disrupted. You obtain one of the mutant strains which contains a
mutation in frizzled and decide to characterize it further.
Wildtype
PCP mutant
A: You first decide to ask whether frizzled is acting autonomously or nonautonomously to coordinate hair orientation. What technique will you use?
Ans: mosaic analysis
B: What is the genotype of the fly that you will analyze?
Ans: wing driver: flp/+; FRT fz/FRT tub:GFP
C: How will you obtain this fly? Assume you have three starting strains consisting
of 1) engrailed::FLP (on the second chromosome) 2) FRT, fz15 (on the third
chromosome) and 3) FRT tubulin:GFP. What fraction of the progeny will be what
you want?
Cross1 en:FLP x FRT tub:GFP
Cross 2 en:FLP/+; FRT tub:GFP/+ x FRT, fz
¼ of progeny will be right can be identified by looking for GFP- clones in the
wing.
D: What do you expect to see if frizzled is cell autonomous or non autonomous?
Note in this example the outlined clone will be dark, and the surrounding cells
will be GFP+. If the gene acts in a purely cell autonomous fashion, then the
mutant phenotype is restricted to the clone.
E: Talking to your lab mates you realize that lots of them are going to be doing the
same experiment you just did only using their favorite genes. As a public service
you decide to make the engrailed:FLP; FRT tubulin-GFP fly line. How will you go
about doing this? For this problem you cannot distinguish by fluorescence one copy
vs two of GFP and the engrailed: FLP is unmarked. Explain at every step how you
will select the flies you want. Hint: your lab has a L/Cyo; TM3/TM6 double balanced
stock.
Ans
En:FLP x L/Cyo; TM3/TM6 select non lobed progeny
En:FLP/Cyo; TM3/+ x L/Cyo; TM3/TM6 select non lobed both TM3 and TM6
progeny
En: FLP/Cyo; TM3/TM6 x L/Cyo; FRT tub:GFP/TM3(these flies obtained from
basically the same series of crosses only using the FRT line) select non lobed,
GFP positive w/ TM6 marker flies
En:FLP/Cyo; FRT tub:GFP/TM6 x to itself; select progeny w/o balancers
2.) You are an ambitious young fly geneticist studying the reproductive system.
After feeding your flies plenty of nasty chemicals, you create balanced stocks of two
interesting mutations. Neither mutations are lethal, but both result in adult female
sterility. Upon sequencing your mutants, you find truncations in two different genes
(Neg1 and Neg2, named for no viable eggs) that show homology to mammalian genes
involved in gonad formation.
A)First, you want to look at Neg1 + 2 protein expression in a wildtype fly. How
would you do this? (Assume that you are an expert at molecular biology, so shuffling
promoters and creating transgenic flies is a cake walk.) There are many possibilities,
but be sure to explain the advantages / disadvantages to any method you choose.
Many options:
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Pneg (= promoter region of Neg gene)  (driving) GFP
o Are you sure you get ALL of the regulatory elements? Maybe you will see
aberrant expression
Pneg  Gal4, UAS-GFP
o You still may miss some regulatory elements
o You may be able to use the Gal4 to drive something else later
o UAS-GFP / RFP / YFP are readily available
Neg-GFP fusion protein at endogenous locus
o More reliable expression pattern
o Fusion may interfere with function
Antibody stain
o Unlikely that a drosophila antibody and protocol exists for your new mutant
B) Your first experiments show Neg2 to be expressed in the posterior part of the
adult germarium (small, reproductive unit of the fly ovary) but you don’t see any
Neg1 expression. You wonder if Neg1 is expressed transiently during development.
Design a fly that would allow you to label cells that transiently express Neg1.


Pneg1  FLP, Tub-FRT-stop-FRT-GFP
Note that not all cells labeled will have expressed Neg1 at some point. You see all
cells that are descended from a transiently-Neg1-expressing cell, even if the
individual cells have never expressed the protein.
C.) You have noticed that Neg1 and Neg2 result in no cell polarization in the
epithelial cells surrounding the egg. You are interested in determining if your
phenotype is cell autonomous or non-cell autonomous, so you want to make
negatively marked clones. What two strains would you need in order to do this
experiment?
Strain 1: hsFLP/ hsFLP ; Neg1[mut], FRT/ cyo;
Strain 2: tub-gal4,UAS::lacZ(or UAS::GFP), FRT/ cyo
D.) For the two chromosomes undergoing recombination draw out what you would
expect to see.
E.) But, looking for absence of your reporter is annoying. What construct could you
make instead that would generate positively marked clones? Why is this sometimes
easier?
For small clones, this is easier to identify.
hsFLP; Neg1[mut], FRT / tub-gal80, FRT; UAS-GFP / tub-Gal4
This is equivalent to the MARCM technique for generating positively marked clones.
F.) Through careful BLAST searching, you discover that Drosophila has a well
conserved ortholog of a mouse gene that is important for germline development
known as Nubn3. You order a line that contains a P-element insertion that is
predicted to disrupt dNubn3. The line comes as a balanced stock
(P[w+]Nubn3/CyO). You cross siblings and recover no non Cy flies. You conclude
that dNubn3 is an essential gene, and the P-element insertion is recessive lethal.
Your PI says that the proper way to do this is to examine the phenotype of flies with
the insertion mutation over a chromosome with a deletion of 15 kb around, and
including dNubn3. Why is this the better method?
When you cross P[w+]/CyO with itself, you’ve homozygoused not only the
insertion, but the entire chromosome. It’s possible that the lethality is due not
to the insertion in dNubn3, but to some other mutation on the chromosome
(remember it’s a balanced stock, so the chromosome can accumulate
additional recessive lethal mutations without a selective disadvantage). This
is especially true for mutations generated by p-element mobilization.
Examining the transheterozygote (P[w+]nubn3/Def) is a more convincing
demonstration that the lethality is due to loss of Nubn3 function. The
assumption here is that the two different null alleles have different genetic
histories, so they are unlikely to share the same linked lethal.
G.) The transheterozygote (P[w+]Nubn3/Def) is still dead, the flies die as larvae.
You're still interested in determining if dNubn3 plays a role in egg production in the
adult female germline. How could you test this?
Use the GAL4/UAS system to drive Nubn3 RNAi specifically in the female
germline. Essentially combining UAS-shRNA (Nubn3) with a female germline
driven Gal4.
Or you could use mosaic analysis to induce mutant clones in the female
germline. Generate cells homozygous for the p-element insertion, in a
heterozygous background. You would need to do this early to allow the
mutant cells to populate most of the germline. Maybe even use the minute
technique to make them faster growing than the competing surrounding
heterozygous cells.
H.) Your analysis reveals that dNubn3 is required for a functional female germline.
Why do we need to be a little careful about over interpreting this result?
There could be a problem with the specificity of the phenotype. We’ve
essentially forced specificity by driving the RNAi/inducing mutant clones only
in the female germline. We need to be careful about what we say about the
actual biology of Nubn3 in the whole organism. For example driving RNAi
against a ribosomal protein in the germline would also give you a female
germline phenotype, even though translation has nothing specifically to do
with germline biology.