Lecture 7 Advanced epistasis Ectopic expression in Drosophila Morphogens: secreted ligands.

Lecture 7

• Advanced epistasis

• Ectopic expression in Drosophila

• Morphogens: secreted ligands

Epistasis

headless twinhead

Epistasis

headless hl twinhead th

hl lf

th lf

phenotype

no head; two tails

no tail; two heads

Epistasis

headless hl twinhead th

hl lf

th lf

phenotype

no head; two tails

no tail; two heads

Head

Tail

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

A B C D EW X Y Z

E is an essential metabolite

Phenotype of mutants in W, X, Y, Z allcan not grow without E in the media.

Mutant

W- X- Y- Z-

A - - - -

B + - - -

C + + - -

D + + + -

AddedtoMedia

E + + + +

Phenotype of mutants in W, X, Y, Z allcan not grow without E in the media.

Double mutants

Z-W- Z-X- Z-Y- Y-W- Y-X-

A - - - - -

B - - - - -

C - - - - -

D - - - + +

AddedtoMedia

E + + + + +

Phenotype of double mutants in W, X, Y, Z.

Order of epistasis

Z epi to Y epi to X epi to W

Example biochemical pathway

A B C D EW X Y Z

What intermediates accumulates in the mutant?

Accumulated intermediates

mutant intermediate

W- A

X- B

Y- C

Z- D

Double mutants

Double mutants intermediateW-X- AW-Y- AW-Z- AX-Y- BX-Z- BY-Z- C

Order of epistasis

W epi to X epi to Y epi to Z

Order of epistasis



Order of epistasis



When you assay along the pathway

When you assay at the end of the pathway

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

YFG

hsp

QualitiesA) high level of expressionB) expressed in all cellsC) expression is transient

Binary or two component system

Fly stock A: UAS line Fly stock B: Driver line

YFG

GAL4 binding sitesUASGAL4

GAL4Promoter

or Enhancer

YFG GAL4

Progeny

Binary or two component system

Fly stock A: UAS line Fly stock B: Driver line

YFG

GAL4 binding sitesUASGAL4

GAL4Promoter

or Enhancer

YFG GAL4

Progeny

GAL4

How do you make different GAL4 drivers?

A. Construct them

Known promoter

GAL4


B. Screen for them by enhancer trapping.

lacZp

P-element transposase promoter:very very weak expression-basically no expression by itself.

Enhancer detector

Enhancer structure of a portion of the genome

Gut

Brain

Gonad

Insertion of a enhancer detector

lacZ

GonadGut

Brain

Insertion of a enhancer detector

GonadGut

Brain

lacZ


B. Screen for them by enhancer trapping.

GAL4p

P-element transposase promoter:very very weak expression-basically no expression by itself.

Enhancer detector

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

YFGFRT FRT

y+, CD2Tubulinpromoter

Flip mediated ectopic expression

FLPhsp

Heat shock and induce FLP expression

YFG

FRT

FRT

y+, CD2

Tubulinpromoter

Flip mediated ectopic expressionHeat shock and induce FLP expression

YFGFRT

FRT

y+, CD2

Tubulinpromoter

Flip mediated ectopic expressionHeat shock and induce FLP expression

Results in a mosaic pattern of expression of YFG: recombination does not occur in all cells

Combination system I

YFGFRT FRT

y+, CD2UASGAL4

FLPhsp

GAL4Promoter

or enhancer


YFGFRT FRT

y+, CD2UASGAL4

FLPhsp

GAL4Promoter

or enhancer

Induce FLP expression


YFGFRT

UASGAL4

GAL4Promoter

or enhancer

What happens?

Cell expressing GAL4 Cells that have undergone a site specific recombination event

What happens?

Combination system II

GAL4FRT FRT

y+, CD2Tubulin

promoter

YFG

UASGAL4

FLPhsp


GAL4FRT FRT

y+, CD2Tubulin

promoter

YFG

UASGAL4

FLPhsp

Induce FLP expression


GAL4FRT

Tubulin

promoter

YFG

UASGAL4


GAL4FRT

Tubulin

promoter

YFG

UASGAL4

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

[morphogen]

Simplified Turing morphogen model

[morphogen]

Simplified Turing morphogen model

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

Tumor growth factor TGF/decapentaplegic dpp

WNT/wingless wg

Sonic hedgehog etc./hedgehog hh

TGF/DPP pathway

TGF/DPP

ligand

One eyed pinhead oepCo-receptor in zebrafish

Type1. thickvein

Type2, punt

Smads

One eyed pinhead

Schier et al. 1997 Development

Get similar phenotypes in mice by altering BMP signalling

Gilbert Developmental Biology 7th ed

TGF/DPP pathway

TGF/DPP

ligand

Type1. thickvein

Type2, punt

Smads

P

TGF/DPP pathway

TGF/DPP

ligand

Type1. thickvein

Type2, punt

Smads

P

P

TGF/DPP pathway

TGF/DPP

ligand

Type1. thickvein

Type2, punt

Smads

P

P

Nucleus

MadMedea

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

Wing imaginal discdpp mRNA

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

OMBSPT

Long range action

DPP expressedin this cell

Long range action


Directly

Affects expression in this cell

Signal relay system


indirectly

TGF/DPP pathway

TGF/DPP

ligand

One eyed pinhead oepCo-receptor

Type1. thickvein

Type2, punt

Smads


tkvQ253D

FRT FRT

y+, CD2UASGAL4

FLPhsp

GAL4ubiquitous

expression

UAS-tkvgf

CD2 expression 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

no tail expression

squint sqt

Zebrafish example

no tail expression

squint sqt

Zebrafish example

one eyed pinhead mutant

no tail expression

squint sqt

Zebrafish example

one eyed pinhead mutantWild-type cell

Lecture 7 Advanced epistasis Ectopic expression in Drosophila Morphogens: secreted ligands.

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Transcript of Lecture 7 Advanced epistasis Ectopic expression in Drosophila Morphogens: secreted ligands.