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Transcript
Lecture 7
• Advanced epistasis
• Ectopic expression in Drosophila
• Morphogens: secreted ligands
Epistasis
headless
twinhead
Epistasis
headless hl
twinhead th
phenotype
hl lf
no head; two tails
th lf
no tail; two heads
Epistasis
headless hl
twinhead th
phenotype
Head
Tail
hl lf
no head; two tails
th lf
no tail; two heads
Advanced Epistasis
Why do I always assume that the most
epistatic step is at the end of the pathway?
Advanced Epistasis
Why do I always assume that the most
epistatic step is at the end of the pathway?
Can we always assume this?
Example biochemical pathway
W
A
X
B
Y
C
Z
D
E is an essential metabolite
E
Phenotype of mutants in W, X, Y, Z all
can not grow without E in the media.
Phenotype of mutants in W, X, Y, Z all
can not grow without E in the media.
Mutant
W
-
X
-
Y
-
-
Z
Added A
to
B
Media
C
-
-
-
-
+
-
-
-
+
+
-
-
D
+
+
+
-
E
+
+
+
+
Phenotype of double mutants in W, X, Y, Z.
Double mutants
-
-
-
ZW ZX
-
-
-
-
-
-
ZY YW YX
Added A
to
B
Media
C
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
D
-
-
-
+
+
E
+
+
+
+
+
-
Order of epistasis
Z epi to Y epi to X epi to W
Example biochemical pathway
W
A
X
B
Y
C
Z
D
E
What intermediates accumulates in the mutant?
Accumulated intermediates
mutant
-
intermediate
W
A
X-
B
-
Y
C
Z-
D
Double mutants
Double mutants
- WX
- WY
- WZ
- XY
X-Z- YZ
intermediate
A
A
A
B
B
C
Order of epistasis
W epi to X epi to Y epi to Z
Order of epistasis
W epi to X epi to Y epi to Z
Z epi to Y epi to X epi to W
Order of epistasis
When you assay along the pathway
W epi to X epi to Y epi to Z
When you assay at the end of the pathway
Z epi to Y epi to X epi to W
Ectopic expression in Drosophila
• Heat-shock promoter fusion genes
• Binary or two component system
• Flip-mediated ectopic expression
• Combination system I
• Combination system II
Heat-shock promoter fusion gene
hsp
YFG
Qualities
A) high level of expression
B) expressed in all cells
C) expression is transient
Binary or two component system
Fly stock A: UAS line
Fly stock B: Driver line
Promoter
YFG
GAL4
or Enhancer
GAL4 binding sites
UASGAL4
GAL4
YFG
Progeny
Binary or two component system
Fly stock A: UAS line
Fly stock B: Driver line
Promoter
YFG
GAL4
or Enhancer
GAL4 binding sites
UASGAL4
GAL4
GAL4
YFG
Progeny
How do you make different GAL4 drivers?
A. Construct them
Known promoter
GAL4
How do you make different GAL4 drivers?
B. Screen for them by enhancer trapping.
Enhancer detector
p
lacZ
P-element transposase promoter:
very very weak expression-basically
no expression by itself.
Enhancer structure of a portion of the genome
Gonad
Gut
Brain
Insertion of a enhancer detector
Gonad
Gut
lacZ
Brain
Insertion of a enhancer detector
Gonad
Gut
lacZ
Brain
How do you make different GAL4 drivers?
B. Screen for them by enhancer trapping.
Enhancer detector
p
GAL4
P-element transposase promoter:
very very weak expression-basically
no expression by itself.
Qualities of Binary system
• Control temporal and spatial pattern of
YFG
• Expression is not transient
• Nonexpression of YFG from UASGAL4
allows transformants to be established
Flip mediated ectopic expression
Tubulin
promoter
y+, CD2
FRT
YFG
FRT
hsp
FLP
Heat shock and induce FLP expression
Flip mediated ectopic expression
Heat shock and induce FLP expression
y+, CD2
FRT
Tubulin
promoter
FRT
YFG
Flip mediated ectopic expression
Heat shock and induce FLP expression
y+, CD2
FRT
Tubulin
promoter
FRT
YFG
Flip mediated ectopic expression
Heat shock and induce FLP expression
y+, CD2
FRT
Tubulin
promoter
YFG
FRT
Results in a mosaic pattern of
expression of YFG: recombination
does not occur in all cells
Combination system I
UASGAL4
y+, CD2
FRT
Promoter
GAL4
or enhancer
YFG
FRT
hsp
FLP
Combination system I
UASGAL4
y+, CD2
FRT
Promoter
GAL4
or enhancer
YFG
FRT
hsp
FLP
Induce FLP expression
Combination system I
UASGAL4
YFG
FRT
Promoter
GAL4
or enhancer
What happens?
Cell expressing GAL4 Cells that have undergone
a site specific
recombination event
What happens?
Combination system II
Tubulin
promoter
hsp
y+, CD2
GAL4
FRT
FRT
UASGAL4
FLP
YFG
Combination system II
Induce FLP expression
hsp
FLP
Tubulin
promoter
y+, CD2
GAL4
FRT
FRT
UASGAL4
YFG
Combination system II
Tubulin
GAL4
promoter
FRT
UASGAL4
YFG
Combination system II
Tubulin
GAL4
promoter
FRT
UASGAL4
YFG
Results in clones of cells that express YFG
Morphogen
Alan Turing-a father of computational sciences
In 1952 publishes a paper entitled “A. M.
Turing on the chemical basis of
morphogenesis.”
Not recommended reading
Simplified Turing morphogen model
[morphogen]
Simplified Turing morphogen model
[morphogen]
Fate A
Fate B
Fate B
Morphogen
• Long range action
• Forms a concentration gradient of activity
• Affect polarity
• Concentration dependent response
• Different fates at different concentrations
Suspected secreted morphogens
bTumor growth factor TGF/decapentaplegic dpp
WNT/wingless wg
Sonic hedgehog etc./hedgehog hh
bTGF/DPP pathway
bTGF/DPP
Type1. thickvein
ligand
Type2, punt
Smads
One eyed pinhead oep
Co-receptor in zebrafish
One eyed pinhead
Schier et al. 1997 Development
Get similar phenotypes in mice by
altering BMP signalling
Gilbert Developmental Biology 7th ed
bTGF/DPP pathway
bTGF/DPP
P
ligand
Type1. thickvein
Type2, punt
Smads
bTGF/DPP pathway
bTGF/DPP
P
ligand
Type1. thickvein
Type2, punt
P
Smads
bTGF/DPP pathway
bTGF/DPP
P
ligand
Type1. thickvein
Type2, punt
P
Smads
Mad
Medea
Nucleus
DPP a morphogen?
• Long range action YES
• Forms a concentration gradient of activity YES
• Affect polarity YES
• Concentration dependent response YES
• Different fates at different concentrations YES
Wing imaginal discs
Wing imaginal discs
A
P
DPP is expressed in gradient
dpp mRNA
Wing imaginal disc
DPP protein
Spalt is a DPP responsive gene
Increase [DPP] and spalt response widens
Spalt and octomotor blind are DPP responsive
SPT
OMB
Spalt and octomotor blind are DPP responsive
SPT
OMB
SPT
OMB
Long range action
DPP expressed
in this cell
Long range action
Directly
DPP expressed
in this cell
Affects expression
in this cell
Signal relay system
indirectly
DPP expressed
in this cell
bTGF/DPP pathway
bTGF/DPP
Type1. thickvein
ligand
Type2, punt
Smads
One eyed pinhead oep
Co-receptor
Combination system I
UASGAL4
y+, CD2
FRT
ubiquitous
GAL4
expression
tkvQ253D
FRT
hsp
FLP
CD2 expression
UAS-tkvgf
OMB expression
CD2 expression
UAS-tkvgf
OMB expression
FLP mediated mitotic recombination
with a tkvlf allele
FLP mediated mitotic recombination
with a tkvlf allele
TKV is required right out to the
edge of OMB expression domain
Signal relay system
indirectly
DPP expressed
in this cell
Zebrafish example
squint sqt
no tail expression
Zebrafish example
squint sqt one eyed pinhead mutant
no tail expression
Zebrafish example
one eyed pinhead mutant
Wild-type
squint sqt
cell
no tail expression
Zebrafish example
one eyed pinhead mutant
Wild-type
squint sqt
cell
no tail expression