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

Epistasis headlesstwinhead

Epistasis headless hltwinhead th hl lf th lf phenotype no head; two tails no tail; two heads

Epistasis headless hltwinhead 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 ABCDE WXY Z E is an essential metabolite

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.

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 ABCDE WXY Z What intermediates accumulates in the mutant?

Accumulated intermediates

Double mutants

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 W epi to X epi to Y epi to Z Z epi to Y epi to X epi to W 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 Qualities A) high level of expression B) expressed in all cells C) expression is transient

Binary or two component system Fly stock A: UAS lineFly stock B: Driver line YFG GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer YFG GAL4 Progeny

Binary or two component system Fly stock A: UAS lineFly stock B: Driver line YFG GAL4 binding sites UASGAL4 GAL4 Promoter or Enhancer YFG GAL4 Progeny GAL4

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. lacZ p 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 Gonad Gut Brain

Insertion of a enhancer detector Gonad Gut Brain lacZ

How do you make different GAL4 drivers? B. Screen for them by enhancer trapping. GAL4 p 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

YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression FLP hsp Heat shock and induce FLP expression

YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression

YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression

YFG FRT y+y+, CD2 Tubulin promoter Flip mediated ectopic expression Heat shock and induce FLP expression Results in a mosaic pattern of expression of YFG: recombination does not occur in all cells

Combination system I YFG FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 Promoter or enhancer

Combination system I YFG FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 Promoter or enhancer Induce FLP expression

Combination system I YFG FRT UASGAL4 GAL4 Promoter or enhancer

What happens? Cell expressing GAL4Cells that have undergone a site specific recombination event

What happens?

Combination system II GAL4 FRT y+y+, CD2 Tubulin promoter YFG UASGAL4 FLP hsp

Combination system II GAL4 FRT y+y+, CD2 Tubulin promoter YFG UASGAL4 FLP hsp Induce FLP expression

Combination system II GAL4 FRT Tubulin promoter YFG UASGAL4

Combination system II GAL4 FRT 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

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 oep Co-receptor in zebrafish Type1. thickvein Type2, punt Smads

One eyed pinhead Schier et al 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 Mad Medea

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

A P

DPP is expressed in gradient Wing imaginal disc dpp 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 SPT

Long range action DPP expressed in this cell

Long range action DPP expressed in this cell Directly Affects expression in this cell

Signal relay system DPP expressed in this cell indirectly

 TGF/DPP pathway  TGF/DPP ligand One eyed pinhead oep Co-receptor Type1. thickvein Type2, punt Smads

Combination system I tkv Q253D FRT y+y+, CD2 UASGAL4 FLP hsp GAL4 ubiquitous expression

UAS-tkv gf CD2 expressionOMB expression

UAS-tkv gf CD2 expressionOMB expression

FLP mediated mitotic recombination with a tkv lf allele

TKV is required right out to the edge of OMB expression domain

Signal relay system DPP expressed in this cell 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 mutant Wild-type cell

no tail expression squint sqt Zebrafish example one eyed pinhead mutant Wild-type cell