C. elegans epistasis
You are interested in studying vulval development in C. elegans! Because you really
truly enjoy examining the bag-of-worms phenotype that results when eggs hatch
inside their mother, and the creepiness of multi-vulval worms delights you!
Yup, pretty cool, right?
You have the genotypes listed on the following table. gf = over-expression (you have
a line with a transgene containing multiple copies of each gene) lf= complete loss of
function)
Single mutants
Multivulval
lin-15(lf)
lin-3(gf)
let-60(gf)
lin-1(lf)
lin-3(lf)
let-23(lf)
let-60(lf)
lin-45(lf)
Vulvaless
lin-3(lf)
let-23(lf)
let-60(lf)
lin-45(lf)
lin-15(lf)
Multivulval
Vulvaless
Vulvaless
Vulvaless
lin-3(gf)
Multivulval
vulvaless
No data
No data
let-60(gf)
Multivulval
Multivulval
Multivulval
Vulvaless
lin-1(lf)
Multivulval
Multivulval
Multivulval
Multivulval
A) Which genes in this pathway are positive regulators of vulval formation? Which
genes are negative regulators of vulval formation?
Negative regulators: lin-15, lin-3, lin-1, lin-45 (lin-45 is tricky because it’s
involved in double inhibition),
Positive regulators: let-60, let-23
B) How would you order these genes in a pathway?
Lin-3 --| lin-15 --| let-23  let-60  lin-45 --| lin-1 --| vulval formation
C) To complicate matters futher – another lab has recently identified another
mutant, glp-1 which lacks a germline altogether. glp-1 mutants also don’t have a
normal vulva. Can you order glp-1 in your pathway?
Purely from an epistatis pathway, yes. glp-1 since it ablates the germline
altogether is technically epistatic to all the lin- genes. However, in reality
because glp-1 specifies cells that are precursors to normal germline
development, glp-1; lin-15 double mutants don’t really tell you much about the
lin-15 pathway. This is similar to the original example Jeff used when
introducing epistatis (ie in a hairless mouse, talking about which gene
specifies coat color is not particularly informative)
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
Problem 1: You first decide to ask
whether frizzled is acting autonomously or non-autonomously to coordinate hair
orientation. What technique will you use?
Ans: mosaic analysis
Problem 2: What is the genotype of the fly that you will analyze?
Ans: wing driver: flp/+; FRT fz/FRT tub:GFP
Problem 3: 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.
Problem 4: What do you expect to see if frizzled is cell autonomous or non
autonomous?
Note: you have to look at the edge of the clone to check for autonomy vs non
autonomy. Fun fact: fz like all the PCP genes has both autonomous and non
autonomous effects.
Problem 5: 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