Advanced epistasis

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Lecture 7

• Advanced epistasis
• Ectopic expression in Drosophila
• Morphogens secreted ligands

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Epistasis
headless
twinhead
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Epistasis
headless hl
twinhead th
phenotype
hl lf
no head two tails
th lf
no tail two heads
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Epistasis
headless hl
twinhead th
phenotype
Head
hl lf
no head two tails
Tail
th lf
no tail two heads
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Advanced Epistasis
Why do I always assume that the most epistatic
step is at the end of the pathway?
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Advanced Epistasis
Why do I always assume that the most epistatic
step is at the end of the pathway? Can we always
assume this?
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Example biochemical pathway
W
X
Y
Z
A
B
C
D
E
E is an essential metabolite
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Phenotype of mutants in W, X, Y, Z all can not
grow without E in the media.
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Phenotype of mutants in W, X, Y, Z all can not
grow without E in the media.
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Phenotype of double mutants in W, X, Y, Z.
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Order of epistasis
Z epi to Y epi to X epi to W
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Example biochemical pathway
W
X
Y
Z
A
B
C
D
E
What intermediates accumulates in the mutant?
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Accumulated intermediates
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Double mutants
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Order of epistasis
W epi to X epi to Y epi to Z
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Order of epistasis
W epi to X epi to Y epi to Z
Z epi to Y epi to X epi to W
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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
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Ectopic expression in Drosophila

• Heat-shock promoter fusion genes
• Binary or two component system
• Flip-mediated ectopic expression
• Combination system I
• Combination system II

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Heat-shock promoter fusion gene
hsp
YFG
Qualities A) high level of expression B)
expressed in all cells C) expression is transient
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Binary or two component system
Fly stock A UAS line
Fly stock B Driver line
Promoter
GAL4
YFG
or Enhancer
GAL4 binding sites UASGAL4
GAL4
YFG
Progeny
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Binary or two component system
Fly stock A UAS line
Fly stock B Driver line
Promoter
GAL4
YFG
or Enhancer
GAL4 binding sites UASGAL4
GAL4
GAL4
YFG
Progeny
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How do you make different GAL4 drivers?
A. Construct them
Known promoter
GAL4
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How do you make different GAL4 drivers?
B. Screen for them by enhancer trapping.
Enhancer detector
lacZ
p
P-element transposase promoter very very weak
expression-basically no expression by itself.
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Enhancer structure of a portion of the genome
Gonad
Gut
Brain
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Insertion of a enhancer detector
Gonad
Gut
lacZ
Brain
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Insertion of a enhancer detector
Gonad
Gut
lacZ
Brain
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How do you make different GAL4 drivers?
B. Screen for them by enhancer trapping.
Enhancer detector
GAL4
p
P-element transposase promoter very very weak
expression-basically no expression by itself.
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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

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Flip mediated ectopic expression
Tubulin promoter
y
, CD2
YFG
FRT
FRT
hsp
FLP
Heat shock and induce FLP expression
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Flip mediated ectopic expression
Heat shock and induce FLP expression
FRT
YFG
Tubulin promoter
FRT
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Flip mediated ectopic expression
Heat shock and induce FLP expression
FRT
YFG
Tubulin promoter
FRT
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Flip mediated ectopic expression
Heat shock and induce FLP expression
FRT
Tubulin promoter
YFG
FRT
Results in a mosaic pattern of expression of YFG
recombination does not occur in all cells
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Combination system I
UASGAL4
y
, CD2
YFG
FRT
FRT
Promoter or enhancer
hsp
FLP
GAL4
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Combination system I
UASGAL4
y
, CD2
YFG
FRT
FRT
Promoter or enhancer
hsp
FLP
GAL4
Induce FLP expression
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Combination system I
UASGAL4
YFG
FRT
Promoter or enhancer
GAL4
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What happens?
Cell expressing GAL4
Cells that have undergone a site specific
recombination event
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What happens?
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Combination system II
Tubulin promoter
y
, CD2
GAL4
FRT
FRT
UASGAL4
hsp
YFG
FLP
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Combination system II
Induce FLP expression
hsp
FLP
Tubulin promoter
y
, CD2
GAL4
FRT
FRT
UASGAL4
YFG
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Combination system II
Tubulin promoter
GAL4
FRT
UASGAL4
YFG
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Combination system II
Tubulin promoter
GAL4
FRT
UASGAL4
YFG
Results in clones of cells that express YFG
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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
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Simplified Turing morphogen model
morphogen
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Simplified Turing morphogen model
morphogen
Fate A
Fate B
Fate B
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Morphogen

• Long range action
• Forms a concentration gradient of activity
• Affect polarity
• Concentration dependent response
• Different fates at different concentrations

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Suspected secreted morphogens
bTumor growth factor TGF/decapentaplegic
dpp WNT/wingless wg Sonic hedgehog
etc./hedgehog hh
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bTGF/DPP pathway
bTGF/DPP
Type1. thickvein
ligand
Type2, punt
Smads
One eyed pinhead oep Co-receptor in zebrafish
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One eyed pinhead
Schier et al. 1997 Development
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Get similar phenotypes in mice by altering BMP
signalling
Gilbert Developmental Biology 7th ed
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bTGF/DPP pathway
bTGF/DPP
P
Type1. thickvein
ligand
Type2, punt
Smads
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bTGF/DPP pathway
bTGF/DPP
P
Type1. thickvein
ligand
Type2, punt
P
Smads
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bTGF/DPP pathway
bTGF/DPP
P
Type1. thickvein
ligand
Type2, punt
P
Smads
Mad Medea
Nucleus
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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

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Wing imaginal discs
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Wing imaginal discs
P
A
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DPP is expressed in gradient
dpp mRNA
Wing imaginal disc
DPP protein
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Spalt is a DPP responsive gene
Increase DPP and spalt response widens
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Spalt and octomotor blind are DPP responsive
SPT
OMB
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Spalt and octomotor blind are DPP responsive
SPT
OMB
SPT
OMB
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Long range action
DPP expressed in this cell
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Long range action
Directly
DPP expressed in this cell
Affects expression in this cell
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Signal relay system
indirectly
DPP expressed in this cell
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bTGF/DPP pathway
bTGF/DPP
Type1. thickvein
ligand
Type2, punt
Smads
One eyed pinhead oep Co-receptor
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Combination system I
UASGAL4
y
, CD2
tkvQ253D
FRT
FRT
ubiquitous expression
hsp
FLP
GAL4
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CD2 expression
OMB expression
UAS-tkvgf
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CD2 expression
OMB expression
UAS-tkvgf
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FLP mediated mitotic recombination with a tkvlf
allele
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FLP mediated mitotic recombination with a tkvlf
allele
TKV is required right out to the edge of OMB
expression domain
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Signal relay system
indirectly
DPP expressed in this cell
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Zebrafish example
squint sqt
no tail expression
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Zebrafish example
squint sqt
one eyed pinhead mutant
no tail expression
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Zebrafish example
one eyed pinhead mutant
Wild-type cell
squint sqt
no tail expression
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Zebrafish example
one eyed pinhead mutant
Wild-type cell
squint sqt
no tail expression